TRI-SPECIFIC BONDING MOLECULES AND METHODS OF THEIR USE

Abstract

The present invention relates to Tri-Specific Binding Molecules, which are multi-chain polypeptide molecules possessing three binding domains and thus capable of mediating coordinated binding to three epitopes. The binding domains can be selected so that the Tri-Specific Binding Molecules are capable of binding to any of three different epitopes. The epitopes can be epitopes of the same antigen or epitopes of two or three different antigens. In a preferred embodiment, one epitope will be capable of binding to CD3, the second epitope will be capable of binding to CD8, and the third epitope will be capable of binding to an epitope of a Disease-Associated Antigen. The invention also provides a novel ROR1-binding antibody, as well as derivatives thereof and uses of such compositions.

Description

TRI-SPECIFIC BINDING MOLECULES AND METHODS OF USE THEREOF Cross Reference to Related Applications

[0001] This application claims priority from United States Patent Applications No. 62 / 008,229 (filed June 5, 2014; pending), 62 / 004,571 (filed May 29, 2014; pending), and 62 / 107,824 (filed January 26, 2015, pending), each of which applications is incorporated herein by reference in its entirety. Reference to the Sequence List:

[0003] This application includes one or more sequence listings in accordance with 37 C.F.R. 1,821 et seq., which are described on the computer-readable medium (file name: 1301_0114PCT_Sequence_Listing_ST25.txt, created on May 18, 2015, and having a size of 244,021 bytes), which file is incorporated herein for reference in its entirety. Background of the Invention: Field of invention:

[0002] The present invention relates to Tri-Specific Binding Molecules, which are multiL77b Ln / nznZ / Ε / ΥΙΛ polypeptide molecules L77b Ln / nznZ / Ε / ΥΙΛ chain possessing three Binding Domains and thus capable of mediating coordinate binding to three epitopes. The Binding Domains can be selected such that the Tri-Specific Binding Molecules are capable of binding to any of three different epitopes. Such epitopes may be epitopes from the same antigen or epitopes from two or three different antigens. In a preferred embodiment, one such epitope will be capable of binding CD3, the second such epitope will be capable of binding CD8, and the third such epitope will be capable of binding an epitope of a disease-associated antigen. The invention also provides a novel ROR1 binding antibody, as well as derivatives thereof and uses for such compositions. Description of Related Art: I. The Mammalian Immune System

[0003] The mammalian immune system serves as a defense against a variety of conditions, including, for example, injury, infection, and neoplasia. The efficiency with which humans and certain mammals mount an immune response to pathogens, foreign substances, and cancer antigens lies in two characteristics: the delicate specificity of the immune response for antigen recognition and immunological memory that allows for more rapid responses. and more vigorous in the re3 L77b Lívnznz / E / YIA activation with the same antigen (Portolés, P. et al. (2009) The TCR / CD3 Complex: Opening the Gate to Successful Vaccination, Current Pharmaceutical Design 15:3290-3300; Guy, C.S. et al. (2009) Organization of Proximal Signal Initiation at the TCR:CD3 Complex, Immunol Rev. 2 32(1).T—21).

[0004] The mammalian immune system is mediated by two separate but interrelated systems: the cellular and humoral immune systems. Generally speaking, the humoral system is mediated by soluble products (antibodies or immunoglobulins) that have the ability to combine with and neutralize products recognized by the system as being foreign to the body. In contrast, the cellular immune system involves the mobilization of certain cells, called T cells, which serve a variety of therapeutic functions. T cells are lymphocytes that are derived from the thymus and circulate between tissues, the lymphatic system, and the circulatory system. In response to the presence and recognition of foreign structures (antigens), T cells become activated to initiate an immune response. In many cases, these foreign antigens are expressed on host cells as a result of neoplasia or infection. Although T cells themselves do not secrete antibodies, they are usually required for the secretion of antibody for the second Lzzb Ln / nznz / E / YiA class of lymphocytes, B cells (derived from bone marrow). Critically, T cells exhibit extraordinary immunological specificity to be able to discern one antigen from another). Two types of T cells, helper T cells and cytotoxic T cells, are of particular relevance.

[0005] Helper T cells are characterized by their expression of the glycoprotein, CD4 (ie, they are CD4+). CD4 + T cells are the essential organizers of most mammalian immune and autoimmune responses (Dong, C. et al. (2003) Immune Regulation by Novel Costimulatory Molecules, Immunolog. Res. 28(1):39-48 ). Activation of CD4+ T cells has been found to be mediated through costimulatory interactions between an antigen complex: major histocompatibility molecule class II (MHC II) that is arranged on the surface of an antigen-presenting cell. (such as a B cell, macrophage, or dendritic cell) and a complex of two molecules, the T cell receptor (TCR) and a CD3 cell surface receptor ligand, which is arranged on the surface of a CD4 T cell +undifferentiated. Activated helper T cells are capable of proliferating into Thl cells that are capable of mediating an inflammatory response to the target cell.

[0006] Cytotoxic T cells are characterized by their Lzzb Ln / nznz / E / YiA expression of CD8 (ie they are CD8+ as well as CD3+). Activation of CD8+ T cells has been found to be mediated through costimulatory interactions between an antigen complex: major histocompatibility molecule class I (MHC I) that is disposed on the surface of a target cell and a complex of CD8 and the T cell receptor, which are arranged on the surface of the CD8+ T cell. Unlike MHC II molecules, which are expressed by only certain cells of the immune system, MHC I molecules are very widely expressed. In this way, cytotoxic T cells are capable of binding to a wide variety of cell types. Activated cytotoxic T cells mediate cell killing through their release of the cytotoxins perforin, granzymes, and granulysin. Through the action of perforin, the granzymes enter the cytoplasm of the target cell and their serine protease function activates the caspase cascade, which is a series of cysteine ​​proteases that eventually lead to apoptosis (programmed cell death) of cells. directed.

[0007] The T cell receptor (TCR) is a covalently linked heterodimer of α and β chains (ΤΟΗαβ). These chains are class I membrane polypeptides of 259 (a) and 296 (β) amino acids in length. The CD3 molecule is a T cell coreceptor composed of five chains of Lzzb Ln / nznz / E / YiA distinct polypeptides (one CD3 γ chain, one CD3 δ chain, two CD3 ε chains and two zeta chains). Individual polypeptide chains associate to form a complex of three dimers (εγ, εδ, ζζ) (Wucherpfennig, K.W. et al. (2010) Structural Biology Of The T Cell Receptor: Insights into Receptor Assembly, Llgand Recognition, And fnitiatlon of Signaling, Cold Spring Harb. Perspect. Biol. 2(4) :a005140; pages 1-14; Chetty, R. et al. (1994) CD3: Structure, Function And The Role Of Immunos tai ning Tn Clinical Practico, J. Pathol 173:303-307 Guy CS et al (2009) Organization of Próxima Signal Initiation at the TCR:CD3 Complex Immunol Rev. 232(1):7-21 Cali ME et al (2007) ) Common Themes In The Assembly And Architecture Of Activating Immune Receptors, Nat. Rev. Immunol. 7:841-850; Weiss, A. (1993) T Cell Antigen Receptor Signal Transduction: A Tale Of Tails And Cytoplasmic Protein-Tyrosine Finases, Cell 73:209-212). The CD3 complex associates with TCRs in order to generate an activation signal in T lymphocytes. In the absence of CD3, TCRs do not assemble properly and degrade (Thomas, S. et al. (2010) Molecular Tmmunology Lessons From Therapeutic T Cell Receptor Gene Transfer, Immunology 129(2):170-177). CD3 is found bound to the membranes of all mature T cells, and virtually no other cell types (see, Janeway, C.A. et al. (2005) In: IMMUNOBIOLOGY: THE IMMUNE SYSTEM IN HEALTH AND L77b Ln / nznZ / Ε / ΥΙΛ DISEASE, 6th ed. Garland Science Publishing, NY, pp. 214216; Sun, Z.J. et al. (2001) Mechanisms Contributing Το T Cell Receptor Signaling And Assembly Revealed By The Solution Structure Of An Ectodomain Fragment Of The CD3s:y Heterodimer, Cell 105(7):913-923; Kuhns, M.S. et al. (2006) Deconstructing The Form And Function Of The TCR / CD3 Complex, Immunity. 2006 Feb;24(2): 133-139).

[0008] The TCR and CD3 complex, together with the CD3 ζ zeta chain (also known as T cell receptor T3 zeta chain or CD247) comprise the TCR complex (van der Merwe, P.A. etc. (epub Dec. 3, 2010 ) Mechanisms For T Cell Receptor Triggering, Nat. Rev. Immunol. 11:47-55; Wucherpfennig, K.W. et al. (2010) Structural Biology of the T cell Receptor: Insights into Receptor Assembly, Ligand Recognition, and Initiation of Signaling, Cold Spring Harb. Perspect. Biol. 2:a005140). The complex is particularly significant, since it contains a large number (ten) of immunoreceptor tyrosine-based activation motifs (ITAMs).

[0009] Two interactions are required for T cell activation (Viglietta, V. et al. (2007) Modulating CoStimulation, Neurotherapeutics 4:666-675; Korman, A.J. et al. (2007) Checkpoint Blockade in Cancer Immunotherapy, Adv Immunol. 90:297-339). In the first interaction, a cell must follow the relevant target antigen bound to the cell's Major Histocompatibility Complex so L77b Ln / nznZ / Ε / ΥΙΛ that can bind to the T cell receptor (TCR) of a naive T cell. In the second interaction, a ligand from the cell must bind to a co-receptor of the T lymphocyte (Dong, C. et al. (2003) Immune Regulation by Novel Costimulatory Molecules, Immunolog. Res. 28(1) : 39-48 Lindley, P. S. et al (2009) The Clinical Utility Of Inhibiting CD28-Mediated Costimulation, Immunol. Rev. 229:307-321). T cells that experience both stimulatory signals are then capable of responding to cytokines (such as interleukin-2 and interleukin-12). In the absence of both costimulatory signals during TCR coupling, T cells enter a functionally unresponsive state, referred to as clonal anergy (Khawli, L.A. et al. (2008) Cytokine, Chemokine, and Co-Stimulatory Fusion Proteins for the Immunotherapy of Solid Tumors, Exper. Pharmacol. 181:291-328). In disease states, T cells are key players in several organ-specific autoimmune diseases, such as type I diabetes, rheumatoid arthritis, and multiple sclerosis (Dong, C. et al. (2003) Immune Regulation by Novel Costimulatory Molecules, Immunolog 28(1):39-48). AND

[0010] The need for two signals to activate T cells such that they elicit an adaptive immune response is believed to provide a mechanism for avoiding responses to self antigens that may be presented in a cell that Lzzb Ln / nznz / E / YiA presents antigen at locations in the system where it can be recognized by a T cell. Where contact of a T cell with a cell results in the generation of only one of two required signals, the T cell it does not become activated and the adaptive immune response does not occur. I. Antibodies and Other Epitope Binding Molecules A. Antibodies

[0011] Antibodies are immunoglobulin molecules capable of binding specifically to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the Variable Domain of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also mutants thereof, naturally occurring variants, fusion proteins comprising a portion of antibody with an antigen recognition site of the required specificity. , humanized antibodies, and chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity. Throughout this application, the numbering of amino acid residues of the light and heavy chains of the antibodies is in accordance with the EU index. Lzzb Ln / nznz / E / YiA as in Kabat et al. (1992) Sequences of Proteins of Immunological Interest, National Institutes of Health Publication No. 91-3242. As used herein, an antigen-binding fragment of an antibody is a portion of an antibody that possesses at least one antigen recognition site. As used herein, the term encompasses fragments (eg, Fab, Fab', F(ab')2Fv), and single-chain molecules (eg, scFv).

[0012] Natural antibodies (such as IgG antibodies) are composed of two light chains complexed with two heavy chains. Each light chain contains a Variable Domain (VL) and a Constant Domain (CL). Each heavy chain contains a Variable Domain (VH), three constant domains (CHI, CH2, and CH3), and a hinge domain located between the CHI and CH2 domains. The basic structural unit of naturally occurring immunoglobulins (eg, IgG) is, is thus a tetramer having two light chains and two heavy chains, usually expressed as a glycoprotein of approximately 150,000 Da. The amino-terminal (N) portion of each chain includes a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal (C) portion of each chain defines a constant region, with light chains having a Lívnznz / E / YIA only constant domain and heavy chains that usually have three constant domains and a hinge region. Thus, the structure of the light chains of an IgG molecule is n-VL-CL-c and the structure of the IgG heavy chains is n-VH-CH1-H-CH2-CH3-c (where H is the region hinge, and n and c represent, respectively, the N-terminus and the C-terminus of the polypeptide).

[0013] The ability of an intact, unmodified antibody (eg, an IgG antibody) to bind to an epitope of an antigen depends on the presence of Variable Domains in the immunoglobulin heavy and light chains (ie, the VL domain and VL domain). VH, respectively). The interaction of an antibody light chain and an antibody heavy chain and, in particular, the interaction of their VL and VH domains form one of the antibody epitope binding sites. The variable regions of the IgG molecule consist of complementarity determining regions (CDRs), which contain the residues in contact with the epitope, and non-CDR segments, referred to as framework segments (FRs), which generally maintain the structure. and determine the positioning of the CDR loops to allow such contact (although certain framework residues can also contact antigen). Thus, the VL and VH Domains have the structure n-FRlCDR1-FR2-CDR2-FR3-CDR3-FR4-c. Polypeptides that are (or Lzzb Ln / nznz / E / YiA can serve as) the first, second and third CDRs of an antibody light chain are designated herein as CDRlI domain, CDRl2 domain and CDRl3 domain respectively. Similarly, polypeptides that are (or can serve as) the first, second, and third CDRs of an antibody heavy chain are designated herein as "CDRhI domain," "CDRh2 domain," and "CDRh3 domain," respectively. Thus, the terms CDRL1 domain, CDRL2 domain, CDRL3 domain, CDRhI domain, CDRh2 domain, and CDRh3 domain are directed to polypeptides that when incorporated into a protein cause the protein to be capable of binding to a specific epitope without consider whether such a protein is an antibody having light and heavy chains or a diabody or a single chain linker (eg, a scFv, a BiTe, etc.), or is another type of protein. In contrast to such antibodies, the scFv construct comprises a VL and VH domain of an antibody contained in a single polypeptide chain where the domains are separated by a flexible linker of sufficient length to allow self-assembly of the two domains into one. functional epitope binding site. Where self-assembly of the VL and VH Domains is rendered impossible due to a linker of insufficient length (less than about 12 amino acid residues), two of the scFv constructs can interact with each other to form a bivalent molecule in the L77b Ln / nznZ / Ε / ΥΙΛ in which the VL of one chain associates with the VH of the other (reviewed in Marvin et al. (2005) Recombinant Approaches To IgG-Líke Bíspecíflc Antibodles, Acta Pharmacol. Sin. 26:649658) .

[0014] In addition to their known uses in diagnostics, antibodies have been shown to be useful as therapeutic agents. The last few decades have seen a revival of interest in the therapeutic potential of antibodies, and antibodies have become one of the leading classes of biotechnology-derived drugs (Chan, C.E. et al. (2009) The Use Of Antibodies In The Treatment Of Infectious Diseases, Singapore Med. J. 50(7) :663-666). Nearly 200 antibody-based drugs have been approved for use or are under development.

[0015] The term "monoclonal antibody" refers to a homogeneous antibody population wherein the monoclonal antibody is comprised of amino acids (naturally occurring and non-naturally occurring) that are involved in the selective binding of an antigen. Monoclonal antibodies are highly specific, being directed against a single epitope (or antigenic site). The term monoclonal antibody encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', Lzzfr Ln / nznz / E / YiA F(ab')2Fv), single chain (scFv), mutants thereof, fusion proteins comprising an antibody portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that it comprises an antigen recognition site of the required specificity and the ability to bind an antigen. It is not intended to be limited with respect to the source of the antibody or the manner in which it is made (eg, by hybridoma, phage selection, recombinant display, transgenic animals, etc.). The term includes complete immunoglobulins, as well as fragments etc. described above under the definition of antibody. Methods for making monoclonal antibodies are known in the art. One method that can be used is the method of Kohler, G. et al. (1975) Continuous Cultures Of Fused Cells Secreting Antibody Of Predefined Specificity, Nature 256:495-497, or a modification thereof. Typically, monoclonal antibodies are developed in mice, rats, or rabbits. Antibodies are produced by immunizing an animal with an immunogenic amount of cells, cell extracts, or protein preparations that contain the desired epitope. The immunogen can be, but is not limited to, primary cells, cultured cell lines, cancer cells, proteins, peptides, nucleic acids, or tissue. The cells Lzzb Ln / nznz / E / YiA used for immunization can be cultured for a period of time (eg, at least 24 hours) prior to use as an immunogen. The cells can be used as immunogens by themselves or in combination with a non-denaturing adjuvant, such as Ribi (see, for example, Jennings, V.M. (1995) Review of Selected Adjuvants Used in Antibody Production, ILAR J. 37(3) :119-125).

[0016] In general, cells should remain intact and preferably viable when used as immunogens. Intact cells may allow antigens to be detected better than cells disrupted by the immunized animal. The use of denaturing or harsh adjuvants, eg, Freud's adjuvant, can disrupt cells and is therefore discouraged. The immunogen can be administered multiple times at periodic intervals, such as twice weekly or weekly, or administered in such a manner as to maintain viability in the animal (eg, in a recombinant tissue). Alternatively, existing monoclonal antibodies and any other equivalent antibodies that are immunospecific for a desired pathogenic epitope can be sequenced and produced recombinantly by any means known in the art. In one embodiment, such an antibody is sequenced and the polynucleotide sequence is then cloned into a vector for expression or propagation. Lzzb Ln / nznz / E / YiA The sequence encoding the antibody of interest can be maintained in a vector in a host cell and the host cell can then be expanded and frozen for future use. The polynucleotide sequence of such antibodies can be used for genetic manipulation to generate a chimeric antibody, a humanized antibody, or a canine antibody, or to improve the affinity, or other characteristics of the antibody. The general principle in humanizing an antibody involves retaining the basic sequence of the antigen-binding portion of the antibody, while displacing the non-human remainder of the antibody with human antibody sequences. There are four general steps to humanizing a monoclonal antibody. These are: (1) determining the predicted nucleotide and amino acid sequence of the light and heavy variable domains of the starting antibody, (2) designing the humanized antibody or canine antibody, i.e., deciding which region of antibody framework to use during the humanization or canonization process, (3) the actual humanization and caninization methodologies / techniques, and (4) the transfection and expression of the humanized antibody. See, for example, US Patent Nos. 4,816,567; 5,807,715; 5,866,692; and 6,331,415.

[0017] The epitope binding domain of such antibodies may comprise either entire Variable Domains Lzzb Ln / nznz / E / YiA fused into Constant Domains or only the complementarity determining regions (CDRs) grafted onto the regions of appropriate structure in the Variable Domains. The antigen binding sites can be wild type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but the possibility of an immune response to the foreign variable region remains (LoBuglio, A.F. et al. (1989) Mouse / Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response, Proc Nati Acad Sci (U.S.A.) 86:4220-4224). Another method focuses not only on providing human-derived constant regions, but on modifying the variable regions, as well to reshape them as closely as possible to the human form. It is known that the variable regions of both heavy and light chains contain three complementarity determining regions (CDRs) that vary in response to the antigens in question and determine binding capacity, flanked by four framework regions (FRs) that are conserved. relatively in a given species and that putatively provide a scaffold for the CDRs. When preparing non-human antibodies to a particular antigen, the variable regions can be reshaped or humanized by grafting non-human antibody-derived CDRs onto the FRs present in the antigen. Lzzb Ln / nznz / E / YiA the human antibody that is modified. The application of this procedure to various antibodies has been reported by Sato, K. et al. (1993) Cancer Res 53:851-856. Riechmann, L. et al. (1988) Reshaping Human Antibodies for Therapy, Nature 332:323-327; Verhoeyen, M. et al. (1988) Reshaping Human Antibodies: Grafting An Antilysozyme Activity, Science 239:1534-1536; Kettleborough, C.A. et al. (1991) Human!zation Of A Mouse Monoclonal Antibody By CDR-Grafting: The Importance Of Framework Residues On Loop Conformation, Protein Engineering 4:773-3783; Maeda, H. et al. (1991) Construction Of Reshaped Human Antibodies With HIV Neutralizing Activity, Human Antibodies Hybridoma 2:124-134; Gorman, S.D. et al. (1991) Reshaping A Therapeutic CD4 Antibody, Proc. nati. Acad. Sci. (U.S.A.) 88:4181-4185; Tempest, PR et al. (1991) Reshaping A Human Monoclonal Antibody To Inhibit Human Respiratory Syncytial Virus Infection in vivo, Bio / Technology 9:266-271; Co, M.S. et al. (1991) Humanized Antibodies For Antiviral Therapy, Proc. nati. Acad. Sci. (U.S.A.) 88:2869-2873; Carter, P. et al. (1992) Humanization Of An Anti-pl85her2 Antibody For Human Cancer Therapy, Proc. nati. Acad. Sci. (U.S.A.) 89:4285-4289; and Co, M.S. et al. (1992) Chimeric And Humanized Antibodies With Specificity For The CD33 Antigen, J. Immunol. 148:1149-1154. In some modalities, humanized antibodies preserve all CDR sequences L77b Ln / nznZ / Ε / ΥΙΛ (eg, a humanized mouse antibody containing all six CDRs of mouse antibodies). In other embodiments, humanized antibodies have one or more CDRs (one, two, three, four, five, or six) that differ in sequence from the parent antibody. B. Bi-Specific Antibodies, Multi-Specific Diabodies, and DART™ Diabodies

[0018] Natural antibodies are capable of binding to only one epitope species (ie, they are monospecific), although they may be capable of binding multiple copies of that species (ie, they may display bi- or multi-valency). A wide variety of recombinant bi-specific antibody formats have been developed (see, for example, PCT Publication Nos. WO 2008 / 003116, WO 2009 / 132876, WO 2008 / 003103, WO 2007 / 146968, WO 2007 / 146968 , WO 2009 / 018386, WO 2012 / 009544, WO 2013 / 070565), most of which use peptide linkers to either fuse the core of antibodies (IgA, IgD, IgE, IgG or IgM) to an additional binding protein (eg, scFv, VL VH, etc.) to, or within, the antibody core, or to fuse multiple antibody portions, or to fuse (eg, two Fab or scFv fragments) to a heterodimerization promoting domain such as the CH2-CH3 Domain or alternative polypeptides (WO L77b Ln / nznZ / Ε / ΥΙΛ 2005 / 070966, WO 2006 / 107786A WO 2006 / 107617A, WO 2007 / 046893). Typically, such procedures involve compromises and exchanges. For example, PCT Publication Nos. WO 2013 / 174873, WO 2011 / 133886 and WO 2010 / 136172 describe the use of linkers that can cause problems in therapeutic settings, and teach a trispecific antibody in which the CL and CH1 domains are moved from their respective positions and the VL and VH Domains have been diversified (WO 2008 / 027236; WO 2010 / 108127) to allow them to bind more than one antigen. Thus, the molecules described in these documents trade binding specificity for the ability to bind additional antigen species. PCT Publication Nos. WO 2013 / 163427 and WO 2013 / 119903 describe modification of the CH2 Domain to contain a fusion protein adduct comprising a binding domain. The paper mentions that the CH2 Domain probably plays only a minimal role in mediating effector function. PCT Publication Nos. WO 2010 / 028797, WO2010028796 and WO 2010 / 028795 describe recombinant antibodies whose Fc Domains have been replaced with additional VL and VH Domains, to form trivalent binding molecules. PCT Publication Nos. WO 2003 / 025018 and WO2003012069 describe recombinant diabodies whose individual chains contain scFv domains. PCT Publication No. WO 2013 / 006544 describes Lzzb Ln / nznz / E / YiA multi-valent Fab molecules that are synthesized as a single polypeptide chain and then subjected to proteolysis to produce heterodimeric structures. Thus, the molecules described in these documents exchange all or some of the ability to mediate effector function for the ability to bind additional antigen species. PCT Publication Nos. WO 2014 / 022540, WO 2013 / 003652, WO 2012 / 162583, WO 2012 / 156430, WO 2011 / 086091, WO 2007 / 075270, WO 1998 / 002463, WO 1992 / 0225831 and WO390391 describe the addition of additional Binding Domains or functional groups to an antibody or antibody portion (for example, the addition of a diabody to the light chain of the antibody, or the addition of additional VL and VH Domains to the light and heavy chains). of the antibody, or the addition of a heterologous fusion protein or chaining of multiple Fab domains together). In this way, the molecules described in these documents exchange the native antibody structure for the ability to bind additional antigen species.

[0019] The art has further mentioned the ability to produce diabodies that differ from such natural antibodies in being capable of binding two or more different epitope species (i.e., displaying bi-specificity or multi-specificity in addition to bi-valency or multivalence) (see, for example, Holliger et al. (1993) Di abodies': Small Bivalent And Bispecific Antibody Fragments, Proc. nati. Acad. Sel. (U.S.A.) 90:6444-6448; US 2004 / 0058400 (Hollinger et al.); US 2004 / 0220388 (Mertens et al.); Alt et al. (1999) FEBS Lett. 454(1-2):90-94; Lu, D. et al. (2005) A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity, J. Biol. Chem. 280(20) : 19 6 65-19 672 ; WO 02 / 02781 (Mertens et al.); Olafsen, T. et al. (2004) Covalent Disulfide-Linked Anti-CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting Applications, Protein Eng Des Sel. 17(1):21-27,- Wu, A. et al. (2001) Multimerization Of A Chimeric Anti-CD20 Singlechain Fv-Fv Fusion Protein Is Mediated Through Variable Domain Exchange, Protein Engineering 14(2):1025-1033; Asano et al. (2004) A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion Of Human Fe Domain, Abstract 3P-683, J. Biochem. 76(8):992; Takemura, S. et al. (2000) Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, Protein Eng. 13(8):583-588; Baeuerle, P.A. et al. (2009) Bispecific TCell Engaging Antibodies For Cancer Therapy, Cancer Res. 69(12):4941-4944).

[0020] The design of a diabody is based on the structure of single-chain Variable Domain (scFv) fragments. Lzzb Ln / nznz / E / YiA Lzzb Ln / nznz / E / YiA Such molecules are made by linking heavy and / or light chain Variable Domains together via a short peptide linker. Bird et al. (1988) ( Single-Chain AntigenBinding Proteins, Science 242:423-426) describe an example of peptide binding that bridges approximately 3.5 nm between the carboxy terminus of one Variable Domain and the amino terminus of the other Variable Domain. Linkers of other sequences have been designed and used (Bird et al. (1988) Single-Chain Antigen-Binding Proteins, Science 242:423426). Linkers in turn can be modified for additional functions, such as drug binding or binding to solid supports. Single chain variants can be produced either recombinantly or synthetically. For the synthetic production of scFv, an automated synthesizer can be used. For recombinant production of scFv, a suitable plasmid containing polynucleotide encoding the scFv can be introduced into a suitable host cell, either eukaryotic, such as a yeast, plant, insect or mammalian cell, or prokaryotic, such as E. coli. . Polynucleotides encoding the scFv of interest can be made by routine manipulations, such as polynucleotide ligation. The resulting scFv can be isolated using standard protein purification techniques known in the art.

[0021] United States Patent No. 7,585,952 and Lzzb Ln / nznz / E / YiA United States Patent Publication No. 20100,173,978 relate to scFv molecules that are immunospecific for ErbB2. Bi-specific T cell couplers (BiTEs), a type of scFv molecule has been described (WO 05 / 061547; Baeuerle, P et al. (2008) BiTE: A New Class Of Antibodies That Recruit T Cells, Drugs of the Future 33: 137-147 (Bargou, et al. 2008) Tumor Regression in Cancer Patients by Very Low Doses of a T Cell-Engaging Antibody, Science 321: 974-977). Such molecules are composed of a polypeptide chain molecule having two antigen-binding domains, one of which immunospecifically binds to a CD3 epitope and the second of which immunospecifically binds to an antigen present on the surface of a target cell. .

[0022] The provision of non-monospecific diabodies provides a significant advantage: the ability to colligate and co-localize cells expressing different epitopes. Bivalent diabodies that have wide-range applications include therapy and immunodiagnosis. Bivalency allows great flexibility in diabody design and engineering in various applications, providing increased avidity to multimeric antigens, cross-linking of different antigens, and targeting of specific cell types that depend on the presence of both. target antigens. Due to its valence Lzzb Ln / nznz / E / YiA, low dissociation rates, and rapid evacuation from the circulation (for diabodies of small size, at or below ~50 kDa), diabody molecules known in the art have also shown particular use in the field of tumor imaging (Fitzgerald et al. (1997) Improved Tumor Targeting By Disulphide Stablized Diabodies Expressed In Pichia pastoris, Protein Eng. 10:1221). Of particular importance is the co-ligation of different cells, eg the cross-linking of cytotoxic T cells to tumor cells (Staerz et al. (1985) Hybrid Antibodles Can Target Sites For Attack By T Cells, Nature 314:628-631 , and Holliger et al (1996) Specific Killing Of Lymphoma Cells By Cytotoxic T-Cells Mediated By A Bispecific Diabody, Protein Eng. 9:299-305).

[0023] Diabody epitope binding domains can be targeted to a surface determinant of any immune effector cell such as CD3, CD16, CD32, CD64, etc., which are expressed on T lymphocytes, the Natural Killer (NK) cells. ) or other mononuclear cells. In many studies, diabody binding to effector cell determinants, for example, Fe gamma receptors (FcyR), has also been found to activate the effector cell (Holliger et al. (1996) Specific Killing Of Lymphoma Cells By Cytotoxic TCells Mediated By A Bispecific Diabody, Protein Eng 9:299305, Holliger et al (1999) Carcinoembryonic Antigen (CEA)26 Lívnznz / E / YIA Specific T-cell Activation In Colon Carcinoma Induced By Anti-CD3 x Anti-CEA Bispecific Diabodies And B7 x Anti-CEA Bispecific Fusion Proteins, Cancer Res. 59:2909-2916; WO 2006 / 113665; WO 2008 / 157379; WO 2010 / 080538; WO 2012 / 018687; WO 2012 / 162068). Normally, effector cell activation is triggered by the binding of an antigen-bound antibody to an effector cell via the Fc-FcyR interaction pathway; thus, in this regard, diabody molecules may display Ig-like functionality independent of whether they comprise an Fe Domain (for example, as tested in any effector function assay known in the art or exemplified herein (by example, the ADCC assay)). By cross-linking the tumor and effector cells, the diabody not only brings the effector cell into the vicinity of the tumor cell, but leads to effective tumor extermination (see, for example, Cao et al. (2003) Bispecific Antibody Conjugates In Therapeutics, Adv. Drug. Deliv. Rev. 55:171197).

[0024] For example, United States Patent No. 6,171,586, relates to the production of bispecific antibodies by proteolytically cleaving two antibodies to obtain their F(ab')2 fragments, by reducing such fragments under conditions to prevent the formation intermolecular disulfide bond, and then by mixing two Lzzb Ln / nznz / E / YiA fragments to generate the bi-specific antibody). United States Patent Nos. 6,551,592; 6,994,853 and 8,277,806 and PCT Publication Nos. WO 2012 / 156430, WO 2002 / 020039, WO 2000 / 018806 and WO 1998 / 003670 relate to the production of tri-specific antibodies capable of simultaneously binding T cells and other antigens to a tumor cell, and, via the Fe portion of the bispecific antibody, to the Fe receptor of cells bearing such a receptor. PCT Publication Nos. WO 2000 / 018806, WO 1998 / 003670 and WO 2006 / 072152 relate to the production of tri-specific antibodies capable of simultaneously binding T cells and other antigens. US Patent Publication No. 2008-0057054 describes bispecific conjugates, with specificity for a binding element against beta amyloid oligomers and a binding element against the transmembrane protein, telencephalin. United States Patent Publication No. 2010-0291112 relates to bi-specific and tri-specific single-chain Fv molecules that specifically bind to one (or two) tumor antigen(s) and one effector cell antigen. (such as CD3, CD16 CD32, CD64, etc.).

[0025] PCT Publication Nos. WO 1999 / 042597 and WO 1998 / 006749 describe antibody derivatives comprising Human Major Histocompatibility Complex binding domains, with or without bound MHC binding peptides. The L77b Ln / nznZ / Ε / ΥΙΛ PCT Publication No. WO 02 / 072141 relates to multi-specific binding molecules whose on rates (rates at which they bind to target molecules) and off rates (rates at which they release target molecules) differ to preferentially bind a target compared to its binding to the other of such a target molecule. Trispecific molecules, for example molecules that have a first monovalent portion that is an anti-CD3 or antiCD28 antibody, and a second portion that comprises a divalent immune function exerted by the portion that immunospecifically binds one or more target ligands or a diseased cell. target or immune cell.

[0026] United States Patent No. 7,695,936 and Patent Publication 2007 / 0196363 relate to bi-specific antibodies that are formed from the heavy chains of two antibodies, one of which has an engineered bulge on its heavy chain and the second of which possesses an engineered complementary pocket in its heavy chain. The presence of such complementary bumps and holes is believed to preferentially form bispecific heteroantibodies (bearing one heavy chain from each antibody of such class) relative to monospecific homoantibodies conferring two heavy chains from the same antibody. Various bispecific hetero-antibodies are proposed, including those that are immunospecific. L77b Lívnznz / E / YIA for CD3 and a tumor cell antigen. Several tri-specific hetero-antibodies are also proposed, including some that are immunospecific for CD3, CD8, and CD37 (a transmembrane protein expressed predominantly on B cells that is involved in the regulation of T-cell proliferation (Robak, T. et al (2014) Anti-CD37 Antibodies For Chronic Lymphocytic Leukemia, Expert Opin Biol Ther 14(5):651-661), however, no mechanism for its production and no description of its structure is provided.

[0027] PCT Publication WO2012-162561 relates to bispecific, tetravalent binding molecules comprising two polypeptides, each comprising two diabody structures, separated by an intervening CH2-CH3 Domain. The document also relates to tetravalent binding molecules composed of four polypeptide chains in which two of the polypeptide chains contain variable light and variable heavy domains for two antigens, and in which the other two polypeptide chains contain the domains variable heavy and variable light complementary to the antigens and a terminal CH2CH3 domain. Bispecific, tetravalent binding molecules are formed through the association of their respective CH2-CH3 domains. In all four polypeptide chain constructs, the light chains are not linked. Lzzb Ln / nznz / E / YiA covalently to the heavy chains, thus leading to instability (see, Lu, D. et al. (2005) A Fully Human Recombinant IgG-Ilke Bispecific Antibody To Both The Epidemial Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity, J. Biol. Chem. 280(20):19665-19672). The document describes a third construct in which the chains are altered to provide such covalent linkage, but at the cost of eliminating their bispecificity (ie the molecules are monospecific). Molecules that have specificity for CD2, CD3, CD4, CD8, CD161, a chemokine receptor, CD95, CCR5, etc., are described. A bispecific molecule capable of binding to both CD3 and CD8 is not described.

[0028] However, the above advantages come at an outgoing cost. The formation of such non-monospecific diabodies requires successful assembly of two or more distinct and different polypeptides (ie, such formation requires that the diabodies be formed through heterodimerization of different polypeptide chain species). This fact is in contrast to monospecific diabodies, which are formed through the homodimerization of identical polypeptide chains. Because at least two distinct polypeptides (ie, two polypeptide species) must be provided in order to form a non-monospecific diabody, and because the Lzzb Ln / nznz / E / YiA homodimerization of such polypeptides leads to inactive molecules (Takemura, S. et al. (2000) Construction Of A Díabody (Small Recombinant Bispecific Antibody) Using A Refolding System, Protein Eng. 13(8):583-588), the production of such polypeptides must be performed in such a way as to prevent covalent linkage between polypeptides of the same species (Takemura , S. et al (2000) Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, Protein Eng. 13(8):583-588). The art has therefore taught non-covalent association of such polypeptides (see, for example, Olafsen et al. (2004) Covalent Disulfide-Linked Anti-CEA Diabody Allows SíteSpecific Conjugation And Radiolabeling For Tumor Targeting Applications, Prot. Engr. Des Sel 17:21-27, Asano et al (2004) A Daybody For Cancer Immunotherapy And Its Functional Enhancement By Fusion Of Human Fe Domain, Abstract 3P-683, J Biochem 76(8):992, Takemura , S. et al (2000) Construction Of A Díabody (Small Recombinant Bispecific Antibody) Using A Refolding System, Protein Eng. Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity, J. Biol. Chem. 280(20):1966519672).

[0029] However, the art has recognized that Lzzfr Ln / nznz / E / YiA bispecific diabodies composed of non-covalently associated polypeptides are unstable and readily dissociate into non-functional monomers (see, for example, Lu, D. et al. (2005) A Fully Human Recombinant IgG- Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity, J. Biol. Chem. 280(20):1966519672).

[0030] In meeting this challenge, the art has succeeded in developing stable, covalently linked heterodimeric, non-monospecific diabodies termed DART™ (see, for example, US Patent Publication Nos. 2013-0295121 2010-0.174053 and 2009-0060910, European Patent Publications Nos. EP 2714079, EP 2601216, EP 2376109, EP 2158221 and PCT Publication Nos. WO 2012 / 162068, WO 2012 / 018687, WO 2012 / 018530 and WO 2012 / 08 / 20 and 2018. Moore, P.A. et al (2011) Application Of Dual Affinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing Of BCell Lymphoma, Blood 117(17):4542-4551 Veri, M.C et al (2010) Therapeutic Control Of B Cell Activation Via Recruitment Of Fcgamma Receptor Ilb (CD32B) Inhibitory Function With A Novel Bispecific Antibody Scaffold, Arthritis Rheum 62(7):1933-1943 Johnson, S et al (2010) Effector Cell Recruitment With Novel Fv-Based Dual-Affinity Re-Targeting Protein Leads To Potent Tumor Cytolysis And in L77b Ln / nznZ / Ε / ΥΙΛ live B-Cell Depletion, J. Mol. Biol. 399(3):436-449). Such diabodies comprise two or more covalently complexed polypeptides and involve the engineering of one or more cysteine ​​residues in each of the polypeptide species employed that allow disulfide bonds to form and thereby covalently link two polypeptide chains. . For example, the addition of a cisterna residue to the C-terminus of such constructs has been shown to allow disulfide bonding between polypeptide chains, stabilizing the resulting heterodimer without interfering with the binding characteristics of the bivalent molecule.

[0031] There are many forms of DART™. Each of the two polypeptides of the simpler DART™ embodiment comprises three domains (Figure 1A). The first polypeptide comprises: (i) a first domain comprising a binding region of a light chain Variable Domain of a first immunoglobulin (VL1), (ii) a second domain comprising a binding region of a light chain Variable Domain heavy weight of a second immunoglobulin (VH2), and (iii) a third domain containing a cysteine ​​residue (or a cysteine-containing domain) and a heterodimerization-promoting domain that serves to promote heterodimerization with the second polypeptide chain ( Figure IB). The cysteine ​​residue (or a domain that Lzzb Ln / nznz / E / YiA contains cisterna) of the third domain serves to promote covalent attachment of the first polypeptide chain to the second polypeptide chain of the diabody. The second polypeptide contains: (i) a first complementary domain (a VL2-containing domain), (ii) a second complementary domain (a VH1-containing domain), and (iii) a third domain containing a cisterna residue (or a cisterna-containing domain) and optionally, a complementary heterodimerization promoting domain that is complexed with the first chain polypeptide heterodimerization promoting domain in order to promote heterodimerization with the first polypeptide chain. The cysteine ​​residue (or a cysteine-containing domain) of the third domain of the second polypeptide chain serves to promote covalent attachment of the second polypeptide chain to the first polypeptide chain of the diabody. Such molecules are stable, potent, and have the ability to simultaneously bind two or more antigens. They are capable of promoting redirected T cell-mediated killing of cells expressing target antigens.

