Heterodimeric antibodies binding to somatostatin receptor 2

Abstract

The present invention relates to antibodies that bind to somatostatin receptor 2 (SSTR2), comprising a novel antigen binding domain and a heterodimeric antibody.

Description

[0001] This application is a divisional application of the invention patent application filed on June 28, 2017, with application number 201780041351.9 and invention title "Heterodimeric antibody binding to somatostatin receptor 2".

[0002] Cross-reference to related applications

[0003] This application claims the benefits of U.S. Provisional Application No. 62 / 481,065, filed April 3, 2017; U.S. Provisional Application No. 62 / 397,322, filed September 20, 2016; U.S. Provisional Application No. 62 / 355,821, filed June 28, 2016; and U.S. Provisional Application No. 62 / 355,820, filed June 28, 2016, the contents of which are expressly incorporated herein by reference in their entirety.

[0004] sequence list

[0005] This application contains a sequence list, which has been submitted electronically in ASCII format and is hereby incorporated herein by reference in its entirety. The ASCII copy, created on June 28, 2017, is named 067461-5194-WO_SL.txt and has a size of 2,771,347 bytes. Background Technology

[0006] Antibody-based therapeutics have been successfully used to treat a variety of diseases, including cancer and autoimmune / inflammatory conditions. However, further improvements to these drugs are needed, specifically, to enhance their clinical efficacy. One approach being explored is to engineer novel antigen-binding sites into antibody-based drugs so that a single immunoglobulin molecule co-binds to two different antigens. This non-native or substituted antibody form that binds to two different antigens is commonly referred to as a bispecific antibody. A typical approach to achieving bispecificity is to introduce novel variable regions into antibodies, as the considerable diversity of antibody variable regions (Fv) makes it possible to generate Fvs that recognize virtually any molecule.

[0007] Various alternative antibody forms targeting bispecific targets have been explored (Chames and Baty, 2009, mAbs 1[6]:1-9; Holliger and Hudson, 2005, Nature Biotechnology 23[9]:1126-1136; Kontermann, mAbs 4(2):182(2012), all of which are explicitly incorporated herein by reference). First, bispecific antibodies were generated by fusing two cell lines that each produced a single monoclonal antibody (Milstein et al., 1983, Nature 305:537-540). Although the resulting hybridomas or tetralogous hybridomas did produce bispecific antibodies, they were only small populations, and extensive purification was required to isolate the desired antibody. An engineering approach to address this is to use antibody fragments to generate bispecific antibodies. Since such fragments lack the complex quaternary structure of full-length antibodies, variable light and heavy chains can be linked into a single gene construct. Many different forms of antibody fragments have been produced, including bifunctional antibodies, single-chain bifunctional antibodies, tandem scFv and Fab2 bispecific antibodies (Chames and Baty, 2009, mAbs 1[6]:1-9; Holliger and Hudson, 2005, Nature Biotechnology 23[9]:1126-1136; these references are explicitly incorporated herein by reference). Although these forms can be expressed at high levels in bacteria and have a permeation advantage due to their small size, they are rapidly cleared in vivo and present manufacturing barriers related to their preparation and stability. The main reason for these drawbacks is that antibody fragments typically lack antibody constant regions with the relevant functional properties of various Fc receptors and ligands, including larger size, high stability, and binding capacity to maintain a long half-life in serum (i.e., nascent Fc receptor FcRn) or to serve as binding sites for purification (i.e., proteins A and G).

[0008] Recent studies have attempted to address the shortcomings of fragment-based bispecific antibodies by introducing dual-binding engineering into full-length antibody-like forms (Wu et al., 2007, Nature Biotechnology 25

[11] :1290-1297; USSN12 / 477,711; Michaelson et al., 2009, mAbs 1[2]:128-141; PCT / US2008 / 074693; Zuo et al., 2000, Protein Engineering 13[5]:361-367; USSN09 / 865,198; Shen et al., 2006, Journal of Biochemistry (J Biol Chem) 281

[16] :10706-10714; Lu et al., 2005, Journal of Biochemistry 280

[20] :19665-19672; PCT / US2005 / 025472; These references are explicitly incorporated herein by reference. These forms overcome some of the obstacles of bispecific antibodies, mainly because they contain Fc regions. A significant drawback of these forms is that the binding to new antigens is always divalent because they establish new antigen-binding sites on the homodimeric constant chain.

[0009] For many antigens that are compelling as co-targets in bispecific therapeutic modalities, the desired binding is monovalent, not divalent. For many immune receptors, cellular activation is achieved through cross-linking of monovalent binding interactions. Cross-linking mechanisms are typically mediated by antibody / antigen immune complexes or through the binding of effector cells to target cells. For example, low-affinity Fcγ receptors (FcγRs), such as FcγRIIa, FcγRIIb, and FcγRIIIa, bind monovalently to the Fc region of antibodies. Monovalent binding does not activate cells expressing these FcγRs; however, upon immune complexation or cell-to-cell contact, the receptors cross-link and aggregate on the cell surface, leading to activation. For receptors responsible for mediating cell killing (e.g., FcγRIIIa on natural killer (NK) cells), receptor crosslinking and cell activation occur when effector cells bind to target cells in a highly affinity manner (Bowles and Weiner, 2005, J Immunol Methods 304:88-99, which are explicitly incorporated herein by reference). Similarly, the inhibitory receptor FcγRIIb on B cells downregulates B cell activation only when it binds to the cell surface B cell receptor (BCR) to form an immune complex, a mechanism mediated by immune complexation of soluble IgG with the same antigen recognized by the BCR (Heyman, 2003, Immunol Lett 88[2]:157-161; Smith and Clatworthy, 2010, Nature Reviews Immunology 10:328-343; these references are explicitly incorporated herein by reference). As another example, CD3 activation of T cells occurs only when their associated T cell receptor (TCR) binds to the antigen-carrying MHC on the antigen-presenting cell in a highly affinity cell-to-cell synaptic manner (Kuhns et al., 2006, Immunity 24:133-139). In fact, nonspecific bivalent cross-linking of CD3 with anti-CD3 antibodies induces cytokine storms and toxicity (Perruche et al., 2009, J Immunol 183[2]:953-61; Chatenoud and Bluestone, 2007, Nature Review Immunology 7:622-632; these references are explicitly incorporated herein by reference). Therefore, in actual clinical use, the preferred mode of CD3 co-conjugation for redirecting the killing of target cells is monovalent binding, which is activated only upon binding to the co-conjugated target.

[0010] Somatostatin is a neuropeptide that acts as an endogenous inhibitory regulator. It has a wide range of cellular functions, including the inhibition of various secretions, cell proliferation, and cell survival (Patel, 1999, Frontiers in Neuroendocrinology, 20:157-198). Somatostatin is widely distributed in the central nervous system, peripheral nervous system, pancreas, and intestines (see, for example, Watt et al., 2008, Molecular Cell Endocrinology, 286:251-261; Epelbaum, 1986, Progress in Neurobiology, 27:63-100; and Raynor, 1992, Crit. Rev. Neurobiology, 6:273-289). Somatostatin is also expressed in neuroendocrine tumors (NETs), such as medullary thyroid carcinoma, neuroblastoma, gangliomas, glucagonomas, adrenocortical tumors, and tumors found in the lungs, paraganglia, and duodenum, as well as in some other non-NETs (Volante et al., 2008, *Molecular and Cellular Endocrinology* 286: 219-229). Somatostatin can affect target cells by directly activating the somatostatin receptor (SSTR) (Watt et al., 2008, *Molecular and Cellular Endocrinology* 286: 251-261; Pyronnet et al., 2008, *Molecular and Cellular Endocrinology* 286: 230-237).

[0011] Somatostatin receptor (SSTR) belongs to the G protein-coupled receptor (GPCR) superfamily, each containing a single polypeptide chain consisting of an extracellular / intracellular domain and seven transmembrane domains. SSTR is highly expressed in a variety of cultured tumor cells and primary tumor tissues, including NETs (lung cancer, gastrointestinal cancer, pancreatic cancer, pituitary cancer, medullary carcinoma, prostate cancer, pancreatic lung carcinoid, osteosarcoma, etc.) and non-NETs (breast cancer, lung cancer, colorectal cancer, ovarian cancer, cervical cancer, etc.) (Reubi., 2003, Endocr. Rev. 24: 389-427; Volante et al., 2008, Molecular and Cellular Endocrinology 286: 219-229; and Schulz et al., 2003, Gynecol. Oncol. 89: 385-390). To date, five SSTR receptor subtypes have been identified (Patel et al., 1997, Trends in Endocrinology & Metabolism, 8:398-405). In particular, SSTR2 is expressed at high concentrations on various tumor cells (Volante et al., 2008, Molecular & Cellular Endocrinology, 286:219-229; and Reubi et al., 2003, European Journal of Nuclear Medicine & Molecular Imaging, 30:781-793), thus making it a candidate target antigen for bispecific antibody-based cancer targeted therapy. Given the high expression of SSTR2 on various tumors, anti-SSTR2 antibodies are considered suitable for, for example, antitumor therapies (e.g., chemotherapeutic agents and T-cell therapy) targeting tumors expressing SSTR2. For example, bispecific antibodies against SSTR2 and CD3 (which can target CD3+ effector T cells of tumors expressing SSTR2) are considered suitable for cancer therapy. However, bispecific antibodies derived from antibody fragments suffer from biophysical and pharmacokinetic obstacles, while those constructed using full-length antibody-like forms have the disadvantage of binding to auxiliary target antigens in a multivalent manner in the absence of the primary target antigen, leading to nonspecific activation and potential toxicity. This invention addresses this problem by introducing novel bispecific antibodies against SSTR2 and CD3. Summary of the Invention

[0012] Therefore, this article provides the somatostatin receptor 2 (SSTR2) antigen-binding domain and anti-SSTR2 antibodies (e.g., bispecific antibodies).

[0013] On the one hand, this paper provides an SSTR2 “opener” form antibody comprising: a) a first heavy chain comprising i) a first variant Fc domain and ii) a single-chain Fv region (scFv), wherein the scFv region comprises a first heavy chain variable domain, a first light chain variable domain, and a charged scFv linker, wherein the charged scFv linker is covalently linked to the first heavy chain variable domain and the first light chain variable domain; b) a second heavy chain comprising a VH-CH1-hinge-CH2-CH3 monomer, wherein VH is the second heavy chain variable domain and CH2-CH3 is the second variant Fc domain; and c) a light chain comprising a second light chain variable domain and a light chain constant domain. The second variant's Fc domain contains amino acid substitutions of N208D / Q295E / N384D / Q418E / N421D, while the first and second variants' Fc domains each contain amino acid substitutions of E233P / L234V / L235A / G236del / S267K; the first variant's Fc domain contains amino acid substitutions of S364K / E357Q, and the second variant's Fc domain contains amino acid substitutions of L368D / K370S. Additionally, the second heavy chain variable domain contains SEQ ID NO:1071, and the second light chain variable domain contains SEQ ID NO:1076, where the numbering is based on the EU index as described in Kabat.

[0014] In some embodiments of the SSTR2 “opener” form antibody, scFv binds to CD3. In some embodiments, the first heavy chain variable domain and the first light chain variable domain are each selected from the group consisting of: SEQ ID NO:1 and SEQ ID NO:5; SEQ ID NO:10 and SEQ ID NO:14; SEQ ID NO:19 and SEQ ID NO:23; SEQ ID NO:28 and SEQ ID NO:32; SEQ ID NO:37 and SEQ ID NO:41; and SEQ ID NO:46 and SEQ ID NO:50. In some embodiments, the first heavy chain variable domain comprises SEQ ID NO:1 and the first light chain variable domain comprises SEQ ID NO:5.

[0015] In some embodiments of the SSTR2 “opener” form antibody, the CH1-hinge-CH2-CH3 component of the second heavy chain comprises SEQ ID NO:1108, the Fc domain of the first variant comprises SEQ ID NO:1109, and the constant domain of the light chain comprises SEQ ID NO:1110.

[0016] In some embodiments, the first heavy chain contains SEQ ID NO:1080, the second heavy chain contains SEQ ID NO:1070, and the light chain contains SEQ ID NO:1075.

[0017] On the other hand, this article provides a type 2 somatostatin receptor (SSTR2) antigen-binding domain comprising a heavy chain variable domain having SEQ ID NO: 958 and a light chain variable domain having SEQ ID NO: 962.

[0018] On the other hand, this document provides a nucleic acid composition comprising a nucleic acid encoding any heterodimeric antibody or antigen-binding domain described herein.

[0019] On the other hand, this paper provides an expression vector that contains any of the nucleic acids described herein.

[0020] On the one hand, this article provides host cells transformed using any expression vector or nucleic acid described herein.

[0021] On the other hand, this document provides a method for preparing the heterodimeric antibody or antigen-binding domain described herein. The method comprises the steps of culturing host cells transformed with any expression vector or nucleic acid described herein under conditions expressing the antibody or antigen-binding domain, and recovering the antibody or antigen-binding domain.

[0022] On the one hand, this article provides a method for treating cancer, which includes administering any of the subject antibodies described herein to a patient in need. In some embodiments, the cancer is a neuroendocrine carcinoma. Attached Figure Description

[0023] Figures 1A to 1I Several forms of the invention are depicted. The first is a "bottle opener" form having first and second anti-antigen binding domains. Additionally, mAb-Fv, mAb-scFv, central scFv, central Fv, single-arm central scFv, single scFv-mAb, scFv-mAb, and double scFv forms are shown. For all depicted scFv domains, the N- to C-terminal heavy chain can be variable—(optionally, a linker)—the light chain can be variable, or vice versa. Furthermore, for the single-arm scFv-mAb, the scFv can be attached to the N-terminus of either the heavy chain monomer or the light chain. In some embodiments, "anti-antigen 1" in FIG1 refers to the anti-SSTR2 binding domain. In some embodiments, "anti-antigen 1" in FIG1 refers to the anti-CD3 binding domain. In some embodiments, "anti-antigen 2" in FIG1 refers to the anti-SSTR2 binding domain. In some embodiments, "anti-antigen 2" in FIG1 refers to the anti-CD3 binding domain. In some embodiments, "anti-antigen 1" in FIG1 refers to the anti-SSTR2 binding domain and "anti-antigen 2" in FIG1 refers to the anti-CD3 binding domain.

[0024] Figure 2Describe the amino acid sequences of the SSTR2 protein in humans and cynomolgus monkeys (Macaca fascicularis).

[0025] Figures 3A-3F Describing applicable pairs of heterodimeric variant groups (including skewed variants and pI variants). In Figure 3F Above, there are variants without a corresponding "monomer 2" variant; these are pI variants that can, for example, be used alone as a monomer, or included on the Fab side of the opener, and a suitable charged scFv linker can be used for a second monomer utilizing scFv as a second antigen-binding domain. Suitable charged linkers are shown in Figure 7A and 7B middle.

[0026] Figure 4 A series of isosteric variant antibody constant regions and their corresponding substitutions are depicted. pI_(-) indicates a lower pI variant, while pI_(+) indicates a higher pI variant. These variants can optionally and independently be combined with other heterodimeric variants of the present invention (as well as other variant types, as outlined herein).

[0027] Figure 5 Describe applicable ablation variants (sometimes referred to as "gene knockout" or "KO" variants) that bind to FcγR.

[0028] Figure 6 Two particularly applicable embodiments of the present invention are shown.

[0029] Figure 7A and 7B Various charged scFv linkers are described, which are used to increase or decrease the pI of the subject heterodimeric antibodies, as described herein, that utilize one or more scFvs as components. In particular, (+H) positive linkers are used here, especially in conjunction with the anti-CD3 vl and vh sequences shown herein. According to Whitlow et al., *Protein Engineering* 6(8): 989-995 (1993), a single prior art scFv linker with a single charge is called “Whitlow”. It should be noted that such linkers are used to reduce scFv aggregation and enhance the proteolytic stability of scFvs.

[0030] Figure 8 A variety of heterodimer skewed variants with amino acid substitutions that can be used with the heterodimer antibodies described herein are described.

[0031] Figures 9A-9EThe sequence shows several suitable opener-type backbones based on human IgG1 and lacking Fv sequences (e.g., scFv and vh and vl on the Fab side). Opener backbone 1 is based on human IgG1 (356E / 358M allotype) and includes the S364K / E357Q:L368D / K370S skewed variant, the N208D / Q295E / N384D / Q418E / N421D pI variant on the Fab side, and the E233P / L234V / L235A / G236del / S267K ablation variant on both strands. Opener backbone 2 is based on human IgG1 (356E / 358M allotype) and includes various skewed variants, N208D / Q295E / N384D / Q418E / N421D pI variants on the Fab side, and E233P / L234V / L235A / G236del / S267K ablation variants on both strands. Opener backbone 3 is based on human IgG1 (356E / 358M allotype) and includes various skewed variants, N208D / Q295E / N384D / Q418E / N421D pI variants on the Fab side, and E233P / L234V / L235A / G236del / S267K ablation variants on both strands. Opener backbone 4 is based on human IgG1 (356E / 358M allotype) and includes various skewed variants, N208D / Q295E / N384D / Q418E / N421D pI variants on the Fab side, and E233P / L234V / L235A / G236del / S267K ablation variants on both strands. Opener backbone 5 is based on human IgG1 (356D / 358L allotype) and includes S364K / E357Q:L368D / K370S skewed variants, N208D / Q295E / N384D / Q418E / N421D pI variants on the Fab side, and E233P / L234V / L235A / G236del / S267K ablation variants on both strands. Bottle opener backbone 6 is based on human IgG1 (356E / 358M allotype) and includes the skewed variant S364K / E357Q:L368D / K370S, the N208D / Q295E / N384D / Q418E / N421D pI variant on the Fab side, and the E233P / L234V / L235A / G236del / S267K ablation variant on both strands, as well as the N297A variant on both strands. Bottle opener backbone 7 is identical to bottle opener backbone 6, except that the mutation is N297S. Substitution forms of bottle opener backbones 6 and 7 may not include the ablation variant E233P / L234V / L235A / G236del / S267K on both strands.Backbone 8 is based on human IgG4 and includes the skewed variant S364K / E357Q:L368D / K370S, the N208D / Q295E / N384D / Q418E / N421D pI variant on the Fab side, and the E233P / L234V / L235A / G236del / S267K ablation variant on both strands, as well as the S228P (EU designation, which is S241P in Kabat) variant on both strands, with ablation Fab arm interchange as is known in the art. Alternative forms of opener backbone 8 may not include the ablation variant E233P / L234V / L235A / G236del / S267K on both strands. Backbone 9 is based on human IgG2 and contains the S364K / E357Q:L368D / K370S skewed variant, the N208D / Q295E / N384D / Q418E / N421D pI variant on the Fab side. Backbone 10 is based on human IgG2 and contains the S364K / E357Q:L368D / K370S skewed variant, the N208D / Q295E / N384D / Q418E / N421D pI variant on the Fab side, and the S267K variant on both strands.

[0032] As will be understood by those skilled in the art and outlined below, these sequences can be used with any vh and vl pairs outlined herein, where one monomer contains an scFv (optionally containing a charged scFv linker) and the other monomer contains a Fab sequence (e.g., vh linked to a “Fab-heavy chain” and vl linked to a “constant light chain”). That is, any Fv sequence outlined herein for anti-SSTR2 and anti-CD3, whether as an scFv (again, optionally having a charged scFv linker) or as a Fab, can be incorporated into these Figure 9 skeletons in any combination. Figure 9A The constant light chain depicted in the figure can be used for all structures in the figure, although the κ constant light chain can also be replaced.

[0033] It should be noted that these bottle opener frames are used in Figure 1F The central scFv form, wherein an additional second Fab (vh-CH1 and vl-constant light chains) with the same antigen binding as the first Fab is added to the N-terminus of the scFv on the "opener side".

[0034] Each of these backbones contains sequences that are 90%, 95%, 98%, and 99% identical to the listed sequences (as defined herein), and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions (compared to the “parent” of the figure, as will be understood by those skilled in the art, compared to parental human IgG1 (or IgG2 or IgG4, depending on the backbone). That is, the listed backbones may contain additional amino acid modifications (usually amino acid substitutions) in addition to the skewed, pI, and ablation variants contained in the backbone of this figure.

[0035] Figures 10A to 10DThe sequence of the mAb-scFv backbone used in this invention is shown, with the Fv sequence of this invention added. The mAb-scFv backbone 1 is based on human IgG1 (356E / 358M allotype) and includes the S364K / E357Q:L368D / K370S skewed variant, the N208D / Q295E / N384D / Q418E / N421D pI variant on the Fab side, and the E233P / L234V / L235A / G236del / S267K ablation variant on both strands. Backbone 2 is based on human IgG1 (356D / 358L allotypes) and includes the S364K / E357Q:L368D / K370S skewed variant, the N208D / Q295E / N384D / Q418E / N421D pI variant on the Fab side, and the E233P / L234V / L235A / G236del / S267K ablation variant on both strands. Backbone 3 is based on human IgG1 (356E / 358M allotype) and includes the skewed variant S364K / E357Q:L368D / K370S, the N208D / Q295E / N384D / Q418E / N421D pI variant on the Fab side, and the E233P / L234V / L235A / G236del / S267K ablation variant on both strands, as well as the N297A variant on both strands. Backbone 4 is identical to backbone 3, except that the mutation is N297S. The substitution forms of mAb-scFv backbones 3 and 4 may not include the ablation variant E233P / L234V / L235A / G236del / S267K on both strands. Backbone 5 is based on human IgG4 and contains the S364K / E357Q:L368D / K370S skewed variant, the N208D / Q295E / N384D / Q418E / N421D pI variant on the Fab side, and the E233P / L234V / L235A / G236del / S267K ablation variant on both strands, as well as the S228P (EU designation, which is S241P in Kabat) variant on both strands, with ablation Fab arm interchange as is known in the art. Backbone 6 is based on human IgG2 and contains the S364K / E357Q:L368D / K370S skewed variant and the N208D / Q295E / N384D / Q418E / N421D pI variant on the Fab side. Backbone 7 is based on human IgG2 and contains the S364K / E357Q:L368D / K370S skewed variant, the N208D / Q295E / N384D / Q418E / N421D pI variant on the Fab side, and the S267K variant on both strands.

[0036] As will be understood by those skilled in the art and outlined below, these sequences can be used with any vh and vl pairs outlined herein, where one monomer contains Fab and scFv (optionally containing a charged scFv linker) and the other monomer contains a Fab sequence (e.g., vh linked to a “Fab-side heavy chain” and vl linked to a “constant light chain”). That is, any Fv sequence outlined herein for anti-SSTR2 and anti-CD3, whether as scFv (again, optionally having a charged scFv linker) or as Fab, can be incorporated into the backbone of Figure 10 in any combination. Monomer 1 is the Fab-scFv pI negative side and contains heterodimeric variants L368D / K370S, isosteric pI variants N208D / Q295E / N384D / Q418E / N421D, and ablation variants E233P / L234V / L235A / G236del / S267K (all relative to IgG1). The monomer 2-side is the scFv pI positive side and contains the heterodimeric variant 364K / E357Q. However, others can be substituted, especially [S364K / E357Q:L368D / K370S]; [L368D / K370S:S364K]; [L368E / K370S:S364K]; [T411T / E360E / Q362E:D401K]; [L368D / K370S:S364K / E357L], [K370S:S364K / E357Q], [T366S / L368A / Y407V:T366W] and [T366S / L368A / Y407V / Y394C:T366W / S354C].

[0037] Figure 10A The constant light chain depicted in the figure can be used for all structures in the figure, although the κ constant light chain can also be replaced.

[0038] It should be noted that these mAb-scFv skeletons are applied to Figure 1H The mAb-Fv form (one monomer includes vl at the C-terminus and the other monomer includes vh at the C-terminus) and Figure 1E The scFv-mAb form (where the scFv domain is added to the C-terminus of one of the monomers).

[0039] Each of these backbones contains sequences that are 90%, 95%, 98%, and 99% identical to the listed sequences (as defined herein), and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions (compared to the “parent” of the figure, as will be understood by those skilled in the art, compared to parental human IgG1 (or IgG2 or IgG4, depending on the backbone). That is, the listed backbones may contain additional amino acid modifications (usually amino acid substitutions) in addition to the skewed, pI, and ablation variants contained in the backbone of this figure.

