Multivalent and multispecific dr5-binding fusion proteins

Abstract

To provide an isolated polypeptide that binds to death receptor 5.SOLUTION: Provided is an isolated polypeptide that binds to at least death receptor 5 (DR5) and includes multiple DR5 binding domains (DR5BDs), the isolated polypeptide being monospecific, multispecific, and bispecific and including at least a second binding domain that binds to a second antigen, or the multiple DR5BDs binding to the same epitope on DR5.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 193,309, filed Jul. 16, 2015, the entire contents of which are incorporated herein by reference.

[0002] Field of the Invention The present disclosure generally relates to molecules that specifically engage cell death receptor 5 (DR5), which is a member of the tumor necrosis factor receptor superfamily (TNFRSF). More specifically, the present disclosure relates to multivalent and multispecific molecules that bind to at least DR5.

Background Art

[0003] Background of the Invention The tumor necrosis factor receptor superfamily consists of several structurally related cell surface receptors. Activation by multimeric ligands is a common feature of many of these receptors. Many members of the TNFRSF are useful for the treatment of a number of medical conditions if appropriately activated. Importantly, proper stimulation of this receptor family often requires higher order clustering, for which conventional bivalent antibodies are not ideal. Thus, more potent agonist molecules of the TNFRSF are therapeutically needed.

Summary of the Invention

[0004] Summary of the Invention The present disclosure provides multivalent fusion polypeptides that bind to at least death receptor 5 (DR5, also known as TRAIL receptor 2 (TRAILR2) or tumor necrosis factor receptor superfamily member 10B (TNFRSF10B)). These DR5-binding fusion polypeptides are also referred to herein as DR5 target molecules. DR5 is a member of the TNF receptor superfamily (TNFRSF) and is a cell surface receptor of the TNF-receptor superfamily that binds to TNF-related apoptosis-inducing ligand (TRAIL). TRAIL has evolved to play an important role in mammalian development and host defense by selectively eradicating unwanted infected and malignant cells from healthy cell populations. TRAIL induces cell death via caspase-dependent apoptosis when it binds to a member of the TNF receptor family, DR4 or DR5. DR5 appears to be the major receptor on tumor cells that promotes the tumor-biased activity observed in the TRAIL pathway. DR5 is activated by the natural ligand TRAIL, bringing three DR5 receptors into proximity, thereby activating intracellular caspase-8 and initiating the activation of other death-inducing caspases such as caspase-9 and caspase-3. Thus, clustering of DR5 receptors for efficient cell death is required for initiation of this cell death pathway.

[0005] Conventional antibodies targeting members of the TNF receptor superfamily (TNFRSF) have been shown to require exogenous cross-linking to achieve sufficient agonistic activity, as demonstrated by the requirement for Fc gamma receptors (FcγR) for active antibodies against DR4, DR5, GITR, and OX40 (Ichikawa et al 2001 al Nat.Med.7,954-960,Li et al 2008 Drug Dev.Res.69,69-82;Pukac et al 2005 Br.J.Cancer 92,1430-1441;Yanda et al 2008 Ann.Oncol.19,1060-1067 Yang et al 2007 Cancer Lett.251:146-157;Bulliard et al 2013 JEM 210(9):1685; Bulliard et al 2014 Imm unol and Cell Biol 92:475-480). In addition to cross-linking via FcγR, other exogenous agents to which oligomeric ligands or antibody-binding entities (e.g., protein A and secondary antibodies) are added have been demonstrated to enhance anti-TNFRSF antibody clustering and downstream signaling. For example, the in vitro agonistic activity of the CD137 antibody, PF-05082566, requires cross-linking via a secondary antibody (Fisher et al Cancer Immunol Immunother 2012 61:1721-1733). These findings suggest the need for TNFRSF clustering beyond dimerization.

[0006] Efforts to clinically utilize the TRAIL pathway for cancer treatment have relied on recombinant forms of the native ligand TRAIL and antibodies specific for DR5. Antibody agonists targeting DR5 required a cross-linking agent in preclinical in vitro experiments. For example, the addition of the DR5 ligand TRAIL enhanced the apoptosis-inducing ability of the anti-DR5 antibody AMG655 (Graves et al, 2014 Cancer Cell 26:177-189). Conventional antibodies are bivalent and can cluster only two DR5 receptors (one per FAB arm). Clustering of two DR5 receptors, in line with other members of TNFRSF, is insufficient to mediate signaling and activate the cell death pathway in vitro. In vivo administration of DR5-targeted antibodies in preclinical mouse models of human cancer surprisingly showed significant activity in a wide variety of tumor types. This activity was later shown to be dependent on the mouse Fc gamma R (FcγR) receptor. Clinical studies in humans have not been able to reproduce the stable responses seen in these preclinical mouse models. The lack of activity in humans is hypothesized to be due to insufficient antibody cross-linking. This could be due to differences in serum IgG, FcγR, and / or TRAIL between immunodeficient mice and human cancer patients.

[0007] The present disclosure provides a multivalent fusion protein targeting DR5 that can potently act on DR5-mediated signaling for direct cell death. The fusion proteins of the present disclosure can be bivalent, trivalent, tetravalent, pentavalent or hexavalent. Importantly, the fusion proteins of the present disclosure can induce apoptosis of DR5-expressing cells independently of exogenous cross-linking agents.

[0008] In some embodiments, the fusion proteins of the present disclosure incorporate a binding domain that binds to DR5 (DR5BD). In preferred embodiments, the DR5-binding DR5BD does not bind to DR4, decoy R1, decoy R2, osteopontin, or any other TNFRSF member. In preferred embodiments, the DR5-binding DR5BD binds to human and cynomolgus monkey DR5. In some embodiments, the DR5-binding DR5BD interferes with the interaction between DR5 and its ligand TRAIL. In other embodiments, the DR5-binding DR5BD does not interfere with the interaction between DR5 and its ligand TRAIL. In some embodiments, the fusion proteins of the present disclosure incorporate multiple DR5-binding DR5BDs that recognize different epitopes on DR5. In some embodiments, the fusion proteins of the present disclosure incorporate multiple DR5-binding DR5BDs, where some of the DR5BDs interfere with the DR5-TRAIL interaction and others do not. In preferred embodiments, the DR5 targeted by the fusion proteins of the present disclosure induces direct cell death of tumor cells. The DR5-targeted fusion proteins of the present disclosure are useful for treating tumors that are both hematological and solid in nature.

[0009] The present disclosure provides a multivalent DR5-binding fusion protein comprising two or more DR5-binding domains (DR5BDs). In some embodiments, the fusion proteins of the present disclosure are useful for treating neoplasms. In some embodiments, the fusion proteins of the present disclosure bind to DR5 expressed on tumor cells. In some embodiments, the fusion protein comprises two or more different DR5BDs, each of which binds to DR5. In some embodiments, the fusion The protein comprises multiple copies of DR5BD that binds to DR5. For example, in some embodiments, the fusion protein comprises at least two copies of DR5BD that binds to DR5. In some embodiments, the fusion protein comprises at least three copies of DR5BD that binds to DR5. In some embodiments, the fusion protein comprises at least four copies of DR5BD that binds to DR5. In some embodiments, the fusion protein comprises at least five copies of DR5BD that binds to DR5. In some embodiments, the fusion protein comprises at least six copies of DR5BD that binds to DR5. In some embodiments, the fusion protein comprises more than six copies of DR5BD that binds to DR5.

[0010] The multivalent DR5-binding fusion protein of the present disclosure can directly induce cell death in damaged cells, transformed cells, virus-infected cells, or neoplastic cells without the need for an exogenous cross-linking agent. Furthermore, the DR5-binding fusion protein of the present disclosure does not directly induce cell death in normal, non-transformed cells, non-virus-infected cells or non-neoplastic cells. Importantly, the DR5BD of the present disclosure and the fusion proteins composed thereof reduce or eliminate recognition by existing antibodies directed against single domain antibodies present in some human subjects.

[0011] TAS266 is a tetravalent humanized DR5-targeting nanobody-based therapeutic agent that exhibits superior apoptosis-inducing ability compared to bivalent antibodies without the need for further cross-linking by FcγR. (Huet, H.A., et al, Multivalent nanobodies targeting death receptor 5 elicit superior tumor cell killing through efficient caspase induction. mAbs Vol.6, Iss.6, 2014).

[0012] It has previously been predicted that approximately half of healthy human subjects have existing antibodies that recognize human single-domain antibodies known as human anti-VH autoantibodies (HAVH) that target epitopes within the human VH domain (Holland et al. J Clin Immunol (2013) 33:1192-1203)). Therefore, VHHs derived from camelids that have been humanized were expected to be recognized by HAVH autoantibodies as the target epitopes are potential and are located within the human germline framework regions. The interaction of HAVH autoantibodies (also referred to herein as anti-drug antibodies (ADA) or anti-single-domain antibodies (ASDA)) can cause clustering and enhanced activation. Consistent with this hypothesis, in a Phase I clinical trial, administration of TAS266 induced elevated AST and ALT levels indicative of hepatotoxicity. The elevated enzyme levels occurred in 3 out of 4 patients and led to the termination of the TAS266 trial. It was noted that since 3 patients who showed clinical signs of hepatotoxicity had existing ADA, the investigators became suspicious of ADA-induced over-clustering of the DR5 receptor causing the toxicity. It was noted that 1 patient without ADA had no signs of toxicity (Isaacs R, Bilic S, Kentsch K, Huet HA, Hofmann M, Rasco D, Kundamal N, Tang Z, Cooksey J, Mahipal A. Unexpected hepatotoxicity in a Phase I trial of TAS266, a novel tetravalent agonist Nanobody® targeting the DR5 receptor. Papadopoulos KP1, Cancer Chemother Pharmacol. 2015 May; 75(5):887-95.doi:10.1007 / s00280-015-2712-0.Epub 2015 Feb. 27.). To support this idea, it has been well documented that the aggregated form of the DR5 agonist induces hepatotoxicity while the non-aggregated form does not (J Lemke, S von Karstedt, J Zinngrebe and H Walczak. Getting TRAIL back on track for cancer therapy.Cell Death and Differentiation(2014)21,1350-1364).

