HK1136835A - Single-chain multivalent binding proteins with effector function - Google Patents

Description

Single-chain multivalent binding proteins with effector function

Technical Field

The present invention relates generally to the field of multivalent binding molecules and therapeutic applications thereof.

The sequence list is submitted in text form and in a PDF file that meets the application requirements for electronic archiving. The sequence listing was created at 12 days 6 month 2007. The sequence listing is incorporated by reference herein in its entirety.

Background

In healthy mammals, the immune system protects the animal's body from foreign objects and pathogens. In some cases, however, the immune system can be subject to error, resulting in traumatic injury and/or disease. For example, B-cells can produce antibodies that recognize self-proteins rather than foreign proteins, resulting in the production of autoimmune diseases characterized by autoantibodies, such as lupus erythematosus, rheumatoid arthritis, and the like. In other cases, the typical beneficial effects of the immune system in combating foreign substances are counter-acting, such as with organ transplants. The efficacy of the mammalian immune system, and in particular the human immune system, has been confirmed, and efforts have been made to control the system to avoid or ameliorate the deleterious consequences to health that result from the normal functioning of the immune system in abnormal environments (e.g., organ transplantation) or from the abnormal functioning of the immune system in other external normal environments (e.g., autoimmune disease progression). In addition, efforts have been made to utilize the immune system to provide a variety of target-specific diagnostic and therapeutic approaches, depending on the ability of the antibody to specifically recognize and bind specific antigenic targets.

One way in which the immune system protects the body of an animal is to produce specialized cells, called B lymphocytes or B-cells. B-cells produce antibodies that bind to and, in some cases, mediate the destruction of foreign objects or pathogens. In some cases, however, the human immune system (and in particular, the B lymphocytes of the human immune system) can be faulty and cause disease. There are a number of cancers that involve uncontrolled proliferation of B-cells. There are also a number of autoimmune diseases involving B-cell production of antibodies that bind to parts of the animal body rather than to foreign objects and pathogens. In addition, there are a number of pathologies where autoimmune and inflammatory diseases involve B-cells, e.g., via inappropriate presentation of B-cell antigens to T-cells or via other pathways involving B-cells. For example, an autoimmune-susceptible mouse lacking B-cells does not develop autoimmune nephropathy, vasculitis, or autoantibodies. (Shlomchik et al, J exp. Med.1994, 180: 1295-306). Interestingly, the susceptible autoimmune mice with B-cells but lacking immunoglobulin production did not develop autoimmune disease when experimentally induced (Chan et al, J exp. Med.1999, 189: 1639-48), suggesting that B-cells have an essential role in the development of autoimmune disease.

B-cells can be recognized by molecules on their cell surface. CD20 is the first human B-cell profile recognized by monoclonal antibodies to be a specific surface molecule. It is an unglycosylated hydrophobic 35kDa B-cell transmembrane phosphoprotein, the amino-terminus and the carboxy-terminus of which are both located intracellularly. Einfeld et al, EMBO j.1988, 7: 711-17. CD20 is expressed by all normal mature B-cells, but not by precursor B-cells or plasma cells. The natural ligand of CD20 has not been identified and the function of CD20 in B-cell biology is still not fully understood.

Another B-cell profile is that the specific cell surface molecule is CD 37. CD37 is a heavily glycosylated 40-52kDa protein that belongs to the family of tetraspanin proteins of cell surface antigens. It crosses the cell membrane four times, forming two extracellular loops and exposing its amino and carboxy termini to the cytoplasm. CD37 was highly expressed on normal antibody producing (sIg +) B-cells, but not on pre-B-cells or plasma cells. CD37 expression was low on resting and activated T cells, monocytes and granulocytes, and CD37 expression on NK cells, platelets or erythrocytes could not be detected. See Belov et al, Cancer res, 61 (11): 4483-4489 (2001); Schwartz-Albiez et al, j.immunol., 140 (3): 905-914 (1988); and Link et al, j.immunol., 137 (9): 3013-3018(1988). Almost all malignancies of B-cell origin, including CLL, NHL and hairy cell leukemia, are positive for CD37 expression except normal B-cells (Moore et al, 1987; Merson and Brochier 1988; Faure et al, 1990). CD37 is involved in the regulation of B-cell function because mice lacking CD37 were found to have low levels of serum IgG1 and their humoral response to viral antigens and model antigens was found to be attenuated. It appears to act as a non-canonical costimulatory molecule or to directly influence antigen presentation via complex formation with MHC class II molecules. See Knobeloch et al, mol.cell.biol., 20 (15): 5363-5369(2000).

Research and drug development are based on the following concepts: the B-cell profile is that specific cell surface molecules (such as CD37 and CD20) can themselves target antibodies that can bind to and mediate the destruction of B-cells that have CD37 and CD20 on their surface, leading to cancerous and autoimmune diseases. So-called "immunotherapeutic" antibodies prepared in non-human animals that bind to CD37 or CD20 (or based on the antibodies prepared) are provided to patients to deplete B-cells leading to cancerous diseases or autoimmune diseases.

Monoclonal antibody technology and genetic engineering methods are useful in the development of immunoglobulin molecules for the diagnosis and treatment of human diseases. The domain structure of immunoglobulins is consistent with engineering in that the antigen binding domain and the domain conferring effector function may be interchanged between immunoglobulin classes and subclasses. Immunoglobulin structure and function are reviewed, for example, in Antibodies compiled by Harlow et al: a Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (1988). Extensive introduction and detailed information on all aspects of Recombinant antibody technology can be found in the textbook "Recombinant Antibodies" (John Wiley & Sons, NY, 1999). A comprehensive compilation of detailed antibody engineering laboratory protocols can be found in R.Kontermann and S.D. umbel (eds), "The antibody engineering Lab Manual (antibody engineering laboratory Manual)" (Springer Verlag, Heidelberg/New York, 2000).

An immunoglobulin molecule (abbreviated Ig) is a polyprotein that is generally composed of two identical light chain polypeptides and two identical heavy chain polypeptides (H)₂L₂) Consisting of inter-chain disulfide bonds (i.e., covalent bonds between sulfhydryl groups of adjacent cysteine residues) joined into a macromolecular complex. Five classes of human immunoglobulins are defined based on heavy chain composition and are designated IgG, IgM, IgA, IgE and IgD. Antibodies of the IgG and IgA classes are further divided into subclasses, namely IgG1, IgG2, IgG3 and IgG4, and IgA1 and IgA2, respectively. Intrachain disulfide bonds connect different regions in the same polypeptide chain, resulting in the formation of loops that, together with adjacent amino acids, constitute an immunoglobulin domain. Each light chain and each heavy chain has a single variable region at the amino-terminal portion, and the single variable regions of different antibodies show considerable differences in amino acid composition. Light chain variable region V_LHas a single antigen binding domain and binds to the variable region V of the heavy chain_H(also contains a single antigen binding domain) to form the immunoglobulin antigen binding site Fv.

In addition to the variable regions, the full length antibody chains each have a constant region containing one or more domains. The light chain has a structure containing a single Constant regions of the domains. Thus, a light chain has one variable domain and one constant domain. Heavy chains have a constant region with several domains. The heavy chains in IgG, IgA and IgD antibodies have three designations C_H1、C_H2And C_H3(ii) a domain of (a); the heavy chains in IgM and IgE antibodies have four domains: c_H1、C_H2、C_H3And C_H4. Thus, a heavy chain has one variable domain and three or four constant domains. Of note in all known species is the invariant composition of the domains, in which the constant region comprising one or more domains is located at or near the C-terminus of the light and heavy chains of the immunoglobulin molecule, and the variable domains are located towards the N-terminus of the light and heavy chains. Immunoglobulin structure and function are reviewed, for example, in Antibodies compiled by Harlow et al: a Laboratory Manual (antibodies: Laboratory Manual), Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (1988).

The heavy chain of an immunoglobulin can also be divided into three functional regions: fd region (containing V)_HAnd C_H1(i.e., the two N-terminal domains of the heavy chain), a hinge region, and an Fc region (the "crystallizable fragment" region). The Fc region contains domains that interact with immunoglobulin receptors on cells and interact with the initiation elements of the complement cascade. Thus, it is believed that the Fc region or fragment is responsible for effector functions of immunoglobulins, such as ADCC (antibody-dependent cell-mediated cytotoxicity), CDC (complement-dependent cytotoxicity) and complement-binding reactions, binding to Fc receptors, relative to the absence of F _CLonger in vivo half-life for polypeptides of the region, protein a binding and perhaps even placental transfer. Capon et al, Nature, 337: 525-531, (1989). In addition, polypeptides containing an Fc region allow dimerization/multimerization of the polypeptide. The term is also applicable to analogous regions of other immunoglobulins.

Although all human immunoglobulin isotypes contain a common recognizable structure, each isotype displays a different type of effector function. IgG (as a non-exhaustive example) neutralizes toxins and viruses, opsonizes, binds complement (CDC) and participates in ADCC. In contrast, IgM neutralizes blood borne pathogens and is involved in opsonization. IgA is secreted when bound to its secretory fragment and provides the primary defense against microbial infection via the mucosa; it also neutralizes toxins and supports opsonization. IgE mediates inflammatory responses and is primarily involved in the recruitment of other cells required to establish a complete response. IgD is known to provide immunomodulatory functions, controlling B cell activation. The described features of isotype effector function provide a non-comprehensive illustration of the differences that may exist between human isotypes.

The hinge region present in IgG, IgA, IgD and IgE class antibodies acts as a flexible spacer, allowing the Fab portion to move freely in space. In immunoglobulins and subclasses, the hinge domain differs structurally from the constant region, both in sequence and length. For example, the length and flexibility of the hinge region varies within the IgG subclass. The hinge region of IgG1 comprises amino acids 216 and 231, and since it is free to flex, the Fab fragment can rotate around its axis of symmetry and can move within a sphere centered on the first bond of the disulfide bond between the two heavy chains. IgG2 has a shorter hinge than IgG1, and IgG2 has a hinge with 12 amino acid residues and four disulfide bonds. The hinge region of IgG2 lacks glycine residues, is relatively short, and contains a rigid polyproline double helix that is stabilized by additional inter-heavy chain disulfide bonds. This property limits the flexibility of the IgG2 molecule. IgG3 differs from other subclasses by its unique extended hinge region (about four times as long as the IgG1 hinge) which contains 62 amino acids (including 21 prolines and 11 cysteines) forming an inflexible polyproline double helix. In IgG3, the Fab fragment is relatively distant from the Fc fragment, making the molecule more flexible. The elongated hinges in IgG3 also contribute to their higher molecular weight than other subclasses. The hinge region of IgG4 is shorter than that of IgG1 and its flexibility is intermediate between that of IgG1 and that of IgG 2. The flexibility of the hinge region is reported to decrease in the following order: IgG3> IgG1> IgG4> IgG 2. The four IgG subclasses also differ from each other with respect to their effector functions. This difference is related to structural differences, including differences in the interaction between the variable regions, the Fab fragment, and the constant Fc fragment.

According to crystallographic studies, immunoglobulin hinge regions can be further functionally subdivided into three regions: an upper hinge region, a core region, and a lower hinge region. Shin et al, 1992 immunologicals reviews 130: 87. The upper hinge region includes C_H1To the first residue in the hinge that restricts movement (typically the first cysteine residue that forms an interchain disulfide bond between the two heavy chains). The length of the upper hinge region is related to the flexibility of the segment of the antibody. The core hinge region contains an intra-heavy chain disulfide bond, and the lower hinge region connects C_H2Amino terminal to the domain and including C_H2The residue of (1). As before. The core hinge region of human IgG1 contains the sequence Cys-Pro-Cys, which when dimerized by formation of disulfide bonds forms a cyclic octapeptide, resulting in the cyclic octapeptide acting as a pivot, thus imparting flexibility. The hinge region may also contain one or more glycosylation sites, including multiple sites of different structural types for attachment of carbohydrates. For example, IgA1 contains five glycosylation sites within the 17 amino acid segment of the hinge region, thereby conferring tolerance of the hinge region polypeptide to intestinal proteases, a property considered advantageous for immunoglobulin secretion.

Conformational changes permitted by the structure and flexibility of the immunoglobulin hinge region polypeptide sequence may also affect the effector function of the Fc portion of the antibody. Three general classes of effector functions associated with the Fc region include (1) activation of the classical complement cascade; (2) interacting with effector cells; and (3) compartmentalization of immunoglobulins. The different human IgG subclasses differ from each other with respect to their relative potency in binding complement or in activating and amplifying the steps of the complement cascade. See, e.g., Kirschfink, 2001 immunol.rev.180: 177; chakraborti et al, 2000 Cell Signal 12: 607; kohl et al, 1999 mol. Immunol.36: 893; marsh et al, 1999 curr. opin. nephrol. hypertens.8: 557; speth et al, 1999 Wien Klin. Wochenschr.111: 378.

present in camelids (camels, dromedary camels and llamas; Hamers-Casterman et al, 1993 Nature 363: 446; Nguyen et al, 1998 J.mol.biol 275: 413), nurse shark (Roux et al, 199. mol.biol. 275: 413), nurse shark (Roux et al, Boeha et al8 proc.nat.acad.sci.usa 95: 11804) And Spanish mackerel (Nguyen et al, 2002 Immunogenetics 54 (1)): 39-47) of immunoglobulins in which H of conventional antibodies is present₂L₂Exceptions to the architecture. Obviously, the antibody can form an antigen binding region using only the heavy chain variable region, i.e., the functional antibody is a heavy chain-only homodimer (referred to as "heavy chain antibody" or "HCAb"). Although antibody technology has advantages in disease diagnosis and treatment, there are certain disadvantages in developing whole antibody technology as a diagnostic and/or therapeutic agent. Whole antibodies are large protein structures, such as heterotetrameric structures of the IgG isotype that contain two light chains and two heavy chains. The macromolecules are sterically hindered in certain applications. For example, in the treatment of solid tumors, whole antibodies do not readily penetrate into the interior of the tumor. In addition, the relatively large size of whole antibodies can provide priming to ensure that in vivo administration of the molecule does not elicit an immune response. Furthermore, the production of active antibody molecules typically involves culturing recombinant eukaryotic cells that are capable of providing appropriate post-translational processing of nascent antibody molecules, which cells are difficult to culture and difficult to induce in a manner that provides commercially useful yields of active antibodies.

Recently, smaller immunoglobulin molecules have been constructed to overcome the problems associated with the whole immunoglobulin approach. Single chain variable antibody fragments (scFv) comprise an antibody heavy chain variable domain linked via a short peptide to an antibody light chain variable domain (Huston et al, Proc. Natl. Acad. Sci. USA, 1988, 85: 5879-83). Due to the small size of scFv molecules, they appear to penetrate tissues more efficiently than whole immunoglobulins. Anti-tumor scFv showed more rapid tumor penetration and more uniform distribution within tumor masses compared to the corresponding chimeric antibody (Yokota et al, Cancer Res.1992, 52: 3402-08).

Although scFv molecules offer advantages for serum therapy, there are several drawbacks to this therapy. scFv can be cleared rapidly from the circulation, thereby reducing toxic effects on normal cells, but this rapid clearance prevents the delivery of the least effective dose to the target tissue. Manufacturing sufficient quantities of scFv for administration to patients has been challenging due to the adverse effects on yield resulting from difficulties in expressing and isolating scFv. During expression, scFv molecules lack stability and often aggregate due to pairing of the variable regions of different molecules. Furthermore, the low production of scFv molecules in mammalian expression systems limits the potential for efficient manufacture of scFv molecules for therapeutic use (Davis et al, J biol. chem.1990, 265: 10410-18; Traunecker et al, EMBO J1991, 10: 3655-59). Strategies to improve production have been explored, including the addition of glycosylation sites to the variable regions (Jost, C.R. U.S. Pat. No. 5,888,773, Jost et al, J.biol. chem.1994, 69: 26267-73).

Another disadvantage of using scFv therapy is the lack of effector function. scFv that bind to the constant region of immunoglobulin have no cytolytic function, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and are ineffective for treating diseases. Although scFv technology has been developed 12 years ago, no scFv products are currently approved for therapy.

Alternatively, it has been proposed that scFv can be fused to another molecule such as a toxin to deliver the toxin to the target tissue using specific antigen binding activity and small size scFv. Chaudary et al, Nature 1989, 339: 394; batra et al, mol.cell.biol.1991, 11: 2200. thus, binding or fusion of toxins to scFv can be used as an alternative strategy to provide effective antigen-specific molecules, but administration of such conjugates or chimeras is limited by excessive and/or non-specific toxicity (due to the toxin moiety in the formulation). Toxic effects may include a hyper-physiological rise in liver enzymes and vascular leak syndrome, among other adverse effects. In addition, immunotoxins are highly immunogenic themselves after administration to a host, and host antibodies raised against immunotoxins limit the potential effectiveness of repeated therapeutic treatments of individuals.

Non-surgical cancer treatments, such as external irradiation and chemotherapy, have limited efficacy due to the lack of specificity that the treatment exhibits for cancer cells, due to toxic effects on normal tissues and cells. To overcome this limitation, targeted therapeutic approaches have been developed to enhance the specificity of treatment for the cells and tissues in need thereof. An example of the targeting method for in vivo use is the administration of an antibody conjugate, wherein the antibody is designed to specifically recognize a marker associated with a cell or tissue in need of treatment and bind the antibody to a therapeutic agent, such as a toxin (in the case of cancer treatment). Antibodies as systemic agents can circulate to sensitive and undesirable body compartments, such as bone marrow. In acute radiation injury, destruction of the lymphoid and hematopoietic compartments is a major factor in the development of sepsis and subsequent death. In addition, antibodies are large globular proteins that can exhibit poor penetration into the tissue in need of treatment.

Human patients and non-human subjects suffering from a variety of end-stage disease processes often require organ transplantation. However, organ transplantation must cope with the inappropriate immune response of the recipient and avoid immune rejection of the transplanted organ by suppressing the recipient's cellular immune response to the foreign organ with cytotoxic agents that affect the lymphoid and other parts of the hematopoietic system. Transplant acceptance is limited by the recipient's tolerance to such cytotoxic chemicals, many of which are similar to anti-cancer (anti-proliferative) agents. Also, when using cytotoxic antimicrobials (especially antiviral drugs) or when using cytotoxic drugs for the treatment of autoimmune diseases, such as in the treatment of systemic lupus erythematosus, the toxic effects of the therapeutic agents on the body's bone marrow and hematopoietic cells are severely limited.

The use of targeted therapies, such as targeted antibody binding therapies, is designed to localize the largest amount of therapeutic agent as possible to the site of desired action, and whether the therapy is successful is manifested by a relatively high signal to background ratio of the therapeutic agent. Examples of targeting antibodies include antibodies or antibody fragments, cell-or tissue-specific peptides, and diagnostic or therapeutic agent combinations of hormones and other receptor binding molecules. For example, antibodies directed to different determinants associated with pathological and normal cells, as well as those associated with pathogenic microorganisms, have been used to detect and treat a variety of pathological conditions or lesions. In the methods, the targeting antibody is directly coupled to a suitable detection or therapeutic agent as described in, for example, Hansen et al, U.S. Pat. No. 3,927,193 and Goldenberg, U.S. Pat. Nos. 4,331,647, 4,348,376, 4,361,544, 4,468,457, 4,444,744, 4,460,459, 4,460,561, 4,624,846 and 4,818,709.

One problem encountered in direct targeting approaches, i.e., approaches in which a diagnostic or therapeutic agent ("active agent") is directly coupled to a targeting moiety, is that a relatively small portion of the conjugate actually binds to the target site, while most of the conjugate remains in circulation and seeks to detract from the function of the targeted conjugate. To ensure maximum localization of the active agent, an excess of targeting conjugate is typically administered, thereby ensuring that certain conjugates remain unbound and promoting a background level of active agent. Diagnostic conjugates (e.g., radioimmunoassay conjugates or magnetic resonance imaging conjugates that do not bind to their target) can remain in circulation, increasing background and reducing resolution of the diagnostic technique. In the case of therapeutic conjugates having a toxin (e.g., a radioisotope, drug, or toxic compound) as an active agent linked to a long circulating targeting moiety, such as an antibody, the circulating conjugate can produce unacceptable toxicity to the host, such as bone marrow toxicity or systemic side effects.

U.S. patent No. 4,782,840 discloses a method of reducing the effects of high background radiation levels during surgery. The method comprises injecting a patient with an antibody specific for neoplastic tissue, wherein the antibody labeled with a radioisotope (such as iodine-125) has a suitably long half-life. After injection of the radiolabeled antibody, surgery is delayed for at least 7 to 10 days, preferably 14 to 21 days, so that any unbound radiolabeled antibody can be cleared up to a low background amount.

U.S. patent No. 4,932,412 discloses a method for reducing or correcting non-specific background radiation during intraoperative detection. The method comprises administering to a patient who has received a radiolabeled primary antibody a contrast agent, a subtraction agent, or a secondary antibody that binds to the primary antibody.

In addition to producing the above antibodies, the immune system includes a variety of cell types with strong biological effects. During hematopoiesis, bone marrow-derived stem cells differentiate into mature cells of the immune system ("B" cells) or into cell precursors that migrate from the bone marrow to mature in the thymus ("T" cells).

B cells are critical for the humoral component of the immune response. B cells become antibody secreting plasma cells upon activation by appropriate presentation of antigen; antigen presentation also results in clonal expansion of activated B cells. B cells are primarily responsible for the humoral component of the immune response. Plasma cells typically exhibit about 10 on their surface ⁵Antibody molecules (IgD and IgM).

T lymphocytes can be divided into two classes. Cytotoxic T cells, Tc lymphocytes or CTL (CD8+ T cells) kill cells carrying foreign surface antigens associated with class I MHC and can kill cells with intracellular parasites (bacteria or viruses) as long as the infected cells display microbial antigens on their surface. Tc cells kill tumor cells and indicate rejection of implanted cells. The Tc cells recognize, contact the antigen-MHC class I complex on the target cell and release the contents of the particles directly into the target cell membrane, thereby lysing the cells.

The second type of T cell is a helper T cell or Th lymphocyte (CD4+ T cell) that produces lymphokines that act as a "helper" factor in the maturation of B cells into antibody-secreting plasma cells. Th cells also produce certain lymphokines that stimulate differentiation of effector T lymphocytes and macrophage activity. Th1 cells recognize MHC class II-associated antigens on macrophages and are activated (by IL-1) to produce lymphokines, including IFN- γ, which activates macrophages and NK cells. The cells mediate various aspects of cell-mediated immune responses, including delayed-type hypersensitivity reactions. Th2 cells recognize MHC class II-associated antigens on antigen presenting cells or APCs (e.g., migratory macrophages and dendritic cells) and then produce interleukins and other substances that stimulate the proliferation and activity of specific B-and T-cells.

In addition to serving as an APC that triggers T cell interactions, development and proliferation, macrophages are also involved in the expression of cell-mediated immunity because they are activated by IFN- γ produced in cell-mediated immune responses. Activated macrophages have enhanced phagocytic potential and release soluble substances that cause inflammation and destroy a variety of bacteria and other cells. Natural killer cells are cytotoxic cells that can lyse cells bearing a new antigen (whether or not they are MHC-type), and even some cells that do not bear MHC proteins. Natural killer T cells or NK cells are defined in terms of their ability to kill cells displaying foreign antigens (e.g., tumor cells), whether MHC-type or not, and whether pre-sensitized (exposed) to the antigen or not. NK cells can be activated by IL-2 and IFN-gamma, and in the same way as cytotoxic T lymphocytes dissolve cells. Certain NK cells have receptors for the Fc domain of IgG antibodies (e.g., CD16 or Fc γ RIII) and are therefore capable of binding to the Fc portion of IgG on the surface of target cells and releasing cytolytic components that kill the target cells via antibody-dependent cell-mediated cytotoxicity.

Another group of cells are granulocytes or polymorphonuclear leukocytes (PMNs). Neutrophils (a type of PMN) kill bacterial invaders and phagocytose the residue. Eosinophils are another type of PMN and contain particles that exhibit cytotoxicity when released against another cell (such as a foreign cell). Basophils (the third type of PMNs) are important mediators of a strong physiological response (e.g., inflammation) that exert their effects by releasing a variety of biologically active compounds such as histamine, serotonin, prostaglandins, and leukotrienes. Common to all cell types is the ability to exert a physiological effect in an organism, typically by killing and optionally eliminating harmful compositions such as foreign cells.

While various mammalian cells, including cells of the immune system, are capable of directly exerting physiological effects (e.g., cell killing as represented by Tc, NK, certain PMNs, macrophages, and the like), other cells indirectly contribute to physiological effects. For example, initial presentation of antigen to naive T cells of the immune system requires MHC presentation that commands cell-cell contact. In addition, contact between activated T cells and antigen-specific B cells is often required to obtain a particular immunogenic response. A third form of cell-cell contact common in immune responses is contact between activated B cells and follicular dendritic cells. The cell-to-cell contact requirements each complicate targeting of the bioactive agent to the intended target.

Complement Dependent Cytotoxicity (CDC) is considered an important mechanism for the clearance of specific target cells such as tumor cells. CDC is a series of events consisting of activation of a large number of enzymes in cascade with each other. Complement plays an important role in clearing antigens, and is thus achieved by its four major functions: (1) local vessel dilation; (2) attraction of immune cells, especially macrophages (chemotaxis); (3) labeling foreign organisms for phagocytosis (opsonization); and (4) destructive attack of the organism by the membrane attack complex (MAC attack). The main molecule is C3 protein. It is an enzyme that cleaves into two fragments from components of either the classical or alternative pathways. The classical pathway is evoked by antibodies (especially IgG and IgM), while the alternative pathway is non-specifically stimulated by bacterial products such as Lipopolysaccharide (LPS). Briefly, the product of C3 cleavage includes the small peptide C3a, which is chemotactic for phagocytic immune cells and causes local vasodilation by causing the release of the C5a fragment from C5. Another portion of C3b of C3 coats antigens on the surface of foreign organisms and exerts opsonization to the organism to destroy them. C3b also reacts with other components of the complement system to form MAC consisting of C5b, C6, C7, C8 and C9.

There are problems associated with the use of antibodies in human therapy because the immune system reacts to any antigen, even the simplest, "polyclonal" reactions, i.e., the system produces antibodies with a wide range of structures in both their binding and effector regions.

Two approaches have been used in an attempt to reduce the problem of immunogenic antibodies. The first method is to prepare a chimeric antibody in which an antigen-binding portion (variable region) of a mouse monoclonal antibody is fused with an effector portion (constant region) of a human antibody. In the second approach, antibodies have been altered by a technique known as Complementarity Determining Region (CDR) grafting or "humanization". The method is further modified to include variations referred to as: "Reconfiguration" (Verhoeyen et al, 1988 Science 239: 1534-1536; Riechmann et al, 1988 Nature 332: 323-337; Tempest et al, Bio/Technol 19919: 266-271), "Superchimerism" (Queen et al, 1989 Proc Natl Acad Sci USA 86: 10029-10033; Co et al, 1991 Proc Natl Acad Sci USA 88: 2869-2873; Co et al, 1992J Immunol 148: 9-1154), and "facing" (Mark et al, in Metcalf BW, Dalton BJ eds. Cellular phase: molecular analysis to thermal approach point potential. New York: Plenum Press, 1994: 114312).

Since 1986, which is eleven years after the release of monoclonal antibodies, on average, less than one therapeutic antibody was introduced into the market each year. During the decade from 1986 to 1995, five murine monoclonal antibodies were introduced into human medicine, including "Moluomab-CD 3(muromonab-CD 3)" (Orthoclone) for acute rejection of organ transplants) (ii) a Esomelomab for colorectal cancer ""odulimomab" for graft rejection "And "ibritumomab (ibritumomab)" for non-Hodgkin's lymphoma "yiuxetan). Furthermore, the monoclonal Fab "abciximab" is commercially available "Can be used for preventing re-occlusion of coronary artery. Three chimeric monoclonal antibodies have also been proposed: "Rituximab" for the treatment of B cell lymphomas”'basiliximab' for transplant rejection "And infliximab for treating rheumatoid arthritis and Crohn's disease "In addition, "abciximab"(chimeric human-mouse monoclonal antibody 47.6kD Fab fragment) as percutaneous coronary intervention adjuvant for the prevention of percutaneous coronary intervention patients ischemic cardiac complications. Finally, seven "humanized" monoclonal antibodies have been proposed. "Dalizumab" Is used for preventing acute rejection of transplanted kidney; 'palivizumab'Is for RSV; trastuzumab "Binds to HER-2 (a growth factor present on breast cancer cells); "gemtuzumab" (gemtuzumab) "Is for Acute Myeloid Leukemia (AML); and "alemtuzumab"Is for chronic lymphocytic leukemia; "adalimumab" (adalimumab) "((D2E7)) is useful for the treatment of rheumatoid arthritis; and "omalizumab"Is used for treating persistent asthma.

Accordingly, a variety of antibody technologies have received attention in the effort to develop and market more effective therapeutic agents and demulcents. Unfortunately, there are still problems that undermine the prospects of the various therapies. For example, most rituximab-treated cancer patients typically relapse within about 6 to 12 months, and fatal infusion reactions have been reported to occur within 24 hours of rituximab infusion. Acute renal failure requiring dialysis in the presence of fatal outcomes has also been reported to have severe (sometimes fatal) mucocutaneous reactions in treatment with rituximab. In addition, intravenous injection requires high doses of rituximab, since the molecule is large (about 150kDa) and diffusion into lymphoid tissues where large numbers of tumor cells are present is limited.

Trastuzumab administration can lead to the development of ventricular dysfunction, congestive heart failure and severe allergic reactions (including severe allergies), infusion reactions and pulmonary events. Dallizumab immunosuppressive therapy increases the risk of developing lymphoproliferative disorders and opportunistic infections. Deaths due to liver failure, deaths due to severe hepatotoxicity, and deaths due to Venous Occlusive Disease (VOD) have been reported to occur in patients receiving gemtuzumab.

Hepatotoxicity in patients receiving alemtuzumab has also been reported. Patients receiving alemtuzumab therapy have developed severe (and in some rare cases fatal) pancytopenia/myelodysplasia, autoimmune idiopathic thrombocytopenia, and autoimmune hemolytic anemia. Alemtuzumab can also cause severe infusion reactions and opportunistic infections. It has been reported that severe infections and sepsis (including fatal severe infections and sepsis) present with worsening clinical symptoms and/or radiographic signs of demyelinating disease in adalimumab-treated patients, and that adalimumab-treated patients have a higher incidence of lymphoma than expected in the general population in clinical trials. Omalizumab is reported to induce malignant tumors and severe allergy.

Cancer encompasses a wide range of diseases, spread across about one quarter of individuals worldwide. Rapid and unregulated proliferation of malignant cells is a hallmark of a variety of cancer types, including hematological malignancies. Although patients with hematological malignancies have benefited over the past two decades due to advances in cancer therapy (Multani et al, 1998J. Clin. Oncology 16: 3691-3710), and remission has multiplied, most patients have relapsed and die of disease. Disorders for treatment with cytotoxic drugs, including, for example, tumor cell tolerance and high toxicity of chemotherapy, prevent optimal administration to many patients.

It has been reported that treatment of patients with low grade malignancy or follicular B cell lymphoma with a chimeric CD20 monoclonal antibody can induce partial or complete responses in the patient. McLaughlin et al, 1996 Blood 88: 90a (supplement 1, abstract); maloney et al, 1997 Blood 90: 2188-95. However, as noted above, tumor recurrence typically occurs within six months to a year. There is a need for further improved serum therapies to induce a more durable response to, for example, low-grade malignant B-cell lymphomas and to make it possible to effectively treat high-grade malignant lymphomas and other B-cell diseases.

Another approach is to target the radioisotope to B cell lymphoma using a monoclonal antibody specific for CD 20. Despite the reported increase in therapeutic effectiveness, toxicity associated with the long in vivo half-life of the radioactive antibody is also enhanced, often requiring patients to undergo stem cell rescue. Press et al, 1993 n.eng.j.med.329: 1219-; kaminski et al, 1993 n.eng.j.med.329: 459-65. Prior to attachment of the radioisotope, a monoclonal antibody to CD20 has also been cleaved with a protease to yield F (ab')₂Or a Fab fragment. This has been reported to improve the penetration of the radioisotope conjugate into tumors and to reduce the half-life in vivo, thereby reducing toxicity to normal tissues. However, the molecule lacks effector functions, including complement fixation and/or ADCC.

Autoimmune diseases include autoimmune thyroid diseases (including Graves 'disease and Hashimoto's thyroiditis). In the united states alone, about two million people suffer from some form of autoimmune thyroid disease. Autoimmune thyroid disease is caused by the production of autoantibodies that stimulate the thyroid gland leading to hyperthyroidism (Gray's disease) or that destroy the thyroid gland leading to hypothyroidism (Hashimoto's thyroiditis). Stimulation of the thyroid is due to autoantibodies binding to and activating Thyroid Stimulating Hormone (TSH) receptors. Damage to the thyroid gland is caused by autoantibodies reacting with other thyroid antigens. Current therapies for gray's disease include surgery, radioiodine or antithyroid drug therapy. Radioactive iodine is widely used because antithyroid drugs have significant side effects and the rate of disease recurrence is high. The operation is especially suitable for patients with large goiter or patients needing to normalize thyroid function extremely rapidly. There are no therapies that target the production of autoantibodies responsible for stimulating TSH receptors. Current therapy for hashimoto's thyroiditis is levothyroxine sodium (levothyroxine sodium), and is expected to be lifelong therapy due to the low likelihood of remission. Inhibitory therapy has been shown to reduce goiter in hashimoto's thyroiditis, but no therapy is known to reduce autoantibody production to target disease mechanisms.

Rheumatoid Arthritis (RA) is a chronic disease characterized by inflammation of the joints, which results in swelling, pain, and loss of function. It is estimated that two hundred and fifty thousand people in the united states have RA. RA is caused by a combination of events including initial infection or injury, abnormal immune response, and genetic factors. Although self-reactive T cells and B cells are present in RA, RA can be diagnosed by detecting high levels of antibodies (called rheumatoid factors) that accumulate in the joints. Current therapies for RA include a variety of drugs for controlling pain and slowing disease progression. No cure for the disease has been found. The drugs include nonsteroidal anti-inflammatory drugs (NSAIDS) and disease modifying anti-rheumatoid drugs (DMARDS). NSAIDS are indicated for benign disease, but do not prevent joint destruction and weakness that progress to severe RA. Both NSAIDS and DMARDS are associated with significant side effects. Only one new DMARD Leflunomide (Leflunomide) was approved for 10 years. Leflunomide prevents the production of autoantibodies, reduces inflammation and slows the progression of RA. However, the drug also causes severe side effects including nausea, diarrhea, hair loss, rash, and liver damage.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease that results from repeated injury to blood vessels in various organs including the kidney, skin and joints. It is estimated that over 500,000 people in the united states have SLE. In patients with SLE, inappropriate interaction between T cells and B cells results in the production of autoantibodies that attack the nucleus. The antibodies include anti-double stranded DNA and anti-Sm antibodies. Autoantibodies that bind phospholipids are also present in about half of SLE patients and cause vascular damage and low blood count. Immune complexes accumulate in the kidneys, blood vessels, and joints of SLE patients, leading to inflammation and tissue damage. No SLE therapy has been found that can cure the disease. Depending on the severity of the disease, NSAIDS and DMARDS may be used for treatment. Plasmapheresis, which removes autoantibodies by plasma exchange, can provide temporary improvement in SLE patients. It is generally agreed that autoantibodies lead to SLE, and therefore novel therapies that deplete the B cell profile (which restore the immune system when new B cells are generated from precursors) offer promise for sustained benefit in SLE patients.

Sjogren's syndrome is an autoimmune disease characterized by the destruction of body water producing glands. The sjogren's syndrome is one of the most common autoimmune disorders, and it is estimated that up to 4 million people in the united states suffer from this disorder. Approximately half of patients with sjogren's syndrome also have connective tissue disease, such as RA, while the other half have primary sjogren's syndrome, but no other concurrent autoimmune disease. Autoantibodies, including anti-nuclear antibodies, rheumatoid factor, anti-fodrin, and anti-muscarinic receptors are commonly present in patients with sjogren's syndrome. Conventional therapies include corticosteroids, and other more effective therapies are also beneficial.

Immune Thrombocytopenic Purpura (ITP) is caused by autoantibodies binding to platelets and causing their destruction. Some cases of ITP are drug-induced, while others are associated with infection, pregnancy, or autoimmune diseases such as SLE. About half of all cases can be classified as idiopathic origin. Treatment of ITP is determined by the severity of the symptoms. Although immunosuppressive drugs (including corticosteroids or intravenous infusion of immunoglobulins to deplete T cells) are provided in most cases, no treatment is required in some cases. Another treatment that often results in an increase in platelet number is removal of the spleen, which destroys antibody-coated platelets. For patients with severe cases, more potent immunosuppressive drugs are used, including cyclosporine, cyclophosphamide, or azathioprine. Removal of autoantibodies by passing patient plasma through a protein a column can be used as a second line of treatment for patients with severe disease. Other more effective therapies are needed.

Multiple Sclerosis (MS) is also an autoimmune disease. It is characterized by inflammation of the central nervous system and destruction of the myelin sheath, leaving nerve cell fibers in the brain, spinal cord and body isolated. Although the cause of MS is unknown, it is widely believed that autoimmune T cells are the major contributor to the pathogenesis of the disease. However, high levels of antibodies are present in the cerebrospinal fluid of MS patients, and some predict that the B cell response leading to antibody production has an important role in mediating the disease. B cell depletion therapy has not been studied for MS patients and there is no cure for MS. Current therapies are corticosteroids, which can reduce the duration and severity of the challenge, but do not affect the progression of MS over time. Recently, new biotechnological Interferon (IFN) therapies for MS have been approved, but there is still a need for other more effective therapies.

Myasthenia Gravis (MG) is a chronic autoimmune neuromuscular disorder characterized by weakness of any muscle group. About 40,000 people in the united states have MG. MG is due to the binding of autoantibodies to acetylcholine receptors expressed at the neuromuscular junction. Autoantibodies reduce or block acetylcholine receptors and prevent signal transmission from the nerve to the muscle. No cure for MG is known. Common treatments include immunosuppression with corticosteroids, cyclosporin, cyclophosphamide or azathioprine. Surgical removal of the thymus is often used to attenuate the autoimmune response. Plasmapheresis, which is used to reduce autoantibody levels in the blood, is effective for MG, but is transient due to the continued production of autoantibodies. Plasmapheresis is typically used by severely muscle infirm prior to surgery. Novel and effective therapies are also beneficial.

About five million people suffer from psoriasis and are characterized by autoimmune inflammation of the skin. Psoriasis is also associated with arthritis (psoriatic arthritis) in 30% of patients. A variety of therapies have been used, including steroids, ultraviolet retinoids, vitamin D derivatives, cyclosporine, and methotrexate (methotrexate), but it is clear that psoriasis will also benefit from novel and effective therapies. Scleroderma is a chronic autoimmune disease of the connective tissue, also known as systemic sclerosis. Scleroderma is characterized by overproduction of collagen, leading to thickening of the skin, and in the united states about 300,000 people suffer from scleroderma, which will also benefit from novel and effective therapies.

As is apparent from the above discussion, there is a need for improved compositions and methods for treating, ameliorating, or preventing a variety of diseases, disorders, and conditions, including cancer and autoimmune diseases.

SUMMARY

The present invention meets at least one of the above-mentioned needs in the art by providing: a protein comprising at least two specific binding domains, wherein the two domains are linked by a constant sub-region derived from an antibody molecule, the C-terminus of which is linked to a linker, referred to herein as a scorpion linker; and nucleic acids encoding the proteins; and the preparation, diagnostic and therapeutic uses of said proteins and nucleic acids. The constant sub-region comprises a constant region derived from immunoglobulin C _H2Domain of a Domain and preferably derived from immunoglobulin C_H3Domains of the Domain, but not derived from or corresponding to immunoglobulin C_H1Junctions of structural domainsA domain or region. It was previously thought that placing constant regions derived from antibodies inside proteins would interfere with antibody function, such as effector function, and so on, and that placing constant regions of antibodies at the carboxy terminus of the antibody chain would also generally interfere with antibody function. Furthermore, the placement of a scorpion linker (which may be an immunoglobulin hinge-like peptide) at the C-terminus of the constant sub-region is a different construct than that of a naturally occurring immunoglobulin. However, placement of the constant sub-region (where the scorpion linker connects the C-terminus of the constant region) within a polypeptide chain or protein chain according to the present invention renders proteins that exhibit effector functions and multivalent (mono-or multispecific) binding capabilities relatively unhindered by steric hindrance. As will be apparent to those skilled in the art upon consideration of the present disclosure, the protein is designed as a module and can be constructed as follows: by selecting any of the domains as binding domain 1 or binding domain 2 (or as any other binding domain present in a particular protein of the invention) among the various binding domains; by selecting constant subregions with effector function; and by selecting hinge-like or non-hinge-like scorpion linkers (e.g., type II C-lectin receptor stem peptides), wherein the protein exhibits the general organization of N-binding domain 1-constant subdomain-scorpion linker-binding domain 2-C. One skilled in the art will further appreciate that there are a variety of applications for proteins having this structure and nucleic acids encoding such proteins, including medical and veterinary applications.

One aspect of the invention pertains to a multivalent single chain binding protein having an effector function or scorpion linker (the terms are used interchangeably) comprising a first binding domain derived from an immunoglobulin (e.g., antibody) or immunoglobulin-like molecule; a constant sub-region providing an effector function, the constant sub-region being located C-terminal to the first binding domain; a scorpion linker at the C-terminus of the constant subregion; and a second binding domain derived from an immunoglobulin (such as an antibody) or immunoglobulin-like molecule, the second binding domain being C-terminal to the constant sub-region; such that the constant sub-region is positioned between the first binding domain and the second binding domain. Single-chain binding proteins are multispecific (e.g., bispecific) in that they can bind two or more different targets, or they can be monospecific, having two binding sites for the same target. In addition, all domains of the protein are present in a single chain, but the protein can form homomultimers, for example by forming interchain disulfide bonds. In certain embodiments, the first binding domain and/or the second binding domain are variable regions of light and heavy immunoglobulin chains derived from the same or different immunoglobulins (e.g., antibodies). The immunoglobulin may be derived from any vertebrate, such as a mammal, including a human, and may be a chimeric, humanized fragment, variant, or derivative of a naturally occurring immunoglobulin.

The invention encompasses proteins wherein the first and second binding domains are derived from the same or different immunoglobulins (e.g., antibodies) and wherein the first and second binding domains recognize the same or different molecular targets (e.g., cell surface markers, such as membrane bound proteins). Furthermore, the first and second binding domains may recognize the same or different epitopes. The first and second molecular targets may be associated with first and second target cells, viruses, vectors, and/or objects. In a preferred embodiment according to this aspect of the invention, the first binding domain, the second binding domain and the constant sub-region are each derived from a human immunoglobulin, such as an IgG antibody. In other embodiments, a multivalent binding protein with effector function has at least one of a first binding domain and a second binding domain that can recognize at least one cell-free molecular target (e.g., a cell-unrelated protein, such as a depositable protein or a soluble protein). For example, cell-free molecular targets include proteins unrelated to the cell, e.g., administered compounds, such as proteins; and proteins secreted, lysed, present in exosomes or excreted or isolated from the cell.

The target molecules recognized by the first and second binding domains may be present on or associated with the same or different prokaryotic cells, eukaryotic cells, viruses (including bacterial phages), organic or inorganic target molecule carriers, and foreign objects. Furthermore, the target molecule may be located on the same type of cell, virus, vector or object (e.g., two different eukaryotic cells, prokaryotic cells, viruses, or vectors) but physically different, or the target molecule may be located on different types of cells, viruses, vectors, or objects (e.g., eukaryotic cells and viruses). Target cells are those associated with the target molecule recognized by the binding domain and include endogenous or autologous cells as well as exogenous or foreign cells (infectious microbial cells, transplanted mammalian cells (including transfusion cells)). The invention includes targets for the first and/or second binding domains present on the surface of a target cell associated with a disease, disorder or condition in a mammal, such as a human. Exemplary target cells include cancer cells, cells associated with autoimmune diseases or disorders, and infectious cells (e.g., infectious bacteria). Cells of infectious organisms (such as mammalian parasites) are also contemplated as target cells. In certain embodiments, the proteins of the invention are multivalent (e.g., multispecific) binding proteins having effector function, wherein at least one of the first and second binding domains recognizes a target selected from the group consisting of: tumor antigens, B-cell targets, TNF receptor superfamily members, Hedgehog family members, receptor tyrosine kinases, proteoglycan-related molecules, TGF- β superfamily members, Wnt-related molecules, receptor ligands, T-cell targets, dendritic cell targets, NK cell targets, monocyte/macrophage targets, and angiogenesis targets.

In certain embodiments of the above protein, the tumor antigen is selected from the group consisting of: squamous cell carcinoma antigen 1(SCCA-1), (protein T4-A), squamous cell carcinoma antigen 2(SCCA-2), ovarian carcinoma antigen CA125(1A1-3B) (KIAA0049), mucin 1 (tumor-associated mucin), (cancer-associated mucin), (polymorphic epithelial mucin), (PEM), (PEMT), (epithelial mucin), (tumor-associated epithelial cell membrane antigen), (EMA), (H23AG), (peanut-reactive uromucin), (PUM), (breast cancer-associated antigen DF3), CTCL tumor antigen se1-1, CTCL tumor antigen se14-3, CTCL tumor antigen se20-4, CTCL tumor antigen se20-9, CTCL tumor antigen se33-1, CTCL tumor antigen se37-2, CTCL tumor antigen se57-1, CTCL tumor antigen se89-1, prostate specific cell membrane antigen, prostate cancer cell membrane antigen, prostate specific cell membrane antigen, 5T4 carcinoembryonic trophoblast glycoprotein, Orf73 Kaposi's sarcoma-associated herpesvirus (Orf73 Kaposi' ssarcoma-associated herpesvirus), MAGE-C1 (cancer/testis antigen CT7), MAGE-B1 antigen (MAGE-XP antigen) (DAM10), MAGE-B2 antigen (DAM6), MAGE-2 antigen, MAGE-4a antigen, MAGE-4B antigen, colon cancer antigen NY-CO-45, lung cancer antigen NY-LU-12 variant A, cancer-associated surface antigen, adenocarcinoma antigen ART1, paratumor-associated brain-testis-cancer antigen (neural cancer antigen MA 2; paratumor neuron antigen), neural tumor ventral antigen 2(NOVA2), hepatocellular carcinoma gene, tumor-associated antigen CO-029, tumor-associated antigen MAGE-X2, synovial sarcoma, X breakpoint 2, squamous carcinoma antigen recognized by T cells, Serologically defined colon cancer antigen 1, serologically defined breast cancer antigen NY-BR-15, serologically defined breast cancer antigen NY-BR-16, chromogranin A; parathyroid secretory protein 1, DUPAN-2, CA 19-9, CA 72-4, CA 195 and L6.

Embodiments of the above methods comprise a B cell target selected from the group consisting of: CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a/b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138 and CDw 150.

In other embodiments of the above methods, the TNF receptor superfamily member is selected from the group consisting of: 4-1BB/TNFRSF, NGFR/TNFRSF, BAFFR/TNFRSF13, osteoprotegerin/TNFRSF 11, BCMA/TNFRSF, OX/TNFRSF, CD/TNFRSF, RANK/TNFRSF11, CD/TNFRSF, RELT/TNFRSF19, CD/TNFRSF, TACI/TNFRSF13, DcR/TNFRSF 6, TNF RI/TNFRSF1, DcTRAR/TNFRSF, TNF RII/TNFRSF1, DcR/TNFRSF, DcRILRRSF 10, DR/TNFRSF 10, TRAILR/TNFRSF 10, DR/TNFRSF, TRAILR/TNFRSF 10, EDAR/TNFRSF 10, TRAILR/TNFRSF 10, FasDA/TNFRSF, TROY/TNFRRSF, GITR/TNFRSF, TNFRSFRJSFF, TNFRSF, HVTRAIL/TNFRSF 1, TNFRSF/TNFRSF 13, TNFRSF, CD30 ligand/TNFSF 8, TNF- α/TNFSF1A, CD40 ligand/TNFSF 5, TNF- β/TNFSF1B, EDA-A2, TRAIL/TNFSF10, Fas ligand/TNFSF 6, TRANCE/TNFSF11, GITR ligand/TNFSF 18, TWEAK/TNFSF12, and LIGHT/TNFSF 14.

The above methods also include embodiments wherein the Hedgehog family member is selected from the group consisting of Patched and Smoothened. In other embodiments, the proteoglycan-related molecule is selected from the group consisting of proteoglycans and modulators thereof.

Other embodiments of the method are directed to methods wherein the receptor tyrosine kinase is selected from the group consisting of: axl, FGF R4, C1q R1/CD93, FGF R93, DDR 93, Flt-3, DDR 93, HGF R, Dtk, IGF-93, EGF R, IGF-II R, Eph, INSRR, EphA 93, insulin R/CD220, EphA 93, M-CSF R, EphA 93, Mer, EphA 93, MSP R/Ron, EphA 93, MuSK, EphA 93, PDGF R alpha, EphA 93, PDGFR beta, EphA 93, Ret, EphB 93, ROR 93, EphB 93, SCF R/C-kit, EphB 93, Tie-1, EphB 93, Tie-2, ErbB 93, TrkB 93, TrkC, VEGF R/FLR 93, Flt-93, Flt/FLR 93, and Flt/FLR 93.