[0040] Figures 11A to 11G The amino acid sequence of the exemplary subject matter anti-SSTR2 antigen-binding domain described herein comprises: anti-SSTR2 H1.143_L1.30; anti-SSTR2 H1_L1.1; anti-SSTR2 H1.107_L1.30; anti-SSTR2 H1.107_L1.67; anti-SSTR2 H1.107_L1.108; anti-SSTR2 H1.107_L1.111; anti-SSTR2 H1.107_L1.114; anti-SSTR2 H1.107_L1.102; anti-SSTR2 H1.107_L1.110; anti-SSTR2 H1.125_L1.30; anti-SSTR2 H1.125_L1.67; anti-SSTR2 H1.125_L1.108; anti-SSTR2 H1.125_L1.111; anti-SSTR2 H1.125_L1.114; anti-SSTR2 H1.125_L1.102; and anti-SSTR2 H1.125_L1.10. The depicted sequences contain heavy chain variable (vh) and light chain variable (vl) domain sequences for each antigen-binding domain. For each vh sequence, vhCDR1, vhCDR2, and vhCDR3 sequences are underlined and highlighted in blue. For each vl sequence, vlCDR1, vlCDR2, and vlCDR3 sequences are underlined and highlighted in blue. As mentioned herein and indeed for each sequence containing a CDR, the precise identification of CDR locations may vary slightly depending on the numbering method used (as shown in Table 1), and therefore this document includes not only underlined CDRs but also CDRs contained within the vh and vl domains using other numbering systems. Furthermore, regarding all the sequences in the figure, these vh and vl sequences can be used in scFv form or in Fab form.

[0041] Figures 12A to 12FVarious anti-CD3 antigen-binding domains (e.g., anti-CD3 scFv) suitable for use with the subject antibodies presented herein are depicted. CDRs are underlined, and scFv linkers are double-underlined (in the sequence, the scFv linker is a positively charged scFv(GKPGS)4 linker, although this linker can be replaced by other linkers, including uncharged or negatively charged linkers, some of which are depicted in Figure 7, as will be understood by those skilled in the art). As above, the nomenclature indicates the orientation of the scFv from the N-terminus to the C-terminus; in the sequences listed in this figure, they are all oriented in a vh-scFv linker-vl (from N-terminus to C-terminus) manner. These sequences can also be reverse-oriented, i.e., (from N-end to C-end) vl-connector-vh. As mentioned herein and indeed for each sequence containing a CDR, the precise identification of the CDR position may vary slightly depending on the numbering method used (as shown in Table 1), and therefore this document includes not only underlined CDRs but also CDRs contained within the vh and vl fields using other numbering systems. Furthermore, regarding all sequences in the figure, these vh and vl sequences can be used in scFv form or Fab form.

[0042] Figure 12A The sequence of “high CD3” CD3_H1.30_L1.47 resistant constructs is depicted, which include heavy and light chain variable domains (CDRs with underlined lines), individual vl and vhCDRs, and scFv constructs with charged connectors (double underlined lines). This is true for all sequences depicted in the figure; these charged connectors can be replaced with uncharged connectors or different charged connectors as needed.

[0043] Figure 12B The sequence depicts a “high Int#l” anti-CD3_H1.32_L1.47 construct, which includes heavy and light chain variable domains (CDRs with underscores), individual vl and vhCDRs, and scFv constructs with charged connectors (double underscores). All sequences depicted in the figure are like this; these charged connectors can be replaced with uncharged connectors or different charged connectors as needed.

[0044] Figure 12C The sequence depicting the "High Int#2" anti-CD3_H1.89_L1.47 construct includes heavy and light chain variable domains (CDRs with underscores), individual vl and vhCDRs, and scFv constructs with charged connectors (double underscores). This is true for all sequences depicted in the figure; these charged connectors can be replaced with uncharged connectors or different charged connectors as needed.

[0045] Figure 12DThe figure depicts "High Int#3" resistant to CD3_H1.90_L1.47, which includes heavy and light chain variable domains (CDRs with underscores), individual vl and vhCDRs, and scFv constructs with charged connectors (double underscores). This applies to all sequences depicted in the figure; these charged connectors can be replaced with uncharged connectors or different charged connectors as needed.

[0046] Figure 12E The sequence depicting the “Int” anti-CD3_H1.33_L1.47 construct includes heavy and light chain variable domains (CDRs with underscores), individual vl and vhCDRs, and scFv constructs with charged connectors (double underscores). This applies to all sequences depicted in the figure; these charged connectors can be replaced with uncharged connectors or different charged connectors as needed.

[0047] Figure 12F The sequence depicting the “low” resistance to CD3_H1.31_L1.47 constructs includes heavy and light chain variable domains (CDRs with underlined lines), individual vl and vhCDRs, and scFv constructs with charged connectors (double underlined lines). This applies to all sequences depicted in the figure; these charged connectors can be replaced with uncharged connectors or different charged connectors as needed.

[0048] Figure 13A-13Z The amino acid sequences of stable, humanized anti-CD3 variant scFv variants (e.g., anti-SSTR2 X anti-CD3 “bottle opener” antibodies) that can be used with the bispecific antibodies described herein are depicted. 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. As mentioned herein and indeed for each sequence containing a CDR, the precise identification of CDR positions may vary slightly depending on the numbering method used (as shown in Table 1), and therefore, this document includes not only underlined CDRs but also CDRs contained within the vh and vl domains using other numbering systems. Furthermore, for all sequences in the figure, these vh and vl sequences may be used in scFv form or in Fab form.

[0049] Figure 14A and 14BThe amino acid sequences of the exemplary anti-SSTR2×anti-CD3 “bottle opener” bispecific antibody XENP018087 (SSTR2 H1.143_L1.30 and CD3 H1.30_L1.47) described herein are depicted. For the SSTR2 Fab-Fc heavy chain sequence, vhCDR l-3 is underlined and highlighted in blue, and the boundary between the heavy chain variable domain and the CH1-hinge-CH2-CH3 is indicated by a “ / ”. For the CD3 scFv-Fc heavy chain sequence, the boundaries between the various domains are indicated by a “ / ” and are as follows: scFv heavy chain variable domain / scFv linker / scFv light chain domain / Fc domain. vhCDR l-3 and vlCDR 1-3 are underlined in blue. For each scFv-Fc domain, the vhCDR 1-3 and vlCDR 1-3 sequences are underlined and highlighted in blue. For CD3 light chain sequences, vlCDR 1-3 are underlined and highlighted in blue, and the boundary between the light chain variable domain and the light chain constant domain is indicated by a " / ". The charged connector depicted is (GKPGS)4, although other charged or uncharged connectors may be used, such as... Figure 7A and 7B The linkers described herein. Furthermore, the sequences outlined herein may contain or not contain the M428L / N434S variant, which causes a longer half-life in serum, within one or preferably two Fc domains.

[0050] Figure 15A-15R This article describes another exemplary anti-SSTR2×anti-CD3 "bottle opener" bispecific antibody, which contains XENP018907 ( Figure 15A and 15B The amino acid sequences of SSTR2 H1.143_L1.30 and CD3 H1.32_L1.47 are shown. For the SSTR2 Fab-Fc heavy chain sequence, vhCDR l-3 is underlined and highlighted in blue, and the boundary between the heavy chain variable domain and CH1-hinge-CH2-CH3 is indicated by a " / ". For the CD3 scFv-Fc heavy chain sequence, the boundaries between the various domains are indicated by a " / " as follows: scFv heavy chain variable domain / scFv linker / scFv light chain domain / Fc domain. vhCDR l-3 and vlCDR 1-3 are underlined in blue. For each scFv-Fc domain, the vhCDR 1-3 and vlCDR 1-3 sequences are underlined and highlighted in blue. For the CD3 light chain sequence, vlCDR 1-3 is underlined and highlighted in blue, and the boundary between the light chain variable domain and the light chain constant domain is indicated by a " / ". The charged connector described is (GKPGS)4, although other charged or uncharged connectors may be used, such as Figure 7A and 7BThe linkers described herein. Furthermore, the sequences outlined herein may contain or not contain the M428L / N434S variant, which causes a prolonged half-life in serum, within one or preferably two Fc domains.

[0051] Figures 16A-16C This matrix depicts possible combinations of the exemplary bispecific anti-SSTR2×anti-CD3 antibodies described herein. "A" indicates that the CDR of the referenced CD3-binding domain sequence located at the top of the matrix can be combined with the CDR of the SSTR2-binding domain sequence listed on the left side of the matrix. For example, relative to "anti-SSTR2 H1.143_L1.30" and "anti-CD3 H1.30_L1.47", "A" represents a bispecific antibody comprising: a) a CD3-binding domain having a vhCDR with a variable heavy chain CD3 H1.30 sequence and a vlCDR with a variable light chain CD3 L1.47 sequence, and b) an SSTR2-binding domain having a vhCDR with an SSTR2 H1.143 sequence and a vlCDR with an SSTR2 L1.30 sequence. "B" indicates that the CDR of the CD3-binding domain construct can be combined with the heavy chain variable domain and the light chain variable domain of the SSTR2-binding domain construct. For example, relative to "anti-SSTR2 H1.143_L1.30" and "anti-CD3 H1.30_L1.47", "B" indicates a bispecific antibody containing: a) a CD3 binding domain of vhCDR with a variable heavy chain CD3 H1.30 sequence and a variable light chain CD3 L1.47 sequence, and b) an SSTR2 binding domain with an S heavy chain variable domain SSTR2H1.143 sequence and a light chain variable domain SSTR2 L1.30 sequence. "C" indicates a bispecific antibody containing: a) a CD3 binding domain with an anti-CD3 sequence heavy chain variable domain and a light chain variable domain, and b) an SSTR2 binding domain with an anti-SSTR2 sequence CDR. "D" indicates a bispecific antibody containing: an SSTR2 binding domain with a specified anti-SSTR2 sequence on both the variable heavy chain and variable light chain, and a CD3 binding domain with a specified anti-CD3 sequence on both the variable heavy chain and variable light chain. "E" indicates a bispecific antibody containing scFv, wherein the CD3 scFv is used in conjunction with the SSTR2 CDR. "F" indicates a bispecific antibody containing scFv, wherein the CD3 scFv is used in conjunction with both the heavy chain variable domain and the light chain variable domain of the SSTR2 antigen-binding domain. All these combinations can be in opener form, for example, with any of the backbone forms shown in Figure 9, or in substituted forms, such as the mAb-Fv, mAb-scFv, central scFv, central Fv, or dual scFv forms shown in Figure 1, containing... Figure 26The skeleton forms shown are illustrated. For example, “A” (CD3 CDR and SSTR2 CDR) can be added to the bottle opener sequence (including those sequences in Figure 9) or contain different heterodimeric variants, or added to the mAb-scFv skeleton, central scFv, mAb-Fv form, or central Fv form in Figure 10. However, in general, forms containing divalent CD3 binding are disadvantageous.

[0052] Figure 16D-16F Matrix depicting possible combinations of the exemplary bispecific anti-SSTR2 × anti-CD3 opener-form combinations described herein. In these matrices, anti-CD3 scFv is listed on the X-axis and anti-SSTR2 Fab on the Y-axis. “A” indicates that the CDR of the referenced CD3 binding domain sequence located at the top of the matrix can be combined with the CDR of the SSTR2 binding domain sequence listed on the left side of the matrix. For example, relative to “anti-SSTR2 H1.143_L1.30” and “anti-CD3 H1.30_L1.47”, “A” indicates a bispecific opener-form antibody comprising: a) an anti-CD3 scFV having a vhCDR of the variable heavy chain CD3 H1.30 sequence and a vlCDR of the variable light chain CD3 L1.47 sequence, and b) an anti-SSTR2 Fab having a vhCDR of the SSTR2 H1.143 sequence and a vlCDR of the SSTR2 L1.30 sequence. "B" indicates that the CDR of the CD3 binding domain construct can combine with the heavy chain variable domain and light chain variable domain of the SSTR2 binding domain construct. For example, relative to "anti-SSTR2 H1.143_L1.30" and "anti-CD3 H1.30_L1.47", "B" indicates a bispecific opener antibody containing: a) an anti-CD3 scFv with a vhCDR having a variable heavy chain CD3 H1.30 sequence and a vlCDR having a variable light chain CD3 L1.47 sequence, and b) an anti-SSTR2 Fab with an S heavy chain variable domain SSTR2 H1.143 sequence and a light chain variable domain SSTR2 L1.30 sequence. "C" indicates a bispecific opener antibody containing: a) an anti-CD3 scFv with an anti-CD3 sequence heavy chain variable domain and a light chain variable domain, and b) an SSTR2 Fab with an anti-SSTR2 sequence CDR. “D” indicates that the following bispecific opener antibodies are included: anti-SSTR2 Fab with a variable heavy chain and a variable light chain having a specified anti-SSTR2 sequence, and anti-CD3 scFv with a variable heavy chain and a variable light chain having a specified anti-CD3 sequence.

[0053] Figures 17A-17PThis study depicts exemplary cell surface binding assays of anti-SSTR2 antibody and anti-SSTR2×anti-CD3 bispecific antibody in CHO cells transfected with human SSTR2. Binding was measured by flow cytometry using a secondary antibody labeled with phycoerythrin (PE).

[0054] Figures 18A-18D The results of a redirected T cell cytotoxicity (RTCC) analysis were depicted using anti-SSTR2×anti-CD3 bispecific antibodies and human SSTR2-transfected CHO cells.

[0055] Figures 19A-19C The description describes the use of anti-SSTR2 × anti-CD3 bispecific antibodies with TT cells (human thyroid medullary carcinoma cell line, Figures 19A-19C The results of the redirected T cell cytotoxicity (RTCC) analysis.

[0056] Figure 20A and 20B Describing the effect of anti-SSTR2×anti-CD3 bispecific antibody on CD4 in cynomolgus monkeys + and CD8 + T cell activation ( Figure 20A ) and CD4 + and CD8 + T cell distribution ( Figure 20B Research on the impact of ).

[0057] Figures 21A-21D Describing the effect of anti-SSTR2×anti-CD3 bispecific antibody on CD4 in cynomolgus monkeys + and CD8 + T cell activation ( Figure 21A ) and CD4 + + and CD8 + +T cell distribution ( Figure 21B Further research is needed on the effects of glucose tolerance tests (GTT). Figure 21C and 21D This was used to assess the ability of the tested subjects to break down glucose.

[0058] Figures 22A-22F Depicting exemplary anti-SSTR2×anti-CD3 bispecific antibodies against CD4 + and CD8+ T cell activation ( Figure 22A and 22B CD4 + and CD8 + T cell distribution ( Figure 22C and 22D) and serum levels of IL-6 and TNFα ( Figure 22E and 22F (and other studies.)

[0059] Figure 23 A-23C depicts XmAb 18087 and XENP13245 in human SSTR2-transfected CHO cells ( Figure 22A CHO cells transfected with SSTR2 from cynomolgus monkeys ( Figure 22B ) and untransfected parental CHO cells ( Figure 22C Cell surface binding analysis on ).

[0060] Figure 24 A-24C depiction using XmAb18087 (squares) and XENP13245 (circles) with human SSTR2-transfected CHO cells ( Figure 24 A) TT cells (human thyroid medullary cancer cell line) Figure 24 B) or A548 cells (lung adenocarcinoma cell line, Figure 24 Results of the redirected T cell cytotoxicity (RTCC) analysis (C).

[0061] Figure 25 The results of a redirected T-cell cytotoxicity (RTCC) assay using anti-SSTR2×anti-CD3 bispecific and control anti-SSTR2 mAh and anti-RSV×anti-CD3 with TT cells (human thyroid medullary carcinoma cell line) or A548 cells (lung cancer) are depicted.

[0062] Figure 26 A-26B depicted in Figure 2 In the experiment described, CD4 cells cultured from CHO cells transfected with human SSTR2 were cultured 24 hours later. + and CD8 + T cells ( Figure 26 A) and TT cells ( Figure 26 Upward adjustment of CD69 on B). Solid data points indicate CD8. + T cells were present in CD69 MFI, and the empty data points indicated CD4. + CD69 MFI on T cells.

[0063] Figure 27 The design of a mouse study to examine the antitumor activity of XmAb18087 is described.

[0064] Figure 28 A-28B depiction The measured tumor size varies with time and treatment.

[0065] Figure 29 Depicting Bioluminescence image (day 28 after administration of drug #1).

[0066] Figure 30 A-30B depicts XmAb18087 versus CD4 in cynomolgus monkeys. + ( Figure 30 A) and CD8 + ( Figure 30 B) Research on the influence of T cell distribution.

[0067] Figure 31 A-31B depicts XmAb18087 versus CD4 in cynomolgus macaques. + ( Figure 31 A) and CD8 + ( Figure 31 B) Research on the effects of T cell activation.

[0068] Figure 32 A-32B describes the effect of XmAb18087 on serum IL-6 and TNF levels in cynomolgus monkeys.

[0069] Figure 33 Described as by Tumor size was measured in NSG mice transplanted with A549-RedFLuc tumor cells and human PBMCs, and varied with time and treatment with multiple concentrations of XmAb18087. Detailed Implementation

[0070] A. Incorporation of materials

[0071] Figures and legends

[0072] All figures and accompanying illustrations of USSN 62 / 481,065; 62 / 397,322; 62 / 355,821 and 62 / 355,820 are explicitly and independently incorporated herein by reference in their entirety, especially the amino acid sequences depicted therein.

[0073] sequence

[0074] The following is a reference to the attached sequence listing. Anti-SSTR2 sequences suitable for ABD contain the heavy chain variable domain, light chain variable domain, and CDR of the anti-SSTR2 heavy and light chain sequences of SEQ ID NO:958-1069 (Figure 11) and SEQ ID NO:58 to 659. Anti-CD3 sequences suitable for ABD contain the heavy chain variable domain, light chain variable domain, and CDR contained in SEQ ID NO:1-54 (Figure 12) and SEQ ID NO:835 to 938. The heavy chain variable domain, light chain variable domain, and CDR may be contained in the scFv or Fv form of the subject antibody and antigen-binding domain described herein.

[0075] The sequences of exemplary bispecific SSTR2×CD3 antibodies are contained in SEQ ID NO: 1070 to 1088 (Figure 14); and SEQ ID NO: 1089 to 1107 and 660 to 806 (Figure 15).

[0076] B. Overview

[0077] This article presents anti-SSTR2 antibodies suitable for cancer treatment. Because of their high expression in neuroendocrine tumors (NETs, ​​such as lung cancer, gastrointestinal cancer, pancreatic cancer, pituitary cancer, medullary carcinoma, prostate cancer, pancreatic lung carcinoid tumors, osteosarcoma, etc.) and non-NETs (breast cancer, lung cancer, colorectal cancer, ovarian cancer, cervical cancer, etc.), anti-SSTR2 antibodies are considered suitable for targeting antitumor therapeutics (e.g., chemotherapy agents and T cells) against these SSTR2-expressing tumors. Specifically, this article presents a bispecific anti-CD3, anti-SSTR2 antibody. This antibody is used to direct CD3+ effector T cells to SSTR2+ tumors, thereby allowing CD3+ effector T cells to attack and lyse SSTR2+ tumors.

[0078] Anti-bispecific antibodies that co-conjugate to CD3 and tumor antigen targets have been engineered for redirecting T cells to attack and lyse targeted tumor cells. Examples include BiTE and DART forms that monovalently conjugate to CD3 and tumor antigens. While CD3-targeting approaches have shown considerable promise, a common side effect of such therapies is the associated production of cytokines, often leading to toxic cytokine release syndrome. Because the anti-CD3 binding domain of bispecific antibodies binds to all T cells, they recruit a subset of CD4 T cells that produce large amounts of cytokines. Moreover, the CD4 T cell subset includes regulatory T cells, whose recruitment and expansion can potentially cause immunosuppression and negatively impact long-term tumor suppression. Furthermore, these forms lack an Fc domain and exhibit a very short serum half-life in patients.

[0079] While CD3-targeting approaches have shown considerable promise, a common side effect of such therapies is the associated production of cytokines, often leading to toxic cytokine release syndrome. Because the anti-CD3 binding domain of bispecific antibodies binds to all T cells, it recruits a subset of CD4 T cells that produce large amounts of cytokines. Moreover, the CD4 T cell subset includes regulatory T cells, whose recruitment and expansion can potentially cause immunosuppression and negatively impact long-term tumor suppression. One feasible way to reduce cytokine production and potentially reduce CD4 T cell activation is by decreasing the affinity of the anti-CD3 domain for CD3.

[0080] Therefore, in some embodiments, the present invention provides antibody constructs comprising an anti-CD3 antigen-binding domain, which is a "strong" or "high-affinity" binder to CD3 (e.g., one example is a heavy chain variable domain and a light chain variable domain described as H1.30-L1.47 (optionally containing a charged linker (where appropriate))) and also binds SSTR2. In other embodiments, the present invention provides antibody constructs comprising an anti-CD3 antigen-binding domain, which is a "weak" or "low-affinity" binder to CD3. Further embodiments provide antibody constructs comprising an anti-CD3 antigen-binding domain, which has a moderate or "intermediate" affinity for CD3 and also binds CD38. Affinity is typically measured using Biacore assays.

[0081] It should be understood that the "high, medium, and low" anti-CD3 sequences of this invention can be used in various heterodimeric forms. While most of this disclosure uses the "bottle opener" form of heterodimers, these variable heavy and light chain sequences, as well as the scFv sequence (and the Fab sequence containing these variable heavy and light chain sequences), can be used in other forms, such as those described in WO Publication No. 2014 / 145806. Figure 2 The forms described therein, including the diagrams, forms and illustrations, are explicitly incorporated into this text by reference.

[0082] Therefore, this article provides heterodimeric antibodies that bind to two different antigens, for example, "bispecific" because they bind to two different target antigens, typically SSTR2, as described below. These heterodimeric antibodies can bind to these target antigens monovalently (e.g., having a single antigen-binding domain, such as a heavy chain and light chain variable domain pair) or bivalently (having two antigen-binding domains that each independently binds an antigen). The heterodimeric antibodies provided herein are based on the use of different monomers containing amino acid substitutions that "skew" the formation of heterodimers rather than homodimers, as described more fully below; and "pI variants" that allow for easy purification of heterodimers from homodimers, as described similarly below. The provided heterodimeric bispecific antibodies generally rely on the use of engineered Fc domains or variant Fc domains that can self-assemble in production cells to generate heterodimeric proteins; and methods for generating and purifying such heterodimeric proteins.

[0083] C. Nomenclature

[0084] The bispecific antibodies of this invention are listed in several different forms. Each polypeptide is given a unique “XENP” number, but as will be understood in the art, longer sequences may have shorter numbers. For example, for a given sequence, the heavy chain of the scFv side monomer in opener form will have a first XENP number, while the scFv domain will have a different XENP number. Some molecules have three polypeptides, and therefore use the XENP number along with the component as the name. Thus, the molecule XENP18087, which is in opener form, comprises three sequences: “XENP 18087HC-Fab” (…). Figure 14A (referred to as "SSTR2 Fab-Fc heavy chain"); "XENP18087 HC-scFv" ( Figure 14B This is referred to as "CD3 scFv-Fc heavy chain"; and "XENP18087 LC" ( Figure 14A These are referred to as “SSTR2 light chains” or equivalents, although those skilled in the art will be able to easily identify them via sequence alignment. These XENP numbers appear in the sequence listing and identifiers, and are used in the figures. Furthermore, a molecule comprising three components generates multiple sequence identifiers. For example, the sequence listing for the Fab monomer has a full-length sequence, a variable heavy chain sequence, and three CDRs for the variable heavy chain sequence; the light chain has a full-length sequence, a variable light chain sequence, and three CDRs for the variable light chain sequence; and the scFv-Fc domain has a full-length sequence, an scFv sequence, a variable light chain sequence, three light chain CDRs, an scFv linker, a variable heavy chain sequence, and three heavy chain CDRs; it should be noted that all molecules in this document containing an scFv domain use a single charged scFv linker (+H), although others may be used. Furthermore, the terminology for specific variable domains is named using the format “Hx.xx_Ly.yy”, where the number serves as a unique identifier for the specific variable chain sequence. Therefore, the variable domain on the Fab side of XENP18087 is “H1.143_L1.30”, which indicates the combination of the heavy chain variable domain H1.143 and the light chain domain L1.30. When these sequences are used in scFv form, the name “H1.143_L1.30” indicates the combination of the heavy chain variable domain H1.143 and the light chain domain LUO, oriented vh-linker-vl from the N-terminus to the C-terminus. A molecule with sequences consistent with but in the reverse order of the heavy and light chain variable domains would be named “L1.30_H1.143”. Similarly, different constructs may “mix and match” heavy and light chains, as will be evident from the sequence listing and figures.

[0085] D. Definition

[0086] To provide a more comprehensive understanding of this application, several definitions are set forth below. These definitions are intended to cover syntactic equivalents.

[0087] In this context, "ablation" refers to a reduction or removal of activity. Therefore, for example, "ablation of FcγR binding" means that an Fc region amino acid variant has less than 50% initial binding compared to the Fc region without the specific variant, preferably greater than 70-80-90-95-98% activity loss, and generally, activity below the level detectable in Biacore, SPR, or BLI analyses. The specific application of FcγR binding ablation is... Figure 5 Those shown in the diagram are typically added to two monomers.

[0088] As used herein, "ADCC" or "antibody-dependent cell-mediated cytotoxicity" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize bound antibodies on target cells and subsequently cause target cell lysis. ADCC is associated with binding to FcγRIIIa; increased binding to FcγRIIIa leads to increased ADCC activity.