[0013] In some embodiments, the fusion protein comprises at least one DR5BD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-91. In some embodiments, the fusion protein comprises two or more copies of a DR5BD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-91. In some embodiments, the fusion protein comprises three or more copies of a DR5BD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-91. In some embodiments, the fusion protein comprises four or more copies of a DR5BD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-91. In some embodiments, the fusion protein comprises five or more copies of a DR5BD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-91. In some embodiments, the fusion protein comprises six or more copies of a DR5BD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-91.

[0014] In some embodiments, the fusion protein comprises a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 31, 128, 134, 138, 141, 142, 159, 162, 163, 168, 173, 176, 178, 181, and 188; a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 28, 129, 131-133, 135, 137, 139, 143, 160, 164, 166, 167, 169, 171, 172, 174, 177, 179, 182, 184, 185, and 189; and a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 130, 136, 140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190, and contains at least one DR5BD. In some embodiments, the fusion protein comprises a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 31, 128, 134, 138, 141, 142, 159, 162, 163, 168, 173, 176, 178, 181, and 188; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 28, 129, 131-133, 135, 137, 139, 143, 160, 164, 166, 167, 169, 171, 172, 174, 177, 179, 182, 184, 185, and 189; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 130, 136, 140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190, and contains two or more copies of DR5BD.In some embodiments, the fusion protein contains three or more copies of DR5BD, comprising: CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 128, 134, 138, 141, 142, 159, 162, 163, 168, 173, 176, 178, 181, and 188; CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 129, 131-133, 135, 137, 139, 143, 160, 164, 166, 167, 169, 171, 172, 174, 177, 179, 182, 184, 185, and 189; and CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 130, 136, 140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190. In some embodiments, the fusion protein contains four or more copies of DR5BD, comprising: CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 128, 134, 138, 141, 142, 159, 162, 163, 168, 173, 176, 178, 181, and 188; CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 129, 131-133, 135, 137, 139, 143, 160, 164, 166, 167, 169, 171, 172, 174, 177, 179, 182, 184, 185, and 189; and CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 130, 136, 140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190. In some embodiments. In some embodiments, the fusion protein contains five or more copies of DR5BD, comprising: CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 128, 134, 138, 141, 142, 159, 162, 163, 168, 173, 176, 178, 181, and 188; CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 129, 131-133, 135, 137, 139, 143, 160, 164, 166, 167, 169, 171, 172, 174, 177, 179, 182, 184, 185, and 189; and CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 130, 136, 140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190. In some embodiments, the fusion protein contains six or more copies of DR5BD, comprising: CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 128, 134, 138, 141, 142, 159, 162, 163, 168, 173, 176, 178, 181, and 188; CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 129, 131-133, 135, 137, 139, 143, 160, 164, 166, 167, 169, 171, 172, 174, 177, 179, 182, 184, 185, and 189; and CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 130, 136, 140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190.

[0015] In some embodiments, the fusion protein contains at least one DR5BD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 91 and at least one immunoglobulin Fc region polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 5 and 127. In some embodiments, the fusion protein contains two or more copies of a DR5BD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 91 and at least one immunoglobulin Fc region polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 5 and 127. In some embodiments, the fusion protein contains three or more copies of a DR5BD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 91 and at least one immunoglobulin Fc region polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 5 and 127. In some embodiments, the fusion protein contains four or more copies of a DR5BD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 91 and at least one immunoglobulin Fc region polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 5 and 127. In some embodiments, the fusion protein contains five or more copies of a DR5BD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 91 and at least one immunoglobulin Fc region polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 5 and 127. In some embodiments, the fusion protein contains six or more copies of a DR5BD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 91 and at least one immunoglobulin Fc region polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 5 and 127.

[0016] In some embodiments, the fusion protein comprises a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 128, 134, 138, 141, 142, 159, 162, 163, 168, 173, 176, 178, 181, and 188; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 129, 131-133, 135, 137, 139, 143, 160, 164, 166, 167, 169, 171, 172, 174, 177, 179, 182, 184, 185, and 189; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 130, 136, 140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190; and at least one DR5BD comprising at least one immunoglobulin Fc region polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1-5 and 127. In some embodiments, the fusion protein comprises SEQ ID NOs: 31, 128, 134, 138, 141, CDR1 comprising an amino acid sequence selected from the group consisting of 142, 159, 162, 163, 168, 173, 176, 178, 181, and 188; CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 129, 131-133, 135, 137, 139, 143, 160, 164, 166, 167, 169, 171, 172, 174, 177, 179, 182, 184, 185, and 189; and CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 130, 136, 140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190; and at least one immunoglobulin Fc region polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1-5 and 127, containing two or more copies of DR5BD. In some embodiments, the fusion protein comprises CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 128, 134, 138, 141, 142, 159, 162, 163, 168, 173, 176, 178, 181, and 188; CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 129, 131-133, 135, 137, 139, 143, 160, 164, 166, 167, 169, 171, 172, 174, 177, 179, 182, 184, 185, and 189; and CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 130, 136, 140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190; and at least one immunoglobulin Fc region polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1-5 and 127, containing three or more copies of DR5BD.In some embodiments, the fusion protein contains four or more copies of DR5BD and comprises a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 128, 134, 138, 141, 142, 159, 162, 163, 168, 173, 176, 178, 181, and 188; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 129, 131-133, 135, 137, 139, 143, 160, 164, 166, 167, 169, 171, 172, 174, 177, 179, 182, 184, 185, and 189; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 130, 136, 140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190; and at least one immunoglobulin Fc region polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1-5 and 127. In some embodiments, the fusion protein contains five or more copies of DR5BD and comprises a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 128, 134, 138, 141, 142, 159, 162, 163, 168, 173, 176, 178, 181, and 188; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 129, 131-133, 135, 137, 139, 143, 160, 164, 166, 167, 169, 171, 172, 174, 177, 179, 182, 184, 185, and 189; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 130, 136, 140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190; and at least one immunoglobulin Fc region polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1-5 and 127.In some embodiments, the fusion protein comprises a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 128, 134, 138, 141, 142, 159, 162, 163, 168, 173, 176, 178, 181, and 188; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 129, 131-133, 135, 137, 139, 143, 160, 164, 166, 167, 169, 171, 172, 174, 177, 179, 182, 184, 185, and 189; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 130, 136, 140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190; and at least one immunoglobulin Fc region polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1-5 and 127, and contains six or more copies of DR5BD.

[0017] In some embodiments, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 92-124. In some embodiments, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 92-118. In some embodiments, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 119-124.

[0018] The fusion proteins of the present disclosure can enhance the clustering of TNFRSF members as compared to uncrosslinked bivalent antibodies. The enhanced clustering of TNFRSF members mediated by the fusion proteins of the present disclosure induces enhanced TNFRSF-dependent signaling as compared to uncrosslinked bivalent antibodies. In most embodiments, the fusion protein incorporates more than 2, for example 3, 4, 5, or 6, DR5BDs. In some embodiments, the fusion protein incorporates a DR5BD and a binding domain directed to a non-TNFRSF member antigen. In these embodiments, the interaction of the non-TNFRSF antigen can provide an additional crosslinking function, and TNFRSF activation is achieved with only one or two DR5BDs. In these embodiments, the fusion protein is multispecific and binds to two different antigens. In other embodiments, the fusion protein incorporates more than 3 DR5BDs and a binding domain directed to an antigen other than DR5, and the interaction with this additional antigen provides advantages in biological distribution without enhancing DR5 clustering beyond that achieved by the DR5BD-containing portion alone, concentrating the DR5 agonist activity of the fusion protein at specific sites in a subject. For example, a tetravalent DR5-binding fusion protein of the present disclosure may include an additional antigen-binding domain that concentrates activity at specific sites, but does not enhance agonist activity beyond that achieved by a tetravalent DR5-binding fusion protein lacking this additional antigen binding.

[0019] In some embodiments, the DR5BD of the present disclosure is derived from an antibody or antibody fragment such as an scFv, Fab, single domain antibody (sdAb), V NAR or VHH. In preferred embodiments, the DR5BD is a human or humanized sdAb. The sdAb fragment can be derived from a VHH, V NAR , engineered VH or VK domain. VHH can be made from the heavy-chain only antibodies of camelids. V NARIt can be prepared from antibodies of only cartilaginous fish heavy chains. Various methods for producing monomeric sdAbs from conventional heterodimeric VH and VK domains, including interface engineering and selection of specific germline families, have been implemented. In other embodiments, DR5BD is derived from non-antibody scaffold proteins such as, but not limited to, ankyrin repeat proteins (darpins), avimers, anticalins / lipocalins, centyrins, and fibronomers.

[0020] Generally, the fusion proteins of the present disclosure consist of at least two or more DR5BDs operably linked via a linker polypeptide. The use of sdAb fragments as specific DR5BDs in the fusions of the present disclosure has the advantage of avoiding the heavy chain:light chain mispairing problems common to many bispecific / multispecific antibody approaches. Further, the fusion proteins of the present disclosure avoid the use of long linkers required by many bispecific antibodies.