In other embodiments of the method, the Transforming Growth Factor (TGF) - β superfamily member is selected from the group consisting of: activin RIA/ALK-2, GFR alpha-1, activin RIB/ALK-4, GFR alpha-2, activin RIIA, GFR alpha-3, activin RIIB, GFR alpha-4, ALK-1, MIS RII, ALK-7, Ret, BMPR-IA/ALK-3, TGF-beta RI/ALK-5, BMPR-IB/ALK-6, TGF-beta RII, BMPR-II, TGF-beta RIIb, endothelial factor/CD 105, and TGF-beta RIII.

Other embodiments of the method comprise a Wnt-related molecule selected from the group consisting of: frizzled-1, Frizzled-8, Frizzled-2, Frizzled-9, Frizzled-3, sFRP-1, Frizzled-4, sFRP-2, Frizzled-5, sFRP-3, Frizzled-6, sFRP-4, Frizzled-7, MFRP, LRP5, LRP6, Wnt-1, Wnt-8a, Wnt-3a, Wnt-10b, Wnt-4, Wnt-11, Wnt-5a, Wnt-9a, and Wnt-7 a.

In other embodiments of the method, the receptor ligand is selected from the group consisting of: 4-1BB ligand/TNFSF 9, lymphotoxin, APRIL/TNFSF13, lymphotoxin beta/TNFSF 3, BAFF/TNFSF13C, OX40 ligand/TNFSF 4, CD27 ligand/TNFSF 27, TL1 27/TNFSF 27, CD27 ligand/TNFSF 27, TNF-alpha/TNFSF 1 27, CD27 ligand/TNFSF 27, TNF-beta/TNFSF 1 27, EDA-A27, TRAIL/TNFSF 27, Fas ligand/TNFSF 27, TRANCE/TNFSF 27, GITR ligand/TNFSF 27, TWEAK/TNFSF 27, LIGHT/TNFSF 27, Amphiregulin (Amperegulin), NRG 27 isoform GGF 27, beta cell growth factor (Betalucilin), NRG 27 isoform SMG 27, NRG-EGF-beta-regulin, Epidegin-alpha-EGF-alpha/TNFargulin (HRegulin) 27, Epidegin-alpha-EGF-alpha-beta-alpha-beta-regulatory protein (HRegulin), Epidegin-beta-alpha-beta-EGF-alpha-beta-alpha-beta-regulatory (HRegulin-alpha-beta-, TMEF 2, IGF-I, IGF-II, insulin, activin A, activin B, activin AB, activin C, BMP-2, BMP-7, BMP-3, BMP-8, BMP-3B/GDF-10, BMP-9, BMP-4, BMP-15, BMP-5, Dpp protein (Decapentaplegic), BMP-6, GDF-1, GDF-8, GDF-3, GDF-9, GDF-5, GDF-11, GDF-6, GDF-15, GDF-7, Atmin (Artemin), Neurturin (Neurturin), GDNF, praseffin (Persephin), TGF-beta 2, TGF-beta 1, TGF-beta 3, LAP (TGF-beta 1), TGF-beta 5, latent TGF-beta 1, latent TGF-beta 1, TGF-beta 1.2, Lefty, Nodal, MIS/AMH, acidic FGF, FGF-12, basic FGF, FGF-13, FGF-3, FGF-16, FGF-4, FGF-17, FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF-21, FGF-9, FGF-23, FGF-10, KGF/FGF-7, FGF-11, Neuropilin-1 (Neuropilin-1), P1GF, Neuropilin-2, P1GF-2, PDGF-A, VEGF, PDGF-B, VEGF-B, PDGF-C, VEGF-C, PDGF-D, VEGF-D, and PDGF-AB.

In other embodiments, the T-cell target is selected from the group consisting of: 2B/SLAMF, IL-2 Ra, 4-1BB/TNFRSF, IL-2 Rbeta, ALCAM, B-1/CD, IL-4-H, BLAME/SLAMF, BTLA, IL-6, IL-7 Ra, CCR, CXCR/IL-8 RA, CCR, IL-10 Ra, CCR, IL-10 Rbeta, CCR, IL-12 Rbeta 1, CCR, IL-12 Rbeta 2, CD, IL-13 Ralpha 1, IL-13, CD, ILT/CD 85, integrin alpha 4/CD49, CD, integrin alpha E/CD103, CD, integrin alpha M/CD11, CD, integrin alpha X/CD11, integrin beta 2/CD, KIR/CD158, CD/RSF, KIR2DL, CD, KIR2DL3, CD30/TNFRSF8, KIR2DL4/CD158d, CD31/PECAM-1, KIR2DS4, CD40 ligand/TNFSF 5, LAG-3, CD43, LAIR1, CD45, LAIR2, CD83, leukotriene B4R 1, CD84/SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229/SLAMF3, NKG2 3, CD2 3-10/SLAMF 3, NT-4, CD3, B-A/SLAMF 3, common gamma chain/IL-2R gamma, Osteopontin (Osteononen), FastCC/SLAMF 3, PD-1, TAM, CTLA-1, CTLA-GL-1, CTLA-1-RSF-72, SIRAM-1, CTLA-11-CXCR-11, TCAM-3, TCHRSA-11, CRACM-11, TCAM-3, CRACR-11, Fas ligand/TNFSF 6, TIM-4, Fc γ RIII/CD16, TIM-6, GITR/TNFRSF18, TNF RI/TNFRSF1A, Granulysin (Granulysin), TNF RII/TNFRSF1B, HVEM/TNFRSF14, TRAILR1/TNFRSF10A, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM-2/CD102, TRAIL R3/TNFRSF10C, IFN- γ R1, TRAILR4/TNFRSF10D, IFN- γ R2, TSLP, IL-1RI, and TSLP R.

In other embodiments, the NK cell receptor is selected from the group consisting of: 2B4/SLAMF4, KIR2DS4, CD155/PVR, KIR3DL1, CD94, LMIR1/CD300A, CD69, LMIR 69/CD 300 69, CRACC/SLAMF 69, LMIR 69/CD 300 69, DNAM-1, LMIR 69/CD 300 69, Fc epsilon RII, LMIR 69/CD 300 69, Fc gamma RI/CD 69, MICA, Fc gamma RIIB/CD32 69, MICB, Fc gamma RIIC/CD 3632, Fc gamma RILT-1, Fc gamma RIIA/CD32 69, Handulin-2/CD 112, Fc gamma RIII/CD 69, NKG2 69, FcRH 69/IRTA 69, NKG2 69, NKH 69/IRTA 69, RaIRTA 69, RaaN 69/69, IRH 69/69, FcRITA 69, NKP-69, CRACR-69, NKP-69, IRC 69, IRH 69, IRCP 69, IRC 69, IRCP 69, IRC-69, IRCP 69, IRC-69, ILT4/CD85d, TREM-2, ILT5/CD85a, TREM-3, KIR/CD158, TREML1/TLT-1, KIR2DL1, ULBP-1, KIR2DL3, ULBP-2, KIR2DL4/CD158d and ULBP-3.

In other embodiments, the monocyte/macrophage target is selected from the group consisting of: B7-1/CD80, ILT4/CD85d, B7-H1, ILT5/CD85a, common beta chain, integrin alpha 4/CD49d, BLAME/SLAMF8, integrin alpha X/CD11C, CCL 6/C6, integrin beta 2/CD 6, CD155/PVR, integrin beta 3/CD6, CD 6/PECAM-1, laticifin (Latexin), CD 6/SR-B6, leukotriene B4R 6, CD 6/TNFRSF 6, LIMPII/SR-B6, CD6, LMIR6/CD 300/CD 6, CD 6/LMIR 6/CD 6, EMICC CD 6/CD 6, EMAMF 72/CD 6, EMiMCF 6/CD 6, EMICC 6/CD 6, EMiMCF 6, CD 6/CD 6, EMICC/CD 6, EMiMCF 6, EMF/CD 6, EMICC 6, MCF/MCF 6, EMF/MCF 6, EMC 6, EMiMCF/MCF 6, EMC 6, EMiMC, Osteopontin, Fc γ RIIB/CD32, PD-L, Fc γ RIIC/CD32, Siglec-3/CD, Fc γ RIIA/CD32, SIGNR/CD 209, Fc γ RIII/CD, SLAM, GM-CSF Ra, TCCR/WSX-1, ICAM-2/CD102, TLR, IFN- γ R, TLR- γ R, TREM-1, IL-1RII, TREM-2, ILT/CD 85, TREM-3, ILT/CD 85, TREML/TLT-1, 2B/SLAMF, IL-10 Ra, ALCAM, IL-10 Rbeta, aminopeptidase N/ANPEP, ILT/CD 85, common beta chain, ILT/CD 85, C1R/CD, ILT/CD 85, CCR integrin alpha 4/CD49, CCR alpha-11, alpha-11/CD 11, CCR 11 alpha-X11, CCR 11, CD155/PVR, integrin beta 2/CD18, CD14, integrin beta 3/CD61, CD36/SR-B3, LAIR1, CD43, LAIR2, CD45, leukotriene B4R1, CD68, LIMPII/SR-B2, CD84/SLAMF5, LMIR1/CD300A, CD97, LMIR2/CD300c, CD163, LMIR3/CD300LF, blood coagulation factor III/tissue factor, LMIR5/CD300LB, CX3CR1, CX3CL1, LMIR6/CD300 CSF 300LE, CXCR4, LRP-1, CXCR6, M-CSF R, DEP-1/CD148, DNAM-1, MPMM-2, EMIN/CD 147, EMIN/CD 105, MMAM/CSF 105, MMAM-72, HVRIR 36RIR, CD 36RIR, PSRIR 36RIR, CD 36RIR-72, CD 36RIR-36RIR, CD 36RIR, PSRIR-36RIR, CD 36RIR-72, CD 36RIR-19, CD 36RIR-5, CD 36RIR, CD36, ICAM-1/CD54, TCCR/WSX-1, ICAM-2/CD102, TREM-1, IL-6R, TREM-2, CXCR1/IL-8RA, TREM-3 and TREML 1/TLT-1.

In other embodiments of the method, the dendritic cell target is selected from the group consisting of: CD36/SR-B3, LOX-1/SR-E1, CD68, MARCO, CD163, SR-AI/MSR, CD5L, SREC-I, CL-P1/COLEC12, SREC-II, LIMPII/SR-B2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-1BB ligand/TNFSF 9, IL-12/IL-23P40, 4-amino-1, 8-naphthalimide, ILT 40/CD 85 40, CCL 40/6 Ckine, ILT 40/CD 85 40, 8-oxy-dG, ILT 40/CD 85 40, 8D6 40, ILT 40/CD 3685, A2B 40, integrin alpha 4/CD 40, AAIR 40, LACIR-B40/LAMIR 40, LAMIR-IRL 40, LAMIR/CD 40, LAMIR 40, C1qR1/CD93, LMIR3/CD300LF, CCR LF, LMIR LF/CD 300LF, CD LF/TNFRSF LF, MAG/Siglec-4a, CD LF, MCAM, CD LF, MD-1, CD LF, MD-2, CD LF, MDL-1/CLEC5 LF, CD LF/SLAMF LF, MMR, CD LF, NCAM-L LF, CD2 LF-10/SLAMF LF, osteocalcin/GPNMB, Chem LF, PD-L LF, CLEC-1, RP105, CLEC-2, Siglec-2/CD LF, CLEC/SLAMF LF, SigSig-3/CD LF, DC-SIG-SIGC-5, SIGC-CD 299, CLEC-6/CLEC-3/CD LF, CRA-SIGC-3/CD LF, SIGC-5, SIGC-DC-299, CLEC-C-6/CLEC-3, CRA-DCDEC-LF, CRA-7/CLEC-3, CRA-3/SIGC-3, CD LF, CLEC-SIGC-3, CD-3, CRA-3, CD LF, CD-SIGC-3/, SIGNR4, DLEC, SLAM, EMMPRIN/CD147, TCCR/WSX-1, Fc γ RI/CD64, TLR3, Fc γ RIIB/CD32b, TREM-1, Fc γ RIIC/CD32c, TREM-2, Fc γ RIIA/CD32a, TREM-3, Fc γ RIII/CD16, TREML1/TLT-1, ICAM-2/CD102, and capsaicin R1(Vanilloid R1).

In other embodiments of the method, the angiogenesis target is selected from the group consisting of: angiopoietin (Angiopoietin) -1, Angiopoietin-2, Angiopoietin-3, Angiopoietin-7/CDT 6, Angiopoietin-4, Tie-1, Angiopoietin-1, Tie-2, Angiogenin (Angiogenin), iNOS, coagulation factor III/tissue factor, nNOS, CThAA/CCN 2, NOV/CCN3, DANCE, OSM, EDG-1, Plfr, EG-VEGF/PK1, Proliferin (Proliferin), Endostatin (Endostatin), ROBO4, Erythropoietin (Erythropoiin), thrombospondin-1, Kininostatin (Kininostatin), thrombospondin-2, MFG-E8, thrombospondin-4, Nitric Oxide (Nitric Oxide), VG5, VG 383, EpOS 3884, EphB 9638, EphAB 369636, EphAb-9, EphA-7, EphhhA-7, EphhhhhA-7, EphA-2, EphA-7, EphhhhA-7, EphA, EphB2, EphB6, EphB3, Ephrin-A1, Ephrin-A4, Ephrin-A2, Ephrin-A5, Ephrin-A3, Ephrin-B1, Ephrin-B3, Ephrin-B2, acidic FGF, FGF-12, basic FGF, FGF-13, FGF-3, FGF-16, FGF-4, FGF-17, FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF-21, FGF-9, FGF-23, FGF-10, KGF/FGF-7, FGF-11, FGF R1, FGF R4, FGF R2, FGF R5, FGF R3, neurotrophin-1, neurotrophin-2, brachial placidin 3A, brachial placidin 6B, brachial placidin 3C 6, brachial placidin D, brachial placidin 6, brachial placidin D, brachial placoid 6, brachial plate protein, brachial plate D, brachial plate 3, and their derivatives, Bradrin 7A, MMP-11, MMP-1, MMP-12, MMP-2, MMP-13, MMP-3, MMP-14, MMP-7, MMP-15, MMP-8, MMP-16/MT3-MMP, MMP-9, MMP-24/MT5-MMP, MMP-10, MMP-25/MT6-MMP, TIMP-1, TIMP-3, TIMP-2, TIMP-4, ACE, IL-13 Ra 1, IL-13, C1q R1/CD93, integrin alpha 4/CD49d, VE-cadherin, integrin beta 2/CD18, CD31/PECAM-1, KLF4, CD36/SR-B3, LYVE-1, CD151, MCAM, CL-P1/COLEC12, fibronectin-2/CD 112, blood coagulation factor III/tissue factor III, E-selectin, D6, P-selectin, DC-SIGNR/CD299, SLAM, EMMPRIN/CD147, Tie-2, endoglin/CD 105, TNF RI/TNFRSF1A, EPCR, TNF RII/TNFRSF1B, erythropoietin R, TRAIL R1/TNFRSF10A, ESAM, TRAIL R2/TNFRSF10B, FABP5, VCAM-1, ICAM-1/CD54, VEGF R2/Flk-1, ICAM-2/CD102, VEGF R3/Flt-4, IL-1RI, and VG 5Q.

Other embodiments of the method provide multivalent binding proteins, wherein at least one of binding domain 1 and binding domain 2 specifically binds a target selected from the group consisting of: prostate specific cell membrane antigen (folate 1), Epidermal Growth Factor Receptor (EGFR), receptors for advanced glycosylation end products (RAGE; also known as advanced glycosylation end product receptor or AGER), IL-17A, IL-17F, P19(IL23A and IL12B), Dickkopf-1(Dkk1), NOTCH1, NG2 (chondroitin sulfate proteoglycan 4 or CSPG4), IgE (IgHE or IgH2), IL-22R (IL22RA1), IL-21, amyloid beta oligomer (Ab oligomer), amyloid beta preproprotein (APP), NOGO receptor (RTN4R), low density lipoprotein receptor-related protein 5(LRP5), IL-4, myostatin (GDF8), very advanced antigen 4, alpha 4, beta 1 integrin (VLA4 or ITGA4), alpha 4, beta 7 integrin present on leukocytes, and IGF-1R. For example, the VLA4 target may be recognized by a multivalent binding protein in which at least one of binding domain 1 and binding domain 2 is a binding domain derived from Natalizumab (Antegren).

In certain embodiments, the cancer cell is a transformed hematopoietic cell or a cancerous hematopoietic cell. In certain such embodiments, at least one of the first binding domain and the second binding domain recognizes a target selected from the group consisting of: b-cell targets, monocyte/macrophage targets, dendritic cell targets, NK-cell targets, and T-cell targets (each as defined herein). Furthermore, at least one of the first and second binding domains may recognize a bone marrow target, including but not limited to: CD5, CD10, CD11B, CD11c, CD13, CD14, CD15, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD100, CD103, CD111, CD112, CD114, CD115, CD116, CD117, CD118, CD119, CD120 25, CDw123, CDw131, CD141, CD162, CD163, CD177, CD312, IRTA 25, irb 25, and irb 25-B-25.

Other embodiments of the invention are directed to multivalent binding proteins as described herein, comprising a sequence selected from the group consisting of: SEQ ID NO: 2. 4, 6, 103, 105, 107, 109, 332, 333, 334, and 345. Other embodiments are directed to multivalent binding proteins comprising a sequence selected from the group consisting of: SEQ ID NO: 355. 356, 357, 358, 359, 360, 361, 362, 363, 364, and 365.

In other embodiments, multivalent and multispecific binding proteins having effector functions have a first binding domain and a second binding domain that recognize a target pair selected from the group consisting of EPHB4-KDR and TIE-TEK. In such embodiments, the protein has a first binding domain that recognizes EPHB4 and a second binding domain that recognizes KDR, or a first binding domain that recognizes KDR and a second binding domain that recognizes EPHB 4. Similarly, the protein may have a first binding domain that recognizes TIE and a second binding domain that recognizes TEK, or a first binding domain that recognizes TEK and a second binding domain that recognizes TIE.

In a related aspect, the invention provides multivalent binding proteins with effector function, wherein the constant sub-region recognizes effector cell F _CReceptors (e.g. F)_CγRI、F_CγRII、F_CγRIII、F_CAlpha R and F_Cε RI). In particular embodiments, the constant sub-region recognizes an effector cell surface protein selected from the group consisting of: CD2, CD3, CD16, CD28, CD32, CD40, CD56, CD64, CD89, F_CεRI, KIR, thrombospondin R, NKG2D, 2B4/NAIL and 41 BB. The constant sub-regions may comprise C's derived from the same or different immunoglobulins, antibody isotypes or allelic variants_H2Domains and C_H3A domain. In certain embodiments, C_H3The domain is a truncated domain and comprises a C-terminal sequence selected from the group consisting of: SEQ ID NO: 366. 367, 368, 369, 370 and 371. When the linker is a hinge-like peptide derived from an immunoglobulin, C_H2The domains and scorpion linkers are preferably immunoglobulins derived from the same class or from the same subclass.

The invention also encompasses certain proteins further comprising a scorpion linker having at least about 5 amino acids linked to the constant sub-region and to the second binding domain, such that the scorpion linker is positioned between the constant sub-region and the second binding domain. Typically, scorpion linker peptides are between 5 and 45 amino acids in length. Scorpion linkers include hinge-like peptides derived from immunoglobulin hinge regions, such as IgG1, IgG2, IgG3, IgG4, IgA, and IgE hinge regions. Preferably, the hinge-like scorpion linker will retain at least one cysteine that is capable of forming interchain disulfide bonds under physiological conditions. The scorpion linker derived from IgG1 may have 1 cysteine or 2 cysteines, and preferably will retain the cysteine corresponding to the N-terminal hinge cysteine of IgG 1. In certain embodiments, the scorpion linker is extended relative to a homologous immunoglobulin hinge region, and in exemplary embodiments, comprises a sequence selected from the group consisting of seq id NOs: 351. 352, 353, and 354. Non-hinge-like peptides are also contemplated as scorpion linkers, provided that the peptides provide sufficient space and flexibility to result in a single chain protein capable of forming two binding domains, which is located towards each protein end (N and C) relative to a more centrally located constant sub-region domain. Exemplary non-hinge-like scorpion linkers include peptides derived from the stem regions of type II C-lectins (such as the stem regions of CD69, CD72, CD94, NKG2A, and NKG 2D). In certain embodiments, the scorpion linker comprises a sequence selected from the group consisting of SEQ ID NOs: 373. 374, 375, 376 and 377.

The protein may also comprise a linker of at least about 5 amino acids linked to the constant sub-region and to the first binding domain, such that the linker is positioned between the constant sub-region and the first binding domain. In certain embodiments, a linker is present between the constant sub-region and one of the two binding domains, and the linkers may have the same or different sequences and may have the same or different lengths.

The constant sub-region of the protein of the invention provides at least one effector function. Any effector function known in the art associated with immunoglobulins (e.g., antibodies) is contemplated, such as an effector function selected from the group consisting of: antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), relatively prolonged half-life in vivo (relative to the same molecule lacking the constant sub-region), FcR binding, protein a binding, and similar effector functions thereof. In certain embodiments, the extended half-life of a protein of the invention in a human is at least 28 hours. Of course, a protein to be administered to a non-human subject will exhibit a relatively extended half-life in such a non-human subject, which is not necessarily the case in humans.

In general, proteins (including polypeptides and peptides) of the invention are directed to a first binding domain and a second binding domainAt least one is less than 10^-9M or at least 10^-6Binding affinity of M.

Another aspect of the invention pertains to a pharmaceutical composition comprising a protein as described herein and a pharmaceutically acceptable adjuvant, carrier or excipient. Any adjuvant, carrier or excipient known in the art is suitable for use in the pharmaceutical compositions of the present invention.

In a further aspect the present invention provides a method of preparing a protein as described above, the method comprising: the protein is expressed by introducing a nucleic acid encoding the protein into a host cell and incubating the host cell, preferably in an amount of at least 1 mg/l, under conditions suitable for expression of the protein. In certain embodiments, the method further comprises isolating the protein as follows: separating it from at least one protein to which it binds after its expression in the cell. Host cells suitable for expressing a nucleic acid to produce a protein of the invention include, but are not limited to, host cells selected from the group consisting of: VERO cells, Hela cells (HeLa cells), CHO cells, COS cells, W138 cells, BHK cells, HepG2 cells, 3T3 cells, RIN cells, MDCK cells, A549 cells, PC12 cells, K562 cells, HEK293 cells, N cells, Spodoptera frugiperda (Spodoptera frugiperda) cells, Saccharomyces cerevisiae (Saccharomyces cerevisiae) cells, Pichia pastoris (Pichia pastoris) cells, any of a variety of fungal cells, and any of a variety of bacterial cells including, but not limited to, Escherichia coli (Escherichia li), Bacillus subtilis, Salmonella typhimurium (Salmonella typhimurium), and Streptomyces (Streptomyces)).

The present invention also provides a method of preparing a nucleic acid encoding the protein as described above, the method comprising: covalently linking the 3 ' end of a polynucleotide encoding a first binding domain derived from an immunoglobulin variable region to the 5 ' end of a polynucleotide encoding a constant sub-region, covalently linking the 5 ' end of a polynucleotide encoding a scorpion linker to the 3 ' end of a polynucleotide encoding a constant sub-region, and covalently linking a second binding domain derived from an immunoglobulin variable region to the 3 ' end of a polynucleotide encoding a constant sub-regionThe 5 'end of the polynucleotide of the syntdomain is covalently linked to the 3' end of the polynucleotide encoding the scorpion linker, thereby forming a nucleic acid encoding a multivalent binding protein with effector function. Each of the coding regions may be separated by a linker or hinge-like peptide coding region as part of a single-chain structure of the invention. In certain embodiments, the method produces a polynucleotide encoding a single-stranded form of a first binding domain comprising a sequence selected from the group consisting of seq id no: SEQ ID NO: 2 (anti-CD 20 variable region, V)_L-V_HOrientation), SEQ ID NO: 4 (anti-CD 28 variable region, V)_L-V_HOrientation) and SEQ ID NO: 6 (anti-CD 28 variable region, V)_H-V_LOrientation), however, requires that the heteropolymeric protein (including the natural antibody) must be assembled from the separately encoded polypeptides. An exemplary polynucleotide sequence encoding the first binding domain is a polynucleotide comprising SEQ ID NO: 1. 3 or 5.

This aspect of the invention also provides a method of making an encoding nucleic acid further comprising a linker polynucleotide inserted between a polynucleotide encoding a first binding domain and a polynucleotide encoding a constant sub-region, the linker polynucleotide encoding a peptide linker having at least 5 amino acids. In addition, the method produces a nucleic acid further comprising a linker polynucleotide inserted between the polynucleotide encoding the constant sub-region and the polynucleotide encoding the second binding domain, the linker polynucleotide encoding a peptide linker having at least 5 amino acids. Preferably, the encoded peptide linker has between 5 and 45 amino acids.

The identity of the linker regions present between BD1 and EFD or between EFD and BD2 arises from other sequences recognized by cognate-Ig superfamily members. In developing novel linkers derived from existing sequences present in members of the homo-Ig superfamily, it is preferred to avoid the occurrence of stretches similar to those located between the end of the C-like domain and the end of the transmembrane domain, since such sequences are typically substrates for proteolytic cleavage of cell surface receptors to form soluble forms. Sequence alignments between different members of the-Ig superfamily and subfamily can compare similarities between molecules in terms of linker sequences linking multiple V-like domains or linking V-like domains to C-like domains. From this analysis, conserved naturally occurring sequence patterns can occur; the sequences should be more protease resistant when used as linkers between subdomains of multivalent fusion proteins, may contribute to proper folding between Ig loop regions, and are not immunogenic because they occur in the extracellular domain of endogenous cell surface molecules.

The nucleic acids themselves constitute a further aspect of the invention. Nucleic acids encoding any of the proteins of the invention described herein are contemplated. Thus, the nucleic acid of the invention comprises, in order from 5 'to 3', a coding region for the first binding domain, a constant sub-region sequence and a coding region for the second binding domain. Also encompassed are nucleic acids encoding protein variants wherein the two binding domain and constant sub-region sequences have at least 80% and preferably at least 85%, 90%, 95% or 99% identity in amino acid sequence to the combined sequence of known immunoglobulin variable region sequences and known constant sub-region sequences. Alternatively, a protein variant of the invention is encoded by a nucleic acid that hybridizes to a nucleic acid encoding a non-variant protein of the invention under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at 65-68 ℃ or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at 42 ℃. Variant nucleic acids of the invention show hybridization capability under the conditions defined immediately above, or show 90%, 95%, 99% or 99.9% sequence identity with nucleic acids encoding non-variant proteins of the invention.

In related aspects, the invention provides vectors comprising a nucleic acid as described above and host cells comprising a vector or nucleic acid as described herein. Any vector known in the art (e.g., plasmids, phagemids (phagemid), phagemids (phasmid), cosmids, viruses, artificial chromosomes, shuttle vectors, and the like) can be used, and one of skill in the art will know which vector is particularly suited for a given purpose. For example, in a method of producing a protein of the invention, an expression vector is selected that can be manipulated in the host cell of choice. Likewise, any host cell capable of being genetically transformed with a nucleic acid or vector of the invention is contemplated. Preferred host cells are higher eukaryotic host cells, but lower eukaryotic (e.g., yeast) and prokaryotic (bacterial) host cells are also contemplated.

Another aspect of the invention relates to a method of inducing damage to a target cell, the method comprising contacting the target cell with a therapeutically effective amount of a protein as described herein. In certain embodiments, the target cell is contacted in vivo by administering the protein or encoding nucleic acid to an organism in need thereof. Encompassed within this aspect of the invention are methods wherein the multivalent single chain binding protein can induce an additive amount of damage to the target cells, this amount being the amount of damage predicted from the total amount of damage due to the isolated antibody comprising one or the other of the binding domains. Also encompassed are methods wherein the multivalent single chain binding protein can induce a synergistic amount of damage to the target cells compared to the total amount of damage induced by a first antibody comprising the first binding domain but not the second binding domain and a second antibody comprising the second binding domain but not the first binding domain. In certain embodiments, the multivalent single chain binding protein is multispecific and comprises a binding domain pair that specifically recognizes an antigen pair selected from the group consisting of: CD19/CD20, CD20/CD21, CD20/CD22, CD20/CD40, CD20/CD79a, CD20/CD79b, CD20/CD81, CD21/CD79b, CD37/CD79b, CD79b/CD81, CD19/CL II (i.e. class II MHC), CD20/CL II, CD30/CL II, CD37/CL II, CD72/CL II and CD79b/CL II.

This aspect of the invention also includes methods wherein the multispecific multivalent single-chain binding protein can induce an inhibitory amount of damage to a target cell (as compared to the total amount of damage induced by a first antibody comprising the first binding domain but not the second binding domain and a second antibody comprising the second binding domain but not the first binding domain). Exemplary embodiments include methods wherein the multispecific multivalent single-chain binding protein comprises a binding domain pair that specifically recognizes an antigen pair selected from the group consisting of: CD20/CL II, CD21/CD79 21, CD79 21/CD 21, CD21/CL II, CD21/CD 21, CD21/CD 3679 a, CD21/CD 21, CD21/CD 3679 b, CD21/CD 21 and CD21/CD 21.

In a related aspect, the invention provides a method of treating a cell proliferative disorder (e.g., cancer), the method comprising administering to an organism in need thereof a therapeutically effective amount of a protein (as described herein) or an encoding nucleic acid. The identification of organisms in need of treatment is well known to those skilled in the art, including medical and veterinary professionals. Conditions susceptible to treatment encompassed by the present invention include conditions selected from the group consisting of cancer, autoimmune conditions, Rous (Rous) sarcoma virus infection, and inflammation. In certain embodiments, the protein is administered by expressing in vivo a nucleic acid encoding the protein as described herein. The invention also includes administering the protein by a route selected from the group consisting of: intravenous injection, intra-arterial injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, and direct tissue injection.

Another aspect of the invention relates to a method of ameliorating a symptom associated with a cell proliferative disorder, the method comprising administering to an organism in need thereof a therapeutically effective amount of a protein as described herein. The identification of those conditions or diseases or conditions that exhibit symptoms susceptible to amelioration is also well known to those skilled in the art. In certain embodiments, the symptom is selected from the group consisting of pain, heat, swelling, and joint stiffness.

Yet another aspect of the invention relates to a method of treating an infection associated with an infectious agent, the method comprising administering to a patient in need thereof a therapeutically effective amount of a protein of the invention, wherein the protein comprises a binding domain that specifically binds to a target molecule of the infectious agent. According to this aspect of the invention, infectious agents susceptible to treatment include prokaryotic and eukaryotic cells, viruses (including bacteriophages), foreign objects, and infectious organisms such as parasites (e.g., mammalian parasites).

A related aspect of the invention relates to a method of ameliorating the symptoms of an infection associated with an infectious agent, the method comprising administering to a patient in need thereof an effective amount of a protein of the invention, wherein the protein comprises a binding domain that specifically binds to a target molecule of the infectious agent. Skilled medical and veterinary practitioners will be able to determine the effective amount of protein on an individual basis using routine experimentation.

A further related aspect of the invention is a method of reducing the risk of infection by an infectious agent, the method comprising administering to a patient at risk of developing the infection a prophylactically effective amount of a protein of the invention, wherein the protein comprises a binding domain that specifically binds to a target molecule of the infectious agent. One skilled in the relevant art will be able to determine a prophylactically effective amount of a protein on an individual basis using routine experimentation.

Another aspect of the invention pertains to the above multivalent single chain binding protein, wherein at least one of the first binding domain and the second binding domain specifically binds to an antigen selected from the group consisting of: CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and major histocompatibility complex class II peptides.

In certain embodiments, one of the first and second binding domains specifically binds CD20, while in certain such embodiments the other binding domain specifically binds an antigen selected from the group consisting of: CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and major histocompatibility complex class II peptides. For example, in one embodiment, the first binding domain is capable of specifically binding to CD20, while the second binding domain is capable of specifically binding to, for example, CD 19. In another embodiment, the first binding domain binds CD19 and the second binding domain binds CD 20. Embodiments are also contemplated in which both binding domains bind CD 20.

In certain other embodiments according to this aspect of the invention, one of the first and second binding domains specifically binds CD79b, while in certain of the described embodiments the other binding domain specifically binds an antigen selected from the group consisting of: CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and major histocompatibility complex class II peptides. Exemplary embodiments include different multispecific multivalent single-chain binding proteins, wherein the first binding domain: the second binding domain specifically binds to CD79b: CD19 or CD19: CD79 b. Also included are multivalent binding proteins having first and second binding domains that recognize CD79 b.

In certain other embodiments, one of the first and second binding domains specifically binds to a major histocompatibility complex class II peptide, while in certain such embodiments the other binding domain specifically binds to an antigen selected from the group consisting of: CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and major histocompatibility complex class II peptides. For example, in one embodiment, the first binding domain is capable of specifically binding to a major histocompatibility complex class II peptide, and the second binding domain is capable of specifically binding to, for example, CD 19. In another embodiment, the first binding domain binds CD19 and the second binding domain binds a major histocompatibility complex class II peptide. Also encompassed are embodiments wherein both binding domains bind to major histocompatibility complex class II peptides.

In other embodiments according to this aspect of the invention, one of the first and second binding domains specifically binds CD22, while in certain of the described embodiments the other binding domain specifically binds an antigen selected from the group consisting of: CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and major histocompatibility complex class II peptides. Exemplary embodiments include different multispecific multivalent single-chain binding proteins, wherein the first binding domain: the second binding domain specifically binds to CD 22: CD19 or CD 19: CD 22. Also included are multivalent binding proteins having first and second binding domains that recognize CD 22.

A related aspect of the invention pertains to the above multivalent single chain binding protein, wherein the protein has a synergistic effect on target cell behavior (relative to the sum of the effects of the binding domains). In certain embodiments, the protein comprises a binding domain pair that specifically recognizes an antigen pair selected from the group consisting of: CD20-CD19, CD20-CD21, CD20-CD22, CD20-CD40, CD20-CD79a, CD20-CD79b and CD20-CD 81.

The invention further includes a multivalent single chain binding protein as described above, wherein the protein has an additive effect on target cell behavior (relative to the sum of the effects of the binding domains). Embodiments according to this aspect of the invention include multispecific proteins comprising a binding domain pair that specifically recognizes an antigen pair selected from the group consisting of: CD20-CD23, CD20-CD30, CD20-CD37, CD20-CD70, CD20-CD80, CD20-CD86, CD79b-CD37, CD79b-CD81, major histocompatibility complex class II peptide-CD 30 and major histocompatibility complex class II peptide-CD 72.

A further related aspect of the invention is a multivalent single chain binding protein as described above, wherein the protein has an inhibitory effect on target cell behavior (relative to the sum of the effects of the binding domains). In certain embodiments, the protein is multispecific and comprises a binding domain that specifically recognizes an antigen pair selected from the group consisting of: CD 20-major histocompatibility complex class II peptide, CD79b-CD19, CD79b-CD20, CD79b-CD21, CD79b-CD22, CD79b-CD23, CD79b-CD30, CD79b-CD40, CD79b-CD70, CD79b-CD72, CD79b-CD79a, CD79b-CD80, CD79b-CD86, CD79 b-major histocompatibility complex class II peptide, major histocompatibility complex class II peptide-CD 19, major histocompatibility complex class II peptide-CD 20, major histocompatibility complex class II peptide-CD 21, major histocompatibility complex class II peptide-CD 22, major histocompatibility complex II peptide-CD 22, major histocompatibility complex 3679-CD 22, major histocompatibility complex II peptide-CD 46, Major histocompatibility complex class II peptide-CD 79b, major histocompatibility complex class II peptide-CD 80, major histocompatibility complex class II peptide-CD 81, major histocompatibility complex class II peptide-CD 86, CD22-CD19, CD22-CD40, CD22-CD79b, CD22-CD86 and CD 22-major histocompatibility complex class II peptide.

Another aspect of the invention is a method of identifying at least one of the binding domains of a multivalent binding molecule (such as a multispecific binding molecule) described above, the method comprising: (a) contacting an anti-isotype antibody with an antibody that specifically recognizes a first antigen and an antibody that specifically recognizes a second antigen; (b) further contacting a target comprising at least one of the antigens with the composition of step (a); and (c) measuring the activity of the target, wherein the activity is used to identify at least one of the binding domains of the multivalent binding molecule. In certain embodiments, the target is a diseased cell, such as a cancer cell (e.g., a cancerous B-cell) or an autoantibody-producing B-cell.

In each of the above methods of the invention, it is contemplated that the method may further comprise a plurality of multivalent single chain binding proteins. In certain embodiments, the binding domain of the first multivalent single chain binding protein and the binding domain of the second multivalent single chain binding protein may induce a synergistic effect, an additive effect, or an inhibitory effect on the target cell, such as inducing a synergistic, additive, or inhibitory amount of damage to the target cell. The synergistic, additive or inhibitory effect of a plurality of multivalent single chain binding proteins is determined by comparing the effect of the plurality of proteins to the combined effect of an antibody comprising one of the binding domains and an antibody comprising the other binding domain.

A related aspect of the invention is a composition comprising a plurality of multivalent single chain binding proteins as described above. In certain embodiments, the composition comprises a plurality of multivalent single chain binding proteins, wherein the binding domain of a first multivalent single chain binding protein and the binding domain of a second multivalent single chain binding protein are capable of inducing a synergistic effect, an additive effect, or an inhibitory effect on the target cell, such as inducing a synergistic, additive, or inhibitory amount of damage to the target cell.

The invention further relates to a pharmaceutical composition comprising the composition and a pharmaceutically acceptable carrier, diluent or excipient. In addition, the invention includes a kit comprising a composition as described herein and a set of instructions for administering the composition to produce an effect on (such as damaging) a target cell.

Finally, the invention also includes a kit comprising a protein as described herein and a set of instructions for administering the protein to treat a cell proliferative disorder or ameliorate a symptom of a cell proliferative disorder.

Other features and advantages of the present invention will become more apparent by reference to the following detailed description, including examples.

Brief Description of Drawings

Figure 1 shows a schematic representation of a multivalent single chain molecule contemplated by the present invention. The individual subdomains of the fusion protein expression cassette are indicated by separate shapes/squares on the figure. BD1 refers to binding domain 1, linker 1 refers to any potential linker or hinge-like peptide between BD1 and the "effector function domain" (indicated by EFD). The subdomain is typically an engineered form of the Fc domain of human IgG1, but may include other subdomains having one or more effector functions as defined herein. Linker 2 refers to the linker sequence that exists (if present) between the carboxy terminus of the EFD and binding domain 2(BD 2).

FIG. 2 shows a Western blot of non-reduced protein expressed in COS cells. The protein was secreted into the culture medium and after 48-72 hours the culture medium supernatant was separated from the transiently transfected cells by centrifugation. Thirty microliters (30 μ l) of the crude supernatant was loaded into each gel well. Lane marking: 1-molecular weight marker, number indicates kilodaltons (kilodalton); 2-2H7-IgG-STD1-2E12 LH; 3-2H7-IgG-STD1-2E12HL, 4-2H7-IgG-STD2-2E12 LH; 5-2H7-IgG-STD2-2E12 HL; 6-2E12LH SMIP; 7-2E12HL SMIP; 8-2H7 SMIP. "2H 7" refers to a single chain construct in which BD1 encodes the VLVH oriented CD20 specific binding domain (2H 7); "2E 12" refers to a binding domain specific for CD 28; IgG-refers to a single-chain construct with a hinge encoding a sequence (sss) in which all C are mutated to S, and the CH2 domain and the CH3 domain of IgG1 contain mutations that exclude ADCC and CDC effector functions (P238S and P331S); "STD 1" refers to 2E12 (V) inserted adjacent to BD2 in VL-VH orientation _L-V_H) The 20 amino acid linker adjacent (identified as "STD 1 ═ 20 aa" in fig. 7). "STD 1-HL" refers to the following constructs similar to those just described but with the BD2V region in the VH-VL orientation: 2H7-sssIgG (P238/331S) -20 amino acid linker-2E 12 (V)_H-V_L). "STD 2-LH" refers to 2H7-sssIgG (P238/331S) -38 amino acid linker-2E 12 (V)_L-V_H) (ii) a "STD 2-LH" refers to 2H7-sssIgG (P238/331SS) -38 amino acid linker-2E 12 (V)_H-V_L) (ii) a "SMIP" refers to small modular immunopharmaceuticals; and "H" is typically V_HAnd "L" is usually V_L. Unless otherwise indicated, all protein orientations are N-terminal to C-terminal orientations.

FIG. 3 shows two histograms illustrating the binding properties of 2H7-sssIgG (P238S/P331S) -STD1-2e12 LH derivatives and 2H7-sssIgG (P238S/P331S) -STD1-2e12HL derivatives expressed by COS cells. The experiment was performed with crude culture supernatant, not purified protein. Serial dilutions of culture supernatants without 16-fold dilution were incubated with cells expressing CD20 (WIL-2S) or CD28 (CD28 CHO). The binding activity of the supernatant is compared to a control sample (such as TRU-015 or 2e12VLVH or 2e12VHVL SMIP) that is tested for binding of the relevant monospecific SMIP. Binding of each sample was detected at a dilution of 1:100 using a conjugate of Fluorescein Isothiocyanate (FITC) and goat anti-human IgG.

FIG. 4 is a graph showing the frequency distribution of the binding pattern of protein A purified forms of the proteins tested in FIG. 3 to WIL2-S cells. "TRU 015" is a SMIP specific for CD 20. Two multispecific binding proteins with effector functions were also analyzed: "2H 7-2E12 LH" has V_L-V_HOriented binding domain 2 specific for CD 28; "2H 7-2E12 HL" has V_H-V_LOriented binding domain 2 specific for CD 28. The proteins were each tested for binding at 5. mu.g/mL and binding was detected with FITC goat anti-human IgG at 1: 100. For a more complete description of the molecules tested, see the description of FIG. 2 above.

Fig. 5 shows two frequency profiles illustrating the binding of protein a purified multispecific binding protein with effector function to CHO cells expressing CD 28. "2H 7-2E12 LH" has V_L-V_HOriented binding domain 2 specific for CD 28; "2H 7-2E12 HL" has V_H-V_LOriented binding domain 2 specific for CD 28. The proteins were each tested for binding at 5. mu.g/mL and binding was detected with FITC goat anti-human IgG at 1: 100. For a more complete description of the molecules tested, see the description of FIG. 2.

Figure 6A) shows a table identifying linkers connecting the constant sub-region with binding domain 2. Linkers are identified by name, sequence identification number, sequence length, and sequence fused to binding domain 2. B) A table identifying a plurality of constructs identifying elements in an exemplary molecule of the invention is shown. In addition to identifying multivalent binding molecules by name, elements in those molecules are also disclosed in terms of binding domain 1(BD1), constant subregions (hinge and effector domain or EFD), linkers (see fig. 6A for additional information about linkers), and binding domain 2(BD 2). Sequences of various exemplary multivalent binding proteins are provided and identified in the figures by sequence identification numbers. Other multivalent binding proteins have altered elements or sequences of elements, and sequence alterations can be predicted from the disclosed sequences.

Figure 7 shows a combinatorial histogram illustrating that purified protein binds to CD 20-expressing WIL-2S cells and CD 28-expressing CHO cells at a single binding concentration. "H1-H6" means having a H1-H6 linker and V_H-V_L2H7-sss-hIgG-Hx-2e12 molecules of the oriented 2e12V region. "L1-L6" means having a L1-L6 linker and V_L-V_H2H7-sss-hIgG-Lx-2e12 molecules of oriented 2e12V region. All molecules were tested at a concentration of 0.72 μ g/mL and binding was detected at 1:100 using a combination of FITC and goat anti-human IgG. The mean fluorescence intensity for each sample was then plotted as a paired histogram of the two target cell types tested against each multivalent construct (L1-L6 or H1-H6) tested.

FIG. 8 shows photographs of non-reducing and reducing SDS-PAGE gels stained by Coomassie (Coomassie). The gel showed the effect of variant linker sequence/length on the 2H7-sss-hIgG-Hx-2e12HL protein on the amount of two major protein bands visualized on the gel.

FIG. 9 shows a Western blot of [2H7-sss-hIgG-H6-2e12] fusion proteins and related monospecific SMIPs probed with (a) CD28mIgG or (b) Fab specifically reactive with 2H 7. The results show that the presence of the H6 linker results in a cleaved form of the multivalent construct, which lacks CD28 binding specificity.

FIG. 10 shows the binding curves for different linker variants in the form of the [ TRU015-sss-IgG-Hx-2e12HL ] H1-H6 linker. The first panel shows the binding curve for binding to WIL-2S cells expressing CD 20. The second panel shows the binding curves for different forms of binding to CD28CHO cells. The binding curve was generated as follows: protein a purified fusion protein was serially diluted and binding was detected using FITC conjugated to goat anti-human IgG at 1: 100.

FIG. 11 shows a table summarizing the results of SEC separations for 2H7-sss-IgG-2e12HL multispecific fusion proteins with variant linkers H1-H7. The columns in the table list different linker variants of the [2H7-sss-IgG-Hx-2e12-HL ] fusion protein. The retention time of the peak of interest (POI) and the percentage of fusion protein present as POI and the percentage of protein present in other forms are also shown. Also recited is whether the molecule is cleaved, the degree of cleavage being indicated in a qualitative manner, with (yes), yes, or no being four possible choices.

FIG. 12 shows two binding profiles for [2H7-sss-hIgG-Hx-2e12] multispecific fusion proteins with variant linkers H3, H6, and H7 to cells expressing CD20 or CD 28. Protein A-purified fusion proteins were incubated from serial dilutions of 10. mu.g/ml to 0.005. mu.g/ml with either WIL-2S cells expressing CD20 or CD28CHO cells. Binding was detected using a conjugate of FITC and goat anti-human IgG at 1: 100. Panel A shows binding to WIL-2S cells and panel B shows binding to CD28CHO cells.

Figure 13 shows the results of an alternative binding assay produced by the molecule used in figure 12. In this case, the fusion protein was first bound to WIL-2S cells expressing CD20, and then binding was detected with CD28mIgG (5. mu.g/ml) and FITC anti-mouse reagent at 1: 100. The results indicate simultaneous binding to CD20 and CD28 in the same molecule.

FIG. 14 shows the results obtained using another variant of the multi-specific fusion construct. In this case, the specificity of BD2 can be altered so that the V region of the G28-1 antibody is used to form the CD 37-specific binding domain. The two panels shown illustrate the relative ability of the CD20 and/or CD37 antibodies to block the binding of [2H7-sss-IgG-Hx-G28-1] multispecific fusion protein to Ramos or BJAB cells expressing CD20 and CD37 targets. Prior to incubation with the multispecific fusion protein, each cell type was preincubated with either a CD 20-specific antibody (25 μ g/ml) or a CD 37-specific antibody (10 μ g/ml) or both reagents, which were mouse anti-human reagents. The binding of the multispecific fusion protein was then detected at 1:100 with FITC goat anti-human IgG reagent (pre-adsorbed to mice to exclude cross-reactivity).

FIG. 15 shows the results of ADCC assays performed with BJAB target cells, PBMC effector cells and CD20-hIgG-CD37 specific fusion protein as the test agent. For a complete description of this procedure, reference is made to the appropriate examples. This figure is a plot of fusion protein concentration versus the% specific kill rate tested at various doses for a single specific SMIP reagent and [2H7-sss-hIgG-STD1-G28-1] LH with the HL variant. The data series were plotted for the dose-response effect of one of the monospecific or multispecific single-chain fusion proteins.

FIG. 16 shows a table listing the results of co-culture experiments in which PBMCs were cultured in the presence of TRU 015, G28-1SMIP, two molecules together, or [2H7-sss-IgG-H7-G28-1HL ] variant. 20. mu.g/ml of the fusion protein was used and incubated for 24 hours or 72 hours. The samples were then stained with FITC-bound CD3 antibody and PE-bound CD19 or CD 40-specific antibody, followed by flow cytometry. The percentage of cells in each gate was then tabulated.

FIG. 17 shows two histograms of the effect on B cell line apoptosis after 24 hours incubation with [2H7-sss-hIgG-H7-G28-1HL ] molecule or control CD20 and/or CD37 specific SMIP alone or in combination. The percentage of annexin v (annexin v) -propidium iodide positive cells was plotted as a function of the type of test agent used in the co-incubation experiment. Panel a shows the results obtained using Ramos cells and panel B shows the results obtained using Daudi cells. SMIPs for each single CD20 or CD37 are shown at the specified concentrations; in addition, if a combination of two reagents is used, the relative amounts of the various reagents are shown in parentheses. The multispecific CD20-CD37 fusion protein was tested at concentrations of 5. mu.g/ml, 10. mu.g/ml and 20. mu.g/ml.

FIG. 18 shows two graphs of [2H7-hIgG-G19-4] molecular variants and their binding to CD 3-expressing cells (Jurkats) or CD 20-expressing cells (WIL-2S). The molecule comprises [2H7-sss-hIgG-STD1-G19-4HL ], LH and [2H7-csc-hIgG-STD1-G19-4HL ]. Protein A purified fusion protein was titrated to 0.05. mu.g/ml by 20. mu.g/ml and binding was detected at 1:100 using FITC goat anti-human IgG. MFI (mean fluorescence intensity) was plotted as a function of protein concentration.

FIG. 19 shows the results of ADCC assays performed with [2H7-hIgG-STD1-G19-4HL ] molecular variants with SSS or CSC hinges, BJAB target cells and either whole human PBMC as effector cells or NK cell depleted PBMC as effector cells. The kill rate was scored as a function of the concentration of the multi-specific fusion protein. The kill rate observed with the molecule was compared to that seen with G19-4, TRU015, or a combination of the two agents. Each data series was plotted against different test agents, with% specific kill rates plotted as a function of protein concentration.