[0032] In one embodiment, the third domain of the first and second polypeptides each contain a cysteine ​​residue, which serves to join the polypeptides together via a disulfide bond. The third domain of one or both of the polypeptides may additionally possess L77b Ln / nznZ / Ε / ΥΙΛ the sequence of a CH2-CH3 Domain, such that complexation of the diabody polypeptides forms an Fe Domain that is capable of binding to the Fe receptor of cells (such as B lymphocytes, dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and mast cells) (Figures 2A-2B).

[0033] Many variations of such molecules have been described (see, for example, United States Patent Publication Nos. 2013-0295121; 2010-0174053 and 20090060910; European Patent Publication Nos. EP 2714079; EP 2601216; EP 2376109; EP 2158221 and PCT Publication Nos. WO 2012 / 162068; WO 2012 / 018687; WO 2010 / 080538). These Fe-bearing DARTs can comprise three polypeptide chains (eg, Figure 2B). The first polypeptide chain of such a diabody contains three domains: (i) a VL1-containing domain, (ii) a VH2-containing domain, and (iii) a domain containing a cysteine ​​residue (or a cysteine-containing domain) and a heterodimerization promoting domain, and (iv) a cysteine ​​residue (or a cysteine-containing domain and a CH2CH3 domain). The second polypeptide chain of such a DART™ contains: (i) a VL2-containing domain, (ii) a VH1-containing domain, and (iii) a domain containing a cysteine ​​residue (or a cysteine-containing domain) and a heterodimerization promoting domain that promotes heterodimerization with L77b Ln / nznZ / Ε / ΥΙΛ the first polypeptide chain. The cysteine ​​residue (or a cysteine-containing domain) of the third domain of the second polypeptide chain serves to promote covalent attachment of the second polypeptide chain to the first polypeptide chain of the diabody. The third polypeptide of such a DART™ comprises a cysteine ​​residue (or a cysteine-containing domain) and a CH2-CH3 domain. In this way, the first and second polypeptide chains of such a DART™ associate together to form a VL1 / VH1 binding site that is capable of binding to the epitope, as well as a VL2 / VH2 binding site that is capable of binding. bind to the second epitope. The first and second polypeptides are linked to each other via a disulfide bond involving cysteine ​​residues in their respective third domains. Notably, the first and third polypeptide chains are complexed with each other to form an Fe Domain that is stabilized via the disulfide bond pathway. Such diabodies have increased potency. Such Fe-bearing DART™ can have either of two orientations (Table 1): Lzzb Ln / nznz / E / YiA Table 1 First Orientation 3rd Chain NH2-CH2-CH3-COOH First Chain NH2-VL1-VH2-Cys-Heterodimer Promotion Domain-CH2-CH3-COOH 2nd Chain NH2-VL2-VH1-Cys-Heterodimer Promotion Domain- COOH Second Orientation 3rd Chain NH2-CH2-CH3-COOH 1st Chain NH2-CH2-CH3-VL1-VH2-Cys-Heterodimer Promotion Domain-COOH 2nd Chain NH2-VL2-VH1-Cys-Heterodimer Promotion Domain- COOH

[0034] Even more complex DART™ diabodies, called Ig-DART™ (Figures 3A-3B) and Fc-DART™ diabodies (Figure 3C) have been described (WO 2012 / 018687). Fc-DARTs™ have four polypeptide chains. The first and second polypeptide chains of such a diabody contain three Domains: (i) a VL1-containing Domain, (ii) a VH2-containing Domain and (iii) a domain containing a CH2-CH3 sequence. The second and fourth Fc-DART™ polypeptides contain: (i) a VL2-containing Domain, (ii) a VH1-containing Domain, and (iii) a domain that promotes heterodimerization and covalent bonding with the first Fc polypeptide chain. -DART's™. The third and fourth, and the first and second polypeptide chains can be the same or different in a way that allows for tetravalent binding that is either mono-specific, bi-specific, or tetra-specific. Such more complex DART™ molecules also possess Cysteine-containing Domains Lzzb Ln / nznz / E / YiA which function to form a covalently linked complex. Fc-DART™ diabodies contain CHI and CL Domains.

[0035] Alternative constructs are known in the art for applications where a tetravalent molecule is desirable but an Fe is not required including, but not limited to, tetravalent tandem antibodies, also referred to as TandAbs (see, for example, Publications US Patent Nos. 2005-0079170, 2007-0031436, 20100099853, 2011-020667 2013-0189263, European Patent Publication Nos. EP 1078004, EP 2371866, EP 2361936 and EP12935; WO 1999 / 057150, WO 2003 / 025018, and WO 2013 / 013700) which are formed by the homo-dimerization of two identical chains each possessing a VH1, VL2, VH2, and VL2 Domain. III. Re-Targeted Extermination

[0036] As discussed above, interactions between CD8, MHC I, and the T cell receptor lead to the activation of cytotoxic T cells and their ability to kill nearby cells. Bi-specific diabodies that bind CD3 and a tumor antigen can be used to co-localize cytotoxic CD8+ T cells to tumor cells, achieving retargeted killing of such cells (WO 2010 / 080538, WO 2012 / 018687, WO / L77b Ln / nznZ / Ε / ΥΙΛ 2012 / 162068, US 2010 / 0174053, US 2013 / 0295121).

[0037] However, efforts to treat cancer or infectious disease by co-localizing CD3+ T cells to the tumor locus or pathogen cells have not been entirely successful. Antibodies directing CD3 binding to both CD3+CD8+ cytotoxic T cells, CD3+CD4+ helper cells, leading to activation of both such cells. Cytokines produced by activated CD3+CD4+ T helper cells, however, contribute to exacerbating side effects, eg, life-threatening form of cytokines (Ferran, C. et al. (1990) Cytokine-Related Syndrome Following Injection Of Anti-CD3 Monoclonal Antibody: Further Evidence For Transient In Vivo T Cell Activation, Eur. J. Immunol. 20:509-515). Additionally, such anti-CD3 antibodies bind other cell types, including CD3+CD4~ CD8~ double negative T cells, etc., which express cytokines upon activation (Johansson, Martina et al. (2003) A Unique Population of Extrathymically Derived αβΤΟΡ+CD4~ CD8~ T Cells with Regulatory Functions Dominates the Mouse Female Genital Tract, J Immunol 170:1659-1666 Blank C et al (2003) Absence of Program! Death Receptor 1 Alters Thymic Development and Enhances Generation of CD4 / CD8 DoubleNegative TCR-Transgenic T Cells, J Immunol 171:4574-4581 Mclntyre MSF et al (2011) Consequences Of Double Lzzb Ln / nznz / E / YiA Negative Regulatory T Cell And Antigen Presenting Cell Interaction On Immune Responso Suppression, Intl. Immunopharmacol. 11:597-603), and that it suppresses cytotoxicity mediated by CD3+CD8+ T cells (Hillhouse, E.E. (2013) A Comprehensive Review Of The Phenotype And Function Of Antigen-Specific Immunoregulatory Double Negative T Cells, J. Autoimmun. 40 :58-65).

[0038] It has been proposed that the cytokine production associated with the administration of antibodies targeting CD3 could be circumvented by using bi-specific antibodies targeting CD8 and tumor antigen (Michalk, I. et al. (2014) Characterization of a Novel Single-Chain Bispecific Antibody for Retargeting of T Cells to Tumor Cells via the TCR CoReceptor CD8, PLOS One 9(4):e95517, pages 1-8). Anti-CD8 antibodies have therefore been studied to determine if they would be capable of inducing effector function when used alone. Clement, M. et al., reported that six of seven anti-human CD8 antibodies were proven to fail to activate CD8+ T cells, but that such activation could be achieved using very high concentrations (10100 pg / ml) of anti-CD8 antibody. -human OKT8 (Clement, M. et al. (2011) Anti-CD8 Antibodies Can Trigger CD8+T Cell Effector Function In The Absence Of TCR Engagement And Improve Peptide-MHCI Tetramer Staining, J. Immuno!. 187(2): 654-663). The cooperative bond to the two molecules Lzzb Ln / nznz / E / YiA CD8 was required for this effect, since OKT8 F(ab)'2 fragments were found to be able to mediate the effect, while OKT8 Fab were found to be unable to do this.

[0039] Thus, despite such studies, the cytokine-mediated toxicity proposed for the use of anti-CD4 or anti-CD8 antibodies has not been fully understood. Studies have revealed that the cytokine toxicity observed upon anti-CD3 antibody administration is not abolished by depleting CD3+CD4+ T cells or by suppressing CD3+CD8+ T cells. Thus, both CD3+CD4~ T cells and CD3+CD8+ T cells contribute to the toxic effects of anti-CD3 antibodies, and relatively few cells are required to mediate the full effect (Finck, B.K. et al. (1992) The Role Of TCell Subsets In The Response To Anti-CD3 Monoclonal Antibodies, Clin Immunol Immunopathol.1992 Dec;65(3):23441).

[0040] On the other hand, a bi-specific antibody that targets CD8 and a tumor antigen is not specific for CD3+CD8+ T cells and tumor cells, but rather is specific only for CD8+ cells and tumor cells. In particular, the CD3 ~ CD8 + subset of natural killer (NK) cells would be targeted by such an antibody. Such cells, which account for a majority of NK cells are L77b Ln / nznZ / Ε / ΥΙΛ are potent cytokine producers and their activation would likely contribute to a cytokine storm. CD3“ CD8+ NK cells are a primary source of IFN-γ in chimpanzees infected with HIV-1 (Rodríguez, A.R. et al. (2007) Influence Of Interleukin-15 On CD8+ Natural Killer Cells In Human Immunodeficlency Virus Type 1-Infected Chimpanzees, J. Gen. Virol. 88:641-651).

[0041] Consequently, despite all previous advances, a need remains for improved compositions capable of more vigorously directing the body's immune system to attack cancer cells or pathogen-infected cells, especially in lower therapeutic concentrations. As described in detail below, the present invention addresses this need by providing tri-specific binding molecules that bind to: (1) a CD3 epitope, (2) a CD8 epitope, and (3) a CD8 epitope. a disease-associated antigen that is expressed on a target cell (especially a cancer cell, or a cell infected by pathogens) and mediate the coordinated binding of cytotoxic T cells to cells that present the disease-associated antigen. Brief Description of the Invention:

[0042] The present invention relates to Tri-Specific Binding Molecules, which are multiLzzb Ln / nznz / E / YiA chain polypeptide molecules that possess three Binding Domains and are thus capable of mediating coordinate binding to three epitopes. The Binding Domains can be selected such that the Tri-Specific Binding Molecules are capable of binding to any of three different epitopes. Such epitopes may be epitopes from the same antigen or epitopes from two or three different antigens. The invention also provides a novel ROR1 binding antibody, as well as derivatives thereof and uses for such compositions.

[0043] The present invention particularly relates to the modality of such Tri-specific Binding Molecules in which the three epitopes are selected such that one or two of such epitopes are epitopes of a cell of the immune system, and especially, a cell of the cytotoxic lymphocyte (CTL) immune system, and in which the remaining epitope(s) are epitopes of a disease-associated antigen. Such particularly preferred Tri-Specific Binding Molecules are capable of localizing a cytotoxic lymphocyte cell to a cell expressing a disease-associated antigen, and thus facilitate killing of cells expressing the disease-associated antigen. The disease-associated antigen may be a cancer antigen, or it may be an antigen that is characteristic of a pathogen (eg, bacterial, fungal, viral, or protozoal) infection. Lzzb Ln / nznz / E / YiA More particularly, the invention relates to such Tri-specific Binding Molecules that are capable of mediating coordinate binding to: (1) a CD3 epitope, (2) a CD8 epitope, and (3) an epitope of an antigen. associated with the disease. By binding CD3 and CD8, and the disease-associated antigen, such molecules co-localize cytotoxic T cells to cells presenting the disease-associated antigen, leading to activation of such T cells and initiation of a response. Cytotoxic against cells expressing the antigen associated with disease.

[0044] In detail, the invention provides a Tri-specific binding molecule capable of immunospecific binding to three different epitopes, wherein the binding molecule comprises four different polypeptide chains covalently complexed together and comprises: (I) an Antigen I Binding Domain that is capable of immunospecifically binding to an Epitope I present on a first antigen, and an Antigen II Binding Domain that is capable of immunospecifically binding to an Epitope II present on a second antigen, wherein the Antigen I Binding Domain and the Antigen II Binding Domain are both Diabody Binding Domains; (II) a non-type Antigen Binding Domain L77b Lívnznz / E / YIA Diabody III that is capable of immunospecifically binding an Epitope III present on a third antigen; and (III) an Fe Domain that is formed by the association of two CH2-CH3 domains with each other, where the first, second and third antigens are the same antigen, or are independently the same or different from one of the other antigens. antigens

[0045] The invention particularly relates to the embodiment of such Tri-Specific Binding Molecule, wherein one of Epitope I, Epitope II or Epitope III is an epitope of a cellular receptor

[0046] The invention further relates to embodiments of such Tri-specific Binding Molecules, wherein one of Epitope I, Epitope II or Epitope III is an epitope of a disease-associated antigen (and especially, wherein the antigen associated with the disease is a cancer antigen that is displayed on the surface of a cancer cell, or is a pathogen antigen that is displayed on the surface of a pathogen or pathogen-infected cell).

[0047] The invention further relates to the modalities of such Tri-specific Binding Molecules, wherein the Fe Domain is capable of binding to an Fe receptor arranged on the surface of a cell

[0048] The invention especially relates to the Lívnznz / E / YIA embodiments of such Tri-specific Binding Molecules, wherein one of Epitope I, Epitope II or Epitope III is a CD3 epitope, a second of Epitope I, Epitope II or Epitope III is a CD8 epitope, and the third of Epitope I, Epitope II or Epitope III is an epitope of the antigen associated with the disease, and wherein the antigen I, II and III Binding Domains of the Trispecific Binding Molecules by coordinate binding of a T cell cytotoxic and a cell expressing the antigen associated with the disease. The invention particularly relates to embodiments of such Tri-Specific Binding Molecules, wherein CD3, CD8 are arranged on the surface of a T cell and wherein the disease-associated antigen is arranged on the surface of a cell. of cancer, pathogen or pathogen-infected cell, and where immunospecific binding is sufficient to co-localize CD3 and CD8, and the disease-associated antigen, thereby facilitating T-cell activation in the array CD8 against the disease-associated antigen array cell.

[0049] The invention additionally relates to the modalities of the Tri-specific Binding Molecules described above, wherein the non-diabody III binding domain comprises the Fab-type binding domain (VLiii / VHin) which is capable of binding immunospecifically to Lzzb Ln / nznz / E / YiA epitope III, wherein the molecule comprises: (A) A first polypeptide chain: (I) comprising, in the N-terminal to C-terminal direction: (1) an immunoglobulin light chain Variable Domain capable of binding to the first of three epitopes (VLi); (2) a heavy chain Variable Domain of an immunoglobulin capable of binding a second of the three epitopes (VHn); (3) (a) a first cisterna-containing domain; and a heterodimer promotion domain; or (b) a cysteine-containing heterodimer promotion domain; (5) a second domain containing cysteine; and (6) CH2 and CH3 domains of an IgG; or (II) comprising, in the N-terminal to C-terminal direction: (1) a first domain containing cysteine; (2) CH2 and CH3 domains of an IgG; (3) an immunoglobulin light chain Variable Domain capable of binding a first of three epitopes (VLi); (4) a heavy chain Variable Domain of an immunoglobulin capable of binding a second of the three L77b Ln / nznZ / Ε / ΥΙΛ epitopes (VHn) ; (5) (a) a second cysteine-containing domain; and a heterodimer promotion domain; or (b) a cysteine-containing heterodimer promotion domain; (B) a second polypeptide chain comprising, in the N-terminal to C-terminal direction: (1) an immunoglobulin light chain Variable Domain capable of binding the second of the three epitopes (VLn); (2) a heavy chain Variable Domain of an immunoglobulin capable of binding to the first of three epitopes (HIV); (3) (a) a first cysteine-containing domain; and a heterodimer promotion domain; or (b) a cysteine-containing heterodimer promotion domain; wherein the second polypeptide chain heterodimer promoting domain is complementary to the first polypeptide chain heterodimer promoting domain; (C) a third polypeptide chain comprising, in the N-terminal to C-terminal direction: (1) a heavy chain Variable Domain of an immunoglobulin capable of binding a third of the three Lzzb Ln / nznz / E / YiA epitopes (VHin); and (2) a CH1 Domain, a cysteine-containing hinge domain, and a CH2-CH3 Domain of an IgG; and (D) a fourth polypeptide chain comprising, in the N-terminal to C-terminal direction: (1) an immunoglobulin light chain Variable Domain capable of binding to the third of three epitopes (VLm); and (2) a cysteine-containing light chain constant domain (CL); where: (i) the VLi and VHi Domains associate to form a domain capable of binding the I epitope; (ii) the VLn and VHn Domains associate to form a domain capable of binding Epitope II; (iii) the VLm and VHm Domains associate to form a domain capable of binding Epitope III; (iv) the CH2-CH3 Domain of the first polypeptide chain and the CH2-CH3 Domain of the third polypeptide chain are associated to form an Fe Domain; (v) the first and second polypeptide chains are covalently linked to each other; (vi) the first and third polypeptide chains are covalently linked to each other; and (vii) the third and fourth polypeptide chains are L77b Ln / nznZ / Ε / ΥΙΛ are covalently linked to each other.

[0050] The invention additionally relates to the modalities of the Tri-specific Binding Molecules described above, where: (A) the Heterodimer Promotion Domain is an E-helix and the complementary Heterodimer Promotion Domain is a K-helix; or (B) the Heterodimer Promotion Domain is a K-helix and the complementary Heterodimer Promotion Domain is an E-helix. [00 51] The invention additionally relates to the modalities of the Tri-specific Binding Molecules described above, where: (A) The CH2-CH3 domains of the first and third polypeptide chains each have the sequence of SEQ ID NO: 6, such that the Fe Domain formed from their association exhibits normal FcyR-mediated effector function; or (B) the CH2-CH3 Domain of the first and third polypeptide chains comprise at least one amino acid substitution, relative to the sequence of SEQ ID NO: 6, such that the Fe Domain formed from their association exhibits functions altered FcyR-mediated effector.

[0052] The invention additionally relates to the modalities of the Tri-specific Binding Molecules described above, where the CH2-CH3 Domain of the Lzzb Ln / nznz / E / YiA first and third polypeptide chains differ from each other and have an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8.

[0053] The invention additionally relates to the modalities of the Tri-specific Binding Molecules described above, where: (A) Epitope I, Epitope II and Epitope III are, respectively, a CD3 epitope, a CD8 epitope and a Disease Associated Antigen epitope; (B) Epitope I, Epitope II and Epitope III are, respectively, a CD3 epitope, a Disease Associated Antigen epitope and a CD8 epitope; (C) Epitope I, Epitope II, and Epitope III are, respectively, a CD8 epitope, a CD3 epitope, and a Disease Associated Antigen epitope; (D) Epitope I, Epitope II and Epitope III are, respectively, a CD8 epitope, a Disease Associated Antigen epitope and a CD3 epitope; (E) Epitope I, Epitope II, and Epitope III are, respectively, a Disease Associated Antigen epitope, a CD3 epitope, and a CD8 epitope; or (F) Epitope I, Epitope II, and Epitope III are, respectively, a Disease Associated Antigen epitope, a CD8 epitope, and a CD3 epitope.

[0054] The invention additionally relates to the L77b Ln / nznZ / Ε / ΥΙΛ modalities of the Tri-specific Binding Molecules described above, where: (A) the CD3 epitope is a CD3 epitope recognized by the OKT3 antibody, M291, YTH12.5, CD3 mAb 1 or CD3 mAb 2; or (B) the CD8 epitope is a CD8 epitope recognized by the TRX2 or OKT8 antibody.

[0055] The invention further relates to a pharmaceutical composition comprising the Tri-Specific Binding Molecule described above and a pharmaceutically acceptable carrier, excipient or diluent.

[0056] The invention further relates to a method of treating cancer comprising administering an effective amount of the pharmaceutical composition described above to an individual in need thereof, wherein the Disease Associated Antigen is the cancer antigen.

[0057] The invention additionally relates to a method for treating a disease associated with the presence of a pathogen, which comprises administering an effective amount of the pharmaceutical composition of claim 15 to an individual in need thereof, wherein the Associated Antigen with Disease is the pathogen antigen.

[0058] The invention further relates to an anti-ROR1 antibody, or ROR1 binding fragment, wherein the antibody comprises: Lzzfr Ln / nznz / E / YiA (A) a light chain Variable Domain comprising a CDRlI having the sequence of SEQ ID NO: 117, a CDRl2 having the sequence of SEQ ID NO: 118, and a CDRl3 having the sequence of SEQ ID NO: 119; and (B) a heavy chain Variable Domain comprising a CDRhI having the sequence of SEQ ID NO: 120, a CDRh2 having the sequence of SEQ ID NO: 121, and a CDRh3 having the sequence of SEQ ID NO: 121. 122.

[0059] The invention further relates to embodiments of such an anti-ROR1 antibody or ROR1 binding fragment thereof, wherein the antibody has a light chain Variable Domain having the sequence of SEQ ID NO: 51. The invention further relates to embodiments of such anti-ROR1 antibodies or ROR1 binding fragments thereof, wherein the antibody has a heavy chain Variable Domain having the sequence of SEQ ID NO: 52, or both a heavy chain Variable Domain light chain having the sequence of SEQ ID NO: 51 as a heavy chain Variable Domain having the sequence of SEQ ID NO: 52.

[0060] The invention further relates to a BiTe diabody or single chain antibody comprising the ROR1 binding fragment of any such anti-ROR1 antibody claims.

[0061] The invention additionally relates to a Lzzb Ln / nznz / E / YiA pharmaceutical composition comprising any of the anti-ROR1 antibodies described above or ROR1 binding fragments thereof and a pharmaceutically acceptable carrier, excipient or diluent. The invention further relates to a method of treating cancer which comprises administering an effective amount of such a pharmaceutical composition to an individual in need thereof. Brief Description of the Figures:

[0062] Figures 1A-1B show the diagrammatic representation of the DART™ diabody Domains. Figure 1A shows a diagrammatic representation of the Domains of a basic DART™ diabody. Figure IB provides a schematic representation of a covalently linked diabody composed of two polypeptide chains, each having a Heterodimer Promotion Domain. VL and VH Domains that recognize the same epitope are shown using the same shading.

[0063] Figures 2A-2B provide a schematic representation of covalently linked diabodies composed of two polypeptide chains, each having a CH2 and CH3 Domain (Figure 2A) in which only one has a CH2 and CH3 Domain (Figure 2A). 2B) , such that the associated chains form a Domain of Faith that comprises all or part Lzzb Ln / nznz / E / YiA of a naturally occurring Faith Domain. VL and VH Domains that recognize the same epitope are shown using the same shading.

[0064] Figures 3A-3C provide schematic representations showing tetravalent diabodies composed of two pairs of polypeptide chains. The pairs are different, thus resulting in a bi-specific molecule that is bivalent with respect to each of two epitopes, in which one is an epitope of DR5 and the other is an epitope of a molecule present on the surface of an effector cell. One polypeptide of each pair possesses a CH2 and a CH3 Domain, such that the associated chains form an Fe Domain comprising all or part of a naturally occurring Fe Domain. VL and VH Domains that recognize the same epitope are shown using the same shading. Only one pair of epitopes (shown with the same shading) is capable of binding to DR5. Figure 3A shows an Ig diabody. Figure 3B shows an Ig diabody, containing the E-helix and K-helix heterodimer promotion domains. Figure 3C shows an Fc-DART™ diabody containing the antibody CH1 and CL domains. The notation VL1 and VH1 denote, respectively, the variable light chain domain and the variable heavy chain domain that bind to the first epitope. Similarly, the notation VL2 and VH2 denote, respectively, the domain Lzzb Ln / nznz / E / YiA variable light chain and the variable heavy chain domain and link the second epitope.

[0065] Figures 4A-4L provide a diagrammatic representation of the preferred Trispecific Binding Molecule Domains. Figures 4A and 4B, respectively, specifically illustrate Domains of preferred Tri-Specific Binding Molecules in which the Non-Diabody-Like Binding Domain of the Tri-Specific Binding Molecule is a Fab-Like Binding Domain or a Fab-Like Domain. T-Cell Receptor Binding. Figures 4C and 4D, respectively, schematically illustrate preferred Tri-specific Binding Molecule Domains having different Domain orientations in which the Non-Diabody Binding Domain is a T-Cell Receptor Binding Domain. Fab-like or a T Cell Receptor-Like Binding Domain. Figures 4E-4J depict similar molecules having three polypeptide chains. The molecule may possess hinge and CL domains (Figures 4E, 4H) or may contain an alternative peptide linker (Figure 4F, 41). Figures 4K-4L depict similar molecules having five polypeptide chains.

[0066] Figures 5A-5D show the ability of the Tri-Specific Binding Molecule B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1 to bind to A498 target cells (Figure 5A), and cells JIMT-1 target (Figure 5B), CD5+ / CD4-regulated PBCMs (Figure 5C), and CD5+ / CD4+-regulated PBMCs (Figure Lzzb Ln / nznz / E / YiA 5 D) .

[0067] Figures 6A-6C demonstrate the ability of the Tri-specific Binding Molecules of the present invention to mediate the redirected killing of target cells. Figure 6A shows the results of a cell lysis luciferase assay of JIMT-1 cells. Figure 6B shows the results of an LDH assay for cytotoxicity of JIMT-1 cells. Figure 6C shows the results of an LDH assay of cytotoxicity of A498 cells.

[0068] Figures 7A-7D demonstrate the ability of the Tri-Specific Binding Molecules of the present invention to mediate T cell activation in incubation with JIMT-1 cells (Figure 7A: CD4 / CD69 T cells; Figure 7B : CD4 / CD25 T cells; Figure 7C: CD8 / CD69 T cells; Figure 7D: CD8 / CD25 T cells).

[0069] Figures 8A-8D demonstrate the ability of the Tri-Specific Binding Molecules of the present invention to mediate T cell activation in incubation with A498 cells (Figure 8A: CD4 / CD69 T cells; Figure 8B: CD498 cells). CD4 / CD25 T cells; Figure 8C: CD8 / CD69 T cells; Figure 8D: CD8 / CD25 T cells).

[0070] Figures 9A-9B show the population of CD5+CD4+-regulated (Figure 9A) or CD5+CD4-regulated (Figure 9B) cells of human PMBC as a function of increased concentration of Livnznz / E TriL77b Binding Molecules / YIA specific B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1 or the Tri-Specific Binding Molecules B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 2. B7-H3 X CD3 DARTs™ (with and without the Fe Domain) were used as controls.

[0071] Figures 10A-10C show the effect of different CD8 Binding Domains on the cytotoxicity of a Tri-Specific Binding Molecule B7-H3 mAb 1 / CD3 / CD8 mAb 2.

[0072] Figures 11A-11C demonstrate the ability to modulate the binding of the Tri-specific Binding Molecules of the present invention by selecting the A site, B site or C site d for the CD3 Binding Domain. The Tri-Specific Binding Molecules employed were able to immunospecifically bind to the disease-associated antigen, B7-H3. Cytotoxicity is measured using a luciferase assay.

[0073] Figures 12A-12C demonstrate the effect of positional selection (Site A, Site B, or Site C) on cytotoxicity mediated by the Trispecific Binding Molecules of the present invention using an LDH assay.

[0074] Figures 13A-13E show the effect of position variation on cytotoxicity using a Tri-Specific Binding Molecule B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1, a Tri-Specific Binding Molecule Specify CD3 mAb 2 / CD8 mAb 1 / B7-H3 mAb 1 and a TriLzzb Ln / nznz / E / YiA Binding Molecule Specific B7-H3 mAb 1 / CD8 mAb 1 / CD3 mAb 2. A B7-H3 X CD3 DART?M with Fe Domain was used as a control.

[0075] Figures 14A-14B, show the placement of the CD3 Binding Domain at Site C, greatly decreased binding to both CD5+CD4+ cells (Figure 14A) and CD5+CD4 cells (Figure 14B).

[0076] Figures 15A-15B show the populations of human PMBC CD5+ CD4+ regulated (Figure 15A) or CD5+CD4~ regulated (Figure 15B) cells as a function of increased concentration of Trispecific Binding Molecules mAb 2 of 5T4 / CD3 mAb 2 / CD8 mAb 1 or Tri-Specific Binding Molecules 5T4 mAb 2 / CD3 mAb 2 / CD8 mAb 2. 5T4 X CD3 DARTs™ (with and without Faith Domain) were used as controls.

[0077] Figures 16A-16C show the observed effect of position variation on cytotoxicity that was not dependent on the CD8 Binding Domain employed.

[0078] Figures 17A-17C show the ability of the Tri-Specific Binding Molecules of the present invention to mediate the redirected killing of R0R1-expressing target cells.

[0079] Figures 18A-18C show the ability of Tri-Specific Binding Molecules HIV mAb 1 / CD3 mAb 2 / CD8 mAb 1 and HIV mAb 2 / CD3 mAb 2 / CD3 mAb 1 L77b Ln / nznZ / Ε / ΥΙΛ CD8 to bind immobilized, soluble gpl40 protein (Figure 18A), human CD3 (Figure 18B) and both human gpl40 and CD3 protein (Figure 18C).

[0080] Figures 19A-19C show the ability of Tri-Specific Binding Molecules HIV mAb 1 / CD3 mAb 2 / CD8 mAb 1 and HIV mAb 2 / CD3 mAb 2 / CD8 mAb 1 to bind to HEK293 / D375 cells expressing HIV in contrast to a control Tri-Specific Binding Molecule (Figure 19C).

[0081] Figures 20A-20B show the ability of Tri-Specific Binding Molecules HIV mAb 1 / CD3 mAb 2 / CD8 mAb 1 γ HIV mAb 2 / CD3 mAb 2 / CD8 mAb 1 to bind to show specific binding to the CD5+ / CD5- cell population of human PBMCs.

[0082] Figures 21A-21F show the cytotoxic activity mediated by the Tri-Specific Binding Molecule HIV mAb 1 / CD3 mAb 2 / CD8 mAb 1 or HIV mAb 2 / CD3 mAb 2 / CD8 mAb 1 in Jurkat cells in the presence or absence of tetracycline (Figures 21A-21B; Figures 21C-21D). Figures 21E-21F show the cytotoxic activity of a control anti-RSV antibody (Palivizumab; RSV mAb 1) of the Tri-Specific Binding Molecule.

[0083] Figures 22A-22B show the percentage of Jurkat 522 cells expressing HIV in vivo at one day and 2 days after incubation with the purified pan T cells and Lzzfr Ln / nznz / E / YiA Tri-Specific Binding Molecules HIV mAb 1 / CD3 mAb 2 / CD8 mAb 1 or HIV mAb 2 / CD3 mAb 2 / CD8 mAb 1.

[0084] Figures 23A-23C show the results of an estimate of CTL activity of HIV mAb 1 / CD3 mAb 2 / CD8 mAb 1 Trispecific Binding Molecules in 522 FY cells expressing HIV env using T cells. of CD4+, CD8+ or bread.

[0085] Figures 24A-24C show the results of an estimate of CTL activity of HIV mAb 2 / CD3 mAb 2 / CD8 mAb 1 TriSpecific Binding Molecules in Jurkat 522 FY cells expressing HIV env using T cells. CD4+, CD8+ or bread.

[0086] Figures 25A-25C show the binding kinetics for DART™ molecules having the CD3 mAb 2 Binding Domain (Figure 25A), and its CD3 mAb 2 low affinity (Figure 25B) and fast variants. of CD3 mAb 2 (Figure 25C).

[0087] Figures 26A-26B show the populations of human PMBC CD5+ CD4+ regulated (Figure 26A) or CD5+ CD4~ regulated (Figure 26B) cells as a function of increased concentration of TriSpecific Binding Molecule mAb 1 of 5T4 / CD3 mAb 2 / CD8 mAb 1, the 5T4 Tri-Specific Binding Molecule mAb 1 / CD3 Low mAb 2 / CD8 mAb 1, and the 5T4 Tri-Specific Binding Molecule mAb 1 / mAb 2 of Rapid CD3 / mAb 1 of CD8. 5T4 X CD3 Lzzfr Ln / nznz / E / YiA DARTs™ (with wild-type, CD3 Low and Fast specificities) were used as controls.

[0088] Figures 27A-27C show the effect of CD3 mAb variants (CD3 Low mAb 2 and CD3 Fast mAb 2) on the cytotoxicity of a TriSpecific Binding Molecule 5T4 mAb 1 / CD3 mAb 2 / mAb 1 of CD8 and using an LDH assay.

[0089] Figures 28A-28F demonstrate the level of IFN-γ (Figure 28A), TNF-α (Figure 28B), IL-10 (Figure 28C), IL-6 (Figure 28D), IL-4 (Figure 28E ), and IL-2 (Figure 28F) released from the PBMCs of donor 1 in the presence of increased concentrations of 5T4 mAb Trispecific Binding Molecules 1 / CD3 mAb 2 / CD8 mAb 1, Trispecific Binding Molecules mAb 5T4 mAb 1 / CD3 Low mAb 2 / CD8 mAb 1 and Tri-Specific Binding Molecules 5T4 mAb 1 / CD3 Fast mAb 2 / CD8 mAb 1.

[0090] Figures 29A-29F demonstrate the level of IFN-γ (Figure 29A), TNF-α (Figure 29B), IL-10 (Figure 29C), IL-6 (Figure 29D), IL-4 (Figure 29E ), and IL-2 (Figure 29F) released from PBMCs from donor 2 in the presence of increased concentrations of the 5T4 Tri-Specific Binding Molecule mAb 1 / CD3 mAb 2 / CD8 mAb 1, Tri-Specific Binding Molecule mAb 5T4 mAb 1 / CD3 Low mAb 2 / CD8 mAb 1 and Tri-Specific Binding Molecule 5T4 mAb 1 / CD3 Fast mAb 2 / CD8 mAb 1. L77b Ln / nznZ / Ε / ΥΙΛ Detailed description of the invention:

[0091] The present invention relates to Tri-Specific Binding Molecules, which are multi-chain polypeptide molecules that possess three Binding Domains and are thus capable of mediating coordinate binding to three epitopes. The Binding Domains can be selected such that the Tri-Specific Binding Molecules are capable of binding to any of three different epitopes. Such epitopes may be epitopes from the same antigen or epitopes from two or three different antigens. The invention also provides a novel ROR1 binding antibody, as well as derivatives thereof and uses for such compositions. I. General Techniques and General Definitions

[0092] The practice of the present invention shall employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. . Such techniques are fully explained in the literature, such as, molecular CLONING: A LABORATORY MANUAL, Third Edition (Sambrook et al. Eds., 2001) Cold Spring Harbor Press, Cold Spring Harbor, NY; OLIGONUCLEOTIDE SYNTHESIS: METHODS AND APPLICATIONS (Methods in Molecular Biology), Herdewijn, P., Lzzb Ln / nznz / E / YiA Ed., Humana Press, Totowa, NJ; Oligonucleotide Synthesis (Gait, M.J., Ed., 1984); Methods in Molecular Biology, Humana Press, Totowa, NJ; Cell Biology: A Laboratory Notebook (Cellis, J.E., Ed., 1998) Academic Press, New York, NY; Animal Cell Culture (Freshney, R.I., Ed., 1987); Introduction to Cell and Tissue Culture (Mather, J.P. and Roberts, P.E., Eds., 1998) Plenum Press, New York, NY; Cell and Tissue Culture: Laboratory Procedures (Doyle, A. et al., Eds., 19938) John Wiley and Sons, Hoboken, NJ; Methods in Enzymology (Academic Press, Inc.) New York, NY; Weir's Handbook of Experimental Immunology (Herzenberg, L.A. et al. Eds. 1997) Wiley-Blackwell Publishers, New York, NY; Gene Transfer Vectora for Mammalian Cells (Miller, J.M. et al. Eds., 1987) Cold Spring Harbor Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (Ausubel, F.M. et al., Eds., 1987) Greene Pub. Associates, New York, NY; PCR: The Polymerase Chain Reaction, (Mullis, K. et al., Eds., 1994) Birkhauser, Boston MA; Current Protocols in Immunology (Coligan, J.E. et al., eds., 1991) John Wiley and Sons, Hoboken, NJ; Short Protocols in Molecular Biology (John Wiley and Sons, 1999) Hoboken, NJ; Immunobiology 7 (Janeway, C.A. et al. 2007) Garland Science, London, UK; Antibodies (P. Finch, 1997) Stride Publications, Devoran, UK; Antibodies: A Practical Approach (D. Catty., ed., 1989) Oxford University Press, USA, New York NY) ; Monoclonal Antibodies: A Practical Lzzfr Ln / nznz / E / YiA Approach (Shepherd, P. et al. Eds., 2000) Oxford University Press, USA, New York NY; Using Antibodies: A Laboratory Manual (Harlow, E. et al. Eds., 1998) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; The Antibodies (Zanetti, M. et al. Eds. 1995) Harwood Academic Publishers, London, UK); and DeVita, Hellman, and Rosenberg's Cancer: Principles & Practice of Oncology, Eighth Edition, DeVita, V. et al. eds. 2008, Lippincott Williams & Wilkins, Philadelphia, PA. II. Preferred Tri-Specific Binding Molecules of the Present Invention A. Union Capabilities

[0093] Preferred Tri-Specific Binding Molecules of the present invention are capable of simultaneously and coordinately binding three different epitopes. Such preferred Tri-Specific Binding Molecules of the present invention comprise: (I) a Binding Domain I that is capable of immunospecifically binding Epitope I present on a first antigen, and a Binding Domain II that is capable of immunospecifically binding an Epitope II present on a second antigen, wherein the Binding Domain I and Binding Domain II are both Diabody Binding Domains; (II) a non-Type III Union Domain L77b Ln / nznZ / Ε / ΥΙΛ Diabody that is capable of binding immunospecifically to an Epitope III present on a third antigen; and (III) an Fe Domain that is formed by the complexation of two CH2-CH3 Domains with each other.

[0004] Typically, the Tri-Specific Binding Molecules of the present invention will comprise four different polypeptide chains, each having an amino terminus and a carboxyl terminus (see Figure 4A-4D, Figure 5A and Figure 5B), however , the molecules can comprise smaller or larger numbers of polypeptide chains by fusing such polypeptide chains together (eg, via a peptide bond) or by dividing such polypeptide chains to form additional polypeptide chains. Figures 4E-4J illustrate this aspect of the present invention by schematically depicting such molecules having three polypeptide chains. Figure 4K-4L illustrates this aspect of the present invention by schematically depicting molecules having five polypeptide chains.

[0094] Although such Tri-Specific Binding Molecules are particularly preferred, the invention additionally specifically contemplates Tri-Specific Binding Molecules comprising any combination of Binding Domains sufficient to produce a molecule having three binding specificities, of which two are Lívnznz / E / YIA binding specificities directed against cancer antigen, and one is a binding specificity directed against an antigen on the effector cell. Thus, for example, the invention contemplates: a Tri-Specific Binding Molecule comprising three Fab-Like Binding Domains, a Tri-Specific Binding Molecule comprising a bivalent, bi-specific antibody Domain (formed, for example, by by forming in complex two different light chains and two different heavy chains) and a Fav-Like Binding Domain, a Tri-Specific Binding Molecule comprising two bivalent, bi-specific, antibody Domains (formed, for example, by forming in complex four different light chains and two different heavy chains), but in which one of the antibody domains has become inactive, etc.