[0021] In some embodiments, all DR5BDs of the fusion protein recognize the same epitope on DR5. For example, the fusion proteins of the present disclosure can incorporate 2, 3, 4, 5, or 6 DR5BDs having different recognition specificities for different epitopes on DR5. In these embodiments, the fusion proteins of the present disclosure include multiple DR5BDs targeting different regions of DR5. In some embodiments, the DR5BD can recognize different epitopes on DR5 or can recognize an epitope on DR5 and a different antigen. For example, the present disclosure provides multispecific fusion proteins incorporating DR5BDs that bind to DR5 and at least a second antigen.

[0022] In some embodiments, the fusion proteins of the present disclosure are composed of a single polypeptide. In other embodiments, the fusion proteins of the present disclosure are composed of two or more polypeptides. For example, a heterodimerization domain is incorporated into the fusion protein to construct an asymmetric fusion protein. For example, when an immunoglobulin Fc region is incorporated into the fusion protein, the CH3 domain can be used as a homodimerization domain, or the CH3 dimer interface region can be mutated to enable heterodimerization.

[0023] In some embodiments, the fusion protein contains DR5BD at both ends. For example, DR5BD is located at both the amino-terminal (N-terminal) portion and the carboxy-terminal (C-terminal) portion of the fusion protein. In other embodiments, all of the DR5BD are present at the same end of the fusion protein. For example, DR5BD is present at either the amino or carboxyl terminal portion of the fusion protein.

[0024] In some embodiments, the fusion protein contains an immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is an IgG isotype selected from the group consisting of IgG1 isotype, IgG2 isotype, IgG3 isotype, and IgG4 subclass.

[0025] In some embodiments, the immunoglobulin Fc region or its immunologically active fragment is of the IgG isotype. For example, the immunoglobulin Fc region of the fusion protein is of the human IgG1 isotype and has the amino acid sequence: [Table 1] and has.

[0026] In some embodiments, the immunoglobulin Fc region or an immunologically active fragment thereof comprises a human IgG1 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1.

[0027] In some embodiments, to prevent glycosylation of the fusion protein, the human IgG1 Fc region is modified at amino acid Asn297 (in-frame, Kabat numbering), for example Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu235 (in-frame, Kabat numbering) to alter Fc receptor interaction, for example, Leu235Glu (L235E) or Leu235Ala (L235A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (in-frame, Kabat numbering) to alter Fc receptor interaction, for example Leu234Ala (L234A). In some embodiments, the Fc region of the fusion protein is altered at both amino acids 234 and 235, for example Leu234Ala and Leu235Ala (L234A / L235A) or Leu234Val and Leu235Ala (L234V / L235A). In some embodiments, the Fc region of the fusion protein is altered at Gly235 to reduce Fc receptor binding. For example, here the Gly235 is removed from the fusion protein In some embodiments, the human IgG1 Fc region is modified at amino acid Gly236 to enhance interaction with CD32A, for example, Gly236Ala (G236A). In some embodiments, the human IgG1 Fc region lacks Lys447 (EU index of Kabat et al, 1991 Sequences of Proteins of Immunological Interest).

[0028] In some embodiments, to reduce Fc receptor binding, the Fc region of the fusion protein is altered at one or more positions of the following: Leu234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325) or Ala327 (A327). For example, Leu234Ala (L234A), Leu235Ala (L235A), Asp265Asn (D265N), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Asn325Glu (N325E) or Ala327Ser (A327S). In preferred embodiments, the modification within the Fc region first suppresses the effect on binding to the neonatal Fc receptor (FcRn), while reducing binding to the Fc receptor-gamma receptor.

[0029] In some embodiments, the Fc region of the fusion protein lacks an amino acid at one or more of the following positions: Glu233 (E233), Leu234 (L234), or Leu235 (L235) to reduce Fc receptor binding. In these embodiments, the Fc deletion of these three amino acids reduces complement protein C1q binding.

Table 2

[0030] In some embodiments, the fusion or an immunologically active fragment thereof comprises a human IgG2 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2.

[0031] In some embodiments, the immunoglobulin Fc region or an immunologically active fragment of the fusion protein is of the human IgG2 isotype and has the amino acid sequence:

Table 3

[0032] In some embodiments, the fusion or an immunologically active fragment thereof comprises a human IgG2 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 3.

[0033] In some embodiments, the human IgG2 Fc region is modified at amino acid Asn297 (boxed, e.g., Asn297Ala (N297A) or Asn297Asp (N297D) to prevent glycosylation of the antibody. In some embodiments, the human IgG2 Fc region lacks Lys447 (EU index of Kabat et al, 1991 Sequences of Proteins of Immunological Interest).

[0034] In some embodiments, the immunoglobulin Fc region or an immunologically active fragment of the fusion protein is of the human IgG3 isotype and has the amino acid sequence:

Table 4

[0035] In some embodiments, the antibody or an immunologically active fragment thereof comprises a human IgG3 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 4.

[0036] In some embodiments, the human IgG3 Fc region is modified at amino acid Asn297 (in-frame, Kabat numbering), e.g., Asn297Ala (N297A) or Asn297Asp (N297D), to prevent glycosylation of the antibody. In some embodiments, the human IgG3 Fc region is modified at amino acid 435, e.g., Arg435His (R435H), to extend the half-life. In some embodiments, the human IgG3 Fc region lacks Lys447 (Kabat et al, 1991 Sequences of Proteins of Immunological Interest's EU index).

[0037] In some embodiments, the immunoglobulin Fc region or immunologically active fragment of the fusion protein is of the human IgG4 isotype and has the amino acid sequence:

Table 5

[0038] In some embodiments, the antibody or its immunologically active fragment comprises a human IgG4 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 5.

[0039] In some embodiments, the immunoglobulin Fc region or immunologically active fragment of the fusion protein is of the human IgG4 isotype and has the amino acid sequence:

Table 6

[0040] In some embodiments, the antibody or its immunologically active fragment comprises a human IgG4 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 127.

[0041] In other embodiments, the human IgG4 Fc region is modified at amino acid 235, e.g., Leu235Glu (L235E), to alter Fc receptor interaction. In some embodiments, the human IgG4 Fc region is modified at amino acid Asn297 (in-frame, Kabat numbering), e.g., Asn297Ala (N297A) or Asn297Asp (N297D), to prevent glycosylation of the antibody. In some embodiments, the human IgG4 Fc region lacks Lys447 (EU index of Kabat et al, 1991 Sequences of Proteins of Immunological Interest).

[0042] In some embodiments, the human IgG Fc region is modified to enhance FcRn binding. Examples of Fc mutations that enhance binding to FcRn are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E respectively) (Kabat numbering, Dall’Acqua et al, 2006, J. Biol Chem Vol. 281(33)23514 - 23524), Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al, 2010 Nature Biotech, Vol. 28(2)157 - 159), or Met252Ile, Thr256Asp, Met428Leu (M252I, T256D, M428L respectively), (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).

[0043] In some embodiments where the fusion protein of the present disclosure includes an Fc polypeptide, the Fc polypeptide is mutated or modified. In these embodiments, the mutated or modified Fc polypeptide includes the following mutations: Met252Tyr and Met428Leu or Met252Tyr and Met428Val (M252Y, M428L, or M252Y, M428V) using the Kabat numbering system.

[0044] In some embodiments, the human IgG Fc region is modified to alter antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), for example, as reported in Natsume et al, 2008 Cancer Res, 68(10):3863-72; Idusogie et al, 2001 J Immunol, 166(4):2571-5; Moore et al, 2010 mAbs, 2(2):181-189; Lazar et al, 2006 PNAS, 103(11):4005-4010, Shields et al, 2001 JBC, 276(9):6591-6604; Stavenhagen et al, 2007 Cancer Res, 67(18):8882-8890; Stavenhagen et al, 2008 Advan.Enzyme Regul., 48:152-164; Alegre et al, 1992 J Immunol, 148:3461-3468; Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11, amino acid modifications. Examples of mutations that enhance ADCC include modifications at Ser239 and Ile332, such as Ser239Asp and Ile332Glu (S239D, I332E). Examples of mutations that enhance CDC include modifications at Lys326 and Glu333. In some embodiments, the Fc region is modified at one or both of these positions, for example, Lys326Ala and / or Glu333Ala (K326A and E333A) using the Kabat numbering system.

[0045] In some embodiments, the human IgG Fc region is modified to induce heterodimerization. For example, having an amino acid modification within the CH3 domain of Thr366 substituted with a bulkier amino acid, such as Try(T366W), enables preferential pairing with a second CH3 domain having an amino acid modification at the positions of Thr366, Leu368, and Tyr407, for example, Ser, Ala, and Val, respectively (T366S / L368A / Y407V), to result in less bulky amino acids. Heterodimerization by CH3 modification can be further stabilized by the introduction of disulfide bonds, for example, by changing Ser354 to Cys (S354C) and Y349 to Cys (Y349C) on the opposing CH3 domain (reported in Carter, 2001 Journal of Immunological Methods, 248:7-15).

[0046] In some embodiments, the human IgG Fc region is modified to prevent dimerization. In these embodiments, the fusion proteins of the present disclosure are monomeric. For example, modification to a charged residue at residue Thr366, such as Thr366Lys, Thr366Arg, Thr366Asp, or Thr366Glu (T366K, T366R, T366D, or T366E, respectively), prevents CH3-CH3 dimerization.

[0047] In some embodiments, the Fc region of the fusion protein reduces Fc receptor binding are modified at one or more of the following positions: Leu 234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325) or Ala327 (A327). For example, Leu234Ala (L234A), Leu235Ala (L235A), Asp265Asn (D265N), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Asn325Glu (N325E) or Ala327Ser (A327S). In preferred embodiments, the modifications within the Fc region minimize the effect on binding to the neonatal Fc receptor (FcRn), while reducing binding to the Fc receptor-gamma receptor.