[0089] As used herein, “ADCP” or “antibody-dependent cell-mediated phagocytosis” refers to a cell-mediated response in which nonspecific phagocytes expressing FcγR recognize bound antibodies on target cells and subsequently induce phagocytosis of the target cells.

[0090] In this document, "antigen-binding domain" or "ABD" refers to a group of six complementary determining regions (CDRs) that, when present as part of a polypeptide sequence, specifically bind to the target antigen as discussed herein. Therefore, a "checkpoint antigen-binding domain" binds to the target checkpoint antigen as outlined herein. As is known in the art, these CDRs typically exist as a first group of variable heavy chain CDRs (vhCDRs or VHCDRs) and a second group of variable light chain CDRs (vlCDRs or VLCDRs), each comprising three CDRs for the heavy chain: vhCDR1, vhCDR2, and vhCDR3, and each comprising vlCDR1, vlCDR2, and vlCDR3 for the light chain. These CDRs are located in the variable domains of both the heavy and light chains and together form the Fv region. (See Table 1 and the related discussion above for the CDR numbering scheme). Therefore, in some cases, the six CDRs of the antigen-binding domain are provided by the variable domains of both the heavy and light chains. In the “Fab” form, the set of six CDRs is provided by two distinct polypeptide sequences: a heavy chain variable domain (vh or VH; containing vhCDR1, vhCDR2, and vhCDR3) and a light chain variable domain (vl or VL; containing vlCDR1, vlCDR2, and vlCDR3), wherein the C-terminus of the vh domain is attached to the N-terminus of the CH1 domain of the heavy chain, and the C-terminus of the vl domain is attached to the N-terminus of the light chain constant domain (thus forming the light chain). In the scFv form, the vh and vl domains are covalently linked (typically by using linkers as outlined herein) to a single polypeptide sequence, which can be (starting from the N-terminus) vh-linker-vl or vl-linker-vh, with the former generally preferred (containing optional domain linkers on each side, depending on the form used (e.g., according to Figure 1). Generally, in the second monomer, the C-terminus of the scFv domain is attached to the N-terminus of the hinge.

[0091] In this article, "modification" means an amino acid substitution, insertion, and / or deletion in the polypeptide sequence or a change in the portion of the protein chemically linked to it. For example, a modification could be a change in the structure of a carbohydrate or PEG linked to a protein. In this article, "amino acid modification" means an amino acid substitution, insertion, and / or deletion in the polypeptide sequence. For clarity, unless otherwise stated, amino acid modifications always refer to amino acids encoded by DNA, such as the 20 amino acids that have codons in both DNA and RNA.

[0092] "Amino acid substitution" or "replacement" as used herein means replacing an amino acid at a specific position in the parental polypeptide sequence with a different amino acid. Specifically, in some embodiments, the substitution is for a non-naturally occurring amino acid at a specific position, not naturally occurring in or within an organism. For example, substitution of E272Y refers to a variant polypeptide, in this case an Fc variant, where glutamic acid at position 272 is replaced by tyrosine. For clarity, proteins that have been engineered to alter the nucleic acid coding sequence but not the starting amino acid (e.g., replacing CGG (encoding arginine) with CGA (still encoding arginine) to increase expression levels in a host organism) are not "amino acid substitutions"; that is, although a new gene encoding the same protein is created, if the protein has the same amino acid at a specific starting position, then it is not an amino acid substitution.

[0093] As used herein, “amino acid insertion” or “insertion” means the addition of an amino acid sequence at a specific position in the parent polypeptide sequence. For example, -233E or 233E indicates the insertion of glutamic acid after position 233 and before position 234. Furthermore, -233ADE or A233ADE indicates the insertion of AlaAspGlu after position 233 and before position 234.

[0094] As used herein, “amino acid deletion” or “deletion” means the removal of an amino acid sequence at a specific position in the parent polypeptide sequence. For example, E233- or E233#; E233() or E233del indicates the deletion of glutamate at position 233. Furthermore, EDA233- or EDA233# indicates a deletion of the GluAspAla sequence starting at position 233.

[0095] As used herein, “variant protein” or “protein variant” or “variant” means a protein that differs from its parent protein by means of at least one amino acid modification.

[0096] Compared to the parent protein, a protein variant has at least one amino acid modification, but not more, otherwise the variant protein would not be able to be aligned with the parent protein using the alignment procedures described below. Generally, using alignment procedures described below, such as BLAST, variant proteins (such as the variant Fc domain outlined herein) are typically at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the parent protein.

[0097] As described below, in some embodiments, the parental polypeptide, such as the Fc parental polypeptide, is the heavy chain constant domain or Fc region of a human wild-type sequence, such as IgG1, IgG2, IgG3, or IgG4, although variant human sequences can also serve as "parental polypeptides," for example, they may comprise the IgG1 / 2 hybrid of US Publication 2006 / 0134105. Protein variant sequences as used herein will preferably have at least about 80% identity with the parental protein sequence, and most preferably at least about 90% identity, more preferably at least about 95-98-99% identity. Therefore, as used herein, "antibody variant" or "variant antibody" means an antibody that differs from the parental antibody by means of at least one amino acid modification; as used herein, "IgG variant" or "variant IgG" means an antibody that differs from the parental IgG by means of at least one amino acid modification (again, in many cases, derived from a human IgG sequence); and as used herein, "immunoglobulin variant" or "variant immunoglobulin" means an immunoglobulin sequence that differs from the parental immunoglobulin sequence by means of at least one amino acid modification. As used herein, “Fc variant” or “variant Fc” refers to a protein whose Fc domain includes amino acid modifications compared to the Fc domain of human IgG1, IgG2, or IgG4.

[0098] The Fc variants of this invention are defined according to the amino acid modifications constituting them. Thus, for example, N434S or 434S is an Fc variant with a serine substitution at position 434 relative to the parental Fc polypeptide, where the numbering is based on the EU index. Similarly, M428L / N434S defines an Fc variant with substitutions for M428L and N434S relative to the parental Fc polypeptide. The identification of the WT amino acids can be nonspecific, in which case the aforementioned variant is referred to as 428L / 434S. It should be noted that the order of substitutions is arbitrary, that is, for example, N434S / M428L and M428L / N434S are the same Fc variant, and so on. Regarding all antibody-related positions discussed in this invention, unless otherwise indicated, the amino acid position numbering is based on the EU index. The EU index, or as in the Kabat or EU numbering scheme, refers to the number of the EU antibody. Kabat et al. collected numerous primary sequences of the variable regions of the heavy and light chains. Based on sequence conservation, individual primary sequences are classified into CDRs and frames and listed accordingly (see *SEQUENCES OF IMMUNOLOGICAL INTEREST*, 5th edition, NIH Publication No. 91-3242, E.A. Kabat et al., which are incorporated herein by reference in their entirety). See also Edelman et al., 1969, *Proceedings of the National Academy of Sciences of the United States of America*, 63:78-85, which are hereby incorporated herein by reference in their entirety. Modifications may include additions, deletions, or substitutions.

[0099] In this document, "protein" refers to at least two covalently linked amino acids, including proteins, polypeptides, oligopeptides, and peptides. Furthermore, the polypeptides constituting the antibodies of this invention may include synthetic derivatization of one or more side chains or terminals, glycosylation, PEGylation, cyclic arrangement, cyclization, linkers to other molecules, fusion with proteins or protein domains, and the addition of peptide tags or labels.

[0100] As used in this article, “residue” refers to a position in a protein and its associated amino acid identifier. For example, asparagine 297 (also known as Asn297 or N297) is the residue at position 297 in the human antibody IgG1.

[0101] As used herein, “Fab” or “Fab region” refers to a polypeptide comprising the VH, CHI, VL, and CL immunoglobulin domains, which are typically located on two different polypeptide chains (e.g., VH-CH1 on one chain and VL-CL on another). Fab may refer to such a region in the case of isolation or in the case of the bispecific antibody of the present invention. In the case of Fab, Fab also includes the Fv region in addition to the CH1 and CL domains.

[0102] As used herein, “Fv” or “Fv fragment” or “Fv region” refers to a polypeptide that includes the VL and VH domains of ABD. Fv regions can be engineered in Fab and scFv forms (as discussed above, typically two distinct polypeptides that also contain constant regions as outlined above), where the vl and vh domains combine (usually using linkers as discussed herein) to form scFv.

[0103] In this paper, "single-chain Fv" or "scFv" refers to a variable domain of a heavy chain covalently linked to a variable domain of a light chain, typically using scFv linkers as discussed herein to form scFv or scFv structural domains. The scFv structural domain can be oriented in either direction from the N-terminus to the C-terminus (vh-linker-vl or vl-linker-vh). In the sequences depicted in the sequence listing and figures, the order of the vh and vl domains is indicated in the name; for example, H.X_L.Y means that the N-to-C-terminus is vh-linker-vl, and L.Y_H.X means vl-linker-vh.

[0104] As used herein, "IgG subclass modification" or "isotype modification" refers to an amino acid modification that converts one amino acid of an IgG isotype into the corresponding amino acid of a different alignment of the IgG isotype. For example, since IgG1 contains tyrosine at EU position 296 and IgG2 contains phenylalanine, the F296Y substitution in IgG2 is considered an IgG subclass modification.

[0105] As used in this article, “non-naturally occurring modification” means a modification of an amino acid that is not of the same type. For example, because none of the human IgGs contain serine at position 434, the substitution of 434S in IgG1, IgG2, IgG3, or IgG4 (or their hybrids) is considered a non-naturally occurring modification.

[0106] As used in this article, “amino acid” and “amino acid identity” refer to one of the 20 naturally occurring amino acids that are encoded for use in DNA and RNA.

[0107] As used herein, "effective function" refers to a biochemical event resulting from the interaction between the antibody's Fc region and an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP, and CDC.

[0108] As used herein, “IgG Fc ligand” refers to a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an IgG antibody to form an Fc / Fc ligand complex. Fc ligands include, but are not limited to, FcγRI, FcγRII, FcγRIII, FcRn, Clq, C3, mannan-binding lectins, mannose receptors, staphylococcal protein A, streptococcal protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRH), which are a family of Fc receptors homologous to FcγR (Davis et al., 2002, *Immunological Reviews* 190:123-136, which are incorporated herein by reference in their entirety). Fc ligands may include undiscovered Fc-binding molecules. Specific IgG Fc ligands are FcRn and Fcγ receptors. As used herein, “Fc ligand” refers to a molecule from any organism, preferably a polypeptide, that binds to the Fc region of an antibody to form an Fc / Fc ligand complex.

[0109] As used herein, “Fcγ receptor,” “FcγR,” or “FcγR (FcgammaR)” refers to any member of the protein family that binds to the Fc region of an IgG antibody and is encoded by the FcγR gene. In humans, this family includes, but is not limited to, FcγRI (CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including alloforms H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (including alloforms V158 and F158) and FcγRIIIb (including alloforms FcγRIIb-NA1 and FcγRIIb-NA2) (Jefferis et al., 2002, *Immunol Letters*). (Lett) 82:57-65, which is incorporated herein by reference in its entirety, and any undiscovered human FcγR or FcγR isoforms or allotypes. FcγR can be derived from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγR includes, but is not limited to, FcγRI(CD64), FcγRII(CD32), FcγRIII-1(CD16), and FcγRIII-2(CD16-2), and any undiscovered mouse FcγR or FcγR isoforms or allotypes.

[0110] As used herein, “FcRn” or “novice Fc receptor” refers to a protein that binds to the Fc region of an IgG antibody and is at least partially encoded by the FcRn gene. FcRn can originate from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. As is known in the art, functional FcRn proteins comprise two polypeptides, commonly referred to as the heavy chain and the light chain. The light chain is β-2-microglobulin, and the heavy chain is encoded by the FcRn gene. Unless otherwise stated, FcRn or FcRn protein as used herein refers to the complex of the FcRn heavy chain and β-2-microglobulin. Various FcRn variants enhance binding to the FcRn receptor and, in some cases, increase serum half-life. “FcRn variants” enhance binding to the FcRn receptor, and suitable FcRn variants are shown below.

[0111] As used herein, “parental polypeptide” refers to the starting polypeptide that is subsequently modified to produce a variant. A parental polypeptide can be a naturally occurring polypeptide or a variant or engineered version of a naturally occurring polypeptide. Therefore, as used herein, “parental immunoglobulin” refers to an unmodified immunoglobulin that has been modified to produce a variant, and as used herein, “parental antibody” refers to an unmodified antibody that has been modified to produce a variant antibody. It should be noted that “parental antibody” includes known commercially available, recombinantly generated antibodies as outlined below. In this context, “parental Fc domain” will be used relative to the listed variants; thus, “variant human IgG1 Fc domain” is relative to the parental Fc domain of human IgG1, “variant human IgG4 Fc domain” is relative to the parental Fc domain of human IgG4, and so on.

[0112] As used herein, “Fc”, “Fc region”, or “Fc domain” refers to a polypeptide comprising the CH2-CH3 domain of an IgG molecule and, in some cases, a hinge. In EU designations, for human IgG1, the CH2-CH3 domain comprises amino acids 231 to 447, and the hinge is 216 to 230. Therefore, the definition of an “Fc domain” includes amino acids 231-447 (CH2-CH3) or 216-447 (hinge-CH2-CH3) or fragments thereof. In this context, an “Fc fragment” may contain fewer amino acids from either the N-terminus or C-terminus but still retains the ability to form a dimer with another Fc domain or Fc fragment, detectable using standard size-based methods (e.g., non-denaturing chromatography, size exclusion chromatography, etc.). In particular, the human IgG Fc domain is used in this invention and can be an Fc domain derived from human IgG1, IgG2, or IgG4.

[0113] Compared to the parental Fc domain, the "variant Fc domain" contains amino acid modifications. Therefore, compared to the human IgG1 Fc domain, the "variant human IgG1 Fc domain" contains amino acid modifications (usually amino acid substitutions, although in the case of ablation variants, amino acid deletions are included). Generally, the variant Fc domain has at least about 80, 85, 90, 95, 97, 98, or 99% identity with the corresponding parental human IgG Fc domain (using the consensus algorithm discussed below; one embodiment utilizes the BLAST algorithm as known in the art with default parameters). Alternatively, compared to the parental Fc domain, the variant Fc domain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications. Alternatively, the variant Fc domain may have modifications of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids compared to the parent Fc domain. Furthermore, as discussed herein, the variant Fc domain retains the ability to form dimers with another Fc domain, as measured using known techniques as described herein, such as non-denaturing gel electrophoresis.

[0114] In this article, "heavy chain constant region" refers to the CH1-hinge-CH2-CH3 portion of an antibody (or a fragment thereof), excluding the heavy chain variable domain; in the EU designation of human IgG1, this is amino acid 118-447. "Heavy chain constant region fragment" refers to a heavy chain constant region containing fewer amino acids from either the N-terminus or C-terminus but still retaining the ability to form a dimer with another heavy chain constant region.

[0115] As used in this article, “position” refers to a location within a protein sequence. Positions can be numbered sequentially or according to established formats (such as the EU index used for antibody numbering).

[0116] As used herein, “target antigen” means a molecule that binds specifically through an antigen-binding domain comprising a variable region of a given antibody. As discussed below, in the context of this invention, the target antigen is a checkpoint inhibitor protein.

[0117] In the case of monomers in the heterodimer antibodies of the present invention, "chain-like" herein refers to two "matched" strands similar to those in DNA, incorporating heterodimer variants into each monomer to maintain the ability to "match" to form heterodimers. For example, if some pI variants are engineered into monomer A (e.g., to make the pI higher), then spatial variants that are also usable as "charge pairs" will not interfere with the pI variants, for example, placing charge variants that make the pI higher on the same "chain" or "monomer" to retain both functions. Similarly, for "skewed" variants appearing in pairs in a group, as more fully outlined below, those skilled in the art consider the pI when deciding which chain or monomer the pair will enter, so that using the pI of the skewed variant also maximizes pI separation.

[0118] As used in this article, "target cell" refers to a cell that expresses the target antigen.

[0119] In the case of generating bispecific antibodies according to the invention, "host cell" as used herein means a cell containing exogenous nucleic acid encoding components of the bispecific antibody and capable of expressing the bispecific antibody under suitable conditions. Suitable host cells are discussed below.

[0120] As used herein, “variable region” or “variable domain” refers to a region of an immunoglobulin, comprising one or more Ig domains encoded by one of the Vκ, Vλ, and / or VH genes that respectively constitute the κ, λ, and heavy chain immunoglobulin gene loci, and containing a CDR that confers antigen specificity. Thus, “heavy chain variable domains” pair with “light chain variable domains” to form an antigen-binding domain (“ABD”). Furthermore, each variable domain comprises three hypervariable regions (“complementarity-determining regions”, “CDRs”) (vhCDR1, vhCDR2, and vhCDR3 for the heavy chain variable domain, and vlCDR1, vlCDR2, and vlCDR3 for the light chain variable domain) and four frame (FR) regions arranged in the following order from the amino terminus to the carboxyl terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0121] "Wild-type or WT" in this article refers to the amino acid or nucleotide sequence found in nature, including allelic variations. WT proteins have an amino acid or nucleotide sequence that has not been intentionally modified.

[0122] This invention provides various antibody domains with sequence identity to human antibody domains. Sequence identity between two similar sequences (e.g., antibody variable domains) can be measured using algorithms as described in the following literature: Smith, TF and Waterman, MS (1981), “Comparison of Biosequences,” Adv. Appl. Math. 2:482 [Local homology algorithm]; Needleman, SB and Wunsch, CD. (1970), “A General Method Applicable To The Search For Similarities In The Amino Acid Sequence Of Two Proteins,” J. Mol. Biol. 48:443 [Homology alignment algorithm]; Pearson, WR and Lipman, DJ (1988), “Improved Tools For Biological Sequence Comparison.” "Sequence Comparison" in *Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. (USA)* 85:2444 [Similarity Search Methods]; or Altschul, SF et al., (1990), "Basic Local Alignment Search Tool," *Journal of Molecular Biology* 215:403-10, i.e., the "BLAST" algorithm, see https: / / blast.ncbi.nlm.nih.gov / Blast.cgi. When using any of the aforementioned algorithms, default parameters (for window length, gap penalty, etc.) are used. In one embodiment, the BLAST algorithm is used to determine sequence consistency using default parameters.

[0123] The antibodies of this invention are typically isolated or recombinant. “Isolated,” when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and isolated and / or recovered from cells or cell cultures expressing it. Typically, isolated polypeptides are prepared by at least one purification step. “Isolated antibody” refers to an antibody substantially detached from other antibodies having different antigen specificities. “Recombinant” means the production of an antibody in exogenous host cells using recombinant nucleic acid technology, and said antibody may also be isolated.

[0124] "Specific binding" or "specifically binding to" a specific antigen or epitope, or "specific to" a specific antigen or epitope, means that the binding is measurably different from nonspecific interactions. Specific binding can be measured, for example, by comparing the binding of a molecule to that of a control molecule, typically a similarly structured molecule that does not have binding activity. For instance, specific binding can be measured by competing with a control molecule similar to the target.

[0125] Specific binding to a particular antigen or epitope can be achieved, for example, by an antibody with a KD of at least about 10 against the antigen or epitope. -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, or at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 M or greater, where KD refers to the dissociation rate of a specific antibody-antigen interaction. Typically, the KD of an antibody that specifically binds to an antigen is 20, 50, 100, 500, 1000, 5,000, 10,000 or more times greater than the KD of the control molecule relative to the antigen or epitope.

[0126] Furthermore, specific binding to a particular antigen or epitope can be demonstrated, for example, by an antibody whose KA or Ka against the antigen or epitope is at least 20, 50, 100, 500, 1000, 5,000, 10,000, or more times greater than that of a control against the epitope, where KA or Ka refers to the binding rate of the specific antibody-antigen interaction. Binding affinity is typically measured using Biacore, SPR, or BLI assays.

[0127] E. Antibody

[0128] On the one hand, this document provides compositions (e.g., anti-SSTR2 antibodies) that bind to SSTR2. In some embodiments, the antibody binds to human SSTR2 (Figure 11). The subject matter of the anti-SSTR2 antibody includes monospecific SSTR2 antibodies and multispecific (e.g., bispecific) anti-SSTR2 antibodies. In some embodiments, the anti-SSTR2 antibody has the form of any of the antibody forms depicted in Figure 1.

[0129] In some embodiments, the subject composition comprises an SSTR2 binding domain. In some embodiments, the composition comprises an antibody having an SSTR2 binding domain. The antibodies provided herein comprise one, two, three, four, and five or more SSTR2 binding domains. In some embodiments, the SSTR2 binding domain comprises vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 sequences of the SSTR2 binding domain selected from the group of SSTR2 binding domains depicted in FIG11. In some embodiments, the SSTR2 binding domain comprises underlined vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 sequences of the SSTR2 binding domain selected from the SSTR2 binding domains depicted in FIG11. In some embodiments, the SSTR2 binding domain comprises a heavy chain variable domain and a light chain variable domain selected from the SSTR2 binding domains depicted in FIG11. The SSTR2 binding domains depicted in Figure 11 include those resisting SSTR2 H1.143_L1.30; SSTR2 H1_L1.1; SSTR2 H1.107_L1.30; SSTR2 H1.107_L1.67; SSTR2 H1.107_L1.108; SSTR2 H1.107_L1.111; SSTR2 H1.107_L1.114; SSTR2 H1.107_L1.102; SSTR2 H1.107_L1.110; SSTR2 H1.125_L1.30; SSTR2 H1.125_L1.67; SSTR2 H1.125_L1.108; SSTR2 H1.125_L1.111; and SSTR2 H1.125_L1.111. H1.125_L1.114; anti-SSTR2 H1.125 L1.102; and anti-SSTR2 H1.125_L1.10. In one exemplary embodiment, the antibody comprises an anti-SSTR2 H1.143_L1.30 binding domain.

[0130] In some embodiments, the antibody is a bispecific antibody that binds to SSTR2 and CD3. Such an antibody comprises a CD3 binding domain and at least one SSTR2 binding domain. Any suitable SSTR2 binding domain may be included in the anti-SSTR2×anti-CD3 bispecific antibody. In some embodiments, the anti-SSTR2×anti-CD3 bispecific antibody comprises one, two, three, four, or more SSTR2 binding domains, including, but not limited to, those depicted in Figure 11. In some embodiments, the anti-SSTR2×anti-CD3 antibody comprises an SSTR2 binding domain comprising the vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 sequences of an SSTR2 binding domain selected from the group of SSTR2 binding domains depicted in Figure 11. In some embodiments, the anti-SSTR2×anti-CD3 antibody includes an SSTR2 binding domain comprising underlined vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 sequences selected from the group of SSTR2 binding domains depicted in Figure 11. In some embodiments, the anti-SSTR2×anti-CD3 antibody includes an SSTR2 binding domain comprising a heavy chain variable domain and a light chain variable domain selected from the group of SSTR2 binding domains depicted in Figure 11. In one exemplary embodiment, the anti-SSTR2×anti-CD3 antibody includes an anti-SSTR2 H1.143_L1.30 binding domain.

[0131] The anti-SSTR2×anti-CD3 antibody provided herein may contain any suitable CD3-binding domain. In some embodiments, the anti-SSTR2×anti-CD3 antibody contains a CD3-binding domain comprising vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 sequences of a CD3-binding domain selected from the group of CD3-binding domains depicted in Figures 12 and 13. In some embodiments, the anti-SSTR2×anti-CD3 antibody contains a CD3-binding domain comprising vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 sequences of a CD3-binding domain selected from the group of CD3-binding domains depicted in Figures 12 or 13. In some embodiments, the anti-SSTR2×anti-CD3 antibody contains a CD3-binding domain comprising a heavy chain variable domain and a light chain variable domain of a CD3-binding domain selected from the group of CD3-binding domains depicted in Figures 12 or 13. In some embodiments, the CD3 binding domain is selected from anti-CD3 H1.30_L1.47; anti-CD3 H1.32_L1.47; anti-CD3 H1.89_L1.48; anti-CD3 H1.90_L1.47; anti-CD3 H1.33_L1.47; and anti-CD3 H1.31_L1.47.

[0132] As used herein, the term "antibody" is used generally. Antibodies used in this invention can take many forms as described herein, including conventional antibodies as described herein, as well as antibody derivatives, fragments, and mimics.

[0133] Traditional antibody structural units typically comprise tetramers. Each tetramer typically consists of two identical pairs of polypeptide chains, each pair having a "light chain" (typically with a molecular weight of about 25 kDa) and a "heavy chain" (typically with a molecular weight of about 50-70 kDa). Human light chains are classified as κ and λ light chains. This invention pertains to the IgG class, which has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. It should be noted that IgG1 has different allotypes exhibiting polymorphism at 356 (D or E) and 358 (L or M). The sequences depicted herein use the 356D / 358M allotype; however, another allotype is included herein. That is, any sequence containing the IgG1 Fc domain included herein may have 356E / 358L, replacing the 356D / 358M allotype.