[0095] The terms polypeptide, polypeptide chain, and peptide are used interchangeably herein to refer to amino acid polymers of any length, but especially lengths greater than 3, 5, 10, 15, 20, or 25 amino acid residues, where which two, and more preferably all, amino acid residues are linked via an amide (peptide) bond (-NH-C(O)-). However, the polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms Lzzb Ln / nznz / E / YiA also encompass a polymer of amino acids that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a label component. Also included within the definition are, for example, polypeptides containing one or more analogues of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. The polypeptides of this invention can occur as single chains or as complex chains.

[0005] A Diabody-Like Binding Domain is the Epitope Binding Domain of a diabody, and especially, a DART® diabody. The terms diabody and DART® diabody are discussed above, and refer to a molecule comprising at least two polypeptide chains that are preferentially complexed together through covalent interaction to form at least two binding sites. epitope binding, which can recognize the same or different epitopes. Two of the polypeptide chains of a diabody or DART® diabody each comprise the immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region, but these regions do not interact to form an epitope binding site (ie L77b Ln / nznZ / Ε / ΥΙΛ that is, they are not mutually complementary) . Rather, the immunoglobulin heavy chain variable region of one (eg, the first) diabody, or DART® diabody, interacts with the immunoglobulin light chain variable region chains of a different diabody (eg, the first). b) or DART® diabody, the polypeptide chain to form an epitope binding site. Similarly, the immunoglobulin light chain variable region of one (eg, the first) diabody, or DART® diabody, of polypeptide chains interacts with the immunoglobulin heavy chain variable region of a different (eg, , the second) diabody, or DART® diabody, the polypeptide chain to form an epitope binding site. DART® diabody molecules are described in US Patent Publication Nos. 2013-0295121; 2010-0174053 and 20090060910; European Patent Publications EP 2714079; EP 2601216; EP 2376109; EP 2158221 and PCT Publication Nos. WO 2012 / 162068; WO 2012 / 018687; WO 2010 / 080538; WO 2006 / 113665, WO 2008 / 157379 and Moore, P.A. et al. (2011) Application Of Dual Affinity Retargeting Molecules To Achieve Optiinal Redirected T-Cell Killing Of B-Cell Lymphoma, Blood 117(17):4542-4551; Veri, M.C. et al. (2010) Therapeutic Control Of B Cell Activation Via Recruitment Of Fcgamma Receptor Ilb (CD32B) inhibitory Function With A Novel Bl-speclflc Antibody Scaffold, Arthritis Rheum. 62(7):19331943; and Johnson, S. et al. (2010) Effector Cell Recruitment With Novel Fv-Based Dual-Affinity Re-Targeting Protein Leads To Potent Tumor Cytolysis And in vivo B-Cell Depletion, J. Mol. Biol. 399(3):436-449.

[0096] Binding Domain III is preferably a Non-Diabody Binding Domain, which is intended to denote that Binding Domain III does not have the structure of a Diabody Binding Domain. Preferably, Binding Domain III is a non-Diabody Binding Domain that is a Fab Binding Domain or a Recipient Binding Domain. As used herein, the term Fab-Like Binding Domain refers to an epitope binding domain that is formed by the interaction of the VL Domain of an immunoglobulin light chain and a VH Complementation Domain of an immunoglobulin heavy chain. . Fab-Like Binding Domains differ from the Diabody-Like Binding Domain in that the two polypeptide chains that form a Fab-Like Binding Domain comprise only a single epitope Binding Domain, while the two polypeptide chains that form a Diabody-Like Binding Domain comprises at least two epitope Binding Domains. Thus, as used herein, the Fab-Like Binding Domains are distinct from the Diabody-Like Binding Domain. As used herein, Lzzb Ln / nznz / E / YiA the term "Receptor-Like Binding Domain" refers to an epitope binding domain of a cellular receptor that is formed by the interaction of two polypeptides. Receptor-Like Binding Domains are exemplified in for reference to a T-cell Receptor-Like Binding Domain, which is formed from the interaction of a Variable Domain of a T-cell receptor alpha chain and a Variable Domain of a T-cell Receptor-Like Binding Domain. T cell receptor beta chain. Such T cell Receptor Binding Domains recognize peptides displayed in the MHC context and thus are capable of recognizing intracellular epitopes. Although the invention is illustrated with respect to such Receptor-Like Binding Domains, it will be appreciated that Receptor-Like Binding Domains other than T cell Receptor-Like Binding Domains may be employed, and are encompassed by the present invention. Other examples of receptors having Receptor-Like Binding Domains include the IL-2 receptor, IL-4 receptor, IL-7 receptor, IL-9 receptor, IL-15 receptor, receptor of IL21, the insulin receptor, and thymic stromal lymphopoietin.

[0006] The Tri-Specific Binding Molecules of the present invention are thus distinguished from tetravalent binding molecules, such as those produced from the dimerization of a bivalent antibody, and preferably possess three rather than four Binding Domains. as discussed Lzzb Ln / nznz / E / YiA Lzzb Ln / nznz / E / YiA Next, the trispecific molecules of the present invention may possess additional Binding Domains (such as an Albumin Binding Domain, an FcR Binding Domain, etc.). Such additional Binding Domains are not intended to be considered or taken into account as being one of the three Binding Domains of the Tri-Specific Binding Molecules of the present invention.

[0097] As used herein, the terms association or associating with respect to polypeptides (eg, one diabody polypeptide to another, one immunoglobulin light chain to one immunoglobulin heavy chain, one CH2-CH3 Domain to another Domain CH2-CH3, etc.) is intended to denote a non-covalent combination of the polypeptides. The terms complex or complexation are intended to denote a covalent combination of the polypeptides.

[0098] As used herein, the Binding Domains of the Tri-specific Binding Molecules of the invention are said to mediate coordinate binding if at least two of their Binding Domains and preferably all of their Binding Domains , are capable of being concurrently bound to their respective recognized epitopes or binding ligand. Such union can be simultaneous. However, one aspect of the present invention refers to the modification of the activation and / or deactivation rates with which such Domains of Lzzb Ln / nznz / E / YiA Bind to their recognized epitopes. As used herein, the binding knock-out rate is a measure of the affinity with which such Binding Domains recognize and initiate binding to their recognized epitopes. In contrast, the binding knock-out rate is a measure of the degree of stability of the Binding Domain: epitope complex. The rates of activation and / or deactivation of binding can be modified by altering the amino acid sequence of the CDRs of a Binding Domain. As discussed below, independent of any CDR modifications, the degree of coordinate binding of the molecules of the present invention can be modulated by changing the configuration of their Binding Domains such that a particular Binding Domain (i.e., a Domain VHx / VLx) is present as Binding Domain III or as a Diabody Binding Domain internal or external to Binding Domain III (discussed in detail below).

[0099] The rates of activation and deactivation of the Binding Domains in the Tri-specific Binding Molecules of the present invention can be readily measured by methods well known in the art, for example by Biacore® assay (Jason-Moller Swanson, S.J. (2005) Characterization Of An Immune Responso, Dev. Biol. (Basel). 122:95-101; Buijs, J. et al. (2005) SPR-MS In Functional Proteomics, Brief Funct. Genomic Proteomic. 4(1) :39-47; Karlsson, R. et al. (2004) SPR For Molecular Interaction Analysis: A Review Of Emerging Application Areas, J. Mol. Recognize 17(3):151161; Van Regenmortel, M.H. (2003) Improving The Quality Of BIACORE-Based Affinity Measurements, Dev. Biol. (Basel) 112:141-151; Malmqvist, M. (1999) BIACORE: An Affinity Biosensor System For Characterization Of Biomolecular Interactions, Biochem. Soc. Trans. 27(2):335-340; Malmqvist, M. et al. (1997) Biomolecular Interaction Analysis: Affinity Biosensor Technologies For Functional Analysis Of Proteins, Curr. Opinion. Chem. Biol. 1(3):378-383; Fivash, M. et al. (1998) Biacore For Macromolecular Interaction, Curr. Opinion. Biotechnol. 9(1) :97-101; Malmborg, A.C. et al. (1995) Biacore As A Tool In Antibody Engineering, J. Immunol. Methods. 183(1):7-13). The rates of activation and inactivation of the Binding Domains of the Trispecific Binding Molecules of the present invention can be readily altered by random or site-directed mutagenesis of nucleic acid molecules encoding such Binding Domains, followed by routine sorting of the nucleic acid molecules recovered for their ability to encode mutated proteins that exhibit such altered binding kinetics.

[00100] The Binding Domains of the TriLzzb Ln / nznz / E / YiA Binding Molecules L77b Ln / nznZ / Ε / ΥΙΛ of the present invention bind epitopes in an immunospecific manner. As used herein, an antibody, diabody, or other epitope-binding molecule is said to immunospecifically bind a region of another molecule (i.e., an epitope) if it reacts or associates more frequently, more rapidly, with longer duration. and / or with greater affinity with that epitope in relation to alternative epitopes. For example, an antibody that immunospecifically binds to a viral epitope is an antibody that binds this viral epitope with greater affinity, avidity, more readily, and / or with greater duration than it immunospecifically binds other viral epitopes or non-viral epitopes. . It is also understood by reading this definition that, for example, an antibody (or moiety or epitope) that immunospecifically binds a first target may or may not specifically or preferentially bind a second target. As such, specific binding does not necessarily require (although it may include) exclusive binding. Generally, but not necessarily, the reference to join means specific join. Two molecules are said to be capable of binding to each other in a physiospecific manner, if such binding exhibits the specificity with which the receptors bind to their respective ligands.

[00101] In this way, in its simplest modality, the Lzzb Ln / nznz / E / YiA Preferred binding molecules of the present invention are at least tri-specific and are capable of mediating coordinate binding to three different epitopes. Significantly, such molecules have at least three sites that are capable of binding antigen: an outer Diabody-Like Binding Domain that is located additionally to Binding Domain III, an inner Diabody-Like Binding Domain that lies closer to the Domain III. of Union III and the Domain of Union III by itself. The positions of such Domains are designated, respectively, Site A, Site B, and Site C (Figures 4A-4D).

[00102] Binding Domains that bind to Epitopes I, II and III are selected to be different from each other. However, Epitopes I, II and III may be epitopes from the same antigen, from two different antigens, or from three different antigens. Thus, the Trispecific Binding Molecules of the present invention may be capable of coordinately binding 1, 2, 3, or different antigen molecules. The Tri-Specific Binding Molecules of the present invention can be used with respect to any possible epitope and any possible antigen. For example, the Tri-specific Binding Molecules of the present invention may have 1, 2, or 3 Binding Domains that bind an epitope from an effector cell (eg, CD2, CD3, CD16, CD19, CD20, CD22, CD32b , CD64, the B cell receptor Lzzb Ln / nznz / E / YiA (BCR), the T cell receptor (TCR), and the NKG2D receptor), or to an epitope on a cytotoxic T cell (eg, CD8 present on cytotoxic T cells), or to a epitope of a disease-associated antigen, or any combination of such potential Binding Domains.

[00103] As used herein a Disease Associated Antigen is an antigen that is characteristically expressed on a pathogen-infected cell or cancer cell, but characteristically not expressed on a normal cell.

[00104] As used herein, the term "pathogen-infected cell" refers to a cell that has been infected by a bacterium (eg, E. coli, C. difficile, Salmonella thyphimurium, Pseudomonas aeruginosa, Vibrio cholerae, Neisseria gonorrhoeae, Helicobacter pylori, Hemophilus influenzae, Shigella dysenteriae, Staphylococcus aureus, Mycobacterium tuberculosis and Streptococcus pneumonia, etc.), a fungus (for example, Candida, Aspergillus, Cryptococcus, Coccidioides, Histoplasma, Pneumocystis, Stachybotrys, etc.), a protozoan (Amoebozoa, Excavata, Chromalveolata, Entamoeba, Plasmodium, Giardia, Trypanosoma, Coccidia, Besnoitia, Dicrocoelium, Leishmania, etc.) or a virus (and especially an adenovirus, an adeno-associated virus, a B virus (macacine herpesvirus I), a virus BK, a bunyavirus, a chikungunya virus, a cocksackie virus, a L77b Ln / nznZ / Ε / ΥΙΛ coronavirus, cytomegalovirus, eastern equine encephalitis virus, ebola virus, enterovirus, Epstein-Barr virus, hantavirus, hepatitis A virus, hepatitis B virus, a hepatitis C virus, a hepatitis D virus, a hepatitis E virus, a herpes simplex virus 1, a herpes simplex virus 2, a human foamy virus, a human herpes virus 3, a human herpes virus 5 , a human herpes virus 6, a human herpes virus 7, a human immunodeficiency virus, a human papillomavirus, a human β-lymphotropic virus, a human T-cell leukemia virus I, a human T-cell leukemia virus II, an influenza virus, a JC virus, a JEV, a Kaposi sarcoma-associated herpesvirus, a Lassa virus, a lymphocytic choriomenengitis virus, a Marburg virus, a measles virus, a mumps virus, a Nipah virus, a norovirus, Norwalk virus, Orthoreovirus, parainfluenza virus, parvovirus, poliovirus rus, a rabies virus, a reovirus, a respiratory syncytial virus, rhinovirus, Rift Valley fever virus, a rotavirus, rubella virus, a smallpox virus, a St Louis encephalitis virus, a smallpox major virus, a smallpox minor virus, a varicella zoster virus, a West Nile virus, a Western equine encephalitis virus, or a yellow fever virus).

[00105] As used herein, the term "cancer cell" refers to a malignant cell of: an adrenal gland tumor, an AIDS-associated cancer, an alveolar soft part sarcoma, an astrocytic tumor, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, chromophobic renal cell carcinoma, cell carcinoma colon cancer, colorectal cancer, benign cutaneous fibrous histiocytoma, desmoplastic small round cell tumor, ependymoma, Ewing tumor, extraskeletal myxoid chondrosarcoma, ossium fibrogenesis imperfecta, fibrous dysplasia of bone, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, islet cell tumor, a Kaposi's sarcoma, a kidney cancer, a leukemia, a benign lipoma / lipomatous tumor, a malignant liposarcoma / lipomatous tumor, a liver cancer, a lymphoma, a lung cancer, a medulloblastoma, a melanoma, a meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumor, pediatric cancer, thyroid gland tumor Peripheral nerve, L77b Lívnznz / E / YIA Lzzfr Ln / nznz / E / YiA a pheochromocytoma, a pituitary tumor, a prostate cancer, a posterior uveal melanoma, a rare hematological disorder, a metastatic renal cancer, a rhabdoid tumor, a rhabdomysarcoma, a sarcoma, a skin cancer , soft tissue sarcoma, squamous cell cancer, stomach cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, metastatic thyroid cancer, or uterine cancer.

[00106] Examples of antigens that are characteristically expressed by cancer cells include a cancer antigen such as a breast cancer antigen, an ovarian cancer antigen, a prostate cancer antigen, a cervical cancer antigen, a pancreatic carcinoma antigen, a lung cancer antigen, a bladder cancer antigen, a colon cancer antigen, a testicular cancer antigen, a glioblastoma cancer antigen, an antigen associated with a B-cell malignancy, an antigen associated with multiple myeloma, an antigen associated with non-Hodgkin lymphoma, or an antigen associated with chronic lymphocytic leukemia. Exemplary antigens that are characteristically expressed by cancer cells include the antigens: colon cancer antigen 19.9; gastric cancer mucin antigen 4.2; colorectal carcinoma antigen A33 (Almqvist, Y. 2006, Nucí Med Biol Nov;33(8):991-998); ADAM-9 (Publication of Lívnznz / E / YIA United States Patent No. 2006 / 0172350; PCT Publication No. WO 06 / 084075; Oncofetal-alphafetoprotein AFP antigen (Malaguarnera, G. et al. (2010) Serum markers of hepatocellular carcinoma, Dig. Dis. Sci. 55(10) :27442755); ALCAM (PCT Publication No. WO 03 / 093443); BAGE (Bodey, B. 2002 Expert Opin Biol Ther. 2 (6): 577-84); betacatenin (Frange W. et al. 2003 J Pathol 201(2): .250-9); CA125 (Bast, RC Jr. et al. 2005 Int J Gynecol Cancer 15 Suppl 3:274-81); Carboxypeptidase M (US Patent Publication No. 2006 / 0166291); B1 B1 (Egloff, A.M. et al. 2006, Cancer Res. 66(1) :6-9); CD5 (Calin, G.A. et al. 2006 Semin Oncol. 33 (2): 167-73; CD19 (Troussard, X. et al. 1998 Hematol Cell Ther. 40 (4): 139-48); CD20 (Thomas, D.A. et al., 2006 Hematol Oncol Clin North Am. .1-9); CD22 (Kreitman, R.J. 2006 AAPS J. 18;8 (3) :E532-51); CD23 (Rosati, S. et al. 2 0 05 Curr Top Microbiol Immunol. 5; 2 94: 91 -107), CD25 (Troussard, X. et al. 1998 Hematol Cell Ther. 40(4):13948), CD27 (Bataille, R. 2006 Haematologica 91 (9): 1234-4 0), CD28 (Bataille, R 2006 Haematologica 91(9):1234-40) CD30 (Muta, H. et al. (2013) CD30: From Basic Research To Cancer Therapy, Immunol. Res. 57(1-3):151-158); CD33 (Walter, R.B. et al. (2012) Acute myeloid leukemia stem cells and CD33targeted immunotherapy, Blood 119(26) :6198-6208) ;CD36 (Ge, Y. 2005 Lab Hematol. 11(1) :31-7) ; CD40 / CD154 (Messmer, D. et al. 2005 Ann N Y Acad Sci. 1062:51-60); CD45 (Jurcic, J.G. 2005 Curr Oncol Rep. 7(5):339-46); CD56 (Bataille, R. 2006 Haematologic 91(9):1234-40); CD46 (US Patent No. 7,148,038; PCT Publication No. WO 03 / 032814; Russell, S. et al. (2004) CD46: A Complement Regulator And Pathogen Receptor That Mediate Links Between Innate And Acquired Immune Function, Tissue Antigens 64(2):111-118); CD52 (Hoelzer, D. et al. (2013) Targeted therapy with monoclonal antibodies in acute lymphoblastic leukemia, Curr. Opin. Oncol. 2 5(6):701-706),- CD79a / CD79b (Troussard, X. et al 1998 Hematol Cell Ther.40(4):139-48;Chu, P.G. et al.2001 Appl Immunohistochem Mol Morphol.9(2):97-106); CD103 Lzzb Ln / nznz / E / YiA (Troussard, X. et al. 1998 Hematol Cell Ther. 4 0(4):139-48),CD317 (Palma, G. et al. (2012) Plasmacytoids Dendritic Cells Are A Therapeutic Target In Anticancer Immunity, Biochim. Biophys. Acta. 1826(2):407-414; CDK4 (Lee, Y.M. et al. 2006 Cell Cycle 5(18):2110-4); CEA (carcinoembryonic antigen; Mathelin, C. 2006 Gynecol Obstet Fértil.34(7-8):638-46,Tellez-Avila, F.I. et al.2005 Rev Invest Clin.57(6):814-9),CEACAM5 and CEACAM6 (PCT Publication No. WO 2011 / 034660, Zheng, C. et al (2011) A Novel Anti~CEACAM5 Monoclonal Antibody, CC4, Suppresses Colorectal Tumor Growth and Enhances NK Cells-Mediated Tumor Immunity, PLoS One 6(6):e21146, pp. 1-11 );CO17-1A (Adkins, J.C. et al. (1998) Edrecolomab (Monoclonal Antibody 17-1A), Drugs 56(4):619626; CO-43 (Leb for blood group) and CO-514 (Lea for blood group) (Garratty, G. (1995) Blood Group Antlgens As Tumor Markers, Parasitlc / Bacterial / Vlral Receptors, And Their Association With Tmmunologlcally Tmportant Proteins, Immunol 24(1-2):213-232, CTLA-1 and CTLA-4 (Peggs, K.S. et al. 2006 Curr Opin Immunol. 18(2):206-13), Cytokeratin 8 (PCT Publication No. WO 03 / 024191), antigen DI.1 (Dao, T. et al. (2009) Identification Of A Human Cyclin Dl-Derived Peptide That Induces Human Cytotoxic CD4 T Cells, PLoS One. 4(8) :e6730); DR5 (Abdulghani, J. et al. (2010) TRAIL Receptor Signaling And Therapeutics, Expert Opin. ther. Targets 14(10):1091-1108; Andera, L. (2009) Signaling Activated By The Death Receptors Of The TNFR Family'', Biomed. Dad. Med. Fac. Univ. Palacky Olomouc Czech. Repub. 153(3):173-180; Carlo-Stella, C. et al. (2007) Targeting TRAIL Agonistic Receptors for Cancer Therapy, Clin, Cancer 13 (8):2313-2317; Chaudhari, B.R. et al. (2006) Following the TRAIL to Apoptosis, Immunologic Res. 35(3):249-262); The series (blood group B); EGF-R (epidermal growth factor receptor; Adenis, A. et al. 2003 Bull Cancer. 90 Spec No:S228-32); Ephrin receptors (and in particular EphA2 (US Patent No. 7, 569, 672; PCT Publication No. WO 06 / 084226); Erb (ErbBl; ErbB3; ErbB4; Zhou, H. et al. 2002 Oncogene L77b Ln / nznZ / Ε / ΥΙΛ L77b Ln / nznZ / Ε / ΥΙΛ (57) :87 32-4 0; Rimon, E. et al. 2004 Int J Oncol. 24(5):1325-38) ; F3 lung adenocarcinoma antigen (Greulich, H. et al. (2012) Functional analysis of receptor tyrosine kinase mutations in lung cancer identifies oncogenic extracellular domain mutations of ERBB2, Proc. Nati. Acad. Sci. (U.S.A.) 109 (36):14476-14481) ; FC10.2 antigen (Loveless, W. et al. (1990) Developmental Patterning Of The Carbohydrate Antigen FC10.2 During Early Embryogenes is In The Chick, Development 108(1):97-106); GAGE (GAGE-1; GAGE-2; Akcakanat, A. et al. 2006 Int J Cancer. 118(1):123-8); GD2 / GD3 / GD49 / GM2 / GM3 (Livingston, P.O. et al. 2005 Cancer Immunol Immunother. 54(10):1018-25); GICA 19-9 (Herlyn et al. (1982) Monoclonal Antibody Detection Of A Circulating Tumor Associated Antigen. I. Presence Of Antigen In Sera Of Patients With Colorectal, Gastric, And Pancreatic Carcinoma, J. Clin. Immunol. 2:135-140 ); gp37 (human leukemia T cell antigen ((Bhattacharya-Chatterjee et al. (1988) Idiotype Vaccines Against Human T Cell Leukemia. II. Generation And Characterization Of A Monoclonal Idiotype Cascade (Abl, Ab2, and Ab3), J. Immunol 141:1398-1403), gp75 (melanoma antigen) (Vijayasardahl et al. (1990) The Melanoma Antigen Gp75 Is The Human Homologue Of The Mouse B (Brown) Locus Gene Product, J. Exp. Med. 171(4 ):1375-1380); gplOO (Lotem, M. et al. 2006 J Immunother. 2 9 (6): 616-27); HER-2 / neu (Kumar, Pal S et al. 2006 Semin Oncol. 33(4) :386 91); human B lymphoma CD20 antigen (Reff et al. (1994) Depletion Of B Cells In Vivo By A Chinerío Mouse Human Monoclonal Antibody To CD20, Blood 83:435-445); human milk fat globule antigen; human papillomavirus E6 / human papillomavirus-E7 (DiMaio, D. et al. 2006 Adv Virus Res. 66:125-59; HMW-MAA (high molecular weight melanoma antigen) (Natali et al. (1987) Immunohistochemical Detection Of Antigen In Human Primary And Metastatic Melanomas By The Monoclonal Antibody 140.240 And Its Possible Prognostic Significance, Cancer 59:55-63; Mittelman et al. (1990) Active Specific Immunotherapy In Patients With Melanoma. A Clinical Trial With Mouse Antiidiotypic Monoclonal Antibodies Elicited With Syngeneic Anti-HighMolecular-Weight-Melanoma-Associated Antigen Monoclonal Antibodies, J. Clin. Invest. 86:2136-2144); Onco-Developmental Antigens, Nature 314:53-57) such as I(Ma) as found in gastric adenocarcinomas; Integrin Alpha-V-Beta-6 Integrinap6 (ITGB6) (PCT Publication No. WO 03 / 087340); JAM -3 (Publication PCT No. WO 06 / 084078); Interleukin-13 Receptor «2 (IL13Ra2) (Bodhinayake, I. et al. (2014) Targeting A Heterogeneous Tumor: The Promise Of The Interleukin-13 Receptor a2, Neurosurgery 75 (2):N18-9) ; JAM-3 Lzzfr Ln / nznz / Ε / γΐΛ (PCT Publication No. WO 06 / 084078); KID3 (PCT Publication No. WO 05 / 028498); KID3 (PCT Publication No. WO 05 / 028498); KID31 (PCT Publication No. WO 06 / 076584); KID31 (PCT Publication No. WO 06 / 076584); KS 1 / 4 pancarcinoma antigen (Perez et al. (1989) Isolation And Characterization Of A cDNA Encoding The Ksl / 4 Epithelial Carcinoma Marker, J. Immunol. 142:3662-3667; Móller et al. (1991) Bispecific-Monoclonal-Antibody-Directed Lysis Of Ovarian Carcinoma Cells By Activated Human T Lymphocytes, Cancer Immunol. Immunother. 33(4):210-216; Ragupathi, G. 2005 Cancer Treat Res. 123:157-80); pan-carcinoma KS 1 / 4 antigen (Perez et al. (1989) Isolation And Characterization Of A cDNA Encoding The Ksl / 4 Epithelial Carcinoma Marker, J. Immunol. 142:3662-3667; Móller et al. (1991) BispecificMonoclonal -Antibody-Directed Lysis Of Ovarian Carcinoma Cells By Activated Human T Lymphocytes, Cancer Immunol. Immunother. 33(4):210-216; Ragupathi, G. 2005 Cancer Treat Res. 123:157-80); KSA (17-1A) (Ragupathi, G. 2005 Cancer Treat Res. 123:157-80); human lung carcinoma antigens L6 and L20 (Hellstrom et al. (1986) Monoclonal Mouse Antibodies Raised Against Human Lung Carcinoma, Cancer Res. 46:3917-3923); LEA (Velázquez-Márquez, N. et al. (2012) Sialyl Lewis x expression in cervical scrapes of premalignant lesions, J. Biosci. 37 (6): 999-1004); LUCA-2 (United States Patent Publication No. Lzzb Ln / nznz / E / YiA 2006 / 0172349; PCT Publication No. WO 06 / 083852); Ml:22:25:8, M18, M39 (Cambien, L. et al. (2012) M19 Modulates Skeletal Muscle Differentiation And Insulin Secretion In Pancreatic B-Cells Through Modulation Of Respiratory Chain Activity, PLoS One 7(2) :e31815; Pui, C.H. et al. (1991) Characterization of childhood acute leukemia with multiple myeloid and lymphoid markers at diagnosis and at relapse, Blood 78 (5):1327-1337) ; MAGE (MAGE-1; MAGE-3; (Bodey, B. 2002 Expert Opin Biol Ther. 2 (6) : 577-84) ; MART (Kounalakis, N. et al. 2005 Curr Oncol Rep. 7 (5) : 377—82; Myl, MUC-1 (Mathelin, C. 2006 Gynecol Obstet Fertile. 34(7-8):63846); MUM-1 (Castelli, C. et al. 2000 J Cell Physiol. 182(3):323-31); N-acetylglucosaminyltransferase (Dennis, J.W. 1999 Biochim Biophys Acta. 6; 1473(1):21-34); neoglycoprotein (Legendre, H. et al. (2004) Prognostic Stratification Of Dukes B Colon Cancer By A Neoglycoprotein, Int. J. Oncol. 25(2):269-276); NS-10; OFA-1 and OFA-2 (Takahashi, M. (1984) A Study On Clinical Significance Of Oncofetal Antigen-1 In Gynecologic Tumors, Nihon Sanka Fujinka Gakkai Zasshi. 36 (12):2613-2618); Oncostatin M (Oncostatin Receptor Beta) (US Patent No. 7,572,896; PCT Publication No. WO 06 / 084092); pl5 (Gil, J. et al. 2006 Nat Rev Mol Cell Biol. 7(9):667-77); PSA (prostate specific antigen; Cracco, C.M. et al. 2005 Minerva Urol Nefrol. 57 (4): 301-11); PSMA (Ragupathi, G. 2005 Cancer Treat Res. 123:157-80); ΡΕΜΑ Lzzb Ln / nznz / E / YiA (polymorphic epithelial mucin antigen) (Chu, N.J. et al. (2015) Nonviral Oncogenic Antigens and the Inf lamina tory Signáis Driving Early Cancer Development as Targets for Cancer Immunoprevention, Clin. Cancer Res. 21 (7) :1549-1557) ; PIPA (US Patent No. 7,405,061; PCT Publication No. WO 04 / 043239); prosthetic acid phosphate (Tailor et al. (1990) Nucleotide Sequence Of Human Prostatic Acid Phosphatase Determined From A FullLength cDNA Clone, Nuci. Acids Res. 18(16):4928); R24 (Zhou, M. et al. (2008) Constitutive Overexpression Of A Novel 21 Kda Protein By Hodgkin Lymphoma And Aggressive Non-Hodgkin Lymphomas, Mol. Cancer 7:12); ROR1 (US Patent No. 5,843,749); Rabbani, H. et al. (2010) Expression Of ROR1 In Patients With Renal Cancer--A Potentlal Diagnostic Marker, Iran Biomed. J. 14(3) :77-82); sphingolipids (Hakomori, S. (1998) Cancer-Associated Glycosphingolipid Antigens: Their Structure, Organization, And Function, Acta Anat. (Basel) 161 (1-4) :79-90; SSEA-1, SSEA-3 γ SSEA- 4 (Muramatsu, T. et al. (2004) Carbohydrate Antigens Expressed On Stem Cells And Early Embryonic Cells, Glycoconj. J. 21(12) :41-45) ; sTn (Holmberg, L.A. 2001 Expert Opin Biol Ther. 1( 5) :881-91); T-cell receptor-derived peptide (Edelson (1998) Cutaneous T-Cell Lymphoma: A Model For Selective Immunotherapy, Cancer J Sci Am. 4:62-71); T5A7 (Hogg, R.J. et al (1991) A monoclonal antibody exhibiting Lzzb Ln / nznz / E / YiA reactivity with both X-hapten- and lactose-bearing glycolipids, Tissue Antigens 37(1):33-38); TAG-72 (Yokota et al. (1992) Rapid Tumor Penetration Of A Single-Chain Fv And Comparison With Other Immunoglobulin Forms, Cancer Res. 52:3402-3408); TL5 (blood group A) (Gooi, H.C. et al. (1983) Monoclonal antibody reactive with the human epidermal-growth-factor receptor recognizes the blood-group-A antigen, Biosci. Rep. 3(11) :1045-1052) ; TNF receptor (TNF-α receptor, TNF-β receptor; or TNF-γ receptor (van Horssen, R. et al. 2006 Oncologist. 11(4):397-408; Gardnerova, M. et al. 2000 Curr Drug Targets 1 (4) : 32 7-64) TRA-1-85 (blood group H) (Williams, B.P. et al. (1988) Biochemical and genetic analysis of the OKa blood group antigen, Immunogenetics 27 (5) :322 -329); Transferrin Receptor (US Patent No. 7,572,895; PCT Publication No. WO 05 / 121179); TSTA tumor-specific transplantation antigen (Hellstrom et al. (1985) Monoclonal Antibodies To Cell Surface Antigens Shared By Chemically Induced Mouse Bladder Carcinomas, Cancer. Res. 45:2210-2188), VEGF-R (O'Dwyer. P.J. 2006 Oncologist. 11(9):992-8), and hapten Y, LeY(Durrant, L.G. et al (1989) Development Of An ELISA To Detect Early Local Relapse Of Colorectal Cancer, Br. J. Cancer 60(4):533-537).