[0048] In some embodiments, the fusion protein comprises a polypeptide derived from an immunoglobulin hinge region. The hinge region can be selected from any of the human IgG subclasses. For example, the fusion protein can comprise a modified IgG1 hinge having the sequence EPKSSDKTHTCPPC (SEQ ID NO: 6) in which Cys220, which forms a disulfide with the C-terminal cysteine of the light chain, is mutated to serine, for example Cys220Ser (C220S). In other embodiments, the fusion protein comprises a shortened hinge having the sequence DKTHTCPPC (SEQ ID NO: 7).

[0049] In some embodiments, the fusion protein comprises a modified hinge derived from IgG4, such as Ser228Pro (S228P), modified to prevent or reduce chain replacement and having the sequence ESKYGPPCPPC (SEQ ID NO: 8). In some embodiments, the fusion protein comprises a linker polypeptide. In other embodiments, the fusion protein comprises a linker and a hinge polypeptide.

[0050] In some embodiments, the fusion proteins of the present disclosure lack or have a reduced fucose that is bound to the N-linked glycan chain at N297. There are many ways to prevent fucosylation, including, but not limited to, production in FUT8-deficient cell lines; addition of inhibitors to mammalian cell culture media, such as Castanospermine; and metabolic engineering of the production cell line.

[0051] In some embodiments, DR5BD is engineered to preclude recognition by existing antibodies present in humans. In some embodiments, the single domain antibodies of the present disclosure are modified by mutations at position Leu11, such as Leu11Glu (L11E) or Leu11Lys (L11K). In other embodiments, the single domain antibodies of the present disclosure are modified by changes in the carboxy-terminal region, such that, for example, its terminal sequence consists of GQGTLVTVKPGG (SEQ ID NO: 9) or GQGTLVTVEPGG (SEQ ID NO: 10) or a modified form thereof. In some embodiments, the single domain antibodies of the present disclosure are modified by a mutation at position 11 and a change in the carboxy-terminal region.

[0052] In some embodiments, the DR5BD of the fusion proteins of the present disclosure is operably linked via an amino acid linker. In some embodiments, these linkers consist primarily of the amino acids glycine and serine, as shown herein as GS-linkers. The GS-linkers of the fusion proteins of the present disclosure can be of various lengths, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in length.

[0053] In some embodiments, the GS-linker is GGSGGS, i.e., (GGS) 2 (SEQ ID NO: 11); GGSGGSGGS, i.e., (GGS) 3 (SEQ ID NO: 12); GGSGGSGGSGGS, i.e., (GGS) 4 (SEQ ID NO: 13); and GGSGGSGGSGGSGGS, i.e., (GGS) 5 (SEQ ID NO: 14) and is selected from the group consisting of ami comprises an amino acid sequence.

[0054] In some embodiments, the multivalent TNFRSF-binding fusion protein is tetravalent. In some embodiments, the tetravalent TNFRSF-binding fusion protein has the following structure: VHH-linker-VHH-linker-hinge-Fc, wherein the VHH is a humanized or fully human VHH sequence that binds at least DR5.

[0055] In some embodiments, the multivalent TNFRSF-binding fusion protein is tetravalent. In some embodiments, the tetravalent TNFRSF-binding fusion protein has the following structure: DR5BD-linker-DR5BD-linker-hinge-Fc, wherein the DR5BD is a humanized VHH or fully human VHH sequence.

[0056] In some embodiments, the multivalent TNFRSF-binding fusion protein is hexavalent. In some embodiments, the hexavalent TNFRSF-binding fusion protein has the following structure: VHH-linker-VHH-linker-VHH-linker-hinge-Fc, wherein the VHH is a humanized or fully human VHH sequence that binds at least DR5.

[0057] In some embodiments, the multivalent TNFRSF-binding fusion protein is hexavalent. In some embodiments, the hexavalent TNFRSF-binding fusion protein has the following structure: DR5BD-linker-DR5BD-linker-DR5BD-linker-hinge-Fc, wherein the DR5BD is a humanized or fully human VHH sequence.

[0058] In some embodiments, the multivalent fusion proteins targeting DR5 of the present disclosure are operably linked via an amino acid linker. In some embodiments, these linkers are mainly composed of the amino acids glycine and serine, shown herein as GS-linkers. The GS-linkers of the fusion proteins of the present disclosure can be of various lengths, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in length.

[0059] In some embodiments, the GS-linker is GGSGGS, i.e., (GGS) 2 (SEQ ID NO: 11); GGSGGSGGS, i.e., (GGS) 3 (SEQ ID NO: 12); GGSGGSGGSGGS, i.e., (GGS) 4 (SEQ ID NO: 13); and GGSGGSGGSGGSGGS, i.e., (GGS) 5 and includes an amino acid sequence selected from the group consisting of (SEQ ID NO: 14).

[0060] In some embodiments, the multivalent DR5-binding fusion protein is tetravalent. In some embodiments, the tetravalent DR5-binding fusion protein has the following structure: VHH-linker-VHH-linker -hinge-Fc (where VHH is a humanized or fully human VHH sequence). In some embodiments, the VHH sequence is selected from the group consisting of SEQ ID NOs: 15 to 91. In some embodiments, the tetravalent DR5-binding fusion protein includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 92 to 118.

[0061] In some embodiments, the multivalent DR5-binding fusion protein is hexavalent. In some embodiments, the hexavalent DR5-binding fusion protein has the following structure: VHH-linker-VHH-linker-VHH-linker-hinge-Fc (wherein VHH is a humanized or fully human VHH sequence). In some embodiments, the VHH sequence is selected from the group consisting of SEQ ID NOs: 15 to 91. In some embodiments, the hexavalent DR5-binding fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 119 to 124.

Brief Description of the Drawings

[0062]

Figure 1

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Figure 7A

Figure 7B

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Figure 9

[0063] Detailed Description The present disclosure provides molecules that specifically engage the cell death receptor 5 (DR5), which is a member of the TNF receptor superfamily (TNFRSF). More specifically, the present disclosure relates to multivalent molecules that bind at least DR5. These multivalent TNFRSF-binding fusion proteins include two or more TNFRSF-binding domains (DR5BDs), and at least one DR5BD binds to DR5. These molecules are referred to herein as DR5-targeting molecules.

[0064] These DR5-targeting molecules include at least one copy of a single-domain antibody (sdAb) sequence that specifically binds to DR5. In some embodiments, the molecule targeting DR5 includes two or more copies of an sdAb that specifically binds to DR5, such as three or more, four or more, five or more, or six or more copies of an sdAb that specifically binds to DR5.

[0065] A single-domain antibody (sdAb) is an antibody fragment consisting of a single monomeric variable antibody domain that can selectively bind to a specific antigen. Having a molecular weight of only 12 - 15 kDa, a single-domain antibody is much smaller than a small common antibody (150 - 160 kDa) composed of two heavy-chain proteins and two light-chain proteins, and is also smaller than a Fab fragment (about 50 kDa, one light chain and half of a heavy chain) and a single-chain variable fragment (about 25 kDa, two variable domains, one from a light chain and the other from a heavy chain).

[0066] A single-domain antibody is an antibody in which the complementarity-determining region is part of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, antibodies that are naturally lacking in light chains, single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single-domain scaffolds other than those derived from antibodies. Single-domain antibodies can be derived from any species including, but not limited to, mouse, human, camel, llama, goat, rabbit, and / or cow. In some embodiments, the single-domain antibodies used herein are naturally occurring single-domain antibodies known as heavy-chain antibodies lacking light chains. For clarity, this variable domain derived from a heavy-chain antibody that is naturally lacking in a light chain is designated herein as VHH to distinguish it from the conventional VH of a four-chain immunoglobulin. Such VHH molecules can be derived from antibodies produced in Camelidae species such as camels, llamas, dromedaries, alpacas, and guanacos. Other species outside of Camelidae may produce heavy-chain antibodies that are naturally lacking in light chains; such VHHs are within the scope of this disclosure.

[0067] Single-domain antibodies can be obtained by immunizing a human cobra, camel, llama, alpaca, or shark with a desired antigen and subsequently isolating the mRNA encoding the heavy-chain antibody. A gene library of single-domain antibodies containing millions of clones is generated by reverse transcription and polymerase chain reaction. Screening techniques such as phage display and ribosome display are useful for identifying clones that bind to the antigen. (See, e.g., Arbabi Ghahroudi, M.; Desmyter, A. et al (1997), “Selection and identification of single domain antibody fragments from camel heavy-chain antibodies”. FEBS Letters 414(3):521-526.)

[0068] Another approach uses a gene library from non-preimmunized animals. Such naive libraries usually contain only antibodies with low affinity for the desired antigen and require affinity maturation by random mutagenesis as an additional step. (Saerens, D.; et al. (2008). “Single-domain antibodies as building blocks for novel therapeutics”. Current Opinion in Pharmacology 8(5):600 - 608.)

[0069] When the most potent clones are identified, their DNA sequences are optimized, for example, to improve stability against enzymes. Another goal is humanization to prevent an immunological reaction in the human organism against the antibody. Humanization is not a problem since there is homology between the VHH of camelids and the human VH fragment. (See, for example, Saerens, et al. (2008). “Single-domain antibodies as building blocks for novel therapeutics”. Current Opinion in Pharmacology 8(5):600 - 608.). The final step is the translation of the optimized single-domain antibody in Escherichia coli, Saccharomyces cerevisiae or other suitable organisms.