Figure 20 shows the percentage of Ramos B-cells stained positively by annexin v (ann) and/or Propidium Iodide (PI) after overnight incubation with members of a matrix combination of B-cell antibodies (2 μ g/ml) in the presence or absence of anti-CD 20 antibody (present at 2 μ g/ml if added). The goat-anti-mouse secondary antibody is always present at a concentration ratio of two-fold relative to the other antibodies (matrix antibody alone, or matrix antibody to anti-CD 20 antibody). Vertical striped bars-matrix antibody (2. mu.g/ml) and goat anti-mouse antibody (4. mu.g/ml) indicated on the X-axis. Horizontal striped bars-matrix antibody (2. mu.g/ml), anti-CD 20 antibody (2. mu.g/ml) and goat anti-mouse antibody (4. mu.g/ml) indicated on the X-axis. The "step 2" conditions served as controls and included the addition of either goat anti-mouse antibody at 4. mu.g/ml (vertical striped bars) or 8. mu.g/ml (horizontal striped bars), without the addition of either matrix antibody or anti-CD 20 antibody. "CL II" (MHC class II) in the figures refers to monoclonal antibodies that are cross-reactive with HLA DR, DQ, and DP (i.e., with MHC class II antigens).

Figure 21 shows the percentage of Ramos B-cells stained positively by annexin v (ann) and/or Propidium Iodide (PI) after incubation overnight with each member of the matrix combination of B-cell antibodies (2 μ g/ml) in the presence or absence of anti-CD 79B antibody (present at 0.5 or 1.0 μ g/ml if added). For identification of the "CL II" and "step 2" samples, reference is made to the description of FIG. 20. Vertical striped column-matrix antibody (2. mu.g/ml) and goat anti-mouse antibody (4. mu.g/ml); horizontal striped column-matrix antibody (2. mu.g/ml), anti-CD 79b antibody (1.0. mu.g/ml) and goat anti-mouse antibody (6. mu.g/ml); the column-matrix antibody (2. mu.g/ml), anti-CD 79b antibody (0.5. mu.g/ml) and goat anti-mouse antibody (5. mu.g/ml) were spotted.

Figure 22 shows the percentage of Ramos B-cells positively stained with annexin v (ann) and/or Propidium Iodide (PI) after overnight incubation with each member of a matrix combination of B-cell antibodies (2 μ g/ml) in the presence or absence of anti-CL II antibodies (present at 0.25 or 0.5 μ g/ml if added). For identification of the "CL II" and "step 2" samples, reference is made to the description of FIG. 20. Vertical striped column-matrix antibody (2. mu.g/ml) and goat anti-mouse antibody (4. mu.g/ml); horizontal striped column-matrix antibody (2. mu.g/ml), anti-CL II antibody (0.5. mu.g/ml) and goat anti-mouse antibody (5. mu.g/ml); the column-matrix antibody (2. mu.g/ml), anti-CL II antibody (0.25. mu.g/ml) and goat anti-mouse antibody (4.5. mu.g/ml) were spotted.

FIG. 23 shows the percentage of DHL-4B-cells stained positively by annexin V (Ann) and/or Propidium Iodide (PI) after overnight incubation with members of a matrix combination of B-cell antibodies (2 μ g/ml) in the presence or absence of anti-CD 22 antibody (present at 2 μ g/ml if added). For identification of the "CL II" and "step 2" samples, reference is made to the description of FIG. 20. Solid column-matrix antibody (2. mu.g/ml) and goat anti-mouse antibody (4. mu.g/ml); striped column-matrix antibody (2. mu.g/ml), anti-CD 22 antibody (2. mu.g/ml) and goat anti-mouse antibody (8. mu.g/ml).

FIG. 24 provides a graph illustrating the direct growth inhibitory effect of free CD20 SMIP (solid) or monospecific CD20 × CD20 scorpion (hollow) on lymphoma cell lines Su-DHL6 (triangle) and DoHH2 (square).

FIG. 25 is a graph showing direct growth inhibition of lymphoma cell lines Su-DHL-6 (triangles) and DoHH2 (squares) by free anti-CD 37 SMIP (solid) or monospecific anti-CD 37 scorpion (hollow).

FIG. 26 provides a graph showing the direct growth inhibitory effect of a combination of two different monospecific SMIPs (filled) or bispecific CD20-CD37 scorpion (empty) on lymphoma cell lines Su-DHL-6 (triangles) and DoHH2 (squares).

FIG. 27 is a graph demonstrating the direct growth inhibitory effect of free CD20 SMIP in combination with CD37 SMIP (filled) or bispecific CD20xCD37 scorpion (empty) on lymphoma cell lines Su-DHL-6 (triangles) and WSU-NHL (squares).

The histogram provided in fig. 28 shows the cell cycle effect of scorpion molecules. Samples of DoHH2 lymphoma cells were divided into: untreated, treated with SMIP 016 or treated with monospecific CD37 xcd37 scorpion. Blank column: sub-G of the cell cycle₁A period; black column: g₀/G₁A period; a shadow column: a stage S; and stripe columns: stage G2/M.

Fig. 29 provides a data graph demonstrating that treatment of lymphoma cells with scorpion molecules results in enhanced signaling capacity compared to free SMIPs (as measured by calcium ion flux).

FIG. 30 provides a graph illustrating scorpion-dependent cellular cytotoxicity.

Figure 31 shows a data graph illustrating scorpion-like molecule mediated complement dependent cytotoxicity.

Figure 32 provides data in graphical form showing comparative ELISA binding of SMIPs and scorpion molecules to low affinity isoform (B) and high affinity isoform (a) of Fc γ RIII (CD 16).

Fig. 33 provides a graph demonstrating the binding of SMIPs and scorpion to the low affinity (a) and high affinity (B) alleles of Fc γ RIII (CD16) in the presence of target cells.

The histogram of fig. 34 shows the degree of expression of CD20 xcd 20 scorpion in two experiments (flask 1 and flask 2) under six different culture conditions. Solid black column: a flask 1; stripe column: flask 2.

The histogram provided in fig. 35 shows the yield of CD20 xcd 37 scorpion.

Figure 36 provides SDS-PAGE gel images (under reducing and non-reducing conditions) of SMIPs and scorpion molecules.

Figure 37 provides a graph showing the ability of scorpion molecules to retain binding to target cells. Filling a square shape: CD20 SMIP; filling triangles: CD37 SMIP; filling circles: humanized CD20(2Lm20-4) SMIP; blank diamond shape: CD37 × CD37 monospecific scorpion; hollow square: CD20 × CD37 bispecific scorpion-like molecules; and a hollow triangle: humanized CD20(2Lm20-4) × humanized CD20(2Lm20-4) scorpion.

The graph contained in FIG. 38 shows the results of a competitive binding assay demonstrating that the N-terminal and C-terminal scorpion binding domains are involved in target cell binding.

The data presented in graph form in fig. 39 shows that scorpion molecules have a lower off-rate than SMIP.

Figure 40 shows a graph demonstrating the stability of scorpion in vivo, in serum, characterized by reproducible and long circulating half-life.

Figure 41 provides a dose-response graph of CD20x CD37 bispecific scorpion, illustrating the in vivo efficacy of scorpion administration.

FIG. 42 shows the binding of monospecific CD20xCD20 scorpion (S0129) and sugar variants to target B-cells.

Figure 43 provides a graph demonstrating that the CD20xCD20 scorpion (parent antibody and sugar variants) induced ADCC-mediated killing of BJAB B-cells.

The gel image shown in fig. 44 reveals the effect on scorpion stability due to changing the scorpion linker (including changing the sequence of this linker and lengthening the linker by adding H7 sequence to the linker), indicated by a "+" in the H7 line below the gel.

Figure 45 shows the binding of CD20xCD20 scorpion (S0129) and scorpion linker variants thereof to WIL2S cells.

FIG. 46 shows the direct cell killing effect of CD20xCD20 scorpion and CD20SMIP on a variety of B-cells.

FIG. 47 reveals the direct cell killing effect of the monospecific CD20xCD20 scorpion on other B-cell lines.

Figure 48 shows the direct cell killing ability of each of two monospecific scorpion (i.e., CD20xCD20 and CD37 xCD 37) and bispecific CD20xCD37 scorpion, the latter showing different forms of killing rate profiles.

FIG. 49 graphically depicts the response of Su-DHL-6B-cells to each of CD20xCD20(S0129), CD37xCD37, and CD20xCD37 scorpion.

FIG. 50 shows bispecific CD19xCD37 scorpion andthe ability to kill Su-DHL-6B-cells directly.

FIG. 51 provides a histogram showing various Scorpion-like molecules binding CD20 and SMIP andDHL-4B-cells were killed directly (as shown in the figure). Blue column: a living cell; the chestnut columns on the right of each pair: annexin +/PI +.

FIG. 52 provides a graph depicting various Scorpion-like molecules that bind CD20 and SMIPs anddirect cell killing effect (as shown in the figure).

FIG. 53 provides a CD20 binding peptide from various scorpion molecules and SMIPs (as shown in the figure) andgraph of the induced ADCC activity.

FIG. 54 provides a CD20 binding peptide from various scorpion molecules and SMIPs (as shown in the figure) andgraph of induced CDC activity.

Fig. 55 provides a histogram showing the extent to which C1q binds to CD20 binding scorpion bound to Ramos B-cells.

The scatter plot of FACS analysis provided in FIG. 56 shows CD20 binding scorpion (2Lm20-4x2Lm20-4 and 011x2Lm20-4) andresulting in a decrease in mitochondrial membrane potential relative to control (upper panel); a histogram of the percentage of cells with a disruptive mitochondrial membrane potential (disruptive MMP: black bar) is shown in the lower panel.

The histograms provided in fig. 57 show the relative insufficiency of CD 20-binding scorpion (2Lm20-4x2Lm20-4 and 011x2Lm20-4), Rituximab (Rituximab), CD95, and controls for caspase 3(caspase3) activation.

Figure 58 provides a combination of four western blot analyses of poly (ADP-ribose) polymerase and caspases 3, 7 and 9 obtained from B-cells, showing that any of these proteins were minimally degraded by binding of CD 20-binding scorpion molecules to the cells.

FIG. 59 is a gel electrophoresis of chromosomal DNA from B-cells showing the extent of disruption due to binding of CD 20-binding scorpion to cells.

FIG. 60 is a gel electrophoresis image of immunoprecipitates obtained with each of the anti-phosphotyrosine antibody and the anti-SYK antibody. Immunoprecipitates were lysates obtained from B-cells contacted with CD 20-binding scorpion molecules, as shown in the figure.

Figure 61 provides a combination index profile of CD 20-binding scorpion with combination therapy of each of daunorubicin (doxorubicin), vincristine (vincristine), and rapamycin (rapamycin).

Detailed Description

The present invention provides compositions of relatively small peptides having at least two binding regions or domains that can provide one or more binding specificities, variable binding domains derived from immunoglobulins, such as antibodies, positioned terminally with respect to an effector domain comprising at least part of an immunoglobulin constant region (i.e., a source of a constant sub-region as defined herein); and nucleic acids, vectors and host cells involved in the recombinant production of the peptides; and methods of using the peptide compositions in a variety of diagnostic and therapeutic applications, including treating a disorder and ameliorating at least one symptom of the disorder. The peptide compositions advantageously arrange the second binding domain C-terminally to the effector domain, which arrangement unexpectedly provides for binding of at least two binding domains in the peptide without steric hindrance or with less steric hindrance, while maintaining the effector function of the centrally disposed effector domain.

The first binding domain and the second binding domain of the multivalent peptide of the invention may be the same (i.e., have identical or substantially identical amino acid sequences and have monospecificity) or different (and have multispecific). Although the first and second binding domains differ in primary structure, they can recognize and bind to the same epitope of the target molecule and thus will have a monospecificity. However, in many cases, the binding domains will have different structures and will bind to different binding sites, resulting in a multivalent multispecific protein. This different binding site may be present on a single target molecule or on different target molecules. In case the two binding molecules recognize different target molecules, the target molecules may be present, for example, on or in the same structure (e.g. the surface of the same cell), or the target molecules may be present on or in separate structures or locations. For example, multispecific binding proteins of the invention may have binding domains that specifically bind to target molecules on the surface of different cell types. Alternatively, one binding domain may specifically bind to a target on the surface of a cell, and the other binding domain may specifically bind to a target found to be unrelated to the cell, such as an extracellular structural (matrix) protein or a free (e.g., soluble or matrix) protein.

The first and second binding domains are derived from one or more regions of the same or different immunoglobulin protein structure, such as an antibody molecule. The first binding domain and/or the second binding domain may exhibit a sequence identical to the sequence of the immunoglobulin region, or may be a modified form of the sequence to provide, for example, altered binding characteristics or altered stability. Such modifications are known in the art and include amino acid sequence changes that directly result in a change in a property, such as a change in binding, for example, an amino acid sequence change that results in a change in the secondary structure or a higher order structure of the peptide. Also encompassed are modified amino acid sequences resulting from the incorporation of unnatural amino acids, such as unnatural conventional amino acids, non-conventional amino acids, and imino acids. In certain embodiments, altering the sequence results in post-translational processing changes, for example, results in changes in glycosylation patterns.

Any of a variety of binding domains derived from immunoglobulins or immunoglobulin-like polypeptides (e.g., receptors) for scorpion molecules are contemplated. The binding domain derived from an antibody comprises V_LDomains and V_HCDR regions of domains (see, e.g., context using binding domains derived from humanized antibodies). Comprising intact V derived from an antibody _LAnd V_HThe binding domains of the domains may be organized in either orientation. The scorpion molecules of the invention may have any of the binding domains described herein. For scorpion molecules having at least one binding domain that recognizes B-cells, exemplary scorpion molecules have at least one binding domain derived from: CD3, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD21, CD 3538, CD 3655, CD 3538, CD10, CD78, CD20, CD 3655, CD22,CD79a/b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138 or CDw 150. In certain embodiments, the scorpion is a multivalent binding protein comprising at least one binding domain having a sequence selected from the group consisting of seq id no: SEQ ID NO: 2. 4, 6, 103, 105, 107 and 109. In certain embodiments, the scorpion molecule comprises a binding domain comprising a sequence selected from the group consisting of any of: SEQ ID NO: 332-345. In certain embodiments, the scorpion molecule comprises a peptide comprising immunoglobulin derived V_LAnd V_HA binding domain of a sequence of a domain, wherein the sequence is selected from the group consisting of SEQ ID NO: 355-365. The invention further encompasses scorpion molecules comprising a binding domain having a sequence that can be encoded by SEQ ID NO: 355-365 having V _LAnd V_HIn the opposite direction.

For those in which either or both of the binding domains are derived from more than one region of an immunoglobulin (e.g., Ig V)_LDomains and Ig V_HRegion), multiple regions may be linked by linker peptides. In addition, a linker may be used to link the first binding domain to the constant sub-region. The association of the constant sub-region with the second binding domain (i.e. the binding domain 2 is disposed towards the C-terminus of the scorpion) may be achieved by a scorpion linker. The scorpion linker preferably has about 2 to 45 amino acids or 2 to 38 amino acids or 5 to 45 amino acids. For example, the H1 linker was 2 amino acids in length and the STD2 linker was 38 amino acids in length. In addition to length considerations, scorpion linker regions suitable for use in the scorpion molecules of the invention include antibody hinge regions selected from the group consisting of IgG, IgA, IgD and IgE hinges and variants thereof. For example, the scorpion linker may be an antibody hinge region selected from the group consisting of human IgG1, human IgG2, human IgG3, and human IgG4, and variants thereof. In certain embodiments, the scorpion linker region has a single cysteine residue for forming interchain disulfide bonds. In other embodiments, the scorpion linker has two linkers for interchain disulfide formation Cysteine residues of the bond. In certain embodiments, the scorpion linker region is derived from an immunoglobulin hinge region or a C-lectin stem region and comprises a sequence selected from the group consisting of: SEQ ID NO: 111. 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 287, 289, 297, 305, 307, 309, 310, 311, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 327, 328, 329, 330, 331, 346, 351, 352, 353, 354, 373, 374, 375, 376, and 377. More generally, when sequences derived from a hinge region are provided, any amino acid sequence identified in the sequence listing is contemplated for use as a scorpion linker in the scorpion molecules of the invention. Furthermore, the scorpion linker derived from the Ig hinge is a hinge-like peptide domain with at least one free cysteine capable of participating in interchain disulfide bonds. Preferably, the scorpion linker derived from the Ig hinge peptide retains a cysteine corresponding to the hinge cysteine disposed towards the N-terminus of the hinge. Preferably, the scorpion linker derived from the IgG1 hinge has one or two cysteines corresponding to hinge cysteines. In addition, the scorpion linker is the stem region of a type II C-lectin molecule. In certain embodiments, the scorpion molecule comprises a peptide having a sequence selected from the group consisting of SEQ id nos: 373-377.

The centrally disposed constant sub-region is an immunoglobulin-derived constant region. The constant sub-region is usually a C derived from an immunoglobulin in the abstract of the invention_HC of region_H2Moiety, although it may also originate from C_H2-C_H3And (4) partial. The constant sub-region may optionally be derived from the hinge-C of an immunoglobulin_H2Or hinge-C_H2-C_H3In part, a peptide corresponding to the Ig hinge region is placed N-terminal to the constant sub-region and disposed between the constant sub-region and binding domain 1. In addition, portions of the constant subregions may be derived from different immunizationsC of globulin_HAnd (4) a zone. In addition, the peptide corresponding to Ig CH3 may be truncated, leaving a peptide selected from the group consisting of SEQ ID NOS: 366-371. Preferably, however, in embodiments in which the scorpion hinge is a hinge-like peptide derived from an immunoglobulin hinge, the scorpion linker and constant sub-region are derived from the same type of immunoglobulin. The constant sub-region provides at least one C with an immunoglobulin_HRegion-related activities such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), protein A binding, binding to at least one F_CBinding to the receptor, stability of the reproducibility test (relative to the protein of the invention except in the absence of the constant subdomain), and perhaps placental transfer, as will be appreciated by those skilled in the art, wherein secondary transfer of the molecules of the invention is advantageous. Like the binding domains described above, a constant sub-region is an amino acid sequence derived from at least one immunoglobulin molecule and exhibits identity or substantial identity to one or more regions of at least one immunoglobulin. In certain embodiments, the constant sub-region is modified (by substitution of one or more conventional or unconventional unnatural (e.g., synthetic) amino acids or imino acids) via the or the sequence of at least one immunoglobulin to produce a primary structure that may alter secondary or higher structure, properties also changing therewith, or may result in post-translational processing changes, such as glycosylation.

Post-translational modification of the molecules of the invention may result in modified (relative to the immunoglobulin on which they are based) molecules if the binding domain and constant sub-region exhibit identical or substantially identical amino acid sequences to one or more immunoglobulin polypeptides. For example, a host cell, e.g., a CHO cell, can be modified using techniques known in the art in such a way that the glycosylation pattern of the polypeptide is altered relative to that of the polypeptide in an unmodified (e.g., CHO) host cell.

With the molecules and methods of making them recombinantly in vivo, novel approaches to targeted diagnosis and therapy have been developed, for example, to allow targeted recruitment of effector cells of the immune system (e.g., cytotoxic T lymphocytes, natural killer cells, and the like) to cells, tissues, agents, and foreign objects, such as cancer cells and infectious agents, to be destroyed or abrogated. In addition to localizing therapeutic cells to the treatment site, the peptides are useful for localizing therapeutic compounds, such as radiolabeled proteins. In addition, the peptides are also useful for the clearance of harmful compositions, for example by binding harmful compositions such as toxins to cells (e.g., macrophages) that are capable of destroying or excluding the toxin. The molecules of the invention are useful for modulating the activity of binding partner molecules, such as cell surface receptors. This is shown in figure 17, where the molecules of the present invention significantly enhanced apoptosis signaling via CD20 and/or CD 37. The result of this signaling is target cell death. Diseases and conditions in which the exclusion of a defined cell population is beneficial include infectious and parasitic diseases, inflammatory and autoimmune conditions, malignancies and the like. One skilled in the art will appreciate that the method of enhancing apoptotic signaling is not limited. Mitotic signaling and signaling leading to differentiation, activation or inactivation of defined cell populations can be induced by the molecules of the invention via appropriate selection of binding partner molecules. Further understanding of the present disclosure will be facilitated by consideration of the following expressive definitions of terms used herein.

A "single-chain binding protein" is a single contiguous array of covalently linked amino acids, the chain being capable of specifically binding to one or more binding partners that share a determinant for a binding site sufficient to detectably bind to the single-chain binding protein. Exemplary binding partners include proteins, carbohydrates, lipids, and small molecules.

For ease of description, "derivatives" and "variants" of the proteins, polypeptides and peptides of the invention are described in terms of differences from the proteins and/or polypeptides and/or peptides of the invention, meaning that the derivatives and variants (which are the proteins/polypeptides/peptides of the invention) differ in a particular way from the non-derivatized or non-variant proteins, polypeptides or peptides of the invention. Those skilled in the art will appreciate that such derivatives and variants are, per se, proteins, polypeptides and peptides of the invention.

"antibodies" are given the broadest definition consistent with their meaning in the art and include proteins, polypeptides and peptides capable of binding at least one binding partner, such as a proteinaceous or non-proteinaceous antigen. As used herein, "antibody" includes members of the immunoglobulin superfamily of proteins of any species, members of the immunoglobulin superfamily of proteins of single or multiple chain composition, and variants, analogs, derivatives and fragments of such molecules. In particular, "antibody" includes any form of antibody known in the art, including, but not limited to, monoclonal and polyclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, single chain variable fragments, bispecific antibodies, bifunctional antibodies, antibody fusions, and the like.

A "binding domain" is a peptide region that specifically binds to one or more specific binding partners, such as a polypeptide fragment derived from an immunoglobulin (e.g., an antibody). If multiple binding partners are present, the partners share binding determinants sufficient to detectably bind the binding domain. The binding domain is preferably a contiguous amino acid sequence.

An "epitope" is given herein in the general sense of having a single antigenic site, i.e. an antigenic determinant, on a substance (e.g. a protein) that specifically interacts (e.g. by binding) with an antibody. Other terms that have been given definite meanings in immunoglobulin (e.g., antibody) technology, such as "light chain variable region", "heavy chain variable region", "light chain constant region", "heavy chain constant region", "antibody hinge region", "complementarity determining region", "framework region", "antibody isotype", "FC region", "single chain variable fragment", or "scFv", "diabody", "chimera", "CDR-grafted antibody", "humanized antibody", "formed antibody", "antibody fusion", and similar terms, each have definite meanings known in the art, unless specifically stated otherwise herein.

Unless explicitly defined otherwise herein, the present inventionTerms known to those skilled in the art with reference to antibody technology have the meaning found in the art. An example of said term is "V_L'and' V_H", refers to variable binding regions derived from the light and heavy chains of an antibody, respectively; and C_LAnd C_HBy "immunoglobulin constant region" is meant, i.e., a constant region derived from an antibody light or heavy chain, respectively, wherein it is understood that the latter constant region may be further divided into C_H1、C_H2、C_H3And C_H4Constant region domains, depending on the isotype of antibody from which the region is derived (IgA, IgD, IgE, IgG, IgM). CDR means "complementarity determining region". The "hinge region" is derived from a C inserted into a single chain of an antibody_H1Region and C_H2The amino acid sequence between and connecting the two regions, a "hinge region" is known in the art to provide flexibility to the whole antibody in the form of a "hinge".

A "constant sub-region" is a term defined herein to refer to a peptide, polypeptide, or protein sequence that corresponds to or is derived from one or more constant region domains of an antibody. Thus, the constant sub-region may comprise any or all of the following domains: c_H1Domains, hinge regions, C_H2Domain, C_H3Domains (IgA, IgD, IgG, IgE and IgM) and C _H4Domains (IgE, IgM). Thus, a constant sub-region as defined herein may refer to a polypeptide region corresponding to the entire constant region of an antibody or a portion thereof. Typically, the constant sub-region or encoding nucleic acid of the polypeptide of the invention has a hinge, C_H2Domains and C_H3A domain.

An "effector function" is a function associated with or provided by an antibody constant region. Exemplary effector functions include antibody-dependent cell-mediated cytotoxicity (ADCC), complement activation and complement-dependent cytotoxicity (CDC), F_CReceptor binding and increased plasma half-life and placental transfer. The effector function of the composition of the invention is detectable; the specific activity of the composition of the invention having this function is preferably approximately the same as the specific activity of the wild-type antibody in respect of this effector function, i.e.relative to the wild-type antibodyIn other words, the constant sub-region of the multivalent binding molecule preferably does not lose any effector function.

A "linker" is a peptide or polynucleotide that links or connects other peptides or polynucleotides. Typically, a peptide linker is an oligopeptide having about 2 to 50 amino acids, wherein a typical polynucleotide linker encodes the peptide linker and is thus about 6 to 150 nucleotides in length. The linker links the first binding domain to the constant sub-region domain. An exemplary peptide linker is (Gly) ₄Ser)₃. A scorpion linker is used to link the C-terminus of the constant sub-region to the second binding domain. The scorpion linker may be derived from an immunoglobulin hinge region or from the stem region of a type II C-lectin, as described in more detail below.

"target" is assigned more than one meaning, and the context of use defines the unambiguous meaning in each case. In its narrowest sense, a "target" is a binding site, i.e. a binding domain of a binding partner of a peptide composition of the invention. In a broader sense, "target" or "molecular target" refers to the entire binding partner (e.g., protein) that must present a binding site. Specific targets (such as "CD 20", "CD 37" and the like) are each assigned the general meaning of the term as it has been acquired in the art. A "target cell" is any prokaryotic or eukaryotic cell (whether healthy or diseased or not) associated with a target molecule of the invention. Of course, target molecules unrelated to any cell (i.e., cell-free targets) or related to other components, such as viruses (including phages), organic or inorganic target molecule carriers, and foreign bodies, are also found.

Examples of substances associated with the target molecule include autologous cells (e.g., cancer cells or other diseased cells), infectious agents (e.g., infectious cells and infectious viruses), and the like. The target molecule may be associated with enucleated cells, cell membranes, liposomes, sponges, gels, capsules, lozenges, and the like, which may be used to deliver, transport, or localize the target molecule regardless of the intended use (e.g., for drug therapy as a result of deliberate or unintentional measures, or further, for bioterrorism threats). "cell-free", "virus-free", "vector-free", "object-free" and the like refer to target molecules that are not bound to a particular composition or substance.

"binding affinity" refers to the strength of non-covalent binding of a peptide composition of the invention to its binding partner. Binding affinity preferably refers to a quantitative measure of the attraction between members of a binding pair.

An "adjuvant" is a substance that enhances or facilitates the functional effect of a compound with which it is associated (such as in the form of a pharmaceutical composition comprising an active agent and an adjuvant). An "excipient" is an inert substance used as a diluent in the formulation of a pharmaceutical composition. A "carrier" is typically an inert substance that is used to provide a vehicle for delivery of the pharmaceutical composition.

"host cell" refers to any prokaryotic or eukaryotic cell in which a polynucleotide, protein, or peptide of the invention is present.

"introducing" a nucleic acid or polynucleotide into a host cell means that the nucleic acid or polynucleotide enters the cell by any means known in the art, including, but not limited to, in vitro salt-mediated precipitation and other forms of transformation of naked nucleic acids/polynucleotides or vector-carried nucleic acids/polynucleotides, virus-mediated infection and optionally transduction with or without "helper" molecules, ballistic projectile delivery, conjugation, and the like.

By "incubating" a host cell is meant maintaining the cell under environmental conditions known in the art to be suitable for a given use, such as gene expression. Such conditions (including temperature, ionic strength, oxygen tension, carbon dioxide concentration, nutrient composition, and the like) are well known in the art.

By "isolating" a compound (such as a protein or peptide of the invention) is meant separating the compound from at least one different compound with which it is naturally associated that is present in, for example, a host cell expressing the compound to be isolated, e.g., separating spent medium containing the compound from host cells grown in the medium.

An "organism in need thereof" is any organism at risk of or suffering from any disease, disorder, or condition susceptible to treatment or amelioration with the compositions of the present invention, including, but not limited to, any of a variety of cancer forms, any of a variety of autoimmune diseases, radiation poisoning due to radiolabeled proteins, peptides, and similar compounds, ingestible or internally produced toxins, and similar forms thereof, as will become apparent upon review of the entire disclosure. The organism in need thereof is preferably a human patient.

As known in the art, "amelioration" of a symptom of a disease means that the severity of the symptom of the disease is detectably reduced. Exemplary symptoms include pain, heat, swelling, and joint stiffness.

Unless the context clearly indicates otherwise, the terms "protein", "peptide" and "polypeptide" are used interchangeably herein, each referring to at least one contiguous chain of amino acids. Similarly, the terms "polynucleotide", "nucleic acid" and "nucleic acid molecule" are used interchangeably unless the context clearly indicates a particular and not interchangeable meaning.

"pharmaceutically acceptable salt" refers to a salt of a compound of the invention, which is derived from the combination of the compound with an organic or inorganic acid (acid addition salt) or the combination of the compound with an organic or inorganic base (base addition salt).

The following provides a general description of various aspects of the invention using terms as defined above. Following the general description, working examples are described to provide additional proof of the operability and applicability of the invention disclosed herein.

Proteins and polypeptides

In certain embodiments of the invention, any of the multivalent binding proteins with effector function (including binding domain-immunoglobulin fusion proteins) described herein are provided, wherein the multivalent binding protein or peptide with effector function comprises two or more binding domain polypeptide sequences. Each of the binding domain polypeptide sequences is capable of binding or specifically binding to one or more targets, such as one or more antigens, wherein the one or more targets or antigens may or may not be identical. The binding domain polypeptide sequence may be derived from an antigen variable region, or it may be derived from an immunoglobulin-like molecule, such as a receptor that folds in a manner mimicking an immunoglobulin molecule. The antibody from which the binding domain is derived may be a polyclonal antibody (including a monospecific polyclonal antibody), a monoclonal antibody (mAb), a recombinant antibody, a chimeric antibody, a humanized antibody (such as a CDR-grafted antibody), a human antibody, a single chain antibody, a catalytic antibody and any other form of antibody known in the art, as well as fragments, variants or derivatives thereof. In certain embodiments, each of the binding domains of the proteins of the invention is derived from the entire variable region of an immunoglobulin. In a preferred embodiment, the binding domains are each based on human Ig variable regions. In other embodiments, the protein is a fragment derived from an Ig variable region. In such embodiments, preferably, each binding domain polypeptide sequence corresponds to the sequence of each of the complementarity determining regions of a designated Ig variable region. The invention also encompasses binding domains corresponding to fewer than all CDRs of a given Ig variable region, provided that the binding domains retain the ability to specifically bind to at least one target.

Multivalent binding proteins with effector function also have constant sub-region sequences derived from an immunoglobulin constant region (preferably an antibody heavy chain constant region) covalently juxtaposed between two binding domains in a multivalent binding protein with effector function.

Multivalent binding proteins with effector function also have scorpion linkers that link the C-terminus of the constant sub-region to the N-terminus of binding domain 2. The scorpion linker is not a helical peptide and may be derived from the antibody hinge region, from a region linked to an immunoglobulin binding domain or from the stem region of a type II C-lectin. The scorpion linker may be derived from a wild-type hinge region of an immunoglobulin, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgD, or IgE hinge region. In other embodiments, the invention provides multivalent binding proteins with altered hinges. One class of altered hinge regions suitable for inclusion in multivalent binding proteins is the class of hinges in which the number of cysteine residues (particularly those Cys residues known in the art) involved in interchain disulfide bond formation in the immunoglobulin counterpart molecule with the wild-type hinge is altered. Thus, the protein may have an IgG1 hinge in which one of the three Cys residues capable of participating in interchain disulfide bond formation is deleted. To indicate the cysteine substructure of the altered hinge, the Cys subsequence is shown from N-terminus to C-terminus. Using this recognition system, multivalent binding proteins with altered IgG hinges include hinge structures characterized as cxc, xxx, ccx, xxx, xcx, cxx, and xxx. Cys residues may be deleted, or may be substituted with amino acids, forming conservative or non-conservative substitutions. In certain embodiments, the cysteine is replaced with a serine. For proteins with scorpion linkers comprising an IgG1 hinge, the number of cysteines corresponding to hinge cysteines is reduced to 1 or 2, preferably one of those corresponding to hinge cysteines is disposed closest to the N-terminus of the hinge.

For proteins with scorpion linkers containing an IgG2 hinge, 0, 1, 2, 3, or 4 Cys residues may be present. For scorpion linkers comprising an altered IgG2 hinge containing 1, 2, or 3 Cys residues, all possible subsets of Cys residues are encompassed. Thus, for the linker with one Cys, the multivalent binding protein may have the following Cys motif in the hinge region: cxxx, xcxx, xxcx or xxxc. For scorpion linkers comprising IgG2 hinge variants with 2 or 3 Cys residues, all possible combinations of retained Cys residues with substituted (or deleted) Cys residues are contemplated. For multivalent binding proteins with scorpion linkers comprising an altered IgG3 or altered IgG4 hinge region, it is contemplated that the Cys residues in the hinge region are reduced from 1 to less than the full number of Cys residues, whether the reduction is via deletion or via substitution with a conserved amino acid or a non-conserved amino acid (e.g., serine). Multivalent binding proteins having scorpion linkers comprising wild-type IgA, IgD or IgE hinges are likewise contemplated, as are corresponding altered hinge regions having a reduced number of Cys residues, which number extends from 0 to a number less than the total number of Cys residues present in the corresponding wild-type hinge. In certain embodiments having an IgG1 hinge, the first or N-terminal Cys residue of the hinge is retained. For proteins having a wild-type hinge region or an altered hinge region, it is contemplated that the multivalent binding protein is a single chain molecule capable of forming a homomultimer (such as a dimer), for example, by disulfide bond formation. Furthermore, as disclosed herein, the hinge region termini of proteins with altered hinges may be modified, e.g., by deletion or substitution of one or more amino acid residues at the N-terminus, C-terminus, or both termini of a designated region or domain (such as a hinge domain).

In another exemplary embodiment, the constant sub-region is derived from a constant region comprising a native IgD hinge region or an engineered IgD hinge region. Wild-type human IgD hinges have a cysteine that can form a disulfide bond with the light chain in the native IgD structure. In certain embodiments, this IgD hinge cysteine is mutated (e.g., deleted) to create an altered hinge that serves, for example, as a linking region between binding domains of a bispecific molecule. Other amino acid changes or deletions or modifications in the IgD hinge that do not result in poor hinge rigidity are within the scope of the invention. Natural or engineered IgD hinge regions from other species are also within the scope of the invention, as are humanized natural or engineered IgD hinges from non-human species and (other non-IgD) hinge regions from other human or non-human antibody isotypes, such as llama IgG2 hinges.

The invention further includes a constant sub-region linked to a scorpion linker that may be derived from a hinge corresponding to a known hinge region as described above, such as an IgG1 hinge or an IgD hinge. The constant sub-region may comprise (relative to the wild-type) a modified or modified hinge region, which may beWherein at least one cysteine residue known to be involved in interchain disulfide bonding is substituted with another amino acid by conservative substitution (e.g., Ser for Cys) or non-conservative substitution. The constant sub-region does not correspond to immunoglobulin C _H1A peptide region or a peptide domain of a domain.

Alternative hinge and linker sequences that may be used as a linking region are derived from portions of a cell surface receptor that are linked to an immunoglobulin V-like domain or an immunoglobulin C-like domain. Also encompassed are regions between IgV-like domains (wherein the cell surface receptor contains multiple tandem Ig V-like domains) and regions between Ig C-like domains (wherein the cell surface receptor contains multiple tandem IgC-like regions) as connecting regions. Hinge and linker sequences are typically 5 to 60 amino acids long and are not only primarily flexible, but may also provide more rigid characteristics. Furthermore, linkers generally provide a space that facilitates minimization of steric hindrance between binding domains. Preferably, the structure of the hinge and linker peptides is predominantly alpha helical with minimal beta sheet structure. Preferred sequences are stable in plasma and serum and resistant to proteolytic cleavage. Preferred sequences may contain naturally occurring motifs or added motifs such as CPPC motifs that confer disulfide bonds to stabilize dimer formation. Preferably the sequence may contain one or more glycosylation sites. Examples of preferred hinge and linker sequences include, but are not limited to, the interdomain region between Ig V-like and Ig C-like regions of CD2, CD4, CD22, CD33, CD48, CD58, CD66, CD80, CD86, CD150, CD166, and CD 244.

The constant sub-regions may be derived from camelid constant regions, such as llama or camel IgG2 or IgG 3.

In particular, contemplated are those having a C derived from any Ig class or from any IgG subclass, such as IgG1 (e.g., human IgG1)_H2-C_H3A constant sub-region of a region. In a preferred embodiment, the constant sub-region and the scorpion linker derived from an immunoglobulin hinge are both derived from the same Ig class. In other preferred embodiments, the constant sub-region and the scorpion linker derived from an immunoglobulin hinge are both derived from the same Ig subclass. Constant subunitA region may also be a CH3 domain derived from any Ig class or subclass (such as IgG1, e.g., human IgG1), provided that it is functionally associated with at least one immunoglobulin response.

The constant sub-region does not correspond to the entire immunoglobulin constant region of the IgG class (i.e., C)_H1-hinge-C_H2-C_H3). The constant sub-region may correspond to other classes of intact immunoglobulin constant regions, also encompassing IgA constant domains with mutated or deleted tails (such as IgA1 hinge, IgA2 hinge, IgA C)_H2And IgA C_H3Domain) as a constant sub-region. In addition, any light chain constant domain can be used as a constant sub-region, e.g., C_KOr any C_L. The constant sub-region may also include JH or JK with or without a hinge. The constant sub-region may also correspond to an engineered antibody, wherein, as is understood in the art, for example, a loop graft has been constructed by making selected amino acid substitutions using an IgG framework to produce a peptide for native F _CReceptors other than R (CD16, CD32, CD64, F)_Cε R1). An exemplary constant sub-region of this type is an IgG C modified to have a CD89 binding site_H2-C_H3And (4) a zone.

This aspect of the invention provides a multivalent binding protein or peptide having effector function comprising, consisting essentially of, or consisting of: (a) an immunoglobulin-derived binding domain polypeptide sequence disposed N-terminally, fused or linked to (b) an immunoglobulin-derived constant region constant sub-region polypeptide sequence, which preferably comprises a hinge region sequence, wherein the hinge region polypeptide can be as described herein, and can comprise, consist essentially of, or consist of: for example, a surrogate hinge region polypeptide sequence, which in turn is fused or linked to (C) a second natural or engineered binding domain polypeptide sequence derived from an immunoglobulin disposed at the C-terminus.

The centrally disposed constant sub-region polypeptide sequence derived from an immunoglobulin constant region can have at least one selected from the group consisting ofImmunological activity: antibody-dependent cell-mediated cytotoxicity, CDC, complement fixation, and F _CThe receptors bind, and the binding domain polypeptides are each capable of binding or specifically binding a target, such as an antigen, wherein the targets may be the same or different, and may be effectively present in the same physiological environment (e.g., the surface of the same cell) or in different environments (e.g., different cell surfaces, and cell-free locations, such as in solution).

This aspect of the invention also includes variant proteins or polypeptides exhibiting effector function that are at least 80% and preferably 85%, 90%, 95% or 99% identical to the effector function-bearing multivalent protein of a particular sequence as disclosed herein.

Polynucleotide

The invention also provides polynucleotides (isolated or purified polynucleotides or pure polynucleotides) encoding the proteins or peptides of the invention, vectors (including cloning and expression vectors) comprising the polynucleotides, and cells (e.g., host cells) transformed or transfected with the polynucleotides or vectors of the invention. In encoding the proteins or polypeptides of the invention, the polynucleotide encodes a first binding domain, a second binding domain, and F_CDomains, all derived from an immunoglobulin, preferably from a human immunoglobulin. Each binding domain may contain a sequence corresponding to the full-length variable region sequence (heavy and/or light chain) or to a portion thereof, provided that each such binding domain retains the ability to specifically bind. F _CThe domains may have a structure corresponding to full-length immunoglobulin F_CA domain sequence or a sequence corresponding to a partial sequence thereof, with the proviso that F_CThe domain exhibits at least one effector function as defined herein. Furthermore, each of the binding domains may be linked to F via a linker peptide typically at least 8 amino acids and preferably at least 13 amino acids in length_CA domain. Preferred linker sequences are based on Gly₄Sequences of Ser motifs, such as (Gly)₄Ser)₃。

Variants of multivalent binding proteins having effector functions are also encompassed by the invention. A variant polynucleotide has at least 90% and preferably 95%, 99% or 99.9% identity to one of the polynucleotides having a defined sequence as described herein, or it may hybridize to one of those polynucleotides having a defined sequence under stringent hybridization conditions of 65-68 ℃, 0.015M sodium chloride, 0.0015M sodium citrate, or 42 ℃, 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide. The polynucleotide variants retain the ability to encode multivalent binding proteins with effector functions.

The term "stringent" is used to refer to conditions that are generally understood in the art to be stringent. Hybridization stringency is determined primarily by temperature, ionic strength, and concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015M sodium chloride, 0.0015M sodium citrate at 65-68 ℃ or 0.015M sodium chloride, 0.0015M sodium citrate and 50% formamide at 42 ℃. See Sambrook et al, Molecular Cloning: a Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory, (Cold Spring Harbor, N.Y. 1989).

More stringent conditions (such as higher temperature, lower ionic strength, higher formamide or other denaturants) may also be used; however, the rate of hybridization will be affected. In cases where hybridization of deoxyoligonucleotides is involved, other exemplary stringent hybridization conditions include washes in 6-fold SSC, 0.05% sodium pyrophosphate at 37 ℃ (for 14 base oligonucleotides), 48 ℃ (for 17 base oligonucleotides), 55 ℃ (for 20 base oligonucleotides), and 60 ℃ (for 23 base oligonucleotides).

In a related aspect of the invention, there is provided a method of making a polypeptide or protein or other construct of the invention (e.g., including a multivalent binding protein or peptide having effector function), the method comprising the steps of: (a) culturing a host cell as described or provided herein under conditions that allow expression of the construct; and (b) isolating the expression product (e.g., multivalent binding protein or peptide having effector function) from the host cell or host cell culture.

Construct

The invention also relates to vectors each comprising a polynucleotide or nucleic acid of the invention and constructs made from known vectors, particularly to recombinant expression constructs suitable for use in gene therapy, including any of a variety of known constructs, including delivery constructs, including any nucleic acid encoding multivalent (e.g., multispecific, including bispecific) binding proteins and polypeptides having effector functions as provided herein; to host cells genetically engineered with the vectors and/or other constructs of the invention; and to methods of administering expression constructs or other constructs comprising nucleic acid sequences encoding multivalent (e.g., multispecific, including bispecific) binding proteins having effector functions, or fragments or variants thereof, by recombinant techniques.

In the case of use in gene therapy, various constructs of the invention, including multivalent (e.g., multispecific) binding proteins having effector functions, may be expressed in virtually any host cell, including in vivo host cells, under the control of an appropriate promoter, depending on the nature of the construct (e.g., the type of promoter as described above) and depending on the nature of the host cell in question (e.g., post-mitotic terminal differentiation or active division; e.g., the expressible construct is maintained episomally or integrated into the host cell genome).

Suitable Cloning and expression vectors for prokaryotic and eukaryotic hosts are described, for example, in Sambrook et al, Molecular Cloning: a Laboratory Manual, 2 nd edition, Cold spring Harbor, NY, (1989). Exemplary cloning/expression vectors include, but are not limited to, cloning vectors, shuttle vectors, and expression constructs, which may be based on plasmids, phagemids (phagemid), phagemids (phasmid), cosmids, viruses, artificial chromosomes, or any nucleic acid vehicle suitable for amplification, transfer, and/or expression of polynucleotides contained therein as is known in the art. As described herein, in a preferred embodiment of the invention, recombinant expression is performed in mammalian cells that have been transfected, transformed or transduced with a nucleic acid of the invention. See also, e.g., Machida, CA., "Viral Vectors for Gene Therapy: methods and Protocols (viral vectors for gene therapy: Methods and Protocols) "; wolff, JA, "Gene Therapeutics: methods and applications of Direct Gene Transfer (Gene therapy: Methods and applications of Direct Gene Transfer) "(Birkhauser 1994); stein, U and Walther, W (eds), "Gene therapy of Cancer: methods and Protocols (Gene therapy for cancer: Methods and Experimental procedures) "(Humana Press 2000); robbins, PD (eds), "Gene Therapy Protocols" Humana Press 1997); morgan, JR (eds), "Gene therapeutics (Gene protocols)" (Humana Press 2002); meager, A (eds.), "Gene therapy Technologies, Applications and Regulations: from Laboratory to clinical (Gene therapy technology, application and Regulation: Laboratory to clinical) "(John Wiley & Sons Inc.1999); MacHida, CA and Constant, JG, "Viral Vectors for Gene therapy: methods and Protocols (viral vectors for gene therapy: Methods and Experimental procedures) "(Humana Press 2002); "New Methods Of Gene Therapy For genetic metabolism Diseases NIH Guide (New method For Gene Therapy NIH Guide For genetic Metabolic Diseases)" Vol 22, No 35, p 1/10 1993. See also U.S. patent nos. 6,384,210, 6,384,203, 6,384,202, 6,384,018, 6,383,814, 6,383,811, 6,383,795, 6,383,794, 6,383,785, 6,383,753, 6,383,746, 6,383,743, 6,383,738, 6,383,737, 6,383,733, 6,383,522, 6,383,512, 6,383,481, 6,383,478, 6,383,138, 6,380,382, 6,380,371, 6,380,369, 6,380,362, 6,380,170, 6,380,169, 6,379,967 and 6,379, 966.

Expression constructs are typically derived from plasmid vectors. One preferred construct is a modified pNASS vector (Clontech, Palo Alto, Calif.) having a nucleic acid sequence encoding an ampicillin (ampicillin) resistance gene, a polyadenylation signal and a T7 promoter site. Other suitable mammalian expression vectors are well known (see, e.g., Ausubel et al, 1995; Sambrook et al, supra; see also, e.g., the catalogues of Invitrogen, San Diego, Calif.; Novagen, Madison, Wis.; Pharmacia, Piscataway, N.J.). To facilitate increased production of multivalent binding proteins with effector functions, which is caused by gene amplification following application of an appropriate selection agent (e.g., methotrexate), presently preferred constructs comprising a dihydrofolate reductase (DHFR) coding sequence can be prepared under appropriate regulatory control.

Typically, a recombinant expression vector will include an origin of replication and a selectable marker that allows transformation of the host cell, as described above, and a promoter derived from a highly expressed gene to directly transcribe the downstream structural sequences. Operable linkage of a vector to a polynucleotide of the invention produces a cloning construct or an expression construct. Exemplary cloning/expression constructs contain at least one expression control element, such as a promoter, operably linked to a polynucleotide of the invention. Other expression control elements, such as enhancers, factor-specific binding sites, terminators, and ribosome binding sites, are also encompassed in the vectors and cloning/expression constructs of the invention. Heterologous structural sequences of the polynucleotides of the invention are assembled at appropriate times with translation initiation and termination sequences. Thus, for example, a multivalent binding protein-encoding nucleic acid as provided herein may be included in any of a variety of expression vector constructs as a recombinant expression construct for expressing the protein in a host cell. In certain preferred embodiments, the construct is included in a formulation for in vivo administration. Such vectors and constructs include chromosomal, nonchromosomal, and synthetic DNA sequences, e.g., derivatives of SV 40; a bacterial plasmid; phage DNA; a yeast plasmid; vectors derived from a combination of plasmids with phage DNA, viral DNA (such as vaccinia, adenovirus, fowlpox virus and pseudorabies, or replication deficient retroviruses as described below). However, any other vector may be used to prepare the recombinant expression construct, and in a preferred embodiment, the vector is replicable and may be viable in a host.

The appropriate DNA sequence can be inserted into the vector, for example, by various procedures. Generally, the DNA sequence is inserted into the appropriate restriction endonuclease cleavage site by procedures known in the art. Standard techniques for cloning, DNA isolation, amplification and purification, standard techniques for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various isolation techniques are contemplated. Various standard techniques are described, for example, in Ausubel et al (1993 Current Protocols in Molecular Biology, Greene Publ.Assoc.Inc. & John Wiley & Sons, Inc., Boston, Mass.); sambrook et al (1989 Molecular Cloning, 2 nd edition, Cold Spring Harbor Laboratory, Plainview, N.Y.); maniatis et al (1982Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.); glover (eds.) (1985 DNA Cloning, volumes I and II, IRL Press, Oxford, UK); hames and Higgins (eds.), (1985 Nucleic acid hybridization, IRL Press, Oxford, UK); and in other documents.

The DNA sequence in the expression vector is operably linked to at least one appropriate expression control sequence (e.g., a constitutive promoter or a regulatory promoter) to direct mRNA synthesis. Representative examples of such expression control sequences include promoters of eukaryotic cells or viruses thereof as described above. The promoter region may be selected from any desired gene using a CAT (chloramphenicol transferase) vector or other vector with a selectable marker. Eukaryotic promoters include CMV fast early promoter, HSV thymidine kinase, early and late SV40, LTR from retrovirus, and mouse metallothionein-I. Selection of appropriate vectors and promoters is within the level of ordinary skill in the art, and the preparation of certain particularly preferred recombinant expression constructs comprising at least one promoter or regulatory promoter operably linked to a nucleic acid encoding a protein or polypeptide of the present invention is described herein.

Transcription of DNA encoding the proteins and polypeptides of the invention by higher eukaryotic cells may be enhanced by inserting enhancer sequences into the vector. Examples include the SV40 enhancer (bp100 to 270) located proximal to the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer proximal to the origin of replication, and adenovirus enhancers.