[00107] Exemplary antibodies that immunospecifically bind to an epitope of an associated antigen L77b Ln / nznZ / Ε / ΥΙΛ Lzzfr Ln / nznz / Ε / γΐΛ with the disease which can be used to provide the Variable Light Chain Domains, Variable Heavy Chain Domains, Antibody Light Chains or Antibody Heavy Chains of the specific Tri5 Binding Molecules of the present invention they are presented in Table 2. Table 2 Name of the Antibodies Antigen Associated with Diseases Application Therapeutic Objective 3F8 GD2 Neuroblastoma 8H9 B7-H3 Neuroblastoma, Sarcoma, Metastatic Brain Cancer Abagovomab CA-125 Ovarian Cancer abciximab CD41 Platelet aggregation inhibitor Actoxumab Clostridium Difficile Clostridium Difficile Infection TNF -Adalim A Rheumatoid Arthritis, Crohn's Disease, Plaque Psoriasis, Psoriatic Arthritis, Ankylosing Spondylitis, Juvenile Idiopathic Arthritis, Hemolytic Disease of the Newborn Adecatumumab EpCAM Prostate and breast cancer Aducanumab Beta-Amyloid Alzheimer's disease Afelimomab TNF-A Sepsis Afutuzumab CD2 0 Linforna Alacizumab VEGFR2 Cancer Ald518 11-6 Rheumatoid Arthritis Alemtuzumab CD52 Multiple Sclerosis Alirocumab NARP-1 Hypercholesterolemia Altumomab CEA Colon Cancer Amatuximab Mesothelin Cancer Anatumomab Mafenatox TAG-72 Non-Small Cell Lung Carcinoma Anifrolumab Delnterferon Receptor A / B Lupus Erythematosus Systemic Anrukinzumab IL-13 Cancer Apolizumab HLA-DR Hematological cancers Arcitumomab CEA Gastrointestinal cancer Aselizumab L-selectin (CD62L) Patients with severe injuries Table 2 Name of the Antibodies Antigen Associated with Diseases Application Therapeutic Objective Atinumab RTN4 Cancer Atlizumab IL-6 Receptor Rheumatoid Arthritis Atorolimumab Rhesus Factor Hemolytic disease of the newborn Bapineuzumab Beta-Amyloid Alzheimer's disease Basiliximab CD25 Prevention of organ transplant rejection Bavituximab Fos fatidilserine Cancer, Viral infections Bectumomab CD22 Non-Hodgkin lymphoma (Screening) Belimumab BAFF Non-Hodgkin lymphoma Benrali zumab CD125 Asthma Bertilimumab CCL11 (eotaxin-1) Severe allergic disorders Besilesomab CEA-related antigen Inflammatory lesions and metastases (screening) Bevacizumab VEGF-A Metastatic cancer, Retinopathy of Prematurity Bezlotoxumab Clostridium difficile Clostridium difficile infection Biciromab Fibrin II, Beta Chain Thromboembolism (Diagnosis) Bimagrumab ACVR2B Myostatin inhibitor Bivatuzumab CD44 V6 Squamous cell carcinoma Blinatumomab CD19 Cancer Blosozumab SOST O osteoporosis Brentuximab CD30 (TNFRSF8) Hematologic Cancers Briakinumab IL-12, IL-23 Psoriasis, Rheumatoid Arthritis, Inflammatory Bowel Diseases, Multiple Sclerosis Brodalumab IL-17 Inflammatory Diseases Canakinumab IL-1 Rheumatoid Arthritis Cantuzumab Mertansine CanAg Mucin Colon Cancer MUC1 Cantuzumab Caplacizumab VWF Cancers Capromab Cancers Prostate Carcinoma Cells Prostate Cancer (Detection) Carlumab MCP-1 Oncology / Immune Indications Catumaxomab EpCAM, CD3 Ovarian Cancer, Malignant Ascites, Gastric Cancer CC4 9 Tag-72 Tumor Detection Certolizumab TNF -A Crohn's disease Cetuximab EGFR Metastatic colorectal cancer and head and neck cancer Lzzb Ln / nznz / Ε / γΐΛΐ Table 2 Name of the Antibodies Antigen Associated with Diseases Application Therapeutic Target Ch.14.18 Indeterminate Neuroblastoma Citatuzumab EpCAM Ovarian cancer and other solid tumors Cixutumumab IGF-1 receptor Solid Tumors Clazakizumab Oryctolagus Cunic ulus Rheumatoid Arthritis Clivatuzumab Cancer MUC1 Pancreatic Cancer Conatumumab TRAIL-Rumab Concizumab TFPI Bleeding Crenezumab 1-4 O-B-amyloid Alzheimer's Disease Cr6261 Influenza A Hemagglutinin Infectious Diseases / Flu A Dacetuzumab CD40 Cancers OGIC Hematol Daclizumab CD25 Prevention of Organ Transplant Rejection Dalotuzumab Insulin-Like Growth Factor I Receptor Cancer Daratumumab CD38 Cancer Demcizumab DLL4 Cancer Denosumab RANKL Osteoporosis, bone metastases Detumomab B-cell lymphoma Lymphoma Dorlimomab aritox Indeterminate Cancer Drozitumab DR5 Cancer Duligotumab HER3 Cancer Dupilumab IL4 Atopic diseases Dusigitumab ILGF2 Cancer Ecromeximab Ganglioside GD3 Melanom a Malignant Eculizumab C5 Paroxysmal nocturnal hemoglobinuria Edobacomab Endotoxin Sepsis caused by Gram-negative bacteria Edrecolomab EpCAM Colorectal Carcinoma Efalizumab LFA-1 (CDlla) Psoriasis (Cell Migration T Blocks) Efungumab Hsp90 Invasive Candida Infection Eldelumab Interferon-gamma-induced Protein Disease Crohn's, Ulcerative Colitis Elotuzumab SLAMF7 Multiple Myeloma Elsilimomab IL-6 Cancer Enavatuzumab Receptor TWEAK Cancer Enlimomab ICAM-1 (CD54) Cancer Enokizumab IL9 Asthma Lzzb Ln / nznz / Ε / γΐΛΐ Table 2 Name of Antibodies Antigen Associated with Diseases Application Therapeutic Target Enoticumab DLL4 Cancer Ensituximab 5AC Cancer Epitumomab Cituxetan Episialin Cancer Epratuzumab CD22 Cancer, SLE Erli zumab ITGB2 (CD18) Heart attack, stroke, traumatic shock Ertumaxomab HER2 / Neu, CD3 Cancer Breast Etaraci zumab Integrin Av β 3 Melanoma, Prostate cancer, ovarian cancer Etrolizumab Integrin A B ,, Inflammatory disease owel B Evolocumab PCSK9 Hypocholesterolemia Exbivirumab Hepatitis B surface antigen Hepatitis B Fanolesomab CD15 Appendicitis (Diagnostic) Faralimomab Interferon Receptor Cancer Farletuzumab Folate Recipient 1 Ovarian Cancer Fasinumab 1511 HNGF Cancer Fbta05 CD2 0 Chronic Lymphocytic Leukemia Felvizumab Respiratory Syncytial Virus Respiratory Syncytial Virus Infection Fezakinumab IL-22 Rheumatoid Arthritis, Psoriasis Fielatuzumab HGF Cancer Figitumumab IGF-1 Receptor Adrenocortical Carcinoma, Carcinoma of p non-small cell lung Flanvotumab TYRP1 (Glycoprotein 75) Melanoma Fontoli zumab IFN-y Crohn's disease Foravirumab Rabies glycoprotein Virus Rabies (prophylaxis) Fresolimumab TGF-B Idiopathic pulmonary fibrosis, Focal and segmental glomerulosclerosis, Cancer Fulranumab NGF Pain Futuximab EGFR Cancer Galiximab CD80 B cell lymphoma Ganitumab IGF-I Cancer Gantenerumab Beta-Amyloid Alzheimer's disease Gavilimomab CD147 (Basigin) Graft-versus-host disease Gemtuzumab ozogamicin CD33 Acute myelogenous leukemia Gevokizumab IL-1β Diabetes Girentuximab Carbonic anhydrase 9 Renal cell carcinoma Table 2 Name of the Antibodies Antigen Associated with Therapeutic Target Application Diseases (CA-IX) Claras I64J Glembatumumab Vedotin GPNMB Melanoma, breast cancer Golimumab TNF-A Rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis Gomiliximab CD23 (Ige Receptor) Allergic asthma Guselkumab IL13 Psoriasis Ibritumomab tiuxetan CD2 0 Non-Hodgkin lymphoma Icrucumab VEGFR-1 Cancer Igovomab CA-125 Ovarian cancer (Diagnosis) Imab362 Cldnl8.2 Gastrointestinal Adenocarcinomas and pancreatic tumor Imgatuzumab EGFR Cancer Inclacumab Selectin P Cancer Indatuximab Ravtansine SDC1 Cancer Infliximab TNF - Rheumatoid Arthritis, Ankylosing Spondylitis, Psoriatic Arthritis, Psoriasis, Crohn's Disease, Ulcerative Colitis Intetumumab CD51 Solid tumors (Prostate Cancer, Melanoma) Inolimomab CD25 (IL-2 receptor chain) Graft-versus-host disease Inotuzuma b Ozogamycin CD22 Cancer Ipilimumab CD152 Melanoma Iratumumab CD30 (TNFRSF8) Hodgkin lymphoma Itolizumab CD6 Cancer Ixekizumab IL-17A Autoimmune Diseases Keliximab CD4 Chronic Asthma Labetuzumab CEA Colon Cancer Lambrolizumab PDCD1 Antineoplastic Agent Lampali zumab CFD Cancer Lebriki zumab IL-13 Asthma Lemalesomab NCA-90 (Granulocyte Antigen) Diagnostic Agent Lerdelimumab TGF Beta 2 Scar Reduction after glaucoma surgery Lexatumumab TRAIL-R2 Cancer Libivirumab Hepatitis B Hepatitis B antigen L77b Ln / nznZ / Ε / ΥΙΛΙ Table 2 Name of Antibodies Antigen Associated with Diseases Application Target Surface Therapeutics Ligelizumab IGHE Cancer Lintuzumab CD33 Cancer Lirilumab KIR2D Cancer Lodelci zumab PCSK9 Hypercholesterolemia Lorvotuzumab CD56 Cancer Lucatumumab CD40 Multiple myeloma, non-Hodgkin lymphoma, Hodgkin's lymphoma Lumiliximab CD23 Chronic lymphocytic leukemia IL R1 Cancer Margetuximab Ch4d5 Cancer Mavrilimumab GMCSF Receptor Chain Rheumatoid Arthritis Matuzumab EGFR Colorectal, Lung, and Stomach Cancer Mepolizumab IL-5 Asthma and White Blood Cell Diseases Metelimumab TGF Beta 1 Systemic Scleroderma Milatuzumab CD74 Multiple Myeloma and Other Hematologic Malignancies Minretumomab TAG-722 Cancer Mitumomab Ganglioside GD3 Small Cell Lung Carcinoma Mogamulizumab CCR4 Cancer Morolimumab Rhesus Factor Cancer Motavizumab Respiratory Syncytial Virus Respiratory Syncytial Virus (Prevention) Moxetumomab Pasudotox CD22 Cancer Muromonab-CD3 CD3 Pr Prevention of Organ Transplant Rejection Nacolomab Tafenatox C242 Colon Cancer Antigen Namilumab CSF2 Cancer Naptumomab Stafenatox 5T4 Non-Small Cell Lung Carcinoma, Renal Cell Carcinoma Narnatumab RON Cancer Nataliz umab Integrin A4 Multiple Sclerosis, Crohn's Disease Nebacumab Endotoxin Sepsis Necitumumab EGFR Non-Small Cell Lung Carcinoma Nerelimomab TNF-A Cancer Nesvacumab Angiopoietin 2 Cancer Nimotuzumab EGFR Squamous Cell Carcinoma, Table 2 Name of Antibodies Antigen Associated with Diseases Application Therapeutic Objective Head and Neck Cancer, Nasopharyngeal Cancer, Glioma Nivolumab IgG4 Cancer Nofetumomab Merpentan Indeterminate Cancer Ocaratuzumab CD2 0 Cancer Ocrelizumab CD2 0 Rheumatoid Arthritis, Systemic Lupus Erythematosus Odulimomab LFA-1 (CDlla) Prevention of Organ Transplant Rejections, Immunological Diseases Ofatumumab CD2 0 Chronic Lymphocytic Leukemia Olaratumab PDGF-R A Cancer Olokizumab IL6 Cancer Onartuzumab Scatter Human Factor Receptor Kinase Cancer Ontuxizumab TEMI Cancer Oportuzumab Monatox EpCAM Cancer Oregovomab CA-125 Ovarian Cancer Orticumab LDLox Cancer O3tuler7 Cancer Oxelumab OX-4 0 Asthma Ozanezumab Nogo-A ALS and multiple sclerosis Ozoralizumab TNF-A Inflammation Pagibaximab Lipoteichoic acid Sepsis (Staphylococcus) Palivizumab F protein respiratory syncytial virus Respiratory Syncytial Virus (Prevention) Panitumumab EGFR Colon Cancer Pankomab Tumor specific glycosylation of MUGI Ovarian Cancer Panobacumab Pseudomonas Aeruginosa Pseudomonas Aeruginosa infection Parsatuzumab EGFL7 Cancer Pascolizumab IL-4 Asthma Patelizumab LTA TNF Patritumab HER3 Cancer Pemtumomab MUC1 Cancer Peraki zumab IL17A Arthritis Cancer Pertuzumab HER2 / Neu of Pexeli zumab C5 Reduction of side effects cardiac surgery Pidilizumab PD-1 Cancer and Infectious Diseases Table 2 Name of Antibodies Antigen Associated with Diseases Application Therapeutic Target Pinatuzumab Vedotin CD22 Cancer Pintumomab Adenocarcinoma Antigen Adenocarcinoma Placulumab Human TNF Cancer Polatuzumab Vedotin CD79b Cancer Human Ponezumab Beta-Amyloid Alzheimer's Disease Pritoxaximab E. Coli Shiga Type-1 Toxin Cancer Pritumumab Vimentin Brain Cancer Pro 140 CCR5 HIV infection Quilizumab IGHE Cancer Racotumomab N-glycolylneuraminic Acid Cancer Radretumab Fibronectin extra Domain-B Cancer Ra f i vi rumab Rabies glycoprotein Virus Rabies (prophylaxis) Ramucirumab VEGFR2 Solid Tumors Ranibizumab VEGF-A Macular Degeneration (wet form) ) Raxibacumab Anthrax toxin, protective antigen Anthrax (Prophylaxis and Treatment) Regavirumab Cytomegalovirus glycoprotein B Cytomegalovirus infection Reslizumab IL-5 Inflammations of the respiratory tract, Skin and Gastrointestinal Diseases Rilotumumab HGF Solid Tumors Rituximab CD2 0 Linforn As, leukemia, some autoimmune disorders roatumumab igf-1 receiver Cancer ROLEDUMAB RHD CANCER ROMOSOZUMAB SCLOSTinstin Osteoporosis RONTALI ZUMAB IFN-A LUPUS ERITEMATOS Systemic ROVELIZUMAB CD11, CD18 RUPLIZUMAB CD154 Satumomab pendetide TAG-72 Cancer Secukinumab IL -17A Uveitis, Rheumatoid Arthritis Psoriasis Seribantumab ERBB3 Cancer Table 2 Name of Antibodies Antigen Associated with Diseases Application Therapeutic Target Setoxaximab E. Coli Shiga Type-1 Toxin Cancer Sevirumab Cytomegalovirus Cytomegalovirus Infection Sibrotuzumab FAP Cancer 19a CD Sgn- CD19 B-Cell Acute Lymphoblastic Leukemia and Non-Hodgkin Lymphoma 33a CD Sgn - CD33 Acute myeloid leukemia Sifalimumab IFN -A SLE, Dermatomyositis, Polymyositis Siltuximab IL-6 Cancer Simtuzumab LOXL2 Fibrosis Siplizumab CD2 Psoriasis, Graft-versus-host disease (Prevention) Sirukumab IL-6 Rheumatoid Arthritis Solanezumab Beta-Amyloid Alzheimer's Disease Solitomab EpCAM Cancer Sonepcizumab Sphingosine-1-phosphate Conoid and retinal neovascularization Sontuzumab Episialin Cancer Stamulumab Myostatin Muscular Dystrophy Sulesomab NCA-90 (Granulocyte Antigen) Osteomyelitis Suvi zumab HIV-1 Viral Infections Tabalumab BAFF B-cell cancers Tacatuzumab Tetraxetan Al pha-fetoprotein Cancer Tadocizumab Integrin ΑΙΙΒβ3 I ntervención Coronaria Percutánea Tañezumab NGF Dolor Taplitumomab Paptox CD19 Cáncer Tefibazumab Factor A de aglutinación Infección Staphylococcus Aureus Telimomab Indeterminado Cáncer Tenatumomab Tenascina C Cáncer Teneliximab CD40 Cáncer T eprotumumab CD221 Tumores hematológicos Ticilimumab CTLA-4 Cáncer Tildrakizumab IL23 Trastornos inflamatorios Mediados inmunológicamente Tigatuzumab TRAIL-R2 Cáncer Tnx -650 11-13 Hodgkin lymphoma Table 2 Name of Antibodies Antigen Associated with Diseases Application Therapeutic Target Tocilizumab IL-6 Receptor Rheumatoid Arthritis Toralizumab CD154 (CD40L) Rheumatoid Arthritis, Lupus Nephritis Tositumomab CD2 0 Follicular Lymphoma Tovetumab CD140a Cancer Tralokinumab IL-13 Asthma Trastuzumab HER2 / Neu Breast Cancer Trb07 GD2 Melanoma Tremelimumab CTLA-4 Cancer Tucotuzumab Celmoleukin EpCAM Cancer Tuvirumab Hepatitis B Chronic Hepatitis B Virus Ublituximab MS4A1 Cancer Urelumab El 4-1BB Cancer Urtoxazumab Escherichia Coli Diarrhea caused by E. Coli Ustekinumab IL-12, IL-23 Multiple Sclerosis, Psoriasis, Arthritis Psoriatic Vantictumab Frizzled Receptor Cancer Vapaliximab AOC3 (VAP-1) Cancer Vatelizumab ITGA2 Cancer Vedolizumab Integrin Α4β7 Crohn's Disease, Ulcerative Colitis Veltuzumab CD2 0 Non-Hodgkin's Lymphoma Vepalimomab AOC3 (VAP-1) Inflammation Vesencumab NRP1 Cancer Volociximab Integrin Α5β1 Solid Vorsetusb Cancer Votumumab Tumor Antigen CTAA16 .88 Colorectal tumors Zalutumumab EGFR Squamous cell carcinoma of the head and neck Zatuximab HERI Cancer Ziralimumab CD147 Cancer Zolimomab aritox CD5 Systemic lupus erythematosus, Graft-versus-Host Disease Lzzb Ln / nznz / E / YiAi

[00108] A Disease Associated Antigen can characteristically be expressed in a pathogen-infected cell or in cancer cells and processed and displayed on a surface Lzzb Ln / nznz / E / YiA 100 cell in the context of an MHC complex, but characteristically not expressed in a normal cell. Antibodies that recognize such peptide fragments are known in the art or can be generated using well known methods, including those described in WO 2002 / 014870.

[00109] The polypeptides of the Trispecific Binding Molecules of the present invention can be adapted to contain the variable light or variable heavy domains (the case of the first and second polypeptide chains of such molecules) or the heavy or light chains (in the case of the first and second polypeptide chains of such molecules). the case of the third and fourth polypeptide chains of such molecules) of such antibodies. Thus, the antibodies described above can be used to produce Tri-specific Binding Molecules of the present invention whose Site A, Site B or Site C is capable of binding to an epitope of such disease-associated antigens]. B. Preferred Structural Attributes

[00110] Typically, the Tri-Specific Binding Molecules of the present molecules will comprise four different polypeptide chains, each having an amino terminus and a carboxyl terminus (see Figures 4A-4D), however, the molecules may comprise fewer or larger numbers of polypeptide chains by fusing such polypeptide chains 101 Lívnznz / E / YIA polypeptides with each other (for example, via peptide linkage) or by dividing such polypeptide chains to form additional polypeptide chains, or by associating fewer or additional polypeptide chains via linkage. disulfide. Figures 4E-4J illustrate this aspect of the present invention by schematically depicting such molecules having three polypeptide chains. Figures 4K-4L illustrate this aspect of the present invention by schematically depicting molecules having five polypeptide chains.

[00111] The various Immunoglobulin Domains of such molecules can be derived from immunoglobulins of any isotype or allotype including, but not limited to, IgA, IgD, IgG, IgE, and IgM. In preferred embodiments, as discussed below such immunoglobulins are derived from IgG immunoglobulins. In specific embodiments, the IgG isotype used is IgG1, however IgG of other isotypes (eg, IgG2, IgG3 or IgG4 or an allotype thereof) may be used. When using an IgG4 Fe Domain, the present invention encompasses the introduction of a stabilizing mutation such as S228P, as numbered by the EU index as set out in Kabat Kabat (Lu et al., (2008) The Effect Of A Point Mutation On The Stability Of Igg4 As Monitored By Analytical Ultracentrifugation, J. pharmaceutical Sciences 97:960-969) to reduce the L77b Ln / nznZ / Ε / ΥΙΛ 102 thread swap incidence. Other art-known stabilizing mutations can be introduced into an IgG4 Fe Domain (Peters, P et al., (2012) Englneering an Improved IgG4 Molecule with Reduced Disulfide Bond Heterogeneity and Increased Fab Domain Thermal Stability, J. Biol. Chem., 287:24525-24533; PCT Patent Publication No: WO 2008 / 145142). Since the N297A, L234A, L235A, and D265A substitutions abolish effector function, in circumstances in which effector function is desired, these preference substitutions will not be employed.

[00112] Figures 4A-4D provide a diagrammatic representation of preferred Trispecific Binding Molecule Domains. Figures 4A and 4B, respectively, schematically illustrate preferred Tri-specific Binding Molecule Domains in which the non-Diabody-Like Binding Domain is a Fab-Like Binding Domain or a Receptor-Like Binding Domain. Figures 4C and 4D, respectively, schematically illustrate preferred Tri-Specific Binding Molecule Domains having different Domain Orientations in which the Diabody-Like Non-Binding Domain is a Fab-Like Binding Domain or a Binding Domain. of the Receiver Type.

[00113] As indicated above, one of Epitope I, Epitope II or Epitope III that is bound by the Binding Domains of such preferred Tri-specific Binding Molecules, L77b Ln / nznZ / Ε / ΥΙΛ 103 copies may be an epitope of an antigen associated with the disease. More preferably, the Binding Domain of such preferred, exemplary Tri-Specific Binding Molecule that binds to such an epitope of a disease-associated antigen is a Fab-Like Binding Domain. The polypeptides of such Tri-Specific Binding Molecules of the present invention can be adapted to contain the Variable Light or Variable Heavy Domains (the case of the first and second polypeptide chains of such molecules) or the heavy or light chains (in the case of the first and second polypeptide chains of such molecules). case of the third and fourth polypeptide chains of such molecules). Thus, such antibodies can be used to produce Tri-specific Binding Molecules of the present invention whose Site A, Site B or Site C is capable of binding to an epitope of such disease-associated antigens. 1. First Preferred Polypeptide Chain

[00114] A first polypeptide chain of a preferred Tri-Specific Binding Molecule of the present invention will comprise a Variable Light Chain Domain capable of binding to Epitope I (VLi), a Variable Heavy Chain Domain capable of binding to Epitope II (VHn), a cysteine ​​residue or cysteine-containing domain and a Heterodimer Promotion Domain and a CH2-CH3 Domain.

[00115] Since the Variable Light Chain Domains and 104 Lzzb Ln / nznz / E / YiA Variable Heavy Chain of the first polypeptide are directed towards different epitopes, they cannot associate together to form a Binding Domain that is capable of binding either Epitope I or Epitope II. The Variable Light Chain and Variable Heavy Chain Domains of the first polypeptide are spaced apart from each other by an intervening linker peptide that is short enough to substantially prevent association of these Domains. An exemplary linker, designated Linker 1, has the sequence (SEQ ID NO: 1): GGGSGGGG.

[00116] The Variable Heavy Chain Domain of the first polypeptide and the Heterodimer Promotion Domain of that preferred polypeptide are spaced from each other by an intervening linker peptide containing 1, 2, 3 or more cysteine ​​residues. A preferred Cysteine-Containing Domain (Linker 2) has the sequence SEQ ID NO: 2: GGCGGG. Alternatively, or additionally, a Cysteine-containing Heterodimer Promotion Domain may be used, as described below.

[00117] Thus, in some embodiments, one or more cysteine ​​residues (or Cysteine-Containing Domain, such as a cysteine-containing peptide linker) will be incorporated into the first polypeptide chain (and / or into the second, third, fourth or more polypeptide chains of the Tri-Specific Binding Molecules of the present invention) 105 Lzzfr Ln / nznz / Ε / γΐΛ in order to covalently link two such polypeptide chains together, while in equivalent embodiments such cysteine ​​residue(s) may be incorporated into a Heterodimer Promotion Domain, or into another Domain with in order to achieve the same result.

[00118] The Heterodimer Promotion Domain of the first polypeptide and the Heterodimer Promotion Domain of the second polypeptide are coordinately selected. The Domains differ from one another and are designed to associate with one another to promote association of the first and second polypeptide chains. For example, one of the Heterodimer Promotion Domains will be designed to have a negative charge at pH 7, while the other of the two polypeptide chains will be designed to have a positive charge at pH 7. The presence of such charged Domains promotes the association between the first and second polypeptides, and thus strengthens the heterodimerization. It is immaterial which Heterodimer Promotion Domain is provided to which chain, whereas the Domains employed in the first and second polypeptide chains differ to enhance heterodimerization between such chains.

[00119] In a preferred embodiment, the Heterodimer Promotion Domain of the first polypeptide chain is either an E-helix Domain (SEQ ID NO: 3): EVAALEKEVAALEKEVAALEKEVAALEK, or a K-helix Domain (SEQ ID Lzzb Ln / nznz / E / YiA 106 ΝΟ:4): KVAALKEKVAALKEKVAALKEKVAALKE. Most preferably, the first polypeptide chain will possess an E-helix Domain. The first polypeptide chain may contain only a single such spacer helix, or it may contain more than one such spacer helix (eg, two spacers) and may be of the same charge of opposite charge preference.

[00120] In a preferred embodiment, the Heterodimer Promotion Domain of the first chain of the polypeptide will comprise four helical E-helix domains in tandem domains (SEQ ID NO:3: EVAALEK-EVAALEK-EVAALEKEVAALEK), whose glutamate residues will form a negatively charged at pH 7, or four tandem K-helix Domains (SEQ ID NO:4: KVAALKE-KVAALKE-KVAALKE-KVAALKE), whose lysine residues will form a positive charge at pH 7. The presence of such charged domains promotes the association between the first and second polypeptide chains, thus strengthening heterodimerization. Especially preferred is a Heterodimer Promotion Domain in which one of the four tandem E-helix helical domains SEQ ID NO: 3 or SEQ ID NO: 4 has been modified to contain a cysteine ​​residue: EVAACEK-EVAALEK-EVAALEKEVAALEK ( SEQ ID NO: 115) or in which one of the four tandem K-helix helical domains SEQ ID NO: 4 has been modified to contain a cysteine ​​residue: KVAACKE107 L77b Ln / nznZ / Ε / ΥΙΛ KVAALKE-KVAALKE-KVAALKE (SEQ ID NO:116). Other E-helix and K-helix domains that can be used in accordance with the present invention are described in: Woolfson, D.N. (2005) The Design Of Coiled-Coil Structures And Assemblies, Adv. Prot. Chem. 70:79-112, Straussman, R. et al. (2007) Kinking the Coiled Coil - Negatively Charged Residues at the Coiled Coil Interface, J. Molec. Biol. 366:1232-1242; Apostolovic, B. et al. (2008) pH-Sensitivity of the E3 / K3 Heterodimeric Coiled Coil, Biomacromolecules 9:3173-3180; Arndt, K.M. et al. (2001) Helix-stabilized Fv (hsFv) Antibody Fragments: Substituting the Constant Domains of a Fab Fragment for a Heterodimeric Coiled-helix Domain, J. Molec. Biol. 312:221-228; Steinkruger, J.D. et al. (2012) The d'--d--d' Vertical Triad is Less Discriminating Than the a'--a--a' Vertical Triad in the Antiparallel Coiled-helix Dimer Motif, J. Amer. Chem. Soc. 134(5):2626-2633; Ghosh, T.S. et al. (2009) End-To-End And End-To-Middle Interhelical Interactions: New Classes Of Interacting Helix Pairs In Protein Structures, Acta Crystallographica D65:1032-1041; Grigoryan, G. et al. (2008) Structural Specificity In Coiled-Coil Interactions, Curr. Opinion. struc. Biol. 18:477483; Boucher, C. et al. (2010) Protein Detection By Western Blot Via Coiled-Coil Interactions, Analytical Biochemistry 399:138-140; Cachia, P.J. et al. (2004) Synthetic Peptide Vaccine Development: Measurement Of Polyclonal Antibody Lzzb Ln / nznz / E / YiA 108 Affinity And Cross-Reactivity Using A New Peptide Capture And Release System For Surface Plasmen Resonance Spectroscopy, J. Mol. Recognize 17:540-557; De Crescenzo, G.D. et al. (2003) Real-Time Monitoring of the Interactions of TwoStranded de novo Designed Coiled-Coils: Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding, Biochemistry 42:1754-1763; Tripet, B. et al. (2002) Kinetic Analysis of the Interactions between Troponin C and the C-terminal Troponin I Regulatory Region and Validation of a New Peptide Delivery / Capture System used for Surface Plasmon Resonance, J. Molec. Biol. 323:345-362; and Zeng, Y. et al. (2008) A Ligand-Pseudoreceptor System Based On de novo Designed Peptides For The Generation Of Adenoviral Vectors With Altered Tropism, J. Gene Med. 10:355-367.

[00121] Preferably, the employed Heterodimer Promotion Domain and the CH2-CH3 Domain of the first polypeptide chain are spaced from each other by an intervening cysteine-containing linker peptide that provides enhanced stabilization of the Heterodimer Promotion Domain. A preferred cysteine-containing peptide linker (Linker 3) has the amino acid sequence (SEQ ID NO:5): DKTHTCPPCP.

[00122] The amino acid sequence of a wild-type CH2-CH3 Domain is as follows (positioning is as in the EU index as in Kabat et al. (1992) Sequences Of 109 Lzzb Ln / nznz / E / YiA Proteins Of Immunological Interest, National Institutes of Health Publication No. 91-3242) (SEQ ID NO:6): | CH2 -> APELLGGPS VFLFPPKPKD TLMISRTPEV TCWVDVSHE DPEVKFNWYV DGVEVHNAKT 231 240 250 260 270 280 <-CH2 | CH3-> KPREEQYNST YRWSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA K GQPREPQVY 290 300 310 320 330 340 TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK 350 338 370 | LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 410 420 430 440

[00123] In some expression systems the C-terminal amino acid residue of the CH3 Domain can be removed by post-translation. Therefore, the C-terminal residue of the CH3 Domain is an optional amino acid residue.

[00124] The CH2-CH3 Domain of the first polypeptide chain will preferably be modified to promote heterodimerization between the CH2-CH3 Domain of the third polypeptide chain (see below). For example, an amino acid substitution (preferably a substitution with an amino acid comprising a bulky side group that forms a 'bump', eg tryptophan) is L77b Lívnznz / E / YIA 110 can introduce into the CH2 or CH3 Domain of the first polypeptide chain such that spherical interference will prevent interaction with a similar mutated Domain and force the mutated Domain to pair with a Domain in which a complementary mutation has been engineered, or fit, i.e. hole (for example, a substitution with glycine). Such sets of mutations can be engineered into any pair of polypeptides comprising the diabody molecule, and furthermore, engineered into any portion of the polypeptide chains of the pair. Protein engineering methods to favor heterodimerization over homodimerization are well known in the art, particularly with respect to the engineering of immunoglobulin-like molecules, and are encompassed herein (see, for example, US Patent No. 7,695,936 and Patent Publication 2007 / 0196363, Ridgway et al (1996) 'Knobs-Into-Holes' Engineering Of Antibody CH3 Domains For Heavy Chain Heterodimerization, Protein Engr 9:617-621, Atwell et al (1997 ) Stable Heterodimers From Remodeling The Domain Interface Of A Homodimer Using A Phage Display Library, J. Mol. Biol. 270: 26-35, and Xie et al. (2005) 'Ά New Format Of Bispecific Antibody: Highly Efficient Heterodimerization, Expression And Tumor Cell Lysis, J. Immunol Methods 296:95101, each of which is incorporated herein by reference in its entirety. Lzzfr Ln / nznz / Ε / γΐΛ 111 creates by modifying a native IgG Fe Domain to contain the T366W modification. A preferred hole is created by modifying a native IgG Fe Domain to contain the T366S, L368A and Y407V modification. To aid purification of the Tri-Specific Binding Molecules of the present invention, the polypeptide chain containing the hole mutations additionally comprise a substitution at position 435 (H435R) to remove the Protein A binding site. In this way, the polypeptide homodimers containing the hole mutations will not bind to Protein A, while the Tri-Specific Binding Molecules that form as a result of the bulge- and hole-containing heterodimers will retain their ability to bind Protein A. A via the binding of Protein A to the polypeptide chain containing the protrusion mutation.

[00125] The CH2-CH3 Domain of the first polypeptide chain will preferably be modified to reduce or abolish binding to Fe to Fe receptors. Such mutations are well known in the art and include substitutions at positions 234, 235, 265 and 297 (see US Patent No. 5,624,821). Preferred substitutions include one or more of L234A and L235A, D265A, and N297Q.

[00126] Preferably, therefore, the CH2-CH3 Domain of the first polypeptide chain will have a sequence carrying overhang (SEQ ID NO: 7): 112 Lzzb Ln / nznz / E / YiA APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLWCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK o la secuencia que lleva agujero con una sustitución H435R para anular la unión de la proteina A (SEQ ID NO: 8): APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG NVFSCSVMHE ALHNRYTQKS LSLSPGK

[00127] It is preferred that the first polypeptide chain will have a CH2-CH3 sequence bearing overhang such as that of (SEQ ID NO: 7):

[00128] As will be recognized, a hole-bearing CH2-CH3 Domain (eg, SEQ ID NO: 8) could be employed in the first polypeptide chain, in which case, a bulge-bearing CH2-CH3 Domain (eg, For example, SEQ ID NO: 7) would be used in the third polypeptide chain.

[00129] Thus, in summary, a preferred first polypeptide chain of a preferred Tri-Specific Binding Molecule of the present invention will comprise the Domains and Linkers: (Domain VLi) — (Linker D-( Domain Lzzb Ln / nznz / E / YiA 113 VHn)—(Cysteine-Containing Domain (Linker 2))—(E-helix Heterodimer Promotion Domain)—(Linker 3)—(CH2-CH3 domain bearing bulge) or (VLi Domain)—(Linker 1)— (VHn Domain) — (Linker 2) —(Cysteine-Containing Heterodimer E-helix Promotion Domain) —(Linker 3) —(CH2-CH3 Domain bearing protrusion or (VLi Domain) — (Linker 1) —(Domain VHn)—(Linker 2)—(Cysteine-Containing Heterodimer E-helix Promotion Domain)—(Linker 3)—(CH2-CH3 Domain bearing bulge). 2. Second Preferred Polypeptide Chain

[00130] A second polypeptide chain of such preferred Tri-Specific Binding Molecules will comprise, in the N-terminal to C-terminal direction, a Variable Light Chain Domain capable of binding to Epitope II (VLn), a Variable Light Chain Domain Variable Heavy capable of binding to Epitope I (HIV), a cisterna residue or Cisterna-Containing Domain and a Heterodimer Promotion Domain.

[0131] Since the Variable Light Chain and Variable Heavy Chain Domains of the second polypeptide are directed toward different epitopes, they cannot associate together to form a Binding Domain that is capable of binding either Epitope I or Epitope II. The Variable Light Chain and Variable Heavy Chain Domains of the second Lzzb Ln / nznz / E / YiA 114 polypeptides are spaced apart by an intervening linker peptide that is short enough to substantially prevent association of these Domains. Linker 1, having the sequence (SEQ ID NO:1): GGGSGGGG is an exemplary linker for this purpose.

[00132] As in the case of the first polypeptide chain, the Variable Heavy Chain Domain of the second polypeptide and the Heterodimer Promotion Domain of that preferred polypeptide are spaced apart with an intervening Cysteine-containing Domain containing 1 , 2, 3 or more cysteine ​​residues. Linker 2, having the sequence (SEQ ID NO: 2) GGCGGG is an exemplary linker for this purpose. Such cysteine ​​residues can form disulfide bonds with cysteine ​​residues in the cysteine-containing spacer peptide that separates the Variable Heavy Chain Domain of the first polypeptide and the Heterodimer Promotion Domain of that polypeptide. In this manner, the first and second polypeptides of the Tri-Specific Binding Molecules of the present invention are covalently linked to each other. Alternatively, a Cysteine-containing Heterodimer Promotion Domain can be used, as described above.

[00133] As discussed above, the Heterodimer Promotion Domain of the second polypeptide chain is selected to coordinate with the Heterodimer Promotion Domain of the second polypeptide chain. 115 Lívnznz / E / YIA Heterodimer of the first polypeptide chain. Thus, in a preferred embodiment, the Heterodimer Promotion Domain of the first polypeptide chain is either a K-helix Domain (eg, SEQ ID NO: 4 or SEQ ID NO: 116) or an E-Domain. helix (eg, SEQ ID NO: 3 or SEQ ID NO: 115). Since the first preferred polypeptide chain will possess an E-helix domain, the second preferred polypeptide chain contains a K-helix domain.

[00134] As the first and second polypeptide chains are diabody polypeptide chains, they are capable of associating together to form a Domain Binding Domain I (VLa / VHa) that recognizes and immunospecifically binds Epitope I, and a Domain Binding Domain II (VLb / VHb) that immunospecifically recognizes and binds to Epitope II.

[00135] Thus, in summary, a preferred second polypeptide chain of a Tri-Specific Binding Molecule of the present invention will comprise the Domains and Linkers: (VLn Domain) — (Linker 1) — (VHi Domain) — ( Cysteine-Containing Domain (Linker 2) ) — (Heterodimer K-helix Promotion Domain) or (VLn Domain) — (Linker 1) — (VHi Domain) — (Linker 2) — (Heterodimer K-helix Promotion Domain that Contains Cysteine). 3. Third Preferred Polypeptide Chain

[00136] A third polypeptide chain of a Molecule of L77b Ln / nznZ / Ε / ΥΙΛ 116 Preferred Tri-Specific Binding of the present invention is a polypeptide comprising, in the N-terminal to C-terminal direction, a Binding Domain, a Cysteine-Containing Domain which may optionally comprise a CH1 Hinge Domain and a CH2-CH3 Domain. The Binding Domain of the third polypeptide chain of a preferred TriSpecific Binding Molecule of the present invention may be a Variable Heavy Chain Domain capable of binding to Epitope III (VHin), in which case, the fourth polypeptide chain of The preferred Tri-specific Binding Molecules of the present invention (discussed below) is a polypeptide comprising a Variable Light Chain Domain capable of binding to Epitope III (VLm), such that the Binding Domain is capable of binding immunospecifically to a antigen possessing epitope III. Alternatively, the Binding Domain of the third polypeptide chain of the preferred Trispecific Binding Molecules of the present invention may comprise a T-Cell Receptor-Like Binding Domain, in which case, the fourth polypeptide chain of the Molecules The preferred Tri-specific Binding Domain of the present invention (discussed below) is a polypeptide comprising a complementary T-Cell Receptor Type Binding Domain, such that the interaction of two polypeptide chains forms a Binding Domain that is capable of Physiospecific binding to an antigen molecule 117 L77b Ln / nznZ / Ε / ΥΙΛ displayed in the MHC complex arranged on the surface of a cell. The third polypeptide chain can be isolated from naturally occurring antibodies. Alternatively, it can be recombinantly constructed. An exemplary CH1 Domain is a human IgGl CH1 Domain having the amino acid sequence (SEQ ID NO: 9):

[00137] An exemplary Hinge Domain is a human IgGl Hinge Domain having the amino acid sequence (SEQ ID NO: 10): EPKTCTHPPKSCD. As will be recognized, the exemplary Hinge Domain comprises multiple cysteine ​​residues (Elkabetz et al. (2005) Cysteines In CH1 Underlie Retention Of Unassembled Ig Heavy Chains, J. Biol. Chem. 280:14402-14412) that may participate in the interchain covalent bonding. Alternatively, a different cysteine-containing domain can be employed (for example, a peptide having the amino acid sequence: VEPKSC (SEQ ID NO:12), AEPKSC (SEQ ID NO:127), GVEPKSC (SEQ ID NO:133) or GGCGGG (SEQ ID NO:2)).

[00138] Although a wild-type CH2-CH3 Domain can be employed, it is preferred, as described above, to employ a modified CH2-CH3 Domain that promotes heterodimerization with the CH2-CH3 Domain of the first polypeptide chain. 118 Lzzfr Ln / nznz / Ε / γΐΛ

[00139] Preferably, therefore, the CH2-CH3 Domain of the third polypeptide chain will be a hole-bearing CH2-CH3 Domain whose amino acid sequence is complementary to the knob-bearing CH2-CH3 Domain (SEQ ID NO: 7 ) used in the first polypeptide. As discussed above, the preferred hole-bearing CH2-CH3 Domain should comprise a substitution at position 435 (H435R) to remove the Protein A Binding Site. An exemplary hole-bearing CH2-CH3 Domain with the substitution H435R for the third polypeptide is SEQ ID NO: 8.

[00140] As will be recognized, a CH2-CH3 Domain bearing overhang (eg, SEQ ID NO: 7) could be employed in the third polypeptide chain, in which case which, a hole-bearing CH2-CH3 Domain (eg, SEQ ID NO: 8) would be employed in the first polypeptide chain.

[00141] In the embodiment in which the third (and fourth) polypeptide chains of the preferred Trispecific Binding Molecules of the present invention each comprise a polypeptide chain of a T-Cell Receptor Type Binding Domain, gue recognized antigen displayed on a cell surface in the context of MHC class I. Methods are well known in the art for producing such T-cell Receptor-Type Binding Domains (eg US2012 / 0294874A1). 119 Lzzb Ln / nznz / E / YiA

[00142] Thus, in summary, a third polypeptide chain of the preferred Tri-Specific Binding Molecules of the present invention will comprise the Domains and Linkers: (VHm Domain) — (Cysteine-Containing Domain (optionally a CHI Domain and / or a Hinge Domain) — (CH2-CH3 Hole-Carrying Domain), or (Receptor Type Binding Domain; first or second polypeptide thereof) — (Cysteine-Containing Domain (optionally a CHI Domain and / or a Hinge Domain) — (CH2-CH3 Domain Carrying Hole). 4. Preferred Fourth Polypeptide Chain

[00143] A fourth polypeptide chain of the preferred Tri-specific Binding Molecules of the present invention is either a Receptor-Like Binding Domain polypeptide (wherein the third and fourth polypeptide chains of a Receptor-Like Binding Domain Receptor), or more preferably, a polypeptide portion of a light chain of the antibody indicated above that immunospecifically binds to Epitope III and / or is complementary to the Binding Domain of the third polypeptide chain.

[00144] Thus, wherein the third and fourth polypeptides of a Fab-Like Binding Domain such fourth polypeptide chain comprises, in the N-terminal direction a L77b Ln / nznZ / Ε / ΥΙΛ 120 C-terminal, a Variable Light Chain Domain capable of binding to Epitope III (VLm), and a Cysteine-Containing Domain to promote covalent attachment to the third polypeptide chain, or a Binding Domain and such a Cysteine-Containing Domain for promote covalent binding to the third polypeptide chain. Such a Cysteine-Containing Domain may be a CL Domain, or a cysteine-containing portion thereof, such as (SEQ ID NO: 11) FNRGEC or (SEQ ID NO: 128) GFNRGEC or a linker such as Linker 2 (having the sequence (SEQ ID NO.: 2) GGCGGG An exemplary Cysteine-Containing Domain that forms disulfide bonds with such Linker 2 comprises the amino acid sequence VEPKSC (SEQ ID NO: 12) or a Hinge Domain.

[00145] The fourth polypeptide chain can be isolated from naturally occurring antibodies. Alternatively, it can be recombinantly constructed. A preferred CL Domain is a human IgGl kappa Domain having an amino acid sequence (SEQ ID NO: 13): RTVAAPSVFI FPPSDEQLKS GTASWCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

[00146] Alternatively, an exemplary CL Domain is a human Lambda2 IgGl CL Domain having the amino acid sequence (SEQ ID NO: 14): QPKAAPSVTL FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA Lzzfr Ln / nznz / E / YiA 121 GVETTPSKQS NNKYAASSYL SLTPEQWKSH RSYSCQVTHE GSTVEKTVAP TECS

[00147] As will be reported, the CL Domain, or other cysteine-containing Domain, of the fourth polypeptide chain comprises a cysteine ​​residue that is capable of covalently binding to a cysteine ​​residue of the Cysteine-containing Domain. cysteine ​​of the third polypeptide chain (eg, a CH1 Domain) whereby the third and fourth polypeptide chains of the Tri-Specific Binding Molecules of the present invention are covalently complexed with each other. In this manner, the third and fourth polypeptide chains are covalently linked to each other.

[00148] Additionally, cysteine ​​residues of the CH2-CH3 Domain of the first polypeptide chain can form disulfide bonds with cysteine ​​residues of the CH2-CH3 Domain of the third polypeptide chain. In this manner, the first and third polypeptide chains are covalently linked to each other.

[00149] Thus, in summary, a fourth polypeptide chain of the preferred Tri-Specific Binding Molecules of the present invention will comprise the Domains and Linkers: (VLm Domain) — (Cysteine-Containing Domain (optionally a CL Domain) , or (Receptor Type Binding Domain; First or Second Polypeptide Thereof) — (Cysteine-Containing Domain (Optionally a CL Domain) . 122 L77b Ln / nznZ / E / Yli C. First Alternative Polypeptide Chain

[00150] In one embodiment, the orientations of the Domains described above will be in the N-terminal to C-terminal direction. The present invention, however, also contemplates a variation thereof, wherein the orientations of the first polypeptide chain Domains are: NH2- (CH3-CH2 Domain bearing Overhang) - (VLi Domain) — (Linker 1 ) — (VHn Domain) — (Cysteine-Containing Domain Linker 2) — (E-helix Heterodimer Promotion Domain). Preferably, a Cysteine-Containing Domain, N-terminal to such a CH2-CH3 Domain, is present. The sequence of an exemplary peptide is (SEQ ID NO: 5) : DKTHTCPPCP, however alternative linkers may be employed, eg, EPKSCDKTHTCPPCP (SEQ ID NO: 129) or LEPKSSDKTHTCPPCP; SEQ ID NO: 130). Preferably, in this embodiment, the CH3 Domain is spaced from the VLI Domain by an intervening peptide linker, such as one having the amino acid sequence of (SEQ ID NO: 15): APSSS, and more preferably, the sequence of amino acids (SEQ ID NO: 16) APSSSPME, however alternative linkers may be employed, eg, ASTKG (SEQ ID NO: 131), LEPKSS (SEQ ID NO: 132), GGC or GGG. 123 D. Albumin Binding Domain

[0151] As described in WO 2012 / 018687, in order to improve the in vivo pharmacokinetic properties of diabodies, a diabody can be modified to contain a polypeptide portion of a serum binding protein in one or more of the diabody terminals. Such considerations are also applicable to the Tri-Specific Binding Molecules of the present invention. Most preferably, when a polypeptide portion of a serum binding protein is desired to be incorporated into the Tri-Specific Binding Molecules of the present invention, such polypeptide portion will be installed at the C-terminus of one of the polypeptide chains. of the Tri-Specific Binding Molecule.