[0134] Furthermore, many antibodies described herein have at least one cysteine ​​substitution at position 220 replaced with a serine; typically this is on the “scFv monomer” side for most sequences depicted herein, although it can also be on the “Fab monomer” side, or both, to reduce disulfide formation. Particularly included within the sequences described herein are one or both of the cysteine ​​substitution (C220S) sequences.

[0135] Therefore, as used herein, “isotype” means any subclass of immunoglobulin defined by the chemical and antigenic characteristics of its constant region. It should be understood that therapeutic antibodies may also comprise hybrids of isotypes and / or subclasses. For example, as illustrated in US Publication 2009 / 0163699, which is incorporated herein by reference, the present invention includes the use of human IgG1 / G2 hybrids.

[0136] Hypervariable regions typically contain the following amino acid residues: approximately amino acid residues 24-34 (LCDR1; "L" indicates light chain), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable region, and approximately 31-35B (HCDR1; "H" indicates heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable region; Kabat et al., *SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST*, 5th edition, Public Health Service, National Institutes of Health. Health), Bethesda, Md. (1991); and / or those residues forming the hypervariable ring (e.g., residues 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3) in the light chain variable region and 26-32 (HCDR1), 53-55 (HCDR2), and 96-101 (HCDR3) in the heavy chain variable region; Chothia and Lesk (1987) Journal of Molecular Biology 196:901-917. Specific CDRs of the present invention are described below.

[0137] As those skilled in the art will understand, the precise numbering and arrangement of CDRs can differ in different systems. However, it should be understood that the disclosure of variable heavy chain and / or variable light chain sequences includes the disclosure of the associated (inherent) CDRs. Therefore, the disclosure of each heavy chain variable region is the disclosure of vhCDRs (e.g., vhCDR1, vhCDR2, and vhCDR3) and the disclosure of each light chain variable region is the disclosure of vlCDRs (e.g., vlCDR1, vlCDR2, and vlCDR3). A comparison of available CDR numbering is provided below, see Lafranc et al., Developmental and Comparative Immunology (Dev. Comp. Immunol.) 27(l):55-77 (2003):

[0138] Table 1

[0139] Kabat+Chothia IMGT Rabat AbM Chothia Contact Xencor vhCDR1 26-35 27-38 31-35 26-35 26-32 30-35 27-35 vhCDR2 50-65 56-65 50-65 50-58 52-56 47-58 54-61 vhCDR3 95-102 105-117 95-102 95-102 95-102 93-101 103-116 vlCDR1 24-34 27-38 24-34 24-34 24-34 30-36 27-38 vlCDR2 50-56 56-65 50-56 50-56 50-56 46-55 56-62 vlCDR3 89-97 105-117 89-97 89-97 89-97 89-96 97-105

[0140] Throughout this specification, when referring to residues in the variable domain (roughly residues 1-107 in the light chain variable region and residues 1-113 in the heavy chain variable region), the Kabat numbering system is generally used and the EU numbering system is used for the Fc region (e.g., Kabat et al., cite (1991)).

[0141] Another type of Ig domain in the heavy chain is the hinge region. In this document, “hinge,” “hinge region,” “antibody hinge region,” or “hinge domain” refers to a flexible polypeptide comprising the amino acids between the first and second constant domains of the antibody. Structurally, the IgG CH1 domain terminates at EU position 215, and the IgG CH2 domain begins at residue EU position 231. Therefore, in this document, for IgG, the antibody hinge is defined to contain positions 216 (E216 in IgG1) through 230 (p230 in IgG1), where the numbering is based on the EU index as in Kabat. In some cases, a “hinge fragment” is used, which contains fewer amino acids at either the N-terminus or C-terminus of the hinge domain. As mentioned herein, pI variants can also be made within the hinge region.

[0142] Light chains typically contain two domains: the light chain variable domain (containing the light chain CDR and forming the Fv region together with the heavy chain variable domain), and the light chain constant domain (usually called CL or Cκ).

[0143] Another region of interest for further substitution is the Fc region, as outlined below.

[0144] This invention provides a large number of different CDR sets. In this case, a “complete CDR set” comprises three variable light chain CDRs and three variable heavy chain CDRs, such as vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2, and vhCDR3. These may be part of a larger light chain variable domain or a heavy chain variable domain, respectively. Additionally, as summarized more fully herein, when using heavy and light chains (e.g., when using Fab), the heavy chain variable domain and the light chain variable domain may be located on separate polypeptide chains, or, in the case of an scFv sequence, on a single polypeptide chain.

[0145] CDRs facilitate the formation of antigen-binding sites, or more specifically, epitope-binding sites, on antibodies. An "epitaph" is a determinant that interacts with a specific antigen-binding site (called a complementary site) in the variable region of an antibody molecule. Epitopes are clusters of molecules, such as amino acid or sugar side chains, and typically possess specific structural and charge characteristics. A single antigen can have more than one epitope.

[0146] Epitopes can include amino acid residues that directly participate in binding (also known as the immunodominant component of epitopes) and other amino acid residues that do not directly participate in binding, such as amino acid residues that are effectively blocked by specific antigen-binding peptides; in other words, the amino acid residues are located within the coverage area of ​​the specific antigen-binding peptide.

[0147] Epitopes can be conformational or linear. Conformational epitopes are formed by the spatial juxtaposition of amino acids from different segments of a linear polypeptide chain. Linear epitopes are formed by adjacent amino acid residues in a polypeptide chain. The difference between conformational and non-conformational epitopes is that, in the presence of denaturing solvents, binding to conformational epitopes disappears, but binding to non-conformational epitopes does not.

[0148] Epitopes typically comprise at least three, and more usually at least five or eight to ten amino acids in a unique spatial conformation. Antibodies recognizing the same epitope can be examined in simple immunoassays (e.g., “binning”) demonstrating the ability of one antibody to block the binding of another antibody to a target antigen. As summarized below, the present invention includes not only the antigen-binding domains and antibodies listed herein, but also those epitopes that compete for binding with epitopes that have already bound the listed antigen-binding domains.

[0149] Therefore, the present invention provides different antibody domains. As described herein and known in the art, the heterodimeric antibodies of the present invention comprise different domains within the heavy and light chains, which may also overlap. These domains include, but are not limited to, the Fc domain, CH1 domain, CH2 domain, CH3 domain, hinge domain, heavy chain constant domain (CH1-hinge-Fc domain or CH1-hinge-CH2-CH3), heavy chain variable domain, light chain variable domain, light chain constant domain, Fab domain, and scFv domain.

[0150] Therefore, the “Fc structural domain” includes the -CH2-CH3 domain and optionally the hinge domain (-H-CH2-CH3). In the embodiments described herein, when the scFv is connected to the Fc structural domain, the C-end of the scFv construct is connected to all or part of the hinge of the Fc structural domain; for example, it is typically connected to the sequence EPKS that serves as the hinge starting point. The heavy chain includes a heavy chain variable domain and a constant domain, which includes the CH1-optional hinge-Fc structural domain including CH2-CH3. The light chain includes a light chain variable domain and a light chain constant domain. The scFv includes a variable heavy chain, an scFv connector, and a light chain variable domain. In most of the constructs and sequences outlined herein, the C-end of the variable heavy chain is connected to the N-end of the scFv connector, and the C-end of the scFv connector is connected to the N-end of the variable light chain (N-vh-connector-vl-C), although this order can be reversed (N-vl-connector-vh-C).

[0151] Some embodiments of the present invention include at least one scFv domain, which, although not naturally occurring, typically comprises heavy-chain and light-chain variable domains linked together by scFv connectors. As outlined herein, while scFv domains are typically oriented from N-terminus to C-terminus as vh-scFv connector-vl, this orientation can be reversed for any scFv domain (or those constructed using the vh and vl sequences of Fab) to become vl-scFv connector-vh, wherein, depending on the form (see generally Figure 1), the optional connector is located at one or both ends.

[0152] As illustrated herein, various suitable linkers exist for covalently linking the listed domains (used as domain linkers or scFv linkers), containing conventional peptide bonds, which are generated through recombination techniques. In some embodiments, the linker peptide may primarily contain the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should have a length sufficient to link two molecules so that they present the correct conformation relative to each other, thereby retaining the desired activity. In one embodiment, the linker has a length of about 1 to 50 amino acids, preferably about 1 to 30 amino acids. In one embodiment, linkers of 1 to 20 amino acid lengths can be used, and in some embodiments, about 5 to about 10 amino acids have been found to be used. Suitable linkers include glycine-serine polymers, including, for example, (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n (where n is at least one (and typically an integer of 3 to 4)), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Alternatively, a variety of non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyepoxide, or copolymers of polyethylene glycol and polypropylene glycol, can be used as linkers.

[0153] Other linker sequences can contain any sequence of any length of the CL / CH1 domain, but not all residues of the CL / CH1 domain; for example, the first 5-12 amino acid residues of the CL / CH1 domain. Linkers can be derived from immunoglobulin light chains, such as Cκ or Cλ. Linkers can be derived from any isotype of immunoglobulin heavy chain, including, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Linker sequences can also be derived from other proteins, such as Ig-like proteins (e.g., TCR, FcR, KIR); hinge region-derived sequences; and other native sequences from other proteins.

[0154] In some embodiments, a connector is a "domain connector" used to join any two domains together as outlined herein. For example, in Figure 1F In this embodiment, a domain linker may be present that links the C-terminus of the CH1 domain of Fab to the N-terminus of scFv, and another optional domain linker links the C-terminus of scFv to the CH2 domain (although in many embodiments, a hinge is used as this domain linker). While any suitable linker may be used, many embodiments utilize glycine-serine polymers as domain linkers, including, for example, (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is at least one (and typically 3 to 4 to 5) integers, and any peptide sequence that allows recombination of the two domains and has sufficient length and flexibility for each domain to retain its biological function. In some cases, and where there is a focus on the “chain-like” characteristics outlined below, charged domain linkers, as used in some embodiments of the scFv linker, may be used.

[0155] In some embodiments, the connector is an scFv connector for covalently connecting the vh and vl domains as discussed herein. In many cases, the scFv connector is a charged scFv connector, and various such charged scFv connectors are shown in Figure 7. Accordingly, the present invention further provides charged scFv connectors to facilitate pI separation between a first monomer and a second monomer. That is, by incorporating a positively or negatively charged (or both, in the case of a scFv support using different monomers) scFv connector, it is thereby allowed that the monomer including the charged connector changes pI without causing further changes in the Fc structural domain. These charged connectors can be substituted into any scFv containing a standard connector. Furthermore, as those skilled in the art will appreciate, charged scFv connectors can be used on appropriate “chains” or monomers depending on the desired pI change. For example, as discussed in this paper, in order to generate a heterodimeric antibody in the form of triple F, the initial pI of the Fv region of each desired antigen-binding domain is calculated, and one is selected to generate scFv, and a positive or negative linker is selected based on the pI.

[0156] Charged domain connectors can also be used to similarly enhance pI separation of the monomers of the present invention, and therefore the connectors included in FIG7 can be used in any embodiment utilizing connectors herein.

[0157] Specifically, the form depicted in Figure 1 is an antibody, often referred to as a "heterodimeric antibody," meaning that the protein has at least two associated Fc sequences that self-assemble into the heterodimeric Fc domain and at least two Fv regions, whether as Fab or as scFv.

[0158] F. Chimeric and humanized antibodies

[0159] In some embodiments, the antibodies of the present invention comprise heavy chain variable regions from specific germline heavy chain immunoglobulin genes and / or light chain variable regions from specific germline light chain immunoglobulin genes. For example, such antibodies may comprise or be composed of human antibodies comprising heavy or light chain variable regions that are “products” or “derived from” a specific germline sequence. Human antibodies that are “products” or “derived from” a human germline immunoglobulin sequence can be identified by comparing the amino acid sequence of the human antibody with the amino acid sequence of the human germline immunoglobulin and selecting the human germline immunoglobulin sequence that is sequence-closest (i.e., has the highest similarity %) to the sequence of the human antibody (using the methods outlined herein). Compared to germline sequences, human antibodies that are “products” or “derived from” a specific human germline immunoglobulin sequence may contain amino acid differences caused, for example, naturally occurring somatic mutations or deliberately introduced site-directed mutations. However, humanized antibodies typically share at least 90% amino acid sequence identity with the sequence encoded by human germline immunoglobulin genes, and when compared to germline immunoglobulin amino acid sequences from other species (e.g., murine germline sequences), contain amino acid residues that identify the antibody as derived from a human sequence. In some cases, humanized antibodies may share at least 95%, 96%, 97%, 98%, or 99% or even at least 96%, 97%, 98%, or 99% amino acid sequence identity with the sequence encoded by germline immunoglobulin genes. Typically, humanized antibodies derived from a particular human germline sequence will show no more than 10-20 amino acid differences compared to the sequence encoded by human germline immunoglobulin genes (previously, the number of variants was generally low before the introduction of any skewed, pI, and ablation variants herein; i.e., before the introduction of the variants of the present invention). In some cases, humanized antibodies may show no more than 5 or even no more than 4, 3, 2 or 1 amino acid difference compared to the amino acid sequence encoded by germline immunoglobulin genes (again, prior to the introduction of any skewed, pI and ablation variants herein; i.e. prior to the introduction of the variants of the present invention, the number of variants was generally low).

[0160] In one embodiment, the parent antibody has undergone affinity maturation, as is known in the art. Humanization and affinity maturation can be performed using structure-based methods, for example, as described in USSN 11 / 004,590. Selection-based methods can be used to humanize and / or mature the antibody variable region, including, but not limited to, the methods described in the following literature: Wu et al., 1999, Journal of Molecular Biology 294:151-162; Baca et al., 1997, Journal of Biochemistry 272(16):10678-10684; Rosok et al., 1996, Journal of Biochemistry 271(37):22611-22618; Rader et al., 1998, Proceedings of the National Academy of Sciences 95:8910-8915; Krauss et al., 2003, Protein Engineering 16(10):753-759, all of which are incorporated herein by reference in their entirety. Other humanization methods may involve transplanting only a portion of the CDR, including but not limited to the methods described in the following literature: USSN 09 / 810,510; Tan et al., 2002, Journal of Immunology 169:1119-1125; De Pascalis et al., 2002, Journal of Immunology 169:3076-3084, all of which are incorporated herein by reference in their entirety.

[0161] G. Heterodimer antibody

[0162] Therefore, in some embodiments, the subject antibody is a heterodimeric antibody that relies on using two different heavy chain variant Fc sequences. This antibody will self-assemble to form a heterodimeric Fc domain and a heterodimeric antibody.

[0163] This invention relates to novel constructs that provide heterodimeric antibodies that allow binding to more than one antigen or ligand, such as allowing bispecific binding (e.g., binding to both SSTR2 and CD3). The heterodimeric antibody constructs are based on the self-assembly nature of two Fc domains in the heavy chain of the antibody, for example, two “monomers” assembling into a “dimer.” Heterodimeric antibodies are generated by altering the amino acid sequences of the individual monomers, as discussed more fully below. Therefore, this invention generally relates to the construction of heterodimeric antibodies that can co-conjugate with antigens (e.g., SSTR2 and CD3) in several ways, depending on amino acid variants in different constant regions on each chain that promote heterodimer formation and / or allow the purification of heterodimers to be easier than that of homodimers.

[0164] Therefore, the present invention provides bispecific antibodies. In some embodiments, the present invention provides bispecific antibodies comprising an SSTR2 binding domain. In some embodiments, the bispecific antibody is an anti-SSTR2×anti-CD3 bispecific antibody. One problem existing in antibody technology is the desire for “bispecific” antibodies to bind to two different antigens simultaneously, which typically allows different antigens to approach each other and thus generate novel functions and new therapies. Generally, these antibodies are produced by incorporating the genes for each heavy chain and light chain into the host cell. This typically results in the formation of the desired heterodimer (AB) as well as two homodimers (AA and BB (excluding the light chain heterodimer problem)). However, a major obstacle to the formation of bispecific antibodies is the difficulty in purifying heterodimers from homodimers and / or the difficulty in favoring the formation of heterodimers (as opposed to the formation of homodimers).

[0165] Multiple mechanisms exist for generating the heterodimers of this invention. Furthermore, as those skilled in the art will appreciate, these mechanisms can be combined to ensure high heterodimerization. Therefore, the amino acid variants that induce heterodimer formation are referred to as "heterodimer variants." As discussed below, heterodimer variants may include spatial variants (e.g., the "mortar and pestle" or "skewed" variants described below, and the "charge pair" variants described below) and "pI variants," which allow for the purification of homodimers from heterodimers. As generally described in WO2014 / 145806 (incorporated hereinously by reference in its entirety), and specifically as described below in the discussion of “heterodimerization variants”, applicable heterodimerization mechanisms include the “kidney and mortar” (“KIH”; sometimes referred to herein as the “skewed” variant) (see discussion in WO2014 / 145806), “electrostatic orientation” or “charge pairing” as described in WO2014 / 145806, the pI variant as described in WO2014 / 145806, and other general Fc variants as outlined in WO2014 / 145806 and below.

[0166] In this invention, several mechanisms exist for the easy purification of heterodimeric antibodies; one relies on the use of pI variants so that each monomer has a different pI, thereby allowing isoelectric purification of AA, AB, and BB dimeric proteins. Alternatively, some scaffold forms (such as the “triple F” form) also allow for separation based on size. As further outlined below, heterodimers can also be formed “skewed” rather than homodimers. Therefore, combinations of spatial heterodimeric variants with pI or charge-pair variants are particularly suitable for this invention.

[0167] Generally, embodiments of the particular use in this invention rely on a group of variants that include skewed variants that facilitate the formation of heterodimers to form non-homodimemers, which are coupled with pI variants that increase the pI difference between the two monomers to aid in the purification of heterodimers from homodimers.

[0168] Furthermore, as outlined more fully below, depending on the form of the heterodimeric antibody, pI variants may be contained within the constant domain and / or Fc domain of the monomer, or may utilize charged linkers (domain linkers or scFv linkers). That is, scFv-based scaffolds, such as triple F or "opener" forms, may contain charged scFv linkers (positive or negative) that provide further pI enhancement for purification purposes. As those skilled in the art will appreciate, some triple F forms are used only with charged scFv linkers without additional pI modulation, but the present invention also provides pI variants located on one or both monomers and / or charged domain linkers. Additionally, further amino acid engineering for alternative functions can also confer pI variations, such as Fc, FcRn, and KO variants.

[0169] In this invention, which uses pI as a separation mechanism to allow for the purification of heterodimeric proteins, amino acid variants can be introduced into one or both monomeric polypeptides; that is, the pI of one monomer (referred to herein as "monomer A" for simplicity) can be engineered to separate from monomer B, or both monomer A and B can be altered, wherein the pI of monomer A increases while the pI of monomer B decreases. As described more fully below, changes to the pI of either monomer can be made by removing or adding charged residues (e.g., replacing a neutral amino acid with a positively or negatively charged amino acid residue, such as replacing glycine with glutamic acid), changing a charged residue from positive or negative charge to the opposite charge (aspartic to lysine), or changing a charged residue to a neutral residue (e.g., loss of charge; lysine to serine). Several of these variants are shown in the figures.

[0170] Accordingly, this embodiment of the invention results in a sufficient change in the pI of at least one monomer to separate the heterodimer from the homodimer. As those skilled in the art will appreciate, and as further discussed below, this can be achieved by using a constant region of the "wild-type" heavy chain and a variant region engineered to increase or decrease its pI (wt A-+B or wt A--B), or by adding one region and removing another (A+-B- or A-B+).

[0171] Therefore, generally, components of some embodiments of the present invention are amino acid variants in the constant region of the antibody, which involve a change in the isoelectric point (pI) of at least one (if not two) monomers in the dimer protein that forms the “pI antibody”. This is achieved by incorporating amino acid substitutions (“pI variants” or “pI substitutions”) into one or both monomers. As shown herein, if the pI difference between the two monomers is as small as 0.1 pH units (0.2, 0.3, 0.4, and 0.5 or larger pH units are all applicable in the present invention), then the separation of the heterodimer from the two homodimers can be achieved.

[0172] As those skilled in the art will understand, for good separation, the number of pI variants contained on each or both monomers will depend in part on the initial pI of the components, for example, in the triple F form, on the initial pI of the scFv and Fab of interest. That is, to determine which monomer to engineer or in which “direction” (e.g., positive or negative), the Fv sequences of the two target antigens are calculated and the decision is made accordingly. As is known in the art, different Fvs have different initial pIs for use in this invention. Generally, as outlined herein, the pIs are engineered such that the total pI difference for each monomer reaches at least about 0.1 log, preferably 0.2 to 0.5 as outlined herein.

[0173] Additionally, as those skilled in the art will understand and as outlined herein, in some embodiments, heterodimers and homodimers can be separated based on size. For example, as shown in Figure 1, several forms allow for the separation of heterodimers and homodimers based on size.

[0174] This approach provides a more modular method for designing and purifying bispecific proteins (including antibodies) by using a heavy chain constant region and achieving heterodimerization using pI variants. Therefore, in some embodiments, the variable region does not contain heterodimer variants (including skewed and purified heterodimer variants), thus requiring engineering for each individual antibody. Additionally, in some embodiments, the likelihood of pI variants causing immunogenicity is significantly reduced by introducing pI variants from different IgG isotypes to modify the pI without introducing significant immunogenicity. Therefore, another problem to be addressed is the elucidation of low pI constant regions and high human sequence content, such as minimizing or avoiding non-human residues at any specific location.

[0175] One additional benefit of this pI engineering is the extension of serum half-life and enhanced FcRn binding. Specifically, as described in USSN 13 / 194,904 (incorporated herein by reference in its entirety), reducing the antibody constant domain (containing the constant domain found in antibody-Fc fusions) of the pI can prolong in vivo serum retention time. These pI variants used to extend serum half-life also facilitate pI changes for purification.

[0176] Additionally, it should be noted that pI variants of heterodimers offer extra benefits for analytical and quality control methods for bispecific antibodies, as their ability to eliminate, minimize, and distinguish homodimers is significant in the presence of homodimers. Similarly, it is important to reliably test the reproducibility of heterodimer antibodies.

[0177] Heterodimer variant

[0178] This invention provides heterodimeric proteins comprising various forms of heterodimeric antibodies that utilize heterodimeric variants to achieve heterodimer formation and / or purification from homodimers.

[0179] The heterodimer skewed variants contain multiple suitable pairs. These variants appear in pairs, that is, one pair is incorporated into the first monomer and the other pair is incorporated into the second monomer. It should be noted that these pairs do not necessarily exhibit a "mortar and pestle" type variant, where there is a one-to-one correspondence between residues on one monomer and residues on the other monomer; that is, these pairs in the group form an interface between the two monomers, promoting heterodimer formation and inhibiting homodimer formation, thereby resulting in a percentage of spontaneously formed heterodimers exceeding 90% under biological conditions, rather than the expected 50% (25% homodimer A / A: 50% heterodimer A / B: 25% homodimer B / B).

[0180] Spatial variants

[0181] In some embodiments, the addition of spatial variants can promote the formation of heterodimers. That is, by changing the amino acids in each heavy chain, different heavy chains are more likely to associate to form heterodimer structures than homodimers with the same Fc amino acid sequence. Suitable spatial variants are shown in Figure 12.

[0182] One mechanism, commonly referred to in the field as the "mortar and pestle," can optionally be used with so-called amino acid engineering to create spatial effects that favor heterodimer formation and discourage homodimer formation; this is sometimes referred to as the "mortar and pestle," as described in the following references: USSN 61 / 596,846; Ridgway et al., *Protein Engineering* 9(7):617(1996); Atwell et al., *Journal of Molecular Biology* 1997 270:26; and US Patent No. 8,216,805, all of which are hereby incorporated in their entirety by reference. The diagram illustrates the number of "monomer A-monomer B" pairs that depend on the "mortar and pestle." Additionally, as described by Merchant et al., *Nature Biotechnology* 16:677(1998), these "mortar and pestle" mutations can combine with disulfide bonds to skew heterodimer formation.

[0183] Another mechanism for generating heterodimers is sometimes referred to as “electrostatic reversal,” as described by Gunasekaran et al., Journal of Biochemistry 285(25):19637(2010), which is hereby incorporated by reference in its entirety. This is sometimes referred to herein as “charge pair.” In this embodiment, electrostatics are used to skew the formation of heterodimers. As those skilled in the art will appreciate, these can also affect pI and thus purification, and therefore can be considered pI variants in some cases. However, since these are generated to force heterodimer formation and are not used as purification tools, they are classified as “spatial variants.” These include, but are not limited to, D221E / P228E / L368E paired with D221R / P228R / K409R (e.g., these are “monomer counterparts”) and C220E / P228E / 368E paired with C220R / E224R / P228R / K409R.

[0184] Other monomer A and monomer B variants may optionally and independently be combined in any amount with other variants (such as the pI variants outlined herein or other spatial variants shown in Figure 37 of US 2012 / 0149876, the diagrams and legends and SEQ ID NO are expressly incorporated herein by reference).