Also encompassed are gene therapies (including strategies to replace defective genes or add novel genes into cells and/or tissues) using the nucleic acids of the invention, and which are being developed for use in the treatment of cancer, modulation of metabolic disorders, and in the field of immunotherapy. Gene therapy of the invention includes the use of various constructs of the invention, with or without separate vectors or delivery vehicles or constructs, for the treatment of the diseases, disorders and/or conditions described herein. The constructs may also be used as vaccines for the treatment or prevention of the diseases, disorders and/or conditions described herein. DNA vaccines, for example, utilize polynucleotides and nucleic acid determinants encoding immunogenic proteins to stimulate the immune system to protect against pathogens or tumor cells. The strategies may stimulate acquired or innate immunity, or may involve modification of immune function via cytokine expression. In vivo gene therapy generally involves the direct injection of genetic material into a patient or animal to treat, prevent or ameliorate disease or symptoms associated with disease. Vaccines and immunomodulation are systemic therapies. For tissue-specific in vivo therapies, such as those aimed at treating cancer, localized gene delivery and/or expression/targeting systems are preferred. A variety of gene therapy vectors targeting specific tissues are known in the art, and procedures have been developed to fully target specific tissues, for example using catheter-based techniques, all of which are encompassed herein.

Ex vivo gene therapy is also contemplated herein, and the methods involve removal, genetic modification, amplification, and re-administration of autologous cells of an individual (e.g., a human patient). Examples include bone marrow transplantation or genetic modification of lymphoid progenitor cells for cancer therapy. Ex vivo gene therapy is preferably suitable for the treatment of cells (such as blood or skin cells) that are readily available and can survive in culture during the gene transfer process.

Suitable gene therapy vectors include adenoviral vectors, lentiviral vectors, adeno-associated virus (AAV) vectors, Herpes Simplex Virus (HSV) vectors, and retroviral vectors. Gene therapy can also be performed using "naked DNA," liposome-based delivery methods, lipid-based delivery methods (including DNA linked to positively charged lipids), electroporation, and ballistic projection methods.

In certain embodiments (including but not limited to gene therapy embodiments), the vector may be a viral vector, such as a retroviral vector. Miller et al, 1989 BioTechniques 7: 980; coffin and Varmus, 1996 Retroviruses, Cold Spring harbor laboratory Press, NY. For example, retroviruses from which retroviral plasmid vectors are derived include, but are not limited to, murine Moloney (Moloney) leukemia virus, splenic necrosis virus, retroviruses such as Rous (Rous) sarcoma virus, Harvey sarcoma virus, avian leukemia virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, myeloproliferative sarcoma virus, and mammary tumor virus.

Retroviruses are RNA viruses that are replicable and can integrate into the genome of a host cell via DNA intermediates. The DNA intermediate or provirus is stably integrated into the host cell DNA. According to certain embodiments of the invention, the expression construct may comprise a retrovirus having incorporated therein a foreign gene encoding a foreign protein rather than normal retroviral RNA. When retroviral RNA enters the host cell, with infection occurring, the foreign gene is also introduced into the cell, and can then integrate into the host cell DNA as if it were part of the retroviral genome. Expression of this foreign gene in the host results in the expression of the foreign protein.

Most retroviral vector systems that have been developed for gene therapy are based on murine retroviruses. The retrovirus exists in two forms: free viral particles called virions or provirus integrated in the DNA of a host cell. Viruses in the form of virions contain: structural and enzymatic proteins of retroviruses (including reverse transcriptase), two RNA copies of the viral genome and a portion of the source cytoplasmic membrane containing the viral envelope glycoproteins. The retroviral genome is organized into four major regions: long Terminal Repeats (LTRs) containing cis-acting elements necessary for transcription initiation and termination and located 5 'and 3' to the encoding gene; and three genes encoding gag, pol, and env. The three genes gag, pol and env encode internal viral structures, enzymatic proteins (such as integrase) and envelope glycoproteins (designated gp70 and p15e), respectively, which confer infectivity and host-range specificity to the virus and undefined function of the "R" peptide.

For safety concerns associated with the use of retroviruses, including for use in expression constructs, separately packaged cell lines and vector-producing cell lines have been developed. Briefly, this method is the use of two components, a retroviral vector and a Packaging Cell Line (PCL). The retroviral vector contains a Long Terminal Repeat (LTR), foreign DNA to be transferred, and a packaging sequence (y). This retroviral vector cannot be regenerated by itself because the genes encoding the structural and envelope proteins are not included in the vector genome. PCL contains genes encoding gag, pol and env proteins but does not contain the packaging signal "y". Thus, PCL can only form empty virions by itself. In this general approach, retroviral vectors are introduced into the PCL, thereby forming a vector-producing cell line (VCL). This VCL produces viral particles that contain only the foreign genome of the retroviral vector, and thus has previously been considered a safe retroviral vector for therapeutic use.

"retroviral vector construct" refers to a composition that, in a preferred embodiment of the invention, is capable of directing the expression of one or more sequences or genes of interest, such as a nucleic acid sequence encoding a multivalent binding protein. Briefly, the retroviral vector construct must include a 5 'LTR, a tRNA binding site, a packaging signal, a start of second strand DNA synthesis, and a 3' LTR. Various heterologous sequences can be included in the vector construct, including, for example, sequences encoding proteins (e.g., cytotoxic proteins, disease-associated antigens, immune accessory molecules, or replacement genes) or sequences that function as molecules themselves (e.g., as ribonucleases or antisense sequences).

Retroviral vector constructs of the present invention can be readily constructed from a variety of retroviruses including, for example, B, C and the D-type retroviruses as well as foamy and lentiviruses (see, for example, RNA Tumor Viruses, 2 nd edition, Cold spring Harbor Laboratory, 1985). The retrovirus is readily available from a depository or collection, such as the American type culture Collection ("ATCC"; Rockville, Maryland), or isolated from known sources using commonly available techniques. It is known that any of the above-described retroviruses can be readily utilized to assemble or construct the retroviral vector constructs, packaging cells, or producer cells of the present invention (e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory Press, 1989; Kunkle, 1985 Proc. Natl. Acad. Sci. (USA) 82: 488), given the disclosure and standard recombinant techniques provided herein.

Promoters suitable for use in viral vectors may generally include, but are not limited to: a retrovirus LTR; the SV40 promoter; and the human Cytomegalovirus (CMV) promoter (described in Miller et al, 1989 Biotechniques 7: 980-990) or any other promoter (e.g., cellular promoters such as eukaryotic promoters, including but not limited to histone, polIII, and β -actin promoters). Other viral promoters that may be used include, but are not limited to, the adenovirus promoter, the Thymidine Kinase (TK) promoter, and the B19 mini-viral promoter. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein, and may be selected from a self-regulating promoter or a promoter as described above.

Retroviral plasmid vectors are used to transduce packaging cell lines to form producer cell lines. Examples of packaging cells that can be transfected include, but are not limited to, e.g., Miller, Human gene therapy, 1: 5-14(1990) PE501, PA317, psi-2, psi-AM, PA12, T19-14X, VT-19-17-H2, psi CRE, psi CRIP, GP + E-86, GP + envAm12 and DAN cell lines as described in (1). The vector may transduce the packaging cell via any means known in the art. Including but not limited to electroporation, the use of liposomes and CaPO₄And (4) precipitating. In one option, the retroviral plasmid vector can be encapsulated in liposomes, or coupled to lipids, and then administered to the host.

The producer cell line produces infectious retroviral vector particles comprising a nucleic acid sequence encoding a multivalent binding protein having effector function. The retroviral vector particles can then be used to transduce eukaryotic cells in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence encoding the protein or polypeptide. Eukaryotic cells that can be transduced include, but are not limited to, embryonic stem cells and hematopoietic stem cells, hepatocytes, fibroblasts, circulating peripheral blood mononuclear cells and polymorphonuclear cells (including bone marrow mononuclear cells), lymphocytes, myoblasts, tissue macrophages, dendritic cells, Kupffer cells, lymph node and spleen lymphocytes and reticulocytes, keratinocytes, endothelial cells, and bronchial epithelial cells.

Host cell

Another aspect of the invention provides a host cell transformed or transfected with any of the polynucleotides or cloning/expression constructs of the invention, or a host cell containing any of the polynucleotides or cloning/expression constructs of the invention. The polynucleotides and cloning/expression constructs may be introduced into suitable cells using any method known in the art, including transformation, transfection and transduction. Host cells include cells of an individual undergoing ex vivo cell therapy, including, for example, ex vivo gene therapy. Eukaryotic host cells encompassed by one aspect of the invention, when having a polynucleotide, vector or protein of the invention, in addition to including individual's own cells (e.g., human patient's own cells), VERO cells, HeLa cells (HeLa cells), Chinese Hamster Ovary (CHO) cell lines (including modified CHO cells capable of modifying the glycosylation pattern of expressed multivalent binding molecules, see published U.S. patent application No. 2003/0115614 a1, which is incorporated herein by reference), COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, a549, PC12, K562, HEK293 cells, HepG2 cells, N cells, 3T3 cells, spodoptera frugiperda cells (e.g., Sf9 cells), saccharomyces cerevisiae cells, and any other eukaryotic cell known in the art suitable for expressing and optionally isolating a protein or peptide of the present invention. Prokaryotic cells are also contemplated, including but not limited to Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Salmonella typhimurium (Salmonella typhimurium), streptomyces (Streptomycete), or any prokaryotic cell known in the art suitable for expression and optionally isolation of a protein or peptide of the invention. In isolating proteins or peptides from prokaryotic cells, it is specifically contemplated that techniques known in the art for extracting proteins from inclusion bodies can be used. The selection of an appropriate host is within the skill of one of skill in the art, as is known from the teachings herein.

The engineered host cells may be cultured in conventional media modified as appropriate for activating promoters, selecting transformants, or amplifying specific genes. The culture conditions, such as temperature, pH and the like, of the particular host cell selected for expression will be readily apparent to one of ordinary skill in the art. Various mammalian cell culture systems can also be used to express recombinant proteins. Examples of mammalian expression systems include the COS-7 line of monkey kidney fibroblasts (described by Gluzman, 1981 Cell 23: 175) and other Cell lines capable of expressing compatible vectors, such as C127, 3T3, CHO, HeLa Cell lines, and BHK Cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and optionally an enhancer, as well as any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences and 5' flanking nontranscribed sequences, for example as described herein for the preparation of multivalent binding protein expression constructs. DNA sequences derived from SV40 splicing and polyadenylation sites can be used to provide the required nontranscribed genetic elements. Introduction of the construct into the host cell can be accomplished by a variety of Methods well known to those skilled in the art, including, but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, or electroporation (Davis et al, 1986 Basic Methods in Molecular Biology).

In one embodiment, the host cell is transduced by a recombinant viral construct that directs the expression of a protein or polypeptide of the invention. The transduced host cell produces a viral particle containing an expressed protein or polypeptide that is derived from a portion of the host cell membrane that is incorporated by the viral particle during viral budding.

Pharmaceutical compositions

In certain embodiments, compositions of the invention (such as multivalent binding proteins or compositions comprising polynucleotides encoding such proteins as described herein) are suitable for administration for gene therapy and the like under conditions and for a time sufficient to allow expression of the encoded protein in a host cell in vivo or in vitro. The compositions may be formulated as pharmaceutical compositions for administration according to well known methods. Pharmaceutical compositions typically comprise one or more recombinant expression constructs and/or expression products of such constructs, together with a pharmaceutically acceptable carrier, excipient or diluent. The carrier should be non-toxic to the recipient at the dosages and concentrations employed. For nucleic acid-based formulations or for formulations comprising the expression products of the invention, from about 0.01 μ g to about 100mg per kg body weight can be administered, for example, by intradermal, subcutaneous, intramuscular or intravenous routes or by any route known in the art as appropriate under a given set of circumstances. Preferred dosages are, for example, from about 1. mu.g/kg to about 1mg/kg, particularly preferably from about 5. mu.g/kg to about 200. mu.g/kg.

It will be apparent to those skilled in the art that the frequency and frequency of administration will depend on the host response. Pharmaceutically acceptable carriers suitable for therapeutic use are well known in the Pharmaceutical art and are described, for example, in Remingtons Pharmaceutical Sciences, Mack Publishing Co (a.r. gennaro eds., 1985). For example, sterile saline at physiological pH and phosphate buffered saline may be used. Preservatives, stabilizers, dyes and the like may be provided in the pharmaceutical compositions. For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid may be added as preservatives. As above, on page 1449. In addition, antioxidants and suspending agents may be used. As above. The compounds of the present invention may be used in the form of the free base or as a salt, both forms being considered to be within the scope of the present invention.

The pharmaceutical composition containing one or more nucleic acid constructs of the invention or proteins corresponding to the products encoded by the nucleic acid constructs may be in any form that allows the composition to be administered to a patient. For example, the composition may be in the form of a solid, liquid or gas (aerosol). Typical routes of administration include, but are not limited to, oral, topical, parenteral (e.g., sublingual or buccal), sublingual, rectal, vaginal and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal, intracavernosal, intrathecal, intracanal, intraurethral injection or infusion techniques. Pharmaceutical compositions can be formulated such that the active ingredients contained therein are bioavailable upon administration of the composition to a patient. The composition to be administered to a patient may take the form of one or more dosage units, where, for example, a lozenge may be a single dosage unit and a container of one or more compounds of the invention in aerosol form may have a plurality of dosage units.

For oral administration, excipients and/or binders may be present. Examples are sucrose, kaolin, glycerol, starch dextrin, sodium alginate, carboxymethyl cellulose and ethyl cellulose. Coloring and/or flavoring agents may be present. A coated shell may be used.

The compositions may be in liquid form, such as elixirs, syrups, solutions, emulsions or suspensions. The liquid may be for oral administration or delivered by injection, to name two examples. When intended for oral administration, it is preferred that the composition contains one or more of a sweetener, a preservative, a dye/colorant, and a flavoring agent in addition to one or more of the binding domain-immunoglobulin fusion construct or the expression product. In compositions intended for administration by injection, one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents, and isotonic agents may be included.

Liquid pharmaceutical compositions (whether in solution, suspension form, or other similar form) as used herein may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution (preferably physiological saline), Ringer's solution, isotonic sodium chloride, non-volatile oils (such as synthetic mono-or diglycerides which may be used as a solvent or suspending medium, polyethylene glycols, glycerol, propylene glycol or other solvents); antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate or phosphate, and agents for adjusting tonicity such as sodium chloride or dextrose. The parenteral formulations may be packaged in ampoules, disposable cartridges, or multi-dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. The injectable pharmaceutical composition is preferably sterile.

It may also be desirable to include other components in the formulation, such as delivery vehicles, including but not limited to aluminum salts, water-in-oil emulsions, biodegradable oil vehicles, oil-in-water emulsions, biodegradable microcapsules, and liposomes. Examples of immunostimulatory substances (adjuvants) used in the vehicle include N-acetylmuramyl-L-alanine-D-isoglutamine (MDP), Lipopolysaccharide (LPS), dextran, IL-12, GM-CSF, gamma interferon and IL-15.

Although any suitable carrier known to those of ordinary skill in the art may be used in the pharmaceutical compositions of the present invention, the type of carrier will vary depending on the mode of administration and whether sustained release is desired. For parenteral administration (such as subcutaneous injection), the carrier preferably comprises water, saline, alcohol, fat, wax or buffer. For oral administration, any of the above carriers or solid carriers can be used, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, and magnesium carbonate. Biodegradable microspheres (e.g., polylactic galactoside) may also be used as carriers for the pharmaceutical compositions of the invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109. In this regard, the microspheres are preferably greater than about 25 microns.

The pharmaceutical compositions may also contain diluents (such as buffers), antioxidants (such as ascorbic acid), low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates (e.g., glucose, sucrose or dextrin), chelating agents (e.g., EDTA), glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with non-specific serum albumin are exemplary suitable diluents. Preferably, the product is formulated as a lyophilizate using a suitable excipient solution (e.g., sucrose) as a diluent.

The pharmaceutical compositions of the present invention also include stable proteins and stable liquid pharmaceutical formulations according to techniques known in the art, including those disclosed in published U.S. patent application No. 2006/0008415 a1, which is incorporated herein by reference. The techniques include derivatization of proteins, wherein the proteins comprise a thiol group coupled to N-acetyl-L-cysteine, N-ethyl-maleimide, or cysteine.

As described above, the present invention includes compositions capable of delivering nucleic acid molecules encoding multivalent binding proteins with effector functions. Such compositions include recombinant viral vectors such as retroviruses (see WO 90/07936, WO 91/02805, WO 93/25234, WO 93/25698 and WO 94/03622), adenoviruses (see Berkner, 1988 Biotechniques 6: 616-: 7851). In certain embodiments, the DNA can be ligated to an inactivated or inactivated adenovirus (see Curiel et al, 1992 hum. Gene Ther.3: 147-154; Cotton et al, 1992 Proc. Natl. Acad. Sci. USA 89: 6094). Other suitable compositions include DNA-ligands (see Wu et al, 1989J. biol. chem.264: 16985-.

In addition to guiding in vivo procedures, ex vivo procedures may also be used in which cells are removed from a host (e.g., an individual, such as a human patient), modified, and placed in the same or another host animal. Obviously, in an ex vivo environment, any of the compositions described above may be used to introduce the constructs (proteins/polypeptides or nucleic acids encoding them) of the invention into tissue cells. Protocols for viral, physical and chemical uptake methods are well known in the art.

Formation of antibodies

Polyclonal antibodies against antigenic polypeptides can generally be generated in animals (e.g., rabbits, hamsters, goats, sheep, horses, pigs, rats, gerbils, guinea pigs, mice or any other suitable mammal, as well as other non-mammalian species) by means of multiple subcutaneous or intraperitoneal injections of the antigenic polypeptide or fragment thereof and an adjuvant. Adjuvants include, but are not limited to, Freund's complete or incomplete adjuvant (complete or incomplete Freund's adjuvant), mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, and dinitrophenol. BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum are also potentially useful adjuvants. It may be suitable for conjugating the antigenic polypeptide to a carrier protein that is immunogenic in the species to be immunized; typical carriers include keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor. In addition, aggregating agents such as alum may be used to enhance the immune response. Following immunization, the animals are bled and the serum is assayed for anti-antigen polypeptide antibody titer using conventional techniques. Polyclonal antibodies can be used in serum, detected from serum, or purified from serum using, for example, antigen affinity chromatography.

Monoclonal antibodies directed against the antigenic polypeptides can be prepared by any method that produces antibody molecules by continuous cell lines in culture. For example, monoclonal antibodies can be produced by methods such as Kohler et al, Nature 256: 495[1975] by the hybridoma method; human B-cell hybridoma technology (Kosbor et al, Immunol Today 4: 72, 1983; Cote et al, Proc Natl Acad Sci 80: 2026-.

When hybridoma technology is used, myeloma cell lines may be used. Preferably, cell lines suitable for use in hybridoma-producing fusion procedures do not produce endogenous antibodies, have high fusion efficiency, and exhibit enzyme deficiencies that render them incapable of growing in certain selective media that support the growth of only the desired fused cells (hybridomas). For example, if the immunized animal is a mouse, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 41, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG1.7, and S194/5XX0 Bul; in the case of rat, R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B 210; and U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6 can all be used in conjunction with cell fusion.

In an alternative embodiment, human antibodies can be prepared via phage display libraries (Hoogenboom et al, J.mol.biol.227: 381[1991 ]; Marks et al, J.mol.biol.222: 581; see also U.S. Pat. No. 5,885,793). The method mimics immunoselection via the following steps: all antibodies are displayed on the surface of filamentous phage, and then the phage are selected for their binding to the selected antigen. One such technique is described in PCT application No. PCT/US98/17364 in the name of Adams et al, which describes the use of this method to isolate high affinity and functional agonist antibodies to the MPL-receptor and msk-receptor. In this method, a complete list of human antibody genes can be formed by cloning naturally rearranged human V genes in peripheral blood lymphocytes as described previously (Mullinax et al, Proc. Natl. Acad. Sci. (USA) 87: 8095- > 8099[1990 ]).

Alternatively, an intact fully synthetic human heavy chain can be formed from unrearranged V gene segments by combining individual human VH segments with random nucleotide D segments along with human J segments (Hoogenboom et al, J.mol.biol.227: 381-. Similarly, all light chains can be constructed by merging individual human V segments with J segments (Griffiths et al, EMBO J.13: 3245-3260[1994 ]). The nucleotides encoding the intact antibody (i.e., the heavy and light chains) are linked into a single-chain Fv fragment, and the polynucleotide is ligated to nucleotides encoding the filamentous phage minor coat protein. When the fusion protein is expressed on the surface of a phage, polynucleotides encoding specific antibodies can be identified by using immobilized antigen selection.

In addition to typical methods for forming polyclonal and monoclonal antibodies, any method for forming any known antibody format is also contemplated. In addition to polyclonal and monoclonal antibodies, antibody formats also include chimeric antibodies, humanized antibodies, CDR-grafted antibodies, and antibody fragments and variants.

Variants and derivatives of specific binding agents

In one embodiment, insertion variants are provided in which one or more amino acid residues supplement the specific binding agent amino acid sequence. Insertions may be located at either or both ends of the protein, or may be located in internal regions of the amino acid sequence of the specific binding agent. Variant products of the invention also include mature specific binding agent products, i.e., specific binding agent products in which the leader or signal sequence has been removed and the resulting protein has other amino terminal residues. Other amino-terminal residues may be derived from another protein or may include one or more residues that cannot be identified as being derived from a specific protein. Polypeptides having other methionine residues at position-1 (e.g., Met-1-multivalent binding peptides having effector function) are contemplated, as are polypeptides of the invention having other methionine and lysine residues at positions-2 and-1 (Met-2-Lys-1-multivalent binding proteins having effector function). Variants of the polypeptides of the invention having additional Met, Met-Lys or Lys residues (or one or more basic residues in general) are particularly useful for enhancing the production of recombinant proteins in bacterial host cells.

The invention also includes specific polypeptides of the invention having additional amino acid residues resulting from the use of specific expression systems. For example, the use of a commercially available vector expressing a desired polypeptide as part of a glutathione-S-transferase (GST) fusion product can provide the desired polypeptide with an additional glycine residue at position-1 after cleavage of the GST component with the desired polypeptide. Variants produced by expression in other vector systems are also encompassed, including those in which a histidine tag is incorporated into the amino acid sequence, typically at the carboxy-terminus and/or amino-terminus of the sequence.

In another aspect, the invention provides deletion variants in which one or more amino acid residues in the polypeptide of the invention are removed. Deletions may be effected at one or both termini of the polypeptide, or via removal of one or more residues in the amino acid sequence. Deletion variants necessarily include all fragments of the polypeptides of the invention.

An antibody fragment refers to a polypeptide having a sequence corresponding to at least a portion of an immunoglobulin variable region sequence. Fragments can be formed, for example, by enzymatic or chemical cleavage of the polypeptide corresponding to the full-length antibody. Other binding fragments include those formed by synthetic techniques or by recombinant DNA techniques such as expression of recombinant plasmids containing nucleic acid sequences encoding portions of the antibody variable regions. Preferred polypeptide fragments exhibit immunological properties that are characteristic of or specific for the targets as described herein. Fragments of the invention having the desired immunological properties may be prepared by any method known and routinely practiced in the art.

In yet another aspect, the invention provides substituted variants of multivalent binding polypeptides having effector function. Substitution variants include those polypeptides in which one or more amino acid residues in the amino acid sequence are removed and replaced with a substitute residue. In certain embodiments, the substitutions are conservative in nature; however, the present invention includes substitutions that are also non-conservative. Amino acids can be classified according to their physical properties and effects on secondary and tertiary protein structure. Conservative substitutions are understood in the art as the substitution of one amino acid for another with similar properties. Exemplary conservative substitutions are listed in Table A (see WO 97/09433, page 10, published 3/13 1997 (PCT/GB96/02197, filed 96/6/9)), immediately below.

TABLE A

Alternatively, as listed in table B, the conservative amino acids may be as set forth in Lehninger, [ Biochemistry, second edition; worth Publishers, inc. ny: NY (1975), pages 71-77, immediately below.

Table, B:

conservative substitutions II

Amino acids with characteristic side chains

Non-polar (hydrophobic)

A. Aliphatic: ALIVP

B. Aromatic: FW

C. Sulfur-containing: m

D. Boundary: g

Uncharged-polarity

A. Hydroxyl group: STY

B. Amide: NQ

C. Sulfhydryl groups: c

D. Boundary: g

Positively charged (basic) KRH

Negatively charged (acidic) DE

The invention also provides derivatives of specific binding agent polypeptides. Derivatives include specific binding agent polypeptides with modifications other than insertion, deletion, or substitution of amino acid residues. Preferably, the modifications are of a covalent nature and include, for example, chemical bonding to polymers, lipids, other organic moieties, and inorganic moieties. The derivatives of the invention may be prepared to increase the circulating half-life of the specific binding agent polypeptide, or may be designed to improve the ability of the polypeptide to target a desired cell, tissue or organ.

The invention further includes effector-functional multivalent binding proteins that are covalently modified or derivatized to include one or more water-soluble polymer linkers, such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol, as described in U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, and 4,179,337. Other suitable polymers known in the art include monomethoxypolyethylene glycol, dextran, cellulose and other carbohydrate-based polymers, poly (N-vinylpyrrolidinone) -polyethylene glycol, propylene glycol homopolymers, polyoxypropylene/oxyethylene copolymers, polyoxyethylated polyols (e.g. glycerol) and polyvinyl alcohol, and mixtures of such polymers. Polyethylene glycol (PEG) derivatized proteins are particularly preferred. The water-soluble polymer may be bonded at a specific position, for example to the amino terminus of the proteins and polypeptides of the invention, or randomly attached to one or more side chains of the polypeptide. The use of PEG for improving therapeutic ability is described in U.S. patent No. 6,133,426 to Gonzales et al.

Target for immunoglobulin mutagenesis

Certain strategies are available for manipulating the inherent properties of antigen-specific immunoglobulins (e.g., antibodies) that are ineffective for non-immunoglobulin based binding molecules. A good example of the strategy that supports the advantage of, for example, antibody-based molecules over such alternatives is the modulation of the affinity of an antibody for its target in vivo via affinity maturation, which exploits the somatic hypermutation of immunoglobulin genes to produce antibodies of increasing affinity as the immune response progresses. In addition, recombinant techniques have been developed to alter the structure of immunoglobulins and immunoglobulin regions and domains. Thus, antibody-derived polypeptides can be prepared that exhibit altered affinity for a given antigen, and a variety of purification schemes and monitoring screens for identifying and purifying or isolating such polypeptides are known in the art. Using the known techniques, polypeptides comprising binding domains derived from antibodies can be obtained that show reduced or increased affinity for the antigen. Strategies for generating polypeptide variants exhibiting altered affinity include: site-directed or random mutagenesis is used on the DNA encoding the antibody to alter the amino acids present in the protein, followed by a screening step designed to recover antibody variants exhibiting the desired changes (e.g., increased or decreased affinity relative to the unmodified parent or reference antibody).

The amino acid residues most often targeted for affinity alteration in a mutagenesis strategy are those in the Complementarity Determining Regions (CDRs) or hypervariable regions of the light and heavy chain variable regions of an antibody. The region contains residues that undergo physicochemical interactions with the antigen, as well as other amino acids that affect the spatial arrangement of the residues. However, amino acids in variable domain framework regions outside of the CDR regions have also been shown to have a substantial effect on the antigen binding properties of antibodies, and can be targeted to manipulate such properties. See Hudson, p.j.curr.opin.biotech, 9: 395-402(1999) and references therein.

Smaller and more efficient screening libraries of antibody variants can be prepared by site-limited random or site-directed mutagenesis of the CDRs corresponding to regions that tend to be "hypermutated" during the course of somatic affinity maturation. See Chowdhury et al, Nature biotech, 17: 568-572(1999) and references therein. The types of DNA elements known to define hypermutation sites in this manner include direct and inverted repeats, certain consensus sequences, secondary structures, and palindromic sequences. The consensus DNA sequence includes the four base sequence purine-G-pyrimidine-A/T (i.e., A or G-G-C or T-A or T) and the serine codon AGY (where Y can be C or T).

Thus, another aspect of the invention is a set of mutagenesis strategies for modifying the affinity of an antibody for its target. The strategies include mutagenesis of the entire variable region of the heavy and/or light chain, mutagenesis of only the CDR regions, mutagenesis of consensus hypermutation sites within the CDRs, mutagenesis of framework regions, or any combination of the methods ("mutagenesis" in this context can be random mutagenesis or site-directed mutagenesis). The antibodies and antibodies are addressed via techniques known to those skilled in the art (such as X-ray crystallography): the structure of the ligand complex thus allows for the unambiguous description of the CDR regions and the identification of residues comprising the binding site of the antibody. Various methods based on the analysis and characterization of the antibody crystal structure are known to those skilled in the art and can be used to access the CDR regions. Examples of such common methods include Kabat, Chothia, AbM, and contact definitions.

The Kabat definition is the most common definition based on sequence variability and is predictive of CDR regions. Johnson et al, Nucleic Acids Research, 28: 214-8(2000). The Chothia definition is based on the position of the structural loop regions. (Chothia et al, J.mol.biol., 196: 901-17[1986 ]; Chothia et al, Nature, 342: 877-83[1989 ]). The AbM definition is a compromise between the Kabat and Chothia definitions. AbM is the complete set of programs produced by the Oxford university Molecular group (Oxford Molecular group) for antibody structural modeling (Martin et al, Proc. Natl. Acad. Sci (USA) 86: 9268-. The AbM program group models the tertiary structure of antibodies from the primary sequence using a combination of knowledge databases and the ab initio approach. Another definition, called the contact definition, has recently been introduced. See MacCallum et al, j.mol.biol., 5: 732-45(1996). This definition is based on an analysis of the available composite crystal structure.

By convention, the CDR domains in the heavy chain are commonly referred to as H1, H2, and H3, and are numbered sequentially in the order of amino-terminal to carboxy-terminal movement. The CDR regions in the light chain are commonly referred to as L1, L2, and L3 and are numbered sequentially in the order of movement from the amino terminus to the carboxy terminus.

CDR-H1 is about 10 to 12 residues in length and is generally initiated at the 4 th residue after Cys according to the Chothia and AbM definitions, or at the 5 th residue after Cys according to the Kabat definition. H1 is typically followed by Trp, typically Trp-Val, and may be Trp-Ile or Trp-Ala. According to the AbM definition, H1 is about 10 to 12 residues in length, whereas the Chothia definition does not include the last 4 residues.

CDR-H2 generally begins at residue 15 after the end of H1, according to Kabat and AbM definitions. The residue preceding H2 is typically Leu-Glu-Trp-Ile-Gly, but many variations are possible. H2 is typically followed by the amino acid sequence Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala. H2 is about 16 to 19 residues in length according to the Kabat definition, with the AbM definition predicting lengths of typically 9 to 12 residues.

CDR-H3 typically begins at residue 33 after the end of H2 and typically follows the amino acid sequence Cys-Ala-Arg. H3 is typically followed by the amino acid Gly. H3 ranges from 3 to 25 residues in length.

CDR-L1 typically begins at about residue 24 and typically follows Cys. The residues following CDR-L1 are typically Trp and typically begin with one of the following sequences: Trp-Tyr-Gln, Trp-Leu-Gln, Trp-Phe-Gln or Trp-Tyr-Leu. CDR-L1 is about 10 to 17 residues in length.

CDR-L2 begins at about 16 residues after the end of L1. It is typically after the following residues: Ile-Tyr, Val-Tyr, Ile-Lys or Ile-Phe. CDR-L2 is about 7 residues in length.

CDR-L3 typically begins at residue 33 after the end of L2 and typically after Cys. L3 is generally followed by the amino acid sequence Phe-Gly-XXX-Gly. L3 is about 7 to 11 residues in length.

Various methods for modifying antibodies have been described in the art, including, for example, methods of making humanized antibodies in which the sequence of the humanized immunoglobulin heavy chain variable region framework has 65% to 95% identity to the sequence of the donor immunoglobulin heavy chain variable region framework. Each humanized immunoglobulin chain typically comprises, in addition to the CDRs, amino acids from the framework of the donor immunoglobulin, e.g., capable of interacting with the CDRs to achieve binding affinity, such as one or more amino acids immediately adjacent to the CDRs in the donor immunoglobulin or those within about 3 angstroms (angstrom), as predicted by molecular modeling. The heavy and light chains can each be designed by using any or all of a variety of localization criteria. When combined into a whole antibody, the humanized immunoglobulin is substantially non-immunogenic in humans and retains substantially the same affinity for an antigen (such as an epitope-containing protein or other compound) as the donor immunoglobulin.

In one embodiment, methods of making antibodies and antibody fragments having similar binding specificity as a parent antibody, but enhanced human characteristics, are described. Humanized antibodies are obtained by chain replacement using, for example, phage display technology and a polypeptide comprising the heavy or light chain variable region of a non-human antibody specific for the antigen of interest, which is then combined with all human complementary (light or heavy) chain variable regions. Hybrid pairs specific for the antigen of interest are identified, and the human chains in the selected pair are combined with all human complementary variable domains (heavy or light chains). In another embodiment, the components from the non-human antibody in the CDRs are combined with all of the components from the human antibody in the CDRs. Selecting hybrids from the resulting library of antibody polypeptide dimers, and which can be used in a second humanization replacement step; alternatively, this second step is eliminated if the hybrid already has sufficient human characteristics to be of therapeutic value. Modification methods to enhance human characteristics are known in the art.

Another example is a method of making a humanized antibody as follows: the corresponding human CDR amino acid sequences are replaced by CDR amino acid sequences and/or the corresponding human FR amino acid sequences are replaced by FR amino acid sequences.

Another embodiment provides a method for identifying amino acid residues in an antibody variable domain that can be modified without diminishing the natural affinity of the antigen binding domain while reducing its immunogenicity relative to a heterologous species; and methods of making the modified antibody variable regions suitable for administration to a heterologous species.

Modification of an immunoglobulin (such as an antibody) by any method known in the art may be designed to achieve increased or decreased antigen binding affinity and/or to decrease the immunogenicity of the antibody in the recipient and/or to modulate the extent of effector activity. In one approach, humanized antibodies may be modified to exclude glycosylation sites to enhance the affinity of the antibody for its cognate antigen (Co et al, mol. Immunol.30: 1361. 1367[1993 ]). Techniques such as "reconstitution", "superchimerization" and "veneering/resurfacing" have produced humanized antibodies with greater therapeutic potential. Vaswam et al, Annals of Allergy, Asthma, & Immunol 81: 105 (1998); roguska et al, prot. engineer.9: 895-904(1996)]. See also U.S. patent No. 6,072,035, which describes antibody reconstitution methods. Although the technique reduces antibody immunogenicity by reducing the number of foreign residues, it does not prevent anti-idiotypic and anti-allotypic responses after repeated administration of the antibody. An alternative to the described methods for reducing immunogenicity is described in Gilliland et al, j.immunol.62 (6): 3663-71 (1999).

In many cases, humanized antibodies result in a decrease in antigen binding ability. Thus, it is preferred that the humanized antibody be "back mutated" to include one or more amino acid residues present in the original (most commonly rodent) antibody in an attempt to restore the binding affinity of the antibody. See, e.g., salvdanha et al, mol. immunol. 36: 709-19(1999).

Glycosylation of immunoglobulins has been shown to affect effector function, structural stability and rate of secretion from antibody-producing cells (see Leatherbarrow et al, mol. Immunol.22: 407(1985), incorporated herein by reference). The carbohydrate population that results in the property is typically linked to the constant region of the antibody. For example, at C_H2Glycosylation of IgG at Asn297 of the domain promotes the full capacity of IgG to activate complement-dependent cytolysis (Tao et al, J.Immunol.143: 2595 (1989)). At C_H3Glycosylation of IgM at Asn 402 in the domain, for example, contributes to the proper assembly and cytolytic activity of antibodies (Muraoka et al, j.immunol.142: 695 (1989)). In IgA antibody C_H1And C_H3Removal of the glycosylation sites at positions 162 and 419 in the domain results in intracellular degradation and at least 90% inhibition of secretion (Taylor et al, Wall, mol.cell.biol.8: 4197 (1988)). Thus, the molecules of the invention include mutationally altered immunoglobulins that display altered glycosylation patterns due to, for example, mutation of specific residues in the constant sub-region, thereby altering effector function. See Co et al, mol. immunol.30: 1361-1367(1993), Jacqemon et al, j.thromb.haemost.4: 1047-1055(2006), Schuster et al, Cancer Res.65: 7934-: 831-842(2005), each of which is incorporated herein by reference.

The invention also includes multivalent binding molecules having at least one binding domain that has at least 80%, preferably 90% or 95% or 99% identity in sequence to a known immunoglobulin variable region sequence and has at least one residue that is different from the immunoglobulin variable region, wherein the altered residue increases the glycosylation site, alters the position of one or more glycosylation sites, or preferably removes a glycosylation site relative to the immunoglobulin variable region. In certain embodiments, the change removes an N-linked glycosylation site in the framework of the immunoglobulin variable region or removes an N-linked glycosylation site present in the framework of the immunoglobulin heavy chain variable region within a region ranging from about amino acid residue 65 to about amino acid residue 85 (using the numbering convention of Co et al, J.Immunol.148: 1149, (1992)).

Any method known in the art for preparing multivalent binding molecules that exhibit altered glycosylation patterns relative to an immunoglobulin reference sequence is contemplated. For example, any of a variety of genetic techniques can be used to alter one or more particular residues. Alternatively, the host cell used for production may be engineered to produce an altered glycosylation pattern. For example, one approach known in the art provides altered glycosylation as a bisecting nonfucosylated variant that enhances ADCC. The variant is produced by expression in a host cell containing the oligosaccharide modifying enzyme. Alternatively, the Potelligent technique of BioWa/Kyowa Hakko is contemplated to reduce the fucose content in the glycosylated molecules of the present invention. In one known method, CHO host cells for recombinant immunoglobulin production are provided that modify the glycosylation pattern of the FC region of an immunoglobulin via the production of GDP-fucose. This technique can be used to modify the glycosylation pattern of the constant subregions of the multivalent binding molecules of the invention.

In addition to modifying the binding properties of a binding domain, such as that of an immunoglobulin, and in addition to modifications such as humanization, the invention also includes modulating effector functions, such as those of a constant sub-region, by altering or mutating residues that contribute to the effector function. The modification can be achieved using any technique known in the art, such as Presta et al, biochem. 487-490(2001), which is incorporated herein by reference. Exemplary methods would include using the protocols disclosed by Presta et al to modify specific residues known to affect binding in one or more constant subregions corresponding to FC γ RI, FC γ RII, FC γ RIII, FC α R, and FC ∈ R.

In another approach, the Xencor XmAb technique can be used to engineer constant subregions corresponding to FC domains to enhance cell killing effector function. See Lazar et al, proc.natl.acad.sci. (USA)103 (11): 4005 and 4010(2006), which is incorporated herein by reference. For example, F can be formed using this method_CGamma R specific and binding optimization of constant sub-region, thereby enhancing cell killing effect function.

Preparation of multivalent binding proteins with effector function

A variety of expression vector/host systems can be used that contain and express the multivalent binding proteins of the invention (with effector function). Such systems include, but are not limited to, microorganisms, such as bacteria transformed with recombinant phage, plasmids, cosmids, or other expression vectors; yeast transformed with yeast expression or shuttle vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transfected with viral expression vectors (e.g., cauliflower mosaic virus (CaMV); Tobacco Mosaic Virus (TMV)) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Mammalian cells suitable for recombinant multivalent binding protein production include, but are not limited to: VERO cells, HeLa cells, Chinese Hamster Ovary (CHO) cell lines, COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and HEK293 cells. Exemplary experimental protocols for recombinant expression of multivalent binding proteins are described herein below.

The expression vector may comprise a transcription unit comprising a combination of: (1) one or more genetic elements having a regulatory role in gene expression, such as promoters, enhancers or factor-specific binding sites; (2) a structure or sequence encoding a binding agent that is transcribed into mRNA and translated into protein; and (3) appropriate transcription initiation and termination sequences. The building blocks to be used in yeast or eukaryotic expression systems preferably comprise leader sequences enabling the host cell to secrete the translated protein extracellularly. Alternatively, if the recombinant multivalent binding protein is expressed without a leader or transporter sequence, it may comprise an amino terminal methionine residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide the final multivalent binding protein.

For example, a commercially available expression System (e.g., the Pichia expression System (Invitrogen, San Diego, Calif.)) can be used to recombinantly express the multivalent binding protein in yeast according to the manufacturer's instructions. The system also relies on the pre-pro-alpha sequence to direct secretion, but transcription of the insert is driven by the alcohol oxidase (AOX1) promoter under methanol induction. The secreted multivalent binding peptides can be purified from yeast growth media, for example, by methods used to purify peptides from bacterial and mammalian cell supernatants.

Alternatively, the cDNA encoding the multivalent binding peptide can be cloned into the baculovirus expression vector pVL1393(PharMingen, San Diego, CA). The vector was used to infect spodoptera frugiperda cells in SF9 protein free medium and to prepare recombinant proteins according to the manufacturer's instructions (PharMingen). Multivalent binding proteins can be purified from the culture medium and concentrated using a heparin-sepharose column (Pharmacia, Piscataway, NJ). Insect systems for protein expression, such as the SF9 system, are well known to those skilled in the art. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) may be used as a vector for expressing foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae (Trichoplusia arvae). The multivalent binding peptide coding sequence may be cloned into a non-essential region of the virus (such as the polyhedrin gene) and placed under the control of the polyhedrin promoter. Successful insertion of the multivalent binding peptide will render the polyhedrin gene inactive and produce a recombinant virus lacking coat protein. The recombinant virus can be used to infect Spodoptera frugiperda cells expressing the peptide or Trichoplusia ni larvae (Smith et al, J Virol 46: 584, 1983; Engelhard et al, Proc Nat Acad Sci (USA) 91: 3224-7, 1994).

In another example, a DNA sequence encoding a multivalent binding peptide may be amplified by PCR and cloned into an appropriate vector, such as pGEX-3X (Pharmacia, Piscataway, N.J.). pGEX vectors are designed to prepare fusion proteins encoded by the vector, comprising glutathione-S-transferase (GST), and multivalent binding proteins encoded by DNA fragments inserted into the cloning sites of the vector. PCR primers can be formed that include, for example, appropriate cleavage sites. If the multivalent binding protein fusion moiety is used only to facilitate expression, or is not required to be a linker for the peptide of interest, the recombinant multivalent binding protein fusion may be cleaved from the GST portion of the fusion protein. pGEX-3X/multivalent binding peptide constructs were transformed into E.coli XL-1Blue cells (Stratagene, La Jolla CA), and individual transformants were isolated and cultured. Plasmid DNA from individual transformants is purified and can be partially sequenced using an automated sequencer to demonstrate the presence of the desired multivalent binding protein-encoding nucleic acid insert with the proper orientation.

The fusion multivalent binding protein, which can be prepared as insoluble inclusion bodies in bacteria, can be purified as follows. Collecting the host cells by centrifugation; washing in 0.15M NaCl, 10mM Tris (pH8), 1mM EDTA; and treated with 0.1mg/ml lysozyme (Sigma chemical Co.) for 15 minutes at room temperature. The lysate was clarified by sonication and the cell debris was pelleted by centrifugation at 12,000 times gravity for 10 minutes. The pellet containing the multivalent binding protein fusion may be resuspended in 50mM Tris (pH8) and 10mM Tris In EDTA, layers were separated by 50% glycerol and centrifuged at 6000 times for 30 minutes. The pellet can be resuspended without Mg⁺⁺And Ca⁺⁺In standard Phosphate Buffered Saline (PBS). Multivalent binding protein fusions can be further purified by separating the resuspended pellet in a denaturing SDS polyacrylamide gel (Sambrook et al). The gel was soaked in 0.4M KCl to visualize the proteins, excised and electroeluted in gel electrophoresis buffer lacking SDS. If the GST/multivalent binding peptide fusion protein is prepared in bacteria in the form of a soluble protein, it can be purified using the GST purification module (Pharmacia Biotech).

Preferably, the multivalent binding protein fusion is subjected to digestion to cleave GST from the multivalent binding peptide of the invention. The digestion reaction (20-40. mu.g fusion protein, 20-30 units of human thrombin (4000U/mg (Sigma) in 0.5ml PBS) can be incubated at room temperature for 16 to 48 hours and loaded on a denaturing SDS-PAGE gel to separate the reaction products

KCl to visualize protein bands. The identity of the protein band corresponding to the multivalent binding peptide with the expected molecular weight can be demonstrated by amino acid sequence analysis using an automated sequencer (Applied Biosystems Model 473A, Foster City, Calif.). Alternatively, the identity of the peptide may be confirmed by HPLC and/or mass spectrometry analysis.

Alternatively, the DNA sequence encoding the multivalent binding peptide may be cloned into a plasmid containing the desired promoter and optionally a leader sequence (see, e.g., Better et al, Science, 240: 1041-43, 1988). The sequence of this construct can be verified by automated sequencing. The bacteria may then be subjected to CaCl₂Standard procedures for incubation and heat shock treatment (Sambrook et al) plasmids were transformed into a suitable E.coli strain, such as strain MC 1061. The transformed bacteria may be cultured in LB medium supplemented with carbenicillin (carbenicillin) or other suitable forms of the selection as known in the art, and production of the expressed protein induced by culturing in a suitable medium. If present, the leader sequence may effect the partitioning of the multivalent binding peptideSecreted and cleaved during secretion. The secreted recombinant protein can be purified from the bacterial culture medium by the methods described herein below.

Mammalian host systems for the expression of recombinant proteins are well known to those skilled in the art and are preferred systems. Host cell lines may be selected that have the specific ability to process the expressed protein or to produce certain post-translational modifications that provide for protein activity. Such modifications of polypeptides include, but are not limited to: acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Different host cells (such as CHO, hela, MDCK, 293, WI38 and similar host cells) have cellular and characteristic mechanisms specific for the post-translational activity and can be selected to ensure proper modification and processing of foreign proteins.

The transformed cells are preferably useful for long-term high-yield protein production and thus require stable expression. After transformation of the vector containing the at least one selectable marker and the desired expression cassette, the cells are preferably cultured in an enrichment medium for 1-2 days before being transferred to a selection medium. The selectable marker is designed to confer resistance to the selector and its presence allows cells that successfully express the foreign protein to be grown and recovered. The resistant mass of stably transformed cells may be propagated using tissue culture techniques appropriate for the cells.

Transformed cells can be recovered using a variety of selection systems for the production of recombinant proteins. The selection system includes, but is not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyl transferase, and adenine phosphoribosyl transferase genes located in tk-cells, hgprt-cells, or aprt-cells, respectively. Antimetabolite resistance may also be utilized as a basis for selection of: dhfr which confers resistance to methotrexate; gpt conferring mycophenolic acid resistance; neo conferring aminoglycoside G418 resistance and chlorsulfuron (chlorsulfuron) resistance; and hygro conferring hygromycin resistance. Other selectable genes that may be applicable include trpB, which allows the cell to use indole instead of tryptophan; or hisD, which allows the cell to use histidinol instead of histidine. Markers that provide visual indication for the identification of transformants include anthocyanins, β -glucuronidase and its substrate, GUS, and luciferase and its substrate, luciferin.

Protein purification

Protein purification techniques are well known to those skilled in the art. The techniques involve the crude separation of polypeptide and non-polypeptide components at one level. Separation of the multivalent binding polypeptide from at least one other protein may purify the polypeptide of interest, but typically requires further purification using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods which are particularly suitable for the preparation of pure multivalent binding peptides are ion exchange chromatography, exclusion chromatography, polyacrylamide gel electrophoresis and isoelectric focusing. Particularly effective methods for peptide purification are fast protein liquid chromatography and HPLC.

Certain aspects of the invention relate to the purification and in particular embodiments the substantial purification of the encoded multivalent binding protein or peptide. The term "purified multivalent binding protein or peptide" as used herein is intended to refer to a composition that is separable from other components, wherein the multivalent binding protein or peptide is purified to any degree relative to its naturally available state. Thus, a purified multivalent binding protein or peptide also refers to a multivalent binding protein or peptide that is not present in its naturally occurring environment.

Generally, "purified" refers to a multivalent binding protein composition that has been subjected to separation to remove various other components and which substantially retains the biological activity it expresses. If the term "substantially purified" is used, this name refers to a multivalent binding protein composition, wherein the multivalent binding protein or peptide forms a major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or more than 99% by weight of the protein in the composition.

One of skill in the art will be aware of various methods for quantifying the degree of purification of a multivalent binding protein in light of this disclosure. The methods include, for example, determining the specific binding activity of the active component or assessing the amount of multivalent binding polypeptide in the component by SDS/PAGE analysis. A preferred method for assessing the purity of a multivalent binding protein component is to calculate the binding activity of the component, compare it to the binding activity of the initial extract, and thereby calculate the degree of purification (herein assessed by "fold purification"). The actual unit used to represent the amount of binding activity will of course depend on the particular assay technique chosen for purification, whether or not the expressed multivalent binding protein or peptide exhibits detectable binding activity.

Various techniques suitable for purification of multivalent binding proteins are well known to those skilled in the art. Such techniques include, for example: precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase chromatography, hydroxyapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of the described techniques with other techniques. As is generally known in the art, it is contemplated that the order in which the various purification steps are performed may be varied, or that certain steps may be omitted and still remain as suitable methods for preparing a substantially purified multivalent binding protein.