[00152] Albumin is the most abundant protein in plasma and has a half-life of 19 days in humans. Albumin possesses several small molecule binding sites that allow it to bind non-covalently to other proteins and thus prolong their half-lives in serum. The Albumin Binding Domain 3 (ABD3) of the G protein of Streptococcus strain G148 consists of 46 amino acid residues that form a stable three-helical cluster and has broad albumin-binding specificity (Johansson, M.U. et al. ( 2002) Structure, Specificity, And Mode Of Interaction For Bacterial Albumin-Binding Modules, J. Biol. Lzzb Ln / nznz / E / YiA Lzzfr Ln / nznz / E / YiAi 124 Chem. 277(10):8114-8120. Thus, a particularly preferred polypeptide portion of a serum binding protein for improving the in vivo pharmacokinetic properties of a diabody is the Albumin Binding Domain (ABD) of the protein is treptococcal G, and more preferably, the Albumin Binding Domain 3 (ABD3) of the G protein of the Streptococcus strain G148 (SEQ ID NO:123): LAEAKVLANR ELDKYGVSDY YKNLIDNAKS AEGVKALIDE ILAALP.

[00153] As described in WO 2012 / 162068 (incorporated herein by reference), deimmunized variants of SEQ ID NO: 123 have the ability to attenuate or eliminate MHC class II binding. Based on the pooling mutation results, the following combinations of substitutions are considered to be preferred substitutions for forming such a deimmunized Albumin Binding Domain: 66S / 70S+71A; 66S / 70S+79A; 64A / 65A / 71A+66S; 64A / 65A / 71A+66D; 64A / 65A / 71A+66E; 64A / 65A / 79A+66S; 64A / 65A / 79A+66D; 64A / 65A / 79A+66E. The variant ABDS having the 164A, I65A and D79A modifications or the N66S, T70S and D79A modifications. The ABD deimmunized variant having the amino acid sequence: LAEAKVLANR ELDKYGVSDY YKNA64A6sNNAKT VEGVKALIA79E ILAALP (SEQ ID NO:124), or the amino acid sequence LAEAKVLANR ELDKYGVSDY YKNLIS66NAKS70 VEGVKALIA79E ILAALP (SEQ Lzzb Ln / nznz / E / YiA 125 ID NO:125), are particularly preferred as such deimmunized Albumin Binding Domains exhibit substantially wild-type binding while providing attenuated MHC class II binding. Although such Albumin Binding Domains can be incorporated into any of the polypeptide chains of the Tri-Specific Binding Molecules of the present invention, it is preferred to place such a Domain C-terminally to the E-helix (or K-helix) Domain of the first or third polypeptide chain (via a linker intervening between the E-helix (or K-helix) Domain and the Albumin Binding Domain (which is preferably a deimmunized Albumin Binding Domain)). A preferred sequence for such a linker is SEQ ID NO: 126: GGGS. E. Faith Domain Functionality

[00154] In one embodiment, the CH2-CH3 Domain of the first polypeptide chain and the CH2-CH3 Domain of the third polypeptide will complex to form an Fe Domain that is substantially incapable of binding to an Fe receptor (i.e., bind at least 10% of the extent of the wild-type Fe Domain Alternatively, the Fe Domain of such molecules will be capable of binding to the Fe receptor under physiological conditions, such that such Tri-specific Binding Molecules will be tetra-specific, capable of mediate the union Lzzb Ln / nznz / E / YiA 126 coordinated to four molecules (Epitope I, Epitope II and Epitope III, and an Fe Receptor). Most preferably, such molecules capable of binding to the Fe receptor will further mediate the Fe receptor-dependent effector function.

[00155] The invention also encompasses molecules comprising variant Fe Domains comprising one or more amino acid substitutions, insertions, or deletions relative to a comparable wild-type Fe Domain. Molecules comprising variant Fe Domains typically have altered phenotypes relative to molecules comprising wild-type Fe Domains. The variant phenotype can be expressed as altered serum half-life, altered stability, altered susceptibility to cellular enzymes, or altered effector function as assayed in an NK-dependent or macrophage-dependent assay. Modifications of the Fc domain identified as altered effector function are known in the art, including modifications that increase binding to activating receptors (eg, FcyRIIA (CD16A)) and reduce binding to inhibitory receptors (eg, FcyRIIB ( CD32b) (see, for example, Stavenhagen, J.B. et al. (2007) Fe Optimizatíon Of Therapeutic Antibodies Enhances Their Ability To Kill Tumor Cells In Vitro And Controls Tumor Expansion In Vivo Via Low-Affinity Activating Fcgamma 127 Lzzb Ln / nznz / E / YiA Receptors, Cancer Res. 57(18):8882-8890). Exemplary variants of the human Fe IgGl Domains with reduced binding to CD32b and / or increased binding to CD16A contain F243L, R292P, Y300L, V305I or P296L substitutions. These amino acid substitutions can occur in a human IgGl Fe Domain in any combination. In one embodiment, the human IgGl Fe Domain variant contains an F243L, R292P and Y300L substitution. In another embodiment, the human IgGl Fe Domain variant contains an F243L, R292P, Y300L, V305I and P296L substitution. In another embodiment, the human IgGl Fe Domain variant contains an N297Q substitution, the L234A and L235A substitutions, or a D265A substitution, as these mutations nest the FcR junction. III. Exemplification of the Trispecific Binding Molecules of the Present Invention: Trispecific Binding Molecules Having Binding Domains That Bind CD3 and CD8 Epitopes to an Epitope of a Disease-Associated Antigen

[00156] As stated above, the present invention particularly relates to the modality of Tri-specific Binding Molecules in which the three epitopes are selected such that one or two of such epitopes are epitopes of a cell of the immune system, Lzzb Ln / nznz / E / YiA 128 especially, a cytotoxic lymphocyte (CTL) immune system cell, and in which the remaining epitope(s) are epitopes of a disease-associated antigen. In a particularly preferred embodiment of such a Tri-Specific Binding Molecule, the Binding Domains of such a molecule are selected such that Epitope I, Epitope II or Epitope III is an Epitope of CD3, a second of Epitope I, Epitope II or Epitope III is an Epitope of CD8, and the third of Epitope I, Epitope II or Epitope III is an Epitope of a disease-associated antigen, wherein Binding Domains I, II and III of such Tri-Specific Binding Molecules mediate the coordinated union of a cytotoxic T cell and a cell expressing the antigen associated with the disease. Such Tri-Specific Binding Molecules are capable of localizing a cytotoxic lymphocyte cell to a cell expressing a disease-associated antigen, and thus facilitate killing of cells expressing the disease-associated antigen. The disease-associated antigen may be a cancer antigen, or it may be an antigen that is characteristic of an infection from pathogens (eg, bacterial, fungal, viral, or protozoal). More particularly, the invention relates to such Tri-Specific Binding Molecules which are capable of mediating coordinate binding to: (1) an epitope on CD3, (2) an epitope on CD8, and (3) an epitope on an antigen. associated with the L77b Ln / nznZ / Ε / ΥΙΛ 129 disease. By binding to CD3 and CD8, and to the disease-associated antigen, such molecules co-localize cytotoxic T cells to cells presenting the Disease-Associated Antigen, leading to the activation of such cells and the initiation of a cytotoxic response against them. cells expressing the Disease Associated Antigen.

[00157] Heavy chains of an anti-CD3 antibody or anti-CD8 antibody can be employed as the third polypeptide chain of such exemplary Tri-specific Binding Molecules of the present invention. Similarly, the light chains of such antibodies can be used as the fourth polypeptide chain of the Tri-Specific Binding Molecules of the present invention. Alternatively, the light chain variable domains and / or heavy chain variable domains of such antibodies can be combined with other immunoglobulin constant regions to achieve such third and fourth polypeptide chains. In this way, such antibodies can be used to produce Tri-specific Binding Molecules of the present invention whose Site C is capable of binding CD3 or CD8.

[00158] Similarly, such Variable Domains can be incorporated into the Variable Domain portions of the first and third polypeptides of the Tri-Specific Binding Molecules of the present invention to produce Binding Molecules. 130 Lzzb Ln / nznz / E / YiA Tri-specific of the present invention whose Site A is capable of binding CD3 or CD8, or whose Site B is capable of binding CD3 or CD8. 1. Exemplary Anti-CD3 Antibodies

[0159] Any of the exemplary anti-CD3 or anti-CD8 antibodies provided below can be used to make the CD3 or CD8 Binding Domains of the Tri-Specific Binding Molecules of the present invention. OKT3 OKT3 Light Chain Variable Domino (SEQ ID NO:17) (CDRs are shown underlined): QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDT SKLASGVPAH FRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPTFFGSG TKLEINR OKT3 Heavy Chain Variable Domino (SEQ ID NO:18) (CDRs are shown underlined): QVQLQQSGAE LARPGASVKM SCKASGYTFT RYTMHWVKQR PGQGLEWIGY INPSRGYTNY NQKFKDKATL TTDKSSSTAY MQLSSLTSED SAVYYCARYY DDHYCLDYWG QGTTLTVSSA KTTAPSVYPL APVCGDTTGS SVTLGCLVKG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL YTLSSSVTVT SS M291 M291 Light Chain Variable Domino (SEQ ID 131 Lzzfr Ln / nznz / Ε / γΐΛ NO:19) (CDRs are shown underlined): DIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWTYDT SKLASGVPAR FSGSGSGTSY SLTISSMEAE DADTYYCQQW SSNPPTFGSG TKLEIK M291 Heavy Chain Variable Domino (SEQ ID NO:20) (CDRs are shown underlined): QVQLQQSGAE LARPGASVKM SCKASGYTFI SYTMHWVKQR PGQGLEWIGY INPRSGYTHY NQKLKDKATL TADKSSSSAY MQLSSLTSED SAVYYCARSA YYDYDGFAYW GQGTLVTVSA YTH12.5 YTH12.5 Light Chain Variable Domino (SEQ ID NO:21) (CDRs are shown underlined): MGWSCIILFL VATATGVHSD IQLTQPNSVS TSLGSTVKLS CTLSSGNIEN NYVHWYQLYE GRSPTTMIYD DDKRPDGVPD RFSGSIDRSS NSAFLTIHNV AIEDEAIYFC HSYVSSFNVF GGGTKLTVLR YTH12.5 Heavy Chain Variable Domino (SEQ ID NO:22) (CDRs are shown underlined): MGWSCIILFL VATATGVHSE VQLLESGGGL VQPGGSLRLS CAASGFTFSS FPMAWVRQAP GKGLEWVSTI STSGGRTYYR DSVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCAKFRQ YSGGFDYWGQ GTLVTVSS Humanized anti-CD3 antibody 1 (CD3 mAb 1) (US2014 / 0099318A1) Light Chain Variable Domain CD3 mAb 1 132 Lívnznz / E / YIA (SEQ ID NO:23) Variant 1 (CDRs shown underlined): DIQMTQSPSS LSASVGDRVT ITCSASSSVS YMNWYQQKPG KAPKRLIYDS SKLASGVPSR FSGSGSGTEF TLTISSLQPE DFATYYCQQW SRNPPTFGGG TKVEIK mAb 1 CD3 Dominoa Variant NO:2SEQ Variant Light Chain 2 (CDRs are shown underlined): DWMTQSPAI MSAFPGEKVT ITCSASSSVS YMNWYQQKPG KAPKRWIYDS SKLASGVPSR FSGSGSGTEF TLTISSLQPE DFATYYCQQW SRNPPTFGGG TKVEIK Heavy Chain Variable Domino CD3 mAb 1 (SEQ ID NO:25) Variant 1 (CDRs shown underlined): QVQLVQSGAE VKKPGASVKV SCKASGYTFT RSTMHWVRQA PGQGLEWIGY INPSSAYTNY NQKFKDRVTI TADKSTSTAY MELSSLRSED TAVYYCASPQ VHYDYNGFPY WGQGTLVTVS S Humanized Anti-CD3 Antibody 2 (CD3 mAb 2) (US2014 / 0099318A1) Light Chain Variable Domain CD3 mAb 2 (SEQ ID NO:26) (CDRs are shown underlined): QAWTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG Heavy Chain Variable Domino CD3 mAb 2 (SEQ ID NO:27) (CDRs shown underlined): EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR Lzzb Ln / nznz / E / YiA 133 HGNFGNSYVS WFAYWGQGTL VTVSS Heavy Chain Variable Domino CD3 mAb 2 D65G Variant (SEQ ID NO:28) (CDRs are shown underlined): EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKGRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSS 2. Anti-CD8 Exemplary Antibody OKT8 (CD8 mAb 1) OKT8 Light Chain Variable Domain (SEQ ID NO:29) (CDRs are shown underlined): DIVMTQSPAS LAVSLGQRAT IS GRASE SVD SYDNSLMHWY QQKPGQPPKV LIYLASNLES GVPARFSGSG SRTDFTLTID PVEADDAATY YCQQNNEDPY TFGGGTKLEI KR OKT8 Heavy Chain Variable Domain (SEQ ID NO:30) (CDRs are shown underlined): QVQLLESGPE LLKPGASVKM SCKASGYTFT DYNMHWVKQS HGKSLEWIGY IYPYTGGTGY NQKFKNKATL TVDSSSSTAY MELRSLTSED SAVYYCARNF RYTYWYFDVW GQGTTVTVSS TRX2 (CD8 mAb 2) TRX2 Light Chain Variable Domain (SEQ ID NO:31) (CDRs are shown underlined): DIQMTQSPSS LSASVGDRVT ITCKGSQDIN NYLAWYQQKP GKAPKLLIYN TDILHTGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCYQ YNNGYTFGQG TKVEIK 134 L77b Lívnznz / E / YIA TRX2 Heavy Chain Variable Domain (SEQ ID NO:32) (CDRs are shown underlined): QVQLVESGGG WQPGRSLRL SCAASGFTFS DFGMNWVRQA PGKGLEWVAL IYYDGSNKFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKPH YDGYYHFFDS WGQGTLVTVS S 3. Exemplary Binding Domain That Bind Epitopes of Disease-Associated Antigens (a) HIV gp41

[0160] As illustrated the Disease Associated Antigen is HIV gp41. An exemplary gp41 antibody is 7B2 (HIV mAb 1).

[00161] Amino Acid Sequence of 7B2 Variable Domain of Light Chain (SEQ ID NO:35): DIVMTQSPDS LAVSPGERAT IHCKSSQTLL YSSNNRHSIA WYQQRPGQPP KLLLYWASMR LSGVPDRFSG SGSGTDFTLT INNLQAEDVA IYYCHQYSSH PPTFGHGTRV EIK

[00162] Amino acid sequence of 7B2 Heavy Chain Variable Domain (SEQ ID NO:36): QVQLVQSGGG VFKPGGSLRL SCEASGFTFT EYYMTWVRQA PGKGLEWLAY ISKNGEYSKY SPSSNGRFTI SRDNAKNSVF LQLDRLSADD TAVYYCARAD GLTYFSELLQ YIFDLWGQGA RVTVSS (b) HIV gpl20

[00163] A Second Disease Associated Antigen Lzzfr Ln / nznz / E / YiA 135 illustrated is HIV gpl20. An exemplary gpl20 antibody is A32 (HIV mAb 2).

[00164] Amino acid sequence of A32 VL Light Chain Variable Domain (SEQ ID NO:33): QSALTQPPSA SGSPGQSVTI SCTGTSSDVG GYNYVSWYQH HPGKAPKLII SEVNNRPSGV PDRFSGSKSG NTASLTVSGL QAEDEAEYYC SSYTDIHNFV FGGGTKLTVL

[00165] Amino acid sequence of A32 VH Heavy Chain Variable Domain (SEQ ID NO:34): QVQLQESGPG LVKPSQTLSL SCTVSGGSSS SGAHYWSWIR QYPGKGLEWI GYIHYSGNTY YNPSLKSRIT ISQHTSENQF SLKLNSVTVA DTAVYYCARG TRLRTLRNAF DIWGQGTMVT VSS (c) RSV glycoprotein F

[0166] A further illustrated Disease Associated Antigen is RSV F-glycoprotein. An exemplary anti-RSV F-glycoprotein antibody is palivizumab (RSV mAb 1).

[00167] Amino acid sequence of Palivizumab Light Chain Variable Domain (SEQ ID NO:37): DIQMTQSPST LSASVGDRVT ITCRASQSVG YMHWYQQKPG KAPKLLIYDT SKLASGVPSR FSGSGSGTEF TLTISSLQPD DFATYYCFQG SGYPFTFGGG TKLEIK

[00168] Palivizumab Heavy Chain Variable Domain Amino Acid Sequence (SEQ ID NO:38): L77b Ln / nznZ / Ε / ΥΙΛ 136 QVTLRESGPA LVKPTQTLTL TCTFSGFSLS TSGMSVGWIR QPPGKALEWL ADIWWDDKKD YNPSLKSRLT ISKDTSKNQV VLKVTNMDPA DTATYYCARS MITNWYFDVW GAGTTVTVSS (d) B7-H3

[0169] An illustrative, particularly preferred Disease Associated Antigen is B7-H3, which is expressed on a variety of cancer cells (eg, neuroblastomas, non-small cell lung, ovarian and gastric cancers, etc.). The expression of B7-H3 protein has been detected immunohistologically in tumor cell lines (Chapoval, A. et al. (2001) B7-H3: Ά Costimulatory Molecule For T Cell Activation and IFN-γ Production, Nature Immunol. 2: 269-274;Saatian, B. et al.(2004) Expression Of Genes For B7-H3 And Other T Cell Ligands By Nasal Epithelial Cells During Difference And Activation, Amer. J. Physiol. Lung Cell. Mol. Physiol. 287: L217-L225, Castriconi et al (2004) Identification Of 4Ig-B7-H3 As A Neuroblastoma Associated Molecule That Exerts A Protective Role From An NK Cell-Mediated Lysis, Proc. Nati. Acad. Sci. (U.S.A.) 101(34) :12640-12645) ; Sun, M. et al. (2002) Characterization of Mouse and Human B7-H3 Genes, J. Immunol. 168:6294-6297). mRNA expression has been found in heart, kidney, testes, lung, liver, pancreas, prostate, colon, and osteoblast cells (Collins, M. et al. (2005) The B7 Family Of Immune-Regulatory Ligands, Genome Biol. 6:223.1-223.7). 137 L77b Ln / nznZ / Ε / ΥΙΛ At the protein level, B7-H3 is found in human organs such as liver, lung, bladder, testes, prostate, breast, placenta, and lymphoids (Hofmeyer, K. et al. (2008) The Contrasting Role Of B7-H3, Proc Nati. Acad. Sci. (U.S.A.) 105(30):10277-10278). Illustrative antibodies that bind to B7-H3 include humanized BRCA84D, BRCA69D γ PRCA157 ( WO 2011 / 109400 ). Exemplary variable light and heavy chains have the following sequences (CDRs are shown underlined):

[00170] Exemplary humanized BRCA84D-5VL Light Chain Variable Domain amino acid sequence (SEQ ID NO:39): DIQLTQSPSF LSASVGDRVT ITCKASQNVD TNVAWYQQKP GQAPKALIYS ASYRYSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YNNYPFTFGQ GTKLEIK

[00171] Exemplary humanized BRCA84D-2VH Heavy Chain Variable Domain amino acid sequence (SEQ ID NO:40): EVQLVESGGG LVQPGGSLRL SCAASGFTFS SFGMHWVRQA PGKGLEWVAY ISSDSSAIYY ADTVKGRFTI SRDNAKNSLY LQMNSLRDED TAVYYCGRGR ENIYYGSRLD YWGQGTTVTV SS

[00172] Exemplary BRCA69D Light Chain Variable Domain (B7-H3 mAb 1) amino acid sequence (SEQ ID NO:41): DIQMTQSPSS LSASVGDRVT ITCRASQDIS NYLNWYQQKP GKAPKLLIYY TSRLHSGVPS RFSGSGSGTD FTLTISSLQP EDIATYYCQQ GNTLPPTFGG gtkleik Lzzb Ln / nznz / E / YiA 138

[00173] Exemplary BRCA69D Heavy Chain Variable Domain (B7-H3 mAb 1) amino acid sequence (SEQ ID NO:42): QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYWMQWVRQA PGQGLEWMGT IYPGDGDTRY TQKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCARRG IPRLWYFDVW GQGTTVTVSS

[00174] Exemplary PRCA157 Light Chain Variable Domain Amino Acid Sequence (SEQ ID NO:43): DIQMTQSPAS LSVSVGETVT ITCRASESIY SYLAWYQQKQ GKSPQLLVYN TKTLPEGVPS RFSGSGSGTQ FSLKINSLQP EDFGRYYCQH HYGTPPWTFG GGTNLEIK

[00175] Exemplary PRCA157 Heavy Chain Variable Domain Amino Acid Sequence (SEQ ID NO:44): EVQQVESGGD LVKPGGSLKL SCAASGFTFS SYGMSWVRQT PDKRLEWVAT INSGGSNTYY PDSLKGRFTI SRDNAKNTLY LQMRSLKSED TAMYYCARHD GGAMDYWGQG TSVTVSS (e) Tumor Antigen A

[00176] Tumor antigen A33 is another illustrated Disease Associated Antigen. The Amino Acid Sequence of the Light Chain Variable Domain of an exemplary humanized anti-A33 antibody (gpA33 mAb 1) is (SEQ ID NO:45): QIVLTQSPAI MSASPGERVT MTCSARSSIS FMYWYQQKPG SSPRLLIYDT SNLASGVPVR FSGSGSGTSY SLTISRMEAE DAATYYCQQW SSYPLTFGSG TKLELKR 139 L77b Ln / nznZ / Ε / ΥΙΛ

[00177] The Amino Acid Sequence of the Variable Domain of Exemplary humanized anti-A33 Heavy Chain (gpA33 mAb 1) antibody is (SEQ ID NO:46): QVQLQQSGPE LVKPGASVKI SCKASGYTFS GSWMNWVKQR PGQGLEWIGR IYPGDGETNY NGKFKDKATL TADKSSTTAY MELSSLTSVD SAVYFCARIY GNNVYFDVWG AGTTVTVSS (f) Tumor Antigen 5T4

[0178] Tumor antigen 5T4 is another illustrated Disease Associated Antigen. The Amino Acid Sequence of the Light Chain Variable Domain of an exemplary humanized anti-5T4 mAb 1 antibody (5T4 mAb 1) is (SEQ ID NO:47): DIQMTQSPSS LSASVGDRVT ITCRASQGIS NYLAWFQQKP GKAPKSLIYR ANRLQSGVPS RFSGSGSGTD FTLTISSLQP EDVATYYCLQ YDDFPWTFGQ GTKLEIK

[00179] The Amino Acid Sequence of the Heavy Chain Variable Domain of exemplary humanized 5T4 mAb 1 is (SEQ ID NO:48): QVQLVQSGAE VKKPGASVKV SCKASGYTFT SFWMHWVRQA PGQGLEWMGR IDPNRGGTEY NEKAKSRVTM TADKSTSTAY MELSSLRSED TAVYYCAGGN PYYPMDYWGQ GTTVTVSS

[0180] The Amino Acid Sequence of the Light Chain Variable Domain of an exemplary humanized 5T4 mAb 2 second antibody (5T4 mAb 2) is (SEQ ID NO:49): 140 Lzzb Ln / nznz / E / YiA DVLMTQTPLS LPVSLGDQAS ISCRSSQSIV YSNGNTYLEW YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSHVP FTFGSGTKLE IK

[00181] The Amino Acid Sequence of the Variable Domain of Second exemplary humanized 5T4 mAb 2 Heavy Chain is (SEQ ID NO:50): QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYWITWVKQR PGQGLEWIGD IYPGSGRANY NEKFKSKATL TVDTSSSTAY MQLSSLTSED SAVYNCARYG PLFTTWDPN SYAMDYWGQG TSVTVSS (g) ROR1 antigen

[00182] The ROR1 tumor antigen is another illustrated Disease Associated Antigen. Exemplary anti-ROR1 antibodies include 2A2 (WO 2010 / 124188), Rll (WO 2012 / 075158) and R12 (WO 2012 / 075158) antibodies.

[00183] The Amino Acid Sequence of the Variable Domain of Light Chain of the 2A2 antibody is (SEQ ID NO:53): DIVMTQSQKI MSTTVGDRVS ITCKASQNVD AAVAWYQQKP GQSPKLLIYS ASNRYTGVPD RFTGSGSGTD FTLTISNMQS EDLADYFCQQ YDIYPYTFGG GTKLEIK

[00184] The Amino Acid Sequence of the Heavy Chain Variable Domain of the 2A2 antibody is (SEQ ID NO:54): QVQLQQSGAE LVRPGASVTL SCKASGYTFS DYEMHWVIQT PVHGLEWIGA IDPETGGTAY NQKFKGKAIL TADKSSSTAY MELRSLTSED SAVYYCTGYY DYDSFTYWGQ GTLVTVSA 141 Lzzb Ln / nznz / E / YiA

[00185] The Amino Acid Sequence of the Variable Domain of Light Chain of the Rll antibody is (SEQ ID NO:55): ELVMTQTPSS TSGAVGGTVT INCQASQSID SNLAWFQQKP GQPPTLLIYR ASNLASGVPS RFSGSRSGTE YTLTISGVQR EDAATYYCLG GVGNVSYRTS FGGGTEVWK

[00186] The Amino Acid Sequence of the Variable Domain of Heavy Chain of the Rll antibody is (SEQ ID NO:56): QSVKESEGDL VTPAGNLTLT CTASGSDIND YPISWVRQAP GKGLEWIGFI NSGGSTWYAS WVKGRFTISR TSTTVDLKMT SLTTDDTATY FCARGYSTYY GDFNIWGPGT LVTISS

[00187] The Amino Acid Sequence of the Variable Domain of Light Chain of the R12 antibody is (SEQ ID NO:57): ELVLTQSPSV SAALGSPAKI TCTLSSAHKT DTIDWYQQLQ GEAPRYLMQV QSDGSYTKRP GVPDRFSGSS SGADRYLIIP SVQADDEADY YCGADYIGGY VFGGGTQLTV TG

[00188] The Amino Acid Sequence of the Variable Domain of Heavy Chain of the R12 antibody is (SEQ ID NO:58): QEQLVESGGR LVTPGGSLTL SCKASGFDFS AYYMSWVRQA PGKGLEWIAT IYPSSGKTYY ATWVNGRFTI SSDNAQNTVD LQMNSLTAAD RATYFCARDS YADDGALFNI WGPGTLVTIS S

[00189] One aspect of the present invention (discussed in detail below) is the provision of an additional preferred humanized anti-ROR1 antibody (ROR1 mAb 1). This additional preferred ROR1 mAb 1 has a Light Chain Variable Domain having the sequence (SEQ ID L77b Ln / nznZ / Ε / ΥΙΛ 142 NOT :51) : QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VL

[00190] The Amino Acid Sequence of the Variable Domain of Additional preferred humanized ROR1 mAb 1 Heavy Chain is (SEQ ID NO:52): QEQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMSWVRQA PGKGLEWVAT IYPSSGKTYY ADSVKGRFTI SSDNAKNSLY LQMNSLRAED TAVYYCARDS YADDAALFDI WGQGTTVTVS S IV. Binding Site Selection: Site A, Site B and Site C

[00191] As indicated above, preferred Tri-specific Binding Molecules of the present invention are at least tri-specific, having an outer Diabody-Like Binding Domain (Site A) that is located furthest from a Joining Domain III, an internal Diabody Link (Site B) that is located closest to the Joining Domain III and the Joining Domain III itself (Site C). As used herein, a description of a Tri-Specific Binding Molecule, such as X / Y / Z indicates that Binding Domain X is at Site A, Binding Domain Y is at Site B, and Binding Domain Z is at Site C. For example, the Tri-Specific Binding Molecules designation B7-H3 mAb 1 / CD3 mAb 2 / CD3 mAb 1 143 Lzzb Ln / nznz / E / YiA CD8 indicate that the B7-H3 mAb 1 Variable Domain occupies Site A of the Tri-Specific Binding Molecule, the CD3 mAb 2 Variable Domain occupies Site B, and the CD8 mAb 1 Variable Domain occupies Site C of the Tri-Specific Binding Molecule. TriSpecific Binding Molecule.

[00192] The present invention thus allows choice for which of such Sites is to be used to bind a particular desired epitope. One factor in guiding such selection, particularly with Trispecific Binding Molecules that bind CD3, CD8, and a disease-associated antigen, involves a consideration of the effect and desirability of trogocytosis. Trogocytosis is a process through which a cell can acquire a portion of a cell membrane from a cell contact (Masuda, S. et al. (2013) Possible Implication Of Fe γ Receptor-Mediated Trogocytosis In Susceptibility To Systemic Autoimmune Disease , Clin. Dev. Immunol. 2013: Article ID 345745, 6 pages); Dhainaut, M. et al. (2014) Regulation of Immune Reactivity by Intercellular Transfer, Front Immunol. 5:112; Ahmed, K.A. et al. (2011) Mechanisms Of Cellular Communication Through Intercellular Protein Transfer, J. Cell. Mol. Med. 15(7) :1458-1473; Ahmed, K.A. et al. (2008) Intercellular Trogocytosis Plays An Important Role In Modulation Of Immune Responses, Cell. Mol. Immunol. 5(4):261-269; LeMaoult, J. et al. (2007) Exchanges Of 144 Membrane Patches (Trogocytosis) Split Theoretical And Actual Functions Of Immune Cells, Hum. Immunol. 68(4):240-243; caumartin. J et al. (2006) Intercellular Exchanges Of Membrane Patches (Trogocytosis) Highlight The Next Level Of Immune Plasticity, Transpl. Immunol. 17(1):20-22).

[0193] Acquisition of cognate MHC class I ligands by trogocytosis induces cytotoxic T cells to become Acquired Treg cells that mediate the killing (fratricidal) of other cytotoxic T cells, thereby contributing to the killing of CD8 cells +cells (D'Acquisto, F. et al. (2011) CD3+CD4-CD8(Double Negative) T Cells: Saviors Or Villains Of The Immune Response? Biochem. Pharmacol. 82:333-340; Joly, E. et al (2003) What Is Trogocytosis And What Is Its Purposel Nat Immunol 4:815- Hudrisier, D. et al (2007) Capture Of Target Cell Membrane Components Via Trogocytosis Is Triggered By A Selected Set Of Surface Molecules On T Or B Cells, J. Immunol. 178:3637-3647).

[00194] A Tri-Specific Binding Molecule of the present invention possessing a CD3 Binding Domain as its C Site position has the attributes of an anti-CD3 antibody, and likewise such a Tri-Specific Binding Molecule possessing a CD8 Binding Domain its Site C position has the attributes of an anti-CD8 antibody. It has been shown that a neutrophil, monocyte, or macrophage that has Lzzb Ln / nznz / E / YiA 145 Lzzb Ln / nznz / E / YiA an Fe receptor that binds to the Fe domain of an anti-CD8 antibody (which has bound to a CD8 molecule on a T cell) is capable of transferring the antibody and the bound CD8 molecule from the T cell on its own via trogocytosis and then rapidly internalizes the antibody; bystander molecules such as TCR and CD3 can also be transferred in this process (Masuda, S. et al., (2013) Possible Implication of Fcg Receptor-Mediated Trogocytosis in Susceptibility to Systemic Autoimmune Disease, Clin. Develop. Immunol. 2013: Article ID 345745, 6 pages).

[00195] The structures of CD3 and CD8 differ in that CD3 lies close to the cell membrane, whereas CD8 extends beyond the cell membrane. Thus, it is expected that CD3 Fe receptor trogocytosis by an anti-CD3 antibody would be more efficient than CD8 Fe receptor trogocytosis by an anti-CD8 antibody.

[00196] This phenomenon indicates that a TriSpecific Binding Molecule of the present invention that binds CD3, CD8, and a disease-associated antigen whose CD3 Binding Domain is located at the C-site position will exhibit less cytotoxicity than a TriSpecific Binding Molecule of the present invention. Analogous Tri-Specific Binding in which the CD3 Binding Domain is located at either Site A or Site B. Thus, when choosing the site 146 Lzzb Ln / nznz / E / YiA of the CD3 Binding Domain at the position of Site C (as opposed to either Site A or B), the degree of cytotoxicity can be modulated. Additionally, pharmaceutical compositions containing a mixture of a C Site and a CD3 (or B) Site may be combined in order to obtain a preferred degree of cytotoxicity. V. ROR1 Anti-mAb 1 Antibody

[00197] As indicated above, one aspect of the present application is the provision of the highly preferred humanized anti-ROR1 antibody (ROR1 mAb 1), the Light Chain Variable Domain of which has the amino acid sequence (SEQ ID NO: 51): QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VL and whose Heavy Chain Variable Domain has the amino acid sequence (SEQ ID NO:52): QEQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMSWVRQA PGKGLEWVAT IYPSSGKTYY ADSVKGRFTI SSDNAKNSLY LQMNSLRAED TAVYYCARDS YADDAALFDI WGQGTTVTVS S

[00198] The CDRlI, CDRl2, and CDRl3 sequences of the Domain Variable Light Chain of the antibody mAb 1 of R0R1 are: CDRlI Light Chain Variable Domain (SEQ ID NO:117): TLSSGHKTDTID L77b Ln / nznZ / Ε / ΥΙΛ 147 CDR12 Light Chain Variable Domain (SEQ ID NO:118): LEGSGSY CDR13 Light Chain Variable Domain (SEQ ID NO:119): GTDYPGNYL

[00199] The sequences of CDRH1, CDRH2, and CDRH3 of the Heavy Chain Variable Domain of the ROR1 antibody mAb 1 are: CDRhI Heavy Chain Variable Domain (SEQ ID NO:120): GFTFSDYYMS CDRh2 Heavy Chain Variable Domain (SEQ ID NO:121): TIYPSSGKTYYADSVKG CDRh3 Heavy Chain Variable Domain (SEQ ID NO:122): DSYADDAALFDI

[00200] The ROR1 mAb 1 antibody mediates increased cytotoxicity and is less immunogenic relative to prior art anti-ROR1 antibodies (eg, anti-ROR1 R12 antibody).

[00201] The invention encompasses not only such sequences, but also intact R0R1 mAb 1 antibody derivatives (including chimeric or humanized derivatives thereof) possessing 1, 2, or 3 of the CDRs of such a Light Chain Variable Domain ( SEQ ID NO: 51, CDRs are shown underlined) or 1, 2 or 3 of the CDRs of such a Heavy Chain Variable Domain (SEQ ID NO: 52; CDRs are shown underlined), and which immunospecifically bind ROR1. More preferably, such antibodies encompass, 148 Lzzfr Ln / nznz / Ε / γΐΛ chimeric antibodies and humanized antibodies will possess 1, 2 or 3 of the CDRs of such a Light Chain Variable Domain (SEQ ID NO: 51, CDRs are shown underlined) and also 1, 2 or 3 of the CDRs of such a Heavy Chain Variable Domain (SEQ ID NO: 52; CDRs are shown underlined), and will immunospecifically bind to ROR1. More preferably, such encompassed antibodies, chimeric antibodies and humanized antibodies will possess all 3 of the CDRs of such a Light Chain Variable Domain, and all 3 of the CDRs of such a Heavy Chain Variable Domain and are capable of binding immunospecifically to ROR1.

[00202] The invention further encompasses fragments and derivatives of such encompassed ROR1 mAb 1 antibodies, including Fab, Fab', F(ab')2 Fv), single chain (scFv), BITES®, DART™ diabody molecules , mutants thereof, naturally occurring variants, and fusion proteins, all of which comprise 1, 2, or 3 of the CDRs Light Chain Variable Domains, or 1, 2, or 3 of the CDRs Heavy Chain Variable Domains , or 1, 2, or 3 of the Light Chain Variable Domain CDRs, and 1, 2, or 3 of the Heavy Chain Variable Domain CDRs, and which are capable of binding immunospecifically to ROR1.

[00203] In a preferred embodiment, such ROR1 mAb 1 antibodies or their fragments or derivatives may have variant Fc domains. Modifying the Mastery of Faith normally Lzzfr Ln / nznz / E / YiA 149 leads to an altered phenotype, eg altered serum half-life, altered stability, altered susceptibility to cellular enzymes, or altered effector function. It may be desirable to modify the antibody of the invention with respect to effector function, to increase the effectiveness of the antibody in the treatment of cancer, for example. Reduction or elimination of effector function is desirable in certain cases, for example in the case of antibodies whose mechanism of action involves the blocking or antagonism, but not the killing of cells bearing a target antigen. Increased effector function is generally desirable when targeting undesirable cells, such as tumor and foreign cells, where FcyRs are expressed at low levels, for example, tumor-specific B cells with low levels of FcyRIIB (for example, non-FcyRs lymphoma). Hodgkin, CLL, and Burkitt's lymphoma). In embodiments, the molecules of the invention with conferred or enhanced effector function activity are useful for treatment and / or prevention of a disease, disorder, or infection, where enhanced efficacy of effector function activity is desired.

[00204] In certain embodiments, such ROR1 mAb 1 antibodies or their fragments or derivatives comprise one or more modifications to the Fe Domain amino acids, which reduce the affinity and avidity of the Fe Domain such 150 Lzzb Ln / nznz / E / YiA such a molecule for one or more FcyR receptors. In other embodiments, such ROR1 mAb 1 antibodies or fragments or derivatives thereof may comprise one or more Fe Domain amino acid modifications that increase the affinity and avidity of the Fe Domain of such molecule for one or more FcR receptors. In other embodiments, molecules comprise a variant Fe Domain wherein the variant confers or mediates increased ADCC activity and / or increased FcyRIIA binding, relative to a molecule that does not comprise an Fe Domain or comprises an Fe Domain of wild type. In alternate embodiments, the molecules comprise a variant Fe Domain wherein the variant confers or mediates decreased ADCC activity (or other effector function) and / or increased binding to FcyRIIB, relative to a molecule that does not comprise the Fe Domain or comprising a wild-type Fe Domain.

[00205] In some embodiments, the invention encompasses such ROR1 mAb 1 antibodies or their fragments or derivatives that comprise a variant Fe Domain, the variant Fe Domain does not show detectable binding to any Fc and R, relative to a comparable molecule that comprises the wild-type Fe Domain. In other embodiments, the invention encompasses such ROR1 mAb 1 antibodies or fragments or derivatives thereof that comprise a variant Fe Domain, the variant Fe Domain binding only to one 151 L77b Lívnznz / E / YIA only FcyR, preferably one of FcyRIIA, FcyRIIB, or FcyRIIIIA.