[0185] In some embodiments, the spatial variants outlined herein may optionally and independently be incorporated into one or two monomers along with any pI variant (or other variants, such as Fc variants, FcRn variants, etc.), and may be independently and optionally included in or excluded from the proteins of the present invention.

[0186] A series of suitable skewed variants are shown in Figure 3, among which Figure 8Some specific utilities are shown in several embodiments. In several embodiments, pairs of groups including, but not limited to, the following are specifically used: S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411T / E360E / Q362E:D401K; L368D / K370S:S364K / E357L; and K370S:S364K / E357Q. For nomenclature purposes, "S364K / E357Q:L368D / K370S" means that one monomer has a bivariate group S364K / E357Q and another monomer has a bivariate group L368D / K370S.

[0187] pI (isoelectric point) variants for heterodimers

[0188] Generally, as those skilled in the art will understand, pI variants fall into two common categories: variants that increase the protein pI (basicity change) and variants that decrease the protein pI (acidicity change). As described herein, all combinations of these variants are possible: one monomer can be wild-type, or a variant with a pI that is not significantly different from wild-type; and the other monomer can be more basic or more acidic. Alternatively, each monomer can be altered, one becoming more basic and the other more acidic.

[0189] Preferred combinations of pI variants are shown in Figure 4 As outlined in this article and shown in the diagram, these changes are relative to IgG1, and all isotypes and isotype heterozygotes can be altered in this manner. R133E and R133Q can also be used when the heavy chain constant domain is derived from IgG2-4.

[0190] In one embodiment, for example in Figure 1A , 1E In the forms 1F, 1G, 1H, and 1I, a preferred combination of pI variants has a monomer (negative Fab side) comprising the 208D / 295E / 384D / 418E / 421D variants (N208D / Q295E / N384D / Q418E / N421D when relative to human IgG1) and a second monomer (positive scFv side) comprising a positively charged scFv linker (containing (GKPGS)4 (SEQ ID NO: 818)). However, as those skilled in the art will understand, the first monomer contains a CH1 domain, which contains position 208. Therefore, in constructs that do not contain a CH1 domain (e.g., for those not using, for example, the dual scFv form or the "single-arm" form, such as... Figure 1B , 1COr the form depicted in 1D, an antibody on one of the CH1 domains of the domain), preferably a negative pI variant Fc group containing the 295E / 384D / 418E / 421D variant (Q295E / N384D / Q418E / N421D when relative to human IgG1).

[0191] Therefore, in some embodiments, a monomer has from Figure 4 One set of replacements and another monomer has charged connectors (in the form of charged scFv connectors, since the monomer includes scFv, or in the form of charged domain connectors, as indicated by the form, which may be selected from those depicted in Figure 7).

[0192] Isomorph

[0193] Furthermore, many embodiments of the present invention rely on “introducing” a pI amino acid from a specific position of one IgG isoform into another IgG isoform, thereby reducing or eliminating the possibility of introducing unwanted immunogenicity into the variant.

[0194] Several of these variants are shown in Figure 21 of U.S. Publication 2014 / 0370013, which is hereby incorporated herein by reference. Specifically, IgG1 is a common isotype of therapeutic antibodies for various reasons, including high-efficiency function. However, the heavy chain constant region of IgG1 has a higher pI (8.10 vs. 7.31) than the heavy chain constant region of IgG2. Introducing specific IgG2 residues into the IgG1 backbone results in a lower (or higher) pI and, additionally, a longer serum half-life for the resulting monomer. For example, IgG1 has glycine at position 137 (pI 5.97), and IgG2 has glutamate (pI 3.22); the introduction of glutamate affects the pI of the resulting protein. As described below, multiple amino acid substitutions are typically required to significantly affect the pI of variant antibodies. However, it should be noted that, as discussed below, uniform changes within the IgG2 molecule prolong the serum half-life.

[0195] In other embodiments, non-isotype amino acid changes are produced to reduce the overall charge state of the resulting protein (e.g., by changing higher pI amino acids to lower pI amino acids), or to allow structural adaptation to stability, etc., as described further below.

[0196] Furthermore, by engineering the pI of the heavy and light chain constant domains, significant changes can be observed in the individual monomers within the heterodimer. As discussed in this paper, ensuring a pI difference of at least 0.5 between the two monomers allows for separation by ion-exchange chromatography, isoelectric point focusing, or other methods sensitive to isoelectric points.

[0197] Calculate pI

[0198] The pI of each monomer can depend on the pI of the variable heavy chain constant domain and the pI of the total monomer, including the variable heavy chain constant domain and the fusion complex. Therefore, in some embodiments, the pI variation is calculated based on the variable heavy chain constant domain using the graph in Figure 19 of U.S. Publication 2014 / 0370013. As discussed herein, which monomer is engineered is typically determined based on the Fv and the inherent pI of the scaffold region. Alternatively, the pI of each monomer can be compared.

[0199] It also endows the pI variant with better in vivo binding of FcRn.

[0200] When the pI variant reduces the monomeric pI, it can have the additional benefit of improving serum retention time in vivo.

[0201] Although still requiring further verification, it is believed that the Fc region prolongs the in vivo half-life because the binding to FcRn at pH 6 in the endosome isolates Fc (Ghetie and Ward, 1997 Immunol Today 18(12):592-598, which is incorporated herein by reference in its entirety). Subsequently, the endosome compartment allows Fc to recirculate to the cell surface. Once the compartment opens to the extracellular space, a higher pH (approximately 7.4) induces the release of Fc back into the bloodstream. Dall'Acqua et al. showed that in mice, Fc mutants with enhanced FcRn binding at pH 6 and pH 7.4 actually had reduced serum concentrations and the same half-life as wild-type Fc (Dall'Acqua et al., 2002 Journal of Immunology 169:5171-5180, which is incorporated herein by reference in its entirety).

[0202] The enhanced affinity of Fc for FcRn at pH 7.4 is thought to prevent Fc release back into the bloodstream. Therefore, Fc mutations that prolong the in vivo half-life of Fc ideally enhance FcRn binding at lower pH levels while still allowing Fc release at higher pH levels. The charge state of the amino acid histidine changes within the pH range of 6.0 to 7.4. Therefore, the presence of His residues at key positions in the Fc / FcRn complex is not unexpected.

[0203] Recently, it has been proposed that antibodies with variable regions having lower isoelectric points can also have longer serum half-lives (Igawa et al., 2010 PEDS.23(5):385-392, which is incorporated herein by reference in its entirety). However, the mechanism of such antibodies is still poorly understood. Furthermore, the variable region varies among different antibodies. Constant region variants with lower pI and longer half-lives offer a more modular approach to improving the pharmacokinetic properties of antibodies, as described in this paper.

[0204] Other Fc variants for additional functions

[0205] Besides pI amino acid variants, multiple applicable Fc amino acid modifications can be generated for various reasons (including, but not limited to, altering the binding to one or more FcγR receptors, altering the binding to FcRn receptors, etc.).

[0206] Accordingly, the proteins of the present invention may include amino acid modifications, including heterodimeric variants as outlined herein, including pI variants and spatial variants. Each set of variants may be independently and optionally included in or excluded from any particular heterodimeric protein.

[0207] FcγR variant

[0208] Therefore, many suitable Fc substitutions exist to alter the binding to one or more FcγR receptors. Substitutions that induce both enhanced and weakened binding can be applicable. For example, enhanced binding to FcγRIIIa is known to typically induce enhanced ADCC (antibody-dependent cell-mediated cytotoxicity; a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize the bound antibody on a target cell and subsequently induce the lysis of the target cell). Similarly, in some cases, weakened binding to FcγRIIb (an inhibitory receptor) may also be advantageous. Amino acid substitutions used in this invention include those listed in USSN 11 / 124,620 (especially Figure 41), 11 / 174,287, 11 / 396,495, and 11 / 538,406, all of which are expressly incorporated herein by reference in their entirety, and especially the variants disclosed therein. Specific variants that may be used 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.

[0209] In addition, there are other Fc substitutions that can be used to enhance binding to the FcRn receptor and prolong serum half-life, as specifically disclosed in USSN 12 / 341,769 (which is incorporated herein by reference in its entirety), including but not limited to 434S, 434A, 428L, 308F, 259I, 428L / 434S, 259I / 308F, 436I / 428L, 436I or V / 434S, 436V / 428L, and 259I / 308F / 428L.

[0210] ablation variant

[0211] Similarly, another class of functional variants are “FcγR ablation variants” or “Fc gene knockout (FcKO or KO)” variants. In these embodiments, in some therapeutic applications, it is desirable to reduce or eliminate the normal binding of the Fc domain to one or more Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) to avoid other mechanisms of action. That is, for example, in many embodiments, especially when using bispecific antibodies that monovalently bind to CD3, it is generally desirable to ablate FcγRIIIa binding to eliminate or significantly reduce ADCC activity, wherein one of the Fc domains includes one or more Fcγ receptor ablation variants. These ablation variants are depicted in Figure 14 and can each be independently and optionally included or excluded. Preferred ablation variants are selected from the group consisting of: G236R / L328R, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A327G, and E233P / L234V / L235A / G236del. It should be noted that the ablation variants mentioned herein ablate FcγR bindings but generally do not ablate FcRn bindings.

[0212] As is known in the art, the Fc domain of human IgG1 binds most strongly to the Fcγ receptor, and therefore ablative variants can be used when the constant domain (or Fc domain) in the backbone of a heterodimer antibody is IgG1. Alternatively, in the context of IgG1, in addition to ablative variants, for example, mutations at glycosylation position 297 (usually A or S) can also significantly ablate binding to FcγRIIIa. Binding to the Fcγ receptor is naturally reduced in human IgG2 and IgG4, and therefore those backbones can be used with or without ablative variants.

[0213] Combination of heterodimeric variants and Fc variants

[0214] As those skilled in the art will appreciate, all of these heterodimeric variants (including skewed and / or pI variants) can be optionally and independently combined in any manner, as long as they retain their “chain-like” or “monomer-like” configuration. Furthermore, all these variants can be combined in any heterodimeric form.

[0215] In the case of pI variants, although the figure shows examples that can be used specifically, other combinations can be generated based on the basic rule of changing the pI difference between the two monomers to aid purification.

[0216] In addition, any of the heterodimer variants (skewed variants and pI variants) can be combined independently and optionally with Fc ablation variants, Fc variants, and FcRn variants, as broadly outlined in this paper.

[0217] H. Applicable Forms of the Invention

[0218] As those skilled in the art will appreciate, and as will be discussed more fully below, the heterodimeric fusion protein of the present invention can take on a wide variety of conformations, as generally depicted in Figure 1. Some figures depict a “single-terminal” conformation, wherein one “arm” of the molecule exhibits one type of specificity and the other “arm” exhibits a different type of specificity. Other figures depict a “double-terminal” conformation, wherein the “top” of the molecule exhibits at least one type of specificity and the “bottom” of the molecule exhibits one or more different types of specificity. Therefore, the present invention relates to novel immunoglobulin compositions that co-conjugate with different first and second antigens.

[0219] As those skilled in the art will appreciate, the heterodimeric forms of the present invention can have different valences and be bispecific. That is, the heterodimeric antibodies of the present invention can be bivalent and bispecific, wherein one target tumor antigen (e.g., CD3) binds through one binding domain and another target tumor antigen (e.g., SSTR2) binds through a second binding domain. The heterodimeric antibodies can also be trivalent and bispecific, wherein the first antigen binds through both binding domains and the second antigen binds through a second binding domain. As outlined herein, when CD3 is one of the target antigens, CD3 preferably binds only monovalently to reduce potential side effects.

[0220] This invention utilizes a combination of an anti-CD3 antigen binding domain and an anti-SSTR2 binding domain. As those skilled in the art will appreciate, this can be used as shown in any of the figures (see details). Figure 2 Any set of anti-CD3 CDR, anti-CD3 light chain variable domain and heavy chain variable domain, Fab, and scFv as depicted in Figures 7 and 18. Similarly, any anti-SSTR2 antigen-binding domain can be used, as shown in any of the figures (e.g., Figure 8 The CDR, light chain variable domain and heavy chain variable domain, Fab and scFv described in (10) are optionally and independently combined in any combination.

[0221] Bottle opener

[0222] A heterodimer scaffold particularly suitable for use in this invention is as follows: Figure 1AThe “triple F” or “bottle opener” scaffold configuration is shown below. In this embodiment, one heavy chain of the antibody contains a single-chain Fv (“scFv”, as defined below) and the other heavy chain is a “regular” FAb configuration comprising a variable heavy chain and a light chain. This structure is sometimes referred to herein as a “triple F” configuration (scFv-FAb-Fc) or a “bottle opener” configuration because it roughly resembles a bottle opener visually. The two chains are introduced together using amino acid variants in constant regions (e.g., the Fc domain, CH1 domain, and / or hinge region) that facilitate the formation of heterodimeric antibodies, as described more fully below.

[0223] The "triple F" form of this invention has several significant advantages. As is known in the art, antibody analogs dependent on two scFv constructs typically suffer from stability and aggregation problems, which can be mitigated in this invention by adding "regular" heavy chain and light chain pairs. Furthermore, unlike forms dependent on two heavy chains and two light chains, there is no problem of improper pairing of heavy and light chains (e.g., heavy chain 1 paired with light chain 2).

[0224] Many of the embodiments outlined herein typically rely on a bottle opener form comprising a first monomer containing scFv, said scFv comprising heavy chain variable domains and light chain variable domains covalently linked using scFv linkers (which are charged in many cases), wherein the scFv is typically covalently linked to the N-terminus of the first Fc structural domain via domain linkers (which may be uncharged or charged as outlined herein). The second monomer in the bottle opener form is a heavy chain, and the composition additionally comprises a light chain.

[0225] Generally, in many preferred embodiments, scFv is a domain that binds CD3, and Fab forms an SSTR2 binding domain.

[0226] Furthermore, the Fc domain of the present invention typically includes skewed variants (e.g., a group of amino acid substitutions as shown in Figures 3 and 8, and particularly suitable skewed variants are selected from the group consisting of: S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411T / E360E / Q362E:D401K; L368D / K370S:S364K / E357L and K370S:S364K / E357Q); optionally ablation variants (containing...). Figure 5 Those shown in Figure 7); optional charged scFv connectors (including those shown in Figure 7); and heavy chains including pI variants (including those shown in Figure 7). Figure 4 (Those shown).

[0227] In some embodiments, the bottle opener form includes skewed variants, pI variants, and ablation variants. Therefore, some embodiments include a bottle opener form comprising: a) a first monomer (“scFv monomer”) comprising a charged scFv connector (preferably the +H sequence of FIG7 in some embodiments), a skew variant S364K / E357Q, an ablation variant E233P / L234V / L235A / G236del / S267K, and an Fv that binds CD3 as outlined herein; b) a second monomer (“Fab monomer”) comprising a skew variant L368D / K370S, a pI variant N208D / Q295E / N384D / Q418E / N421D, an ablation variant E233P / L234V / L235A / G236del / S267K, and a heavy chain variable domain, which together with the light chain variable domain constitutes an Fv that binds SSTR2 as outlined herein; and c) a light chain.

[0228] Exemplary heavy-chain and light-chain variable domains of the CD3-binding scFv are shown in Figures 12 and 13. Exemplary heavy-chain and light-chain variable domains of the SSTR2-binding Fv are shown in Figure 11. In one exemplary embodiment, the SSTR2 binding domain is the H1.143_L1.30 SSTR2 binding domain, and the CD3-binding scFv includes the heavy-chain and light-chain variable domains of the H1.30_L1.47 CD3 binding domain. Other particularly suitable combinations of SSTR2 and CD3 sequences are disclosed in Figure 16.

[0229] In some embodiments, the opener form includes skew variants, pI variants, ablation variants, and FcRn variants. Thus, some embodiments include an opener form comprising: a) a first monomer (“scFv monomer”) including a charged scFv connector (preferably the +H sequence of FIG7 in some embodiments), a skew variant S364K / E357Q, an ablation variant E233P / L234V / L235A / G236del / S267K, an FcRn variant M428L / N434S, and an Fv incorporating CD3 as outlined herein; b) a second The monomers (“Fab monomers”, which include the skewed variant L368D / K370S, the pI variant N208D / Q295E / N384D / Q418E / N421D, the ablation variant E233P / L234V / L235A / G236del / S267K, the FcRn variant M428L / N434S, and the heavy chain variable domain, which together with the light chain variable domain constitutes the SSTR2-binding Fv as outlined herein; and c) the light chain.

[0230] Exemplary heavy-chain and light-chain variable domains of scFv bound to CD3 are shown in Figures 12 and 13. Exemplary heavy-chain and light-chain variable domains of Fv bound to SSTR2 are shown in Figure 11. In one exemplary embodiment, the SSTR2 binding domain comprises the heavy-chain and light-chain variable domains of the H1.143_L1.30 SSTR2 binding domain, and the scFv bound to CD3 comprises the heavy-chain and light-chain variable domains of the H1.30_L1.47 CD3 binding domain. Other particularly suitable combinations of SSTR2 and CD3 sequences are disclosed in Figure 16.

[0231] Figure 9 illustrates some exemplary bottle opener “skeleton” sequences, the absence of which can be used for the Fv sequence in this invention. In some embodiments, any of the vh and vl sequences depicted herein (including all vh and vl sequences depicted in the figures and sequence listings) can be added to the bottle opener skeleton form of Figure 9 as a “Fab side” using any of the anti-CD3 scFv sequences shown in the figures and sequence listings.

[0232] For the opener skeleton 1 in FIG9 (optionally including the 428L / 434S variant), the CD binding domain sequences particularly suitable in these embodiments include, but are not limited to: CD3 binding domains anti-CD3 H1.30_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 anti-CD3 H1.31_L1.47, as well as those depicted in FIG12 and 13, which serve as scFv-side connections to the skeleton shown in FIG9.

[0233] For the bottle opener skeleton 1 in Figure 9 (optionally including the 428L / 434S variant), the SSTR2 binding domain sequences particularly suitable in these embodiments include, but are not limited to: anti-SSTR2 H1.143_L1.30; anti-SSTR2 H1_L1.1; anti-SSTR2 H1.107_L1.30; anti-SSTR2 H1.107_L1.67; anti-SSTR2 H1.107_L1.108; anti-SSTR2 H1.107_L1.111; anti-SSTR2 H1.107_L1.114; anti-SSTR2 H1.107_L1.102; anti-SSTR2 H1.107_L1.110; anti-SSTR2 H1.125_L1.30; anti-SSTR2 H1.125_L1.67; anti-SSTR2 H1.125_L1.108; anti-SSTR2 H1.125_L1.111; anti-SSTR2 H1.125_L1.114; anti-SSTR2 H1.125_L1.102; and anti-SSTR2 H1.125_L1.10.

[0234] The SSTR2 and CD3 sequence combinations, particularly suitable for use with the bottle opener skeleton 1 in Figure 9 (optionally including the 428L / 434S variant), are disclosed in Figure 16.

[0235] In one exemplary embodiment, the bottle opener antibody comprises the bottle opener "backbone" 1 in FIG9, the SSTR2 binding domain comprising the heavy chain variable domain and the light chain variable domain of the SSTR2 binding domain H1.143_L1.30, and the CD3-binding scFv comprising the heavy chain variable domain and the light chain variable domain of the CD3 binding domain H1.30_L1.47.

[0236] mAb-Fv

[0237] A heterodimer scaffold particularly suitable for use in this invention is Figure 1H The mAb-Fv form is shown in this embodiment. In this embodiment, the form relies on the C-terminus of an “extra” heavy chain variable domain connected to one monomer and the C-terminus of an “extra” light chain variable domain connected to another monomer to form a third antigen-binding domain, wherein the Fab portions of both monomers bind SSTR2 and the “extra” scFv domain binds CD3.

[0238] In this embodiment, the first monomer comprises a first heavy chain including a first heavy chain variable domain and a first heavy chain constant domain including a first Fc structural domain, wherein the first light chain variable domain is covalently connected to the C-terminus of the first Fc structural domain using domain connectors (vh1-CH1-hinge-CH2-CH3-[optional connector]-vl2). The second monomer comprises a second heavy chain variable domain belonging to a second heavy chain constant domain including a second Fc structural domain, and a third heavy chain variable domain covalently connected to the C-terminus of the second Fc structural domain using domain connectors (vj1-CH1-hinge-CH2-CH3-[optional connector]-vh2). The two C-terminus-connected variable domains constitute an Fv bonded to CD3 (since it is less preferred to have a bivalent CD3 bond). This embodiment further uses a shared light chain including a light chain variable domain and a light chain constant domain, which associates with the heavy chain to form two identical Fabs bonded to SSTR2. For many of the embodiments described herein, these constructs include desired and described herein skewed variants, pI variants, ablation variants, additional Fc variants, etc.

[0239] This invention provides CD binding domain sequences as shown in Figures 12 and 13 and the mAb-Fv form in the sequence listing. This invention also provides SSTR2 binding domain sequences as shown in Figure 11 and the mAb-Fv form in the sequence listing. Particularly suitable SSTR2 and CD3 sequence combinations for use with the mAb-Fv form are disclosed in Figure 16.

[0240] In addition, the Fc domain in mAb-Fv form includes skewed variants (e.g., a set of amino acid substitutions as shown in Figures 3 and 8, with particularly suitable skewed variants selected from the group consisting of: S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411T / E360E / Q362E:D401K; L368D / K370S:S364K / E357L; K370S:S364K / E357Q; T366S / L368A / Y407V:T366W; and T366S / L368A / Y407V / Y349C:T366W / S354C); optionally ablation variants (containing Figure 5 Those shown in Figure 7); optional charged scFv connectors (including those shown in Figure 7); and heavy chains including pI variants (including those shown in Figure 7). Figure 4 (Those shown).

[0241] In some embodiments, the mAb-Fv form comprises a skewed variant, a pI variant, and an ablation variant. Therefore, some embodiments include the mAb-Fv form comprising: a) a first monomer comprising the skewed variant S364K / E357Q, the ablation variant E233P / L234V / L235A / G236del / S267K, and a first heavy chain variable domain (which, together with the first light chain variable domain of the light chain, constitutes an Fv binding SSTR2) and a second heavy chain variable domain; b) a second monomer comprising the skewed variant L368D / K370S, the pI variant N2, and... 08D / Q295E / N384D / Q418E / N421D, ablation variant E233P / L234V / L235A / G236del / S267K, and a first heavy chain variable domain (which together with the first light chain variable domain constitutes an SSTR2-binding Fv as outlined herein) and a second variable light chain (which together with the second heavy chain variable domain forms a CD3-binding Fv(ABD); and c) a light chain comprising a first light chain variable domain and a light chain constant domain.

[0242] In some embodiments, the mAb-Fv form comprises a skewed variant, a pI variant, an ablation variant, and an FcRn variant. Therefore, some embodiments include the mAb-Fv form comprising: a) a first monomer comprising the skewed variant S364K / E357Q, the ablation variant E233P / L234V / L235A / G236del / S267K, the FcRn variant M428L / N434S, and a first heavy chain variable domain (which, together with the first light chain variable domain of the light chain, constitutes an SSTR2-binding Fv) and a second heavy chain variable domain; b) a second monomer comprising the skewed variant L368D / K370S, the pI variant N208D / Q295E / N384D / Q418E / N421D, ablation variant E233P / L234V / L235A / G236del / S267K, FcRn variant M428L / N434S, and a first heavy chain variable domain (which, together with the first light chain variable domain, constitutes an Fv that binds SSTR2 as outlined herein), and a second variable light chain, which, together with the second heavy chain variable domain of the first monomer, forms an Fv (ABD) that binds CD3; and c) a light chain comprising a first light chain variable domain and a light chain constant domain.

[0243] For mAb-Fv sequences similar to mAb-Fv backbone 1 in Figure 10 (optionally including the M428L / 434S variant), CD3 binding domain sequences particularly suitable in these embodiments include, but are not limited to: anti-CD3 H1.30_L1.47, anti-CD3H1.32_L1.47, anti-CD3 H1.89_L1.47, anti-CD3 H1.90_L1.47, anti-CD3 H1.33_L1.47, and anti-CD3H1.31_L1.47, as well as those depicted in Figures 12 and 13.

[0244] For mAb-Fv sequences similar to mAb-Fv backbone 1 in Figure 10 (optionally including the M428L / 434S variant), the SSTR2 binding domain sequences particularly suitable in these embodiments include, but are not limited to: anti-SSTR2 H1.143_L1.30; anti-SSTR2 H1_L1.1; anti-SSTR2 H1.107_L1.30; anti-SSTR2 H1.107_L1.67; anti-SSTR2 H1.107_L1.108; anti-SSTR2 H1.107_L1.111; anti-SSTR2 H1.107_L1.114; anti-SSTR2 H1.107_L1.102; anti-SSTR2 H1.107_L1.110; anti-SSTR2 H1.125_L1.30; anti-SSTR2 H1.125_L1.67; anti-SSTR2 H1.125_L1.108; anti-SSTR2 H1.125_L1.111; anti-SSTR2 H1.125_L1.114; anti-SSTR2 H1.125_L1.102; and anti-SSTR2 H1.125_L1.10, as well as those listed in Figures 11 and 15 and SEQ ID NO: 68 to 659.