It is generally not necessary to always provide the multivalent binding protein in the most purified state. Indeed, it is contemplated that multivalent binding proteins that are not substantially purified may be used in certain embodiments. Partial purification can be achieved by using fewer purification steps in combination, or by using different forms of the same conventional purification scheme. For example, it will be appreciated that cation exchange column chromatography using an HPLC apparatus generally results in greater purification than the same technique using a low pressure chromatography system. Methods that exhibit a relatively low degree of purification have advantages in terms of the total amount of multivalent binding protein product recovered or in terms of maintaining the binding activity of the expressed multivalent binding protein.

It is known that migration of polypeptides may vary, sometimes significantly, under different conditions of SDS/PAGE (Capaldi et al, biochem. Biophys. Res. Comm., 76: 425, 1977). It will therefore be appreciated that the apparent molecular weight of a purified or partially purified multivalent binding protein expression product may vary under different electrophoretic conditions.

Effector cell

Effector cells that induce, for example, ADCC, ADCP (antibody-dependent cellular phagocytosis) and the like against target cells include human leukocytes, macrophages, monocytes, activated neutrophils, activated Natural Killer (NK) cells, and eosinophils. Effector cell expression of F_Cα R (CD89), Fc γ RI, Fc γ RII, Fc γ RIII and/or F_Cε R1 and includes, for example, monocytes and activated neutrophils. It has been found that expression of, for example, Fc γ RI can be upregulated by interferon γ (IFN- γ). This enhanced expression may enhance the cytotoxic activity of monocytes and neutrophils against the target cells. Thus, effector cells can be activated by (IFN-. gamma.) or other cytokines (e.g., TNF-. alpha.or.beta., colony stimulating factor, IL-2) to increase the presence of Fc. gamma.RI on the cell surface prior to contact with the multivalent proteins of the present invention.

The multivalent proteins of the present invention provide antibody effector functions, such as antibody-dependent effector cell-mediated cytotoxicity (ADCC), for use in defending against target cells. Multivalent proteins with effector function can be administered alone as taught herein, or after conjugation to effector cells, thereby forming "activated effector cells. An "activated effector cell" is an effector cell as defined herein linked to a multivalent protein having effector function, also as defined herein, so as to effectively provide the effector cell with targeting function prior to administration.

Activated effector cells can be administered in vivo as a suspension of cells in a physiologically acceptable solution. The number of cells administered is about 10⁸-10⁹On the order of magnitude of each, but varies depending on the purpose of the treatment. Generally, the amount will be sufficient to obtain localization of the effector cell at the target cell and to provide a desired degree of effector cell function at this location, such as cell killing by ADCC and/or phagocytosis. The term physiologically acceptable solution as used herein is intended to includeAny carrier solution that is stable to targeted effector cells for in vivo administration includes, for example, saline and buffered aqueous solutions, solvents, antibacterial and antifungal agents, isotonic agents, and the like.

Accordingly, a further aspect of the invention provides a method of inducing specific antibody effector function (such as ADCC) against cells in an individual, the method comprising administering to the individual a multivalent protein (or encoding nucleic acid) in a physiologically acceptable medium or activating effector cells. As is known in the art, the route of administration can vary and the appropriate route of administration will be determined by one of skill in the art based on consideration of case-specific variables and routine procedures.

No cell effect

Cell-free effects are also provided by the multivalent molecules of the invention, for example by providing CDC functionality. The complement system is a biochemical cascade of the immune system that helps to clear foreign objects (such as pathogens) from the body. It is derived from many small plasma proteins that work together to induce lysis of target cells by disrupting their plasma membranes. The complement system consists of more than 35 soluble and cell-binding proteins, 12 of which are directly involved in the complement pathway. The proteins are active in three biochemical pathways leading to activation of the complement system: the classical complement pathway, the alternative complement pathway, and the mannose-binding lectin pathway. Antibodies (particularly IgG1 class antibodies) can also "bind" complement. Such pathways are well understood in the art and are not repeated here, but it is noteworthy that complement-dependent cytotoxicity does not depend on the interaction of binding molecules with cells of the immune system (e.g., B cells). It is also noteworthy that the complement system is regulated by complement regulatory proteins. The protein is present in the blood plasma at a higher concentration than the complement protein. Complement regulatory proteins are present on the surface of self-cells and provide a mechanism to prevent self-cells from being targeted by complement proteins. The complement system is expected to play a role in several diseases with an immune component, such as Barraquer-Simon syndrome, Alzheimer's disease, asthma, lupus erythematosus, various forms of arthritis, autoimmune heart disease, and multiple sclerosis. The lack of terminal pathways predisposes individuals to autoimmune diseases and infectious diseases (particularly meningitis).

Diseases, disorders and conditions

The present invention provides effector function multivalent binding proteins, and variants and derivatives thereof, that bind to one or more binding partners and that are useful for treating, preventing or ameliorating a condition associated with a disease, disorder or pathological condition, preferably a human afflicting disease, disorder or pathological condition. In a preferred embodiment of the method, multivalent (and multispecific) binding proteins with effector functions can bind cells with targets (such as tumor-specific cell surface markers) to effector cells (such as cells exhibiting cytotoxic activity in the immune system). In other embodiments, a multispecific, multivalent binding protein having effector function specifically binds two different disease-specific, disorder-specific, or state-specific cell surface markers to ensure that the correct target binds to an effector cell (such as a cytotoxic cell of the immune system). In addition, multivalent binding proteins with effector functions can be used to induce or enhance antigenic activity or inhibit antigenic activity. Multivalent binding proteins with effector function are also suitable for combination therapy and palliative therapy.

In one aspect, the invention provides compositions and methods useful for treating or preventing diseases and conditions characterized by an abnormal amount of antigen activity associated with cells. The diseases include cancer and other hyperproliferative states such as hyperplasia, psoriasis, contact dermatitis, immunological disorders, and infertility. Various cancers, including solid tumors and leukemias, are susceptible to the compositions and methods disclosed herein. Types of cancers that can be treated include, but are not limited to: adenocarcinoma of the breast, prostate and colon; all forms of bronchopulmonary carcinoma; medullary tumor; melanoma; hepatoma; neuroblastoma; papilloma; ammonia precursor uptake and decarboxylation cytoma (apudoma); a granuloma; gill primary tumor; malignant carcinoid syndrome; carcinoid heart disease; and cancers (e.g., Walker's cancer (Walker), basal cell carcinoma, basal squamous cell carcinoma, buers-sebaceous (Brown-pearl) carcinoma, ductal carcinoma, Ehrlich (Ehrlich) carcinoma, Krebs 2 carcinoma, merkel (merkel) cell carcinoma, mucinous carcinoma, non-small cell lung carcinoma, oat cell carcinoma, papillary carcinoma, solid carcinoma, bronchiolar carcinoma, bronchial carcinoma, squamous cell carcinoma, and metastatic cell carcinoma). Other cancer types that may be treated include: a tissue cell disorder; leukemia; malignant tissue cell proliferation; hodgkin's disease; small immunoproliferative disorders; non-hodgkin's lymphoma; a plasmacytoma; reticuloendotheliosis; melanoma; chondroblastoma; chondroma; chondrosarcoma; fibroids; fibrosarcoma; giant cell tumor; a histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma; myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngioma; clonal cell tumors; hamartoma; interstitial tumors; middle kidney tumor; myosarcoma; amelogblastoma; cementoma; dental tumors; teratoma; thymoma; leaf tumor. Furthermore, the following cancer types susceptible to treatment are also encompassed: adenoma; biliary duct tumors; cholesteatoma; a cylindrical tumor; cystic carcinoma; a cystic tumor; a granulosa cell tumor; amphoterial blastoma; hepatoma; sweat gland adenoma; islet cell tumor of pancreas; leigh (Leydig) cell tumors; papilloma; a sertoli (sertoli) cell tumor; a follicular membrane cell tumor; leiomyoma; leiomyosarcoma; myoblastoma; myoma; myosarcoma; rhabdomyoma; rhabdomyosarcoma; ependymoma; ganglionic neuroma; a glioma; medulloblastoma; meningioma; schwannoma; neuroblastoma; neuroepithelial tumors; neurofibromas; neuroma; paragangliomas; non-chromotropic paragangliomas. Types of cancers that can be treated also include, but are not limited to: angiokeratoma; hyperlymphoproliferative vascular disease with eosinophilia; sclerosing hemangioma; vascular tumor diseases; glomus; vascular endothelioma; hemangioma; vascular endothelial cell tumor; angiosarcoma; lymphangioma; lymphangioleiomyomata; lymphangioleiomyosarcoma; pineal tumor; a carcinosarcoma; chondrosarcoma; phyllocystic sarcoma; fibrosarcoma; angiosarcoma; leiomyosarcoma; leukemic sarcoma; liposarcoma; lymphangioleiomyosarcoma; myosarcoma; myxosarcoma; ovarian cancer; rhabdomyosarcoma; a sarcoma; neoplasma; neurofibroma; and cervical dysplasia. The invention further provides compositions and methods useful for treating other conditions in which cells become immortalized or hyperproliferative due to abnormally high expression of antigens.

Various hyperproliferative disorders susceptible to the compositions and methods of the invention are exemplified by B-cell cancers, including B-cell lymphomas (such as various forms of hodgkin's disease, non-hodgkin's lymphoma (NHL), or central nervous system lymphoma), leukemias (such as Acute Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), hairy cell leukemia, and chronic myoblast leukemia), and myelomas (such as multiple myeloma). Other B cell cancers include small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma/leukemia, B-cell proliferation of tumors of undetermined malignant potential, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorder.

Disorders characterized by the production of autoantibodies are generally considered autoimmune diseases. Autoimmune diseases include, but are not limited to: arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, polychondritis, psoriatic arthritis, psoriasis, dermatitis, polymyositis/dermatomyositis, inclusion body myositis, inflammatory myositis, toxic epidermal necrolysis, systemic scleroderma and sclerosis, acroscleroderma syndrome (CREST syndrome), reactions accompanying inflammatory bowel disease, Crohn's disease, ulcerative colitis, respiratory distress syndrome, Adult Respiratory Distress Syndrome (ARDS), meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, eczema, asthma, conditions involving T-cell infiltration and chronic inflammatory reactions, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, Systemic Lupus Erythematosus (SLE), subacute cutaneous lupus erythematosus, discoid wolfsbane disease, psoriasis, Lupus osteomyelitis, lupus cerebritis, juvenile onset diabetes, multiple sclerosis, allergic encephalomyelitis, neuromyelitis optica, rheumatic fever, Sydenham's chorea, immune reactions associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis (including Wegener's granulomatosis and Churg-schus disease), agranulocytosis, vasculitis (including anaphylactic vasculitis/vasculitis, ANCA and rheumatoid vasculitis), aplastic anemia, Diamond-back anemia, immune hemolytic anemia (including autoimmune hemolytic anemia (AIHA)), pernicious anemia, erythrodysgenesis (PRCA), factor VIII deficiency, sjogren's disease, rheumatoid arthritis, and autoimmune hemolytic anemia (including autoimmune hemolytic anemia (AIHA), Hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte extravasation, Central Nervous System (CNS) inflammatory disorders, multiple organ injury syndrome, myasthenia gravis, antigen-antibody complex mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Behcet's (Behcet) disease, Cashmean's syndrome, Goodpasture's syndrome, Lambda-Lanceos myasthenia gravis syndrome, Renaud's syndrome, Sjorgen's syndrome, Steven-Johnson's syndrome, solid organ transplant rejection, graft-versus-host disease (GVHD), bullous pemphigoid, pemphigus, autoimmune polyendocrine disease, Seronegative spondyloarthropathy, Reiter's disease, generalized myolepsy syndrome, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathy or IgM mediated neuropathy, Idiopathic Thrombocytopenic Purpura (ITP), Thrombotic Thrombocytopenic Purpura (TTP), Henoch-Schonlein purpura, autoimmune thrombocytopenia, autoimmune diseases of the testis and ovary (including autoimmune orchitis and oophoritis), primary thyroid dysfunction; autoimmune endocrine diseases including autoimmune thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Grave's disease, autoimmune polyglandular syndrome (or polyglandular endocrine syndrome), type I diabetes (also known as Insulin Dependent Diabetes Mellitus (IDDM)), and schiehan's syndrome; autoimmune hepatitis, lymphoid interstitial pneumonia (HIV), bronchiolitis obliterans (non-transplant) associated NSIP, Guillain-Barre syndrome, macrovasculitis (including polymyalgia rheumatica and giant cell (Takayasu's)) arteritis, intermediate vasculitis (including Kawasaki's disease and polyarteritis nodosa), polyarteritis nodosa (PAN), ankylosing spondylitis, Berger's disease (IgA nephropathy), rapidly progressive glomerulonephritis, primary biliary cirrhosis, celiac disease (gliosis), cryoglobulinemia with hepatitis, Amyotrophic Lateral Sclerosis (ALS), coronary artery disease, familial mediterranean fever, microscopic polyangiitis, Cogan's syndrome, wiskott-Aldrich syndrome, and obstructive thromboangiitis.

Rheumatoid Arthritis (RA) is a chronic disease characterized by inflammation of the joints, which results in swelling, pain, and loss of function. Patients with long-term RA often show progressive joint damage, deformity, disability and even premature death. In addition to RA, inflammatory diseases, disorders and conditions are generally susceptible to treatment, prevention or amelioration of symptoms associated with inflammatory processes (e.g., fever, pain, swelling, congestion), and the compositions and methods of the invention are useful for treating, preventing or ameliorating abnormal or abnormal inflammatory processes, including RA.

Crohn's disease and related diseases ulcerative colitis are two major diseases belonging to a group of diseases known as Inflammatory Bowel Disease (IBD). Crohn's disease is a chronic condition that causes inflammation of the digestive or Gastrointestinal (GI) tract. Although it may involve any region of the gastrointestinal tract from the mouth to the anus, it most often affects the small intestine and/or colon. In ulcerative colitis, the involvement of the GI is limited to the colon. Crohn's disease is characterized by antibodies against neutrophil antigens, i.e., "perinuclear anti-neutrophil antibodies" (pANCA); and antibodies against Saccharomyces cerevisiae, i.e., "anti-Saccharomyces cerevisiae antibodies" (ASCA). Many ulcerative colitis patients have pANCA antibodies in their blood but no ASCA antibodies; while many crohn's disease patients show the ASCA antibody and do not show the pANCA antibody. One method of assessing Crohn's disease is to use the Crohn's Disease Activity Index (CDAI), which is based on 18 predictor variable scores collected by the physician. CDAI values below 150 and 150 are associated with static disease; values above 150 indicate active disease, and values above 450 are considered to be extremely severe disease [ Best et al, "Development of a crohn's disease activity index.)" Gastroenterology 70: 439-444(1976)]. However, since the beginning of the study, some researchers used a "subjective value" of 200 to 250 as a health score.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease that results from repeated injury to blood vessels in a variety of organs including the kidney, skin and joints. In patients with SLE, inappropriate interaction between T cells and B cells results in the production of autoantibodies that attack the nucleus. It is generally agreed that autoantibodies lead to SLE, and therefore novel therapies that deplete the B cell lineage (the immune system can revert as new B cells are formed from precursor cells) provide the hope of lasting benefit to SLE patients.

Multiple Sclerosis (MS) is also an autoimmune disease. It is characterized by inflammation of the central nervous system and destruction of the myelin sheath, which insulates nerve cell fibers in the brain, spinal cord, and body. Although the cause of MS is not yet known, autoimmune T cells are widely recognized as the causative agent of the pathogenesis of the disease. However, high levels of antibodies are present in the cerebrospinal fluid of MS patients and some theories predict that the B cell response leading to antibody production plays an important role in mediating the disease.

Autoimmune thyroid disease is caused by the production of autoantibodies that stimulate the thyroid gland leading to hyperthyroidism (Gray's disease) or that destroy the thyroid gland leading to hypothyroidism (Hashimoto's thyroiditis). Stimulation of the thyroid gland is due to autoantibodies binding to and activating Thyroid Stimulating Hormone (TSH) receptors. Destruction of the thyroid gland is caused by autoantibodies reacting with other thyroid antigens.

Other diseases, disorders and conditions that may be affected by the benefits provided by the compositions and methods of the invention include the Shetland syndrome, which is an autoimmune disease characterized by the destruction of the body's moisture-producing glands. In addition, Immune Thrombocytopenic Purpura (ITP) is caused by autoantibodies binding to platelets and causing their destruction, and this state is suitable for application of the materials and methods of the present invention. Myasthenia Gravis (MG), a chronic autoimmune neuromuscular disorder that results in weakness in voluntary muscle groups characterized by autoantibodies binding to acetylcholine receptors expressed at neuromuscular junctions, is a disease with symptoms that can be treated using the compositions and methods of the invention, and it is expected that the invention will be beneficial in the treatment and/or prevention of MG. Furthermore, using the compositions and methods of the invention, it is contemplated that Rous (Rous) sarcoma virus infection can be affected by the treatment or amelioration of at least one symptom.

Another aspect of the invention is the use of the materials and methods of the invention to prevent and/or treat any hyperproliferative state of the skin, including psoriasis and contact dermatitis or other hyperproliferative diseases. Psoriasis is characterized by autoimmune inflammation of the skin and is also associated with arthritis (in 30% of cases) and scleroderma, inflammatory bowel disease (including crohn's disease and ulcerative colitis). Patients with psoriasis and contact dermatitis have been shown to have increased antigenic activity within the lesions (Ogoshi et al, J.Inv.Dermatol., 110: 818-23[1998 ]). Multispecific multivalent binding proteins can deliver cytotoxic cells of the immune system directly to cells within, for example, a lesion that expresses high levels of antigen. Multivalent (e.g., multispecific) binding proteins can be administered subcutaneously or to the vicinity of a lesion using any of the various routes of administration described herein, as well as other routes of administration well known to those skilled in the art.

Treatment of Idiopathic Inflammatory Myopathies (IIMs), including Dermatomyositis (DM) and Polymyositis (PM), is also contemplated. Inflammatory myopathies can be classified using a variety of classification schemes. The Miller's classification schema (Miller, Rheum Dis Clin North am.20: 811-826, 1994) identifies 2 Idiopathic Inflammatory Myopathies (IIM): polymyositis (PM) and Dermatomyositis (DM).

Polymyositis and dermatomyositis are chronic, debilitating inflammatory diseases involving muscle and (in the case of DM) skin. The condition is a rare condition, and annual incidence rates of about 5 to 10 per million adults and 0.6 to 3.2 per million children in the United states are reported (Targoff, Curr Probl Dermatol.1991, 3: 131-. Idiopathic inflammatory myopathy is associated with significant morbidity and mortality, and it has been noted that as many as half of the affected adults have suffered severe injury (Gottdiener et al, Am J Cardiol.1978, 41: 1141-49). Miller (Rheum Disclin North am.1994, 20: 811-826 and Arthritis and Allied Conditions, Chapter 75, edited by Koopman and Moreland, Lippincott Williams and Wilkins, 2005) lists five types of criteria for diagnosing IIM, namely, the evaluation of the Idiopathic Inflammatory Myopathy Criteria (IIMC), including signs of muscle weakness, biopsy of muscle degeneration, elevated serum levels of muscle-associated enzymes, electromagnetic triads of myopathy, signs of skin rash in dermatomyositis, and also signs of autoantibodies as a second criterion.

IIM-related factors, including muscle-related enzymes and autoantibodies, include, but are not limited to, Creatine Kinase (CK), lactate dehydrogenase, aldolase, C-reactive protein, aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), and antinuclear autoantibodies (ANA), myositis-specific antibody (MSA), and antibodies to extractable nuclear antigens.

Preferred autoimmune diseases susceptible to the methods of the invention include Crohn's disease, Guillain-Barre syndrome (GBS; also known as acute inflammatory demyelinating polyneuropathy, acute idiopathic polyradical neuritis, acute idiopathic polyneuritis, and Landric (Landry's) upgoing paralysis), lupus erythematosus, multiple sclerosis, myasthenia gravis, optic neuritis, psoriasis, rheumatoid arthritis, hyperthyroidism (e.g., Gray's disease), hypothyroidism (e.g., Hashimoto's disease), Order (Ord's) thyroiditis (thyroiditis similar to Hashimoto's disease), diabetes (type 1), aplastic anemia, Retle's syndrome, autoimmune hepatitis, primary biliary cirrhosis, antiphospholipid antibody syndrome (Marek's), Ocular clonus-myoclonus syndrome (OMS); APS, Temporal arteritis (also known as "giant cell arteritis"), Acute Disseminated Encephalomyelitis (ADEM), Goodpasture's syndrome, Wegener's granulomatosis, celiac disease, pemphigus, canine polyarthritis, warm autoimmune hemolytic anemia. Furthermore, the present invention encompasses methods for treating or ameliorating symptoms associated with: endometriosis, interstitial cystitis, neuromuscular rigidity, scleroderma, leukoderma, vulvitis, Chagas' disease leading to enlarged heart disease (cardiac hypertrophy), sarcoidosis, chronic fatigue syndrome and autonomic dysfunction.

The complement system is believed to play a role in many diseases with an immune component, such as alzheimer's disease, asthma, lupus erythematosus, various forms of arthritis, autoimmune heart disease, and multiple sclerosis, all of which are encompassed by the present invention as diseases, disorders, or conditions susceptible to treatment or conditions that can be improved using the methods of the present invention.

Depending on the specific effector function or functions exhibited by the multivalent single-stranded binding molecule, certain constant subregions are preferred. For example, for complement activation, IgG (IgG1, IgG2, or IgG3) and IgM are preferred; any subtype of IgG is preferred for opsonization and toxin neutralization; for pathogen binding, IgA is preferred; and IgE is preferred for binding by parasites such as helminths.

For example, there are F on human leukocytes which recognize the constant region of IgG antibodies as three different types of Fc γ receptors_CR, which can be distinguished according to structural and functional properties and according to the antigenic structure detected by the CD monoclonal antibody. They are called Fc γ RI, Fc γ RII and Fc γ RIII and are differentially expressed on (overlapping) subsets of leukocytes.

FcgRI (CD64) (a high affinity receptor expressed on monocytes, macrophages, neutrophils, bone marrow precursor cells and dendritic cells) comprises isoforms 1a and 1 b. FcgRI has high affinity for monomeric human IgG1 and IgG 3. Its affinity for IgG4 was about 10-fold lower, while it did not bind IgG 2. FcgRI does not show genetic polymorphism.

Fc γ RII (CD32) (comprising isoforms lla, llb1, llb2, llb3 and llc) is the most widely distributed human Fc γ R type, expressed on most types of blood leukocytes and on Langerhans cells, dendritic cells and platelets. Fc γ RII is a low affinity receptor that binds only aggregated IgG. Only the Fc γ R class is able to bind IgG 2. Fc γ RIIa displays genetic polymorphisms that yield two distinct allotypes: fc γ Rlla-H131 and Fc γ Rlla-R131. This functional polymorphism can be attributed to a single amino acid difference at position 131: histidine (H) or arginine (R) residues, which are critical for IgG binding. Fc γ Rlla binds readily to human IgG and IgG3 and does not appear to bind IgG 4. Fc γ Rlla-H131 has a much higher affinity for complex IgG2 than the Fc γ Rlla-R131 allotype.

Fc γ RIII (CD16) has two isotypes or alleles, both of which are capable of binding IgG1 and IgG 3. Fc γ RIIa with moderate affinity for IgG is expressed on macrophages, monocytes, Natural Killer (NK) cells and T cell subsets. Fc γ RIIIb is a receptor with low affinity for IgG that is selectively expressed on neutrophils. High mobility receptors can work effectively with other membrane receptors. Studies with myeloma IgG dimers have demonstrated that only IgG1 and IgG3 bind Fc γ RIIIb (binding with low affinity), while no binding of IgG2 and IgG4 was found. Fc γ RIIIb has a co-dominant biallelic polymorphism, and the allotypes are designated NA1 (neutrophil antigen) and NA 2.

Yet another aspect of the invention is the use of the materials and methods of the invention to combat infections caused by any of a variety of infectious agents by treating, preventing or reducing the effects of the infection. The multivalent multispecific binding molecules of the present invention are designed to efficiently and effectively recruit the immune system of a host organism to fight an infection from a foreign organism, cell, virus, or inanimate foreign object. For example, a multispecific binding molecule may have one binding domain that specifically binds a target on an infectious agent and another binding domain that specifically binds a target on an antigen presenting cell (such as CD40, CD80, CD86, DC-SIGN, DEC-205, CD83, and the like). Alternatively, each binding domain of the multivalent binding molecule can specifically bind to the infectious agent, thereby neutralizing the infectious agent more effectively. Furthermore, the invention encompasses multispecific multivalent binding molecules that specifically bind to a target on an infectious agent and bind to a non-cell related binding partner, which multispecific multivalent binding molecules can bind to effector functions of the multispecific binding molecules to effectively treat or prevent infection by the infectious agent.

Infectious cells encompassed by the present invention include any known infectious cells, including, but not limited to, any of a variety of bacteria (e.g., pathogenic escherichia coli (e), salmonella typhimurium(s), pseudomonas aeruginosa (p. aeruginosa), bacillus anthracis (b. antrhacis), clostridium botulinum (c. botulium), clostridium refractory (c. difficile), clostridium perfringens (c. perfringens), helicobacter pylori (h. pylori), vibrio cholerae (v. cholerae) and the like), mycobacteria, mycoplasma, fungi (including yeasts and molds), and parasites (including Protozoa (Protozoa), Trematoda (Trematoda), Cestoda (Cestoda), and any known parasitic member of the Nematoda class (Nematoda)). Infectious viruses include, but are not limited to, eukaryotic viruses (e.g., adenovirus (adenovirus), bunyavirus (bunyavirus), herpesvirus (herpesvirus), papovavirus (papovavirus), paramyxovirus (paramyxovirus), picornavirus (picornavirus), poxvirus (poxvirus), reovirus (reovirus), retrovirus (retrovirus), and the like), and bacteriophages. Foreign objects include objects that enter an organism (preferably a human being) regardless of the manner of entry and regardless of whether there is an intentional injury. In view of the increasing prevalence of multi-drug resistant infectious agents (e.g., bacteria), particularly nosocomial infectious pathogens, the materials and methods of the present invention can provide a treatment to avoid the difficulties caused by the increased antibiotic resistance.

Diseases, conditions or disorders associated with infection and susceptible to treatment (prophylactic or therapeutic) by the substances and methods disclosed herein include, but are not limited to, anthrax, aspergillosis, bacterial meningitis, bacterial pneumonia (e.g., chlamydia pneumoniae), blastomycosis, botulism, brucellosis (brucellosis), candidiasis (candidiasis), cholera, cerebral cryptococcosis, diarrheal, enterohemorrhagic, or enterotoxigenic escherichia coli, diphtheria, melilotosis, histoplasmosis, wood louse, leprosy, listeriosis (listeriosis), nocardiosis (nocardiosis), pertussis, salmonellosis (salmonellosis), scarlet fever, sporotrichosis, streptococcal laryngitis, toxic shock syndrome, travelers, and typhoid fever.

Other aspects and details of the present invention will be apparent from the following examples, which are intended to be illustrative and not limiting. Example 1 describes recombinant cloning of immunoglobulin heavy and light chain variable regions. Example 2 describes the construction of small modular immunopharmaceuticals. Example 3 describes the construction of prototype cassettes for multivalent binding proteins with effector function. Example 4 describes the study of binding and expression of this initial prototype molecule. Example 5 describes the construction of an alternative construct derived from this initial prototype molecule in which the sequence of the linker region between the EFD and BD2 has been altered in length and sequence. Furthermore, an alternative form is described in which the orientation of the V region in binding domain 2 is also changed. Example 6 describes subsequent binding and functional studies of the alternative constructs with variant linker forms, identifying cleavage of the linker region in several of the derived forms; and novel sequence variants developed to address this problem. Example 7 describes the construction of a preferred alternative embodiment of a multispecific multivalent fusion protein in which both BD1 and BD2 bind antigens on the same cell type (CD20 and CD37) or another multispecific fusion protein in which the antigen with binding specificity for BD2 has been changed to human CD3 instead of CD 28. Example 8 describes binding and functional studies with a CD20-hIgG-CD37 multispecific construct. Example 9 describes binding and functional studies with a CD20-hIgG-CD3 multivalent fusion protein construct. Example 10 discloses multivalent binding molecules with linkers based on specific regions of the extracellular domain of a member of the immunoglobulin superfamily. Example 11 discloses assays that identify binding domains that are expected to be effective in multivalent binding molecules to achieve at least one beneficial effect identified as being associated with the molecule (e.g., treatment of a disease).

Example 1

Cloning of immunoglobulin heavy and light chain variable regions

Antibodies to a given antigen target may be induced using any method known in the art. In addition, the immunoglobulin light and/or heavy chain variable regions, as well as one or more antibody constant subregions, can be cloned using any method known in the art. The following methods provide exemplary cloning methods.

A. Isolation of Total RNA

To clone the immunoglobulin heavy and light chain variable or constant sub-regions, total RNA is isolated from hybridoma cells that secrete the appropriate antibodies. Cells obtained from hybridoma cell line (2X 10)⁷) Washed 1 times with PBS and passed through a 12X 75mm round bottom polypropylene tube (Falcon No. 2059)The precipitate was centrifuged. Add TRIzol to each tube^TMTotal RNA isolation reagents (GibcoBRL, Life Technologies, Cat. No. 15596-018) (8ml) and cells were lysed by repeated pipetting. The lysate was incubated for 5 minutes at room temperature, after which 1.6ml (0.2 volume) of chloroform was added and shaken vigorously for 15 seconds. After standing at room temperature for 3 minutes, the lysate was centrifuged at 9,000rpm for 15 minutes in a Beckman JA-17 spinner pre-cooled at 4 ℃ to separate the aqueous phase from the organic phase. The upper aqueous phase (about 4.8ml) was transferred to a new tube and gently mixed with 4ml isopropanol. After incubation at room temperature for 10 minutes, RNA was precipitated by centrifugation at 9,000rpm for 11 minutes in a JA-17 spinner at 4 ℃, the RNA precipitate was washed with 8ml of ice-cold 75% ethanol, and re-precipitated by centrifugation at 7,000 × rpm for 7 minutes in the JA-17 spinner at 4 ℃. The ethanol wash was decanted and the RNA pellet air dried for 10 minutes. The RNA pellet was resuspended in 150. mu.l of Diethylpyrocarbonate (DEPC) -treated ddH ₂In O, every 1ml of DEPC-treated ddH₂O contained 1. mu.l of RNase inhibitor (catalog No. 799017; Boehringer Mannheim/Roche). The pellet was resuspended by gentle aspiration and incubated at 55 ℃ for 20 minutes. By measuring the OD of a diluted aliquot_260nm(1.0OD_260nmUnit 40 μ g/ml RNA) to quantify the RNA samples.

Rapid amplification of cDNA Ends

5' RACE was performed to amplify the ends of the heavy and light chain variable or constant sub-regions. The 5 'RACE System, version 2.0 of the Rapid amplification cDNA Ends kit (Life technologies, Cat. No. 18374-058) was used according to the manufacturer's instructions. Degenerate 5' RACE oligonucleotide primers were designed to match the constant regions of, for example, two types of commonly used mouse immunoglobulin heavy chains (IgG1 and IgG2b) using the oligonucleotide design program Oligo version 5.1 (Molecular Biology instruments, Cascade CO). Primers were also designed to match the constant region of mouse IgG kappa light chain. This is the only class of immunoglobulin light chain and thus no degeneracy in primer design is required. The sequences of the primers are as follows:

the name sequence SEQ ID NO.

Heavy chain GSP1

5′AGGTGCTGGAGGGGACAGTCACTGAGCTGC3′ 7

Nested heavy chain

5′GTCACWGTCACTGRCTCAGGGAARTAGC3′ 8

(W ═ A or T; R ═ A or G)

Light chain GSP1

5′GGGTGCTGCTCATGCTGTAGGTGCTGTCTTTGC3′ 9

Nested light chain 5' CAAGAAGCACACGACTG

AGGCACCTCCAGATG3′ 10

5' Race simplified anchor primer

5′GGCCACGCGTCGACTAGTACGG

GNNGGGNNGGGNNG3′ 11

To amplify the mouse immunoglobulin heavy chain component, a reverse transcriptase reaction was performed in a 0.2ml thin-walled PCR tube containing 2.5 picomoles (pmole) of heavy chain GSP1 primer (SEQ ID NO: 7), 4. mu.g of total RNA isolated from a suitable hybridoma clone (e.g., clone 4A5 or clone 4B5), and 12. mu.l of DEPC-treated ddH₂And O. Similarly, for the mouse light chain component, the reverse transcriptase reaction was performed in a 0.2ml thin-walled PCR tube containing 2.5 picomoles of light chain GSP1 primer (SEQ ID NO: 9), 4. mu.g of total RNA obtained from the appropriate hybridoma clone (e.g., clone 4A5 or clone 4B5), and 12. mu.l of DEPC-treated ddH₂O。

The reaction was carried out in a PTC-100 programmable thermal cycler (MJ research Inc., Waltham, Mass.). The mixture was incubated at 70 ℃ for 10 minutes to denature the RNA and then chilled on wet iceAnd then cooled for 1 minute. The tube was briefly centrifuged to collect water from the tube cap. Subsequently, the following components were added to the reaction: 2.5. mu.l of 10 XPCR buffer (200mM Tris-HCl (pH8.4), 500mM KCl), 2.5. mu.l of 25mM MgCl₂1. mu.l of 10mM dNTP mix and 2.5. mu.l of 0.1M DTT. After mixing the tubes by gentle pipetting, the tubes were placed in a PTC-100 thermocycler at 42 ℃ for 1 minute to pre-warm the mixture. Subsequently, 1. mu.l (200 units) of SuperScript was added ^TMII reverse transcriptase (Gibco-BRL; catalog No. 18089-011) was added to each tube, gently mixed by pipetting, and incubated at 42 ℃ for 45 minutes. The reaction was circulated to 70 ℃ for 15 minutes to terminate the reaction, and then to 37 ℃. Next, a ribonuclease mixture (1. mu.l) was added to each reaction tube, gently mixed, and incubated at 37 ℃ for 30 minutes.

First strand cDNA produced by the reverse transcriptase reaction was purified using a GlassMAX DNA isolation spin cartridge (Gibco-BRL) according to the manufacturer's instructions. To each first strand reaction was added 120. mu.l of 6M NaI binding solution. The cDNA/NaI solution was then transferred to a GlassMAX spin cartridge and centrifuged at 13,000 times gravity for 20 seconds. The cartridge insert is carefully removed and the flow portion in the tube is discarded. The spin cartridges were then placed back into the empty tube and 0.4ml of cold (4 ℃)1 × wash buffer was added to each spin cartridge. The tube was centrifuged at 13,000 times gravity for 20 seconds and the flow portion discarded. This washing step was repeated three more times. The GlassMAX cartridge was then washed 4 times with 0.4ml of cold (4 ℃) 70% ethanol. After discarding the flow portion of the last 70% ethanol wash, the cartridge was placed back into the tube and centrifuged at 13,000 times gravity for an additional 1 minute to completely dry the cartridge. The spin cartridge insert was then transferred to a fresh sample recovery tube, where 50 μ Ι of 65 ℃ (preheated) DEPC-treated ddH was added ₂O was added rapidly to each rotary filter cartridge. The cartridge was centrifuged at 13,000 times gravity for 30 seconds to elute the cDNA.

C. Terminal deoxynucleotidyl transferase (TdT) tailing

For each first strand cDNA sample, the following components were added to a 0.2ml thin-walled PCR tube:6.5 μ l DEPC treated ddH₂O, 5.0. mu.l of 5 Xtailing buffer, 2.5. mu.l of 2mM dCTP and 10. mu.l of the appropriate cDNA sample purified with GlassMAX. Each 24. mu.l reaction was incubated in a thermocycler at 94 ℃ for 2-3 minutes to denature the DNA and cooled on wet ice for 1 minute. The contents of the collection tube were collected by simple centrifugation. Subsequently, 1. mu.l of terminal deoxynucleotidyl transferase (TdT) was added to each tube. The tubes were mixed via gentle pipetting and incubated in a PTC-100 thermocycler at 37 ℃ for 10 minutes. After this 10 minute incubation, TdT was heat inactivated by cycling to 65 ℃ for 10 minutes. The reaction was cooled on ice and the TdT-tailed first strand cDNA was stored at-20 ℃.

D. PCR of dC-tailed first Strand cDNA

Double-repeat PCR amplification (two independent PCR reactions for each dC-tailed first strand cDNA sample) was performed in a 50. mu.l volume containing: 200. mu.M dNTPs, 0.4. mu.M of a 5' RACE simplified anchor primer (SEQ ID NO: 11) and either 0.4. mu.M of nested heavy chain GSP2(SEQ ID NO: 8) or nested light chain GSP2(SEQ ID NO: 10), 10mM Tris-HCl (pH8.3), 1.5mM MgCl ₂50mM KCl, 5. mu.l of dC-tailed cDNA, and 5 units of Expand^TMHi-Fi DNA polymerase (Roche/Boehringer Mannheim GmbH, Germany). PCR reactions were amplified in a PTC-100 programmable thermal cycler (MJ Research Inc.) using a "step-down/step-up" annealing temperature protocol under the following conditions: initial 95 ℃ denaturation for 40 seconds, 5 cycles as follows: 20 seconds at 94 ℃, 20 seconds at 61-2 ℃/cycle, 40 seconds at 72 ℃ plus 1 second/cycle; the following 5 cycles follow: 25 seconds at 94 ℃, 20 seconds at 53 ℃ plus 1 ℃/cycle, 46 seconds at 72 ℃ plus 1 second/cycle; the following 20 cycles follow: 25 seconds at 94 ℃, 20 seconds at 55 ℃, 51 seconds at 72 ℃ and 1 second/cycle; and finally incubation at 72 ℃ for 5 minutes.

TOPO TA-cloning

The resulting PCR products were gel-purified on a 1.0% agarose gel using a qiaguick gel purification system (QIAGEN inc., Chatsworth, CA) and TOPO TA was usedKit (Invitrogen, San Diego, CA, catalog No. K4550-40) its TA-clones into pcr2.1 and transformed into e.coli TOP10F 'cells (Invitrogen) according to the manufacturer's instructions. Clones with inserts were identified by blue/white screening according to the manufacturer's instructions, where white clones were considered positive clones. 3.5ml of a culture of liquid Luria Broth (LB) containing 50. mu.g/ml ampicillin were inoculated with white colonies and incubated overnight (about 16 hours) at 37 ℃ with shaking at 225 rpm.

Plasmid DNA in the culture was purified using a QIAGEN Plasmid miniprep kit (QIAGEN Plasmid miniprep kit, QIAGEN inc., catalog No. 12125) according to the manufacturer's instructions. Plasmid DNA was suspended in 34. mu.l of 1 × TE buffer (pH8.0) and then positive clones were sequenced by fluorescent dideoxynucleotide sequencing and automated detection as before using ABI Big Dye Terminator 3.1 reagent at dilutions between 1:4 and 1:8 and analyzed using ABI 3100 DNA sequencer. The sequencing primers used included T7(5 'GTAATACGACTCACTATAGG 3'; SEQ ID NO: 12) and M13 reverse (5 'CAGGAAACAGCTATGACC 3'; SEQ ID NO: 13) primers. Sequencing results will demonstrate that the clones correspond to mouse IgG sequences.

F. De novo gene synthesis using overlapping oligonucleotide extension PCR

The method involves the synthesis of immunoglobulin V-regions or other genes using overlapping oligonucleotide primers and PCR, using a high fidelity DNA polymerase or mixture of polymerases. Starting in the middle of the V-region sequence, 40 to 50 base primers are designed to amplify the growing strand 20 to 30 bases in either direction, with a minimum of 20 bases overlapping with the adjacent primers. Each PCR step requires two primers, one priming the antisense strand (forward primer or sense primer) and one priming the sense strand (reverse primer or antisense primer), to form a growing double-stranded PCR product. During primer design, the nucleotide sequence of the final product may be altered to form restriction enzyme sites, disrupt existing restriction enzyme sites, add flexible linkers, alter, delete or insert bases that alter the amino acid sequence, optimize the overall DNA sequence to enhance primer synthesis and to comply with the codon usage rules of the organism intended for expression of the synthetic gene.

Primers were paired and diluted so that the first pair was 5 μ M, after which each pair had a concentration of 2-fold or more, up to 80 μ M. mu.L of the sample from each of the primer mixtures was amplified in a 50. mu.L PCR reaction using Platinum PCR Supermix-High Fidelity (Invitrogen, San Diego, Calif., Cat. No. 12532-016). After initial denaturation at 94 ℃ for 2 min, 30 cycles of PCR were performed using the following cycling protocol: at 94 ℃ for 20 seconds, at 60 ℃ for 10 seconds and at 68 ℃ for 15 seconds. The PCR products were purified using Qiaquick PCR purification columns (qiagen inc., catalog No. 28704) to remove excess primers and enzymes. The PCR product was then re-amplified with the next similarly diluted primer pair using the PCR conditions as described exactly above (except that the time to 68 ℃ for each cycle was extended to 30 seconds). The resulting PCR product was purified again with primers and enzymes as described above, cloned with TOPO-TA and sequenced as described exactly in section E above.

Example 2

Construction of Small Module Immunopharmaceuticals (SMIPs)

A multi-specific multivalent binding protein with effector function containing binding domain 1 was constructed as a single-chain recombinant (murine/human) scFv and named 2H7 (VL-linker-VH). scFv2H7 is a Small Modular Immunopharmaceutical (SMIP) that specifically recognizes CD 20. The binding domain is based on a publicly available human CD20 antibody sequence with GenBank accession number M17953 for VH and M17954 for VL. CD 20-specific SMIPs are described in commonly owned U.S. patent publications 2003/133939, 2003/0118592 and 2005/0136049, which are incorporated herein by reference in their entirety. The peptide linker separating VL and VH is a 15 amino acid linker encoding the following sequence: Asp-Gly ₃Ser-(Gly₄Ser)₂. Binding Domain 1 is located at the N-terminus of the multispecific binding proteinThe C-terminus is directly linked to the N-terminus of a constant sub-region comprising a hinge, C_H2Domains and C_H3Domain (amino to carboxyl orientation). The constant sub-region was derived from an IgG1 antibody, which was isolated by PCR amplification of human IgG1 from human PBMC. The hinge region is modified by: the three Ser residues were substituted for the three Cys residues present in the hinge domain of wild-type human IgG1, which hinge region was defined by the 15 amino acid sequence: EPKSCDKTHTCPPCP (SEQ ID NO: 14; the three Cys residues substituted by Ser residues are indicated in bold). In alternative embodiments, the hinge region is modified at one or more cysteines so as to form SSS and CSC-type hinges. Furthermore, proline is sometimes finally substituted by serine, as well as cysteine.

C_H3The C-terminus of the domain is covalently linked to a series of alternative linker domains juxtaposed between the C-terminus of the constant sub-region and the amino-terminus of the binding domain 2. Depending on the folding properties of BD2, a preferred multivalent binding protein with effector function will have one of the linkers such that the constant subdomain is spaced from binding domain 2, although this linker is not an essential component of the composition of the invention. For certain specific multivalent molecules, the linker may be important for domain isolation, while for others, the linker may be of minor importance. This linker was linked to the N-terminus of scFv 2E12 which specifically recognizes CD28 (V) _Hlinker-V_L). The linker separating the VH domain from the VL domain of the scFv 2E12 portion of the multivalent binding molecule is a 20 amino acid linker (Gly₄Ser)₄Rather than the standard (Gy) which is usually inserted between the V domains of scFv₄Ser)₃A linker. It was observed that longer linkers may improve the binding properties of 2e12 scFv in VH-VL orientation.

The constructed multispecific multivalent binding molecule contains a binding domain 1, which comprises: from SEQ ID NO: 171 from amino acids 1-23 of the 2E12 leader peptide sequence; shown in SEQ ID NO: 171 the 2H7 murine anti human CD20 light chain variable region at position 24; starting from SEQ ID NO: 171 residue ofAsp-Gly of group 130₃-Ser-(Gly₄Ser)₂A linker; 2H7 murine anti-human CD20 heavy chain variable region wherein the amino acid at residue 11 of the variable domain of VH is replaced by leucine to serine (VHL11S) and the binding molecule has a single serine residue (i.e. VTVS wherein the canonical sequence is VTVSs) at the end of the heavy chain region (Genbank accession number M17953), and inserted between the two binding domains BD1(2H7) and BD2(2E12) is a human IgG1 constant sub-region comprising a modified hinge region comprising a "CSC" or "SSS" sequence, and a wild-type C1 constant sub-region_H2And C_H3A domain. The nucleotide and amino acid sequences of multivalent binding proteins with effector function are represented in SEQ ID NOs: 228 and 229, and for the SSS form in SEQ ID NOs: 170 and 171.

Stably expressing cell lines were formed by: the uncut or linearized recombinant expression plasmid was transfected into chinese hamster ovary cells (CHO DG44 cells) via electroporation, followed by selection in methotrexate-containing medium. The bulk culture and master wells that produce the highest amounts of multivalent binding protein were expanded in increasing amounts of methotrexate, and adapted cultures were subsequently cloned by limiting dilution. Transfected CHO cells producing multivalent binding protein were cultured in a bioreactor or corrugated bag using serum-free medium (Excell 302, catalog # 14324-. Other serum-free CHO basal media, such as CD-CHO and its analogs, can also be used for preparation.

The fusion protein was purified from the supernatant of the consumed CHO culture by protein a affinity chromatography. The multivalent binding protein is purified using a series of chromatography and filtration steps, including a viral reduction filter. The cell culture supernatant was filtered and then subjected to protein a sepharose affinity chromatography on a GE Healthcare XK 16/40 column. After the protein binding column, the column was washed with dPBS, then 1.0M NaCl, 20mM sodium phosphate (ph6.0), and then 25mN NaOAc (ph5.0) to remove non-specifically bound proteins. The bound protein was eluted from the column with 100mM glycine (Sigma) (pH3.5) and brought to pH5.0 with 0.5M2- (N-morpholino) ethanesulfonic acid (MES) (pH 6.0). Protein samples were concentrated to 25mg/mL for GPC purification. Size exclusion chromatography was performed on a GE Healthcare AKTA Explorer 100Air apparatus using a GE Healthcare XK column and Superdex 200 preparative grade (GE Healthcare).

The material was then concentrated and formulated with 20mM sodium phosphate and 240mM sucrose, resulting in a pH of 6.0. The composition was filtered and then filled into sterile vials at various concentrations depending on the amount of material recovered.

Example 3

Construction of Scorpion-shaped molecular expression cassette

A nucleic acid containing synthetic 2H7 scFv (anti-CD 20; SEQ ID NO: 1) linked to a constant sub-region as described in example 2 was designated TRU-015. TRU-015 nucleic acids, as well as synthetic scFv 2E12 (anti-CD 28 VL-VH; SEQ ID NO: 3) and synthetic scFv 2E12 (anti-CD 28 VH-VL; SEQ ID NO: 5) nucleic acids encoding small modular immunopharmaceuticals were used as templates for PCR amplification of various components of the scorpion cassette. The template or backbone of binding domain 1 and constant sub-region is provided by TRU-015 (a nucleic acid encoding scFv 2H7 (anti-CD 20) linked to a constant sub-region), and this template is constructed in expression vector pD 18. The above-mentioned polymer having two orientations (V)_L-V_HAnd V_H-V_L) The nucleic acid of scFv 2E12 in either orientation provides a coding region for binding domain 2.

TRU 015 SSS hinge C for BD 2/connector insert_H2C_H3

The scorpion box was formed using synthetic 2H7 scFv IgG1 type containing SSS hinge by acting as a template to add EcoRI site to replace existing stop codon and XbaI site. This molecule was amplified by PCR using primer 9(SEQ ID NO: 23; see Table 1) and primer 87(SEQ ID NO: 40; see Table 1) and Platinum PCR Hi-Fi cocktail (Invitrogen). The resulting 1.5Kbp fragment was purified and cloned into the vector pCR2.1-TOPO (Invitrogen) and transformed into the E.coli strain TOP10(Invitrogen) to verify the DNA sequence.