[00206] Such R0R1 mAb 1 antibodies or fragments or derivatives thereof may comprise altered affinities for Fcy receptor activation and / or inhibition. In one embodiment, the antibody or molecule comprises a variant Fe Domain that has increased affinity for FcyRIIB and decreased affinity for FcyRIIIIA and / or FcyRIIA, relative to a molecule comparable to a wild-type Fe Domain. In another embodiment, such ROR1 mAb 1 antibodies or fragments or derivatives thereof may comprise a variant Fe Domain having decreased affinity for FcyRIIB and increased affinity for FcyRIIIIA and / or FcyRIIA, relative to a molecule comparable to an Fe Domain of wild type. In yet another embodiment, such ROR1 mAb 1 antibodies or fragments or derivatives thereof comprise a variant Fe Domain having decreased affinity for FcyRIIB and decreased affinity for FcyRIIIIA and / or FcyRIIA, relative to a molecule comparable to an Fe Domain of wild type. In yet another embodiment, such ROR1 mAb 1 antibodies or their fragments or derivatives may comprise a variant Fc Domain having unchanged affinity for FcyRIIB and decreased (or increased) affinity for FcyRIIIIA and / or FcyRIIA, relative to a comparable molecule. with a wild-type Faith Mastery. 152 Lzzfr Ln / nznz / Ε / γΐΛ

[00207] In certain embodiments, the invention encompasses such ROR1 mAb 1 antibodies or their fragments or derivatives that comprise a variant Fc Domain with an altered affinity for FcyRIIA and / or FcyRIIA such that the immunoglobulin has increased effector function, for example , ADCC. Non-limiting examples of effector cell functions include ADCC, antibody-dependent cell phagocytosis (ADCP), phagocytosis, opsonization, opsonophagocytosis, cell attachment, rosetting, Clq binding, and CDC.

[00208] In a preferred embodiment, the alteration in effector affinity or function is at least 2-fold, preferably at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, for at least 8-fold, at least 9-fold, at least 10-fold, at least 50-fold, or at least 100-fold, relative to a comparable molecule comprising a wild-type Fe Domain. In other embodiments of the invention, the variant Fe Domain specifically binds one or more FcRs with at least 65%, preferably at least 70%, 75%, 80%, 85%, 90%, 95%, 100 %, 125%, 150%, 175%, 200%, 225%, or 250% greater affinity relative to a molecule comprising a wild-type Fe Domain. Such measurements may be in vivo or in vitro assays, and in a preferred embodiment are in vitro assays such as ELISA or surface plasma resonance assays. Lzzfr Ln / nznz / Ε / γΐΛ 153

[00209] In different embodiments, such ROR1 mAb 1 antibodies or their fragments or derivatives comprise a variant Fc Domain wherein the variant agonizes at least one FcyR receptor activity, or antagonizes at least one FcR receptor activity. from FcyR. In a preferred embodiment, the molecules comprise a variant that agonizes (or antagonizes) one or more activities of FcyRIIB, eg, B-cell receptor-mediated signaling, B-cell activation, B-cell proliferation, antibody production. , B-cell intracellular calcium influx, cell cycle progression, FcyRIIB-mediated inhibition of FcsRI signaling, FcyRIIB phosphorylation, SHIP recruitment, SHIP phosphorylation and Shc association, or activity of one or more downstream molecules (for example, MAP kinase, JNK, p38, or Akt) in the FcyRIIB signal transduction pathway. In another embodiment, the molecules comprise a variant that agonizes (or antagonizes) one or more FcsRI activities, eg, mast cell activation, calcium mobilization, degranulation, cytokine production, or serotonin release.

[00210] In certain embodiments, such ROR1 mAb 1 antibodies or fragments thereof comprise a Domain comprising the Fe Domain of two or more IgG isotypes (eg, IgG1, IgG2, IgG3, and IgG4). The different IgG isotypes exhibit 154 Lívnznz / E / YIA different physical and functional properties including serum half-life, complement fixation, FcyR binding affinities, and effector function activities (eg, ADCC, CDC, etc.) due to differences in the amino acid sequences of their hinge and / or Fe domains, for example as described in Flesch, B.K. and Neppert, J. (1999) Functions Of The Fe Receptors For Immunoglobulin G”, J. Clin. Lab. Anal. 14:141-156; Chappel, M.S. et al. (1993) Identification Of A Secondary Fe Gamma RI Binding Site Within A Genetically Engineered Human IgG Antibody, J. Biol. Chem. 33:25124-25131; Chappel, M.S. et al. (1991) Identification Of The Fe Gamma Receptor Class I Binding Site In Human IgG Through The Use Of Recombinant IgGl / IgG2 Hybrid And Point-Mutated Antibodies, Proc. nati. Acad. Sci. (U.S.A.) 88:9036-9040; Brüggemann, M. et al. (1987) Comparison Of The Effector Functions Of Human Immunoglobulins Using A Matched Set Of Chimeric Antibodies, J. Exp. Med 166:1351-1361. This type of variant Fe Domain can be used alone, or in combination with an amino acid modification, to affect Fe-mediated effector function and / or binding activity. In combination, the amino acid modification of the IgG Hinge / Fc Domain may display similar functionality (eg, increased affinity for FcyRIIA) and may act additively or, more preferably, synergistically to 155 L77b Ln / nznZ / Ε / ΥΙΛ modify effector functionality in the molecule of the invention, relative to a molecule of the invention comprising a wild-type Fe Domain. In other embodiments, the amino acid modification and the IgG Fe Domain may display opposite functionality (eg, increased and decreased affinity for FcyRIIA, respectively) and may act to selectively moderate or reduce specific functionality in the molecule of the invention, with respect to a molecule of the invention which does not comprise an Fe Domain or which comprises a wild-type Fe Domain of the same isotype.

[00211] In a specific preferred embodiment, such ROR1 mAb 1 antibodies or fragments thereof comprise a variant Fe Domain, wherein the variant Fe Domain comprises at least one amino acid modification relative to a wild-type Fe Domain , such that the molecule has altered affinity for an FcR, provided that the variant Fe Domain does not have a substitution at positions that make direct contact with FcR based on crystallographic and structural analysis of Fc- FcR such as groins described by Sondermann, P. et al. (2000) The 3.2-A Crystal Structure Of The Human IgGl Fe Fragment-Fc GammaRIII Complex, Nature 406:267-273. Examples of positions within the Fe Domain that make direct contact with FcR are the residues of 156 Lzzb Ln / nznz / E / YiA amino acids 234-239 (Hinge Domain), amino acid residues 265-269 (B / C loop), amino acid residues 297299 (C' / E loop), and amino acid residues 327-332 (F / G loop) . In some embodiments, the molecules of the invention comprise variant Fe Domains that comprise the modification of at least one residue that does not make direct contact with an FcyR based on structural and crystallographic analysis, for example, it is not within the Binding Site. of Fc-FcyR.

[00212] Variant Fe Domains are well known in the art, and any known Fe variants can be used in the present invention to confer or modify the effector function displayed by such ROR1 mAb 1 antibodies or fragments thereof comprising a Fe Domain (or portion thereof) as functionally assayed, eg, in an NK-dependent or macrophage-dependent assay. For example, the Fe Domain variants identified as altering effector function are described in PCT Publication Nos. WO 04 / 063351; WO 06 / 088494; WO 07 / 024249; WO 06 / 113665; WO 07 / 021841; WO 07 / 106707; WO 2008 / 140603, and any suitable variants described therein can be used in the present molecules.

[00213] In certain embodiments, such ROR1 mAb 1 antibodies or fragments thereof comprise a variant Fe Domain, having one or more amino acid modifications in one or more 157 Lzzb Ln / nznz / E / YiA more sites, than the altering modification(s) (relative to a wild-type Fe Domain). The ratio of affinities of the variant Fe Domain to an activating FcyR (such as FcyRIIA or FcyRIIIIA) relative to an inhibiting FcyR (such as FcyRIIB): Change from Wild Type to Variant in Affinity to FcyRactiVation Relationship of affinities=___________________________________ Change from Wild Type to Variant in Affinity to FcyRinhibition Where an Fe variant has a ratio of affinities greater than 1, the methods of the invention are of particular use in providing a therapeutic or prophylactic method of a disease, disorder or infection, or in alleviation of a symptom thereof, where efficacy is increased. of FcyR-mediated effector cell (eg, ADCC) function is desired, eg, cancer or infectious disease. Where an Fe variant has an affinity ratio of less than 1, the methods of the invention are of particular use in providing therapeutic or prophylactic treatment of a disease or disorder, or alleviation of a symptom thereof, where a decreased efficacy of the function FcyR-mediated effector cell depletion is desired, eg, autoimmune or inflammatory disorders. Table 3 lists exemplar single, double, triple, quadruple, and quintuple mutations by their affinity ratio that is greater or less than 158 1, and further information concerning these mutations can be found in PCT Publication Nos. WO 04 / 063351; WO 06 / 088494; WO 07 / 024249; WO 06 / 113665; WO 07 / 021841; WO 07 / 106707; WO 2008 / 140603. Lzzb Ln / nznz / E / YiA 159 Table 3: List of Single and Multiple Copy Mutations to Provide Affinities Ratio Single Double Triple Quadruple Quintuple > 1 F243L F243L & F243L, P247L & L234F, F243L, L235V, D270E R292G R292P F243L Y300L & N421K F243L2, Y300 &L2P R202 , F243L, R292P & Y300L F243L, R292P, Y300L P396L & R292P F243L & F243L, R292P & L235Q, F243L, L235P, F243L, P396L V305I R292P & Y300L D270E & F243L, R292P & F243L, P247L, R292P, P396L P396L D270E & N421K Y300L & R292P & F243L, Y300L & F243L, R255L, P396L Y300L P396L D270E & P396L F243L, R292P V305I & P247L, N421K D270E & F243L, D270E, G316D & R416G R292P, V305I, Y300L & R292P & R255L, D270E & F243L, D270E , P396L P396L P396L K392T & P396L Y300L & D270E, G316D & F243L, D270E, P396L R416G P396L & Q419H P396L & D270E, K392T & F243L, R292P, Q419H P396L Y300L, & P396L D270E, P396L & F243L, R292P, Q419H V305I & P396L V284M, R292L & P247L, D270E, K370N Y300L & N421K R292P, Y300L & R255L, D270E, P396L R292G & P396L R255L, D270E, Y300L & P396L D270E, G316D, P396L & R416G < 1 Y300L F243L & F243L, R292P & P396L P396L V305I P247L N421K & R255L P396L & R292P V305I & K392T P396L & P396L Q419H & Lzzfr Ln / nznz / Ε / γΐΛΐ 160 L77b Ln / nznZ / Ε / ΥΙΛ

[00214] In a specific embodiment, in variant Fe Domains, any of the amino acid modifications (eg, substitutions) at any of positions 235, 240, 241, 243, 244, 247, 262, 263, 269, 298, 328, or 330 and preferably one or more of the following residues: A240, 1240, L241, L243, H244, N298, 1328 or V330. In a different specific embodiment, in the variant Fe Domains, any of the amino acid modifications (eg, substitutions) at any of positions 268, 269, 270, 272, 276, 278, 283, 285, 286, 289, 292 and, preferably one or more of the following residues: H280, Q280, y280, G290, S290, T290, Y290, N294, K295, P296, D298, N298, P298, V298, 1300 or L300.

[00215] In a preferred embodiment, in variant Fc Domains that bind an FcyR with altered affinity, any of the amino acid modifications (eg, substitutions) in any of the positions 255, 256, 258, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 300, 301, 303, 305, 307, 309, 312, 320, 322, 326, 329, 330, 332, 331, 333, 334, 335, 337, 338, 339, 340, 359, 360, 373, , 376, 416 , 419, 430, 434, 435, 437, 438 or 439. Preferably, the variant Fe Domain has any of the following residues: A256, N268, Q272, 161 L77b Ln / nznZ / Ε / ΥΙΛ D286, Q286, S286, Α290, S290, Α298, Μ301, Α312, Ε320,Μ320, Q320, R320, Ε322, Α326, d326, E326, N326, S326, K330,T339, A333, A334, E334, H334, L334, M334, Q334, V334, K335,Q335, A359, A360 or A430.

[00216] In a different embodiment, in variant Fe Domains that bind an FcyR (via its Fe Domain pathway) with reduced affinity, any of the amino acid modifications (eg, substitutions) at any of the 252 positions , 254, 265, 268, 269, 270, 278, 289,292, 293, 294, 295, 296, 298, 300, 301, 303, 322, 324, 327,329, 333, 335, 338, 340, 373, 376, 382, ​​388, 389, 414, 416,419, 434, 435, 437, 438, or 439.

[00217] In a different embodiment, in variant Fe Domains that bind to an FcyR (via its Fe Domain pathway) with increased affinity, any of the amino acid modifications (eg, substitutions) at any of the positions 280, 283, 285, 286, 290, 294, 295, 298, 300, 301, 305, 307, 309, 312, 315, 331, 333, 334, 337, 340, 360, 378, 398, or 430. In a different embodiment, in the variant Fe Domains that FcyRIIA binds with increased affinity, any of the following residues: A255, A256, A258, A267, A268, N268, A272, Q272, A276, A280, A283, A285, A286, D286, Q286, S286, A290, S290, M301, E320, M320, Q320, R320, E322, A326, d326, E326, S326, K330, A331, Q335, A337 or A430.

[00218] Preferred variants include one or more 162 L77b Ln / nznZ / Ε / ΥΙΛ modifications in any of the positions: 228,230, 231, 232, 233, 234, 235, 239, 240, 241, 243, 244, 245,247, 262, 263, 264, 265, 266, 271, 273, 275, 281, 284, 291,296, 297, 298, 299, 302, 304, 305, 313, 323, 325, 326, 328,330 or 5332.

[00219] Particularly preferred variants include one or more modifications selected from the groups A-AI: To 228E, 228K, 228Y or 228G; B 230A, 230E, 230Y or 230G; C 231E, 231K, 231Y, 231P or 231G; D 232E, 232K, 232Y, 232G; E 233D; F 2341 or 234F; G 235D, 235Q, 235P, 2351 or 235V; H 239D, 239E, 239N or 239Q; I 240A, 2401, 240M or 240T; J 243R, 243, 243Y, 243L, 243Q, 243W, 243H or 2431; K244H; L 24 5A; M 247G, 247V or 247L; No. 262A, 262E, 2621, 262T, 262E, or 262F; 0 263A, 2631, 263M or 263T; P 264F, 264E, 264R, 2641, 264A, 264T or 264W; Q 265F, 265Y, 265H, 2651, 265L, 265T, 265V, 265N or 265Q; R 266A, 2661, 266M or 266T; S 271D, 271E, 271N, 271Q, 271K, 271R, 271S, 271T, 271H, 271A, 271V, 271L, 2711, 271F, 271M, 271Y, 271W or 271G; T-2731; U 275L or 275W; V 281D, 281K, 281Y or 281P; W 284E, 284N, 284T, 284L, 284Y or 284M; X 291D, 291E, 291Q, 291T, 291H, 2911 or 291G; Y 299A, 299D, 299E, 299F, 299G, 299H, 2991, 299K, 299L, 299M, 299N, 299P, 299Q, 299R, 299S, 299V, 299W or 299Y; Z3021; AA 304D, 304N, 304T, 304H or 304L AB 3051; AC 313F; 163 AD 3231; AE 325A, 325D, 325E, 325G, 325H, 3251, 325L, 325K, 325R, 325S, 325F, 325M, 325T, 325V, 325Y, 325W, or 325P; AF 328D, 328Q, 328K, 328R, 328S, 328T, 328V, 3281, 328Y, 328W, 328P, 328G, 328A, 328E, 328F, 328H, 328M, or 328N; AG 330L, 330Y, 3301 or 330V; AH 332A, 332D, 332E, 332H, 332N, 332Q, 332T, 332K, 332R, 332S, 332V, 332L, 332F, 332M, 332W, 332P, 332G, or 332Y; and AI 336E, 336K or 336Y L77b Ln / nznZ / E / YIA

[00220] Even more particularly preferred variants include one or more modifications selected from the groups 1-105: Variant Group Variant Group 130L / I332E 54 S239D / D265L / N297D / I332E 2 D265F / N297E / I332E 55 S239D / D265T / N297D / I332E 3 D265Y / N297D / I332E 56 S239D / D265V / N2 / I332E 57 S239D / D265Y / N297D / I332E 5 F241E / F243Q / V262T / V264F 58 S239D / I332D 6 F241E / F243Q / V262T / V264E / I332E 59 S239D / I332E 7 F241E / F243R / V262E / V264R 60 S239D / I332E / A330I 8 F241E / F243R / V262E / V264R / I332E 61 S239D / I332N 9 F241E / F243Y / V262T / V264R 62 S239D / I332Q 10 F241E / F243Y / V262T / V264R / I332E 63 S239D / N297D / I332E 11 F241L / F243L / V262I / V264I 64 S239D / N297D / I332E / A330Y 12 F241L / V262I 65 S239D / N297D / I332E / A330Y / F241S / F243H / V262T / V264T 13 F241R / F243Q / V262T / V264R 66 S239D / N297D / I332E / K326E 14 F241R / F243Q / V262T / V264R / I332E 67 S239D / N297D / I332E / L235D 164 15 F241W / F243W / V262A / Ν284Ά 68 S239D / S298A / I332E 16 F241Y / F243Y / V262T / V264T 69 S239D / V264I / A330L / I332E 17 F241Y / F243Y / N262T / V264T / N297D / I332E 70 S239D / V264I / I332E 18 F243L / V262I / V264W 71 S239D / V264I / S298A / I332E 19 P243L / V264I 72 S239E / D265N 20 L328D / I332E 73 S239E / D265Q 21 L328E / I332E 74 S239E / I332D 22 L328H / I332E 7 5 S239E / I332E 23 L328I / I332E 76 S239E / I332N 24 L328M / I332E 77 S239E / I332Q 25 L328N / I332E 78 S239E / N297D / I332E 26 L328Q / I332E 79 S239E / V264I / A330Y / 1332 E 27 L328T / I332E 80 S239 S239E / V264I / S298A / A330Y / I332E 29 N297D / A330Y / I332E 82 S239N / A330L / I332E 30 N297D / I332E 83 S239N / A330Y / I332E 31 N297D / I332E / S239D I 332E 85 S239N / I332E 33 N297D / T299L / I332E 86 S239N / I332N 34 N297D / T299F / I332E / N297D / T299H / I332E 87 S239N / I332Q 35 N297D / T299I8 / I329E1 S328N 32E 36 N297D / T299L / I332E 89 S239Q / I332D 37 N297D / T299V / I332E 90 S239Q / I332E 38 N297E / I332E 91 S239Q / I332N 39 N297S / I332E 92 S239Q / I332Q 40 P230A / E233D / I332E 93 S239Q / V264I / I332E 41 P244H / P245A / P247V 94 S298A / I332E 42 S239D / A330L / I332E 95 V264E / N297D / I332E 43 S239D / A330Y / I332E 96 V264I / A330L / I332E 44 S239D / A330Y / I332E / K326E 97 V264I / A330Y / I332E 45 S239D / A330Y / I332E / K326T 98 V264I / I332E 46 S239D / A330Y / I332E / L234I 99 V264I / S298A / I332E 47 S239D / A330Y / I332E / L2 3 5D 100 Y296D / N297D / I332E Lzzb Ln / nznz / Ε / γΐΛΐ 165 48 S239D / A330Y / I332E / V24 0I 101 Y296E / N297D / 1332 E 49 S239D / A330Y / I332E / V264T 102 Y296H / N297D / I332E 50 S239D / A330Y / I332E / V266I 103 / I332E 104 Y296Q / N297I / I332E 52 S239D / D265H / N297D / I332E 105 Y296T / N297D / I332E 53 S239D / D265I / N297D / I332E

[00221] In one embodiment, such R0R1 mAb 1 antibodies or fragments thereof will comprise a variant Fe Domain having at least one modification in the Fe Domain. In certain embodiments, the variant Fe Domain comprises at least one substitution selected from the group consisting of L235V, F243L, R292P, Y300L, V305I, and P396L, where the numbering is that of the EU index as in Kabat. In a specific modality, the variant Domain of Faith comprises: (A) at least one substitution selected from the group consisting of F243L, R292P, Y300L, V305I, and P396L; (B) at least two substitutions selected from the group consisting of: (1) F243L and P396L; (2) F243L and R292P; and (3) R292P and V305I; (C) at least three substitutions selected from the group consisting of: 166 Lzzb Ln / nznz / E / YiA (1) F243L, R292P and Y300L; (2) F243L, R292P and V305I; (3) F243L, R292P and P396L; and (4) R292P, V305I and P396L; D) at least four substitutions selected from the group consisting of: (1) F243L, R292P, Y300L, and P396L; and (2) F243L, R292P, V305I, and P396L; or E) at least five substitutions selected from the group consisting of: (1) F243L, R292P, Y300L, V305I and P396L; and (2) L235V, F243L, R292P, Y300L, and P396L.

[00222] In another specific modality, the variant Faith Domain comprises substitutions of: (A) F243L, R292P, and Y300L; (B) L235V, F243L, R292P, Y300L, and P396L; or (C) F243L, R292P, Y300L, V305I, and P396L.

[00223] In other embodiments, such R0R1 mAb 1 antibodies or fragments thereof may possess any Fe variants known in the art, such as those described in Jefferis, R. et al. (2002) Interaction Sites On Human IgG-Fc For FcgammaR: Current Models, Immunol. Lett. 82:57-65; Presta, L.G. et al. (2002) Engineering Therapeutic Antibodies For Improved Function, Biochem. Soc. Trans. 30:487-90; Idusogie, E.E. et al. (2001) Engineered Antibodies With Increased 167 Activity To Recruit Complement, J. Immunol. 166:2571-75; Shields, R. L. et al. (2001) High Resolution Mapping Of The Binding Site On Human IgGl For Fe Gamma RI, Fe Gamma RII, Fe Gamma RUI, And FcRn And Design Of IgGl Varlants With Improved Binding To The Fe gamma R , J. Biol. Chem. 276: 65916604; Idusogie, E.E. et al. (2000) Mapping Of The Clq Binding Site On Rituxan, A Chimeric Antibody With A Human IgG Fe, J. Immunol. 164:4178-84; Reddy, M.P. et al. (2000) Elimination Of Fe Receptor-Dependent Effector Functions Of A Modified IgG4 Monoclonal Antibody To Human CD4, J. Immunol. 164:1925-1933; Xu, D. et al. (2000) In Vitro Characterization of Five Humanized OKT3 Effector Function Variant Antibodies, Cell. Immunol. 200:16-26; Armor, K. L. et al. (1999) Recombinant human IgG Molecules Lacking Fcgamma Receptor I Binding And Monocyte Triggering Activities, Eur. J. Immunol. 29:2613-24; Jefferis, R. et al. (1996) Modulation Of Fe(Gamma)R And Human Complement Activation By IgG3-Core Oligosaccharide Interactions, Immunol. Lett. 54:101-04; Lund, J. et al. (1996) Multiple Interactions Of IgG With Its Core Oligosaccharide Can Modulate Recognition By Complement And Human Fe Gamma Receptor I And Influence The Synthesis Of Its Oligosaccharide Chains, J. Immunol. 157:4963-4969; Hutchins et al. (1995) Improved Biodistribution, Tumor Targeting, And Reduced Immunogenicity In Mice With A Gamma 4 Variant Of Campath-IH, L77b Lívnznz / E / YIA Lzzb Ln / nznz / E / YiA 168 Proc. nati. Acad. Sel. (U.S.A.) 92:11980-84; Jefferis, R. et al. (1995) Recognition Sites On Human IgG For Fe Receptor Gamma: The Role Of Glycosylation, Immunol. Lett. 44:11117; Lund, J. et al. (1995) Oligosaccharide-Protein Interactions Tn IgG Can Modulate Recognition By Fe Gamma Receptors, FASEB J. 9:115-19; Alegre, M. L. et al. (1994) A Non-Activating Humanized Anti-CD3 Monoclonal Antibody Retains Immuno-suppressive Properties In Vivo, Transplantation 57:1537-1543; Lund et al. (1992) Multiple Binding Sites On The CH2 Domain Of IgG For Mouse Fe Gamma Rll, Mol. Immunol. 29:53-59; Lund et al. (1991) Human Fe Gamma RI And Fe Gamma RII Interact With Distinct But Overlapping Sites On Human IgG, J. Immunol. 147:2657-2662; Duncan, A.R. et al. (1988) Localization Of The Binding Site For The Human High-Affinity Fe Receptor On IgG, Nature 332:563-564; US Patent Nos. 5,624,821; 5,885,573; 6,194,551; 7,276,586; and 7,317,091; and PCT Publications WO 00 / 42072 and PCT WO 99 / 58572.

[00224] In some embodiments, such ROR1 mAb 1 antibodies or fragments thereof may further comprise one or more glycosylation sites such that one or more carbohydrate moieties are covalently attached to the molecule. Preferably, such ROR1 mAb 1 antibodies or their fragments with one or more glycosylation sites and / or one or more Fe Domain modifications confer or have a Lzzb Ln / nznz / E / YiA 169 increased antibody-mediated effector function, eg, increased ADCC activity, compared to unmodified ROR1 mAb 1 antibodies or fragment. In some embodiments, the invention further encompasses such ROR1 mAb 1 antibodies or fragments thereof comprising one or more amino acid modifications that are known directly or indirectly to interact with a carbohydrate portion of the Fe Domain, including but not limited to amino acids in positions 241, 243, 244, 245, 245, 249, 256, 258, 260, 262, 264, 265, 296, 299, and 301. Amino acids that directly or indirectly interact with a carbohydrate portion of an Fe Domain are known in the art, see, for example, Jefferis, R. et al. (1995) Recognition Sites On Human IgG For Fe Gamma Receptors: The Role Of Glycosylation, Immunol. Lett. 44:111-17.

[00225] In another embodiment, the invention encompasses such ROR1 mAb 1 antibodies or fragments thereof that have been modified by introducing one or more glycosylation sites at one or more sites of the molecules, preferably without altering the functionality of the molecules, eg, FcyR target antigen binding activity. Glycosylation sites can be introduced into the variable and / or constant region of the molecules of the invention. As used herein, glycosylation sites include any specific amino acid sequence in an antibody to which a 170 Lzzfr Ln / nznz / Ε / γΐΛ oligosaccharide (ie, carbohydrates containing two or more simple sugars linked together) will bind specifically and covalently. Oligosaccharide side chains are typically linked to the main chain of an antibody via N- or O-linkage. N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxyamino acid, eg, serine, threonine. Such ROR1 mAb 1 antibodies or fragments thereof may comprise one or more glycosylation sites, including N-linked or O-linked glycosylation sites. Any glycosylation site for N-linked or O-linked glycosylation known in the art used in accordance with the present invention. An exemplary N-linked glycosylation site is the amino acid sequence: Asn-X-Thr / Ser, where X can be any amino acid and Thr / Ser indicates a threonine or a serine. Such a site or sites can be introduced into a molecule of the invention using methods well known in the art to which the invention pertains (see, for example, In vitro Mutagenesis, Recombinant DNA: A Short Course, J. D. Watson, et al. W.H. Freeman and Company, New York, 1983, chapter 8, pp. 106-116, which is incorporated herein by reference in its entirety An exemplary method of introducing a site 171 Lzzfr Ln / nznz / Ε / γΐΛ glycosylation in such antibodies ROR1 mAb 1 or its fragments may comprise: modification or mutation of an amino acid sequence of the molecule such that it is derived from the desired Asn-X-Thr / Ser sequence , or expression of an antibody mAb 1 to ROR1 encoding the nucleic acid molecule having such a sequence.

[00226] In some embodiments, the invention encompasses methods of modifying the carbohydrate content of such ROR1 mAb 1 antibodies or fragments thereof by adding or deleting a glycosylation site. Methods for modifying the carbohydrate content of antibodies (and molecules comprising antibody domains, eg, Fe Domain) are well known in the art and encompassed within the invention, see, for example, US Pat. No. 6,218,149; EP 0 359 096 Bl; United States Publication No. US 2002 / 0028486; WO 03 / 035835; US Publication No. 2003 / 0115614; US Patent No. 6,218,149; US Patent No. 6,472,511; all of which are incorporated herein by reference in their entirety. In other embodiments, the invention encompasses methods of modifying the carbohydrate content of such ROR1 mAb 1 antibodies or fragments thereof by deleting one or more endogenous carbohydrate portions of the molecule. In a specific embodiment, the invention encompasses displacement of the glycosylation site Lzzb Ln / nznz / E / YiA 172 of the Fe Domain of an antibody, by modifying positions adjacent to 297. In a specific embodiment, the invention encompasses the modification and position 296 such that position 296 and not position 297 is glycosylated.

[00227] Effector function can be modified by techniques such as those described in PCT Publication Nos. WO 04 / 063351; WO 06 / 088494; WO 07 / 024249; WO 06 / 113665; WO 07 / 021841; WO 07 / 106707; WO 2008 / 140603, or by other means. For example, the cysteine ​​residue(s) can be introduced into the Fe Domain, thereby allowing interchain disulfide bond formation in this region, resulting in the generation of a homodimeric antibody that may have internalization capacity. enhanced and / or increased complement-mediated cell killing and ADCC. See Carón, P.C. et al. (1992) Engineered Humanized Dimeric Ponas Of IgG Are More Effective Antibodies, J. Exp. Med. 176:1191-1195; Shopes, B. (1992) A Genetically Engineered Human IgG Mutant Wlth Enhanced Cytolytic Activity, J. Immunol. 148(9):2918-2922. Homodimeric antibodies with increased antitumor activity can also be prepared using heterobifunctional crosslinkers as described in Wolff, E.A. et al. (1993) Monoclonal Antibody Homodimers: Enhanced Antitumor Activity In Nude Mice, Cancer Research 53:2560-2565. Alternatively, an antibody can be designed so that 173 Lzzb Ln / nznz / E / YiA has dual Fe Domains and thus may have increased complement lysis and ADCC capabilities (Stevenson, G.T. et al. (1989) A Chímenle Antibody Wlth Dual Fe Domales (bisFabFc) Prepared By Manípulatlons At The IgG Hinge, AntiCancer Drug Design 3:219-230.

[00228] The fact that a single amino acid alteration of a CDR residue can result in loss of functional binding (Rudikoff, S. etc. (1982) Single Amino Acid Substitution Altering Antigen-Binding Specificity, Proc. Nati. Acad. Sci. (USA) 79(6):1979-1983) provides a means of systematically identifying alternative functional CDR sequences. In a preferred method of obtaining such variant CDRs, a polynucleotide encoding the CDRs is mutagenized (for example, via random mutagenesis or by a site-directed method (for example, polymerase chain-mediated amplification with primers encoding the mutated locus)) to produce a CDR having a substituted amino acid residue. By comparing the identity of the relevant residue in the original (functional) CDR sequence to the identity of the substituted (non-functional) variant CDR sequence, the BLOSUM62.iij substitution record for that substitution can be identified. The BLOSUM system provides a matrix of amino acid substitutions created by scanning a sequence database for cumulative alignments (Eddy, S.R. 174 (2004) Where Did The BL0SUM62 Alignment Score Matrix Come From?, Nature Biotech. 22(8) :1035-1036; Henikoff, J.G. (1992) Amino acid substitution matrices from protein blocks, Proc. nati. Acad. Sci. (USA) 89:10915-10919; Karlin, S. et al. (1990) Methods For Assessing The Statistical Significance Of Molecular Sequence Features By Using General Scoring Schemes, Proc. nati. Acad. Sci. (USA) 87:2264-2268; Altschul, S.F. (1991) Amino Acid Substitution Matrices From An Information Theoretic Perspective, J. Mol. Biol. 219, 555-565. Currently, the most advanced BLOSUM database is the BLOSUM62 database (BLOSUM62 . ii j ). Table 4 presents the BLOSUM62.iij substitution logs (the higher the log, the more conservative the substitution and thus the more likely the substitution will not affect function). If an antigen-binding fragment comprising the CDR fails to bind to ROR1, for example, then the BLOSUM62.iij substitution record is considered to be insufficiently conservative, and a new candidate substitution is selected and produced to have an antigen-binding record. higher substitution. Thus, for example, if the original residue was glutamate (E), and the surrogate non-functional residue was histidine (H), then the BLOSUM62.iij substitution score will be 0, and more conservative changes are preferred (eg, to aspartate, asparagine, glutamine, or Usina) . Lzzb Ln / nznz / E / YiA 175 L77b Ln / nznZ / Ε / ΥΙΛΙ Table 4 A R N D C Q E G H I L K M F P S T W Y V A -1 -2 -2 0 -1 -1 0 -2 -1 -1 -1 -1 -2 -1 + 1 0 -3 -2 0 R -1 sil 0 -2 -3 + 1 0 -2 0 -3 -2 +2 -1 -3 -2 -1 -1 -3 -2 -3 N -2 0 + 1 -3 0 0 0 + 1 -3 -3 0 -2 -3 -2 +1 0 - 4 -2 -3 D -2 -2 + 1 -3 0 +2 -1 -1 -3 -4 -1 -3 -3 -1 0 -1 -4 -3 -3 C 0 -3 -3 -3 1Í -3 -4 -3 -3 -1 -1 -3 -1 -2 -3 -1 -1 -2 -2 -1 Q -1 + 1 0 0 -3 +2 -2 0 -3 -2 + 1 0 -3 -1 0 -1 -2 -1 -2 E -1 0 0 +2 -4 +2 -2 0 -3 -3 + 1 -2 -3 -1 0 -1 -3 -2 -2 G 0 -2 0 -1 -3 -2 -2 1! -2 -4 -4 -2 -3 -3 -2 0 -2 -2 -3 -3 H -2 0 + 1 -1 -3 0 0 -2 -3 -3 -1 -2 -1 -2 - 1 -2 -2 +2 -3 I -1 -3 -3 -3 -1 -3 -3 -4 -3 +2 -3 +1 0 -3 -2 -1 -3 -1 +3 L -1 -2 -3 -4 -1 -2 -3 -4 -3 +2 je -2 +2 0 -3 -2 -1 -2 -1 + 1 K -1 +2 0 -1 -3 +1 + 1 -2 -1 -3 -2 -1 -3 -1 0 -1 -3 -2 -2 M -1 -1 -2 -3 -1 0 -2 -3 -2 + 1 +2 -1 0 -2 -1 -1 -1 -1 + 1 F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 Hit -4 -2 -2 + 1 +3 -1 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 -1 -1 -4 -3 -2 S + 1 -1 + 1 0 -1 0 0 0 - 1 -2 -2 0 -1 -2 -1 + 4 + 1 -3 -2 -2 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 - 1 + 1 -2 -2 0 w -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 + 1 -4 -3 -2 +2 -3 Y -2 -2 -2 -3 -2 -1 -2 -3 +2 -1 -1 -2 -1 +3 -3 -2 -2 +2 -1V 0 -3 -3 -3 -1 -2 -2 -3 -3 +3 + 1 -2 +1 -1 -2 -2 0 -3 -1 lili

[00229] The invention thus contemplates the use of random mutagenesis to identify improved CDRs. Phage display technology, alternatively, can be used to increase (or decrease) CDR affinity. This technology, referred to as affinity maturation, employs the mutagenesis or CDR pathway and uses reselection of the target antigen or an antigenic fragment thereof to identify antibodies that have CDRs that bind with higher (or lower) affinity to the antigen. when compared to the initial or parental antibody (See, eg, Glaser et al. (1992) J. Immunology 149:3903). Mutagenization of entire codons before individual nucleotides results in a semi-randomized repertoire of amino acid mutations. Libraries will L77b Ln / nznZ / Ε / ΥΙΛ 176 can be constructed to consist of a pool of variant clones each differing by a single amino acid alteration in a single CDR and containing variants representing every possible amino acid substitution for each residue or CDR. Mutants with increased (or decreased) binding affinity for the antigen can be sorted by contacting the immobilized mutants with the labeled antigen. Any sorting method known in the art can be used to identify mutant antibodies with increased or decreased affinity to the antigen (for example, ELISA) (see See Wu et al. 1998, Proc. Nati. Acad. Sel. (U.S.A.) 95: 6037; Yelton et al., 1995, J. Immunology 155:1994). The CDR pathway that randomizes the Light Chain can possibly be used (see, Schier et al., 1996, J. Mol. Bio. 263:551).

[00230] Methods for performing such affinity maturation are described for example in: Krause, J.C. et al. (2011) An Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function Of A Human Antibody, MBio. 2(1) pii: e00345-10. doi: 10.1128 / mBio.00345-10; Kuan, C.T. et al. (2010) Affini ty-Matured Anti-Glycoprotein NMB Recombinant Immunotoxins Targeting Malignant Gliomas And Melanomas, Int. J. Cancer 10.1002 / ijc.25645; Hackel, B.J. et al. (2010) Stability And CDR Composition Blases Enrích Binder Functionality Landscapes, J. Mol. Biol. 401(1):84-96; 177 Montgomery, D. L. et al. (2009) Affinity Maturation And Characterization Of A Human Monoclonal Antibody Against HIV-1 gp41, MAbs 1 (5):462-474; Gustchina, E. et al. (2009) Affinity Maturation By Targeted Diversification Of The CDRH2 Loop Of A Monoclonal Fab Derived From A Synthetic Naive Human Antibody Library And Directed Against The Internal Trimeric Coiled-Coil Of Gp41 Yields A Set Of Fabs With Improved HLV-1 Neutralization Potency And Breadth, Virology 393(1):112-119; Finlay, W.J. et al. (2009) Affinity Maturation Of A Humanized Rat Antibody For Anti-RAGE Therapy: Comprehensive Mutagenesis Reveals A High Level Of Mutational Plasticity Both Inside And Outside The ComplementarityDetermining Regions, J. Mol. Biol. 388(3):541-558; Bostrom, J. et al. (2009) Improving Antibody Binding Affinity And Specificity For Therapeutic Development, Methods Mol. Biol. 525:353-376; Steidl, S. et al. (2008) In Vitro Affinity Maturation Of Human GM-CSF Antibodies By Targeted CDR Diversification, Mol. Immunol. 46(1):135-144; and Barderas, R. et al. (2008) Affinity maturation of antibodies assisted by in silico modelling, Proc. nati. Acad. Sci. (USA) 105(26):9029-9034. As an example, multiwell plates can be coated with a selected ROR1 mAb 1 antibody (eg, 100 ng / well in carbonate buffer at room temperature for 2 hours) and subsequently incubated with soluble ROR1 added to Lzzb Ln / nznz / E / YiA L77b Ln / nznZ / Ε / ΥΙΛ 178 a 1 / 10 dilution and incubate at room temperature for 16 hours or dilute to a concentration of 50 ng / ml in PBS-TBSA (0.05 ml added to each well and incubate for at least 2 h at room temperature). The plate is then washed and recombinant antibodies are diluted starting at 0.5 pg / ml in PBS-T-BSA then added and incubated for 1 hour at room temperature. The binding of the recombinant antibodies to the captured antigen is then measured using, for example, an anti-human IgG-HRP conjugate and TMB substrate. After arrest of color development using dilute sulfuric acid, the plate is read at 450 nM and the highest affinity antibodies are identified (see, for example, US Patent No. 7,351,803). SAW. Pharmaceutical Compositions

[00231] In one embodiment, the present invention includes pharmaceutical compositions for the treatment of a cancer or disease characterized by the presence of a disease-associated antigen. Such compositions include bulk drug compositions useful in the manufacture of pharmaceutical compositions (eg, impure or non-sterile compositions) and pharmaceutical compositions (ie, compositions that are suitable for administration to a subject or patient) that can be 179 Lzzb Ln / nznz / E / YiA Use in the preparation of unit dosage forms. Such compositions comprise a prophylactically or therapeutically effective amount of a modified diabody of the present invention, or a combination of such agents and a pharmaceutically acceptable carrier. Preferably, the compositions of the invention comprise a prophylactically or therapeutically effective amount of one or more molecules of the invention and a pharmaceutically acceptable carrier. The invention also encompasses pharmaceutical compositions comprising such modified diabodies and a second therapeutic antibody that is specific for a particular disease antigen, and a pharmaceutically acceptable carrier.