[0245] The SSTR2 and CD3 sequence combinations that are particularly suitable for use with mAb-Fv sequences similar to mAb-Fv backbone 1 (optionally including the 428L / 434S variant) in Figure 10 are disclosed in Figure 16.

[0246] mAb-scFv

[0247] One heterodimeric scaffold particularly suitable for this invention is the mAb-Fv form shown in Figure 1. In this embodiment, the form relies on the C-terminus of the scFv being connected to one of the monomers to form a third antigen-binding domain, wherein the Fab portions of both monomers bind SSTR2 and an “additional” scFv domain binds CD3. Thus, the first monomer comprises a first heavy chain (including a heavy chain variable domain and a constant domain) and a C-terminus covalently connected to the scFv, which includes a scFv light chain variable domain, an scFv connector, and a scFv heavy chain variable domain, oriented (vh1-CH1-hinge-CH2-CH3-[optional connector]-vh2-scFv connector-vl2 or vh1-CH1-hinge-CH2-CH3-[optional connector]-vl2-scFv connector-vh2). This embodiment further utilizes a shared light chain including a light chain variable domain and a light chain constant domain, which associates with the heavy chain to form two identical Fabs that bind SSTR2. For many of the embodiments described herein, these constructs include desired and described herein skewed variants, pI variants, ablation variants, additional Fc variants, etc.

[0248] This invention provides CD binding domain sequences as shown in Figures 12 and 13 and the mAb-scFv form in the sequence listing. This invention also provides SSTR2 binding domain sequences as shown in Figure 11 and the mAb-scFv form in the sequence listing. Particularly applicable SSTR2 and CD3 sequence combinations for use with the mAb-scFv form are disclosed in Figure 16.

[0249] In addition, the Fc domain of the mAb-scFv form includes skew variants (e.g., a set of amino acid substitutions as shown in Figures 3 and 8, with particularly suitable skew variants selected from the group consisting of: S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411T / E360E / Q362E:D401K; L368D / K370S:S364K / E357L; K370S:S364K / E357Q; T366S / L368A / Y407V:T366W; and T366S / L368A / Y407V / Y349C:T366W / S354C); optionally ablation variants (containing Figure 5 Those shown in Figure 7); optional charged scFv connectors (including those shown in Figure 7); and heavy chains including pI variants (including those shown in Figure 7). Figure 4 (Those shown).

[0250] In some embodiments, the mAb-scFv form comprises a skew variant, a pI variant, and an ablation variant. Therefore, some embodiments include the mAb-scFv form comprising: a) a first monomer comprising the skew variant S364K / E357Q, the ablation variant E233P / L234V / L235A / G236del / S267K, a heavy chain variable domain (which, together with a light chain variable domain sharing a light chain, constitutes an SSTR2-binding Fv as outlined herein), and a CD3-binding scFv domain; b) a second monomer, which This includes the skewed variant L368D / K370S, the pI variant N208D / Q295E / N384D / Q418E / N421D, the ablation variant E233P / L234V / L235A / G236del / S267K, and the heavy chain variable domain (which, together with the light chain variable domain of the shared light chain, constitutes the Fv of SSTR2 as outlined herein); and c) the shared light chain, including the light chain variable domain and the light chain constant domain.

[0251] In some embodiments, the mAb-scFv form comprises a skew variant, a pI variant, an ablation variant, and an FcRn variant. Thus, some embodiments include the mAb-scFv form comprising: a) a first monomer comprising the skew variant S364K / E357Q, the ablation variant E233P / L234V / L235A / G236del / S267K, a heavy chain variable domain (which, together with a light chain variable domain sharing a light chain, constitutes an SSTR2-binding Fv as outlined herein), and a CD3-binding scFv domain; b) a second monomer comprising the skew variant L368. D / K370S, pI variant N208D / Q295E / N384D / Q418E / N421D, ablation variant E233P / L234V / L235A / G236del / S267K, FcRn variant M428L / N434S, and heavy chain variable domains (which, together with the light chain variable domains of the shared light chain, constitute the Fv of SSTR2 as outlined herein); and c) shared light chains, which include light chain variable domains and light chain constant domains.

[0252] In the mAb-scFv backbone 1 (optionally comprising M428L / N434S) in Figure 10 (optionally comprising 428L / 434S variant CD3), the CD3 binding domain sequences particularly suitable in these embodiments include, but are not limited to: anti-CD3 H1.30_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 anti-CD3 H1.31_L1.47, as well as those depicted in Figures 12 and 13.

[0253] In the mAb-scFv backbone 1 (optionally comprising M428L / N434S) in Figure 10 (optionally comprising the 428L / 434S variant), the SSTR2 binding domain sequences particularly suitable in these embodiments include, but are not limited to: anti-SSTR2 H1.143_L1.30; anti-SSTR2 H1_L1.1; anti-SSTR2 H1.107_L1.30; anti-SSTR2 H1.107_L1.67; anti-SSTR2 H1.107_L1.108; anti-SSTR2 H1.107_L1.111; anti-SSTR2 H1.107_L1.114; anti-SSTR2 H1.107_L1.102; anti-SSTR2 H1.107_L1.110; anti-SSTR2 H1.125_L1.30; anti-SSTR2 H1.125_L1.67; anti-SSTR2 H1.125_L1.108; anti-SSTR2 H1.125_L1.111; anti-SSTR2 H1.125_L1.114; anti-SSTR2 H1.125_L1.102; and anti-SSTR2 H1.125_L1.10, as well as those listed in Figures 11 and 15 and SEQ ID NO: 68 to 659.

[0254] Center scFv

[0255] One type of heterodimer scaffold particularly suitable for this invention is the central scFv form shown in Figure 1. In this embodiment, the form relies on the use of an inserted scFv domain to form a third antigen-binding domain, wherein the Fab portions of both monomers bind SSTR2 and the “additional” scFv domain binds CD3. The scFv domain is inserted between the Fc domain and the CH1-Fv region of one of the monomers, thereby providing the third antigen-binding domain.

[0256] In this embodiment, one monomer includes a first heavy chain comprising a first heavy chain variable domain, a CH1 domain (and optional hinge), and an Fc structural domain, and the scFv includes an scFv light chain variable domain, an scFv connector, and an scFv heavy chain variable domain. The scFv is covalently connected between the C-end of the heavy chain constant domain CH1 and the N-end of the first Fc structural domain using optional domain connectors (vh1-CH1-[optional connector]-vh2-scFv connector-vl2-[optional connector including hinge]-CH2-CH3, or scFv reverse orientation vh1-CH1-[optional connector]-vl2-scFv connector-vh2-[optional connector including hinge]-CH2-CH3). The other monomer is the standard Fab side. This embodiment further uses a shared light chain comprising a light chain variable domain and a light chain constant domain, which is associated with the heavy chain to form two identical Fabs bonded to SSTR2. For many of the embodiments described herein, these constructs include desired and described herein skewed variants, pI variants, ablation variants, additional Fc variants, etc.

[0257] This invention provides a CD3 binding domain sequence as shown in Figures 12 and 13, and a central scFv form as shown in the sequence listing. This invention also provides an anti-SSTR2 sequence as shown in Figure 11 and a central scFv form as shown in the sequence listing. Combinations of SSTR2 and CD3 sequences particularly suitable for use with the central scFv form are disclosed in Figure 16.

[0258] In addition, the Fc domain of the central scFv form includes skewed variants (e.g., a set of amino acid substitutions as shown in Figures 3 and 8, and particularly suitable skewed variants are selected from the group consisting of: S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411T / E360E / Q362E:D401K; L368D / K370S:S364K / E357L; K370S:S364K / E357Q; T366S / L368A / Y407V:T366W; and T366S / L368A / Y407V / Y349C:T366W / S354C); optionally ablation variants (containing Figure 5 Those shown in Figure 7); optional charged scFv connectors (including those shown in Figure 7); and heavy chains including pI variants (including those shown in Figure 7). Figure 4 (Those shown).

[0259] In some embodiments, the central scFv form includes skew variants, pI variants, and ablation variants. Therefore, some embodiments include a central scFv form comprising: a) a first monomer comprising the skew variant S364K / E357Q, the ablation variant E233P / L234V / L235A / G236del / S267K, and a heavy chain variable domain (which, together with the light chain variable domain of the light chain, constitutes an SSTR2-binding Fv as outlined herein) and a CD3-binding scFv domain; b) a second monomer comprising the skew variant L368D / K370S, the pI variant N208D / Q295E / N384D / Q418E / N421D, the ablation variant E233P / L234V / L235A / G236del / S267K, and a heavy chain variable domain (which, together with the light chain variable domain of the light chain, constitutes an SSTR2-binding Fv as outlined herein); and c) a light chain comprising a light chain variable domain and a light chain constant domain.

[0260] In some embodiments, the central scFv form includes skew variants, pI variants, ablation variants, and FcRn variants. Therefore, some embodiments include a central scFv form comprising: a) a first monomer comprising the skew variant S364K / E357Q, the ablation variant E233P / L234V / L235A / G236del / S267K, a heavy chain variable domain (which, together with the light chain variable domain, constitutes an SSTR2-binding Fv as outlined herein), and a CD3-binding scFv domain; b) a second monomer comprising the skew variant L368. D / K370S, pI variant N208D / Q295E / N384D / Q418E / N421D, ablation variant E233P / L234V / L235A / G236del / S267K, FcRn variant M428L / N434S, and heavy chain variable domains (which, together with the light chain variable domains of the light chains, constitute the Fv of SSTR2 as outlined herein); and c) light chains, which include light chain variable domains and light chain constant domains.

[0261] For a central scFv sequence (optionally including M428L / 434S) similar to / using the opener skeleton 1 in Figure 9, the CD3 binding domain sequences particularly suitable in these embodiments include, but are not limited to: anti-CD3H1.30_L1.47; anti-CD3H1.32_L1.47, 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, as well as those depicted in Figures 12 and 13.

[0262] For a central scFv sequence (optionally including the M428L / 434S variant) similar to / using the opener skeleton 1 in Figure 9, the SSTR2 binding domain sequences particularly suitable in these embodiments include, but are not limited to: anti-SSTR2 H1.143_L1.30; anti-SSTR2 H1_L1.1; anti-SSTR2 H1.107_L1.30; anti-SSTR2 H1.107_L1.67; anti-SSTR2 H1.107_L1.108; anti-SSTR2 H1.107_L1.111; anti-SSTR2 H1.107_L1.114; anti-SSTR2 H1.107_L1.102; anti-SSTR2 H1.107_L1.110; anti-SSTR2 H1.125_L1.30; anti-SSTR2H1.125_L1.67; anti-SSTR2 H1.125_L1.108; anti-SSTR2 H1.125_L1.111; anti-SSTR2 H1.125_L1.114; anti-SSTR2 H1.125_L1.102; and anti-SSTR2 H1.125_L1.10, as well as those listed in Figures 11 and 15 and SEQ ID NO: 68 to 659.

[0263] Center Fv

[0264] A heterodimer scaffold particularly suitable for use in this invention is Figure 1G The central Fv form is shown in the illustration. In this embodiment, the form relies on the use of an inserted Fv domain (i.e., the central Fv domain) to form a third antigen-binding domain, wherein the Fab portions of both monomers bind SSTR2 and the "central Fv" domain binds CD3. An scFv domain is inserted between the Fc domain and the CH1-Fv region of the monomer to provide the third antigen-binding domain, wherein each monomer contains an scFv component (e.g., one monomer contains a heavy chain variable domain and the other contains a light chain variable domain).

[0265] In this embodiment, one monomer comprises a first heavy chain, which includes a first heavy chain variable domain, a CH1 domain, an Fc structural domain, and an additional light chain variable domain. The light chain domain is covalently connected between the C-terminus of the CH1 domain of the heavy chain constant domain and the N-terminus of the first Fc structural domain using domain connectors (vh1-CH1-[optional connector]-vl2-hinge-CH2-CH3). Another monomer comprises a first heavy chain, which includes a first heavy chain variable domain, a CH1 domain, an Fc structural domain, and an additional heavy chain variable domain (vh1-CH1-[optional connector]-vh2-hinge-CH2-CH3). The light chain domain is covalently connected between the C-terminus of the CH1 domain of the heavy chain constant domain and the N-terminus of the first Fc structural domain using domain connectors.

[0266] This embodiment further utilizes a shared light chain comprising a light chain variable domain and a light chain constant domain, which associates with the heavy chain to form two identical Fabs that bind SSTR2. For many embodiments herein, these constructs include desired and described herein skewed variants, pI variants, ablation variants, additional Fc variants, etc.

[0267] This invention provides a CD3 binding domain sequence as shown in Figures 12 and 13, and a central Fv form as shown in the sequence listing. This invention also provides an SSTR2 binding domain sequence as shown in Figure 11, and a central Fv form as shown in the sequence listing. Particularly applicable combinations of SSTR2 and CD3 sequences used with the central Fv form are disclosed in Figure 16.

[0268] For the central Fv form, in these embodiments, the CD3 binding domain sequences particularly suitable include, but are not limited to: anti-CD3 H1.30_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 anti-CD3 H1.31_L1.47, as well as those depicted in Figures 12 and 13.

[0269] For the central Fv form, the SSTR2 binding domain sequences particularly suitable in these embodiments include, but are not limited to: anti-SSTR2 H1.143_L1.30; anti-SSTR2 H1_L1.1; anti-SSTR2 H1.107_L1.30; anti-SSTR2 H1.107_L1.67; anti-SSTR2 H1.107_L1.108; anti-SSTR2 H1.107_L1.111; anti-SSTR2 H1.107_L1.114; anti-SSTR2 H1.107_L1.102; anti-SSTR2 H1.107_L1.110; anti-SSTR2 H1.125_L1.30; anti-SSTR2 H1.125_L1.67; anti-SSTR2 H1.125_L1.108; anti-SSTR2 H1.125_L1.111; anti-SSTR2 H1.125_L1.114; anti-SSTR2 H1.125_L1.102; and anti-SSTR2 H1.125_L1.10, as well as those listed in Figures 11 and 15 and SEQ ID NO:68 to 659.

[0270] Single-arm center scFv

[0271] One type of heterodimer scaffold particularly suitable for this invention is the single-arm central scFv form shown in Figure 1. In this embodiment, one monomer contains only the Fc domain, while the other monomer uses the inserted scFv domain to form a second antigen-binding domain. In this form, the Fab portion binds SSTR2 and the scFv binds CD3, or vice versa. The scFv domain is inserted between the Fc domain and the CH1-Fv region of one of the monomers.

[0272] In this embodiment, a monomer comprises a first heavy chain including a first heavy chain variable domain, a CH1 domain, and an Fc structural domain, and the scFv includes an scFv light chain variable domain, an scFv linker, and an scFv heavy chain variable domain. The scFv is covalently connected between the C-terminus of the CH1 domain of the heavy chain constant domain and the N-terminus of the first Fc structural domain using domain linkers. A second monomer comprises an Fc structural domain. This embodiment further uses a light chain including a light chain variable domain and a light chain constant domain, which is associated with the heavy chain to form a Fab. For many embodiments herein, these constructs include desired and described herein skewed variants, pI variants, ablation variants, additional Fc variants, etc.

[0273] This invention provides a CD3 binding domain sequence as shown in Figures 12 and 13, and a central Fv form as shown in the sequence listing. This invention also provides an SSTR2 binding domain sequence as shown in Figure 11, and a central Fv form as shown in the sequence listing. Particularly applicable combinations of SSTR2 and CD3 sequences used with the central Fv form are disclosed in Figure 16.

[0274] Furthermore, the Fc domain in the form of a single-arm central scFv generally includes skew variants (e.g., a group of amino acid substitutions as shown in Figures 3 and 8, with particularly suitable skew variants selected from the group consisting of: S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411T / E360E / Q362E:D401K; L368D / K370S:S364K / E357L; K370S:S364K / E357Q; T366S / L368A / Y407V:T366W and T366S / L368A / Y407V / Y349C:T366W / S354C); optionally ablation variants (including...) Figure 5 Those shown in Figure 7); optional charged scFv connectors (including those shown in Figure 7); and heavy chains including pI variants (including those shown in Figure 7). Figure 4 (Those shown).

[0275] In some embodiments, the single-arm central scFv form includes skew variants, pI variants, and ablation variants. Therefore, some embodiments of the single-arm central scFv form include: a) a first monomer comprising: a skew variant S364K / E357Q, an ablation variant E233P / L234V / L235A / G236del / S267K, a heavy chain variable domain (which, together with the light chain variable domain of the light chain, constitutes an SSTR2-binding Fv as outlined herein), and a CD3-binding scFv structural domain; b) a second monomer comprising an Fc structural domain having a skew variant L368D / K370S, a pI variant N208D / Q295E / N384D / Q418E / N421D, an ablation variant E233P / L234V / L235A / G236del / S267K; and c) a light chain comprising a light chain variable domain and a light chain constant domain.

[0276] In some embodiments, the single-arm central scFv form comprises a skewed variant, a pI variant, an ablation variant, and an FcRn variant. Thus, some embodiments of the single-arm central scFv form include: a) a first monomer comprising the skewed variant S364K / E357Q, the ablation variant E233P / L234V / L235A / G236del / S267K, the FcRn variant M428L / N434S, and a heavy chain variable domain (which, together with the light chain variable domain, constitutes an SSTR2-binding Fv as outlined herein) and a CD3-binding scFv junction. b) a second monomer comprising an Fc domain having skewed variants L368D / K370S, pI variants N208D / Q295E / N384D / Q418E / N421D, ablation variants E233P / L234V / L235A / G236del / S267K, and FcRn variant M428L / N434S; and c) a light chain comprising a light chain variable domain and a light chain constant domain.

[0277] For the single-arm central scFv form, particularly suitable CD3 binding domain sequences include, but are not limited to: anti-CD3 H1.30_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 anti-CD3 H1.31_L1.47, as well as those depicted in Figures 12 and 13.

[0278] For the single-arm central scFv form, the SSTR2 binding domain sequences that are particularly suitable include, but are not limited to: anti-SSTR2H1.143_L1.30; anti-SSTR2 H1_L1.1; anti-SSTR2 H1.107_L1.30; anti-SSTR2 H1.107_L1.67; anti-SSTR2 H1.107_L1.108; anti-SSTR2 H1.107_L1.111; anti-SSTR2 H1.107_L1.114; anti-SSTR2H1.107_L1.102; anti-SSTR2 H1.107_L1.110; anti-SSTR2 H1.125_L1.30; anti-SSTR2 H1.125_L1.67; anti-SSTR2 H1.125_L1.108; anti-SSTR2 H1.125_L1.111; anti-SSTR2 H1.125_L1.114; anti-SSTR2 H1.125_L1.102; anti-SSTR2 H1.125_L1.10, and those listed in Figures 11 and 15 and SEQ ID NO:68 to 659.

[0279] Single-arm scFv-mAb

[0280] A heterodimer scaffold particularly suitable for use in this invention is Figure 1D The single-arm scFv-mAb form is shown. In this embodiment, one monomer includes only the Fc domain, while the other monomer uses the scFv domain attached to the N end of the heavy chain, typically by using a connector: vh-scFv connector-vl-[optional domain connector]-CH1-hinge-CH2-CH3 or (in opposite orientation) vl-scFv connector-vh-[optional domain connector]-CH1-hinge-CH2-CH3. In this form, the Fab portion is each bonded to SSTR2 and the scFv is bonded to CD3. This embodiment further uses a light chain including a light chain variable domain and a light chain constant domain, which is associated with the heavy chain to form the Fab. For many embodiments herein, these constructs include desired and as described herein skewed variants, pI variants, ablation variants, additional Fc variants, etc.

[0281] This invention provides a single-arm scFv-mAb form in which the CD3 binding domain sequence is shown in Figures 12 and 13 and in the sequence listing. This invention also provides a single-arm scFv-mAb form in which the SSTR2 binding domain sequence is shown in Figure 11 and in the sequence listing. A combination of SSTR2 and CD3 sequences, particularly suitable for use with the single-arm scFv-mAb form, is disclosed in Figure 16.

[0282] Furthermore, the Fc domain in the single-arm scFv-mAb form generally includes skewed variants (e.g., a group of amino acid substitutions as shown in Figures 3 and 8, with particularly suitable skewed variants selected from the group consisting of: S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411T / E360E / Q362E:D401K; L368D / K370S:S364K / E357L; K370S:S364K / E357Q; T366S / L368A / Y407V:T366W and T366S / L368A / Y407V / Y349C:T366W / S354C); optionally ablation variants (including...) Figure 5 Those shown in Figure 7); optional charged scFv connectors (including those shown in Figure 7); and heavy chains including pI variants (including those shown in Figure 7). Figure 4 (Those shown).

[0283] In some embodiments, the single-arm scFv-mAb form includes skew variants, pI variants, and ablation variants. Therefore, some embodiments of the single-arm scFv-mAb form include: a) a first monomer comprising: a skew variant S364K / E357Q, an ablation variant E233P / L234V / L235A / G236del / S267K, a heavy chain variable domain (which, together with the light chain variable domain of the light chain, constitutes an SSTR2-binding Fv as outlined herein), and a CD3-binding scFv domain; b) a second monomer comprising an Fc domain having a skew variant L368D / K370S, a pI variant N208D / Q295E / N384D / Q418E / N421D, an ablation variant E233P / L234V / L235A / G236del / S267K; and c) a light chain comprising a light chain variable domain and a light chain constant domain.

[0284] In some embodiments, the single-arm scFv-mAb form comprises a skewed variant, a pI variant, an ablation variant, and an FcRn variant. Thus, some embodiments of the single-arm scFv-mAb form include: a) a first monomer comprising the skewed variant S364K / E357Q, the ablation variant E233P / L234V / L235A / G236del / S267K, the FcRn variant M428L / N434S, and a heavy chain variable domain (which, together with the light chain variable domain, constitutes an SSTR2-binding Fv as outlined herein) and a CD3-binding scFv. a) a structural domain; b) a second monomer comprising an Fc structural domain having skewed variants L368D / K370S, pI variants N208D / Q295E / N384D / Q418E / N421D, ablation variants E233P / L234V / L235A / G236del / S267K, and FcRn variant M428L / N434S; and c) a light chain comprising a light chain variable domain and a light chain constant domain.

[0285] For the single-arm scFv-mAb form, particularly suitable CD3 binding domain sequences include, but are not limited to: anti-CD3 H1.30_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 anti-CD3 H1.31_L1.47, as well as those depicted in Figures 12 and 13.

[0286] For single-arm scFv-mAb forms, particularly suitable SSTR2 binding domain sequences include, but are not limited to: anti-SSTR2H1.143_L1.30; anti-SSTR2 H1_L1.1; anti-SSTR2 H1.107_L1.30; anti-SSTR2 H1.107_L1.67; anti-SSTR2 H1.107_L1.108; anti-SSTR2 H1.107_L1.111; anti-SSTR2 H1.107_L1.114; anti-SSTR2H1.107_L1.102; anti-SSTR2 H1.107_L1.110; anti-SSTR2 H1.125_L1.30; anti-SSTR2 H1.125_L1.67; anti-SSTR2 H1.125_L1.108; anti-SSTR2 H1.125_L1.111; anti-SSTR2 H1.125_L1.114; anti-SSTR2 H1.125_L1.102; and anti-SSTR2 H1.125_L1.10, as well as those listed in Figures 11 and 15 and SEQ ID NO:68 to 659.

[0287] scFv-mAb

[0288] A heterodimer scaffold particularly suitable for use in this invention is Figure 1E The mAb-scFv form is shown in this embodiment. In this embodiment, the form relies on the N-terminus of the scFv being attached to one of the monomers to form a third antigen-binding domain, wherein the Fab portions of both monomers bind SSTR2 and the “additional” scFv domain binds CD3.

[0289] In this embodiment, the first monomer includes a first heavy chain (comprising a heavy chain variable domain and a constant domain) and is covalently connected to an N-terminus scFv, which includes a scFv light chain variable domain, a scFv connector, and a scFv heavy chain variable domain, oriented in the following directions: ((vh1-scFv connector-vl1-[optional domain connector]-vh2-CH1-hinge-CH2-CH3) or (scFv in the opposite orientation) ((vl1-scFv connector-vh1-[optional domain connector]-vh2-CH1-hinge-CH2-CH3)). This embodiment further utilizes a shared light chain comprising a light chain variable domain and a light chain constant domain, which is associated with the heavy chain to form two identical Fabs that bind to SSTR2. For many embodiments herein, these constructs include desired and described herein skewed variants, pI variants, ablation variants, additional Fc variants, etc.

[0290] This invention provides CD3 binding domain sequences as shown in Figures 12 and 13, and in the scFv-mAb form shown in the sequence listing. This invention also provides SSTR2 binding domain sequences as shown in Figure 11 and in the scFv-mAb form shown in the sequence listing. Particularly suitable combinations of SSTR2 and CD3 sequences for use with the scFv-mAb form are disclosed in Figure 16.