[0070] Single-domain antibody fragments can also be derived from conventional antibodies. In some embodiments, single-domain antibodies can be made from common murine or human IgG having four chains. (Holt, L.J. et al. (2003), “Domain antibodies: proteins for therapy”. Trends in Biotechnology 21(11):484-490). This process is similar and involves a gene library from immunized or naive donors and display technology for the identification of the most specific antigens. The problem with this approach is that the binding region of common IgG consists of two domains (VH and VL) that tend to dimerize or aggregate due to their lipophilicity. Monomerization is usually achieved by replacing lipophilic amino acids with hydrophilic amino acids, but often results in a loss of affinity for the antigen. (See, e.g., Borrebaeck, C. A. K.; Ohlin, M. (2002). “Antibody evolution beyond Nature”. Nature Biotechnology 20(12):1189-90). If the affinity can be retained, single-domain antibodies can be produced similarly in E. coli, S. cerevisiae or other organisms.

[0071] Monovalent single-domain antibodies can be made multivalent by several methods. For example, the cDNA encoding the first sdAb can be genetically fused to a linker encoding a DNA sequence, followed by a second cDNA encoding an sdAb, etc. Alternatively, the cDNA encoding the sdAb can be fused to a cDNA encoding a second protein or a fragment thereof that naturally multimerizes or has been engineered to multimerize. For example, fusion of an sdAb to the IgG Fc region dimerizes the sdAb. If the constructed tandem sdAb-encoding is linked to the Fc-encoding construct, the resulting fusion protein, once expressed, is tetravalent. If a construct encoding three sdAbs is linked to the Fc-encoding construct, the resulting fusion protein, once expressed, is hexavalent. The present disclosure contemplates the use of additional multimerization domains such as collagen homotrimeric domains and heterotrimeric domains, leucine zipper domains, p53 tetramerization domains, c-Jun:Fos heterodimeric peptide sequences, cartilage oligomeric matrix protein (COMP48), trimeric adiponectin, trimeric surfactant protein D, and / or synaptic acetylcholinesterase tetramer, etc.

[0072] Targeting death receptor 5 (TRIAL-R2, TNFRSF10B) Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has evolved to play an important role in mammalian development and host defense by selectively eradicating unwanted infected and malignant cells from healthy cell populations. TRAIL induces cell death via caspase-dependent apoptosis when it binds to the members DR4 or DR5 of the TNF receptor family. DR5 (TNFRSF10B) appears to be the major receptor on tumor cells that promotes the tumor-biased activity observed in the TRAIL pathway. DR5 is activated by the natural ligand TRAIL, which brings three DR5 receptors into proximity, thereby activating intracellular caspase-8 and initiating the activation of other death-inducing caspases such as caspase-9 and caspase-3. Thus, the initiation of this cell death pathway requires clustering of DR5 receptors for efficient cell death.

[0073] Efforts to clinically utilize the TRAIL pathway for cancer treatment have relied on recombinant forms of the natural ligand TRAIL and antibodies specific for DR5. Antibody agonists targeting DR5 required a cross-linking agent in preclinical in vitro experiments. This was due to the fact that conventional antibodies brought about clustering of two DR5 receptors (one for each heavy and light chain). The two DR5 receptors are insufficient to activate the cell death pathway and thus the cross- A bridging agent is required. In vivo administration of DR5-targeted antibodies in preclinical mouse models of human cancer surprisingly showed significant activity in a wide variety of tumor types. This activity was later shown to be dependent on the mouse Fc gamma R (FcγR) receptor. Clinical studies in humans have not been able to reproduce the robust responses seen in these preclinical mouse models. The lack of activity in humans is hypothesized to be due to insufficient antibody cross-linking. This could be due to differences in serum IgG, Fc gamma R (FcγR), and / or TRAIL concentrations between immunodeficient mice and human cancer patients.

[0074] The present disclosure provides a multivalent fusion protein targeting DR5 that can potently stimulate DR5 signaling mediating direct cell death. The fusion proteins of the present disclosure can be trivalent, tetravalent, pentavalent, or hexavalent. Importantly, the fusion proteins of the present disclosure can induce apoptosis of DR5-expressing cells independently of exogenous bridging agents.

[0075] In some embodiments, the fusion proteins of the present disclosure incorporate a DR5BD that binds to DR5. In preferred embodiments, the DR5-binding DR5BD does not bind to DR4, decoy R1, decoy R2, osteopontin, or any other TNFRSF member. In preferred embodiments, the DR5-binding DR5BD binds to human and cynomolgus DR5. In some embodiments, the DR5-binding DR5BD interferes with the interaction between DR5 and its ligand TRAIL. In other embodiments, the DR5-binding DR5BD does not interfere with the interaction between DR5 and its ligand TRAIL. In some embodiments, the fusion proteins of the present disclosure incorporate multiple DR5-binding DR5BDs that recognize different epitopes on DR5. In some embodiments, the fusion proteins of the present disclosure incorporate multiple DR5-binding DR5BDs, where some of the DR5BDs interfere with the DR5-TRAIL interaction and others do not. In preferred embodiments, the DR5-targeted fusion proteins of the present disclosure induce direct cell death of tumor cells. The DR5-targeted fusion proteins of the present disclosure are inherently useful for treating both hematological and solid tumors.

[0076] Examples of DR5-binding sdAbs The DR5 VHH (derived from llama) and humanized sequences are shown below, and the CDR sequences are shown below each sequence. In some embodiments, the DR5-binding sdAb is fused to the IgG Fc region, and in these embodiments, the fusion protein is bivalent and has two DR5-binding domains per molecule. In some embodiments, two DR5-binding sdAbs (2x) are fused to the IgG Fc region, and in these embodiments, the fusion protein is tetravalent and has four DR5-binding domains per molecule. In some embodiments, three DR5-binding sdAbs (3x) are fused to the IgG Fc region, and in these embodiments, the fusion protein is hexavalent and has six DR5-binding domains per molecule. [Table 7] JPEG2025084923000009.jpg232166JPEG2025084923000010.jpg227166JPEG2025084923000011.jpg227167JPEG2025084923000012.jpg227166JPEG2025084923000013.jpg228168JPEG2025084923000014.jpg227165JPEG2025084923000015.jpg228165JPEG2025084923000016.jpg228165JPEG2025084923000017.jpg228166JPEG2025084923000018.jpg228169JPEG2025084923000019.jpg228164JPEG2025084923000020.jpg228166JPEG2025084923000021.jpg228167JPEG2025084923000022.jpg228164JPEG2025084923000023.jpg228167JPEG2025084923000024.jpg228167JPEG2025084923000025.jpg228167JPEG2025084923000026.jpg223167JPEG2025084923000027.jpg214167JPEG2025084923000028.jpg207167JPEG2025084923000029.jpg253167JPEG2025084923000030.jpg249167TIFF2025084923000031.tif201170JPEG2025084923000032.jpg234168JPEG2025084923000033.jpg244170JPEG2025084923000034.jpg254168JPEG2025084923000035.jpg69168

[0077] The DR5 target proteins described herein are useful in various therapeutic, diagnostic, and prophylactic conditions. For example, the DR5 target proteins are useful for treating various diseases and disorders in a subject. In some embodiments, the DR5 target proteins are useful for treating a disease or disorder, reducing its symptoms, improving and / or delaying its progression, in a subject suffering from or considered to be suffering from an inflammatory disease or disorder. In some embodiments, the DR5 target proteins are useful for treating cancer or other neoplastic conditions, improving its symptoms, alleviating and / or delaying its progression. In some embodiments, the cancer is bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system neoplasms, lymphoma, leukemia, myeloma, sarcoma, mesothelioma, leukemia, lymphoma, myeloma, and virus-related cancer. In certain embodiments, the cancer is metastatic cancer, refractory cancer, or recurrent cancer. In some embodiments, the DR5 target proteins are useful for reducing or depleting the number of regulatory T cells in the tumor of a subject in need thereof. In some embodiments, the DR5 target proteins are useful for stimulating an immune response in a subject. In some embodiments, the DR5 target proteins are useful for treating an autoimmune disease or disorder, reducing its symptoms, improving and / or delaying its progression. In some embodiments, the DR5-targeted proteins are useful for treating viral, bacterial, and parasitic infections, alleviating their symptoms, improving and / or delaying their progression.

[0078] The therapeutic formulations of the present disclosure comprising the DR5 targeting molecules are used to treat or alleviate symptoms associated with diseases or disorders related to abnormal activity and / or expression of DR5 in a subject. The treatment regimen is carried out by identifying a subject, e.g., a human patient suffering from (or at risk of developing) a disease or disorder related to abnormal activity and / or expression of DR5, using standard methods including various clinical and / or laboratory procedures. The term patient includes human and veterinary subjects. The term subject includes humans and other mammals.

[0079] The effectiveness of the treatment is determined in relation to any known method for diagnosing or treating a particular disease or disorder related to abnormal activity and / or expression of DR5. Alleviation of one or more symptoms of a disease or disorder related to abnormal activity and / or expression of DR5 indicates that the DR5 targeting molecule confers a clinical benefit.

[0080] The therapeutic use of the DR5 targeting molecules of the present disclosure may also include administration of one or more additional agents.

[0081] In some embodiments, the DR5 targeting molecule is administered during and / or after treatment in combination with one or more additional agents. In some embodiments, the DR5 targeting molecule and the additional agent are formulated into a single therapeutic composition and the DR5 targeting molecule and the additional agent are administered simultaneously. Alternatively, the DR5 targeting molecule and the additional agent are separate from each other, e.g., each is formulated into a separate therapeutic composition, and the DR5 targeting molecule and the additional agent are administered simultaneously or the DR5 targeting molecule and the additional agent are administered at different times during the treatment regimen. For example, the DR5 targeting molecule is administered before the administration of the additional agent, the DR5 targeting molecule is administered after the administration of the additional agent, or the DR5 targeting molecule and the additional agent are administered alternately. As described herein, the DR5 targeting molecule and the additional agent are administered in a single dose or multiple doses.