TABLE 1

SEQ

Number name sequence 5 '-3' ID NO

PCR primer

GCGATAAAGCTTGCCGCCATGGAA

1 hVK3L-F3H3 GCACCAGCGCAGCTTCTCTTCC 15

ACCAGCGCAGCTTCTCTTCCTCCTG

2 hVK3L-F2 CTACTCTGGCTCCCAGATACCACCG 16

GGCTCCCAGATACCACCGGTCAAAT

3 hVK3L-F1-2H7VL TGTTCTCTCCCAGTCTCCAG 17

GCGATAGCTAGCCAGGCTTATCTAC

4 2H7VH-NheF AGCAGTCTGG 18

GCGATAGCTAGCCCCACCTCCTCCA

5 G4S-NheR GATCCACCACCGCCCGAG 19

GCGTACTCGAGGAGACGGTGACCGT

6 015VH-XhoR GGTCCCTGTG 20

GCAGTCTCGAGCGAGCCCAAATCTTG

7 G1H-C-XHO TGACAAAACTC 21

GCAGTCTCGAGCGAGCCCAAATCTTC

8 G1H-S-XHO TGACAAAACTC 22

GCGTGAGAATTCTTACCCGGAGACAGG

9 CH3R-EcoR1 GAGAGGCTC 23

GCGACGTCTAGAGTCATTTACCCGGAG

10 G1-XBA-R ACAGG 24

AATTATGGTGGCGGTGGCTCGGGCGGT

11 G4SLinkR1-S GGTGGATCTGGAGGAGGTGGGAGTGGG 25

AATTCCCACTCCCACCTCCTCCAGATCCA

12 G4SLinkR1-AS CCACCGCCCGAGCCACCGCCACCAT 26

GCGTGTCTAGATTAACGTTTGATTTCCAG

13 2E12VLXbaR CTTGGTG 27

GCGATGAATTCTGACATTGTGCTCACCCA

14 2E12VLR1F ATCTCC 28

GCGATGAATTCTCAGGTGCAGCTGAAGGA

15 2E12VHR1F GTCAG 29

GCGAGTCTAGATTAAGAGGAGACGGTGAC

16 2E12VHXbaR TGAGGTTC 30

17 2e12VHdXbaF1 GGGTCTGGAGTGGCTGGGAATGATATG 31

18 2e12VHdXbaR1 ATTCCCAGCCACTCCAGACCCTTTCCTG 32

19 IgBsrG1F GAGAACCACAGGTGTACACCCTG 33

20 IgBsrG1R GCAGGGTGTACACCTGTGGTTCTCG 34

SEQ

Number name sequence 5 '-3' ID NO

Sequencing primer

82 M13R CAGGAAACAGCTATGAC 35

83 M13F GTAAAACGACGGCCAGTG 36

84 T7 GTAATACGACTCACTATAGG 37

85 pD18F-17 AACTAGAGAACCCACTG 38

86 pD18F-20 GCTAACTAGAGAACCCACTG 39

87 pD18F-1 ATACGACTCACTATAGGG 40

88 pD18R-s GCTCTAGCATTTAGGTGAC 41

89 CH3seqF1 CATGAGGCTCTGCACAAC 42

90C H3seqF2 CCTCTACAGCAAGCTCAC 43

91 CH3seqR1 GGTTCTTGGTCAGCTCATC 44

92 CH3seqR2 GTGAGCTTGCTGTAGAGG 45

Table 1: oligonucleotide primers were used to construct the CD20-CD28 scorpion cassette. The primers were divided into 2 groups: PCR group and sequencing group. PCR primers were used to construct the cassette and sequencing primers were DNA sequences used to demonstrate all intermediate and final constructs.

n2H7 V_KAnd human V_K3Leader sequence fusions

Using primers 3 and 5 in table 1, an agei (acccggt) restriction site was introduced 5 'to the coding region of TRU 015 VK and a Nhe I (GCTAGC) restriction site was introduced (G4S) 3' to the coding region of the 3 linker using oligonucleotide-directed PCR mutagenesis. Since primer 3 also encodes the last 6 amino acids of the human VK3 leader (gb: X01668), the N-terminal sequence of the leader (including the consensus Kozak box and HinDIII (AAGCTT) restriction sites) was added sequentially by overlap PCR using primers 1, 2 and 5 in Table 1.

n2H7 IgG1 SSS hinge-C_H2C_H3Construct

Re-amplification of TRU-015V Using primers 4 and 6 (SEQ ID NOS: 18 and 20; Table 1, respectively)_HWherein V of NheI site 5' to TRU-015 is caused to react with_KFusion and Xho I (5 ' -CTCGAG-3 ') site at the 3 ' end hinged-C to IgG1_H2C_H3Domain joining. Similarly, IgG1 hinge-C was amplified using primers 8 and 9 in Table 1_H2-C_H3Region, thereby introducing a 5 'XhoI site, replacing the existing 3' end with an EcoRI (5 '-GAATTC-3') site for cloning, and disrupting the stop codon to allow translation of the binding domain 2 linked downstream of the CH3 domain. This scorpion-like molecular box type is different from the above-mentioned box whose prefix is "n".

In addition to the multivalent binding proteins described above, the proteins of the invention may have binding domains (binding domains 1 or 2, or both) corresponding to a single variable region of an immunoglobulin. Exemplary embodiments of this aspect of the invention will include V corresponding to a camelid antibody_HBinding domains of domains, or capable of bindingSingle modified or unmodified V regions of antibodies of other species to the target antigen, although any single variable domain suitable for use in the proteins of the invention is contemplated.

2E12 VL-VH and VH-VL constructs

To prepare a 2E12 scFv compatible with the cassette, the internal Xba I (5 '-TCTAGA-3') site was disrupted using the overlapping oligonucleotide primers 17 and 18 of Table 1. The two primers were used in combination with primer pair 14/16(VL-VH) or 13/15(VH-VL) to amplify two oppositely oriented binding domains so that they carry an EcoRI site and an XbaI site at their 5 'and 3' ends, respectively. Primers 13 and 16 also encoded a stop codon (TAA) immediately before the Xba I site.

2H7 SSS IgG 12 e12 LH/HL construct

Effector-binding Domain 2 linker addition (STD linker-STD 1 and STD2)

Complementary primers 11 and 12 in Table 1 were combined, heated to 70 ℃ and slowly cooled to room temperature to allow the two strands to adhere. The 5 'phosphate group was added using the manufacturer's protocol using T4 polynucleotide kinase (Roche) in 1X ligation buffer (Roche) with 1mM ATP. The resulting double stranded linker was then ligated to the DNA ligase (Roche) positioned at IgG1C using T4 DNA ligase_H3The coding region at the end and the EcoRI site between the beginning of the binding domain 2. The resulting DNA constructs were screened for the presence of the EcoRI site at the junction of the linker-BD 2 and for C_H3-the presence of the nucleotide sequence GAATTA at the linker junction. The appropriate STD1 linker construct was then re-digested with EcoRI, and linker ligation was repeated to generate a molecule with a linker consisting of two (STD2) iterations of identity of the Lx1 sequence. The DNA constructs were again screened as above.

Example 4

Expression study

Expression studies were performed on the above-described nucleic acids encoding multivalent binding proteins with effector functions. A nucleic acid encoding a multivalent binding protein is transiently transfected into COS cells and the transfected cells are maintained under well-known conditions that allow expression of a heterologous gene in the cells. DNA was transiently transfected into COS cells using PEI or DEAE-dextran as described above (PEI. Boussif O. et al, PNAS 92: 7297-. Multiple independent transfections of each novel molecule were performed in order to determine the average degree of expression of each novel form. If transfected by PEI, COS cells were plated on 60mm tissue culture plates in DMEM/10% FBS medium and incubated overnight to allow them to grow to approximately 90% on the day of transfection. The medium was changed to serum-free DMEM without antibiotics and incubated for 4 hours. Transfection medium (4 ml/plate) contained serum-free DMEM with 50. mu.g PEI and 10-20. mu.g DNA plasmid of interest. The transfection medium was mixed by vortexing, incubated at room temperature for 15 minutes, and added to the plate after the existing medium was withdrawn. The culture was incubated for 3-7 days and the supernatant was collected. Protein expression of culture supernatants was determined by SDS-PAGE, western blot, binding was verified by flow cytometry and function was determined using various assays including ADCC, CDC and co-culture experiments.

SDS-PAGE analysis and Western blot analysis

Samples were prepared from crude culture supernatant (typically 30 μ l/well) or purified protein aliquots containing 8 μ g protein per well, and 2 × Tris-glycine SDS buffer (Invitrogen) was added to 1-fold final concentration. 10 microliters of SeeBlue marker (Invitrogen, Carlsbad, Calif.) was electrophoresed to provide MW size standards. Multivalent binding (fusion) protein variants were subjected to SDS-PAGE analysis on a 4-20% Novex Tris-glycine gel (Invitrogen, San Diego, CA). After heating at 95 ℃ for 3 min, the samples were loaded with Novex Tris-glycine SDS sample buffer (2X) under reducing or non-reducing conditions followed by electrophoresis at 175V for 60 min. Electrophoresis was performed using 1X Novex Tris-glycine SDS electrophoresis buffer (Invitrogen).

After electrophoresis, a semi-dry electroblotting apparatus (E) was usedllard, Seattle, WA) transferred the protein to PVDF membrane at 100mAmp for 1 hour. Western transfer buffers included the following three buffers present on saturated Whatman filter paper: no. 1 contains 36.34 g/l Tris (pH10.4) and 20% methanol; no. 2 contains 3.02 g/l Tris (pH10.4) and 20% methanol; no. 3 contains 3.03 g/l Tris (pH9.4), 5.25 g/l ε -aminocaproic acid and 20% methanol. Membranes were blocked overnight in BLOTTO (═ 5% skim milk) in PBS with stirring. The cell membranes were incubated with 5. mu.g/ml of HRP-conjugated goat anti-human IgG (Fc-specific, Caltag) for one hour in BLOTTO followed by 3 washes in PBS-0.5% Tween 20 for 15 minutes each. The wet membranes were incubated with ECL solution for 1 minute followed by exposure to X-omat membrane for 20 seconds. FIG. 2 shows a Western blot of proteins expressed in COS cell culture supernatants (30. mu.l/well) electrophoresed under non-reducing conditions. Lanes are indicated with markers 1-9 and contain the following samples: lane 1 (cut-off See Blue marker, kDa for the blot side, Lane 2 ═ 2H7-sssIgG P238S/P331S-STD1-2e12 VLVH; Lane 3 ═ 2H 7-ssIgGP 238S/P331S-STD1-2e12 VHVL; Lane 4 ═ 2H 7-ssIgG P238S/P331S-STD2-2e12 VLVH; Lane 5 ═ 2H 7-ssIgG P238S/P331S-STD2-2e12 VHVL; Lane 6 ═ 2e12VH SMIP; Lane 7 ═ 2e12 VL SMIP; Lane 8 ═ 2H7 SMIP; the SMIP construct is always V7 _LV_HOrientation, sssIgG indicates the properties of the hinge/linker at linker position 1 (as shown in FIG. 5), P238S/P331S indicates the human IgG1 pattern mutated from wild type (first aa shown) to mutant (second aa shown) and its presence in wild type human IgGl C_H2And C_H3Amino acid positions in the domain, STD1 indicates the 20 amino acid (18+ restriction site) linker at linker position 2 (as shown in figure 5), and STD2 indicates the 38 amino acid (36+ restriction site) linker at linker position 2 (as shown in figure 6).

Binding study

Binding studies were performed to evaluate the bispecific binding properties of CD20/CD28 multispecific multivalent binding peptides. Initially, WIL2-S cells were added to 96-well plates and centrifuged into pelleted cells. The CD20/CD28 purified protein was added to the inoculated plates using a two-fold titration method (20. mu.g/ml titration to 0.16. mu.g/ml) on the whole plate. Two-fold serial dilutions of TRU-015 (source of binding domain 1) purified protein (TRU-015 concentration ranging from 20 μ g/ml to 0.16 μ g/ml) were also added to the inoculated plate wells. One well without protein served as background control.

The protein containing inoculum plates were incubated on ice for one hour. Subsequently, the wells were washed once with 200 μ l 1% FBS (in PBS). Goat anti-human antibody labeled with 1:100 fitc (fcsp) was then added to each well and the plates were incubated on ice for an additional hour. The plate was then washed once with 200 μ l 1% FBS (in PBS), and the cells were resuspended in 200 μ l 1% FBS and analyzed by FACS.

To evaluate the anti-CD 28 peptide 2E 12V_HV_LThe binding properties of (a), CHO cells expressing CD28 were coated by plating into individual wells of a culture plate. CD20/CD28 purified protein was then added to individual wells using a two-fold dilution protocol (concentration ranging from 20. mu.g/ml to 0.16. mu.g/ml). Again using a two-fold dilution scheme (i.e., 20 μ g/ml to 0.16 μ g/ml), 2E12IgG-VHVL SMIP purified protein was added to individual inoculum wells. One well received no protein to provide a background control. The plates were then incubated on ice for one hour, washed once with 200 μ l 1% FBS (in PBS), and goat anti-human antibody labeled with FITC (Fc Sp, CalTag, Burlingame, CA) at 1:100 was added to each well. The plates were incubated on ice for an additional hour and then washed once with 200 μ l 1% FBS (in PBS). FACS analysis was performed after resuspending the cells in 200. mu.l of 1% FBS. The results show that multivalent binding proteins with N-terminal CD20 binding domain 1 bind CD 20; having N-V_H-V_LThose proteins that bind domain 2 to C-terminal CD28 in the C-orientation also bind CD 28.

Flow cytometry (FACS) showed that the expressed protein can bind to CD20 presented on WIL-2S cells (see fig. 3) and can bind to CD28 presented by CHO cells (see fig. 3), demonstrating that BD1 or BD2 can function to bind to specific target antigens. The data presented graphically in fig. 3 show that serial dilutions of different multivalent binding (fusion) proteins have binding properties within the indicated titration range. Data obtained using the initial construct indicated that at equivalent concentrations, multivalent binding (fusion) proteins with binding domain type 2 (using VHVL-oriented 2e12) expressed better and bound CD28 better than the VLVH-oriented version.

Figure 4 shows a graphical representation of the results of binding studies performed with purified protein obtained from each of the transfectants/constructs. The figure shows the binding profile of the protein to WIL-2S cells expressing CD20, demonstrating that the multivalent molecule binds CD20, as well as a monospecific SMIP at the same concentration. The top and bottom panels of figure 5 show the profile of BD2 specific (2e12 ═ CD28) binding to CD28 CHO cells. For binding of binding domain 2 to CD28, the orientation of the V region affects binding of 2e 12. The 2H7-sss-hIgG-STD1-2e12 multivalent binding protein with VH-vl (hl) -oriented 2e12 showed binding to an extent equivalent to monospecific SMIPs, while the 2e12LH molecule showed low binding efficiency at the same concentration.

Example 5

Construction of various linker forms of multivalent fusion proteins

This example describes the construction of the different linker forms listed in the table shown in FIG. 6.

C_H3Construction of the BD2 linker H1 to H7

To explore C_H3Effect of BD2 linker length and composition on expression and binding of scorpion molecules, experiments were designed to compare the existing molecule 2H7 ssigg1-Lx1-2e12HL with a larger set of similar constructs with different linkers. A series of PCR reactions were performed using 2H7sssIgG1-Lx1-2e12HL as a template and the primers listed in Table 2 for the oligonucleotides to form linkers varying from 0 to 16 amino acids in length. The linker can be constructed as a nucleic acid fragment that spans the C at the BsrGI site _H3The coding region was to the end of the nucleic acid encoding the linker-BD 2 junction at the EcoRI site.

TABLE 2

SEQ ID

The numbering name sequence 5 '-3' NO.

PCR primer

GCGATAGAATTCCCAGATCCACCACCGCCCGA

1 L1-11R GCCACCGCCACCATAATTC 46

GCGATAGAATTCCCAGAGCCACCGCCACCATA

2 L1-6R ATTC 47

GCGTATGAATTCCCCGAGCCACCGCCACCCTTA

3 L3R CCCGGAGACAGG 48

GCGTATGAATTCCCAGATCCACCACCGCCCGAG

4 L4R CCACCGCCACCCTTAC 49

GCGTATGAATTCCCGCTGCCTCCTCCCCCAGATC

5 L5R CACCACCGCC 50

6 IgBsrG1F GAGAACCACAGGTGTACACCCTG 51

GCGATAGAATTCGGACAAGGTGGACACCCCTTAC

7 L-CPPCPR CCGGAGACAGGGAGAG52

Table 2: sequences of primers for forming CH3-BD2 linker variants

Figure 6 illustrates the schematic structure of a multivalent binding (fusion) protein and shows the orientation of the V-regions of each binding domain, the sequence present at linker position 1 (only Cys residues listed) and the sequence and identifier of the linker at linker position 2 of the molecule.

Example 6

Binding and functional Studies against the variant linker form of the 2H7-IgG-2e12 prototype multivalent fusion protein

This example shows the results of a series of expression and binding studies on a "prototype" 2H7-sssIgG-Hx-2e12 VHVL construct with various linkers (H1-H7) present in linker position 2. Each of the proteins was expressed by large-scale COS transient transfection and purification of the molecules using protein a affinity chromatography, as described in the examples above. The purified proteins were then subjected to analyses including SDS-PAGE, western blotting, analytical studies of binding by flow cytometry and functional assays for biological activity.

Binding studies comparing different BD2 orientations

Binding studies were performed as described in the examples above, except that protein a purified material was used and a constant amount of binding (fusion) protein, i.e. 0.72 μ g/ml, was used for each variant studied. Figure 7 shows a bar graph comparing the binding properties of each linker variant and 2e12 orientation variant for CD20 and CD28 target cells. H1-H6 refers to a construct with a H1-H6 linker and 2e12 in VH-VL orientation. L1-L6 refers to a construct with a H1-H6 linker and 2e12 in VL-VH orientation. The data show that binding domain 2 specific for 2e12 binds more efficiently when present in the HL orientation (samples H1-H6) than when present in the LH orientation (samples L1-L6). As shown in the next set of figures, the effect of linker length was found to be complicated by the fact that molecules with longer linkers contain some monospecific cleavage molecules that lack CD28 binding specificity at the carboxy terminus. Additional experiments were performed to determine binding of the selected linkers, and the results are shown in fig. 10, 12, and 13.

SDS-PAGE analysis of purified H1-H7 linker variants

Samples were prepared from aliquots of purified protein, each well containing 8 μ g of protein, and 2 × Tris-glycine SDS buffer (Invitrogen) was added to 1 × final concentration. For reduced samples/gels, 10X reduction buffer plus Tris-glycine SDS buffer was added to the sample to 1X. 10 μ l of SeeBlue marker (Invitrogen, Carlsbad, Calif.) was electrophoresed to provide MW size standards. Multivalent binding (fusion) protein variants were subjected to SDS-PAGE analysis on 4-20% Novex Tris-glycine gel (Invitrogen, San Diego, Calif.). After heating at 95 ℃ for 3 min, the samples were loaded with Novex Tris-glycine SDS sample buffer (2X) under reducing or non-reducing conditions followed by electrophoresis at 175V for 60 min. The buffer (Invitrogen) was electrophoresed using 1X Novex Tris-glycine SDS. After electrophoresis, the gel was stained in Coomassie (Coomassie) SDS PAGE R-250 stain for 30 minutes with stirring and destained for at least one hour. FIG. 8 shows a non-reduced and reduced Coomassie stained gel of [2H7-sss-hIgG P238S/P331S-Hx-2e12VHVL ] multivalent binding (fusion) protein variants and TRU-015 and 2e12 HLSMIP as control samples. As the linker length increases, the amount of protein electrophoresed at about SMIP size (or 52kDa) increases. An increase in the amount of protein in this band corresponds to a decrease in the amount of protein in the band above electrophoresis at about 90 kDa. Gel data show that the full length molecule is cleaved at or near the linker to form a molecule lacking the BD2 region. The absence of the smaller BD2 fragment indicates (1) that the nucleotide sequence in the linker sequence may form a cryptic splice site, allowing the smaller fragment to be removed from the spliced RNA transcript; or (2) the protein is proteolytically cleaved after translation of the full-length polypeptide, and the smaller fragment of BD2 is unstable, i.e., susceptible to proteolytic processing. Western blot analysis of some of the molecules showed that the proteins all contained the CD20 BD1 sequence, but the smaller band lacked CD28 BD2 reactivity. No migration of the smaller band in the "naked" scFv size (25-27kDa) was observed on any gel or blot, indicating the absence of this smaller peptide fragment in the sample.

Western blot analysis of binding of BD1 and BD2 to 2H 7-specific Fab or CD28mIg

FIG. 9 shows the results of Western blot analysis of 2H7-sss-hIgG-H6 multivalent binding (fusion) protein compared to each monospecific SMIP.

Electrophoresis was performed under non-reducing conditions and the sample was not boiled prior to loading. After electrophoresis, proteins were transferred to PVDF membranes for 1 hour at 100mAmp using a semi-dry electroblotting apparatus (Ellard, Seattle, WA). Membranes were blocked in BLOTTO (5% skim milk in PBS) overnight with stirring. FIG. 9A: membranes were incubated with 5. mu.g/mL of AbyD02429.2 (Fab against 2H7 antibody) for one hour in BLOTTO, followed by 3 washes in PBS-0.5% Tween 20 for 5 minutes each. The membrane was then incubated in 6 × His-HRP at a concentration of 0.5 μ g/ml for one hour. The blot was washed three times in PBST for 15 minutes each. The wet membranes were incubated with ECL solution for 1 minute followed by exposure to X-omat membrane for 20 seconds.

FIG. 9B: membranes were incubated with 10. mu.g/ml CD28Ig (Ancell, Bayport, MN) in BLOTTO followed by three 15 minute washes in PBS-0.5% Tween 20. The membranes were then incubated in BLOTTO with 1:3000 goat anti-mouse HRP conjugate (CalTag, Burlingame, Calif.). The membrane was washed three times for 15 minutes each, followed by incubation in ECL solution for 1 minute, followed by exposure to X-omat membrane for 20 seconds. Western blot analysis results indicated that the CD28 binding domain is present as a multivalent "monomeric" component (which migrates at about 90 kDa) and is present in higher order forms. No band migration was observed at the expected position for a single SMIP or naked scFv size fragment. When using CD20 anti-idiotypic Fab, SMIP-sized fragments were detected, indicating the presence of a peptide fragment containing the (2H7-sss-hIgG) and lacking the CD28 scFv BD2 portion of the fusion protein.

Binding studies of selected linkers

FIG. 10 shows the results of binding studies performed on purified 2H7-sss-hIgG-Hx-2e12 fusion protein. Binding studies were performed to evaluate the bispecific binding properties of CD20/CD28 multispecific binding peptides. WIL2-S cells were initially coated using conventional techniques. CD20/CD28 purified protein was added to the inoculated plates using a two-fold titration method (20. mu.g/ml to 0.16. mu.g/ml) on the whole plate. Two-fold serial dilutions of TRU-015 (source of binding domain 1) purified protein (TRU-015 concentration ranging from 20 μ g/ml to 0.16 μ g/ml) were also added to the inoculated plate wells. One well without protein served as background control.

The inoculated plates containing the protein were incubated on ice for one hour. Subsequently, the wells were washed once with 200 μ l 1% FBS (in PBS). Goat anti-human antibody labeled with 1:100 fitc (fcsp) was then added to each well and the plates were incubated on ice for an additional hour. The plate was then washed once with 200 μ l 1% FBS (in PBS), and the cells were resuspended in 200 μ l 1% FBS and analyzed by FACS.

To evaluate the anti-CD 28 peptide 2E 12V_HV_LThe binding properties of (a), CHO cells expressing CD28 were coated by plating into individual wells of a culture plate. CD20/CD28 purified protein was then added to individual wells using a two-fold dilution protocol (concentration ranging from 20. mu.g/ml to 0.16. mu.g/ml). Again using a two-fold dilution protocol (i.e., 20 μ g/ml to 0.16 μ g/ml), 2E12IgGvHvL SMIP purified protein was added to individual inoculum wells. One well received no protein to provide a background control. The plates were then incubated on ice for one hour, washed once with 200 μ l 1% FBS (in PBS), and goat anti-human antibody labeled with 1:100 fitc (fc sp) was added to each well. The plates were incubated on ice for an additional hour and then washed once with 200 μ l 1% FBS (in PBS). After the cells are re-cultured After suspending in 200. mu.l of 1% FBS, FACS analysis was performed. Flow cytometry (FACS) showed that the expressed protein can bind to CD20 presented on WIL-2S cells (see fig. 10A) and can bind to CD28 presented on CHO cells (see fig. 10B), indicating that BD1 or BD2 can function to bind to specific target antigens. Furthermore, the linker used (H1-H6) was not found to have a significant effect on the affinity for binding to the target antigen.

SEC separates multivalent binding (fusion) proteins. The binding (fusion) protein was purified from the cell culture supernatant by protein a sepharose affinity chromatography on a GE Healthcare XK 16/40 column. After the proteins were bound to the column, the column was washed with dPBS, then 1.0M NaCl, 20mM sodium phosphate (pH6.0), and then 25mM NaCl, 25mN NaOAc (pH5.0) to remove non-specifically bound proteins. The bound protein was eluted from the column with 100mM glycine (Sigma) (pH3.5) and brought to pH5.0 with 0.5M 2- (N-morpholino) ethanesulfonic acid (MES) (pH 6.0). Protein samples were concentrated to 25mg/ml using conventional techniques for GPC purification. Size Exclusion Chromatography (SEC) was performed on a GE Healthcare AKTA Explorer 100Air apparatus using a GE Healthcare XK column and Superdex 200 preparative grade (GE Healthcare).

Figure 12 shows a table summarizing the results of SEC separation of different binding (fusion) proteins. As linker length increases, the complexes of molecules in solution also increase, making it difficult to separate the peak (or POI) of interest from higher order forms by HPLC. The H7 linker appears to resolve this majority of the complex into a more homogeneous form in solution, so that the soluble form migrates mostly as a single POI at about 172 kDa.

Other binding studies

A second series of experiments (see fig. 12 and 13) was performed on a smaller subset of multivalent binding (fusion) proteins, this time comparing linkers H3, H6, and H7. The data show that the degree of binding to CD28 is more pronounced than to CD20, but both decrease slightly as the linker length increases. In addition, the data shows that the H7 linker showed the highest degree of binding to both antigens. The data were obtained using multivalent binding (fusion) proteins purified with protein a, but without further purification by SEC, so that various forms of the molecule can be present in solution. The results also indicate that the truncated form is less stable than the actual multivalent polypeptide, as the binding curve does not appear to fully reflect the large number of monospecific forms of linker H6 present in solution.

Demonstration of Multi-specific binding of Single molecules

An alternative binding assay was performed (see fig. 13) in which CD20 binding to the WIL-2S cell surface was detected with reagents specific for CD28 BD2, indicating that binding to both target antigens can occur simultaneously for BD1 and BD2 on the same multi-specific binding (fusion) protein (see fig. 12). This assay demonstrates the multispecific binding characteristics of the protein.

Example 7

Construction of a Multispecific binding (fusion) protein of BD2 with alternative specificity

In addition to the prototype CD20-CD28 multispecific binding molecule, two other forms having 2 regions of an alternative binding domain (including the CD37 binding domain and the CD3 binding domain) can be made. Molecules with several linker domains as described for the [2H7-sss-IgG-Hx/STDx-2e12HL ] multispecific binding (fusion) protein can also be prepared. The construction of such other multispecific binding (fusion) molecules is described below.

anti-CD 37 binding domain constructs

TABLE 3

Numbering name sequence SEQ ID NO.

ACTGCTGCAGCTGGACCGCGCT

23 G281LH-NheR AGCTCCGCCGCCACCCGAC 53

GGCGGAGCTAGCGCGGTCCAGC

24 G281LH-NheF TGCAGCAGTCTGGACCTG 54

GCGATCACCGGTGACATCCAGAT

25 G281-LH-LPinF GACTCAGTCTCCAG 55

GCGATACTCGAGGAGACGGTGAC

26 G281-LH-HXhoR TGAGGTTCCTTGAC 56

GCGATCGAATTCAGACATCCAGAT

27 G281-LH-LEcoF GACTCAGTCTCCAG 57

GCGATTCTAGATTAGGAAGAGACG

28 G281-LH-HXbaR GTGACTGAGGTTCCTTGAC 58

GCGATAACCGGTGCGGTCCAGCTG

29 G281-HL-HF CAGCAGTCTGGAC 59

GACCCACCACCGCCCGAGCCACCG

CCACCAGAAGAGACGGTGACTGAGG 60

30 G281-HL-HR3 TTC

ACTCCCGCCTCCTCCTGATCCGCCG

31 G281-HL-HR2 CCACCCGACCCACCACCGCCCGAG 61

GAGTCATCTGGATGTCGCTAGCACTC

32 G281-HL-HNheR CCGCCTCCTCCTGATC 62

ATCAGGAGGAGGCGGGAGTGCTAGC

33 G281-HL-LNheF GACATCCAGATGACTCAGTC 63

GCGATACTCGAGCCTTTGATCTCCAG

34 G281-HL-LXhoR TTCGGTGCCTC 64

GCGATATCTAGACTCAACCTTTGATCT

35 G281-HL-LXbaR CCAGTTCGGTGCCTC 65

GCGATAGAATTCGCGGTCCAGCTGCA

36 G281-HL-EcoF GCAGTCTGGAC 66

Table 3: oligonucleotide primers for forming the G28-1 anti-CD 37 binding domain of SMIP and scorpion molecules.

G28-1scFv (SEQ ID NO: 102) was converted to G28-1LH SMIP by PCR using the primers in Table X above. Primers 23 and 25 were combined with 10ng G28-1scFv and VK was amplified in an ABI 9700 thermocycler using a Platinum PCR Supermix Hi-Fidelity PCR mixer (Invitrogen, Carlsbad, Calif.) (30 cycles: 94 ℃ for 20 seconds, 58 ℃ for 15 seconds, 68 ℃ for 15 seconds). This PCR product had the restriction site pinai (agei) at the 5' end of VK and NheI at the end of the scFv (G4S)3 linker. VH was similarly altered by combining primers 24 and 26 with 10ng G28-1scFv in a PCR run under the same conditions as VK above. This PCR product has a restriction site NheI at the 5 'end of VH and XhoI at the 3' end. Since the important sequence identity overlap has been engineered as primers 23 and 24, VK and VH were diluted 5-fold, followed by addition to PCR using flanking primers 25 and 26 at a 1:1 ratio and full-length scFv amplified as above by extending 68 ℃ duration from 15 seconds to 45 seconds. This PCR product represents the entire G28-1scFv as the PinAI-XhoI fragment and can be purified by MinElute column (Qiagen) to remove excess primers, enzymes and salts. The eluate was digested with pinai (invitrogen) and xhoi (Roche) in a volume of 50 μ L of 1X H buffer (Roche) at 37 ℃ for 4 hours until completion. The digested PCR products were then electrophoresed in a 1% agarose gel, the fragments were removed from the gel and repurified using buffer QG on a MinElute column, and the gel-buffer mixture was incubated at 50 ℃ for 10 minutes with intermittent mixing to dissolve the agarose before purification on the column as for PCR after primer removal. mu.L of PinAI-XhoI digested G28-1LH was combined with 1. mu.L of PinAI-XhoI digested pD18-n2H7 ssIgG1 SMIP and 5. mu.L of 2X LigaFast ligation buffer (Promega, Madison, Wis.) and 1. mu. L T4 DNA ligase (Roche) to 10. mu.L of reaction, mixed well and incubated at room temperature for 10 min. mu.L of this ligation was then transformed into competent TOP 10(Invitrogen) using the manufacturer's protocol. The transformants were spread on LB agar plates with 100. mu.g/ml carbenicillin (Teknova) and incubated overnight at 37 ℃. After 18 hours of incubation, colonies were picked and inoculated in 1mlT-Broth (Teknova) containing 100. mu.g/ml carbenicillin in a deep-well 96-well plate and incubated overnight in a shaking incubator at 37 ℃. After 18 to 24 hours of culture, DNA was isolated from each overnight culture on a BioRobot8000(Qiagen) using the QIAprep 96 Turbo kit (Qiagen). Then 10. mu.L of each clone was digested with HindIII and XhoI restriction enzymes in 1X B buffer in a reaction volume of 15. mu.L. The digested DNA was electrophoresed on a 1% agarose E-gel (Invitrogen, CA) for restriction site analysis. Clones containing appropriately sized HindIII-XhoI fragments were sequence verified. The G28-1HL SMIP was constructed in a similar manner by positioning the PinAI site at the 5' end using primers 29, 30, 31 and 32 in Table X above and terminating the (G4S)4 linker at the Nhe I site of the G28-1 VH. VK was altered by PCR using primers 33 and 34 in Table X, such that the NheI site was introduced at the 5 'end and XhoI was introduced at the 3' end of the VK. The PCRs were then combined as above and amplified with flanking primers 29 and 34 to generate the entire G28-1scFv DNA in VH-VL orientation, just as the G28-1LH SMIP cloned into PinAI-XhoI digested pD18- (n2H7) sssIgG1 SMIP.

2H7sssIgG1-STD1-G28-1 LH/HL construct

The LH and HL anti-CD 37 binding domains were altered by PCR using G28-1LH and G28-1HL SMIP as templates to make their flanking restriction sites compatible with the scorpion cassette. An EcoRI site was introduced to the 5 'end of each scFv using primer 27(LH) or 36(HL), and a stop codon/XbaI site was introduced to the 3' end using primer 28(LH) or 35 (HL). The resulting DNA was cloned into EcoRI-XbaI digested pD18-2H7sssIgG-STD 1.

2H7sssIgG1-Hx-G28-1HL construct

The 2H7sssIgG1-Hx-2e12HL DNA was digested with BsrGI and EcoRI and a 325bp fragment consisting of the C-terminus of IgG1 and the linker. The STD1 linker was replaced with the equivalent region in 2H7 ssigg1-STD1-G19-4HL by removing the STD1 linker using BsrGI-EcoRI, and replacing it with the corresponding linker in 2H7 ssigg1-Hx-2e12HL clone.

anti-CD 3 binding domain constructs

TABLE 4

SEQ ID

Number name sequence NO.

GCGTATGAACCGGTGACATCCAGAT

37 194-LH-LF1 GACACAGACTACATC 67

ATCCAGATGACACAGACTACATCCTC

38 194-LF2 CCTGTCTGCCTCTCTGGGAGACAG 68

GTCTGCCTCTCTGGGAGACAGAGTCA

39 194-LF3 CCATCAGTTGCAGGGCAAGTCAGGAC 69

GTTGCAGGGCAAGTCAGGACATTCGC

40 194-LF4 AATTATTTAAACTGGTATCAGCAG 70

ATTTAAACTGGTATCAGCAGAAACCAG

41 194-LF5 ATGGAACTGTTAAACTCCTGATC 71

GAACTGTTAAACTCCTGATCTACTACA

42 194-LF6 CATCAAGATTACACTCAGGAGTC 72

CAAGATTACACTCAGGAGTCCCATCAA

43 194-LF7 GGTTCAGTGGCAGTGGGTCTGGAAC 73

CAGGTTGGCAATGGTGAGAGAATAATC

44 194-LR7 TGTTCCAGACCCACTGCCACTGAAC 74

GCAAAAGTAAGTGGCAATATCTTCTGGT

45 194-LR6 TGCAGGTTGGCAATGGTGAGAG 75

GAACGTCCACGGAAGCGTATTACCC

46 194-LR5 TGTTGGCAAAAGTAAGTGGCAATATC 76

CGTTTGGTTACCAGTTTGGTGCCTCCAC

47 194-LR4 CGAACGTCCACGGAAGCGTATTAC 77

ACCACCGCCCGAGCCACCGCCACC

48 194-LR3 CCGTTTGGTTACCAGTTTGGTG 78

GCTAGCGCTCCCACCTCCTCCAGATCCA

49 194-LR2 CCACCGCCCGAGCCACCGCCAC 79

GTTGCAGCTGGACCTCGCTAGCGCT

50 194-LH-LR1 CCCACCTCCTCCAGATC 80

GATCTGGAGGAGGTGGGAGCGCTAGC

51 194-LH-HF1 GAGGTCCAGCTGCAACAGTCTGGACCTG 81

AGCTGCAACAGTCTGGACCTGAACT

52 194-HF2 GGTGAAGCCTGGAGCTTCAATGAAG 82

AGCCTGGAGCTTCAATGAAGATTTCC

53 194-HF3 TGCAAGGCCTCTGGTTACTCATTC 83

GCAAGGCCTCTGGTTACTCATTCACT

54 194-HF4 GGCTACATCGTGAACTGGCTGAAGCAG 84

ATCGTGAACTGGCTGAAGCAGAGCC

55 194-HF5 ATGGAAAGAACCTTGAGTGGATTGGAC 85

GAACCTTGAGTGGATTGGACTTATTA

56 194-HF6 ATCCATACAAAGGTCTTACTACCTAC 86

AATGTGGCCTTGCCCTTGAATTTCTG

57 194-HR6 GTTGTAGGTAGTAAGACCTTTGTATG 87

CATGTAGGCTGTGCTGGATGACTTGT

58 194-HR5 CTACAGTTAATGTGGCCTTGCCCTTG 88

ACTGCAGAGTCTTCAGATGTCAGACTG

59 194-HR4 AGGAGCTCCATGTAGGCTGTGCTGGATG 89

ACCATAGTACCCAGATCTTGCACAG

60 194-HR3 TAATAGACTGCAGAGTCTTCAGATGTC 90

GCGCCCCAGACATCGAAGTACCAGTC

61 194-HR2 CGAGTCACCATAGTACCCAGATCTTG 91

GCGAATACTCGAGGAGACGGTGACCG

62 194-LH-HR1 TGGTCCCTGCGCCCCAGACATCGAAG 92

GCGTATGAACCGGTGAGGTCCAGC

631 94-HL-HF1 TGCAACAGTCTGGACCTG 93

ACCGCCACCAGAGGAGACGGTGACCGT

641 94-HL-HR1 GGTCCCTGCGCCCCAGACATCGAAGTAC 94

ACCTCCTCCAGATCCACCACCGCCCG

651 94-HL-HR0 AGCCACCGCCACCAGAGGAGACGGTG 95

GCGGGGGAGGTGGCAGTGCTAGCGA

661 94-HL-LF1 CATCCAGATGACACAGACTACATC 96

GCGAATACTCGAGCGTTTGGTTACCA

671 94-HL-LR3Xho GTTTGGTG 97

GCGATATCTAGATTACCGTTTGGTTAC

68 194-HL-LR3Xba CAGTTTGGTG 98

GCGTATGAGAATTCAGAGGTCCAGCTG

69 194-HL-HF1R1 CAACAGTCTGGACCTG 99

GCGTATGAGAATTCTGACATCCAGA

70 194-LH-LF1R1 TGACACAGACTACATC 100

GCGTATCTAGATTAGGAGACGGTGACC

71 194-LH-HR1Xba GTGGTCCCTGCGCCCCAGACATCGAAG 101

Table 4: an oligonucleotide for forming an anti-CD 3 binding domain of the G19-4scFv sequence.

The G19-4 binding domain was synthesized by extending overlapping oligonucleotide primers as described above. Light chain PCR was performed in two steps, starting with 30 cycles of 5. mu.M, 10. mu.M, 20. mu.M and 40. mu.M primers 43/44, 42/45, 41/46 and 40/47, respectively, in Platinum PCR Supermix Hi-Fidelity combined as follows: at 94 ℃ for 20 seconds, at 60 ℃ for 10 seconds, and at 68 ℃ for 15 seconds. mu.L of the resulting PCR product was re-amplified using the same PCR conditions (except that 68 ℃ duration was increased to 25 seconds) using 39/48 (10. mu.M), 38/49 (20. mu.M) and 37/50 (40. mu.M) primer mix (for LH orientation) or 66/67 (40. mu.M) primer mix (for HL orientation). VK with LH orientation is bound at the 5 'end by PinAI and at the 3' end by NheI, whereas VK with HL orientation is bound at the 5 'end by NheI and at the 3' end by XhoI.

To synthesize the heavy chain, a primer mixture with the same concentration as above was prepared by combining the primers 56/57, 55/58, 54/59, and 53/60 in the first PCR step. In the second PCR, primers 52/61 (20. mu.M) and 51/62 (50. mu.M) were amplified at 1. mu.l from the first PCR using the same PCR conditions as the second PCR for the light chain to form an LH orientation with NheI at the 5 'end and XhoI at the 3' end. In a second PCR, primers 52/61 (10. mu.M), 63/64 (20. mu.M), 63 (20. mu.M)/65 (40. mu.M) and 63 (20. mu.M)/5 (80. mu.M) were combined with 1. mu.L from the previous PCR to form an HL-oriented heavy chain with PinAI at the 5 'end and NheI at the 3' end. As with the constructs above, sufficient overlap was designed as primers centered at the NheI site so that G19-4LH was synthesized by PCR combining the heavy and light chains with LH orientation and reamplifying with flanking primers 37 and 62, and G19-4HL was synthesized by PCR combining HL and reamplifying with primers 63 and 67.

The full-length G19-4LH/HL PCR product was isolated by agarose gel electrophoresis, excised from the gel and purified using Qiagen MinElute columns as described above. The DNA was then TOPO cloned into pcr2.1(Invitrogen), transformed into TOP10, and colonies were first screened for EcoRI fragment size followed by DNA sequencing. G19-4LH/HL was then cloned into pD18-IgG1 via PinAI-XhoI for expression in mammalian cells.

2H7sssIgG1-STD1-G19-4LH/HL construct

The LH and HL anti-CD 3 binding domains were altered by PCR using G19-4LH and G19-4HL SMIP as templates to make their flanking restriction sites compatible with the scorpion cassette. An EcoRI site was introduced to the 5 'end of each scFv using primer 27(LH) or 36(HL), and a stop codon/XbaI site was introduced to the 3' end using primer 28(LH) or 35 (HL). The resulting DNA was cloned into EcoRI-XbaI digested pD18-2H7sssIgG-STD 1.

2H7sssIgG1-Hx-G19-4HL construct

The 2H7sssIgG1-Hx-2e12HL DNA was digested with BsrGI and EcoRI and a 325bp fragment consisting of the C-terminus of IgG1 and the linker. The STD1 linker was replaced with the equivalent region in 2H7 ssigg1-STD1-G19-4HL by removing the STD1 linker using BsrGI-EcoRI, and replacing it with the corresponding linker in 2H7 ssigg1-Hx-2e12HL clone.

The features of the molecules that form the molecules of the invention can be readily combined as will be apparent upon consideration of the various multivalent binding proteins disclosed herein. This feature includes binding domain 1, constant sub-regions (including hinge or hinge-like domains), linker domains and binding domain 2. The inherent modularity in the design of the novel binding proteins allows one of skill in the art to manipulate DNA sequences directly at the N-terminus and/or C-terminus of any desired module, such that the sequences can be inserted into virtually any position to form novel molecules that exhibit modified or enhanced functionality as compared to the parent molecule from which they were derived. For example, any binding domain derived from a member of the immunoglobulin superfamily is contemplated as binding domain 1 or binding domain 2 of the molecule of the invention. The derivatized binding domain includes: having amino acid sequences and even domains encoding polynucleotide sequences that correspond one-to-one to the sequences of immunoglobulin superfamily members, and variants and derivatives that preferably share 80%, 90%, 95%, 99% or 99.5% sequence identity with immunoglobulin superfamily members. The binding domains (1 and 2) are preferably linked to the other modules of the molecules of the invention via linkers of different sequence and length as described elsewhere herein, provided that the linkers are sufficient to provide any space and flexibility necessary for the molecule to achieve a functional tertiary structure. Another module of multivalent binding proteins is the hinge region, which may correspond not only to the hinge region of immunoglobulin superfamily members, but also to variants thereof, such as the "CSC" or "SSS" hinge regions described herein. In addition, the constant sub-region comprises a module of a protein of the invention, which may correspond to a sub-region of a constant region of a member of the immunoglobulin superfamily, such as hinge-C _H2-C_H3The structure of the constant subregions is illustrated. Also encompassed are variants and derivatives of the constant sub-regions, which preferably have an amino acid sequence sharing 80%, 90%, 95%, 99% or 99.5% sequence identity with a member of the immunoglobulin superfamily.

Has the advantages ofExemplary primary structures of the molecular features are shown in table 5, which discloses the polynucleotide and homologous amino acid sequences of illustrative binding domains 1 and 2, as well as the primary structure of the constant sub-region (including the hinge or hinge-like domain) and a linker that can be inserted between the C-terminus of the constant sub-region and the N-terminus of the binding domain 2 region of, for example, a multivalent binding protein. Other examples of molecules of the invention include the above features wherein, for example, either or both of binding domain 1 and binding domain 2 comprise a V derived from a member of the immunoglobulin superfamily_LOr V_LLike domains and V derived from the same or different members of the immunoglobulin superfamily_HOr V_HA domain of a like domain, which domains may be separated by a linker as represented by any of the linkers disclosed herein. Covering wherein the orientation of BD1 and/or BD2 in the domain is V_L-V_HOr V_H-V_LThe molecule of (1). A more complete representation of the primary structure of the various features of the multivalent binding molecules of the invention is presented in the table appended at the end of this disclosure. The invention further includes polynucleotides encoding the molecules.

TABLE 5

Binding domains	Nucleotide sequence	Amino acid sequence	SEQ ID NOS (amino acid sequence)
				2H7 LH	atggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtcggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctct	mdfqvqifsfllisasvimsrgqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaesvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvs	1(2)

Binding domains	Nucleotide sequence	Amino acid sequence	SEQ ID NOS (amino acid sequence)
				2e12 LH	atggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgctagcaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagactttagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccatggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggatccggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactatcgatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgcccgagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctct	MDFQVQIFSFLLISASVIMSRGVDIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKRGGGGSGGGGSGGGGSQVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKMNSLQTDDTARYYCARDGYSNFHYYVMDYWGQGTSVTVSS	3(4)
2e12 HL	atggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtccaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactatcgatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgcccgagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctctgggggtggaggctctggtggcggtggatccggcggaggtgggtcgggtggcggcggatctgacattgt	MDFQVQIFSFLLISASVIMSRGVQVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKMNSLQTDDTARYYCARDGYSNFHYYVMDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSDIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKR	5(6)

Binding domains	Nucleotide sequence	Amino acid sequence	SEQ ID NOS (amino acid sequence)
					gctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgctagcaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagactttagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccatggacgttcggtggaggcaccaagctggaaatcaaacgt
G28-1LH	accggtgacatccagatgactcagtctccagcctccctatctgcatctgtgggagagactgtcaccatcacatgtcgaacaagtgaaaatgtttacagttatttggcttggtatcagcagaaacagggaaaatctcctcagctcctggtctcttttgcaaaaaccttagcagaaggtgtgccatcaaggttcagtggcagtggatcaggcacacagttttctctgaagatcagcagcctgcagcctgaagattctggaagttatttctgtcaacatcattccgataatccgtggacgttcggtggaggcaccgaactggagatcaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgctagcgcagtccagctgcagcagtctggacctgagctggaaaagcctggcgcttcagtgaagatttcctgcaaggcttctggttactcattcactggctacaatatgaactgggtgaagcagaataatggaaagagccttgagtggattggaaatattgatccttattatggtggtactacctacaaccggaagttcaagggcaaggccacattgactgtagacaaatcctccagcacagcctacatgcagctcaagagtctgacatctgaggactctgcagtctattactgtgcaagatcggtcggccctatggactactggggtcaaggaacctcagtcaccgtctcgag	DIQMTQSPASLSASVGETVTITCRTSENVYSYLAWYQQKQGKSPQLLVSFAKTLAEGVPSRFSGSGSGTQFSLKISSLQPEDSGSYFCQHHSDNPWTFGGGTELEIKGGGGSGGGGSGGGGSASAVQLQQSGPELEKPGASVKISCKASGYSFTGYNMNWVKQNNGKSLEWIGNIDPYYGGTTYNRKFKGKATLTVDKSSSTAYMQLKSLTSEDSAVYYCARSVGPMDYWGQGTSVTVS	102(103)
				G28-1HL	accggtgaggtccagctgcaacagtctggacctgaactggtgaagcctggagcttcaatgaagatttcctgcaaggcctctggttactcattcactggctacatcgtgaactggctgaagcagagccatggaaagaaccttgagtggattggacttattaatccatacaaaggtcttactacctacaaccagaaattcaagggcaaggccacattaactgtagacaagtcatccagcacagcctacatggagctcctcagtctgacat	EVQLQQSGPELVKPGASMKISCKASGYSFTGYIVNWLKQSHGKNLEWIGLINPYKGLTTYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSGGGGSASDIQMTQTTSSLSASLGDRVTISCRASQDIR	104(105)

Binding domains	Nucleotide sequence	Amino acid sequence	SEQ ID NOS (amino acid sequence)
					ctgaagactctgcagtctattactgtgcaagatctgggtactatggtgactcggactggtacttcgatgtctggggcgcagggaccacggtcaccgtctcctctggtggcggtggctcgggcggtggtggatctggaggaggtgggagcgggggaggtggcagtgctagcgacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattcgcaattatttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctactacacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattgccaacctgcaaccagaagatattgccacttacttttgccaacagggtaatacgcttccgtggacgttcggtggaggcaccaaactggtaaccaaacgctcgag	NYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTIANLQPEDIATYFCQQGNTLPWTFGGGTKLVTKRS
G19-4LH	accggtgacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattcgcaattatttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctactacacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattgccaacctgcaaccagaagatattgccacttacttttgccaacagggtaatacgcttccgtggacgttcggtggaggcaccaaactggtaaccaaacggggtggcggtggctcgggcggtggtggatctggaggaggtgggagcgctagcgaggtccagctgcaacagtctggacctgaactggtgaagcctggagcttcaatgaagatttcctgcaaggcctctggttactcattcactggctacatcgtgaactggctgaagcagagccatggaaagaaccttgagtggattggacttattaatccatacaaaggtcttactacctacaaccagaaattcaagggcaaggccacattaactgtagacaagtcatccagcacagcctacatggagctcctcagtctgacatctgaagactctgcagtctattactgtgcaagatctgggtactatggtgactcggactggtacttcgatgtctggggcgcagggaccacggtcaccgtctcctcgag	DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTIANLQPEDIATYFCQQGNTLPWTFGGGTKLVTKRGGGGSGGGGSGGGGSASEVQLQQSGPELVKPGASMKISCKASGYSFTGYIVNWLKQSHGKNLEWIGLINPYKGLTTYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVSS	106(107)
				G19-4HL	accggtgaggtccagctgcaacagtctggacctgaactggtgaagcctggagcttcaatgaagattt	EVQLQQSGPELVKPGASMKISCKASGYSFTGYIVNWLKQSHGK	108(109)

Binding domains	Nucleotide sequence	Amino acid sequence	SEQ ID NOS (amino acid sequence)
					cctgcaaggcctctggttactcattcactggctacatcgtgaactggctgaagcagagccatggaaagaaccttgagtggattggacttattaatccatacaaaggtcttactacctacaaccagaaattcaagggcaaggccacattaactgtagacaagtcatccagcacagcctacatggagctcctcagtctgacatctgaagactctgcagtctattactgtgcaagatctgggtactatggtgactcggactggtacttcgatgtctggggcgcagggaccacggtcaccgtctcctctggtggcggtggctcgggcggtggtggatctggaggaggtgggagcgctagcgacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattcgcaattatttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctactacacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattgccaacctgcaaccagaagatattgccacttacttttgccaacagggtaatacgcttccgtggacgttcggtggaggcaccaaactggtaaccaaacgctcgag	NLEWIGLINPYKGLTTYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSASDIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTIANLQPEDIATYFCQQGNTLPWTFGGGTKLVTKRS

Hinge region	Nucleotide sequence	Amino acid sequence	(amino acid sequence) of SEQ ID NO
				sss(s)-hIgG1	gagcccaaatcttctgacaaaactcacacatctccaccgagctca	EPKSSDKTHTSPPSS	230(231)
csc(s)-hIgG1	gagcccaaatcttgtgacaaaactcacacatctccaccgtgctca	EPKSCDKTHTSPPCS	232(233)
				ssc(s)-hIgG1	gagcccaaatcttctgacaaaactcacacatctccaccgtgctca	EPKSSDKTHTSPPCS	110(111)
scc(s)-hIgG1	gagcccaaatcttctgacaaaactcacacatgtccaccgtgctca	EPKSSDKTHTCPPCS	112(113)
				css(s)-hIgG1	gagcccaaatcttgtgacaaaactcacacatctccaccgagctca	EPKSCDKTHTSPPSS	114(115)
scs(s)-hIgG1	gagcccaaatcttgtgacaaaactcacacatgtccaccgagctca	EPKSSDKTHTCPPSS	116(117)
				ccc(s)-hIgG1	gagcccaaatcttgtgacaaaactcacacatgtccaccgtgctca	EPKSCDKTHTSPPCS	118(119)
ccc(p)-	Gagcccaaatcttgtgacaaaactcacacatgt	EPKSCDKTHTSPPCP	120(121)

hIgG1	ccaccgtgccca
				sss(p)-hIgG1	gagcccaaatcttctgacaaaactcacacatctccaccgagccca	EPKSSDKTHTSPPSP	122(123)
csc(p)-hIgG1	gagcccaaatcttgtgacaaaactcacacatctccaccgtgccca	EPKSCDKTHTSPPCP	124(125)
				ssc(p)-hIgG1	gagcccaaatcttctgacaaaactcacacatctccaccgtgccca	EPKSSDKTHTSPPCP	126(127)
scc(p)-hIgG1	gagcccaaatcttctgacaaaactcacacatgtccaccgtgccca	EPKSSDKTHTCPPCP	128(129)
				css(p)-hIgG1	gagcccaaatcttgtgacaaaactcacacatctccaccgagccca	EPKSCDKTHTSPPSP	130(131)
scs(p)-hIgG1	gagcccaaatcttgtgacaaaactcacacatgtccaccgagccca	EPKSSDKTHTCPPSP	132(133)
				scppcp	agttgtccaccgtgccca	SCPPCP	134(135)
EFD	Nucleotide sequence	Amino acid sequence	Sequence identifier (amino acid sequence)
				hIgG1(P238S)CH2CH3	gcacctgaactcctgggtggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga	APELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK	142(143)
hIgG1(P331S)CH2CH3	gcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgt	APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELT	144(145)

	cctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga	KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
				hIgG1(P238S/P331S)CH2CH3	gcacctgaactcctgggtggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga	APELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK	146(147)
Connector	Nucleotide sequence	Amino acid sequence	Sequence identifier
				STD1	aattatggtggcggtggctcgggcggtggtggatctggaggaggtgggagtgggaattct	NYGGGGSGGGGSGGGGSGNS	148(149)
STD2	aattatggtggcggtggctcgggcggtggtggatctggaggaggtgggagtgggaattatggtggcggtggctcgggcggtggtggatctggaggaggtgggagtgggaattct	NYGGGGSGGGGSGGGGSGNYGGGGSGGGGSGGGGSGNS	150(151)
				H1	aattct	NS	152(153)
H2	ggtggcggtggctcggggaattct	GGGGSGNS	154(155)

H3	aattatggtggcggtggctctgggaattct	NYGGGGSGNS	156(157)
				H4	ggtggcggtggctcgggcggtggtggatctgggaattct	GGGGSGGGGSGNS	158(159)
H5	aattatggtggcggtggctcgggcggtggtggatctgggaattct	NYGGGGSGGGGSGNS	160(161)
				H6	ggtggcggtggctcgggcggtggtggatctgggggaggaggcagcgggaattct	GGGGSGGGGSGGGGSGNS	162(163)
H7	gggtgtccaccttgtccgaattct	GCPPCPNS	164(165)
				(G4S)3	ggtggcggtggatccggcggaggtgggtcgggtggcggcggatct	GGGSGGGSGGGS	166(167)
(G4S)4	ggtggcggtggctcgggcggtggtggatctggaggaggtgggagcgggggaggtggcagt	GGGSGGGSGGGSGGGGS	168(169)

Table 5: primary Structure with exemplary features of multivalent binding molecules (Polynucleotide and homologous amino acid sequences)

Example 8

Binding and functional studies of alternative multispecific fusion proteins

Parallel experiments with experiments for multispecific binding (fusion) of each of the other multivalent binding molecules described above with the prototype CD20-IgG-CD28 described above were performed. In general, the data obtained for the other molecules is similar to that observed for the prototype molecule. Some significant results of the experiments are disclosed below. Figure 14 shows the results of blocking studies performed on one of the novel molecules, where both BD1 and BD2 bind to target antigens on the same cell or cell type (in this case CD20 and CD 37). The multispecific multivalent binding (fusion) protein is designed to have a binding domain 1 that binds CD20 (2H 7; VLVH orientation) and a binding domain 2 that binds CD37 (G28-1VL-VH (LH) or VH-VL (HL)). The experiments were performed to demonstrate the multispecific nature of the proteins.