[0291] Furthermore, the Fc domain in the scFv-mAb form generally includes skewed variants (e.g., a group of amino acid substitutions as shown in Figures 3 and 8, with particularly suitable skewed variants selected from the group consisting of: S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411T / E360E / Q362E:D401K; L368D / K370S:S364K / E357L; K370S:S364K / E357Q; T366S / L368A / Y407V:T366W and T366S / L368A / Y407V / Y349C:T366W / S354C); optionally ablation variants (including...) Figure 5 Those shown in Figure 7); optional charged scFv connectors (including those shown in Figure 7); and heavy chains including pI variants (including those shown in Figure 7). Figure 4 (Those shown).

[0292] In some embodiments, the scFv-mAb form comprises a skewed variant, a pI variant, and an ablation variant. Therefore, some embodiments include the scFv-mAb form comprising: a) a first monomer comprising the skewed variant S364K / E357Q, the ablation variant E233P / L234V / L235A / G236del / S267K, a heavy chain variable domain (which, together with a light chain variable domain sharing a light chain, constitutes an SSTR2-binding Fv as outlined herein), and a CD3-binding scFv domain; b) a second monomer, which This includes the skewed variant L368D / K370S, the pI variant N208D / Q295E / N384D / Q418E / N421D, the ablation variant E233P / L234V / L235A / G236del / S267K, and the heavy chain variable domain (which, together with the light chain variable domain of the common light chain, constitutes the Fv of SSTR2 as outlined herein); and c) the shared light chain, including the light chain variable domain and the light chain constant domain.

[0293] In some embodiments, the scFv-mAb form comprises a skewed variant, a pI variant, an ablation variant, and an FcRn variant. Therefore, some embodiments include the scFv-mAb form comprising: a) a first monomer comprising the skewed variant S364K / E357Q, the ablation variant E233P / L234V / L235A / G236del / S267K, the FcRn variant M428L / N434S, a heavy chain variable domain (which, together with a light chain variable domain sharing a light chain, constitutes an SSTR2-binding Fv as outlined herein), and a CD3-binding scFv domain; b) a second monomer comprising... This includes the skewed variant L368D / K370S, the pI variant N208D / Q295E / N384D / Q418E / N421D, the ablation variant E233P / L234V / L235A / G236del / S267K, the FcRn variant M428L / N434S, and the heavy chain variable domain (which, together with the light chain variable domain of the shared light chain, constitutes the Fv of SSTR2 as outlined herein); and c) the shared light chain, which includes the light chain variable domain and the light chain constant domain.

[0294] For the mAb-scFv form skeleton 1 in Figure 10 (optionally including M428L / N434S), the particularly suitable CD3 binding domain sequences include, but are not limited to: anti-CD3 H1.30_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 anti-CD3 H1.31_L1.47, as well as those depicted in Figures 12 and 13.

[0295] For the mAb-scFv form skeleton 1 in Figure 10 (optionally including M428L / N434S), the particularly suitable SSTR2 binding domain sequences include, but are not limited to: anti-SSTR2 H1.143_L1.30; anti-SSTR2 H1_L1.1; anti-SSTR2 H1.107_L1.30; anti-SSTR2 H1.107_L1.67; anti-SSTR2 H1.107_L1.108; anti-SSTR2 H1.107_L1.111; anti-SSTR2 H1.107_L1.114; anti-SSTR2 H1.107_L1.102; anti-SSTR2 H1.107_L1.110; anti-SSTR2 H1.125_L1.30; anti-SSTR2 H1.125_L1.67; anti-SSTR2 H1.125_L1.108; anti-SSTR2 H1.125_L1.111; anti-SSTR2 H1.125_L1.114; anti-SSTR2 H1.125_L1.102; and anti-SSTR2 H1.125_L1.10, as well as those listed in Figures 11 and 15 and SEQ ID NO: 68 to 659.

[0296] Dual SCFV form

[0297] The present invention also provides, as known in the art and as... Figure 1B The dual scFv form is shown. In this embodiment, the SSTR2×CD3 heterodimeric bispecific antibody consists of two scFv-Fc monomers (both monomers are in the form of (vh-scFv linker-vl-[optional domain linker]-CH2-CH3) or (vl-scFv linker-vh-[optional domain linker]-CH2-CH3), or one monomer is oriented in one direction and the other monomer is oriented in another direction).

[0298] This invention provides a double scFv form in which the CD3 binding domain sequence is shown in Figures 12 and 13 and in the sequence listing. This invention also provides a double scFv form in which the SSTR2 binding domain sequence is shown in Figure 11 and in the sequence listing. A combination of SSTR2 and CD3 sequences, particularly suitable for use with the double scFv form, is disclosed in Figure 16.

[0299] In some embodiments, the dual scFv form comprises a skew variant, a pI variant, and an ablation variant. Therefore, some embodiments include a dual scFv form comprising: a) a first monomer comprising the skew variant S364K / E357Q, the ablation variant E233P / L234V / L235A / G236del / S267K, and a first scFv combining CD3 or SSTR2; and b) a second monomer comprising the skew variant L368D / K370S, the pI variant N208D / Q295E / N384D / Q418E / N421D, the ablation variant E233P / L234V / L235A / G236del / S267K, and a second scFv combining CD3 or SSTR2.

[0300] In some embodiments, the dual scFv form includes a skew variant, a pI variant, an ablation variant, and an FcRn variant. Therefore, some embodiments include a dual scFv form, comprising: a) a first monomer comprising the skewed variant S364K / E357Q, the ablation variant E233P / L234V / L235A / G236del / S267K, the FcRn variant M428L / N434S, and a first scFv bound to CD3 or SSTR2; and b) a second monomer comprising the skewed variant L368D / K370S, the pI variant N208D / Q295E / N384D / Q418E / N421D, the ablation variant E233P / L234V / L235A / G236del / S267K, the FcRn variant M428L / N434S, and a second scFv bound to CD3 or SSTR2.

[0301] For the dual scFv form, the CD3 binding domain sequences that are particularly suitable include, but are not limited to: anti-CD3 H1.30_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 anti-CD3 H1.31_L1.47, as well as those depicted in Figures 12 and 13.

[0302] For the double scFv form, the SSTR2 binding domain sequences that are particularly suitable include, but are not limited to: anti-SSTR2 H1.143_L1.30; anti-SSTR2 H1_L1.1; anti-SSTR2 H1.107_L1.30; anti-SSTR2 H1.107_L1.67; anti-SSTR2 H1.107_L1.108; anti-SSTR2 H1.107_L1.111; anti-SSTR2 H1.107_L1.114; anti-SSTR2 H1.107_L1.102; anti-SSTR2 H1.107_L1.110; anti-SSTR2 H1.125_L1.30; anti-SSTR2 H1.125_L1.67; anti-SSTR2 H1.125_L1.108; anti-SSTR2 H1.125_L1.111; anti-SSTR2 H1.125_L1.114; anti-SSTR2 H1.125_L1.102; and anti-SSTR2 H1.125_L1.10, as well as those listed in Figures 11 and 15 and SEQ ID NO:68 to 659.

[0303] Monospecific monoclonal antibodies

[0304] As those skilled in the art will appreciate, the novel Fv sequences outlined herein can also be used for monospecific antibodies (e.g., “conventional” monoclonal antibodies) or non-heterodimeric bispecific forms. Therefore, the present invention provides monoclonal (monospecific) antibodies comprising the six CDRs and / or vh and vl sequences shown in the figure, typically having a constant region of IgG1, IgG2, IgG3, or IgG4, with IgG1, IgG2, and IgG4 (including an IgG4 constant region comprising an S228P amino acid substitution) being particularly suitable in some embodiments. That is, any sequence with the designation “H_L” herein can be linked to the constant region of a human IgG1 antibody.

[0305] I. Antigen-binding domain of the target antigen

[0306] The bispecific antibodies of the present invention have two distinct antigen-binding domains (ABDs) that bind to two different target checkpoint antigens (“target pairs”) in a bivalent, bispecific, or trivalent bispecific form, generally as shown in Figure 1. It should be noted that, for each pair, these bispecific antibodies are typically named “anti-SSTR2 × anti-CD3” or commonly simplified or conveniently (and therefore interchangeably) as “SSTR2 × CD3”, etc. It should be noted that, unless specifically specified herein, the order of the antigen list in the name does not assign a structure; that is, the SSTR2 × CD3 opener antibody may have an scFv that binds to either SSTR2 or CD3, although in some cases, the order specifies the indicated structure.

[0307] As outlined more fully in this paper, these ABD combinations can take various forms, as summarized below, typically a combination where one ABD is in a Fab form and the other in an scFv form. As discussed in this paper and shown in Figure 1, some forms use a single Fab and a single scFv (…). Figure 1A , 1C And 1D), and some forms use two Fabs and a single scFv ( Figure 1E , 1F And 1I).

[0308] antigen-binding domain

[0309] As discussed in this paper, thematic heterodimers contain two antigen-binding domains (ABD), each binding to either SSTR2 or CD3. As outlined in this paper, these heterodimers can be bispecific and bivalent (e.g., using...). Figure 1A The forms described herein, where each antigen binds to a single ABD, or bispecific and trivalent (e.g., as shown in the image) Figure 1F As depicted, one antigen binds to a single ABD and another antigen binds to both.

[0310] Furthermore, generally, one of the ABDs includes an scFv as outlined herein, which is oriented from the N-terminus to the C-terminus in a vh-scFv linker-vl or vl-scFv linker-vh configuration. One or both of the other ABDs, according to the aforementioned form, are typically Fabs, which include a vh domain on one protein chain (typically a component of the heavy chain) and a vl domain on another protein chain (typically a component of the light chain).

[0311] This invention provides a variety of ABDs that bind to a variety of different checkpoint proteins, as outlined below. As those skilled in the art will appreciate, any set of six CDRs or vh and vl domains can be in scFv or Fab form, which are subsequently added to heavy chain constant domains and light chain constant domains, wherein the heavy chain constant domain includes variants (contained within the CH1 domain and the Fc domain). The scFv sequences included in the sequence listing use specific charged linkers, but as outlined herein, uncharged linkers or other charged linkers, including those depicted in Figure 7, can be used.

[0312] Furthermore, as discussed above, the number used to identify CDRs in the sequence listing is Kabat; however, different numbers can be used, which will change the amino acid sequence of the CDRs as shown in Table 1.

[0313] Other variations can be prepared for all heavy chain and light chain variable domains listed herein. As outlined herein, in some embodiments, the set of six CDRs may have 0, 1, 2, 3, 4, or 5 amino acid modifications (particularly applicable are amino acid substitutions), and variations in the frame regions of the heavy and light chain variable domains, provided that the frame (excluding the CDR) retains at least about 80, 85, or 90% identity with the human germline sequence selected from the sequence listed in Figure 1 of U.S. Patent No. 7,657,380 (the figures and illustrations of which are incorporated herein by reference in their entirety). Thus, for example, the same CDR as described herein can be combined with different frame sequences from human germline sequences, provided that the frame region retains at least about 80, 85, or 90% identity with the human germline sequence selected from the sequence listed in Figure 1 of U.S. Patent No. 7,657,380. Alternatively, the CDR may have amino acid modifications (e.g., 1, 2, 3, 4, or 5 amino acid modifications in the group of CDRs; i.e., the CDR may be modified, provided that the total number of variations in the group of 6 CDRs is less than 6 amino acid modifications, wherein the modified CDRs are in any combination; for example, one variation may be present in vlCDR1, two variations may be present in vhCDR2, no variation may be present in vhCDR3, etc.)), and frame region variations, provided that the frame region retains at least 80, 85, or 90% identity with the human germline sequence selected from the sequence listed in Figure 1 of U.S. Patent No. 7,657,380.

[0314] SSTR2 antigen-binding domain

[0315] In some embodiments, one of the ABDs is combined with SSTR2. The suitable six CDR groups and / or vh and vl domains, as well as the scFv sequence, are depicted in Figure 11 and the sequence listing. Particularly applicable SSTR2 binding domain sequences include, but are not limited to: anti-SSTR2H1.143_L1.30; anti-SSTR2 H1_L1.1; anti-SSTR2 H1.107_L1.30; anti-SSTR2 H1.107_L1.67; anti-SSTR2 H1.107_L1.108; anti-SSTR2 H1.107_L1.111; anti-SSTR2 H1.107_L1.114; anti-SSTR2H1.107_L1.102; anti-SSTR2 H1.107_L1.110; anti-SSTR2 H1.125_L1.30; anti-SSTR2 H1.125_L1.67; anti-SSTR2 H1.125_L1.108; anti-SSTR2 H1.125_L1.111; anti-SSTR2 H1.125_L1.114; anti-SSTR2 H1.125_L1.102; and anti-SSTR2 H1.125_L1.10, as well as those listed in Figures 11 and 15 and SEQ ID NO:68 to 659.

[0316] As those skilled in the art will appreciate, a suitable SSTR2 binding domain may include a set of six CDRs as depicted in the sequence listing and figures, in an underlined form, or, in the case of using different numbering schemes as described herein and shown in Table 1, the CDRs may be identified using other alignments within the vh and vl sequences of the sequence depicted in Figure 11. A suitable ABD may also comprise the entire vh and vl sequences as depicted in these sequences and figures, used as an scFv or as a Fab. In many embodiments herein containing an Fv for SSTR2, the Fab monomer binds to SSTR2.

[0317] In addition to the parental CDR set for forming the SSTR2 ABD disclosed in the figures and sequence listings, the present invention provides a variant CDR set. In one embodiment, a set of six CDRs may have 1, 2, 3, 4, or 5 amino acid variations of the parental CDRs, as long as the SSTR2 ABD is still able to bind to the target antigen, as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biomembrane interference assay, e.g., Octet analysis), the latter being particularly applicable in many embodiments.

[0318] In addition to the parental heavy chain variable domain and light chain variable domain of the ABD forming SSTR2 disclosed herein, the present invention provides variant vh and vl domains. In one embodiment, the variant vh and vl domains may each have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations from the parental vh and vl domains, provided that the ABD remains capable of binding to the target antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biomembrane interferometry, e.g., Octet analysis), the latter being particularly suitable in many embodiments. In another embodiment, the variant vh and vl are at least 90, 95, 97, 98, or 99% identical to the corresponding parental vh and vl, provided that the ABD remains capable of binding to the target antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biomembrane interferometry, e.g., Octet analysis), the latter being particularly suitable in many embodiments.

[0319] A particularly preferred embodiment includes the H1.143_L1.30 SSTR2 antigen-binding domain, which is included as “Fab” within any of the bottle opener-type skeletons in FIG9.

[0320] CD3 antigen-binding domain

[0321] In some embodiments, one of the ABDs binds to CD3. Suitable six CDR groups and / or vh and vl domains, as well as scFv sequences, are depicted in Figures 12 and 13 and in the sequence listing. Particularly suitable CD3 binding domain sequences include, but are not limited to: anti-CD3H1.30_L1.47, anti-CD3 H1.32_L1.47, anti-CD3 H1.89_L1.47, anti-CD3 H1.90_L1.47, anti-CD3H1.33_L1.47, and anti-CD3 H1.31_L1.47, as well as those depicted in Figures 12 and 13.

[0322] As those skilled in the art will appreciate, a suitable CD3 binding domain may include a set of six CDRs as depicted in the sequence listing and figures, in an underlined form, or, in the case of using different numbering schemes as described herein and shown in Table 1, the CDRs may be identified using other alignments within the vh and vl sequences of the sequence depicted in Figure 11. A suitable ABD may also comprise the entire vh and vl sequences as depicted in these sequences and figures, used as scFv or as Fab. In many embodiments herein containing an Fv targeting CD3, the scFv monomer binds CD3.

[0323] In addition to the parental CDR set for CD3-forming ABD disclosed in the figures and sequence listings, the present invention provides a set of variant CDRs. In one embodiment, a set of six CDRs may have 1, 2, 3, 4, or 5 amino acid variations of the parental CDRs, as long as the CD3 ABD is still able to bind to the target antigen, as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biomembrane interferometry, such as Octet analysis), the latter being particularly applicable in many embodiments.

[0324] In addition to the parental heavy chain variable domain and light chain variable domain of the CD3-forming ABD disclosed herein, the present invention provides variant vh and vl domains. In one embodiment, the variant vh and vl domains may each have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations from the parental vh and vl domains, provided that the ABD remains capable of binding to the target antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biomembrane interferometry, e.g., Octet analysis), the latter being particularly suitable in many embodiments. In another embodiment, the variant vh and vl are at least 90, 95, 97, 98, or 99% identical to the corresponding parental vh and vl, provided that the ABD remains capable of binding to the target antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biomembrane interferometry, e.g., Octet analysis), the latter being particularly suitable in many embodiments.

[0325] A particularly preferred embodiment includes the H1.30_L1.47 CD3 antigen-binding domain, which is included as “Fab” within any of the bottle opener-type skeletons in Figure 9.

[0326] J. Applicable Examples

[0327] In one embodiment, a specific combination of the skewed variant and pI variant applicable in this invention is T366S / L368A / Y407V:T366W (optionally containing a bridging disulfide, T366S / L368A / Y407V / Y349C:T366W / S354C), where one monomer comprises Q295E / N384D / Q418E / N481D and the other monomer comprises a positively charged scFv linker (when the form contains an scFv domain). As will be understood in the art, the "mortar and pestle" type variant does not change the pI and is therefore applicable to either monomer.

[0328] K. Nucleic acid of the present invention

[0329] The present invention further provides a nucleic acid composition encoding the anti-SSTR2 antibody provided herein, comprising, but not limited to, an anti-SSTR2×anti-CD3 bispecific antibody and an SSTR2 monospecific antibody.

[0330] As those skilled in the art will understand, the nucleic acid composition will depend on the form and scaffold of the heterodimeric protein. Thus, for example, when the form requires three amino acid sequences, such as for the tri-F form (e.g., the first amino acid monomer includes an Fc domain and scFv, and the second amino acid monomer includes a heavy chain and a light chain), the three nucleic acid sequences can be incorporated into one or more expression vectors for expression. Similarly, some forms (e.g., the dual scFv form disclosed in Figure 1) require only two nucleic acids; again, they can be placed in one or two expression vectors.

[0331] As is known in the art, depending on the host cell used to generate the heterodimeric antibody of the present invention, the nucleic acid encoding the component of the present invention can be incorporated into an expression vector as known in the art. Generally, the nucleic acid is operatively linked to any number of regulatory elements (promoter, origin of replication, optional marker, ribosome binding site, inducer, etc.). The expression vector can be an additional chromosome or an integration vector.

[0332] The nucleic acid and / or expression vector of the present invention are then transformed into any number of different types of host cells (including mammalian, bacterial, yeast, insect and / or fungal cells) well known in the art, with mammalian cells (e.g., CHO cells) being suitable in many embodiments.

[0333] In some embodiments, according to the described form, the nucleic acid encoding each monomer and the optional nucleic acid encoding the light chain are each contained in a single expression vector, where appropriate (typically under the control of different or the same promoter). In embodiments particularly used in this invention, each of the two or three nucleic acids is contained on different expression vectors. As shown herein and in 62 / 025,931 (incorporated herein by reference), heterodimer formation can be driven using different vector ratios. That is, unexpectedly, although the protein comprises a 1:1:2 ratio of first monomer: second monomer: light chain (in many embodiments herein, where there are three polypeptides constituting the heterodimeric antibody), these are not the ratios that yield optimal results.

[0334] The heterodimeric antibodies of this invention are generated by culturing host cells comprising expression vectors well known in the art. Once generated, conventional antibody purification steps are performed, including ion exchange chromatography. As discussed herein, ensuring that the pIs of the two monomers differ by at least 0.5 allows separation by ion exchange chromatography, isoelectric point focusing, or other methods sensitive to isoelectric point. That is, pI substitutions that alter the isoelectric point (pI) of each monomer, such that each monomer has a different pI and the heterodimer also has a different pI, facilitate isoelectric purification of the “triple F” heterodimer (e.g., anion exchange column, cation exchange column). These substitutions also facilitate the determination and monitoring of any bis-scFv-Fc and mAb homodimers contaminated after purification (e.g., IEF gel, cIEF, and analytical IEX column).

[0335] Biological and biochemical functions of L. heterodimer checkpoint antibodies

[0336] In general, the efficacy of the bispecific SSTR2×CD3 antibody of this invention administered to cancer patients can be assessed in a variety of ways as described herein. Therefore, while standard efficacy analyses, such as assessments of cancer burden, tumor size, presence or extent of metastasis, can be performed, immuno-oncology therapy can also be evaluated based on immune status assessments. This can be done in many ways, including in vitro and in vivo analyses. For example, changes in immune status (e.g., the presence of ICOS+CD4+ T cells after IPI treatment) can be assessed in conjunction with "conventional" measurements such as tumor burden, size, invasiveness, LN involvement, metastasis, etc. Therefore, the inhibitory effects of checkpoints on CD4+ T cell activation or proliferation, CD8+ T (CTL) cell activation or proliferation, CD8+ T cell-mediated cytotoxicity and / or CTL-mediated cell elimination, NK cell activity, and NK-mediated cell elimination can be assessed.

[0337] In some embodiments, treatment is assessed by using, for example, the CFSE dilution method, Ki67 intracellular staining of immune effector cells, and the 3H-thymidine incorporation method to evaluate immune cell proliferation.

[0338] In some embodiments, treatment is assessed by evaluating the increase in gene expression or the increased protein levels of activation-related markers and cell degranulation as measured by surface expression of CD107A, the markers comprising one or more of the following: CD25, CD69, CD137, ICOS, PD1, GITR, OX40.

[0339] Generally, gene expression analysis is performed as is known in the art.

[0340] Generally, protein expression measurements are performed in a similar manner, as is known in the field.

[0341] In some embodiments, treatment is evaluated by assessing a number of cellular parameters, such as enzyme activity (including protease activity), cell membrane permeability, cell adhesion, ATP production, coenzyme production, and nucleotide uptake activity, to assess cytotoxic activity as measured by a target cell viability assay. Specific examples of these analyses include, but are not limited to, trypan blue or PI staining, 51Cr or 35S release assays, LDH activity, MTT and / or WST analysis, calcein-AM analysis, luminescence-based analysis, and other analyses.

[0342] In some embodiments, treatment is assessed by evaluating T cell activity measured by cytokine production, using cytokines, employing well-known techniques, and measuring intracellular culture supernatant, wherein the cytokines include, but are not limited to: IFNγ, TNFα, GM-CSF, IL2, IL6, IL4, IL5, IL10, and IL13.

[0343] Therefore, treatment can be evaluated using one or more of the following analyses: (i) increased immune response; (ii) increased activation of αβ and / or γδ T cells; (iii) increased cytotoxic T cell activity; (iv) increased NK and / or NKT cell activity; (v) relief of αβ and / or γδ T cell suppression; (vi) increased secretion of pro-inflammatory cytokines; (vii) increased IL-2 secretion; (viii) increased interferon-γ production; (ix) increased Th1 response; (x) decreased Th2 response; (xi) decreased or eliminated the number and / or activity of at least one regulatory T cell (Treg).

[0344] Analysis of measurement effectiveness

[0345] In some embodiments, T cell activation is assessed using a mixed lymphocyte reaction (MLR) assay as known in the art. Increased activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0346] In one embodiment, signal transduction pathway analysis measures an increase or decrease in the immune response, such as by phosphorylation or dephosphorylation of different factors or by measuring other post-translational modifications. Increased activity indicates immune-stimulating activity. Appropriate increases in activity are outlined below.

[0347] In one embodiment, signal transduction pathway analysis measures an increase or decrease in the activation of αβ and / or γδ T cells, such as through cytokine secretion, proliferation, or changes in the expression of activation markers such as CD137, CD107a, PD1, etc. Increased activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0348] In one embodiment, signal transduction pathway analysis measures an increase or decrease in cytotoxic T cell activity, such as by directly killing target cells, such as cancer cells, or by cytokine secretion, or by proliferation, or by changes in the expression of activating markers such as CD137, CD107a, PD1, etc. Increased activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0349] In one embodiment, signal transduction pathway analysis measures an increase or decrease in NK and / or NKT cell activity, such as by directly killing target cells, such as cancer cells, or by changes in cytokine secretion or by changes in the expression of activating markers, such as CD107a. Increased activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0350] In one embodiment, signal transduction pathway analysis measures an increase or decrease in αβ and / or γδ T cell suppression, such as through cytokine secretion, proliferation, or changes in the expression of activating markers such as CD137, CD107a, PD1, etc. Increased activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0351] In one embodiment, signal transduction pathway analysis measures an increase or decrease in the secretion of pro-inflammatory cytokines, such as by ELISA, or by bead-based Luminex or Multiplex methods (Luminex / Multiplex bead-based methods), or by intracellular staining and FACS analysis, or by methods such as Alispot. Increased activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0352] In one embodiment, signal transduction pathway analysis measures an increase or decrease in IL-2 secretion, such as by ELISA, or by bead-based Luminex or Multiplex methods, or by intracellular staining and FACS analysis, or by methods like Alispot. Increased activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0353] In one embodiment, signal transduction pathway analysis measures an increase or decrease in interferon-γ production, such as by ELISA, bead-based Luminex or Multiplex methods, intracellular staining and FACS analysis, or measurements by Alispot, etc. Increased activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0354] In one embodiment, signal transduction pathway analysis measures an increase or decrease in the Th1 response, such as through changes in cytokine secretion or through changes in the expression of activation markers. Increased activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0355] In one embodiment, signal transduction pathway analysis measures an increase or decrease in the Th2 response, such as through changes in cytokine secretion or through changes in the expression of activation markers. Increased activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0356] In one embodiment, signal transduction pathway analysis measures an increase or decrease in the number and / or activity of at least one of regulatory T cells (Tregs), such as by flow cytometry or by IHC. A decreased response indicates immunostimulatory activity. Appropriate decreases are equivalent to increases, as outlined below.