[0082] In some embodiments, the DR5 target molecule and the additional agent(s) are administered simultaneously. For example, the DR5 target molecule and the additional agent(s) can be formulated in a single composition or administered as two or more separate compositions. In some embodiments, the DR5 target molecule and the additional agent(s) are administered sequentially or the DR5 target molecule and the additional agent are administered at different times during a treatment regimen.

[0083] Methods for screening for DR5 target molecules having the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), enzyme assay, flow cytometry, and other immunologically mediated techniques known in the art.

[0084] The present disclosure further provides nucleic acid sequences, particularly DNA sequences, encoding the fusion proteins of the invention. Preferably, the DNA sequence is carried by a vector suitable for extrachromosomal replication such as a phage, virus, plasmid, phagemid, cosmid, YAC or episome. In particular, the DNA vector encoding the desired fusion protein can be used to facilitate the methods for preparing the DR5 target molecules described herein and a significant amount of the fusion protein can be obtained. The DNA sequence can be inserted into a suitable expression vector, i.e., a vector containing the elements necessary for transcription and translation of the inserted protein coding sequence. A variety of host-vector systems can be utilized to express the protein coding sequence. These include mammalian cell lines infected with a virus (e.g., vaccinia virus, adenovirus, etc.); insect cell lines infected with a virus (e.g., baculovirus); microorganisms such as yeast containing a yeast vector, or bacteria transformed with bacteriophage DNA, plasmid DNA or cosmid DNA. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.

[0085] The present disclosure also provides a method for producing a DR5 target molecule by culturing cells under conditions that result in the expression of the polypeptide, wherein the cells comprise an isolated nucleic acid molecule encoding the DR5 target molecule described herein, and / or a vector comprising these isolated nucleic acid sequences. The present disclosure provides a method for producing a DR5 target molecule by culturing cells under conditions that result in the expression of the DR5 target molecule, wherein the cells comprise an isolated nucleic acid molecule encoding the DR5 target molecule described herein, and / or a vector comprising these isolated nucleic acid sequences.

[0086] The fusion proteins of the present disclosure (also referred to herein as "active compounds"), and their derivatives, fragments, analogs and homologs (isologs) can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the fusion protein and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like that are compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington’s Pharmaceutical Sciences, a standard reference in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, physiological saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and fixed oils can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Their use in the compositions is intended, except in cases where any conventional media or agent is incompatible with the active compound. Supplementary active compounds can also be incorporated into the compositions.

[0087] The pharmaceutical compositions of the present disclosure are formulated to be compatible with the intended route of administration. Examples of routes of administration include parenteral administrations such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administrations. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffering agents such as acetate, citrate, or phosphate, and tonicity adjusting agents such as sodium chloride or dextrose. The pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral formulations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0088] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water-soluble), dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, Cremophor EL (trademark) (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must have a fluidity that allows easy passage through a hypodermic needle. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium including, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycols, etc.) and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include in the composition isotonic agents such as sugars, polyalcohols such as mannitol and sorbitol, sodium chloride. Sustained absorption of the injectable composition can be brought about by including in the composition agents that delay absorption, for example, aluminum monostearate and gelatin.

[0089] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound in an appropriate solvent, with one or a combination of the ingredients enumerated above as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound in a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze drying that yield a powder of the active ingredient and any additional desired ingredients from its previously sterile filtered solution.

[0090] Oral compositions generally contain an inert diluent or an edible carrier. These can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compounds can be mixed with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared with a fluid carrier for use as a mouthwash, and the compounds in this liquid carrier are orally applied, swished in the mouth, and then spat out or swallowed. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, etc. can contain any of the following ingredients, or compounds of similar nature: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose, disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterote; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavor.

[0091] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as a gas like carbon dioxide or a nebulizer.

[0092] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, suitable penetration enhancers are used in the formulation for the barrier to be penetrated. Such penetration enhancers are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by the use of nasal sprays or suppositories. In the case of transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams generally known in the art.

[0093] The compounds can also be prepared in the form of suppositories (e.g., having conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0094] In one embodiment, the active compounds are prepared with a carrier that protects the compound against rapid elimination from the body, such as in a controlled release formulation including implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials are also commercially available and can be obtained from, for example, Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, as described, for example, in U.S. Patent No. 4,522,811.

[0095] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate oral or parenteral compositions in dosage unit form. As used herein, a dosage unit form refers to physically discrete units suitable as unit dosages for the subjects to be treated; each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier. The specification for the dosage unit forms of the present disclosure is determined by and directly depends on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the inherent limitations in the art of compounding such active compounds for the treatment of individuals.

[0096] The pharmaceutical compositions can be included in a kit, container, pack, or dispenser, together with instructions for administration. These pharmaceutical compositions can be included in a diagnostic kit together with instructions for use.

[0097] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless specifically required by context, the singular terms shall include the plural, and the plural terms shall include the singular. In general, the nomenclature utilized in connection with cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization as described herein is the well-known and commonly used nomenclature in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzyme reactions and purification techniques are performed according to the manufacturer's specifications, or as commonly practiced in the art, or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry as described herein, as well as the experimental procedures and techniques of these chemistries, are those well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and patient delivery and treatment. The term patient includes human and veterinary subjects.

[0098] When utilized in accordance with the present disclosure, the following terms shall be understood to have the following meanings, unless otherwise indicated:

[0099] As used herein, the terms “target fusion protein” and “antibody” may be synonymous. As used herein, the term “antibody” refers to an immunoglobulin molecule and an immunologically active portion of an immunoglobulin (Ig) molecule, i.e., a molecule that specifically binds (immunoreacts) with an antigen, including an antigen-binding site. “Specifically binds” or “immunoresponds” or “is directed against” means that an antibody reacts with one or more antigenic determinants of a desired antigen and does not react with or binds with much lower affinity to other polypeptides (K d >10 -6 ). Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single-chain, Fab, Fab ’ and F(ab’) 2 fragments, F v , scFvs, Fab expression libraries, and single domain antibody (sdAb) fragments, such as V H H, V NAR , engineered V H or V K .

[0100] The basic antibody structural unit is known to comprise a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, and each pair has one “light” chain (about 25 kDa) and one “heavy” chain (about 50 - 70 kDa). The amino-terminal portion of each chain contains a variable region of about 100 - 110 or more amino acids that is mainly involved in antigen recognition. The carboxy-terminal portion of each chain defines a constant region that is mainly responsible for effector functions. Generally, antibody molecules obtained from humans are associated with one of IgG, IgM, IgA, IgE, and IgD, which differ from each other depending on the nature of the heavy chains present in the molecule. Specific classes further have subclasses (also called isotypes) such as IgG 1 , IgG 2 , etc. Furthermore, in humans, the light chain can be a kappa chain or a lambda chain.

[0101] As used herein, the term "monoclonal antibody" (mAb) or "monoclonal antibody composition" refers to a population of antibody molecules that contains only one molecular species of an antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. A MAb contains an antigen binding site that can immunoreact with a specific epitope of an antigen characterized by a unique binding affinity for the antigen.

[0102] The term "antigen binding site" or "binding moiety" refers to the part of an immunoglobulin molecule involved in antigen binding. The antigen binding site is formed by the amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains, called "hypervariable regions", are inserted between more conserved adjacent stretches known as "framework regions" or "FRs". Thus, the term "FR" refers to the naturally occurring amino acid sequences between and adjacent to the hypervariable regions of an immunoglobulin. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged in relation to each other in three-dimensional space to form an antigen binding surface. The antigen binding surface is complementary to the three-dimensional surface of the bound antigen, and each of the three hypervariable regions of the heavy and light chains is called a "complementarity determining region" or "CDR". The assignment of each domain of amino acids follows the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989).

[0103] The single domain antibody (sdAb) fragment portion of the fusion protein of the present disclosure is interchangeably referred to herein as the target polypeptide of the present specification.

[0104] As used herein, the term "epitope" includes any protein determinant that can be specifically bound by an immunoglobulin or fragment thereof, or by a T cell receptor. The term "epitope" includes any protein determinant that can be specifically bound by an immunoglobulin or a T cell receptor. Epitope determinants usually consist of chemically active surface groups of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is ≦1 μM, for example ≦100 nM, preferably ≦10 nM, more preferably ≦1 nM.

[0105] As used herein, the terms "immunological binding" and "immunological binding properties" and "specific binding" refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction can be represented by the dissociation constant (K d ), and a smaller K d represents a greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method requires measuring the rates of formation and dissociation of the antigen-binding site / antigen complex, and these rates depend on the concentrations of the complex partners, the affinity of the interaction, and the geometric parameters that equally affect the rates in both directions. Thus, both the "on-rate constant" (k on ) and the "off-rate constant" (k off ) can be determined by calculation of the concentrations and actual rates of association and dissociation. (See Nature 361:186-87 (1993)). The ratio of k off / k on allows elimination of all parameters not related to affinity and is equal to the dissociation constant K d . (Generally, see Davies et al. (1990) Annual Rev Biochem 59:439-473). The antibodies of the present invention have an equilibrium binding constant (K d) is said to specifically bind to an antigen when it is ≦1 μM, preferably ≦100 nM, more preferably ≦10 nM, and most preferably ≦100 pM to about 1 pM. This equilibrium binding constant (K d ) is measured by an assay such as a radioligand binding assay, surface plasmon resonance (SPR), flow cytometry binding assay, or a similar assay known to those skilled in the art.

[0106] Preferably, the non-identical residue positions differ by conservative amino acid substitutions.