[00232] As used herein, the terms "treatment" or "treating" denote a procedure to obtain a beneficial or desired result, including and preferably a beneficial or desired clinical result. Such beneficial or desired clinical results include, but are not limited to, one or more of the following: reduction in the proliferation of (or destruction of) infected cells or other diseased cells, decrease in symptoms resulting from the disease, increase in the quality of life of those who suffer from the disease, decrease in the dose of other medications required to treat the disease, retardation of the progression of the disease, and / or prolongation of 180 Lívnznz / E / YIA survival of companion animal recipients.

[00233] In a specific embodiment, the term pharmaceutically acceptable means approved by a federal or state government regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans (see, for example, Remington: The Science and Practice of Pharmacy (2012) Alien, Loyd V., Jr. (Ed.) 22F Edition, Pharmaceutical Press, London UK). The term "carrier" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete), excipient, or vehicle in which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.Water is a preferred carrier when the pharmaceutical composition is administered intravenously.Saline solutions and solutions Aqueous dextrose and glycerol may also be used as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, monostearate glycerol, talc, sodium chloride, milk 181 Lzzb Ln / nznz / E / YiA dried skim, glycerol, propyl, glycol, water, ethanol and the like. The composition, if desired, may also contain minor amounts of wetting or emulsifying agents, or buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like.

[00234] Generally, the ingredients of the compositions of the invention are administered either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule. or sachet indicating the amount of active agent. Where the composition is to be administered by infusion, it may be supplied with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration.

[00235] The compositions of the invention may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include, but are not limited to, those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric, etc., and 182 those formed with cations such as those derived from hydroxides of sodium, potassium, ammonium, calcium, ferric, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[00236] The invention also provides a pharmaceutical pack or kit comprising one or more containers containing a modified diabody of the present invention, alone or with such a pharmaceutically acceptable carrier. Additionally, one or more other prophylactic or therapeutic agents useful for the treatment of a disease may also be included in the pharmaceutical package or kit. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with the recipient(s) may be a notification in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceutical substances or biologics, the notification reflecting approval by the manufacturing, use, or sale agency for human administration.

[00237] The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises one or more molecules of the invention. In another embodiment, a kit further comprises one or more other prophylactic or therapeutic agents useful for the treatment of Lzzfr Ln / nznz / Ε / γΐΛ Lzzfr Ln / nznz / Ε / γΐΛ 183 cancer or a disease characterized by the presence of an antigen associated with the disease, in one or more vessels. In another embodiment, a kit further comprises one or more antibodies or diabodies that bind one or more disease-associated antigens. In certain embodiments, the other prophylactic or therapeutic agent is a chemotherapeutic. In other embodiments, the prophylactic or therapeutic agent is a biological or hormonal therapeutic agent. VII. Methods for Producing the Tri-Specific Binding Molecules of the Present Invention

[00238] The Tri-Specific Binding Molecules of the present invention are most preferably produced through recombinant expression of nucleic acid molecules encoding such polypeptides, as is well known in the art.

[00239] Polypeptides of the invention can be conveniently prepared using solid phase peptide synthesis (Merrifield, B. (1986) Solid Phase Synthesis, Science 232(4748):341-347; Houghten, R.A. (1985) General Method For The Rapid Solid-Phase Synthesis Of Large Numbers Of Peptides: Specificity Of Antigen-Antibody Interaction At The Level Of Individual Amino Acids, Proc. Nati. Acad. Sci. (E.U.A.) 82 (15): 5131-5135; Ganesan, A. (2006) Solid-Phase Synthesis In The Twenty-First Century, Mini Rev. Med. Chem. L77b Ln / nznZ / Ε / ΥΙΛ 184 6(1):3-10).

[00240] In an alternative, the antibodies can be made recombinantly and expressed using any method known in the art. Antibodies can be made recombinantly by first isolating the antibodies made from host animals, obtaining the gene sequence, and using the gene sequence to express the antibody recombinantly in host cells (eg, CHO cells). Another method that can be employed is to express the antibody sequence in transgenic plants (eg tobacco) or milk. Suitable methods for expressing antibodies recombinantly in plants or milk have been described (see, for example, Peeters et al. (2001) Production Of Antibodies And Antibody Fragmenta In Plants, Vaccine 19:2756; Lonberg, N. et al. (1995) Human Antibodies From Transgenic Mice, Int Rev Immunol 13:65-93 and Pollock et al (1999) Transgenic Milk As A Method For The Production Of Recombinant Antibodies, J Immunol Methods 231:147-157). Suitable methods for making antibody derivatives, eg, chimeric, humanized, single chain, etc., are known in the art. In another alternative, the antibodies can be made recombinantly by phage display technology (see, for example, US Patent Nos. 5,565,332; 5,580,717; 5,733,743; 6,265,150; and Winter, G. et al. (1994) Making L77b Ln / nznZ / Ε / ΥΙΛ 185 Antibodies By Phage Display Technology, Annu. Rev. Immunol. 12,433-455).

[00241] Antibodies or protein of interest can be subjected to sequencing using Edman degradation, which is well known to those of skill in the art. Peptide information generated from mass spectrometry or Edman degradation can be used to design probes or primers that are used to clone the protein of interest.

[00242] An alternative method of cloning the protein of interest is by immunoadsorption using purified proteins or portions thereof to cells expressing the antibody or protein of interest. The immunoadsorption procedure can be carried out by obtaining a cDNA library from tissues or cells expressing or overexpressing the desired cDNAs in a second cell type, and sorting transfected cells of the second cell type for specific binding to the desired protein. Detailed descriptions of the methods used in the cloning of mammalian genes encoding cell surface proteins by immunoadsorption can be found in the art (see, for example, Aruffo, A. et al. (1987) Molecular Cloning Of A CD28 cDNA By A High-Efficiency COS Cell Expression System, Proc. Nati. Acad. Sci. (U.S.A.) 84:8573-8577 and Stephan, J. Lzzb Ln / nznz / E / YiA 186 et al. (1999) Selective Cloning Of Cell Surface Proteins Involved In Organ Development: Epithelial Glycoprotein Is Involved In Normal Epithelial Differentiation, Endocrinol. 140:5841-5854) .

[00243] cDNAs encoding antibodies, and other peptide agonists, antagonists, and modulators can be obtained by reverse transcribing mRNAs from a particular cell type according to standard methods in the art. Specifically, mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook et al. supra or extracted by commercially available nucleic acid binding resins following the accompanying instructions provided by the manufacturers (eg, Qiagen, Invitrogen, Promega). The synthesized cDNAs are then introduced into an expression vector to produce the antibody or protein of interest in cells of a second type. It is implied that an expression vector must be replicable in host cells either as episomes or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, and cosmids.

[00244] Vectors containing the polynucleotides of interest can be introduced into the host cell by 187 any of a number of suitable means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (eg, where the vector is an infectious agent, such as vaccinia virus). The choice to introduce vectors or polynucleotides will often depend on the characteristics of the host cell.

[00245] Any of the host cells capable of overexpressing heterologous DNA can be used for the purpose of isolating the genes encoding the antibody, polypeptide or protein of interest. Non-limiting examples of suitable mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. Preferably, the host cells express the cDNAs at a level approximately 5-fold, more preferably 10-fold, more preferably 20-fold, more preferably 50-fold, more preferably 100-fold than the endogenous antibody. corresponding or protein of interest, if present, in the host cells. Sorting of host cells for specific binding to a desired protein is preferably done by immunoassay or FACS. A cell that overexpresses the antibody or protein of interest can be identified in this way.

[00246] Various techniques are also available L77b Ln / nznZ / Ε / ΥΙΛ Lzzb Ln / nznz / E / YiA 188 which can now be used to produce mutant peptide agonists, antagonists, and modulators, which encode additions, deletions, or changes in the amino acid sequence of the resulting protein relative to the original agonist, antagonist, or modulator peptide molecule.

[00247] The invention includes modifications to the Tri-Specific Binding Molecules of the invention that do not significantly affect their properties and variants that have increased or decreased activity. Modification of polypeptides is a routine practice in the art. Examples of modified polypeptides include polypeptides with conservative amino acid residue substitutions, one or more amino acid deletions or additions that do not significantly and detrimentally change functional activity, or the use of chemical analogues. Amino acid residues that can be conservatively substituted for one another include but are not limited to: glycine / alanine; valine / isoleucine / leucine; asparagine / glutamine; aspartic acid / glutamic acid; serine / threonine; lysine / arginine; and phenylalanine / tyrosine. These polypeptides also include glycosylated and unglycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation, and phosphorylation. Preferably, the 189 Lzzb Ln / nznz / E / YiA amino acid substitutions may be conservative, ie, the substituted amino acid may possess similar chemical properties as those of the original amino acid. Such conservative substitutions are known in the art, and examples have been provided above. Amino acid modifications can range from changing or modifying one or more amino acids to completely redesigning a region, such as the Variable Domain. Changes to the Variable Domain may alter the binding affinity and / or specificity. Other methods of modification include using coupling techniques known in the art, including, but not limited to, enzymatic means, oxidative substitution and chelation. Modifications can be used, for example, for attachment of labels for immunoassay, such as attachment of radioactive moieties for radioimmunoassay. Modified polypeptides are made using established procedures in the art and can be screened using standard assays known in the art.

[00248] The invention also encompasses fusion proteins comprising one or more fragments or regions of the polypeptides and antibodies of this invention. In one embodiment, a fusion polypeptide is provided comprising at least 10 contiguous amino acids from the variable Light Chain region and at least 10 amino acids from the 190 Lzzb Ln / nznz / E / YiA variable Heavy Chain region. In another embodiment, the fusion polypeptide contains a heterologous immunoglobulin constant region. In another embodiment, the fusion polypeptide contains a Light Chain Variable Domain and a Heavy Chain Variable Domain from an antibody produced from a publicly deposited hybridoma. For purposes of this invention, an antibody fusion protein contains one or more polypeptide domains that specifically bind to a desired viral epitope or to a desired activation receptor of an effector immune cell or a protein present on the surface of an effector cell. immune system expressing such an activation receptor and another amino acid sequence to which the native molecule does not bind, eg, a heterologous sequence or a sequence homologous from another region.

[00249] The invention includes polypeptides comprising an amino acid sequence of the antibodies of this invention. The polypeptides of this invention can be made by procedures known in the art. Polypeptides can be produced by proteolytic or other degradation of antibodies, by recombinant methods (ie, single or fusion polypeptides) as described above, or by guimic synthesis. Antibody polypeptides, especially shorter polypeptides up to about 50 191 Lzzb Ln / nznz / E / YiA amino acids, are conveniently made by chemical synthesis. Chemical synthesis methods are known in the art and are commercially available. For example, such a polypeptide could be produced by an automated polypeptide synthesizer using the solid phase method. VIII. Uses of the Compositions of the Invention

[00250] The present invention encompasses compositions, including pharmaceutical compositions, comprising the Tri-Specific Binding Molecules of the invention, polypeptides derived from such molecules, polynucleotides comprising sequences encoding such molecules or polypeptides, and other agents as described at the moment.

[00251] The Tri-Specific Binding Molecules of the present invention have the ability to coordinately bind three epitopes, and thus find substantial use in diagnostics, chemical separation, and therapeutic agents not involving such epitopes. For example, such molecules can be used as a reagent in an intercalation immunoassay.

[00252] In the embodiment in which such Tri-Specific Binding Molecules bind to an epitope of a Disease Associated Antigen, such molecules can be used to treat the disease or condition associated with or characterized Lzzb Ln / nznz / E / YiA 192 for the expression of such Disease Associated Antigen. Thus, without limitation, pharmaceutical compositions comprising such molecules can be used in the diagnosis or treatment of cancer, and diseases caused by infection by pathogens (eg, bacterial, fungal, viral, or protozoan). IX. Administration Methods

[00253] The compositions of the present invention may be provided for the treatment, prophylaxis, and amelioration of one or more symptoms associated with a disease, disorder, or infection by administering to a subject an effective amount of a pharmaceutical composition of the invention. In a preferred aspect, such compositions are substantially purified (ie, substantially free of substances that limit their effect or cause unwanted side effects). In a specific embodiment, the subject is an animal, preferably a mammal such as a non-primate (eg, bovine, equine, feline, canine, rodent, etc.), or a primate (eg, monkey such as a cynomolgus monkey). , human, etc.) . In a preferred embodiment, the subject is a human.

[00254] Various delivery systems are known and can be used to deliver the compositions of the invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of 193 to express the antibody or fusion protein, receptor-mediated endocytosis (see, for example, Wu et al. (1987) Receptor-Mediated In Vitro Gene Transformation By A Soluble DNA Carrier System, J. Biol. Chem. 262:4429 -4432), construction of a nucleic acid as part of a retroviral or other vector, etc.

[00255] Methods for administering the TriSpecific Binding Molecules of the present invention include, but are not limited to, parenteral (eg, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal (eg, intranasal routes). and oral). In a specific embodiment, the molecules of the invention are administered intramuscularly, intravenously, or subcutaneously. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (for example, oral mucosa, rectal and intestinal mucosa, etc.), and can be administered in conjunction with other biologically active agents. Administration can be systemic or local. In addition, pulmonary administration, for example, through the use of an inhaler or nebulizer, and formulation with an aerosolizing agent, can also be employed. See, for example, US Patent Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; Lívnznz / E / YIA Lzzfr Ln / nznz / Ε / γΐΛ 194 5,874,064; 5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO 92 / 19244; WO 97 / 32572; WO 97 / 44013; WO 98 / 31346; and WO 99 / 66903, each of which is incorporated herein by reference in its entirety.

[00256] The invention also allows the Tri-Specific Binding Molecules of the present invention to be packaged in a hermetically sealed container such as a vial or sachet indicating the quantity of such molecules. In one embodiment, the TriSpecific Binding Molecules of the present invention are supplied as a dry sterilized lyophilized powder or water-free concentrate in a hermetically sealed container and can be reconstituted, for example, with water or saline at the appropriate concentration for administration to a subject. Preferably, the Tri-Specific Binding Molecules of the present invention are supplied as a sterile dry lyophilized powder in a hermetically sealed container at a unit dosage of at least 5 pg, more preferably at least 10 pg, at least 15 HP, at least 25 HP, at least 50 HP, at least 100 HP, or at least 200 HP.

[00257] The lyophilized Tri-Specific Binding Molecules of the present invention should be stored at 2-8oC in their original container and the molecules should be administered within 12 hours, preferably in a 195 Lzzb Ln / nznz / E / Yi Within 6 hours, within 5 hours, within 3 hours, or within 1 hour after reconstitution. In an alternative embodiment, the TriSpecific Binding Molecules of the present invention are supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of molecules, fusion protein, or conjugated molecule. Preferably, the liquid form of the Tri-Specific Binding Molecules of the present invention is supplied in a hermetically sealed container in which the molecules are present in a concentration of at least 1 pg / ml, more preferably at least 2.5 pg / ml, at least 5 pg / ml, at least 10 pg / ml, at least 50 pg / ml, or at least 100 pg / ml.

[00258] As used herein, an effective amount of a pharmaceutical composition, in one embodiment, is an amount sufficient to effect beneficial or desired results, including, without limitation, clinical results such as amelioration of symptoms resulting from the disease that attenuate a symptom of infection (for example, viral load, fever, pain, sepsis, etc.) or a symptom of cancer (for example, the proliferation of cancer cells, presence of tumors, tumor metastasis, etc. .), in order to increase the quality of life of those who suffer from the disease, decreasing the dose of other medications required to treat the disease, improving Lzzfr Ln / nznz / E / YiA 196 the effect of other medication such as targeting and / or internalization, delaying the progression of the disease, and / or prolonging the survival of individuals.

[00259] An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to reduce the proliferation of (or the effect of) the viral presence and / or reduce and / or retard the development of the viral disease, either directly or indirectly. In some embodiments, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an effective amount may be considered in the context of administering one or more chemotherapeutic agents, and a single agent may be considered to be given an effective amount if, together with one or more additional agents, it can or can be used. achieve a desirable result. Although individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typical dosages for administration of antibodies comprise one or more unit doses between 0.1 to 100 mg / kg / body weight.

[00260] The amount of the Tri-Specific Binding Molecule of the present invention will be effective in the treatment, 197 prevention or amelioration of one or more symptoms associated with a disorder that can be determined by standard clinical techniques. The precise dose that is employed in the formulation will also depend on the route of administration, and the severity of the condition, and must be decided according to the good judgment of the professional and each of the patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. For the TriSpecific Binding Molecules of the present invention, the dosage administered to a patient is typically at least about 0.01 pg / kg / day, at least about 0.05 pg / kg / day, at least about 0.1 pg / kg / day, at least about 0.2 pg / kg / day, at least about 0.5 pg / kg / day, at least about 1 pg / kg / day, at least about 2 pg / kg / day, at least about 5 pg / kg / day, at least about 10 pg / kg / day, at least about 20 pg / kg / day, at least about 50 pg / kg / day, at least about 0.1 pg / kg / day day, or more than the subject's body weight.

[00261] Preferably, the dosage administered to a patient is between about 0.01 pg / kg / day and about 0.1 pg / kg / day, more preferably, between about 0.01 pg / kg / day and about 50 Lzzb Ln / nznz / E / YiA 198 pg / kg / day, more preferably between about 0.01 pg / kg / day and about 50 pg / kg / day, more preferably between about 0.01 pg / kg / day and about 10 pg / kg / day, of more preferably, between about 0.01 pg / kg / day and about 1 pg / kg / day, more preferably, between about 0.01 pg / kg / day and about 0.5 pg / kg / day, and most preferably, between about 0.01 pg / kg / day and approximately 0.1 pg / kg / day of the subject's body weight. The dosage and frequency of administration of the Tri-Specific Binding Molecules of the invention can be reduced or altered by increasing the uptake and tissue penetration of the Tri-Specific Binding Molecules by modifications such as, for example, lipidation.

[00262] In another embodiment, the patient is administered a treatment regimen comprising one or more doses of such prophylactically or therapeutically effective amount of the Tri-Specific Binding Molecules encompassed by the invention, wherein the treatment regimen is administered for 2 days, 3 days, 4 days, 5 days, 6 days or 7 days. In certain embodiments, the treatment regimen comprises intermittently administering doses of the prophylactically or therapeutically effective amount of the TriSpecific Binding Molecules encompassed by the invention (eg, administering a dose on day 1, day 2, day 3, and day 4 of a Lzzfr Ln / nznz / Ε / γΐΛ Lzzb Ln / nznz / E / YiA 199 given week and not administering doses of the prophylactically or therapeutically effective amount of the Tri-Specific Binding Molecules encompassed by the invention on day 5, day 6 and day 7 of the same week). Typically, there are 1, 2, 3, 4, 5, or more treatment cycles. Each cycle can be of the same regimen or a different regimen.

[00263] In another embodiment, the administered dose is intensified during the first quarter, first half, or first two-thirds or three-quarters of the regimen(s) (eg, during the first, second, or third 4-cycle treatment regimens ) until the prophylactically or therapeutically effective daily amount of the Tri-Specific Binding Molecules encompassed by the invention is reached.

[00264] In one embodiment, the dosage of the Tri-Specific Binding Molecules of the present invention administered to a patient can be calculated for use as a single agent therapy. In another embodiment, the Tri-Specific Binding Molecules of the present invention are used in combination with other therapeutic compositions and the dosage administered to a patient is less than when such Tri-Specific Binding Molecules are used as a single agent therapy.

[00265] In a specific modality, it may be desirable to administer the pharmaceutical compositions of the invention L77b Ln / nznZ / Ε / ΥΙΛ 200 locally to the area in need of treatment; this can be accomplished, for example, and not by way of limitation, by local infusion, by injection, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes , or fibers. Preferably, when administering a molecule of the invention, care should be taken to use materials to which the molecule does not absorb.

[00266] In another embodiment, the compositions can be delivered in a vesicle, in particular a liposome (see Langer (1990) New Methods Of Drug Delivery, Science 249:1527-1533 ); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pages 3 17-327; see generally ibid.).

[00267] In yet another embodiment, the compositions can be delivered in a controlled release or sustained release system. Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more molecules of the invention. See, for example, US Patent No. 4,526,938; PCT Publication WO 91 / 05548; PCT Publication WO 96 / 20698; Ning et al. (1996) Intratumoral Radioimmunotheraphy Of A Human Colon Cancer 201 Xenograft Using A Sustained-Release Gel, Radiotherapy & Oncology 39:179-189, Song et al. (1995) Antibody Mediated Lung Targeting Of Long-Circulating Emulsions, PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek et al. (1997) Biodegradable Polymeric Carriers For A bFGF Antibody For Cardiovascular Application, Pro. Int'l. symp. Control. I laughed. Bioact. Mater. 24:853-854; and Lam et al. (1997) Microencapsulation Of Recombinant Humanized Monoclonal Antibody For Local Delivery, Proc. Int'l. symp. Control I laughed. Bioact. Mater. 24:759-760, each of which is incorporated herein by reference in its entirety. In one embodiment, a pump can be used in a controlled release system (see Langer, supra; Sefton, (1987) Implantable Pumps, CRC Crit. Rev. Biomed. Eng. 14:201-240; Buchwald et al. (1980 ) Long-Term, Continuous Intravenous Heparin Administration By An Implantable Infusion Pump In Ambulatory Patients With Recurrent Venous Thrombosis, Surgery 88:507-516 and Saudek et al (1989) A Preliminary Trial Of The Programmable Implantable Medication System For Insulin Delivery, N. Engl. J. Med. 321:574-579). In another embodiment, polymeric materials can be used to achieve controlled release of antibodies (see, for example, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and L77b Ln / nznZ / Ε / ΥΙΛ Lzzb Ln / nznz / E / YiA 202 Performance, Smolen and Ball (eds.), Wiley, New York (1984) ; Levi et al. (1985) Inhibition Of Calcification Of Bioprosthetic Heart Valves By Local Controlled-Release Diphosphonate, Science 228:190-192; During et al. (1989) Controlled Release Of Dopamine From A Polymeric Brain Implant: Ln Vivo Characterization, Ann. Neurol. 25:351-356; Howard et al. (1989) Intracerebral Drug Delivery In Rats With Lesion-Induced Memory Deficits, J. Neurosurg. 7(1):105112 ); US Patent No. 5,679,377; US Patent No. 5,916,597; US Patent No. 5,912,015; US Patent No. 5,989,463; US Patent No. 5,128,326; PCT Publication No. WO 99 / 15154; and PCT Publication No. WO 99 / 20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly( methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-coglycolides) (PLGA), and polyorthoesters. In yet another embodiment, a controlled-release system can be placed in close proximity to the therapeutic target (for example, the lungs), thereby requiring only a fraction of the systemic dose (see, for example, Goodson, in Medical L77b Ln / nznZ / Ε / ΥΙΛ 203 Applications of Controlled Release, supra, vol. 2, pages 115-138 (1984)). In another embodiment, polymeric compositions useful as controlled release implants are used according to Dunn et al. (See U.S. 5, 945, 155). This particular method is based on the therapeutic effect of the in situ controlled release of the bioactive material from the polymer system. Implantation can generally occur anywhere within the body of the patient in need of therapeutic treatment. In another embodiment, a non-polymeric sustained delivery system is used, whereby a non-polymeric implant in the subject's body is used as a drug delivery system. On implantation in the body, the organic solvent of the implant will dissipate, disperse, or leach from the composition into the surrounding tissue fluid, and the non-polymeric material will gradually coagulate or precipitate to form a solid, microporous matrix (see U.S. 5,888,533) .

[00268] Controlled release systems are discussed in the review by Langer (1990, New Methods Of Drug Delivery, Science 249:1527-1533). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more therapeutic agents of the invention. See, for example, US Patent No. 4,526,938; the Lzzb Ln / nznz / E / YiA 204 International Publication Nos. WO 91 / 05548 and WO 96 / 20698; Ning et al. (1996) Intratumoral Radioimmunotherapy Of A Human Colon Cancer Xenograft Using A Sustained-Release Gel, Radiotherapy & Oncology 39:179-189, Song et al. (1995) Antibody Mediated Lung Targeting Of Long-Circulating Emulsions, PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek et al. (1997) Biodegradable Polymeric Carriers For A bFGF Antibody For Cardiovascular Application, Pro. Int'l. symp. Control. I laughed. Bioact. Mater. 24:853-854; and Lam et al. (1997) Microencapsulation Of Recombinant Humanized Monoclonal Antibody For Local Delivery, Proc. Int'l. symp. Control I laughed. Bioact. Mater. 24:759-760, each of which is incorporated herein by reference in its entirety.

[00269] In a specific embodiment where the composition of the invention is a nucleic acid encoding a Tri-Specific Binding Molecule of the present invention, the nucleic acid can be administered in vivo to promote expression of its Tri-Specific Binding Molecule of the present invention. Encoded specific, by constructing it as part of a suitable nucleic acid expression vector and by administering it so as to become intracellular, for example, by use of a retroviral vector (see US Patent No. 4,980,286), or by direct injection, or through the use of microparticle bombardment (for example, a gene gun; Biolistics, 205 L77b Ln / nznZ / Ε / ΥΙΛ Dupont), or coating with lipids or cell surface receptors or transfection agents, or by administering it in ligation to a homeobox peptide known to enter the nucleus (see for example, Joliot et al. (1991) Antennapedia Homeobox Peptide Requintes Neural Morphogenesis, Proc. Nati. Acad. Sci. (U.S.A.) 88:18641868), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated into the host cell's DNA for expression via homologous recombination.

[00270] Treatment of a subject with a therapeutically or prophylactically effective amount of the Tri-Specific Binding Molecules of the present invention may include a single treatment or, preferably, may include a series of treatments. In a preferred example, a subject is treated with molecules of the invention once a week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even longer. preference for approximately 4, 5, or 6 weeks. In other embodiments, the pharmaceutical compositions of the invention are administered once a day, twice a day, or three times a day. In other embodiments, the pharmaceutical compositions are administered once a week, twice a week, once every two weeks, once a month, once every six 206 weeks, once every two months, twice a year or once a year. It will also be appreciated that the effective dosage of the molecules used for treatment may increase or decrease during the cycle of a particular treatment.

[00271] Having now generally described the invention, the same will be more readily understood by reference to the following Examples. Such Examples are provided by way of illustration and are not intended to be limiting of the present invention unless otherwise specified. Example 1 Production and Properties of Exemplary Anti-CD3, Anti-CD8, Anti-B7-H3 Tri-Specific Binding Molecules

[00271] In order to develop a therapeutic molecule that may display greater specificity to CD8+ T cells, and more potent retargeted killing , Tri-Specific Binding Molecules that have the ability to coordinately bind CD3, CD8 and a Disease Associated Antigen were constructed. The produced Tri-Specific Binding Molecule additionally presented an Fe Domain to improve the lifetime of the Tri-Specific Binding Molecule in vivo. The general structures of the TriSpecific Binding Molecules are shown in Figures 4A-4D. An exemplary Tri-Specific Binding Molecule specific for the Disease Associated Antigen B7-H3 was constructed. the molecule of L77b Lívnznz / E / YIA 207 Tri-Specific Binding is referred to as B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1 to denote the relative positions of the Binding Domains within the Tri-Specific Binding Molecule. The Binding Domain of B7-H3 occupies position 5 of Site A, the Binding Domain of CD3 occupies the position of Site B and the CD8 Binding Domain occupies the position of the Site C (Figure 4A). The Tri-Specific Binding Molecule B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1 was composed of Lzzb Ln / nznz / E / YiA four different polypeptide chains (Table 5). Table 5 Polypeptide Chain Domains Binding Affinity 1 VL(B7-H3 mAb 1)-VH(CD3 mAb 2)-E-Helix-(CH2-CH3) Light Chain: B7-H3 Heavy Chain: CD3 2 VL( CD3 mAb 2)-VH(B7-H3 mAb 1)-K-Helix Light Chain: CD3 Heavy Chain: B7-H3 3 CD8 mAb 1 from CD8 Heavy Chain 4 CD8 mAb 1 from CD8 Light Chain

[00273] The amino acid sequence of the first polypeptide chain of the B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1 Tri-Specific Binding Molecule is (SEQ ID NO:59): DIQMTQSPSS LSASVGDRVT ITCRASQDIS NYLNWYQQKP GKAPKLLIYY TSRLHSGVPS RFSGSGSGTD FTLTISSLQP EDIATYYCQQ GNTLPPTFGG GTKLEIKGGG SGGGGEVQLV ESGGGLVQPG GSLRLSCAAS GFTFSTYAMN WVRQAPGKGL EWVGRIRSKY NNYATYYADS VKDRFTISRD DSKNSLYLQM NSLKTEDTAV YYCVRHGNFG NSYVSWFAYW GQGTLVTVSS GGCGGGEVAA LEKEVAALEK EVAALEKEVA ALEKGGGDKT HTCPPCPAPE AAGGPSVFLF 208 Lzzb Ln / nznz / E / YiA PPKPKDTLMI SRTPEVTCW VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRW SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SREEMTKNQV SLWCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK

[00274] In the first polypeptide chain, VL (B7-H3 mAb 1) has the amino acid sequence of SEQ ID NO:41, VH (CD3 mAb 2) has the amino acid sequence of SEQ ID NO:27, E-helix has the amino acid sequence of SEQ ID NO:3 and (CH2-CH3) has the amino acid sequence of the overhanging amino acid sequence of SEQ ID NOT: 7 .

[00275] The amino acid sequence of the second polypeptide chain of the B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1 Tri-Specific Binding Molecule is (SEQ ID NO:60): QAWTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGGQV QLVQSGAEVK KPGASVKVSC KASGYTFTSY WMQWVRQAPG QGLEWMGTIY PGDGDTRYTQ KFKGRVTITA DKSTSTAYME LSSLRSEDTA VYYCARRGIP RLWYFDVWGQ GTTVTVSSGG CGGGKVAALK EKVAALKEKV AALKEKVAAL KE

[00276] In the second polypeptide chain, VL (mAb 2 of CD3) has the amino acid sequence of SEQ ID NO:26, VH (B7-H3 mAb 1) has the amino acid sequence of SEQ ID 209 Lzzb Ln / nznz / E / YiA NO:42, and K-helix has the amino acid sequence of SEQ ID NO:4.

[00277] The amino acid sequence of the third polypeptide chain of the B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1 Tri-Specific Binding Molecule is (SEQ ID NO:61): EVQLQQSGAE LVKPGASVKL SCTASGFNIK DTYIHFVRQR PEQGLEWIGR IDPANDNTLY ASKFQGKATI TADTSSNTAY MHLCSLTSGD TAVYYCGRGY GYYVFDHWGQ GTTLTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSWTVP SSSLGTQTYI CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAAGGPS VFLFPPKPKD TLMISRTPEV TCVWDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRWSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLSCAV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLVSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPGK

[00278] In the third polypeptide chain, the amino acid sequence of the CD8 mAb1 Heavy Chain Variable Domain employed has the amino acid sequence of SEQ ID NO:30, a Hinge Domain, a CH1 Domain and the CH2Ch3 Domain which carries a hole with an H435R substitution to remove the protein A binding site (SEQ ID NO:8).

[00279] The amino acid sequence of the fourth polypeptide chain of the B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1 Tri-Specific Binding Molecule is (SEQ ID NO:62): 210 Lzzfr Ln / nznz / Ε / γΐΛ DVQINQSPSF LAASPGETIT INCRTSRSIS QYLAWYQEKP GKTNKLLIYS GSTLQSGIPS RFSGSGSGTD FTLTISGLEP EDFAMYYCQQ HNENPLTFGA GTKLELRRTV AAPSVFIFPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC

[00280] In the fourth polypeptide chain, the amino acid sequence of the CD8 mAb 1 Light Chain Variable Domain employed has the amino acid sequence of SEQ ID NO:29 and a kappa Light Chain Constant Domain.

[00281] Expressed B7H3 MAb 1 / CD3 mAb 2 / CD8 mAb 1 Tri-Specific Binding Molecule was loaded onto MSA resin, washed with 10 mM NaPCU (pH6); 10 mM NaPO4, 1M NaCI (pH6) and 10mM NaPO4 (pH6). Polypeptides were eluted from the resin with 50mM glycine (pH3) and neutralized with 1M Tris (pH8). Expression was found to be 1.7 mg / L; the preparation of the TriSpecific Binding Molecule was 0.6 mg / ml, having a final yield of 0.42 mg.

[00282] The TriSpecific Binding Molecule properties of B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1 were compared to those of a B7-H3 X CD3 DART and a B7-H3 X CD3 DART with an Fe Domain. As shown in Figures 5A-5B, the Tri-Specific Binding Molecule of B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1 showed similar target cell binding (A498 cells ( Figure 5A) ; JIMT-1 (Figure 211 Lzzb Ln / nznz / E / YiA 5Β))) compared to B7-H3 X CD3 DART and B7-H3 X CD3 DART with an Fe Domain. However, as shown in Figures 5C-5D, the Tri-Specific Binding Molecule of mAb 1 of B7-H3 / CD3 mAb 2 / CD8 mAb 1 demonstrated increased binding to CD8+ T cells compared to CD4+ T cells.

[00283] In order to demonstrate the ability of the B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1 Tri-Specific Binding Molecules of the present invention to mediate retargeted killing of target cells, such molecules were incubated in the presence of T cells and either JIMT-1 or A498 target cells. JIMT-1 cells are a trastuzumab-resistant carcinoma line (Tanner, M. et al. (2004) Characterizatlon Of A Novel Cell Line Established From A Patient With Herceptin-Resistant Breast Cancer, Mol. Cancer Ther. 3(12) :1585-1592) . The A498 cell line is a renal carcinoma cell line (Gogh, J. (1978) Cultivation, Characterization, And Identification Of Human Tumor Cells With Emphasis On Kidney, Testis, And Bladder Tumors, Nati. Cancer Inst. Monogr. 49 :5-9). As shown in Figures 6A-6C, retargeted killing of target cells was observed. Unexpectedly, such killing was substantially more potent than that observed for B7-H3 X CD3 DARTs and corresponding B7-H3 X CD3 DARTs with an Fe Domain. The observed redirected killing is summarized 212 Lzzfr Ln / nznz / E / YiA in Table 6. Table 6 Binding Molecule Re-Targeted Killing JIMT-1 Cells A498 Cells LDH LDH Luci Ferase Assay LDH LDH Maximum Kill (%) EC50 (pM) EC50 (pM) Maximum Kill (%) EC50 (pM) B7-H3 DART™ X CD3 60.72 27 22 61.95 11 B7-H3 DART™ X CD3 with Fe Domain 59.95 343 245 63.2 168 B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1 TriSpecific Binding Molecule 54.88 0.4 0.6 53.5 4

[00284] Figures 7A-7D show the capacity of the Tri-Specific Binding Molecules of B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1 to mediate T cell activation after incubation with JIMT-1 cells. Figures 8A-8D show the ability of B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1 Tri-Specific Binding Molecules to mediate T cell activation after incubation with A498 cells. In both cases, such activation was unexpectedly superior to the activation observed with comparative B7-H3 X CD3 DART™ and B7-H3 X CD3 DART™ with an Fe Domain. Table 7 summarizes the EC50 results. 213 Table 7 Tumor Cells T Cell Subset Tri-Specific Binding Molecule EC50 Values ​​of B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1 Compared to DARTs DART™ DART™ with Fe Domain Tri-Spec Binding Molecule ica A498 CD4 / CD69 51 95 75 CD4 / CD25 75 291 22 CD8 / CD69 70 185 4 CD8 / CD25 115 339 4 CTL 10 48 4 JIMT-1 CD4 / CD69 116 339 253 CD4 / CD25 257 1034 185 CD8 / CD69 185 3 CD8 / CD25 140 678 37 CTL 7 68 1 Lzzb Ln / nznz / E / YiAi

[00285] The expressed B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1 Tri-Specific Binding Molecule showed much higher (13-fold) cytolytic activity using CD8+ effector cells compared to CD4+ effector cells. The B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1 Tri-Specific Binding Molecule also showed greatly (85-fold) increased overall potency using 'pan' T cells as effectors compared to DART™ . Example 2 Effect of the CD8 Binding Domain on Redirected Cytotoxicity

[00286] In order to estimate the effect of the specificity of CD8, a second Tri-Specific Binding Molecule, 214 Ln / nznZ / E / YIA specific for Disease Associated Antigen B7-H3 was constructed using a different CD8 Antibody Variable Domain Sequence. The specificities of the B7-H3 Variable Domain and the specificities of the CD3 5 Variable Domain were identical to those used to construct the Tri-Specific Binding Molecule of mAb 1 from B7-H3 / mAb 2 from CD3 / mAb 1 from CD8. The Tri-Specific Binding Molecule is designated as B7-H3 mAb 1 / CD3 2 / CD8 MAb 2 and was composed of four different polypeptide chains (Table 10 8 ). Table 8 Polypeptide Chain Domains Binding Affinity 1 VL(B7-H3 mAb 1)-VH(CD3 mAb 2)-E-Helix-(CH2-CH3) Light Chain: B7-H3 Heavy Chain: CD3 2 VL( CD3 mAb 2)-VH(B7-H3 mAb 1)-K-Helix Light Chain: CD3 Heavy Chain: B7-H3 3 CD8 MAb 2 from CD8 Heavy Chain 4 CD8 MAb 2 from CD8 Light Chain

[00287] The amino acid sequence of the first polypeptide chain of the B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 2 Tri-Specific Binding Molecule is (SEQ ID NO:63): DIQMTQSPSS LSASVGDRVT ITCRASQDIS NYLNWYQQKP GKAPKLLIYY TSRLHSGVPS RFSGSGSGTD FTLTISSLQP EDIATYYCQQ GNTLPPTFGG GTKLEIKGGG SGGGGEVQLV ESGGGLVQPG GSLRLSCAAS GFTFSTYAMN WVRQAPGKGL EWVGRIRSKY NNYATYYADS VKDRFTISRD DSKNSLYLQM 215 Lzzb Ln / nznz / E / YiA NSLKTEDTAV YYCVRHGNFG NSYVSWFAYW GQGTLVTVSS GGCGGGEVAA LEKEVAALEK EVAALEKEVA ALEKGGGDKT HTCPPCPAPE AAGGPSVFLF PPKPKDTLMI SRTPEVTCW VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRW SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SREEMTKNQV SLWCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK

[00288] The amino acid sequence of the second polypeptide chain of the B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 2 Tri-Specific Binding Molecule is (SEQ ID NO:64): QAWTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGGQV QLVQSGAEVK KPGASVKVSC KASGYTFTSY WMQWVRQAPG QGLEWMGTIY PGDGDTRYTQ KFKGRVTITA DKSTSTAYME LSSLRSEDTA VYYCARRGIP RLWYFDVWGQ GTTVTVSSGG CGGGKVAALK EKVAALKEKV AALKEKVAAL KE

[00289] The amino acid sequence of the third polypeptide chain of the B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 2 Tri-Specific Binding Molecule is (SEQ ID NO:65): QVQLVESGGG WQPGRSLRL SCAASGFTFS DFGMNWVRQA PGKGLEWVAL IYYDGSNKFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKPH YDGYYHFFDS WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSSLGTQ TYICNVNHKP SNTKVDKRVE PKSCDKTHTC PPCPAPEAAG GPSVFLFPPK PKDTLMISRT PEVTCVWDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY 216 Lzzb Ln / nznz / E / YiA NSTYRWSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLS CAVKGFYPSD ​​IAVEWESNGQ PENNYKTTPP VLDSDGSFFL VSKLTVDKSR WQQGNVFSCS VMHEALHNRY TQKSLSLSPG K

[00290] The amino acid sequence of the fourth polypeptide chain of the B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 2 Tri-Specific Binding Molecule is (SEQ ID NO:66): DIQMTQSPSS LSASVGDRVT ITCKGSQDIN NYLAWYQQKP GKAPKLLIYN TDILHTGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCYQ YNNGYTFGQG TKVEIKRTVA APSVFIFPPS DEQLKSGTAS WCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC

[00291] To compare the ability of Tri-Specific Binding Molecules of B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1 (construct and sequence described above) or B7-H3 mAb 1 / mAb 2 of CD3 / CD8 mAb to bind T cells, human PBMC from healthy donors were purified with Ficoll, washed twice with PBS and resuspended in FACS buffer containing 10% human AB serum and incubated at room temperature. room for 20 min, the cell was spun down and 4 x 10 6 cells / mL cells were resuspended in FACS buffer. 50 μΐ Trispecific Binding Molecules of B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1 or B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 2 titrated serially or DART™ (B7- H3 X CD3 or B7-H3 X CD3 with Mastery of Faith) 217 Lzzfr Ln / nznz / Ε / γΐΛ were added to the wells of a 96-well deep plate. 50 µL (4 x 10 6 cells / mL) of cells mixed into the wells in FACS buffer containing 0.01% sodium azide were then added into the corresponding wells and mixed thoroughly using a pipet. The plate was incubated in the dark for approximately 45 minutes at 2-8°C. At the end of the incubation, cells were washed twice by adding 300 pL of FACS buffer to each well, centrifuging the plate at 1,200 rpm for 5 minutes, and discarding the supernatant. Cell pellets were resuspended in a mixture of 100 pL PE-conjugated goat anti-Human Fcy, 1:500 diluted, CD5-APC and CD4-PerCP5.5 in FACS buffer containing 0.01% sodium azide, and incubated in the dark for approximately 45 minutes at 2-8°C. At the end of the incubation, cells were washed, resuspended with FACS buffer, and analyzed with a BD Caliber flow cytometer. Cells were confined to CD5+ CD4+ (Figure 9A) or CD5+CD4 (Figure 9B). Differential staining was observed in the CD5+ CD4~ population compared to Binding Molecules that either exhibited or lacked CD8 specificity.