Blocking studies: ramos or BJAB B lymphoblastoid cells (2.5X 10)⁵) Preincubated in 96-well V-plates together with murine anti-CD 20(25 μ g/ml) antibody or murine anti-CD 37(10 μ g/ml) antibody in staining medium (PBS with 2% mouse serum) either together or separately on ice in the dark for 45 minutes. Blocking antibodies were preincubated with cells at room temperature for 10 minutes, after which multispecific binding (fusion) protein was added at the indicated concentration range (typically 0.02 μ g/ml to 10 μ g/ml) and incubated on ice for an additional 45 minutes in the dark. Cells were washed 2 times in staining medium and incubated with Caltag (Burlingame, CA) FITC goat anti-human IgG (1:100) on ice for one hour in staining medium to detect binding of multispecific binding (fusion) protein to cells. The cells were then washed 2 times with PBS and washed with 1% trioxymethylene (catalog No. 19943, USB, Cleveland,ohio) fixed. Cells were analyzed by flow cytometry using a FACsCalibur instrument and CellQuest software (BD Biosciences, San Jose, CA). Each data set depicts the binding of 2H7-sss-hIgG-STD1-G28-1HL fusion protein in the presence of CD20, CD37, or both CD20 and CD37 blocking antibodies. Although this experiment used one of the split linkers, the presence of the two blocking antibodies completely excluded binding to the multispecific binding (fusion) protein, demonstrating that most molecules have a binding function to CD20 and CD 37. The data tested in panels a and B for two cell lines Ramos and BJAB are similar, with the CD20 blocking antibody being more effective than the CD37 blocking antibody in reducing the extent of observed binding to the multispecific binding (fusion) protein.

ADCC assay

Figure 15 shows the results of ADCC assays performed on CD20-CD37 multispecific binding (fusion) proteins. ADCC assays were performed using BJAB lymphoblastoid B cells as targets and human PBMCs as effector cells. BJAB cells were plated at 37 ℃ with 500. mu. Ci/ml in IMDM/10% FBS⁵¹Cr sodium chromate (250. mu. Ci/. mu.g) was labeled for 2 hours. The labeled cells were washed three times in RPMI.10% FBS and at 4X 10⁵Individual cells/ml were resuspended in RPMI. Heparinized human whole blood was obtained from anonymous laboratory donors and PBMCs were isolated by fractionation via a gradient of lymphocyte separation media (LSM, ICN biological). Buffy coats (Buffy coats) were harvested and washed twice in RPMI/10% FBS, followed by 5X 10⁶The final concentration of individual cells/ml was resuspended in RPMI/10% FBS. Cells were counted by trypan blue exclusion using a hemocytometer prior to use in subsequent assays. Reagent samples were added 4 times at final concentration to RPMI medium with 10% FBS and triplicate 10-fold serial dilutions were made for each reagent. The reagents were then added to a 96-well U-shaped plate at 50 μ l/well to the final concentrations indicated. Will be passed ⁵¹Cr-labeled BJAB cells were cultured at 50. mu.l/well (2X 10)⁴Individual cells/well) were added to the plate. PBMC were then assayed at 100. mu.l/well (5X 10)⁵Individual cells/well) were added to the plate, resulting in effector (PBMC): final ratio of target (BJAB)Is 25: 1. Effectors and targets were added to the simple medium to measure background kill rates. Will be passed⁵¹Cr-labeled cells added to simple Medium for measurement⁵¹Spontaneous release rate of Cr and added to medium with 5% NP40 (catalog No. 28324, Pierce, Rockford, IL) to measure⁵¹Maximum release rate of Cr. The reaction was performed in triplicate in wells of a 96-well plate. The multispecific binding (fusion) protein is added to the wells at a final concentration in the range of 0.01 μ g/ml to 10 μ g/ml (as shown). Each data set depicts different multi-specific binding (fusion) proteins or corresponding monospecific SMIPs within the titration range. The reaction was allowed to react at 37 ℃ in 5% CO before harvesting and counting₂For 6 hours. Then 25. mu.l of the supernatant from each well was transferred to a Luma Plate 96 (catalog No. 6006633, Perkin Elmer, Boston, Mass) and dried overnight at room temperature. The released CPM was measured on a PackardTopCounNXT. The specific percent kill was calculated by subtracting (cpm of samples { average of triplicate samples } -cpm spontaneous release rate)/(cpm maximum release rate-cpm spontaneous release rate) × 100. Data are plotted as% specific kill versus protein concentration. The data demonstrate that multi-specific binding (fusion) proteins are able to mediate ADCC activity against cells expressing one or more target antigens as well as the monospecificity of SMIPs to CD20 and/or CD37, but do not show an increased degree of this effector function.

Co-culture experiments

Figure 16 shows the results of experiments designed with a view to other properties of this type of multi-specific binding (fusion) protein, where a synergistic effect can be produced by expressing two binding domains against a target on the same cell or cell type by signaling/binding through two bound surface receptors. Co-culture experiments were performed using PBMCs isolated as described above for the ADCC assay. Mixing said PBMC at 2x10⁶Cells/ml, resuspended in medium at a final volume of 500. mu.l/well, and cultured alone, or using the H7 linker [2H7-sss-IgG-H7-G28-1HL]Monospecific S with CD20, CD37, CD20+ CD37 or multispecific binding (fusion) proteinsMIPs are incubated together. Each test agent was added at a final concentration of 20. mu.g/ml. After 24 hours of culture, no actual difference in% of B cells in the culture was observed; however, when the cells were subjected to flow cytometry, cell clumps were visible in the FWD X90 staining pattern of cultures containing multispecific binding (fusion) proteins, suggesting that B cells expressing the two target antigens are involved in homotypic adhesion. After 72 hours of culture, the multispecific binding (fusion) protein causes almost all B cells present to die. The combination of two monospecific SMIPs also dramatically reduced the percentage of B cells, but this amount was not seen for multispecific binding molecules. The data indicate that the addition of both the binding domain of CD20 and the binding domain of CD37 to the same multispecific molecule can produce homotypic adhesion between B cells and can also bind CD20 and CD37 antigens to the same cell. While not wishing to be bound by theory, the synergistic effect of clearing the target cells may be due to (1) binding to the same cell type via binding domains 1 and 2; and/or (2) in PBMC cultures, the effector functional domain (constant sub-region) of the multivalent binding molecule interacts with monocytes or other cell types such that killing is delayed. The kinetics of this killing effect are not rapid, requiring more than 24 hours to complete, suggesting that it may be a secondary effect that requires the production of cytokines or other molecules before the effect is observed.

Apoptosis assay

FIG. 17 shows the results of studies on the use of [2H7-sss-hIgG-H7-G28-1HL]Results of experiments designed to induce apoptosis following treatment of B cell lines with multispecific multivalent binding (fusion) proteins or monospecific CD20 and/or CD37SMIPS alone and in combination with each other. At 37 ℃ in 5% CO₂In Iscoves (Gibco) complete medium with 10% FBS and 5. mu.g/ml, 10. mu.g/ml or 20. mu.g/ml fusion protein, Ramos cells (panel A; ATCC No. CRL-1596) and Daudi cells (panel B; ATCC No. CCL-213) were cultured at 3X 10⁵The cells/ml were incubated overnight (24 hours). If the combined experiments were performed with monospecific SMIPs, the proteins were used at the following concentrations: TRU-015 (SMIP for CD 20) 10 μ G/ml, G28-1LH (SMIP for CD 37) 5 μ G/ml. OrThus, 20. mu.g/ml of TRU-015 was combined with 10. mu.g/ml of G28-1 LH. The cells were then stained with annexin V-FITC (annexin V-FITC) and propidium iodide using the BD Pharmingen apoptosis detection kit (cat # 556547) and processed according to the kit instructions. Cells were gently swirled, incubated in the dark at room temperature for 15 minutes, and diluted in 400 μ l binding buffer before analysis. Samples were analyzed by flow cytometry on a FACsCalibur (Becton Dickinson) instrument using Cell Quest software (Becton Dickinson). Data are shown as a bar graph depicting the percentage of annexin V/propidium iodide positive cells versus treatment type. Clearly, the multispecific binding (fusion) protein is capable of inducing a significantly higher degree of apoptosis in both cell lines than a monospecific agent, even when used together. This enhanced functional activity reflects a coordinated binding interaction of the BD1 and BD2 (specific for CD20 and CD 37) receptors on target cells.

Example 9

Binding and functional Properties of 2H7-hIgG-G19-4 Multi-specific binding (fusion) proteins

This example describes the binding and functional properties of a 2H7-hIgG-G19-4 multi-specific fusion protein. The construction of the molecule is described in example 7. Expression and purification was as described in the examples above.

Binding experiments were performed as described for the above molecules, except that the target cells used to measure CD3 binding were Jurkat cells expressing CD3 on their surface. Referring to FIG. 18, the top panel shows the binding curves obtained for CD20-CD3 multispecific molecules binding to Jurkat cells using purified protein serially diluted from 20 μ g/ml to 0.05 μ g/ml. G19-4 with the HL orientation appeared to bind specifically to CD3 antigen better than LH orientation. The lower panel shows the binding curves obtained for BD1 (recognizing the binding domain of CD 20). All molecules bound well and to an extent approximately equivalent to SMIP with a single specificity to CD 20.

ADCC assay

For the data shown in figure 19, ADCC assays were performed as described in the examples above. In this case, the fusion proteins were all combinations of 2H7-hIgG-G19-4 variants or monospecific SMIPs (2H 7 specific for CD 20) or antibodies (G19-4 specific for CD 3). In addition, for the data shown in the lower panel of FIG. 19, NK cells were depleted from PBMC prior to use by a magnetic bead depletion method using a MACS (Miltenyi Biotec, Auburn, CA) column separation device and NK cell-specific CD16 magnetic microbeads (catalog No. 130-045-701). The data shown in both figures demonstrate that all CD20-hIgG-CD3 multispecific molecules mediate ADCC regardless of whether NK cells are depleted or whether total PBMCs are used in the assay. For TRU015 or the combination of G19-4 and TRU015, only NK cell containing cultures may mediate ADCC. Although G19-4 could bind to NK T cells expressing CD3 and activate the cells in the first assay shown, G19-4 did not function well in any of the assays against BJAB targets, it did not express CD 3. The killing effect of the multi-specific binding (fusion) proteins observed in the lower panels is likely mediated via cytotoxicity of activated T cell populations by binding to CD3, as opposed to BJAB targets expressing CD20 antigen. This killing activity appears to be relatively insensitive to the dose of the molecule over the range of concentrations used, and even at concentrations of 0.01 μ g/ml, is still significantly different from the other molecules tested.

Example 10

Multivalent binding molecules

Other embodiments include linker domains derived from immunoglobulins. More specifically, the source sequence of the linker is a sequence obtained as follows: comparing the regions present between the V-like domains of other members of the immunoglobulin superfamily or between the V-like domains and the C-like domains. Because the sequences are typically expressed as part of the extracellular domain of a cell surface receptor, they are expected to be more stable to proteolytic cleavage and should also not be immunogenic. One type of sequence that is not expected to be suitable as a linker for multivalent binding (fusion) proteins, but is present in the intervening region between the C-like domain and the transmembrane domain, is the type of sequence expressed on surface-expressed members of the-Ig superfamily. Many of these molecules were observed to have a soluble form and to cleave at the insertion region near the cell membrane, indicating that the sequence is more susceptible to cleavage than the remaining molecules.

As described herein, the above linkers are inserted into monospecific SMIPs, between the binding domain and the effector function domain, or into one of two possible linker positions of a multivalent binding (fusion) protein.

The complete sequence listing disclosed in this application is herewith appended and incorporated by reference in its entirety. Color coding indicates that the sequence of various regions or domains in particular polynucleotides and polypeptides are useful for identifying corresponding regions or domains in the sequence of any of the molecules disclosed herein.

Example 11

Screening matrix for B-cell targeted scorpion candidates

Introduction to the design reside in

As a method to identify combinations of paired monoclonal antibody binding domains that are likely to produce useful and potent multivalent binding molecules or scorpion-like molecules against a target population, a series of monoclonal antibodies against B cell antigens were tested in an anti-B cell line combinatorial matrix representing various non-hodgkin's lymphomas. To ensure that all possible pair-wise comparisons of antibodies known or expected to bind to cells of interest are determined, a two-dimensional matrix of antibodies can be used to guide the study design using a given cell type. Known monoclonal antibodies against various B cell antigens according to their cluster name (CD) are recorded in the left column. Certain of the antibodies (named after the antigen or antigens to which they specifically bind), namely CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and CL II (MHC class II), are incubated with antigen-positive target cells, either alone or in combination with other members of the monoclonal antibody panel. The variable domains of the antibodies are contemplated as binding domains in exemplary embodiments of multivalent binding molecules. Those skilled in the art are able, using knowledge of the art and routine procedures, to identify appropriate antibody sequences (nucleic acid coding sequences as well as amino acid sequences) in, for example, publicly available databases, to form appropriate antibodies or fragments thereof (e.g., by hybridization-based cloning, PCR, peptide synthesis, and the like), and to construct multivalent binding molecules using the compounds. Sources of exemplary antibodies to obtain binding domains as described herein are provided in table 6. Cloning or synthetic strategies for obtaining CDR regions of antibody chains will generally be used, but any antibody, fragment or derivative thereof that retains the ability to specifically bind to a target antigen is still contemplated.

In more detail, the variable regions of the heavy and/or light chains from the hybridoma cloned antibodies are standard in the art. The sequence of the variable region of interest need not be known in order to obtain this region using conventional cloning techniques. See, e.g., Gilliland et al, Tissue antibodies 47 (1): 1-20(1996). To prepare single-chain polypeptides comprising a variable region that recognizes mouse or human leukocyte antigens, a method for rapid cloning and expression is devised that produces functional proteins within two to three weeks of isolating RNA from hybridoma cells. The variable region is cloned by poly-G tailing the first strand cDNA followed by a pre-poly-C anchor primer and a reverse primer anchor PCT specific for the constant region sequence. Both primers contained flanking restriction endonuclease sites for insertion into pUC 19. Formation of V for isolation of mouse, hamster and rat_LAnd V_HPCR primer set for gene. In the determination of specificity V_LAnd V_HAfter the consensus sequence of the pair, by encoding an intervening peptide linker (usually encoding (Gly)₄Ser)₃) DNA of (a) to (b) V_LAnd V_HThe genes are linked, and V is_Llinker-VH gene cassette transfer into pCDM8 mammalian expression vector. Constructs were transfected into COS cells and sFv was recovered from conditioned medium supernatant. The method has been successfully used to form antibodies against human CD2, CD3, CD4, CD8, CD28, CD40, CD45 from hybridomas producing murine, rat or hamster antibodies Functional sFv and functional sFv against murine CD3 and gp 39. Initially, sFv was expressed as hinge-C with human IgG1_H2-C_H3The fusion protein of the structural domain is favorable for rapid characterization and purification by using a goat anti-human IgG reagent or protein A. Active sfvs can also be expressed with small peptides (e.g., tags) or in a tailless form. Expression of the tailless form of the CD3(G19-4) sFv demonstrated enhanced cell signaling activity and revealed that the sFv has the potential to activate the receptor.

Alternatively, the identification of the primary amino acid sequence of the variable domain of a monoclonal antibody can be achieved directly as follows: e.g., to limit proteolysis of the antibody, followed by N-terminal peptide sequencing, e.g., using the Edman degradation method or by fragmentation mass spectrometry. N-terminal sequencing methods are well known in the art. After determining the primary amino acid sequence of the variable domain, the cDNA encoding this sequence is assembled by synthetic nucleic acid synthesis methods (e.g., PCR), followed by scFv formation. Such necessary or preferred nucleic acid manipulation methods are standard in the art.

Fragments, derivatives and analogues of the antibodies described above are also contemplated as suitable binding domains. Furthermore, any of the constant sub-regions described above are contemplated, including constant sub-regions comprising any of the hinge regions described above. Furthermore, the multivalent single-chain binding molecule described in this embodiment can include any or all of the linkers described herein.

Monoclonal antibodies were initially exposed to cells and then cross-linked using goat anti-mouse second step antibodies (step 2). Optionally, the antibody can be crosslinked prior to contacting the cell with the antibody (e.g., by crosslinking the antibody in solution). As another alternative, monoclonal antibodies can be crosslinked in solid phase as follows: by adsorption to the plastic bottom layer of the tissue culture wells or by means of goat anti-mouse antibodies adsorbed to the plastic, "trapping" on the plastic, followed by assessment of e.g. growth disruption or cell survival by well plate based assays.

The reversion of phosphatidylserine through the cytosolic side of the cell membrane to the outer cell surface of the plasma membrane is an acceptable indicator of pro-apoptotic events. Progression to apoptosis results in loss of cell membrane integrity, which can be detected by entry of a cell-impermeable intercalating dye, such as Propidium Iodide (PI). Following exposure of the cells to monoclonal antibodies, alone or in combination, dual pro-apoptotic assays were performed, and the treated cell populations were scored for cell surface positive annexin V (ANN) and/or PI inclusion.

Annexin V binding/propidium iodide internalization assay

Cells and cell culture conditions. The experiments were aimed at testing the effect of cross-linking two different monoclonal antibodies against the expressed target on four human B-cell lines. The effect on cell lines was measured by determining the extent of ANN and/or PI staining after exposure. At 37 ℃ in 5% CO ₂In (2), human B cell lines BJAB, Ramos (ATCC # CRL-1596), Daudi (ATCC # CCL-213) and DHL-4(DSMZ # ACC495) were incubated in Iscoves (Gibco) complete medium with 10% FBS for 24 hours. Before the study, the cell density was maintained at 2X 10⁵To 8X 10⁵Individual cells/ml and survival rates are generally high>95％。

At 2X 10⁵Cell density of individual cells/ml and 2. mu.g/ml of each of the comparative monoclonal antibodies were tested via a matrix against B-cell antigens. Each comparative monoclonal antibody was added at 2. mu.g/ml alone or also separately at 2. mu.g/ml in combination with each matrix monoclonal antibody. Table 6 lists the catalogue numbers and sources of monoclonal antibodies used in the experiments. For cross-linking the monoclonal antibodies in solution, goat anti-mouse IgG (Jackson Labs cat # 115-001-008) is added to each well at a concentration ratio of 2:1 (goat anti-mouse antibody: each monoclonal antibody), e.g., wells with only one monoclonal antibody (2. mu.g/ml) may have goat anti-mouse antibody added at a final concentration of 4. mu.g/ml, while wells with the comparative monoclonal antibody (2. mu.g/ml) and monoclonal antibody from matrix (2. mu.g/ml) may have goat anti-mouse antibody added at 8. mu.g/ml.

Cells were incubated at 37 ℃ in 5% CO₂After 24 hours of incubation, the cells were incubatedThe cell surface was stained with annexin V-FITC and propidium iodide using BD Pharmingen annexin V-FITC apoptosis assay kit I (# 556547). Briefly, cells were washed twice with cold PBS and at 1 × 10⁶Individual cells/ml were resuspended in "binding buffer". 100 μ l of cells in binding buffer were then stained with 5 μ l annexin V-FITC and 5 μ l propidium iodide. Cells were gently mixed and incubated in the dark at room temperature for 15 minutes. Then 400 microliters of binding buffer was added to each sample. The samples were then read on a FACsCalibur (Becton Dickinson) and analyzed using Cell Quest software (Becton Dickinson).

TABLE 6

Name (R) Directory number Suppliers of goods

anti-CD 19 #C2269-74 US Biological(Swampscott，MA)

anti-CD 20 #169-820 Ancell Corp(Bayport，MN)

anti-CD 21 #170-820 Ancell Corp(Bayport，MN)

anti-CD 22 #171-820 Ancell Corp(Bayport，MN)

anti-CD 23 #172-820 Ancell Corp(Bayport，MN)

anti-CD 30 #179-820 Ancell Corp(Bayport，MN)

anti-CD 37 #186-820 Ancell Corp(Bayport，MN)

anti-CD 40 #300-820 Ancell Corp(Bayport，MN)

anti-CD 70 #222-820 Ancell Corp(Bayport，MN)

Name (R) Directory number Suppliers of goods

anti-CD 72 #C2428-41B1 US Biological(Swampscott，MA)

anti-CD 79a #235-820 Ancell Corp(Bayport，MN)

anti-CD 79b #301-820 Ancell Corp(Bayport，MN)

anti-CD 80 #110-820 Ancell Corp(Bayport，MN)

anti-CD 81 #302-820 Ancell Corp(Bayport，MN)

anti-CD 86 #307-820 Ancell Corp(Bayport，MN)

anti-CLII DR, DQ, DP #131-820 Ancell Corp(Bayport，MN)

Table 6: antibodies against B cell antigens and their sources were used in this study.

The addition of cross-linked antibodies (e.g., goat anti-mouse antibodies) to monoclonal antibody a alone resulted in enhanced cell sensitivity, suggesting that multivalent binding molecules or scorpion molecules constructed with two binding domains that recognize the same antigen may be effective in enhancing cell sensitivity. Without wishing to be bound by theory, this enhanced sensitivity may be due to antigen aggregation and modified signaling. TNF receptor family members, for example, require homomultimerization for signal transduction, and scorpion molecules with equivalent binding domains at each end of the molecule can facilitate this interaction. The aggregation and subsequent signaling of CD40 is an example of this phenomenon in the B cell system.

As shown in figures 20, 21 and 22, the addition of monoclonal antibody a and monoclonal antibody B against different antigens may produce an additive pro-apoptotic effect on the treated cells, or in some combinations a greater than additive (i.e., synergistic) pro-apoptotic effect. In FIG. 20, for example, anti-CD 20 in combination with monoclonal antibodies against other B cell antigens all gave varying degrees of enhancement in cell sensitivity. However, certain combinations (such as anti-CD 20 in combination with anti-CD 19, or anti-CD 20 in combination with anti-CD 21) produced greater than additive pro-apoptotic effects, suggesting that multivalent binding molecules or scorpion molecules consisting of the binding domains may be particularly effective in clearing transformed B cells. Referring to fig. 20, the percentage of cells showing pro-apoptotic activity when exposed to anti-CD 20 antibody alone was about 33% (corresponding to the vertical bar of "20", i.e., anti-CD 20 antibody); the percentage of pro-apoptotic cells was about 12% when exposed to anti-CD 19 antibody (vertical bar corresponding to "19" in figure 20, i.e. anti-CD 19 antibody); and the percentage of pro-apoptotic cells when exposed to anti-CD 20 and anti-CD 19 antibodies was about 73% (horizontal bar corresponding to "19" in figure 20). After exposure to both antibodies, the number of pro-apoptotic cells was 73%, significantly greater than 45% (33% + 12%) of the sum of the effects produced by each antibody alone, indicating that the anti-CD 19 and anti-CD 20 antibody pairs may produce a synergistic effect. Suitable multivalent binding molecules include molecules in which two binding domains can exert an additive effect on B-cell behavior and multivalent binding molecules in which two binding domains can exert a synergistic effect on B-cell behavior. In certain embodiments, one binding domain will have no detectable effect on the measured cell behavior parameter, with each of the paired binding domains contributing to a different aspect of multivalent binding molecule activity, such as a multispecific multivalent binding molecule (e.g., binding domain a binds to a target cell and promotes apoptosis, while binding domain B binds to a soluble therapeutic agent, such as a cytotoxin). Depending on the design of the multivalent binding molecule, the type of combined effect (additive, synergistic, or inhibitory effect) produced by the two binding domains on the target cell may not be relevant, as one of the binding domains may be specific for a non-cellular (e.g., soluble) binding partner or specific for a cell-related binding partner, but not for a different cell type.

Exemplary binding domain pairings that produce additive, synergistic, or inhibitory effects (as shown in figures 20-23) are apparent from tables 7 and 8. Table 7 provides quantitative data on the percentage of cells staining positive for ANN and/or PI extracted from each of figures 20-23. Table 8 provides values calculated using the data of table 7, based on which it can be determined whether the interaction of a given antibody pair produces an additive, synergistic or inhibitory effect, as assessed by the percentage of cells staining positive for ANN and/or PI.

TABLE 7

Name (R)	anti-CD 20	anti-CD 79b	anti-CL II	anti-CD 22
					anti-CD 19	13/73*	18/76/66	14/47/46	12/11
anti-CD 20	33/NA	42/94/92	33/71/76	28/33
					anti-CD 21	14/75	22/50/76	18/24/40	11/11
anti-CD 22	8/55	12/39/33	12/19/17	10/12
					anti-CD 23	8/41	12/63/55	14/22/17	10/12
anti-CD 30	8/38	14/72/61	12/56/61	10/11
					anti-CD 37	15/45	19/92/86	20/60/62	19/20
anti-CD 40	10/48	12/44/30	13/21/28	14/13
					anti-CD 70	9/40	12/56/39	15/21/15	10/10
anti-CD 72	NA	16/60/64	30/78/63	17/17

Name (R)	anti-CD 20	anti-CD 79b	anti-CL II	anti-CD 22
					anti-CD 79a	21/66	43/42/50	28/55/51	14/14
anti-CD 79b	46/88	70/70/68	45/80/76	26/16
					anti-CD 80	7/41	14/35/30	15/19/17	11/11
anti-CD 81	14/65	25/86/83	25/54/43	19/20
					anti-CD 86	7/38	16/58/42	15/24/18	14/11
anti-CL II	53/77	52/96/98	47/52/43	72/57

^*The values in columns 2-4 of table 7 reflect the height of the histogram bars in fig. 20-22, respectively, the first value for each cell represents the height of the vertical bar, the second value represents the height of the horizontal bar, and the third value (if present) reflects the height of the stippled bar. In column 5, the first value reflects the height of a solid bar and the second value reflects the height of a diagonal bar in fig. 23.

TABLE 8

Name (R)	anti-CD 20	anti-CD 79b	anti-CL II	anti-CD 22
					anti-CD 19	S：13+33＝46*	A：18+56＝74A：18+43＝61	S：14+26＝40S：14+18＝32	I：12+10＝22
anti-CD 20	NA	A：42+56＝98A：42+43＝85	S：33+26＝59S：33+18＝51	A/I：28+10＝38
					anti-CD 21	S：14+33＝47	I：22+56＝78S：22+43＝65	I：18+26＝44A：18+18＝36	I：11+10＝21
anti-CD 22	S：8+33＝41	I：12+56＝68I：12+43＝55	I：12+26＝38I：12+18＝30	NA
					anti-CD 23	A：8+33＝41	A：12+56＝68A：12+43＝55	I：14+26＝40I：14+18＝32	I：10+10＝20
anti-CD 30	A：8+33＝41	A：14+56＝70A：14+43＝57	S：12+26＝38S：12+18＝30	I：10+10＝20
					anti-CD 37	A：15+33＝48	S：19+56＝75S：19+43＝62	S：20+26＝46S：20+18＝38	I：19+10＝29
anti-CD 40	A/S：10+33＝43	I：12+56＝68I：12+43＝55	I：13+26＝39A：13+18＝31	I：14+10＝24
					anti-CD 70	A：9+33＝42	I：12+56＝68I：12+43＝55	I：15+26＝41I：15+18＝33	I：10+10＝20
anti-CD 72	NA	I：16+56＝72A：16+43＝59	S：30+26＝56S：30+18＝48	I：17+10＝27

Name (R)	anti-CD 20	anti-CD 79b	anti-CL II	anti-CD 22
					anti-CD 79a	S：21+33＝54	I：43+56＝99I：43+43＝86	A：28+26＝54A：28+18＝46	I：14+10＝24
anti-CD 79b	S：46+33＝79	NA	S：45+26＝71S：45+18＝63	I：26+10＝36
					anti-CD 80	A：7+33＝40	I：14+56＝70I：14+43＝57	I：15+26＝41I：15+18＝33	I：11+10＝21
anti-CD 81	S：14+33＝47	A：25+56＝81S：25+43＝68	A：25+26＝51A：25+18＝43	I：19+10＝29
					anti-CD 86	A：7+33＝40	I：16+56＝72I：16+43＝59	I：15+26＝41I：15+18＝33	I：14+11＝25
anti-CL II	I：53+33＝86	A：52+56＝108A：52+43＝95	NA	I：72+10＝82

"A" means that an "addition" effect is observed;

"S" means that a "synergistic" effect is observed;

"I" means that an "inhibitory" effect is observed;

^*schematic equation: a + B ═ C, where "a" is the percentage of ANN and/or PI positive cells due to matrix antibody alone, "B" is the percentage of ANN and/or PI positive cells due to common antibodies (anti-CD 20 in fig. 20, anti-CD 79B in fig. 21, anti-CLII in fig. 22 and anti-CD 22 in fig. 23), and "C" is the predicted additive effect. (see Table 7 above for quantitative data corresponding to FIGS. 20-23). If two equations exist in the cell, the upper equation reflects the results obtained with the higher indicated concentration of common antibody; the following equation reflects the results obtained using a lower indicated concentration of common antibody.

In certain embodiments, the two binding domains interact in an inhibitory, additive, or synergistic manner in a sensitized (or desensitized) target cell, such as a B cell. Figure 23 shows the protective or inhibitory effect of combining an anti-CD 22 antibody with a potent pro-apoptotic monoclonal antibody, such as an anti-CD 79b antibody or an anti-MHC class II (i.e., anti-CL II) antibody. For example, fig. 23 and table 7 show that anti-CD 22 antibody alone induced no more than about 10% of cells exhibiting pro-apoptotic behavior (the solid bars corresponding to "22" in fig. 23) and anti-CD 79b induced about 26% of pro-apoptotic cells (the solid bars corresponding to "CD 79 b" in fig. 23). However, the combination of anti-CD 22 and anti-CD 79b induced only about 16% of pro-apoptotic cells (diagonal bars corresponding to "79 b" in fig. 23). Thus, the combined antibodies induced 16% of pro-apoptotic cells, which was less than 38% of the sum of the effects produced by anti-CD 22 (12%) and anti-CD 79b (26%) alone. Examination of fig. 23 and/or tables 7-8 using this approach revealed that anti-CD 22 antibodies and multispecific multivalent binding molecules comprising an anti-CD 22 binding domain via extension, will achieve an inhibitory overall effect when used in independent combination with each of the following antibodies (or corresponding binding domains): anti-CD 19, anti-CD 20, anti-CD 21, anti-CD 23, anti-CD 30, anti-CD 37, anti-CD 40, anti-CD 70, anti-CD 72, anti-CD 79a, anti-CD 79b, anti-CD 80, anti-CD 81, anti-CD 86 and anti-MHC class II antibodies/binding domains.

Without wishing to be bound by theory, the data can be explained as follows: demonstrating that an anti-CD 22 antibody or a multispecific multivalent binding molecule comprising an anti-CD 22 binding domain can prevent or mitigate any of the antibody effects listed immediately above. More generally, multispecific multivalent binding molecules comprising an anti-CD 22 binding domain will inhibit the effects of interaction with any one of CD19, CD20, CD21, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and MHC class II molecules. As can be seen in fig. 23 and table 8, the anti-CD 22 antibody and any antibody/binding domain that recognizes a B-cell surface marker (such as a CD antigen) will function to inhibit or mitigate the activity via the extension binding domain (comprising the anti-CD 22 binding domain). Multivalent binding molecules, including multispecific multivalent binding molecules, are expected to be useful in improved treatment regimens for a variety of diseases where it is desirable to attenuate or control the activity of the binding domain.

In addition to the two binding domains that interact with the target cell, typically producing an inhibitory, additive, or synergistic combined effect via binding of cell surface ligands, the experimental results disclosed herein demonstrate that a given binding domain pair can provide different types of combined effects depending on the relative concentrations of the two binding domains, thereby enhancing the versatility of the present invention. For example, table 8 reveals that anti-CD 21 interacts in an inhibitory manner with anti-CD 79b at higher test concentrations of anti-CD 79b, but the two antibodies interact in a synergistic manner at lower test concentrations of anti-CD 79 b. Although certain embodiments will use a single type of multivalent binding molecule, i.e., a monospecific multivalent binding molecule comprising, for example, a single CD21 binding domain and a single CD79b binding domain, the present invention includes mixtures of multivalent binding molecules that will allow the relative binding domain concentrations to be adjusted to achieve a desired effect, such as an inhibitory, additive, or synergistic effect. Furthermore, the methods of the invention comprise the use of a single multivalent binding molecule in combination with another binding molecule (such as a conventional antibody molecule) to adjust or optimize the relative concentration of the binding domains. One skilled in the art will be able to determine the applicable relative concentrations of binding domains using standard techniques (e.g., by designing two experimental serial dilutions of the matrix, one for each binding domain).

Without wishing to be bound by theory, it is understood that binding of one ligand may induce or modulate surface expression of a second ligand on the same cell type, or it may alter the surface environment of the second ligand thereby altering its sensitivity to binding to a specific binding molecule (such as an antibody or multivalent binding molecule).

Although the use of B cell lines and antigens is exemplified herein, the methods of optimally determining effective multivalent binding molecules (i.e., scorpion molecules) are applicable to other disease contexts and target cell populations, including other normal cells, their abnormal cellular counterparts, including chronically stimulated hematopoietic cells, cancer cells, and infected cells.

Other signaling phenotypes (such as Ca) may also be used in the method of screening for direct effects of monoclonal antibody combinations²⁺Moving; tyrosine phosphorylation regulation; caspase (caspase) activation; NF-KB activation; cytokine, growth factor or chemokine processing; or gene expression (e.g., in a reporter gene system)).

As an alternative to using secondary antibodies to crosslink the primary antibody and mimic multivalent binding molecules or scorpion structures, other molecules that bind the Fc portion of the antibody, including soluble Fc receptors, protein a, complement components (including C1q), mannose-binding lectins, beads or matrices containing reagents or crosslinkers, bifunctional chemical crosslinkers, and plastic adsorbents can be used to crosslink multiple monoclonal antibodies against the same or different antigens.

Example 12

Multivalent binding proteins with effector function or scorpion-like molecule structure

The general schematic structure of a scorpion polypeptide is H2N-binding domain 1-scorpion linker-constant sub-region-binding domain 2. Scorpion molecules may also have a hinge-like region, typically a peptide region derived from an antibody hinge, disposed at the N-terminus of binding domain 1. In certain scorpion embodiments, binding domain 1 and binding domain 2 are each derived from an immunoglobulin binding domain, e.g., from V_LAnd V_H。V_LAnd V_HTypically via a linker. Experiments have aimed to demonstrate that scorpion polypeptides may have binding domains that differ from the immunoglobulin binding domains, including the Ig binding domain from which the scorpion binding domain is derived, by amino acid sequence differences resulting in a sequence divergence of typically less than 5% and preferably less than 1% relative to the Ig binding domain from which it is derived.

Typically, sequence differences result in single amino acid changes, such as substitutions. Preferred positions for the amino acid changes are in one or more regions of the corresponding scorpion binding domain or show at least 80% and preferably 85% or 90% sequence identity to the Ig Complementarity Determining Regions (CDRs) of the Ig binding domain from which the scorpion binding domain is derived. Further guidance is provided by comparing models of peptides binding to the same target (such as CD 20). Relative to CD20, epitope mapping has revealed that 2H7 antibodies that bind CD20 recognize the Ala-Asn-Pro-Ser (ANPS) motif of CD20, and that scorpion molecules that bind CD20 are also expected to recognize this motif. Amino acid sequence changes resulting from deep embedding of the ANPS motif in the pocket formed by the scorpion binding domain region (corresponding to the Ig CDR) are expected to be functional binders to CD 20. Model studies have also revealed that the corresponding CDR3 (V) _L)、CDR1-3(V_H) Scorpion breedingThe conformational region contacts CD20, and changes that are expected to maintain or contribute to the contact can result in scorpion molecules that bind CD 20.

In addition to promoting the interaction of scorpion with its target, it is contemplated that promotion in the binding domain of scorpion corresponds to Ig V_LAnd V_HSequence changes in the interaction between scorpion binding domain regions of the domains (relative to homologous Ig binding domain sequences). For example, in the case of corresponding to V_LIn the region of the scorpion binding CD20, the sequence SYIV may be altered by substituting Val (V33) with an amino acid such as His, resulting in the sequence SYIH. This variation is expected to improve for V_LAnd V_HInteractions between scorpion-like molecular regions of the domains. In addition, it is expected to correspond to V_HThe addition of residues at the N-terminus of the scorpion region of CDR3 will alter the orientation of this scorpion region, which may affect its binding characteristics, since V_HThe N-terminal Ser of CDR3 in contact with CD 20. Conventional assays reveal that those orientations can produce the desired changes in binding characteristics. It is also expected that corresponding to V_H-CDR2 and/or V_HMutations in the scorpion region of CDR3 will form potential novel contacts with targets such as CD 20. For example, based on model studies, the expected substitution corresponds to V_HAny of Y105 and W106 (present in the sequence NSYW) in the region of CDR3 will alter the binding characteristics of the scorpion in a manner that is amenable to routine determination for use in identifying scorpions with modified binding characteristics. Also for example, it is expected that changes in the sequence of the scorpion binding domain corresponding to Ig VL-CDR3, such as Trp (W) in the sequence CQQW, will affect binding. Typically, for those scorpion molecules that show enhanced affinity for the target, changes in the scorpion region corresponding to the Ig CDRs are screened.

Based on the model structure of humanized CD20 scFv binding domain 20-4, based on published information relating to the extracellular loop structure of CD20 (Du et al, J biol. chem.282 (20): 15073-80(2007)), and based on the CD20 binding epitope recognized by the mouse 2H7 antibody, which is the source of the CDRs of the humanized 20-4scFv binding domain, engineered mutations in the CDR regions of the 2Lm20-4x2Lm20-4 scorpion in order to improve its affinity for binding to CD 20. First, mutations were designed that affect the 20-4CDR configuration and promote more efficient binding to the CD20 extracellular loop. Second, an introductive variation was designed that could provide a novel intermolecular interaction between the 2Lm20-4x2Lm20-4 scorpion and its target. The mutation comprises: VL CDR 1V 33H (i.e. substitution of Val with His at position 33 of CDR1 of the VL region), VL CDR 3W 90Y, VH CDR 2D 57E, VH CDR3 insert V, VH CDR 3Y 101K, VH CDR 3N 103G, VH CDR 3N 104G and VH CDR 3Y 105D after residue S99. Since it is expected that combining some of these mutations will produce a synergistic effect, 11 mutants combining different mutations as shown in table 9 were designed (the residues introduced by the mutations are bolded and underlined).

TABLE 9

VL CDR1	VL CDR3	VH CDR2	VH CDR3
				RASSSVSYIH	QQWSFNPPT	AIYPGNGDTSYNQKFKG	SVYYSNYWYFDL
RASSSVSYIH	QQWSFNPPT	AIYPGNGDTSYNQKFKG	SVYYGGYWYFDL
				RASSSVSYIH	QQWSFNPPT	AIYPGNGDTSYNQKFKG	SYYSNSDWYFDL
RASSSVSYIH	QQWSFNPPT	AIYPGNGDTSYNQKFKG	SYYSGGDWYFDL
				RASSSVSYIV	QQWSFNPPT	AIYPGNGDTSYNQKFKG	SYKSNSYWYFDL
RASSSVSYIV	QQWSFNPPT	AIYPGNGETSYNQKFKG	SYYSNSYWYFDL
				RASSSVSYIV	QQYSFNPPT	AIYPGNGDTSYNQKFKG	SYYSNSYWYFDL
RASSSVSYIH	QQWSFNPPT	AIYPGNGDTSYNQKFKG	SYKSNSDWYFDL
				RASSSVSYIH	QQWSFNPPT	AIYPGNGETSYNQKFKG	SYYSNSDWYFDL
RASSSVSYIH	QQYSFNPPT	AIYPGNGDTSYNQKFKG	SYYSNSDWYFDL
				RASSSVSYIH	QQYSFNPPT	AIYPGNGETSYNQKFKG	SYKSGGDWYFDL

Mutations were introduced into the binding domain of a scorpion molecule of CD20xCD20 (2Lm20-4x2Lm20-4) by PCR mutagenesis using primers encoding altered sequence regions. After sequence verification, the DNA fragment encoding the 2Lm20-4scFv fragment with the corresponding mutation was cloned into a conventional expression vector containing the coding region of the constant sub-region of the scorpion, thereby generating a polynucleotide containing the complete DNA sequence of the novel 2Lm20-4x2Lm20-4 scorpion. Variants of 2Lm20-4x2Lm20-4 scorpion with CDR mutations were generated by expression in a transient COS cell system and purified via protein a and Size Exclusion (SEC) chromatography. The binding properties of 2Lm20-4x2Lm20-4 scorpion variants were evaluated by FACS analysis using primary B-cells and WIL2-S B-lymphoma cell lines.

Other mutants have also been formed using similar methods to optimize the CD20 binding domain. The CD20SMIP designated TRU015 serves as a substrate for mutant formation and, unless stated to the contrary, all domains are human domains. The following mutants were found to contain a functional CD20 binding domain suitable for use. 018008 molecules contain Q in place of S at position 27 in CDR1 in VL (one letter amino acid encoding), S in place of T at position 28 in CDR1 of VH and L in place of V at position 102 in CDR3 of VH. Following the modular design of scorpion molecules, the following partial scorpion linker sequences corresponding to the CCCP sequence in the IgG1 hinge were independently combined with mutated VL and VH: CSCS, SCCS, and SCCP. 018009 molecules contain a Q substituted for S at position 27 of CDR1 of VL, a S substituted for T at position 28 of CDR1 of VH, and in CDR3 of VH a S substituted for V at position 96, a L substituted for V at position 102 and a V deleted at position 95. 018009 the same scorpion linker subsequence as found in the scorpion linker used in 018008 was used. 018010 molecules contain, in the CDR1 of VL, Q with a substitution of S at position 27, I with a substitution of M at position 33 and V with a substitution of H at position 34, and S with a substitution of T at position 28 of CDR1 of VH and L with a substitution of V at position 102 of CDR3 of VH. The scorpion linker defined by the CSCS and SCCS subsequences is suitable for use at 018010. 018011 contains the same mutations as described at 018010 in the CDR1 of VL and in the CDR1 of VH, and in the CDR3 of VH, V is deleted at position 95, S with substitution V at position 96 and L with substitution V at position 102. Scorpion linkers, defined by the CSCS, SCCS and SCCP subsequences, were used in the 018011 molecule. 018014VL is an unmutated mouse VL in which the human VH contains a change in the S-substituted T at 28 of CDR1 and a change in the L-substituted V at 102 of CDR 3. 018015 also contains an unmutated mouse VL and a human VH containing a change in S-substituted T at 28 of CDR1, and in CDR3, V is deleted at 95, S substituted V at 96 and L substituted V at 102. The 2Lm5 molecule had Q substituted for S at 27 in the CDR1 of VL; in CDR1 of VH, F with substitution Y at 27 and S with substitution T at 30; and in CDR3 of VH, V is deleted at 95, S with substitution V at 96, and L with substitution V at 102. The scorpion connectors defined by CSCS, SCCS and SCCP are used independently in each of 018014 and 018015. 2Lm5-1 was identical to 2Lm5, except that 2Lm5-1 had no mutations in the CDRs 1 of the VH and only scorpion linkers defined by CSSS subsequences were used. 2Lm6-1 had a mutation of 2Lm5 and in CDR3 of VL, T with substitution S at 92 and S with substitution F at 93, and only scorpion linker defined by CSSS subsequence was used. Only the mutation in 2Lm16 was a mutation in the CDR3 of the VH listed above for 2Lm 5-1. The scorpion linker defined by subsequences CSCS, SCCS and SCCP was used independently in 2Lm 16. 2Lm16-1 has Q substituted for S at 27 in CDR1 of VL; and in CDR3 of VL, T with substitution S at 92 and S with substitution F at 93; and VH, in CDR3, lacks V at 95, has S in place of V at 96, and has L in place of V at 102; only scorpion linkers defined by CSSS subsequences were used. 2Lm19-3 has, in CDR1 of VL, Q with substitution S at 27, I with substitution M at 33 and V with substitution H at 34, and mutations in CDR3 with the VH listed for 2Lm 16-1. The scorpion linker defined by subsequences CSCS, SCCS and SCCP was used independently in 2Lm 19-3. The 2Lm20-4 molecule contained mutations in the CDR1 of VL, I substituted for M at 33 and V substituted for H at 34, and in the CDR3 for the VH listed for 2Lm 16-1. For 2Lm5-1, 2Lm6-1, 2Lm16, 2Lm16-1, 2Lm19-3 and 2Lm20-4, S is also present in place of L at position 11 of the framework regions of the VH. The scorpion linker defined by the CSCS, SCCS and SCCP subsequences was used independently in 2Lm 20-4. Finally, S substituted for P is present at position 331 in the following mutants: 018008 wherein the scorpion linker is defined by CSCS; 018009 wherein each scorpion linker is defined by CSCS and SCCP; 018010 wherein the scorpion linker is defined by CSCS; 018011 wherein the scorpion linker is defined by SCCP; 018014 wherein the scorpion linker is defined by CSCS; 018015 wherein the scorpion linker is defined by CSCS; 2Lm16 wherein the scorpion connector is defined by any one of CSCS, SCCS and SCCP; wherein the scorpion linker is 2Lm19-3 defined by CSCS or SCCP; and 2Lm20-4 where the scorpion linker is defined by CSCS or SCCP.