[0107] Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, the group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; the group of amino acids having amide-containing side chains is asparagine and glutamine; the group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids having basic side chains is lysine, arginine, and histidine; and the group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0108] As discussed herein, minor changes in the amino acid sequence of an antibody or immunoglobulin molecule are contemplated to be encompassed by the present disclosure, provided that the changes in the amino acid sequence retain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions are those that occur within an amino acid family related by side chain. Genetically encoded amino acids are generally classified into families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other amino acid families include (i) serine and threonine, the aliphatic-hydroxy family; (ii) asparagine and glutamine, the amide-containing family; (iii) alanine, valine, leucine, and isoleucine, the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, the aromatic family. For example, an isolated substitution of leucine with isoleucine or valine, an isolated substitution of aspartic acid with glutamic acid, an isolated substitution of threonine with serine, or a similar substitution of an amino acid with a structurally related amino acid is reasonably expected not to have a major effect on the binding or properties of the resulting molecule, especially if the substitution does not involve an amino acid within the backbone portion. Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein.Fragments or analogs of an antibody or immunoglobulin molecule can be readily prepared by one of ordinary skill in the art. The preferred amino and carboxy termini of the fragment or analog occur near the boundaries of the functional domains. Structural and functional domains can be identified by comparison of nucleotide and / or amino acid sequence data with public or proprietary sequence databases. Computerized comparison methods are preferably used to identify sequence motifs or predicted protein conformation domains that occur in other proteins having known structures and / or functions. Methods for identifying protein sequences folded into known three-dimensional structures are known. Bowie et al. Science 253:164 (1991). Thus, the foregoing examples demonstrate that one of ordinary skill in the art can recognize sequence motifs and structural conformations that can be used to define structural and functional domains in accordance with the present disclosure.

[0109] Preferred amino acid substitutions are (1) substitutions that reduce susceptibility to proteolysis, (2) substitutions that reduce susceptibility to oxidation, (3) substitutions that change the binding affinity for the formation of protein complexes, (4) substitutions that change the binding affinity, and (4) such kind Substitutions that can confer or modify other physicochemical or functional properties of the analog. The analogs can include various mutations of sequences other than the naturally occurring peptide sequences. For example, in a naturally occurring sequence (preferably, the portion of the polypeptide outside the domain(s) forming intermolecular contacts), single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made. Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (e.g., the substituted amino acid should not tend to disrupt a helix that occurs in the parent sequence and should not disrupt other types of secondary structure that characterize the parent sequence). Examples of polypeptide secondary and tertiary structures recognized in the art are described in Proteins, Structures and Molecular Principles (Creighton, Ed, W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al. Nature 354:105 (1991).

[0110] As used herein, the term "polypeptide fragment" refers to a polypeptide having amino-terminal and / or carboxy-terminal deletions but having the same amino acid sequence as the corresponding position of a native sequence (e.g., from a full-length cDNA sequence) where the remaining amino acid sequence is deduced. Fragments are typically at least 5, 6, 8 or 10 amino acids in length, preferably at least 14 amino acids in length, more preferably at least 20 amino acids in length, usually at least 50 amino acids in length, and even more preferably at least 70 amino acids in length. As used herein, the term "analog" refers to a polypeptide consisting of a segment of at least 25 amino acids that is substantially identical to a portion of the deduced amino acid sequence and has specific binding to DR5 under appropriate binding conditions. Typically, polypeptide analogs contain conservative amino acid substitutions (or additions or deletions) relative to the naturally occurring sequence. Analogs are typically at least 20 amino acids in length, preferably at least 50 amino acids in length or more, and can often be the length of the full-length native polypeptide.

[0111] Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs having properties similar to those of the template peptide. These types of non-peptide compounds are referred to as "peptide mimetics" or "peptidomimetics". Fauchere, J. Adv.Drug Res.15:29(1986),Veber and Freidinger TINS p.392(1985);Evans et al.J.Med.Chem. 30:1229(1987). Such compounds are often developed using computerized molecular modeling. Peptidomimetics that are structurally similar to therapeutically useful peptides can be used to produce equivalent therapeutic or prophylactic effects. In general, peptidomimetics are structurally similar to paradigm polypeptides such as human antibodies (i.e., polypeptides having biochemical properties or pharmacological activities), but optionally, by methods well known in the art, the following: -CH 2 NH-, -CH 2 S-, -CH 2 -CH 2-, -CH=CH- (cis and trans), -COCH 2 -, CH(OH)CH 2 - and CH 2 It has one or more peptide bonds substituted by a bond selected from the group consisting of 3 SO-. One or more amino acids of the consensus sequence can be systematically substituted with D-amino acids of the same type (e.g., D-lysine instead of L-lysine) to produce a more stable peptide. Further, a constrained peptide containing the consensus sequence or a substantially identical consensus sequence variation can be produced by methods known in the art (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992)); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges that cyclize the peptide.

[0112] The term "agent" is used herein to denote a compound, a mixture of compounds, a biological macromolecule, and / or an extract made from a biological material.

[0113] As used herein, the terms "label" or "labeled" refer to the incorporation of a detectable marker, for example, by incorporation of a radiolabeled amino acid or by attachment of a biotin moiety to a polypeptide that can be detected by a prominent avidin (e.g., streptavidin containing a fluorescent marker or enzyme activity that can be detected by optical or calorimetric methods). In certain situations, the label or marker can also be therapeutic. Various methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of polypeptide labeling include, but are not limited to: radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131I), Fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors, etc.), enzyme labels (e.g., horseradish peroxidase, peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites of secondary antibodies, metal binding domains, epitope tags). In some embodiments, to reduce potential steric hindrance, the label is attached by spacer arms of various lengths. As used herein, the term "pharmaceutical or agent" refers to a compound or chemical composition that can induce a desired therapeutic effect when appropriately administered to a patient.

[0114] As used herein, the terms "treat", "treating", "treatment", etc. refer to reducing and / or ameliorating a disorder and / or associated symptoms. "Alleviate" and / or "alleviating" mean, for example, reducing, suppressing, attenuating, regressing, arresting and / or stabilizing the onset or progression of a disease such as cancer. Although not excluded, it will be understood that treatment of a disorder or condition does not require that the associated disorder, condition or symptoms be completely eliminated.

[0115] In the present disclosure, terms such as "comprises", "comprising", "containing", "having", etc. can have the meanings ascribed to them in the United States Patent Law and can mean "includes", "including", etc.; the term "consisting essentially of" or "consists essentially" similarly has the meaning ascribed to it in the United States Patent Law. It is obvious to those skilled in the art that these terms are unrestricted and allow for more than the recited elements, provided that the basic or novel features of the recited elements are not changed by the presence of more than the recited elements and do not exclude aspects of the prior art.

[0116] "Effective amount" means the amount necessary to improve the symptoms of a disease as compared to an untreated patient. The effective amount of the active compound(s) used to practice the present disclosure for the therapeutic treatment of a disease will vary depending on the method of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosing regimen. Such an amount is referred to as an "effective" amount.

[0117] "Subject" means a mammal including, but not limited to, a human or a non-human mammal such as a cow, horse, dog, rodent, sheep, primate, camelid, or cat.

[0118] As used herein, the term "administering" refers to any manner of transferring, delivering, introducing, or transporting a therapeutic agent to a subject in need of treatment with such an agent. Such manners include, but are not limited to, oral, topical, intravenous, intraperitoneal, intramuscular, intradermal, intranasal, and subcutaneous administration.

[0119] ​"Fragment" means a part of a polypeptide or nucleic acid molecule. This part preferably comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the full length of the reference nucleic acid molecule or polypeptide. Fragments can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0120] The ranges provided herein are to be understood as being in a simplified form that includes all values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0121] Unless otherwise specified or clear from the context, as used herein, the terms "a", "an" and "the" are understood to be singular or plural. As used herein, unless otherwise specified or clear from the context, the term "or" is understood to be inclusive.

[0122] As used herein, unless specifically stated otherwise or clear from the context, the term "about" is understood to be within the normal acceptable range in the art, for example within 2 standard deviations of the mean. "About" can be understood to be within about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term "about".

[0123] The present invention will be further described in the following examples, but does not limit the scope of the disclosure described in the claims.

Example

[0124] Example 1: Binding assay The binding of the DR5 target fusion protein was evaluated by flow cytometry using CHO cell lines stably transfected with cDNA encoding full-length DR5 or cDNA encoding a cancer cell line that endogenously expresses DR5. A titration series of the fusion protein was performed in a 96-well plate in FACS buffer (PBS 1% BSA, 0.1% NaN 3 pH 7.4) at 4°C for 30 minutes using DR5-expressing cell lines (approximately 2.5 - 5×10 4 cells / well). After washing three times in FACS buffer, an APC-conjugated anti-human Fcγ-specific secondary antibody (Jackson ImmunoResearch) was added and incubated at 4°C for 30 minutes. After three additional washing steps in FACS buffer, the bound antibody was detected via flow cytometry (IQue Intellicyte). The binding of the fusion protein to cynomolgus DR5 (cynoDR5) was determined by ELISA in which a recombinant protein (mFc) corresponding to the extracellular domain (ECD) of cynoDR5 fused to the murine Fc region was immobilized on a Medisorp 96-well plate (Nunc). After sufficient blocking and washing steps, the bound fusion protein was detected using an HRP-conjugated anti-human Fcγ-specific secondary antibody (Jackson ImmunoResearch), TMB reagent, and A 650nm absorbance readings at were used.