[00292] Cytotoxicity of B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1 Tri-Specific Binding Molecule was compared to that of B7-H3 mAb 1 / mAb 2 Tri-Specific Binding Molecule of CD3 / MAb 2 of CD8. They used a L77b Ln / nznZ / Ε / ΥΙΛ 218 luciferase assay and one LDH assay. The results of the two trials were consistent. The two TriSpecific Binding Molecules caused equivalent retargeted cytotoxicity in the presence of either CD8+ T-cell or pan-activating T-cell populations. The Tri-Specific Binding Molecule having a CD8 mAb 1 Binding Domain showed greater retargeted cytotoxicity in the presence of CD8+ cell populations or pan T cells compared to B7H3 X CD3 DART (Figures 10A-10C).

[00293] An increase (60-fold) in EC50 for CD8+ effector cells was also observed for the Tri-Specific Binding Molecule of B7-H3 mAb 1 / CD3 mAb 2 / CD8 MAb 2 compared to the Tri-Specific Binding Molecule. Tri-Specific Binding of B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1. For the B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 2 Tri-Specific Binding Molecule, the increased potency resulted in a decrease in EC50 of more than 100-fold that observed when cells T pan were used as effector cells. Example 3 Effect of Domain Positions on Redirected Cytotoxicity

[00294] In order to estimate the effect of position for a given Binding Domain (CD3, CD8 and Antigen Associated with the 219 L77b Ln / nznZ / Ε / ΥΙΛ Disease) within the Tri-Specific Binding Molecule (Site A, Site B and Site C), several Additional Tri-Specific Binding Molecules. Table 9 shows the Tri-Specific Binding Molecules and the location (Site A, Site B and Site C) of the various Binding Domains (CD3, CD8 and Disease Associated Antigen). Table 9 Tri-Specific Binding Molecule Site A Site B Site C Tri-Specific Binding Molecule of B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1 B7-H3 mAb 1 CD3 mAb 1 mAb CD8 Tri-Specific Binding Molecule of CD3 mAb 2 / CD8 mAb 1 / B7-H3 mAb 1 CD3 mAb 2 CD8 mAb 1 B7H3 mAb 1 Tri-Specific Binding Molecule of B7-H3 mAb 1 / CD8 mAb 1 / CD3 mAb 2 B7-H3 mAb 1 CD8 mAb 1 CD3 mAb 2

[00295] The Construction and Sequence of the Binding Molecule Tri-Specific B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1 were described above. For the two additional Tri-Specific Binding Molecules (CD3 mAb 2 / CD8 mAb 1 / B7-H3 mAb 1 and B7-H3 mAb 1 / CD8 mAb 1 / CD3 mAb 2), the Domain specificities Variable B7-H3, CD3 Variable Domain specificities and CD8 Variable Domain specificities were identical to those used to construct the B7-H3 mAb 1 / CD3 mAb 2 / CD8 mAb 1 Tri-Specific Binding Molecule. The Tri-Specific Binding Molecule of CD3 mAb 2 / CD8 mAb 1 / mAb 220 L77b Ln / nznZ / E / YIA from B7-H3 was composed of four different polypeptide chains (Table 10). Table 10 Polypeptide Chain Domains Affinity and Binding 1 VL(CD3 mAb 2)-VH(CD8 mAb 1)-E-Helix-(CH2-CH3) Light Chain: CD3 Heavy Chain: CD8 2 VL(CD8 mAb 1 )-VH(mAb 2 of CD3)-K-Helix Light Chain: CD8 Heavy Chain: CD3 3 mAb 1 of B7-H3 of Heavy Chain B7-H3 4 mAb 1 of B7-H3 of Light Chain B7-H3

[00296] The amino acid sequence of the first polypeptide chain of the CD3 mAb 2 / CD8 mAb 1 / B7-H3 mAb 1 Tri-Specific Binding Molecule is (SEQ ID NO:67): DVQINQSPSF LAASPGETIT INCRTSRSIS QYLAWYQEKP GKTNKLLIYS GSTLQSGIPS RFSGSGSGTD FTLTISGLEP EDFAMYYCQQ HNENPLTFGA GTKLELRGGG SGGGGEVQLV ESGGGLVQPG GSLRLSCAAS GFTFSTYAMN WVRQAPGKGL EWVGRIRSKY NNYATYYADS VKGRFTISRD DSKNSLYLQM NSLKTEDTAV YYCVRHGNFG NSYVSWFAYW GQGTLVTVSS GGCGGGEVAA LEKEVAALEK EVAALEKEVA ALEKGGGDKT HTCPPCPAPE AAGGPSVFLF PPKPKDTLMI SRTPEVTCW VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRW SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SREEMTKNQV SLWCLVKGFY PSDIAVEWES NGQPENNYKT 221 L77b Ln / nznZ / Ε / ΥΙΛ TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK

[00297] The amino acid sequence of the second polypeptide chain of the CD3 mAb 2 / CD8 mAb 1 / B7-H3 mAb 1 Tri-Specific Binding Molecule is (SEQ ID NO:68): QAWTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGGEV QLQQSGAELV KPGASVKLSC TASGFNIKDT YIHFVRQRPE QGLEWIGRID PANDNTLYAS KFQGKATITA DTSSNTAYMH LCSLTSGDTA VYYCRGGYGY YVFDHWGQGT TLTVSSGGCG GGKVAALKEK VAALKEKVAA LKEKVAALKE

[00298] The amino acid sequence of the third polypeptide chain of the CD3 mAb 2 / CD8 mAb 1 / B7-H3 mAb 1 Tri-Specific Binding Molecule is (SEQ ID NO:69): QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYWMQWVRQA PGQGLEWMGT IYPGDGDTRY TQKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCARRG IPRLWYFDVW GQGTTVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPEAAGG PSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRWSVLT VLHQDWLNGKPREPKTISKI EYPKIE VYTLPPSREE MTKNQVSLSC AVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLV SKLTVDKSRW QQGNVFSCSV MHEALHNRYT QKSLSLSPGK 222 Lzzb Ln / nznz / B / Yi

[00299] The amino acid sequence of the fourth polypeptide chain of the CD3 mAb 2 / CD8 mAb 1 / B7-H3 mAb 1 Tri-Specific Binding Molecule is (SEQ ID NO:70): DIQMTQSPSS LSASVGDRVT ITCRASQDIS NYLNWYQQKP GKAPKLLIYY TSRLHSGVPS RFSGSGSGTD FTLTISSLQP EDIATYYCQQ GNTLPTFGG GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC The CD3 mAb 2 / CD8 mAb 1 / B7-H3 mAb 1 Tri-Specific Binding Molecule was composed of four different polypeptide chains (Table 11). Table 11 Polypeptide Chain Domains Binding Affinity 1 VL(B7-H3 mAb 1)-VH(CD8 mAb 1)-E-Helix-(CH2-CH3) Light Chain: B7-H3 Heavy Chain: CD8 2 VL( CD8 mAb 1)-VH(B7-H3 mAb 1)-K-Helix Light Chain: CD8 Heavy Chain: B7-H3 3 CD3 mAb 2 from CD3 Heavy Chain 4 CD3 mAb 2 from CD3 Light Chain

[00301] The amino acid sequence of the first polypeptide chain of the B7-H3 mAb 1 / CD8 mAb 1 / CD3 mAb 2 Tri-Specific Binding Molecule is (SEQ ID NO:71): 223 Lzzb Ln / nznz / E / YiA DVQINQSPSF LAASPGETIT INCRTSRSIS QYLAWYQEKP GKTNKLLIYS GSTLQSGIPS RFSGSGSGTD FTLTISGLEP EDFAMYYCQQ HNENPLTFGA GTKLELRGGG SGGGGQVQLV QSGAEVKKPG ASVKVSCKAS GYTFTSYWMQ WVRQAPGQGL EWMGTIYPGD GDTRYTQKFK GRVTITADKS TSTAYMELSS LRSEDTAVYY CARRGIPRLW YFDVWGQGTT VTVSSGGCGG GEVAALEKEV AALEKEVAAL EKEVAALEKG GGDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLMISRTPE VTCVWDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRWSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM TKNQVSLWCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

[00302] The amino acid sequence of the second polypeptide chain of the B7-H3 mAb 1 / CD8 mAb 1 / CD3 mAb 2 Tri-Specific Binding Molecule is (SEQ ID NO:72): DIQMTQSPSS LSASVGDRVT ITCRASQDIS NYLNWYQQKP GKAPKLLIYY TSRLHSGVPS RFSGSGSGTD FTLTISSLQP EDIATYYCQQ GNTLPPTFGG GTKLEIKGGG SGGGGEVQLQ QSGAELVKPG ASVKLSCTAS GFNIKDTYIH FVRQRPEQGL EWIGRIDPAN DNTLYASKFQ GKATITADTS SNTAYMHLCS LTSGDTAVYY CGRGYGYYVF DHWGQGTTLT VSSGGCGGGK VAALKEKVAA LKEKVAALKE KVAALKE

[00303] The amino acid sequence of the third polypeptide chain of the B7-H3 mAb 1 / CD8 mAb 1 / CD3 mAb 2 Tri-Specific Binding Molecule is (SEQ ID NO:73): EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSSASTKG PSVFPLAPSS KSTSGGTAAL 224 Lzzfr Ln / nznz / E / YiA GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSWTVPSSS LGTQTYICNV NHKPSNTKVD KRVEPKSCDK THTCPPCPAP EAAGGPSVFL FPPKPKDTLM ISRTPEVTCV WDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLSCAVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLVSKLTV DKSRWQQGNV FSCSVMHEAL HNRYTQKSLS LSPGK

[00304] The amino acid sequence of the fourth polypeptide chain of the Tri-Specific Binding Molecule of B7-H3 mAb 1 / CD8 mAb 1 / CD3 mAb 2 is (SEQ ID NO:74): QAWTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG RTVAAPSVFI FPPSDEQLKS GTASWCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQSFGNGVT

[00305] The results of this investigation are shown in Figures 11A-11C, Figures 12A-12C, Figures 13A-13E, and in Table 26. Figures 11A-11C and Figures 12A-12C independently demonstrate that placing a CD3 Binding Domain at Site C of TriSpecific Binding Molecules reduces the cytotoxicity of the molecule. Figures 13A-13E show that this decreased cytotoxicity is observed regardless of whether any of the T cells, CD4 + , CD8 + or pan, were used. 225 Lzzb Ln / nznz / E / YiA

[00306] As shown in Figures 14A-14B, placement of the CD3 Binding Domain at Site C, greatly decreased binding to both CD5+ CD4+ cells (Figure 14A) and CD5+ CD4- cells ( Figure 14B). Notably, however, without considering the placement of the Joining Domain of CD3, all Tri-Specific Binding Molecules were able to mediate retargeted cytotoxicity Example 4 Production and Properties of Exemplary Anti-CD3, Anti-CD8, Anti-5T4 Tri10-Specific Binding Molecules

[00307] Additional exemplary Tri-Specific Binding Molecules specific for the 5T4 Disease Associated Antigen were constructed. The 5T4 mAb 2 / CD3 mAb 2 / CD8 mAb 1 Tri-Specific Binding Molecule was composed of four different polypeptide chains (Table 12). Table 12 Polypeptide Chain Domains Binding Affinity 1 VL(5T4 mAb 2)-VH(CD3 mAb 2)-E-Helix-(CH2-CH3) Light Chain: 5T4 Heavy Chain: CD3 2 VL(CD3 mAb 2 )-VH(mAb 2 of 5T4)-K-Helix Light Chain: CD3 Heavy Chain: 5T4 3 mAb 1 from CD8 from CD8 Heavy Chain 4 mAb 1 from CD8 from CD8 Light Chain 226 Lzzb Ln / nznz / E / YiA

[00308] The amino acid sequence of the first polypeptide chain of the 5T4 mAb 2 / CD3 mAb 2 / CD8 mAb 1 Tri-Specific Binding Molecule is (SEQ ID NO:75): DVLMTQTPLS LPVSLGDQAS ISCRSSQSIV YSNGNTYLEW YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSHVP FTFGSGTKLE IKGGGSGGGG EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKGRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSSGGCGG GEVAALEKEV AALEKEVAAL EKEVAALEKG GGDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLMISRTPE VTCVWDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRWSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM TKNQVSLWCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

[00309] The amino acid sequence of the second polypeptide chain of the 5T4 mAb 2 / CD3 mAb 2 / CD8 mAb 1 Tri-Specific Binding Molecule is (SEQ ID NO:76): QAWTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGGQV QLQQPGAELV KPGASVKMSC KASGYTFTSY WITWVKQRPG QGLEWIGDIY PGSGRANYNE KFKSKATLTV DTSSSTAYMQ LSSLTSEDSA VYNCARYGPL FTTWDPNSY AMDYWGQGTS VTVSSGGCGG GKVAALKEKV AALKEKVAAL KEKVAALKE 227 Lzzb Ln / nznz / E / YiA

[00310] The amino acid sequence of the third polypeptide chain of the 5T4 mAb 2 / CD3 mAb 2 / CD8 mAb 1 Tri-Specific Binding Molecule is (SEQ ID NO:77): EVQLQQSGAE LVKPGASVKL SCTASGFNIK DTYIHFVRQR PEQGLEWIGR IDPANDNTLY ASKFQGKATI TADTSSNTAY MHLCSLTSGD TAVYYCGRGY GYYVFDHWGQ GTTLTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSWTVP SSSLGTQTYI CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAAGGPS VFLFPPKPKD TLMISRTPEV TCVWDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRWSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLSCAV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLVSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPGK

[00311] The amino acid sequence of the fourth polypeptide chain of the 5T4 mAb 2 / CD3 mAb 2 / CD8 mAb 1 Tri-Specific Binding Molecule is (SEQ ID NO:78): DVQINQSPSF LAASPGETIT INCRTSRSIS QYLAWYQEKP GKTNKLLIYS GSTLQSGIPS RFSGSGSGTD FTLTISGLEP EDFAMYYCQQ HNENPLTFGA GTKLELRRTV AAPSVFIFPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC

[00312] The 5T4 mAb 2 / CD3 mAb 2 / CD8 mAb 2 Tri-Specific Binding Molecule was composed of four different polypeptide chains (Table 13). 228 Table 13 Polypeptide Chain Domains Binding Affinity 1 VL(5T4 mAb 2)-VH(CD3 mAb 2)-E-Helix-(CH2-CH3) Light Chain: 5T4 Heavy Chain: CD3 2 VL(CD3 mAb 2 )-VH(mAb 2 of 5T4)-K-Helix Light Chain: CD3 Heavy Chain: 5T4 3 mAb 2 from CD8 from CD8 Heavy Chain 4 mAb 2 from CD8 from CD8 Light Chain Lzzb Ln / nznz / E / YiAi

[00313] The amino acid sequence of the first polypeptide chain of the 5T4 mAb 2 / CD3 mAb 2 / CD8 mAb 2 Tri-Specific Binding Molecule is (SEQ ID NO:79): DVLMTQTPLS LPVSLGDQAS ISCRSSQSIV YSNGNTYLEW YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSHVP FTFGSGTKLE IKGGGSGGGG EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKGRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSSGGCGG GEVAALEKEV AALEKEVAAL EKEVAALEKG GGDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLMISRTPE VTCWVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRWSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM TKNQVSLWCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

[00314] The amino acid sequence of the second polypeptide chain of the 5T4 mAb 2 / CD3 mAb 2 / CD8 mAb 2 Tri-Specific Binding Molecule is (SEQ ID NO:80): QAWTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF 229 Lzzfr Ln / nznz / Ε / γΐΛ GGGTKLTVLG GGGSGGGGQV QLQQPGAELV KPGASVKMSC KASGYTFTSY WITWVKQRPG QGLEWIGDIY PGSGRANYNE KFKSKATLTV DTSSSTAYMQ LSSLTSEDSA VYNCARYGPL FTTWDPNSY AMDYWGQGTS VTVSSGGCGG GKVAALKEKV AALKEKVAAL KEKVAAL

[00315] The amino acid sequence of the third polypeptide chain of the 5T4 mAb 2 / CD3 mAb 2 / CD8 mAb 2 Tri-Specific Binding Molecule is (SEQ ID NO:81): QVQLVESGGG WQPGRSLRL SCAASGFTFS DFGMNWVRQA PGKGLEWVAL IYYDGSNKFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKPH YDGYYHFFDS WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSSLGTQ TYICNVNHKP SNTKVDKRVE PKSCDKTHTC PPCPAPEAAG GPSVFLFPPK PKDTLMISRT PEVTCVWDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRWSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLS CAVKGFYPSD ​​IAVEWESNGQ PENNYKTTPP VLDSDGSFFL VSKLTVDKSR WQQGNVFSCS VMHEALHNRY TQKSLSLSPG K

[00316] The amino acid sequence of the fourth polypeptide chain of the 5T4 mAb 2 / CD3 mAb 2 / CD8 mAb 2 Tri-Specific Binding Molecule is (SEQ ID NO: 82): DIQMTQSPSS LSASVGDRVT ITCKGSQDIN NYLAWYQQKP GKAPKLLIYN TDILHTGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCYQ YNNGYTFGQG TKVEIKRTVA APSVFIFPPS DEQLKSGTAS WCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC 230 Lzzfr Ln / nznz / Ε / γΐΛ

[00317] Tri-Specific Binding Molecules of 5T4 mAb 2 / CD3 mAb 2 / CD8 mAb 1 and 5T4 mAb 2 / CD3 mAb 2 / CD8 MAb 2 were expressed and purified as described above. former. The ability of these two Tri-Specific Binding Molecules to bind to CD5+ / CD4+ and CD5+ / CD4- confined human PBMCs were compared to that of a 5T4 X CD3 DART with an Fe Domain. As shown in the Figures 15A-15B, Tri-Specific Binding Molecules of 5T4 / CD3 mAb 2 / CD8 mAb 1 and 5T4 / CD3 mAb 2 / CD8 mAb 2 demonstrated greatly increased binding to CD8 + T cells (Figure 15B) compared to CD4+ T cells (Figure 15A).

[00318] In order to demonstrate the ability of 5T4 mAb 2 / CD3 mAb 2 / CD8 mAb 1 and 5T4 mAb 2 / CD3 mAb 2 / CD8 mAb 2 Tri-Specific Binding Molecules to mediate For redirected killing of target cells, such molecules were incubated in the presence of T cells and JIMT-1 target cells. As shown in Figures 16A-16C, redirected killing of target cells was observed. As observed for the B7-H3 Tri-Specific Binding Molecules described above, the killing was substantially more potent than that observed for the corresponding 5T4 X CD3 DART containing an Fe Domain. Similarly, as observed for Tri-Specific Binding Molecules B7-H3, the use of different Domains 231 L77b Ln / nznZ / Ε / ΥΙΛ Variables CD8 had no effect on the ability of 5T4 Tri-Specific Binding Molecules to redirect CD8+ T cells to target cells. The TriSpecific Binding Molecule of 5T4 mAb 2 / CD3 mAb 2 / CD8 MAb 2 was also found to be highly active and demonstrated much greater CTL activity using CD8+ vs. effector cells. CD4+ (23 times lower than EC50). 5T4 mAb 2 / CD3 mAb 2 / CD8 mAb 1 Tri-Specific Binding Molecule was 22-fold more potent using pan T effector cells compared to a 5T4 X CD3 DART™ and mAb Tri-Specific Binding Molecule 2 of 5T4 / mAb 2 of CD3 / MAb 2 of CD8 was 25 times more potent. Example 5 Properties of Exemplary AntiCD3, Anti-CD8, Anti-ROR1 Tri-Specific Binding Molecules

[00319] Additional exemplary Tri-Specific Binding Molecules specific for the Disease Associated Antigen R0R1 were constructed. The ROR1 aMb 1 / CD3 mAb 2 / CD8 mAb 1 Tri-Specific Binding Molecule was composed of four different polypeptide chains (Table 14). Table 14 Chain of Polypeptides Domains Binding Affinity 1 VL(aMb 1 from RORl)-VH(mAb 2 from CD3)-E-Helix-(CH2-CH3) Light Chain: ROR1 Heavy Chain: CD3 2 VL( mAb 2 from CD3)-VH(aMb 1 from ROR1)-K-Helix Light Chain: CD3 Heavy Chain: ROR1 232 Ln / nznZ / Ε / ΥΙΛΙ 3 CD8 mAb 1 from CD8 Heavy Chain 4 CD8 mAb 1 from CD8 Light Chain

[00320] The amino acid sequence of the first polypeptide chain of the ROR1 aMb 1 / CD3 mAb 2 / CD8 mAb 1 Tri-Specific Binding Molecule is (SEQ ID NO:83): QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLGGGGSGGG GEVQLVESGG GLVQPGGSLR LSCAASGFTF STYAMNWVRQ APGKGLEWVG RIRSKYNNYA TYYADSVKGR FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYV SWFAYWGQGT LVTVSSGGCG GGEVAALEKE VAALEKEVAA LEKEVAALEK GGGDKTHTCP PCPAPEAAGG PSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLWC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

[00321] The amino acid sequence of the second polypeptide chain of the ROR1 aMb 1 / CD3 mAb 2 / CD8 mAb 1 Tri-Specific Binding Molecule is (SEQ ID NO:84): QAWTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGGQE QLVESGGGLV QPGGSLRLSC AASGFTFSDY YMSWVRQAPG KGLEWVATIY PSSGKTYYAD SVKGRFTISS DNAKNSLYLQ MNSLRAEDTA VYYCARDDISS GALTVGTVGALGDA KEKVAALKEK VAALKEKVAA LKE 233 L77b Ln / nznZ / Ε / ΥΙΛ

[00322] The amino acid sequence of the third polypeptide chain of the ROR1 aMb 1 / CD3 mAb 2 / CD8 mAb 1 Tri-Specific Binding Molecule is (SEQ ID NO: 85): EVQLQQSGAE LVKPGASVKL SCTASGFNIK DTYIHFVRQR PEQGLEWIGR IDPANDNTLY ASKFQGKATI TADTSSNTAY MHLCSLTSGD TAVYYCGRGY GYYVFDHWGQ GTTLTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSWTVP SSSLGTQTYI CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAAGGPS VFLFPPKPKD TLMISRTPEV TCWVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRWSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLSCAV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLVSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPG

[00323] The amino acid sequence of the fourth polypeptide chain of the ROR1 aMb 1 / CD3 mAb 2 / CD8 mAb 1 Tri-Specific Binding Molecule is (SEQ ID NO:86): DVQINQSPSF LAASPGETIT INCRTSRSIS QYLAWYQEKP GKTNKLLIYS GSTLQSGIPS RFSGSGSGTD FTLTISGLEP EDFAMYYCQQ HNENPLTFGA GTKLELRRTV AAPSVFIFPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC

[00324] The ROR1 aMb 1 / CD3 mAb 2 / CD8 mAb 2 Tri-Specific Binding Molecule was composed of four different polypeptide chains (Table 15). 2. 3. 4 Lzzb Ln / nznz / E / YiA Table 15 Polypeptide Chain Domains Binding Affinity 1 VL (ROR1 aMb 1)-VH(CD3 mAb 2)-E-Helix-(CH2-CH3) Light Chain: ROR1 Heavy Chain: CD3 2 VL(CD3 mAb 2 )-VH(aMb 1 of ROR1)-K-Helix Light Chain: CD3 Heavy Chain: ROR1 3 mAb 2 of CD8 of Heavy Chain CD8 4 mAb 2 of CD8 of Light Chain CD8

[00325] The amino acid sequence of the first polypeptide chain of the ROR1 aMb 1 / CD3 mAb 2 / CD8 mAb 2 Tri-Specific Binding Molecule is (SEQ ID NO:87): QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLGGGGSGGG GEVQLVESGG GLVQPGGSLR LSCAASGFTF STYAMNWVRQ APGKGLEWVG RIRSKYNNYA TYYADSVKGR FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYV SWFAYWGQGT LVTVSSGGCG GGEVAALEKE VAALEKEVAA LEKEVAALEK GGGDKTHTCP PCPAPEAAGG PSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLWC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 235 Lzzb Ln / nznz / E / YiA

[00326] The amino acid sequence of the second polypeptide chain of the ROR1 aMb 1 / CD3 mAb 2 / CD8 mAb 2 Tri-Specific Binding Molecule is (SEQ ID NO:88): QAWTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGGQE QLVESGGGLV QPGGSLRLSC AASGFTFSDY YMSWVRQAPG KGLEWVATIY PSSGKTYYAD SVKGRFTISS DNAKNSLYLQ MNSLRAEDTA VYYCARDSYA DDAALFDIWG QGTTVTVSSG GCGGGKVAAL KEKVAALKEK VAALKEKVAA LKE

[00327] The amino acid sequence of the third polypeptide chain of the ROR1 aMb1 / CD3 mAb 2 / CD8 mAb 2 Tri-Specific Binding Molecule is (SEQ ID NO:89): QVQLVESGGG WQPGRSLRL SCAASGFTFS DFGMNWVRQA PGKGLEWVAL IYYDGSNKFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKPH YDGYYHFFDS WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSSLGTQ TYICNVNHKP SNTKVDKRVE PKSCDKTHTC PPCPAPEAAG GPSVFLFPPK PKDTLMISRT PEVTCVWDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRWSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLS CAVKGFYPSD ​​IAVEWESNGQ PENNYKTTPP VLDSDGSFFL VSKLTVDKSR WQQGNVFSCS VMHEALHNRY TQKSLSLSPG K

[00328] The amino acid sequence of the fourth polypeptide chain of the ROR1 aMb 1 / CD3 mAb 2 / CD8 mAb 2 Tri-Specific Binding Molecule is (SEQ ID NO:90): 236 Ln / nznZ / Ε / ΥΙΛ DIQMTQSPSS LSASVGDRVT ITCKGSQDIN NYLAWYQQKP GKAPKLLIYN TDILHTGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCYQ YNNGYTFGQG TKVEIKRTVA APSVFIFPPS DEQLKSGTAS WCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC

[00329] The TriSpecific Binding Molecule properties of ROR1 aMb 1 / CD3 mAb 2 / CD8 mAb 1 and ROR1 aMb 1 / CD3 mAb 2 / CD8 MAb 2 were compared to those of ROR1 DART™ X CD3 containing an Fe Domain. The DART™ and Tri-Specific Binding Molecules of ROR1 aMb 1 / CD3 mAb 2 / CD8 mAb 1 and ROR1 aMb 1 / CD3 mAb 2 / CD8 MAb 2 were constructed using the Fv sequences of an anti-ROR1 monoclonal antibody, ROR1 aMb1, which binds to the ROR-1 antigen.

[00330] The two Tri-Specific Binding Molecules of ROR1 aMb 1 / CD3 mAb 2 / CD8 mAb 1 and ROR1 aMb 1 / CD3 mAb 2 / CD8 MAb 2 and DART were all active in the DART assays. CTL against JIMTl-luc and A549 target cells. However, the Tri-Specific Binding Molecules of ROR1 aMb 1 / CD3 mAb 2 / CD8 mAb 1 and ROR1 aMb 1 / CD3 mAb 2 / CD8 M...

Claims

CLAIMS 1. A Tri-Specific Binding Molecule capable of immunospecifically binding to three different epitopes, wherein the binding molecule consists of four different polypeptide chains covalently complexed together and characterized in that they comprise: (I) an Antigen Binding Domain I capable of immunospecifically binding to an Epitope I present on the first antigen and an Antigen Binding Domain II capable of immunospecifically binding to an Epitope II present on a second antigen, wherein the Antigen Binding Domain I and the Antigen Binding Domain II are Diabody-Type Binding Domains; (II) a Non-Diabody-Type Antigen Binding Domain III capable of immunospecifically binding to an Epitope III present on a third antigen; and (III) an Fe Domain formed by the association of two CH2-CH3 Domains to one another.wherein the first, second, and third antigens are the same antigen, or are independent of each other, or different from any other antigen. 266 L77b Ln / nznZ / E / YIL 2. The Tri-Specific Binding Molecule according to claim 1, characterized in that one of Epitope I, or Epitope II, or Epitope III is an epitope of a cell receptor.

3. The Tri-Specific Binding Molecule according to any one of claims 1-2, characterized in that one of Epitope I, Epitope II, or Epitope III is a Disease-Associated Antigen epitope.

4. The Tri-Specific Binding Molecule according to claim 3, characterized in that the Disease-Associated Antigen is a cancer antigen that is located on the surface of a cancer cell. 5.The Tri-Specific Binding Molecule according to claim 3, characterized in that the Disease-Associated Antigen is a pathogen antigen that is disposed on the surface of a pathogen or a cell infected by the pathogen.

6. The Tri-Specific Binding Molecule according to any one of claims 1 to 5, characterized in that the Fe Domain is capable of binding to an Fe Receptor disposed on the surface of a cell. 7.The Tri-Specific Binding Molecule according to any one of claims 3 to 6, characterized in that one of Epitope I, Epitope II or Epitope III is a CD3 epitope, a second of Epitope I, Epitope II or Epitope III is a CD8 epitope, and the third of Epitope I, Epitope II or Epitope III is a Disease-Associated Antigen epitope, and wherein the Antigen-Binding Domains I, II and III of the Tri-Specific Binding Molecules mediate the coordinated binding of a cytotoxic T cell and a cell expressing the Disease-Associated Antigen. 8.The Tri-Specific Binding Molecule according to claim 7, characterized in that CD3 and CD8 are arranged on the surface of a T cell and the Disease-Associated Antigen is arranged on the surface of a cancer cell, pathogen, or pathogen-infected cell, and wherein the immunospecific binding is sufficient to colocalize CD3 and CD8 and the Disease-Associated Antigen, thereby facilitating the activation of the CD8-arranged T cell against the Disease-Associated Antigen-arranged cell. 9.The Tri-Specific Binding Molecule according to any one of claim 18, characterized in that the Non-Diabody Type Binding Domain III comprising the Fab Type Binding Domain (VLiii / VHm) is capable of immunospecifically binding Epitope III, characterized in that the molecule comprises: (A) a first polypeptide chain: Lzzfr Ln / nznz / E / γAL 268 (I) comprising in the N-terminal to C-terminal direction: (1) an immunoglobulin Light Chain Variable Domain capable of binding to a first of the three epitopes (VLT); (2) a Heavy Chain Variable Domain of an immunoglobulin capable of binding to a second of the three epitopes (VHn); (3) (a) a first Cysteine-Containing Domain; and a Heterodimer Promotion Domain; or (b) a Cysteine-Containing Heterodimer Promotion Domain; (5) a second Cysteine-Containing Domain; and (6) CH2 and CH3 Domains of an IgG. OR (II) comprising in the N-terminal to C-terminal direction: (1) a first Cysteine-Containing Domain. (2) CH2 and CH3 Domains of an IgG. (3) a Light Chain Variable Domain of an immunoglobulin capable of binding to a first of three epitopes (VLi); Ln / nznZ / E / YIL 269 (4) a Heavy Chain Variable Domain of an immunoglobulin capable of binding to a second of three epitopes (VHII); (5) (a) a second Cysteine-Containing Domain; and a Heterodimer Promotion Domain; or (b) a Cysteine-Containing Heterodimer Promotion Domain; (B) a second polypeptide chain comprising, in the N-terminal to C-terminal direction: (1) an immunoglobulin Light Chain Variable Domain capable of binding a second of the three epitopes (VLn); (2) an immunoglobulin Heavy Chain Variable Domain capable of binding to the first of three epitopes (VHT); (3) (a) a first(a) a Cysteine-Containing Domain; and a Heterodimer Promotion Domain; or (b) a Cysteine-Containing Heterodimer Promotion Domain; wherein the Heterodimer Promotion Domain of the second polypeptide chain is complementary to the Heterodimer Promotion Domain of the first polypeptide chain. (c) a third polypeptide chain comprising, from N-terminal to C-terminal: (1) a Heavy Chain Variable Domain of an immunoglobulin capable of binding to a third of the three epitopes (VHm); and (2) a CHi Domain, a Cysteine-Containing Hinge Domain, and a CH2CH3 Domain of an IgG; and (d) a fourth polypeptide chain comprising, from N-terminal to C-terminal: (1) a Light Chain Variable Domain of an immunoglobulin capable of binding to the third of the three epitopes (VLm); and (2) a Cysteine-Containing Light Chain Constant Domain (CL); wherein: (i) the VLi and VHi domains are associatedto form a Domain capable of binding to Epitope I; (ii) Domains VLi and VHi associate to form a Domain capable of binding to Epitope II; Lzzfr Ln / nznz / E / YiA 271 (iii) Domains VLi and VHi associate to form a Domain capable of binding to Epitope III; (iv) the CH2-CH3 Domain of the first polypeptide chain and the CH2-CH3 Domain of the third polypeptide chain associate to form an Fe Domain. (v) the first and second polypeptide chains are covalently linked to each other; (vi) the first and third polypeptide chains are covalently linked to each other; and (VII) the third and fourth polypeptide chains are covalently linked to each other.

10. The Tri-Specific Binding Molecule according to claim 9, characterized in that: (A) the Heterodimer Promotion Domain is an E-helix and the complementary Heterodimer Promotion Domain is a K-helix; or (B) the Heterodimer Promotion Domain is a K-helix and the Promotion Domain ofComplementary heterodimer is an E-helix. Lívnznz / E / YIA 272 11. The Tri-Specific Binding molecule according to any one of claims 9-10, characterized in that: (A) the CH2-CH3 Domains of the first and third polypeptide chains each have the sequence SEQ ID NO: 6, such that the Fe Domain formed from their association exhibits an FcyR-mediated effector function; or (B) the CH2-CH3 Domain of the first and third polypeptide chains comprises at least one amino acid substitution, relative to the sequence SEQ ID NO: 6, such that the Fe Domain formed from their association exhibits the altered FcyR-mediated effector function.

12. The Tri-Specific Binding molecule according to any of claims 9-11, characterized in that the CH2-CH3 domain of the first and third polypeptide chains differ from each other and have an amino acid sequence selected from the group consisting of SEQ ID NO:7 and SEQID NO:

8. L77b Ln / nznZ / E / YIL 273 13. The Tri-Specific Binding Molecule according to any of claims 9-12, characterized in that: (A) Epitope I, Epitope II and Epitope III are, respectively, a CD3 epitope, a CD8 lymphocyte epitope and a Disease-Associated Antigen epitope. (B) Epitope I, Epitope II and Epitope III are, respectively, a CD3 epitope, a Disease-Associated Antigen epitope and a CD8 epitope; (C) Epitope I, Epitope II and Epitope III are, respectively, a CD8 epitope, a CD3 epitope, and a Disease-Associated Antigen epitope. (D) Epitope I, Epitope II, and Epitope III are, respectively, a CD8 epitope, a Disease-Associated Antigen epitope, and a CD3 epitope; (E) Epitope I, Epitope II, and Epitope III are, respectively, a Disease-Associated Antigen epitope, a CD3 epitope, and a CD8 epitope; 274 Lzzb Ln / nznz / E / YiA (F) Epitope I, Epitope II, andEpitope III are, respectively, a Disease-Associated Antigen epitope, a CD8 epitope, and a CD3 epitope.

14. The Tri-Specific Binding Molecule according to any one of claims 7-13, characterized in that: (A) the CD3 epitope is a CD3 epitope recognized by the antibody OKT3, M291, YTH12.5, CD3 mab 1, or CD3 mab 2; or (B) the CD8 epitope is a CD8 epitope recognized by the antibody TRX2 or OKT8.

15. A pharmaceutical composition characterized in that it comprises the Tri-Specific Binding Molecule of any one of claims 7-14, and a pharmaceutically acceptable carrier, diluent, or excipient.

16. A method for treating cancer comprising administering an effective quantity of the pharmaceutical composition according to claim 15 to a person in need thereof, characterized in that the Disease-Associated Antigen is the cancer antigen.

17. A method for treating the disease associated withThe presence of a pathogen comprising administering an effective quantity of the pharmaceutical composition according to claim 15 to a person in need thereof, wherein the Disease-Associated Antigen is the pathogen antigen.

18. An anti-ROR1 antibody or ROR1-binding fragment, characterized in that the antibody comprises: (A) a Light Chain Variable Domain comprising CDR1 having the sequence SEQ ID NO:117, a CDR12 having the sequence SEQ ID NO:118, and a CDR13 having the sequence SEQ ID NO:119; and (B) a Heavy Chain Variable Domain comprising a CDR1 having the sequence SEQ ID NO:120, a CDR2 having the sequence SEQ ID NO:121, and a CDR3 having the sequence SEQ ID NO:

122.

19. The anti-ROR1 antibody or ROR1 binding fragment thereof according to claim 18, characterized in that the antibody has a Light Chain Variable Domain having the20. The anti-ROR1 antibody or ROR1 binding fragment thereof according to any one of claims 18-19, characterized in that the antibody has a Heavy Chain Variable Domain having the sequence SEQ ID NO:

52.

21. A diabody, BiTe, or single-chain antibody characterized in that it comprises the Lzzb Ln / nznz / E / YiA 276 R0R1 binding fragment according to any one of claims 18-20.

22. A pharmaceutical composition characterized in that it comprises the anti-ROR1 antibody or ROR1 binding fragment thereof according to any one of claims 19-21, and a pharmaceutically acceptable carrier, diluent, or excipient.

23. A method for treating cancer characterized in that it comprises administering an effective amount of the pharmaceutical composition according to claim 22 to an individual in need thereof.