[0357] In one embodiment, signal transduction pathway analysis measures an increase or decrease in the number of M2 macrophages, such as by flow cytometry or by IHC. A decreased response indicates immunostimulatory activity. Appropriate decreases are equivalent to increases, as outlined below.

[0358] In one embodiment, signal transduction pathway analysis measures an increase or decrease in the pro-tumorigenic activity of M2 macrophages, such as by changes in cytokine secretion or by changes in the expression of activating markers. A decreased response indicates immunostimulatory activity. Appropriate decreases are equivalent to increases, as outlined below.

[0359] In one embodiment, signal transduction pathway analysis measures an increase or decrease in N2 neutrophils, such as by flow cytometry or by IHC. A decreased response indicates immunostimulatory activity. Appropriate decreases are equivalent to increases, as outlined below.

[0360] In one embodiment, signal transduction pathway analysis measures an increase or decrease in N2 neutrophil tumorigenic activity, such as by changes in cytokine secretion or by changes in the expression of activating markers. A decreased response indicates immunostimulatory activity. Appropriate decreases are equivalent to increases, as outlined below.

[0361] In one embodiment, signal transduction pathway analysis measures an increase or decrease in the inhibition of T cell activation, such as through cytokine secretion, proliferation, or changes in the expression of activation markers such as CD137, CD107a, and PD1. Increased activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0362] In one embodiment, signal transduction pathway analysis measures an increase or decrease in the inhibition of CTL activation, such as by, for example, directly killing target cells such as cancer cells, or by, through, cytokine secretion, or by, through, proliferation, or by, changes in the expression of activating markers such as, for example, CD137, CD107a, PD1, etc. Increased activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0363] In one embodiment, signal transduction pathway analysis measures an increase or decrease in αβ and / or γδ T cell exhaustion, such as by changes in the expression of activation markers. A decreased response indicates immunostimulatory activity. Appropriate decreases are equivalent to increases, as outlined below.

[0364] In one embodiment, signal transduction pathway analysis measures an increase or decrease in αβ and / or γδ T cell responses, such as through cytokine secretion, proliferation, or changes in the expression of activating markers such as CD137, CD107a, PD1, etc. Increased activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0365] In one embodiment, signal transduction pathway analysis measures an increase or decrease in stimulation of an antigen-specific memory response, such as through changes in the expression of cytokine secretion, proliferation, or activation markers such as CD45RA and CCR7. Increased activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0366] In one embodiment, signal transduction pathway analysis measures an increase or decrease in apoptosis or lysis of cancer cells, such as by cytotoxicity assays (e.g., MTT, Cr release, Calcine AM) or by flow cytometry-based assays (e.g., CFSE dilution or propidium iodide staining). Increased activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0367] In one embodiment, signal transduction pathway analysis measures an increase or decrease in stimulation that has cytotoxic or inhibitory effects on cancer cells, such as by cytotoxicity assays (e.g., MTT, Cr release, Calcine AM) or by flow cytometry-based assays (e.g., CFSE dilution or propidium iodide staining). Increased activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0368] In one embodiment, signal transduction pathway analysis measures an increase or decrease in the direct killing of cancer cells, such as by cytotoxicity analysis (e.g., MTT, Cr release, Calcine AM) or by flow cytometry-based analysis (e.g., CFSE dilution or propidium iodide staining). Increased activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0369] In one embodiment, signal transduction pathway analysis measures an increase or decrease in Th17 activity, such as through changes in cytokine secretion, proliferation, or the expression of activation markers. Increased activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0370] In one embodiment, signal transduction pathway analysis measures an increase or decrease in the induction of complement-dependent cytotoxicity and / or antibody-dependent cell-mediated cytotoxicity, such as by cytotoxicity assays (e.g., MTT, Cr release, Calcine AM) or by flow cytometry-based assays (e.g., CFSE dilution or propidium iodide staining). Increased activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0371] In one embodiment, T cell activation is measured, for example, by directly killing target cells such as cancer cells, or by cytokine secretion, or by proliferation, or by changes in the expression of activation markers such as CD137, CD107a, PD1, etc. Regarding T cells, increases in proliferation, cell surface activation markers (e.g., CD25, CD69, CD137, PD1), cytotoxicity (the ability to kill target cells), and cytokine production (e.g., IL-2, IL-4, IL-6, IFNγ, TNF-α, IL-10, IL-17A) will indicate immune regulation consistent with enhanced cancer cell killing.

[0372] In one embodiment, NK cell activation is measured, for example, by directly killing target cells such as cancer cells, or by changes in cytokine secretion, or by changes in the expression of activation markers such as CD107a. Regarding NK cells, increases in proliferation, cytotoxicity (the ability to kill target cells and increase the expression of CD107a, granzyme, and perforin), cytokine production (e.g., IFNγ and TNF), and cell surface receptor expression (e.g., CD25) will indicate immune regulation consistent with enhanced cancer cell killing.

[0373] In one embodiment, γδT cell activation is measured, for example, by changes in cytokine secretion, proliferation, or expression of activation markers.

[0374] In one embodiment, Th1 cell activation is measured, for example, by changes in cytokine secretion or by changes in the expression of activation markers.

[0375] An appropriate increase (or appropriate decrease, as outlined above) in activity or response is a percentage increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98% to 99% compared to a reference or control sample, which is, for example, a test sample not containing the antibody of the present invention. Similarly, an increase of at least one, two, three, four, or five times compared to a reference or control sample indicates efficacy.

[0376] M. Treatment

[0377] The compositions of the present invention can be used in a variety of applications after preparation. SSTR2 is highly expressed in neuroendocrine tumors (NETs, ​​such as lung cancer, gastrointestinal cancer, pancreatic cancer, pituitary cancer, medullary carcinoma, prostate cancer, pancreatic lung carcinoid, osteosarcoma, bronchoma, thymoma) and non-NETs (breast cancer, lung cancer, colorectal cancer, ovarian cancer, cervical cancer).

[0378] Accordingly, the heterodimer compositions of the present invention can be used to treat such SSTR2-positive cancers.

[0379] Antibody compositions for in vivo administration

[0380] Formulas of antibodies used according to the invention are prepared by mixing antibodies of desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed.

[1980] ), for storage in lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosage and concentration used and contain buffers such as phosphates, citrates, and other organic acids; antioxidants containing ascorbic acid and methionine; and preservatives such as octadecyl dimethylbenzyl ammonium chloride; hexahydroxyquaternary ammonium chloride; benzalkonium chloride; and benzyl chloride. Chloride); phenols, butanol, or benzyl alcohol; alkyl p-hydroxybenzoates, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming ions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN. TM PLURONICS TM Or polyethylene glycol (PEG).

[0381] Application mode

[0382] The antibodies and chemotherapeutic agents of the present invention are administered to subjects according to known methods, such as intravenous injection or by continuous infusion over a period of time.

[0383] Treatment mode

[0384] In the method of the present invention, the therapy is used to provide a positive therapeutic response relative to the disease or condition. A “positive therapeutic response” is an expected improvement in the disease or condition, and / or an improvement in symptoms associated with the disease or condition. For example, a positive therapeutic response would refer to an improvement in one or more of the following: (1) a reduction in the number of neoplasms; (2) an increase in neoplasm death; (3) inhibition of neoplasm survival; (5) inhibition of tumor growth (i.e., a certain degree of slowing, preferably interruption); (6) an increase in patient survival; and (7) a certain degree of relief from one or more symptoms associated with the disease or condition.

[0385] A positive therapeutic response in any given disease or condition can be measured using standardized response indicators specific to that disease or condition. Screening techniques such as magnetic resonance imaging (MRI), X-ray imaging, computed tomography (CT), bone scan imaging, endoscopy, and tumor biopsy sampling (including bone marrow aspiration (BMA) and circulating tumor cell count) can be used to assess tumor response based on changes in tumor morphology (i.e., total tumor burden, tumor size, etc.).

[0386] In addition to these positive therapeutic responses, subjects undergoing therapy may benefit from improvements in disease-associated symptoms.

[0387] The treatment according to the present invention comprises the use of a "therapeuticly effective amount" of medicine. "Therapeuticly effective amount" refers to the amount that effectively achieves the desired therapeutic result at the necessary dosage and within the necessary time.

[0388] Therapeutic effective doses can vary depending on factors such as disease state, individual age, sex, weight, and the ability of the drug to elicit the desired response in the individual. A therapeutic effective dose is also the amount at which the beneficial therapeutic effect outweighs any toxic or harmful effects of the antibody or antibody fraction.

[0389] The "therapeutic effective dose" of an oncology therapy can also be measured by its ability to stabilize disease progression. The ability of a compound to inhibit cancer can be evaluated in animal model systems where its efficacy against human tumors can be predicted.

[0390] Alternatively, this property of the composition can be assessed by examining the compound's ability to inhibit cell growth or induce apoptosis using in vitro analysis known to an experienced physician. A therapeutically effective amount of the therapeutic compound can reduce tumor size or otherwise improve the subject's symptoms. A person skilled in the art will be able to determine this amount based on factors such as the subject's size, the severity of the subject's symptoms, and the specific composition or route of administration chosen.

[0391] Dosing regimens can be adjusted to provide the best expected response (e.g., a therapeutic response). For example, as indicated by an emergency situation in the treatment, a single bolus injection can be administered, several fractionated doses can be administered over time, or the dose can be reduced or increased proportionally. Parenteral compositions can be formulated in unit dosage forms for ease of administration and to ensure uniform dosing. As used herein, unit dosage form refers to a physically dispersed unit suitable for use as a single dose in a subject to be treated; each unit contains a predetermined amount of active compound calculated to bind with the desired drug carrier to produce the desired therapeutic effect.

[0392] The specifications of the unit dosage form of the present invention are specified by and directly depend on the following: (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved; and (b) the inherent limitations in the field of treatment of individual sensitivities when such therapeutic compounds are mixed.

[0393] The effective dose and dosing regimen of the bispecific antibody used in this invention depend on the disease or condition to be treated and can be determined by those skilled in the art.

[0394] The exemplary, non-limiting range of therapeutically effective amounts of the bispecific antibodies used in this invention is about 0.1 to 100 mg / kg.

[0395] All references are explicitly incorporated into this paper in full.

[0396] While specific embodiments of the invention have been described above for illustrative purposes, those skilled in the art will understand that various changes to the details may be made without departing from the invention as described in the appended claims.

[0397] Example

[0398] Examples are provided below to illustrate the invention. These examples are not intended to limit the invention to any particular application or operational theory. For all constant region locations discussed in this invention, reference is made to the EU index, such as that in Kabat (Kabat et al., 1991, Protein Sequences of Immunological Concern, 5th Edition, U.S. Public Health Service, National Institutes of Health, Bethesda, which is incorporated herein by reference in its entirety). Those skilled in the art of antibodies will understand that this specification consists of: non-sequential numbering of specific regions of an immunoglobulin sequence, and standardized designations for conserved locations within the immunoglobulin family. Accordingly, any designated immunoglobulin location as defined by the EU index does not necessarily correspond to its sequential sequence.

[0399] U.S. Publications 2015 / 0307629, 2014 / 0288275, and WO2014 / 145806 outline general and specific science and technology, all of which are explicitly incorporated in their entirety (and especially in the form of the technologies outlined therein).

[0400] Example 1: Generation of anti-SSTR2 × anti-CD3 bispecific antibodies

[0401] 1A: Generation of SSTR2-resistant Fab arms

[0402] The parental variable region of the engineered anti-SSTR2 antibody was used as a component of the anti-STTR2×anti-CD3 bispecific antibody of the present invention. Humanization of the murine VH and VL regions was performed as previously described in U.S. Patent No. 7,657,380, issued February 2, 2010. Amino acid substitutions were performed via QuikChange (Stratagene, Cedar Creek, Tx.) mutagenesis to attempt to identify variants by means of modified properties.

[0403] 1B: Bispecific antibody production

[0404] A cartoon illustration of the anti-SSTR2×anti-CD3 bispecific form is shown in Figure 1. Exemplary antibodies were generated using the anti-SSTR2 Fab arm and anti-CD3 scFv arm, derived from engineered anti-SSTR2 antibodies as described above. Exemplary anti-SSTR2×anti-CD3 opener antibodies XENP018087 and XENP018907 are shown in Figures 14 and 15, respectively. DNA encoding the three strands required for bispecific expression was generated either through gene synthesis (Blue Heron Biotechnology, Bothell, Wash.) and its standard subcloning into expression vectors using pTT5 technology, or through QuikChange mutagenesis. The DNA was transfected into HEK293E cells for expression, and the resulting protein was purified from the supernatant using protein A affinity analysis (GE Healthcare) and cation exchange chromatography. Cation exchange chromatography purification was performed using a HiTrap SP HP column (GE Healthcare) with a wash / equilibration buffer of 50 mM MES at pH 6.0 and an elution buffer of 50 mM MES at pH 6.0 with a linear gradient of +1 M NaCl.

[0405] 1C: Bispecific binding of anti-SSTR2 antibody.

[0406] Cell surface binding of anti-SSTR2 antibody and exemplary anti-SSTR2×anti-CD3 bispecific antibody was assessed using CHO cells transfected with human SSTR2. Cells were incubated on ice for 45 min with the designated test sample and then centrifuged. Cells were resuspended in staining buffer containing a phycoerythrin (PE)-labeled secondary antibody (μg / mL; goat anti-human IgG) and then incubated on ice for 45 min. Cells were centrifuged twice and then resuspended in staining buffer. Binding was measured by flow cytometry. Figures 17A-17P ).

[0407] Example 2: Characterization of exemplary anti-SSTR2×anti-CD3 bispecific antibody

[0408] 2A: In vitro characterization of exemplary anti-SSTR2×anti-CD3 bispecific antibodies

[0409] Targeting CHO cells transfected with SSTR2 ( Figures 18A-18D ) and SSTR2-positive TT cells (human thyroid medullary cancer cell line; Figures 19A-19C The exemplary anti-SSTR2×anti-CD3 Fab-scFv-Fc bispecific antibody was characterized in vitro by redirected T-cell cytotoxicity (RTCC). RTCC was determined by measuring lactate dehydrogenase (LDH) levels. As shown in these figures, the anti-SSTR2×anti-CD3 Fab-scFv-Fc bispecific antibody exhibited a high percentage of RTCC in SSTR2-transfected CHO cells and human cancer cell lines compared to the control group.

[0410] 2B: In vivo characterization of exemplary anti-SSTR2×anti-CD3 bispecific antibodies

[0411] In the first study, cynomolgus macaques (n=3) were administered 0.03 mg / kg XENP18087 or 1 mg / kg XENP18088 intravenously (iv) twice (at weeks 0 and 3). These anti-SSTR2×anti-CD3 bispecific antibodies were subsequently evaluated for their effectiveness against CD4. + and CD8 + The effects of T cell activation (e.g., on CD69 expression) Figure 20A ) and CD4 + and CD8 + T cell distribution ( Figure 20B )instruct).

[0412] In the second study, cynomolgus macaques (n=3) were administered a single intravenous (iv) dose of anti-SSTR2×anti-CD3 bispecific antibodies: 0.06 mg / kg XENP18087, 0.1 mg / kg XENP18907, 0.5 mg / kg XENP18907, or 2 mg / kg XENP18907. The effects of these anti-SSTR2×anti-CD3 bispecific antibodies on CD4 were evaluated. + and CD8 + T cell activation (CD69 upregulation) Figure 21A ) and CD4 + and CD8 + T cell redistribution (cell count, Figure 21B The impact of ) . In addition, a glucose tolerance test (GTT) was performed ( Figure 21C and 21DThis was used to assess the ability of the tested subjects to break down glucose. For the GTT, blood samples were collected at eight different time points: before administration, and 5, 10, 20, 30, 40, 60, and 90 minutes after administration of dextran. As shown in these studies, CD4 levels were [data missing] during each treatment period. + and CD8 + It is rapidly redistributed in the blood and subsequently recovers and normalizes after administration. Figure 21B T cells are activated immediately upon administration. Figure 21A And then it subsides while T cells redistribute.

[0413] In the third study, cynomolgus macaques (n=3) were administered XENP18087 twice intravenously (iv) at weeks 0 and 1. The expression of these anti-SSTR2×anti-CD3 bispecific antibodies against CD4 was subsequently assessed using CD69 expression (a marker of T cell activation). + and CD8 + T cell activation ( Figures 22A-22B ) and CD4 + and CD8 + T cell redistribution ( Figure 22C-22D The effects of IL-6 and TNF levels on these monkeys were analyzed. Figure 22E-22F As these studies show, CD4 during each processing period + and CD8 + T cells rapidly redistribute in the blood and subsequently recover and normalize after administration. With each dose-dependent administration, T cells are immediately activated and then deactivate while redistributing. The release of IL-6 and TNF cytokines is associated with T cell activation.

[0414] Example 3: Evaluation of XmAb18087

[0415] 3A: Specific binding of XmAb18087 to SSTR2 in humans and cynomolgus monkeys

[0416] Cell surface binding of XmAb18087 and the control anti-RSV×anti-CD3 bispecific antibody (XENP13245) was assessed using human SSTR2-transfected CHO cells and cynomolgus monkey SSTR2-transfected CHO cells. Parental CHO cells were also used as a control group for cell surface binding assessment. Binding was measured by flow cytometry using a phycoerythrin (PE)-labeled secondary antibody as generally described in Example 1C.

[0417] XmAb18087 not only binds to human SSTR2 on the cell surface (… Figure 23 A) also related to the crab-eating macaque SSTR2 ( Figure 23B) Cross-reactivity was observed, while the control group's anti-RSV×anti-CD3 bispecific antibody XENP13245 did not bind to human SSTR2 or cynomolgus monkey SSTR2-transfected CHO cells. Further data showed that XmAb18087 did not bind to untransfected parental CHO cells ( Figure 23 C) demonstrates the specificity of XmAb18087.

[0418] 3B: Redirected T cell cytotoxicity induced by XmAb18087

[0419] CHO cells transfected with SSTR2 ( Figure 24 A) SSTR2-positive TT cells (thyroid medullary cancer cell line); Figure 24 B and 25), A549 cells (lung adenocarcinoma cell line); Figure 24 C and 25) and untransfected parental CHO cells as the control group ( Figure 24 A) Redirected T cell cytotoxicity (RTCC) in vitro characterization of XmAb18087. Containing anti-RSV×anti-CD3 bispecific antibody (XENP13245) and bivalent anti-SSTR2 mAh as a control group ( Figure 25 ).

[0420] Target cells and human PBMCs were cultured together with XmAb18087 or XENP13245 at an E:T ratio of 10 or 20:1 for 24 hours. RTCC was determined by measuring lactate dehydrogenase (LDH) content.

[0421] As shown in these figures, compared to the control group anti-CD3 bispecific antibody XENP13245 and the control group bivalent anti-SSTR2 mAh ( Figure 25 XmAb18087 in SSTR2-transfected CHO cells ( Figure 24 A) and human cancer cell lines ( Figure 24 XmAb18087 showed a high percentage of RTCCs in B-24C and 25 cells. Furthermore, data showed that XmAb18087 did not show RTCCs in untransfected parental CHO cells. Figure 24 A).

[0422] In the experiment, SSTR2-transfected CHO cells and TT cells were also used to assess CD8 using flow cytometry. + and CD4 + To investigate T cell activation induced by XmAb18087 by the surface expression of CD69 on T cells. Figure 26 A-26B). As shown in the figure, XmAb18087 makes CD8 + and CD4 +T cell activation levels were significantly higher than those achieved by the control group using the anti-CD3 bispecific antibody XENP13245. This result indicates that XmAb18087 eliminates SSTR2 by inducing T cell activation. + Target cells.

[0423] 3C:XmAb18087 exhibited antitumor activity in NSG mice transplanted with A549 lung cancer cells and human PBMCs.

[0424] On day 7, 1 × 10⁸ scisic acid gamma (NOD scid gamma, NSG) saturates were transplanted into each of the twenty-five non-obese diabetic severe combined immunodeficiency gamma (NOD scid gamma, NSG) mice. 6 A549-RedFLuc tumor cells (0.1 mL volume, subcutaneous injection). Day 0, 10 × 10⁶ cells were injected. 6 Human PBMCs were intraperitoneally transplanted into mice. Following PBMC transplantation on day 0, mice were administered XmAb18087 weekly (days 0, 7, and 14) via intraperitoneal injection at 3.0 mg / kg (control mice received PBS). The study design is further outlined in [reference needed]. Figure 27 In China, by using an in vitro imaging system ( Lumina III measures the total throughput of each mouse to monitor tumor growth.

[0425] like Figure 28 and 29 As shown, treatment with 3 mg / kg XmAb18087 significantly inhibited local tumor growth of A549 compared to treatment with PBS.

[0426] 3D: Representation of XmAb18087 in cynomolgus monkeys

[0427] In further studies, cynomolgus monkeys (n=3) were administered a single intravenous (iv) dose of XmAb18087 or a control group of anti-RSV×anti-CD3 bispecific antibody (XENP13245). The effects of these bispecific antibodies on CD4 were evaluated. + and CD8 + T cell activation (CD69 upregulation); Figure 31 A-31B) and the release of cytokines (IL-6 and TNF) Figure 32 The impact of A-32B).

[0428] As shown in the figure, when processing CD4 using XmAb18087 + and CD8 + T cells are rapidly redistributed from the bloodstream. Figure 30A and 30B, compared to treatment with XENP13245, showed normalization and recovery after administration. Upon administration of XmAb18087, T cells were immediately activated (compared to XENP13245) and subsequently deactivated with T cell redistribution. Furthermore, the release of IL-6 and TNF cytokines was associated with T cell activation. Figure 32 A-32B).

[0429] 3E: Characterization of XmAb18087 in NSG mice

[0430] In another dose-response study, on day 7, 60 NSG mice were transplanted with 1×10⁶ A549-RedFLuc tumor cells (0.1 mL volume subcutaneously). On day 0, mice were grouped according to total flux and intraperitoneally transplanted with 10×10⁶ human PBMCs, and administered test sample at specified concentrations (#1, 12 mice per concentration). Administration of #2 and #3 was administered on days 8 and 15, respectively. As described above, tumor growth was monitored by measuring total flux per mouse using an in vitro imaging system two to three times per week, such as... Figure 33 As described in the text. In addition, tumor volume was measured with calipers once or twice a week on days 18 and 22 after administration of #1.

Claims

1. An SSTR2 antigen-binding domain, comprising: a) A variable heavy chain domain comprising vhCDR1 consisting of the amino acid sequence of SEQ ID NO: 959, vhCDR2 consisting of the amino acid sequence of SEQ ID NO: 960, and vhCDR3 consisting of the amino acid sequence of SEQ ID NO: 961; and b) A variable light chain domain comprising vlCDR1 consisting of the amino acid sequence of SEQ ID NO: 963, vlCDR2 consisting of the amino acid sequence of SEQ ID NO: 964, and vlCDR3 consisting of the amino acid sequence of SEQ ID NO:

965.

2. The SSTR2 antigen-binding domain according to claim 1, wherein the variable heavy chain domain is composed of the amino acid sequence of SEQ ID NO: 958, and the variable light chain domain is composed of the amino acid sequence of SEQ ID NO:

962.

3. A nucleic acid composition comprising: a) A first nucleic acid encoding the variable heavy chain domain of claim 1 or 2; and b) A second nucleic acid encoding the variable light chain domain of claim 1 or 2.

4. An expression vector composition comprising: a) a first expression vector comprising the first nucleic acid of claim 3; and b) A second expression vector comprising the second nucleic acid of claim 3.

5. A host cell comprising the nucleic acid composition according to claim 3 or the expression vector composition according to claim 4.

6. A method for preparing an SSTR2 antigen-binding domain, comprising culturing a host cell according to claim 5 under conditions in which the SSTR2 antigen-binding domain is expressed, and recovering the SSTR2 antigen-binding domain.

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