[0125] Example 2: Apoptosis assay Antibody-mediated direct killing of cells was determined by measuring the amount of ATP present after a 16 - 48-hour treatment period using CellTiter-Glo® (Promega G7572). Cancer cells were seeded at 7×10 in a 96-well flat-bottom tissue culture-treated plate.4 Cells / well were seeded at 1.5 - 3×10 4 cells / well. Another method for measuring cell death is to fluorescently stain the cells with the IncuCyte™ Caspase-3 / 7 apoptosis reagent (Essen BioScience 4440) during antibody treatment and quantify the fluorescent cells using the IncuCyte® ZOOM system, and in some embodiments the fusion protein comprises a polypeptide. Cell lines used include Colo-205 (ATCC® CCL-222™), Panc-1 (ATCC® CRL-1469™), HCT-116 (ATCC® CCL-247™), JL-1 (DSMZ ACC 596), NCI-H28 (ATCC® CRL-5820™), NCI-H460 (ATCC® HTB-177™), HT-29 (ATCC HTB-38™). MSTO-211H (ATCC® CRL-2081™). In some experiments, the DR5-targeted fusion protein of the present disclosure was cross-linked and further clustered using an anti-human IgG Fcγ-specific secondary (Jackson ImmunoResearch) antibody. In other experiments, cells were sensitized to DR5-mediated apoptosis using 6 μM doxorubicin.

[0126] Example 3: Existing autoantibodies recognizing sdAb Existing human anti-VH (HAVH) in human plasma or IVIG (purified IgG derived from pooled human plasma, trade name Gamunex®-C) was measured by ELISA. The test article (TAS266, a fusion protein or a therapeutic antibody) was coated on ELISA plates in PBS, the plates were blocked with 3% BSA in PBS, and then human plasma or IVIG (as a natural HAVH source) was dissolved in PBS + 0.1% polysorbate-20 (PBST) and bound to the plates. After washing the plates with PBST, the bound plasma antibody (HAVH) was detected with a secondary anti-light chain antibody conjugated to HRP (anti-human IgKappa or anti-IgLambda) and developed with TMB substrate. This strategy of detecting HAVH with a secondary anti-light chain antibody is compatible with TAS266 and test articles lacking light chains including the multivalent sdAbs described, and facilitates the detection of HAVH of any isotype. Therapeutic antibodies with kappa or lambda light chains were coated and used as 100% binding reference data points to normalize the data and as control IgG against the opposite secondary antibody.

[0127] Example 4: Hepatotoxicity assay Primary human hepatocytes or HepRG™ (Thermo Fisher Scientific), terminally differentiated hepatocytes derived from a hepatic progenitor cell line, were used to evaluate DR5 agonist-mediated apoptosis of hepatocytes. All assays were performed in the same manner as the apoptosis assay using cancer cell lines (Example 2). Human IgG pooled from multiple donors, IVIG (Gamunex®-C, Grifols), was used as a source of natural sdAb-directed autoantibodies, also called human anti-VH (HAVH) autoantibodies. In some experiments, IVIG was titrated or used at a constant concentration. In some assays, FIX-TAS266, a modified version of TAS266 designed to avoid recognition by HAVH autoantibodies, was included. FIX-2TAS66 contains modifications of Leu11 and the C-terminal region of each of the four DR5 sdAbs of TAS266.

Claims

1. An isolated polypeptide that binds to at least death receptor 5 (DR5) and comprises a plurality of DR5 binding domains (DR5BDs).

2. The isolated polypeptide of claim 1, wherein the isolated polypeptide is monospecific.

3. The isolated polypeptide of claim 1, wherein the isolated polypeptide is multispecific.

4. The isolated polypeptide of claim 1, wherein the isolated polypeptide is bispecific.

5. The isolated polypeptide of claim 1, wherein the polypeptide comprises at least a second binding domain (BD2) that binds to a second antigen.

6. The isolated polypeptide of claim 1, wherein the multiple DR5BDs bind to the same epitope on DR5.

7. The isolated polypeptide of claim 1, wherein at least two of the DR5BDs bind to different epitopes on DR5.

8. 8. The isolated polypeptide of any one of claims 1, 6 or 7, wherein the multiple DR5BDs comprise at least two DR5BDs.

9. 8. The isolated polypeptide of claim 1, 6 or 7, wherein the multiple DR5BDs comprise at least four DR5BDs.

10. 8. The isolated polypeptide of claim 1, 6 or 7, wherein the multiple DR5BDs comprise at least six DR5BDs.

11. 2. The isolated polypeptide of claim 1, wherein at least one DR5BD of the plurality of DR5BDs comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-91.

12. 2. The isolated polypeptide of claim 1, wherein each of the DR5BDs of the plurality of DR5BDs comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-91.

13. 2. The isolated polypeptide of claim 1, wherein each of the DR5BDs in the plurality of DR5BDs comprises the same amino acid sequence selected from the group consisting of SEQ ID NOs: 15-91.

14. At least one of the DR5BDs of the plurality of DR5BDs comprises a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 128, 134, 138, 141, 142, 159, 162, 163, 168, 173, 176, 178, 181, and 188; a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 169, 171, 172, 174, 177, 179, 182, 184, 185, and 189; and a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 130, 136, 140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190.

2. The isolated polypeptide of claim 1.

15. each of the DR5BDs of the plurality of DR5BDs comprises a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 128, 134, 138, 141, 142, 159, 162, 163, 168, 173, 176, 178, 181, and 188; 134, 135, 136, 137, 139, 140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190. The isolated polypeptide of claim 1, comprising a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 130, 136, 140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190.

16. each of the DR5BDs of the plurality of DR5BDs comprises a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 128, 134, 138, 141, 142, 159, 162, 163, 168, 173, 176, 178, 181, and 188; 77, 179, 182, 184, 185 and 189; and a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 130, 136, 140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190.

17. The isolated polypeptide of claim 1, wherein each of the DR5BDs of the plurality of DR5BDs is operably linked via a linker polypeptide.

18. The isolated polypeptide of claim 1 , wherein the isolated polypeptide comprises at least one binding domain that binds to a second target.

19. The isolated polypeptide of claim 1 , wherein the isolated polypeptide comprises an immunoglobulin Fc region polypeptide.

20. 20. The isolated polypeptide of claim 19, wherein the immunoglobulin is an IgG1 Fc region polypeptide, an IgG2 Fc region polypeptide, an IgG3 Fc region polypeptide, or an IgG1 Fc region polypeptide.

21. 20. The isolated polypeptide of claim 19, wherein the immunoglobulin Fc region polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5 or 127.

22. 22. The isolated polypeptide of any one of claims 1-21, wherein at least one DR5BD of the plurality of DR5BDs comprises an antibody or an antigen-binding fragment thereof.

23. 22. The isolated polypeptide of any one of claims 1-21, wherein each DR5BD in the plurality of DR5BDs comprises an antibody or antigen-binding fragment thereof.

24. The antibody or antigen-binding fragment thereof may be an scFv, Fab, single domain antibody (sdAb), V ＮＡＲ 24. The isolated polypeptide of claim 22 or claim 23, which is a VHH.

25. 24. The isolated polypeptide of claim 22 or 23, wherein the antibody or antigen-binding fragment is an sdAb.

26. 26. The isolated polypeptide of claim 25, wherein the sdAb is a human or humanized sdAb.

27. The sdAb is ＮＡＲ 26. The isolated polypeptide of claim 25, which is an engineered VH domain or an engineered VK domain.

28. 28. The isolated polypeptide of claim 27, wherein the sdAb is generated from a camelid heavy chain only antibody.

29. 28. The isolated polypeptide of claim 27, wherein the sdAb is generated from a cartilaginous heavy chain only antibody.

30. 30. The isolated polypeptide of any one of claims 1 to 29, wherein at least one of said DR5 binding domains comprises a non-antibody scaffold protein.

31. 31. The isolated polypeptide of claim 30, wherein the non-antibody scaffold protein is an ankyrin repeat protein, a DARPin, an avimer, an anticalin / lipocalin, a centrin or a finomer.

32. The isolated polypeptide of claim 1, wherein the polypeptide is tetravalent.

33. 33. The isolated polypeptide of claim 32, wherein the polypeptide comprises the structure: DR5BD-linker-DR5BD-linker-hinge-Fc, wherein the DR5BD is a humanized or fully human VHH sequence.

34. 34. The isolated polypeptide of claim 33, wherein the polypeptide comprises two or more copies of an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-91.

35. 34. The isolated polypeptide of claim 33, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 92-118.

36. the polypeptide comprises a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 128, 134, 138, 141, 142, 159, 162, 163, 168, 173, 176, 178, 181, and 188; 34. The isolated polypeptide of claim 33, comprising: a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 130, 136, 140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190.

37. The isolated polypeptide of claim 1, wherein the polypeptide is hexavalent.

38. 38. The isolated polypeptide of claim 37, wherein the polypeptide comprises the structure: DR5BD-linker-DR5BD-linker-DR5BD-linker-hinge-Fc, wherein the DR5BD is a humanized VHH sequence or a fully human VHH sequence.

39. 38. The isolated polypeptide of claim 37, wherein the polypeptide comprises three or more copies of an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-91.

40. 38. The isolated polypeptide of claim 37, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:119-124.

41. the polypeptide comprises a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 128, 134, 138, 141, 142, 159, 162, 163, 168, 173, 176, 178, 181, and 188; 38. The isolated polypeptide of claim 37, comprising a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 130, 136, 140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190.

42. a complementarity determining region 1 (CDR1) that binds at least death receptor 5 (DR5) and comprises two or more copies of an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-91 or an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 128, 134, 138, 141, 142, 159, 162, 163, 168, 173, 176, 178, 181, and 188; 67, 169, 171, 172, 174, 177, 179, 182, 184, 185 and 189; and a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 130, 136, 140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190.

43. An isolated polypeptide that binds to at least death receptor 5 (DR5) and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 92-124.

44. An isolated polypeptide that binds to at least death receptor 5 (DR5), and wherein the isolated polypeptide is not recognized by one or more human anti-VH autoantibodies (HAVH).

45. Use of a polypeptide according to any one of claims 1 to 44 for treating a neoplasm.

46. Use of a polypeptide according to any one of claims 1 to 44 for modulating immune cells to enhance tumor destruction.

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