In addition, two of the binding domains are contemplatedIndividual domains (e.g. scorpion binding domains corresponding to Ig V_LAnd V_HRegion(s) of the linker length. For example, a C-terminal Asp in which the interdomain linker was found to be removed is expected to affect the binding characteristics of the scorpion, as is the substitution of Gly for Asp.

Also contemplated are scorpion molecules with extended scorpion linkers (inserted between the C-terminus and the constant sub-region and between the N-terminus and the binding domain 2) relative to the hinge region of Ig, in which amino acid residues are added between the C-terminus and any cysteine (corresponding to Ig hinge cysteine) in the scorpion molecule, wherein the scorpion molecule cysteine is capable of forming interchain disulfide bonds. Scorpion-shaped molecules containing such characteristics have been constructed and characterized below.

Efforts have been made to improve the expression, stability and efficacy of scorpion molecules by optimizing scorpion linkers that covalently link constant subregions to C-terminally disposed binding domains 2. Prototype scorpion molecules for optimization studies contained N-terminal pairs derived from IgG 1C_H2And C_H3An anti-CD 20 scFV (binding Domain 1) fused to the constant sub-region of (A) and a secondary antibody-CD 20 scFv fused C-terminally to this constant sub-region. The scorpion molecules (e.g., immunoglobulin molecules) are expected to bind via constant regions (or subregions) to form homodimeric complexes with peptide chains linked by disulfide bonds. To obtain a high degree of expression of the tetravalent stable molecule with high affinity for its CD20 target, the scorpion linker between the constant sub-region and the second binding domain must satisfy the following considerations. First, steric hindrance between the homologous binding domains carried by the two scFv fragments (one scFv fragment on each of the two scorpion monomers) should be minimized in order to facilitate maintenance of the native conformation of each binding domain. Second, the configuration and orientation of the binding domains should be such that each domain can productively bind and each binding domain can bind with high affinity to its target. Third, the scorpion linker itself should be relatively protease resistant and non-immunogenic.

In the exemplary CD20xCD20 scorpion construct S0129, C_H3C-terminal of (2) and second antibody-CThe D20scFV domains were linked via a 2H7 scorpion linker (a peptide derived from and corresponding to a fragment of the native human hinge sequence of IgG 1). The 2H7 scorpion linker served as the basis for design studies using computer-assisted models aimed at improving the expression of scorpion molecules and improving the binding characteristics of the expressed molecules.

To analyze the 2H7 scorpion linker, the 3-dimensional structure of the dimeric form of the human IgG1 hinge was modeled using the Insight II software. Having a V_H-V_LThe crystal structure of the oriented anti-CD 20scFV was chosen as the reference structure for the 20-4 binding domain (RCSB protein database entry code: 1A 14). In intact IgG1, the hinge makes C_H1C-terminal and C of the Domain_H2The domains are linked at their N-termini, and the configuration of each domain is such that the hinge cysteine residues can be paired to form a homodimer. In an exemplary scorpion, the hinge-derived 2H7 linker was derived from IgG 1C_H3C-terminal of Scorpion-like molecular Domain of Domain and derived from IgG 1V_H2The N-terminal end of this portion of scorpion binding domain 2 of the domain is linked. Using V_H-V_LThree-dimensional model structure of scFV, the optimal distance between the C-termini of 2H7 linkers is expected to be influenced by three considerations. First, hinge stability must be maintained, and stability can be supported by dimerization (e.g., homodimerization), meaning that the hinge cysteines must be able to pair in the presence of two fold binding domains. Second, two binding domains (e.g., scFV) must meet the requirement that the C-terminus of the 2H7 linker does not create steric interference so that the protein can fold properly. Third, the CDRs of each binding domain should be able to face the same direction (as in a natural antibody) because each binding domain of the prototype scorpion can bind to a proximity receptor (CD20) on the same cell surface. For this consideration, the distance between the two N-termini of the scFv is expected to be about 28 . In the dimeric scorpion form, the distance between the C-termini of the theoretically designed 2H7 linkers is expected to be about 16. To meet the distance expected to be required to optimize the potency of the scorpion, the C-terminus of the 2H7 linker was extended by at least 3 amino acids. This extension is expected to allow the formation of a disulfide bond between the cysteine residues of the 2H7 linker, allowing the C-terminal binding domain 2 to fold properly and contribute to the correct orientation of the CDRs. Furthermore, in intact IgG1, due to the presence of a C between the hinge and the binding domain_H1And V_L1The distance between the domains, and therefore the binding domains carried by the two chains, is further increased and is expected to further facilitate cross-linking of adjacent receptors on the same cell surface. In view of the above considerations, a set of linkers with different lengths was designed (table 10). To minimize immunogenicity, C was used_H2The 2H7 scorpion linker was extended by the addition of a sequence to the C-terminus of the scorpion linker to the natural residues (Ala-Pro-Glu-Leu or APEL) present at the N-terminus of the domain. The longer construct contains one or more (Gly4Ser) linker units known to be protease tolerant and flexible.

C in constant sub-region_H3The CD20 xcd 20 scorpion construct containing an expanded scorpion linker between the domain and the C-terminal scFv binding domain was constructed using PCR mutagenesis and subcloned into conventional mammalian expression vectors. The effect of linker length on CD20 × CD20 scorpion expression can be analyzed by comparing the yield of secreted protein in transient expression experiments using COS or HEK293 cells, or by Western blot analysis or using [35 ] ]Pulse-chase studies of S-tagged methionine/cysteine protein synthesis and accumulation were analyzed to analyze the effect of linker length on CD20 × CD20 scorpion expression.

Watch 10

Construct numbering	Scorpion-shaped linker core (2H 7)) Sequence of	Spreading sequences	Expanded scorpion linker sequences
				1	GCPPCPNS	APEL	GCPPCPNSAPEL
2	GCPPCPNS	APELGGGGS	GCPPCPNSAPELGGGGS
				3	GCPPCPNS	APELGGGGSGGGGS	GCPPCPNSAPELGGGGSGGGGS
4	GCPPCPNS	APELGGGGSGGGGSGGGGS	GCPPCPNSAPELGGGGSGGGGSGGGGS

Glycosylated scorpion molecules are also contemplated, and in the present context, host cells expressing scorpion molecules can be cultured in the presence of a carbohydrate modifier, defined herein as a small molecule organic compound, preferably having a molecular weight of less than 1000 daltons, which can inhibit the activity of enzymes involved in the addition, removal or modification of sugars which are part of carbohydrates attached to polypeptides, such as occurs during maturation of N-linked carbohydrates of proteins. Glycosylation is a complex process that occurs in the endoplasmic reticulum ("core glycosylation") and the Golgi apparatus ("end glycosylation"). Various glycosidase and/or mannosidase inhibitors provide one or more of the following desired effects: enhanced ADCC activity, enhanced Fc receptor binding and altered glycosylation patterns. Exemplary inhibitors include, but are not limited to, castanospermine (castanospermine) and inhibition of alpha-mannosidase (kifunensine). The effect of expressing scorpion in the presence of at least one such inhibitor is disclosed in the examples below.

Example 13

Scorpion-shaped molecular protein expression degree and characterization

The extent of scorpion protein expression was determined and the expressed protein was characterized to demonstrate that protein design can produce products with practical benefits. Monospecific CD20 × CD20 scorpion and bispecific CD20 × CD37 scorpion were expressed in CHO DG44 cells in culture using conventional techniques.

A baseline degree of stable expression of the CD20 × CD20 scorpion molecule S0129(21m20-4 × 21m20-4) was observed in CHO DG44 cells cultured in the presence of various supplemental feeds, as shown in FIG. 34. All media contained 50nM methotrexate, at a concentration that maintained the copy number of the scorpion encoding polynucleotide. The polynucleotide contains a coding region for a scorpion protein that is not codon optimized for expression in CHO DG44 cells. The polynucleotides were introduced into cells using the pD18 vector. As is apparent from FIG. 34, a degree of expression of about 7-46. mu.g/ml was obtained.

The extent of expression following amplification of the polynucleotide encoding the bispecific CD20xCD37 scorpion was also determined. The CD20xcd37 scorpion coding region was cloned using the pD18 vector and the plasmid was introduced into CHO DG44 cells. Amplification of the encoded polynucleotides is achieved using the dhfr-methotrexate technique known in the art, in which increasing concentrations of MTX are used to select for increased copy numbers of the dihydrofolate reductase gene (dhfr) and thereby co-amplify the tightly linked polynucleotides of interest. FIG. 35 shows that stable expression of bispecific CD20 × CD37 scorpion was observed to a degree typically of about 22-118 μ g/ml. Yield variations were observed under different conditions (including methotrexate concentration for amplification), but could easily be optimized by the skilled person. Various other scorpion molecules described herein were also subjected to expression analysis in CHO and/or COS cells, with the results of the analysis provided in table 11 below. The results demonstrate that high yields of scorpion protein can be obtained using conventional techniques and conventional amplification optimization techniques.

The expressed proteins were also characterized by SDS-PAGE analysis to assess the degree of homology and integrity of the expressed proteins and to verify the molecular weight of the monomeric peptides. Denaturing polyacrylamide gels (4-20% Tris glycine) were electrophoresed under reducing and non-reducing conditions. The results shown in figure 36 reveal a single protein band for each of 2Lm20-4 SCC SMIPs and S1000(CD20(21m20-4) xCD20(21m20-4) monospecific scorpion S0126) with the expected monomer molecular weight under reducing conditions. The data indicate that SMIPs and scorpion molecules are susceptible to purification in intact form. Under non-reducing conditions, trace peptides were observed to be consistent with monomeric SMIPs of the expected size, with most of the protein appearing as a single distinct band consistent with the dimeric structure. Under the non-reducing conditions, the monospecific scorpion protein showed a single distinct band with a molecular weight consistent with the dimer structure. The dimeric structure of SMIPs and scorpion molecules is identical to their monomeric structure and each contains a hinge-like scorpion linker containing at least one cysteine capable of participating in disulfide bond formation.

The effect of scorpion linkers on the expression and integrity of scorpion molecules was also evaluated and the results are shown in table 12. This table lists scorpion linker variants of monospecific CD20xCD20(2Lm20-4x2Lm20-4) S0129 scorpion and CD20xCD 28S 0033 scorpion (2H7sccpig 1-H7-2e12) with the same integrity as single stranded molecules and their degree of transient expression in COS cells relative to the parent scorpion S0129 or S0033 with H7 linker (set to 100%) as required. Table 13 provides data from the evaluation of scorpion linker variants incorporated into CD20 × CD20 scorpion molecules, as well as similar data for CD20 × CD28 scorpion molecules. Table 13 provides data from the evaluation of S0129 variants containing scorpion linkers other than hinge-like linkers containing at least one cysteine capable of forming a disulfide bond; in contrast, the scorpion linker in the molecule is derived from the type II C-lectin stem region. As is apparent from the data shown in table 13, the hinge-like scorpion linker can bind to scorpion molecules expressed to a greater or lesser extent than the unmodified parent scorpion linker in transient expression assays. In addition, certain linker variants exhibit greater resistance to proteolytic cleavage than the unmodified parent linker, which correlates to all or nearly all of the expressed protein. The data of table 13 show that non-hinge-like linkers, such as linkers derived from the stem region of type II C-lectins, are present in scorpion molecules that exhibit slightly different binding characteristics than scorpion molecules containing hinge-like scorpion linkers. Furthermore, scorpion molecules containing non-hinge-like scorpion linkers exhibit equivalent or superior effector function (ADCC) as compared to ADCC associated with scorpion molecules having hinge-like scorpion linkers.

TABLE 11

Linker name	Upstream (CH3) sequence	S0129(2Lm20-4 x 2Lm 20-4) linker variant-amino acid sequence1	Based on	#AAs	Expression of COS2	Cracking3	Expression of CHO2
								H7	QKSLSLSPGK	GCPPCPNS	H7	18	100	-	100
H16	QKSLSLSPGK	LSVKADFLTPSIGNS	CD80-	25	174	+
								H18	QKSLSLSPGK	LSVLANFSQPEIGNS	CD86	25	165	++
H19	QKSLSLSPGK	LSVLANFSQPEISCPPCPNA	CD86+H7	30	161	+	109
								H26	QKSLSLSPGK	RIHQMNSELSVLANS	CD86	25	170	++
H32	QKSLSLSPGK	RIHLNVSERPFPPNS	CD22	25	184	++
								H47	QKSLSLSPG	LSVKADFLTPSIGNS	H16	24	141	-	206

H48	QKSLSLSPG	KADFLTPSIGNS	H16	21	137	-
								H50	Q	LSVLANFSQPEIGNS	H18	16	21	-
H51	QKS	LSVLANFSQPEIGNS	H18	18	110	-
								H52	QKSLSLSPG	SQPEIVPISNS	H18	20	95	-
H53	QKSLSL	SQPEIVPISCPPCPNS	H19	26	96	-
								H54	Q	SVLANFSQPEISCPPCPNS	H19	21	72	+/-
H55	OKSLSLSPG	RIHQMNSELSVLANS	H26	24	118	+
								H56	QKSLSLSPG	QMNSELSVLANS	H26	21	130	-	163
H57	QKSLSLSPG	VSERPFPPNS	H32	19	118	-
								H58	QKSLSLSPG	KPFFTCGSADTCPNS	CD72	24	103	-
H59	CKSLS	KPFFTCGSADTCPNS	CD72	20	94	-

¹NFS is a glycosylation consistent motif

²Transient expression in COS (6W plates) or CHO (Single flask) relative to SO129-H7 (%)

³Cleavage products observed by SDS-PAGE/silver staining: -, none, + -fuzzy band; a main band of +++, a main band of ++++, a main band of>50% splitting)

TABLE 12

Watch 13

Protein	Description of the invention	Yield (μ g purified protein/ml supplement)	POI% (M.wt. expressed as Kd, according to MALS)	Improvement of S0129wt POI	Bound Ramos	ADCC assay	Sequences of scorpion-like linkers
								S0129wt	H7 linker	1.6	67(167)	-	-	-	GCPPC
S0129-CD69	CD69 Stem region	2.9	66(167)	1.8	Weaker than S0129wt	*Is slightly superior to S0129wtPOI	QYNCPGQYTFSM
								S0129-CD72	CD72 truncated stem region	2.0	69(165)	1.2	Similar to S0129wt	*Is slightly superior to S0129wtPOI	PFFTCGSADTC
S0129-CD94	CD94 Stem region	2.9	67(171)	1.8	Similar to S0129wt	*Is slightly superior to S0129wtPOI	EPAFTPGPNIELQKDSDC
								S0129-NKG2A	NKG2A Stem region	2.5	93(170)	2.2	Slightly better than S0129wt	Similar to S0129wtPOI	QRHNNSSLNTRTQKARHC
S0129-NKG2D	NKG2D Stem region	1.9	70(166)	1.2	Similar to S0129wt	*Is slightly superior to S0129wtPOI	NSLFNQEVQIPLTESYC

As described in the examples above, it is contemplated that scorpion molecules are prepared by expression in a culture broth containing a carbohydrate modifier. In exemplary embodiments, castanospermine (MW189.21) is added to the culture medium to a final concentration of about 200 μ M (corresponding to about 37.8 μ g/mL), or to a concentration range of greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 μ M, and up to about 300, 275, 250, 225, 200, 175, 150, 125, 100, 75, 60, or 50 μ g/mL. For example, ranges of 10-50 μ M or 50-200 μ M or 50-300 μ M or 100-300 μ M or 150-250 μ M are contemplated. In other exemplary embodiments, DMJ, e.g., DMJ-HCl (MW199.6), is added to the culture medium to a final concentration of about 200 μ M (corresponding to about 32.6 μ g DMJ/mL) or to a concentration range of greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 μ M, and up to about 300, 275, 250, 225, 200, 175, 150, 125, 100, 75, 60, or 50 μ g/mL. For example, ranges of 10-50 μ M or 50-200 μ M or 50-300 μ M or 100-300 μ M or 150-250 μ M are contemplated. In other exemplary embodiments, the inhibitory alpha-mannosidase (MW232.2) is added to the culture medium until the final concentration is about 10 μ M (corresponding to about 2.3 μ g/mL) or a concentration range greater than about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μ M, and up to about 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, or 11 μ M. For example, ranges of 1-10 μ M or 1-25 μ M or 1-50 μ M or 5-10 μ M or 5-25 μ M or 5-15 μ M are contemplated.

In one experiment, monospecific CD20xCD20 scorpion (S0129) were expressed from cells cultured in 200 μ M castanospermine (S0129 CS200) or 10 μ M (excess) inhibition of α -mannosidase (S0129 KF 10) and the binding or staining of WIL2S cells by the expressed scorpion was measured as shown in figure 42. Furthermore, in comparative binding studies, the glycosylated S0129 scorpion bound CD16(FC γ RIII) more than about three times greater than the non-glycosylated S0129 scorpion.

In another study, ADCC mediated killing of BJAB-cells by a humanized CD20xCD20 scorpion (S0129) was explored. The results shown in figure 43 demonstrate that scorpion when expressed in cells cultured in the presence of castanospermine or inhibition of alpha-mannosidase results in significantly stronger ADCC-mediated BJAB-cell death at a given concentration of scorpion exposure.

Example 14

Scorpion-shaped molecular binding

a. Distance between structural domains

Bispecific scorpion molecules are capable of binding at least two targets simultaneously using a pair of binding domains located at the N-terminus and C-terminus of the molecule. In this case, for a cell surface target, the composition may crosslink with the target or cause a physically synergistic approach to the target. One skilled in the art will appreciate that activation of various receptor systems by this cross-linking results in induction of a signal that causes a change in the cell phenotype. The compositions disclosed herein are designed, in part, to maximize this signaling and control the phenotype produced.

The approximate size of each domain in the scorpion-like molecular composition and the expected value of inter-domain flexibility expressed in terms of inter-domain angular spread should be understood and considered in the design of scorpion-like molecular architecture. For scorpion molecules using scFv binding domains (for binding domains 1 and 2(BD1 and BD 2)), IgG 1N-terminal hinge (H1), and the H7PIMS linker described herein, the N-terminal binding domain can be spaced from the C-terminal binding domain by a maximum of aboutAnd the minimum can be separated by aboutThe binding domains at the N-terminus can be maximally spaced apart by aboutAnd the minimum can be separated by about(Deisenhofer et al, 1976, Hoppe-Seyler's Z, Physiol, chem, Bd.357, S.435-445; gregory et al, 1987, mol. Immunol.24 (8): 821-9; poljak et al, 1973, proc.natl.acad.sci., 1973, 70: 3305-3310; bongini et al, 2004, proc.natl.acad.sci.101: 6466-6471; kienberger et al, 2004, EMBO Reports, 5: 579-583, each of which is incorporated herein by reference). The dimensions are selected, in part, such that the receptor-to-receptor distance in the receptor complex bound by the scorpion is less than about 50This is because distances less than this distance may be optimal for maximal signaling of certain receptor oligomers (Paar et al, 2002, J.Immunol., 169: 856-864, which is incorporated herein by reference), while allowing incorporation of the F required for effector function _CAnd (5) structure.

The binding domains at the N-and C-termini of the scorpion are designed to be flexible structures to facilitate target binding and allow for a range of geometric sizes of the bound target. One skilled in the art will also appreciate the flexibility between the N-terminal or C-terminal binding domains (BD 1 and BD2, respectively) and the binding domains of the molecules and F_CThe flexibility between domains, as well as the maximum and minimum distance between receptors to which BD1 and/or BD2 bind, can be modified, for example, by selecting an N-terminal hinge domain (H1) and a scorpion linker domain (H2) that is positioned closer to the C-terminus by structural simulation. For example, hinge domains of IgG1, IgG2, IgG3, IgG4, IgE, IgA2, synthetic hinges, and hinge-like C of IgM_H2The domains exhibit varying degrees of flexibility and varying lengths. Those skilled in the art will appreciate that the optimal choice of H1 and scorpion linker (H2) will depend on the following factors: the scorpion is designed to interact with its receptor system and the desired signaling phenotype induced by the binding of the scorpion.

In certain embodiments, the scorpion molecule has a scorpion linker (H2) that is a hinge-like linker corresponding to an Ig hinge, such as an IgG1 hinge. Such embodiments include amino acid sequences having scorpion hinges Scorpion molecules of the series, the amino acid sequence being a sequence that is N-terminally extended relative to, for example, the H7 sequence or the wild-type IgG1 hinge sequence. An exemplary scorpion linker of this type would have a linker of H₂N-APEL(x)_y-CO₂H7 hinge sequence N-terminally extended, wherein x is a Gly4Ser linker unit and y has a value from 0 to 3. The effect of scorpion linkers on scorpion stability was exemplarily studied using two scorpion molecules (a bispecific CD20xCD28 scorpion and a monospecific CD20xCD20 scorpion). For each of the two scorpion designs, various scorpion linkers were inserted. In particular, the main difference between scorpion linker H16 and H17 is that H17 has the sequence of H16 with the sequence of H7 attached to the C-terminus, and that scorpion linker H18 differs from H19 similarly in that in forming H19, the sequence of H7 is attached to the C-terminus of H18. For each of the two scorpion backbones (20 × 28 and 20 × 20), each of the four scorpion linkers described above was inserted in place. Transient expression of the construct was obtained in COS cells and scorpion proteins present in culture supernatant were purified on protein a/G-coated wells (Pierce size IP kit). The purified proteins were separated on SDS-PAGE gels and visualized by silver staining. Examination of fig. 44 reveals that the additional H7 sequence in the scorpion linker increases the stability of each type of scorpion linker and each type of scorpion protein. In other words, attaching H7 to the C-terminus of H16 or H18 increases the stability of the scorpion, and this observation holds true regardless of whether the scorpion is CD20xCD28 or CD20xCD 20. In terms of target binding, the scorpion protein with the CD20 × CD20 architecture showed similar binding properties to the parent monospecific humanized CD20 × CD20 scorpion S0129, as shown in fig. 45.

However, in addition to the above embodiments, it is desirable to prevent bound receptors from approaching approximately 50 a to one anotherThe second-largest signal was intentionally formed internally (Paar et al, J.Immunol., 169: 856-864). In this case, it is contemplated to choose shorter linkers than those described aboveAnd less flexible H1 and scorpion linker (H2) are suitable.

The same spacing considerations apply to non-hinge-like scorpion connectors. The scorpion linker is, for example, a peptide having the amino acid sequence of the stem region of C-lectin. Exemplary scorpion hinges comprising a C-lectin stem region are scorpion hinges derived from the CD72, CD94 and NKG2A stem regions. Scorpion-shaped molecules containing the scorpion hinge were constructed and characterized for expression, sensitivity to cleavage, and purification flexibility. The data are shown in table 14.

TABLE 14

Linker name	G4S codon optimization1	End of CH3	S0129 Scorpion linker variant amino acid sequence	Linker sequences based on	Expression (% S0129)2	Cracking3	Laboratory bench% purification POI
								H7	N	K	GCPPCPNS	H7	100	-	70
H60	Y(17)	K	GCPPCPNS	H7	114	-	ND
								H61	Y(15)	K	GCPPCPNS	H7	90	-	66

H62	N	G	QRHNNSSLNTRTQKARHCPNS	NKG2A Stem region	129	-	89
								H63	Y(17)	G	QRIINNSSLNTRTQKARHCPNS	NKG2A Stem region	100	-	85
H64	Y(15)	G	QRHNNSSLNTRTQKARHCPNS	NKG2A Stem region	81	-	93
								H65	N	G	EPAFTPGPNIELQKDSDCPNS	CD94 Stem region	133	-	66
H66	Y(17)	G	EPAFTPGPNIELQKDSDCPNS	CD94 Stem region	200	-	64
								H67	Y(15)	G	EPAFTPGPNIELQKDSDCPNS	CD94 Stem region	129	-	65
H68	N	G	RTRYLQVSQQLQQTNRVLEVTNSSLRQQLRLKITQLGQSAEDLQGSRRELAQSQEALQVEQRAHQAAEGQLQACQADRQKTKETLQSEEQQRRALEQKLSNMENRLKPFFTCGSADT	CD72 intact Stem region	110	-	75

¹Gly₄Codon optimization of Ser linker with (17) or without (15) restriction sites

²Evaluation of expression in COS based on recovery of protein from bench purification

³One or more cleavage products observed by SDS-PAGE/Coomassie blue staining of purified protein

binding of N-terminal and C-terminal binding domains

N-terminal and C-terminal domains involved in target cell binding

The target cell binding capacity of CD20SMIP (TRU015), CD37SMIP (SMIP016), combinations of CD20 and CD37SMIPs (TRU015+ SMIP016) and CD20xCD37 bispecific scorpion molecule (015x016) was assessed by measuring the ability of each of said molecules to block the binding of an antibody specifically competing for binding to the relevant target (CD37 or CD 20). The competing antibody is optionally a FITC-labeled monoclonal anti-CD 37 antibody or a PE-labeled monoclonal anti-CD 20 antibody. Ramos B-cells provide the target.

PBS (#100-113, Gemini Bio-Products, Wes) with 5% mouse serumRamos B-cells (1.2X 10) in tSacramento, CA (staining Medium)⁷Individual cells/ml) were added to a 96-well V-shaped bottom plate (25 μ l/well). Various SMIPs and scorpion molecules were diluted to 75 μ g/ml in staining medium and 4-fold to the concentrations shown in figure 38. The diluted compound was added to the cells of the coated plate (except for the medium used for control wells alone). Cells were incubated with the compound for 10 minutes, and then 5 μ g/ml of FITC anti-CD 37 antibody (#186-040, Ancell, Bayport, MN) was added to 25 μ l of staining medium in wells together with 3 μ g/ml PE anti-CD 20 antibody (#555623, BD Pharmingen, San Jose, CA) (pure). The cells were incubated on ice for 45 minutes in the dark and then washed 2.5 times with PBS. Cells were fixed with 1% trioxymethylene (# 199431 LT, USB Corp, Cleveland, OH) and then run on FACs Calibur (BD Biosciences, San Jose, Calif.). Data were analyzed using Cell Quest software (BD Biosciences, San Jose, CA). The results shown in fig. 38 demonstrate that all SMIPs, SMIP combinations, and scorpion molecules containing a binding site for CD20 successfully competed with PE-labeled anti-CD 20 antibody for binding to Ramos B-cells (upper panel); all SMIPs, SMIP combinations and scorpion molecules containing a binding site for CD37 successfully competed with FITC-labeled anti-CD 37 antibody for binding to Ramos B-cells (lower panel). Thus, bispecific CD20xcd37 scorpion molecules were shown to have operable N-terminal and C-terminal binding sites for targets on B-cells.

c. Persistence of cell surface

The study of cell surface persistence of the bound SMIP and scorpion (monospecific and bispecific) on the B-cell surface revealed that scorpion showed stronger cell surface persistence than SMIP. Staining media (2.5% goat serum in PBS, 2.5% mouse serum) 6X 10⁶Individual cells/ml Ramos B-cells (3X 10)⁵Individual cells/well) were added to a 96-well V-shaped bottom plate. Test reagents at two-fold final concentrations were prepared by 5-fold serial dilution of 500nM starting material in staining medium and then added to Ramos B-cells at 1: 1. In addition, a control medium was also applied. Cells were incubated on ice for 45 min in the darkA clock. Plates were then washed 3.5 times with cold PBS. A second reagent FITC goat anti-human IgG (# H10501, Caltag/Invitrogen, Carlsbad, Calif.) was then added to the staining medium at a 1:100 dilution. Cells were incubated on ice for 30 minutes in the dark. The cells were then washed 2.5 times with cold PBS by centrifugation, fixed with 1% paraformaldehyde solution (#199431LT, USB Corp, Cleveland, OH), and then run on FACs Calibur (BD Biosciences, San Jose, CA). Data were analyzed using CellQuest software (BD Biosciences, San Jose, Calif.). The results of the data analysis are shown in fig. 37, which shows the binding of several SMIPs (monospecific CD20 × CD20 scorpion and bispecific CD20 × CD37 scorpion) to their targets on Ramos B cells.

Will have Ramos B-cells (7X 10)⁵Individual cells/ml) were incubated on ice for 30 minutes and each of the two compounds (i.e., humanized CD20(2Lm20-4) SMIP and humanized CD20 xcd 20(2Lm20-4 x 2Lm20-4) scorpion) were studied (25 μ g/ml each in Iscoves media with 10% FBS). At the end of the incubation period, both tubes were washed 3 times by centrifugation. One tube of cells (in 150. mu.l Iscoves medium) was then incubated at 2X 10⁵Individual cells/well were plated in 96-well flat-bottom plates, then one plate was placed in a 37 ℃ incubator and the other plate was incubated on ice. The second tube of each set of cells was resuspended in cold PBS (staining medium) with 2% mouse serum and 1% sodium azide and at 2X 10⁵Individual cells/well were plated in 96-well V-shaped plates for rapid staining with a secondary antibody, i.e., FITC goat anti-human IgG (# H10501, Caltag/Invitrogen, Carlsbad, CA). Secondary antibody was added to staining medium at a 1:100 final dilution and cells were stained on ice for 30 minutes in the dark. The cells were then washed 2.5 times with cold PBS and fixed with 1% paraformaldehyde (#199431LT, USB Corp, Cleveland, OH).

At the time points designated in fig. 39, samples were harvested from 96-well flat-bottom plates, incubated at 37 ℃ or on ice, and placed in 96-well V-bottom plates (2 × 10) ⁵Individual cells/well). Cells were washed once with cold staining medium, resuspended, and secondary antibody 1100 final dilution was added to the dyeing medium. The cells were incubated on ice for 30 minutes in the dark. The cells were then washed 2.5 times in cold PBS by centrifugation and subsequently fixed with 1% trioxymethylene. Samples were run on a FACS Calibur (BD Biosciences, San Jose, CA) and data were analyzed with CellQuest software (BD Biosciences, San Jose, CA). The results shown in figure 39 demonstrate that SMIPs and scorpion binding to the B-cell surface lasted at least six hours, with monospecific hu CD20xCD20(2Lm20-4x2Lm20-4) scorpion persisted to a greater extent than huCD20(2Lm20-4) SMIPs.

Example 15

Direct cell killing of monospecific and bispecific scorpion molecules

The experiments were aimed at assessing the ability of monospecific and bispecific scorpion molecules to kill lymphoma cells directly, i.e. without involving ADCC or CDC to kill the cells. In particular, the Su-DHL-6 and DoHH2 lymphoma cell lines were subjected to monospecific scorpion molecules (i.e., CD20 × CD20 scorpion or CD37 × CD37 scorpion) alone or to bispecific CD20 × CD37 scorpion.

Culture solutions of Su-DHL-6, DoHH2, Rec-1, and WSU-NHL lymphoma cells were established using conventional techniques, and then some of the cultures were individually exposed to monospecific CD20 SMIPs, monospecific scorpion (CD20 × CD20 or CD37 × CD37), or bispecific scorpion (CD20 × CD37 or CD19 × CD 37). Exposure of the cells to the SMIP or scorpion is performed under conditions that do not result in cross-linking. The cells were kept in contact with the molecule for 96 hours, after which their growth was measured by detecting ATP, as known in the art. The cell killing effect produced by the CD20SMIP and CD20xCD20 monospecific scorpion molecules is evident from figure 24 and table 15. The cell killing ability of the CD37 xcd 37 monospecific scorpion was apparent from fig. 25 and table 15, the ability of the CD20xcd 37 bispecific scorpion killed lymphoma cells was apparent from fig. 26 and table 15 and the ability of the CD19 xcd 37 bispecific scorpion killed lymphoma cells was apparent from fig. 27 and table 15. Will threeData from independent experiments were combined and each point represents mean ± Standard Error (SEM). IC's in Table 15, as noted by the symbols in Table 15₅₀Values were determined by the curves in fig. 24, 25 and 26 and were defined as the concentration that produced 50% inhibition compared to untreated cultures. The data in the figures and tables demonstrate that the efficiency of scorpion killing the cell line is more than 10-fold greater than free SMIP using the same binding domain.

Watch 15

^*The data is derived from figure 24.

^**The data are derived from figure 25.

^***The data is derived from figure 26.

^****Data are derived from figure 27.

Additional experiments with the humanized CD20xCD20 scorpion S0129 were performed in the following cells: Su-DHL-4, Su-DHL-6, DoHH2, Rec-1, and WSU-NHL cells. The results are shown in fig. 46 and 47. The data presented in the figures extends the above conclusion, demonstrating that scorpion molecules have the ability to kill various cell lines directly.

The above conclusions can be extended to other monospecific and bispecific scorpion molecules, various of which demonstrate the ability to kill B cells directly. DoHH 2B-cells were exposed in vitro to either a monospecific CD20 × CD20 scorpion, a monospecific CD37 × CD37 scorpion, or a bispecific CD20 × CD37 scorpion. The results shown in figure 48 demonstrate that bispecific scorpion molecules have a killing rate curve that is in a different form than monospecific scorpion molecules.

Culturing Su-DHL-6 cells in the presence of 70nM CD20xCD20 scorpion (S0129), CD20xCD37 scorpion, or CD37xCD37 scorpion also produced direct B-cell killing in an in vitro environment (fig. 49). Similarly, Su-DHL-6 cells were exposed to bispecific CD19xCD37 scorpion or to bispecific CD19xCD37 scorpion Direct cell killing resulted, while the bispecific scorpion showed lethality at lower doses, as revealed in figure 50.

Another demonstration of direct cell killing was provided by exposing DHL-4 cells to four separate monospecific scorpion molecules that recognize CD 20. Two versions of the CD20xCD20 scorpion were designed to incorporate two 20-4 binding domains (20-4x20-4 and S0129) and two other hybrids that incorporate 011 and 20-4 binding domains. All four independently constructed and purified versions of the two CD20XCD20 scorpion design ((20-4X 20-4 and S0129) and hybrids (011X 20-4 and 011X 20-4. delta. Asp)) effectively killed DHL-4 cells in a direct manner. For this study, DHL-4 cells were treated in vitro with 1. mu.g/ml of the indicated protein for 24 hours. The cells were then stained with annexin V and propidium iodide (early and late markers of cell death), respectively, and the cell population was quantified by FACS. The results shown in figure 51 demonstrate the direct killing ability of each CD20x CD20 construct, as evidenced by the enhanced staining shown by the black bars. Furthermore, the results demonstrate that the hybrid 011x20-4 protein shows a slight enhancement in direct cell killing over the 20-4x20-4 based scorpion, although the scorpion each monospecifically recognizes CD 20. The dose-response of the four independent scorpion constructs was determined by FACS analysis of annexin V stained cell populations and propidium iodide stained cell populations in separate experimental groups. The results shown in fig. 52 demonstrate an increase in dose-responsiveness in cell death caused by treatment of DHL-4 cells with various independent scorpion constructs.

Example 16

Collateral functions mediated by scorpion (ADCC and CDC)

a. Scorpion-like molecule-dependent cell cytotoxicity

The experiment aims to determine whether the scorpion-shaped molecule mediates the killing effect of the BJAB lymphoma cells. It was observed that BJAB B lymphoma cells were killed by CD20 and/or CD37 scorpion molecules.

Initially, BJABB-cells (1X 10) were cultured in Iscoves medium with 10% FBS at 37 ℃⁷One/ml) with 500. mu. Ci/ml⁵¹Cr sodium chromate (# CJS1, Amersham biosciences, Piscataway, NJ) was labeled for 2 hours. Then will be loaded with⁵¹Cr BJAB B cells were washed 3 times in RPMI medium with 10% FBS and at 4X 10⁵One/ml was resuspended in RPMI. Peripheral Blood Mononuclear Cells (PBMC) from laboratory donors were separated from heparinized whole blood by centrifugation in lymphocyte separation media (#50494, MP Biomedicals, Aurora, Oh), washed 2 times with RPMI media and at 5X 10⁶One/ml was resuspended in RPMI with 10% FBS. Reagent samples were added to RPMI media with 10% FBS at 4-fold final concentration and triplicate 10-fold serial dilutions were made for each reagent. The reagents were then added to a 96-well U-shaped plate at 50 μ l/well to the final concentrations specified. Then will be passed through ⁵¹Cr-labeled BJAB cells were cultured at 50. mu.l/well (2X 10)⁴Individual cells/well) were added to the plate. PBMC were then assayed at 100. mu.l/well (5X 10)⁵Individual cells/well) were added to the plate, resulting in effector (PBMC): the final ratio of target (BJAB) was 25: 1. Effectors and targets were added to separate media to measure background kill rates. Will be passed⁵¹Cr-labeled BJAB addition to separate media for measurement⁵¹Spontaneous release rate of Cr and added to media with 5% NP40(#28324, Pierce, Rockford, I11) to measure⁵¹Maximum release rate of Cr. The plates were incubated at 37 ℃ in 5% CO₂And (4) incubating for 6 hours. Then 50. mu.l (25. mu.l are also suitable) of the supernatant from each well are transferred to LumaPlate-96(#6006633, Perkin Elmer, Boston, Mass) and dried overnight at room temperature.

After drying, the radioactive emission was quantified in cpm on a Packard TopCount-NXT. The sample value is the average of three samples. Percent specific kill was calculated using the following equation: the killing% ((sample-spontaneous release rate)/(maximum release rate-spontaneous release rate)) × 100. The curves in figure 30 show that BJAB B cells were killed by monospecific scorpion molecules CD20 xcd 20 and CD37 xcd 37. The combination of CD20SMIP and CD37SMIP also killed BJAB B cells. The results demonstrate that scorpion showed scorpion-dependent cellular cytotoxicity and that this functionality is expected to be provided by the constant sub-region of scorpion providing ADCC activity.

b. Role of Scorpion-like molecules in complement-dependent cytotoxicity

Experiments also demonstrate that scorpion molecules possess Complement Dependent Cytotoxicity (CDC) activity. As described below and shown in fig. 31, the experiment involved exposing Ramos B-cells to CD19 and/or CD37 SMIPs and scorpion molecules.

The experiment was initiated by mixing 50. mu.l of 5X 10 in Iscoves medium (without FBS)⁵To 2.5X 10⁵Individual Ramos B-cells were added to the wells of a 96-well V-shaped bottom plate. Test compounds in Iscoves (or Iscoves alone) were added to wells at the indicated final concentration in 50 μ Ι portions. Cells and reagents were incubated at 37 ℃ for 45 minutes. Cells were washed 2.5 times with FBS-free Iscoves and resuspended at the indicated concentration in human serum in 96-well plates (# a113, Quidel, San Diego, CA). The cells were then incubated at 37 ℃ for 90 minutes. Cells were washed by centrifugation and resuspended in 125 μ l cold PBS. The cells were then transferred to FAC cluster tubes (#4410, CoStar, Corning, NY) and 125. mu.l PBS with 5. mu.g/ml propidium iodide (# P-16063, Molecular Probes, Eugene, OR) was added. Cells were incubated with propidium iodide for 15 minutes at room temperature in the dark and then placed on ice, quantified, and analyzed on a FACsCalibur with CellQuest software (Becton Dickinson). The results shown in figure 31 demonstrate that CD19SMIP (but not CD37SMIP) shows CDC activity and that the combination of the two SMIPs shows about the same degree of CDC activity as CD19SMIP alone. However, it is not limited to The CD19xCD37 scorpion showed significantly stronger CDC activity than either SMIP alone or SMIP combination, suggesting that the scorpion architecture may provide a greater degree of complement dependent cytotoxicity than other molecular designs.

ADCC/CDC Activity of CD20 × CD20 monospecific scorpion

ADCC and CDC functionality was tested on three different CD20xCD20 monospecific scorpion molecules as well as appropriate control molecules. ADCC was determined using conventional techniques and the results are shown in figure 53. Significant (but non-uniform) ADCC activity associated with each CD20 × CD20 monospecific scorpion tested is evident from the figure.

To assess CDC, Ramos B-cell samples (4X 10) were assayed at 37 deg.C⁵) Incubate with each CD20xcd20 scorpion (0, 0.5, 5, 50 and 500nM) and serum (10%) for 3.5 hours. Cell death was assessed by 7-AAD staining and FACS analysis. The results are shown in figure 54, which reveals that the scorpion displayed some CDC activity. In a similar experiment, Ramos B-cell samples (4X 10) were taken at 37 deg.C⁵) Incubate with CD20xcd20 scorpion protein (5, 50, 100nM) and serum (10%) for 2 hours. Cells were washed 2 times and incubated with anti-human C1qFITC antibody. The bound C1q was assessed by FACS analysis and the results are shown in figure 55. The results are consistent with those shown in figure 54, indicating that each CD20xCD20 monospecific scorpion is associated with some CDC activity, but less than the activity associated with CD20 SMIP.

d. Scorpion-shaped molecule and F_CInteraction of gamma RIII

ELISA studies showed that scorpion molecules bind to low Fc γ RIII (CD16) (low affinity isoform or allele) to an enhanced extent in the absence of target cells. The ELISA plates were initially coated with low affinity or high affinity CD16mIgG using conventional techniques. The ability of the immobilized fusion protein to capture CD20SMIP or CD20 xcd 20 monospecific scorpion was evaluated. Bound SMIP and scorpion were detected with goat anti-human IgG (HRP) secondary antibody and Mean Fluorescence Intensity (MFI) was determined. PBS alone (negative control) is shown as a single dot. The results are shown in fig. 32A (captured by CD16 high affinity isoform fusion) and fig. 32B (captured by CD16 low affinity isoform fusion). As is apparent from consideration of fig. 32A and 32B, the CD20SMIP and CD20 xcd 20 monospecific scorpion molecules showed enhanced binding to high and low affinity CD16 isoform fusions, with the CD20 xcd 20 scorpion molecule showing significantly enhanced binding to low affinity isoform fusions with increasing protein concentration.

Binding of scorpion molecules to Fc γ RIII isoforms in the presence of target cells was also assessed. The data show that binding of scorpion to Fc γ RIII (CD16) low and high affinity isoforms or alleles increases with increasing protein concentration in the presence of target cells.

In conducting the experiment, CD20 positive target cells were exposed to CD20SMIP or CD20 x CD20 monospecific scorpion under conditions that allow binding of SMIP or scorpion to CD20 positive target cells. Subsequently, target cells carrying SMIPs or scorpion molecules are exposed to the mouse IgFc-labeled CD16 high-or low-affinity isoform. The labeled goat anti-mouse Fc was then added as a secondary antibody to label the immobilized CD16 linked to the mouse IgFc. Cells were then detected by flow cytometry on FACs Calibur (BD Biosciences, San Jose, CA) and analyzed with Cell Quest software (BD Biosciences, San Jose, CA). As shown in figure 33, increasing the concentration of each of the CD20SMIP and CD20 xcd 20 monospecific scorpion in the presence of target cells resulted in enhanced binding to the CD16 isoform, with the enhancement of binding of the CD20 xcd 20 scorpion being more pronounced than that seen for the CD20 SMIP.

Example 17

Cell cycle effects of Scorpion-like molecules on target lymphoma cells

The cell cycle effects of scorpion-like molecules were evaluated by exposing lymphoma cells to SMIP, monospecific scorpion-like molecules and bispecific scorpion-like molecules. More particularly DoHH2 lymphoma cells (0.5X 10)⁶) Treatment with 0.4nM rituximab (rituximab), CD20 XCD 37 scorpion, TRU-015(CD20SMIP) + SMIP-016 combination (0.2 nM each), 100 nSMIP-016 or 100nM CD37 XCD 37 scorpion for 24 hours. IC shown by the concentration in the 96-hour growth inhibition assay₅₀IC of scorpionic molecules₅₀The values were about 10 times greater (see fig. 24-27). Cultures were labeled with 10. mu.M BrdU (bromodeoxyuridine) for 20 minutes at 37 ℃. After fixation, cells were stained with anti-BrdU-FITC antibody and counterstained with propidium iodide. The values in FIG. 28 are the mean +/-SD of 4 replicate cultures from 2 to 3 independent experiments. All sample data were analyzed simultaneously and displayed in combination using BrdU and PI merge point curves. The graphs demonstrate that the main effects of scorpion treatment are depletion of cells in S-phase, and G₀/G₁The interval is increased.

Example 18

Physiological effects of Scorpion-like molecules

a. Mitochondrial potential

As revealed in the JC-1 assay, the CD20 × CD20 scorpion induced a decrease in mitochondrial membrane potential of DHL 4B-cells. JC-1 is a cationic carbocyanine dye (from Molecular Probes) showing potential-dependent accumulation of mitochondriaJC-1 flow cytometry assay kit). JC-1 is more specific for mitochondrial membranes than plasma membranes and is used to determine changes in mitochondrial membrane potential. Accumulation of mitochondria can be indicated by a shift in fluorescence from green (529nm) to red (590 nm).

In carrying out the experiment, DHL-4B-cells (5X 10) were initially taken⁵Individual cells/ml) were cultured in 24-well plates and in a standard tissue culture incubator at 37 ℃ in 5% CO₂The cells were treated with 1. mu.g/ml of CD20 × CD20 scorpion, rituximab, IgG control antibody or 5. mu.M staurosporine (staurosporine) for 24 hours. JC-1 dye (10. mu.l/ml, 2. mu.M final concentration) was added and the temperature was 37 deg.CNext, the cells were incubated for another 30 minutes. Cells were harvested by centrifugation (1200rpm, 5 min), washed with 1ml PBS and resuspended in 500. mu.l PBS. Cells were analyzed by flow cytometry (FACSCalibur, BD) via 488nM excitation and 530nM and 585nM emission filters. For the representative scatter plot shown in fig. 56, red fluorescence was measured on the Y-axis and green fluorescence was measured on the X-axis. Depolarization of the mitochondrial membrane is measured as a reduction in red fluorescence as seen with the positive control CCCP (carbonyl cyanide 3-chlorophenylhydrazone), a known mitochondrial membrane potential perturber. To verify that JC-1 has a response to changes in membrane potential, DHL-4B-cells were incubated at 37 ℃ with 5% CO₂Next, two concentrations of CCCP (50. mu.M and 250. mu.M) were used for 5 minutes. Another positive control was cells treated with the broad-spectrum kinase inhibitor staurosporine to induce apoptosis. The results shown in fig. 56 are a plot of 10,000 points with red fluorescence plotted on the Y-axis and green fluorescence plotted on the X-axis. A generalized histogram with the percentage of cells perturbing mitochondrial membrane potential (perturbing MMP: black bars) is shown in FIG. 56. The results demonstrate that treatment with either the 20-4X 20-4 scorpion or the 011X 20-4 scorpion results in a decrease in mitochondrial membrane potential associated with cell death.

b. Calcium flux

Using Ca⁺⁺Fluidity (a common characteristic of cell signaling) was used as a measure to analyze the effect of scorpion molecules on cell signaling pathways. SU-DHL-6 lymphoma cells were labeled with calcium 4 dye and treated with test molecules identified below. The cells were read for 20 seconds to determine background fluorescence, and then SMIP/scorpion molecules (first dashed line in figure 28) were added and fluorescence was measured for up to 600 seconds. At 600 seconds, an 8-fold excess of crosslinked goat anti-human F (ab)' 2 was added and fluorescence was measured for an additional 300 seconds. Panel (a) in figure 28 shows the results obtained with the combination of CD20 SMIP and CD37 SMIP (red line) compared to unstimulated cells (blue line); results obtained with CD20 × CD37 bispecific scorpion (black line). In panel B of FIG. 28, the result of treating cells with CD20 SMIP alone (red line) resulted in Ca⁺⁺Flow phenomenon, howeverAnd not as strong as the signal (black line) generated by the monospecific CD20 xcd 20 scorpion. Ca of FIG. 28⁺⁺The flow chart shows fluorescence obtained from three wells treated with equimolar amounts of scorpion and SMIP/SMIP combination.

c. Caspases 3, 7 and 9

The ability of scorpion molecules binding to CD20 to directly kill B-cells, as demonstrated by increased annexin V and propidium iodide staining and decreased mitochondrial membrane potential, led to further studies of other apoptosis-related effects of scorpion molecules binding to CD20 on B-cells. The method used was Apo1 assay of DHL-4B-cells exposed to CD20 × CD20 scorpion or appropriate controls. The Apo1 assay is based on synthetic peptide substrates for caspases 3 and 7. Assay components are available from Promega (R) Homologous caspase-3/7 assay). Caspase-mediated cleavage of the labeled peptide Z-DEVD-Rhodamine 110(Rhodamine 110) releases the fluorescent Rhodamine 110 marker, which is measured using 485nm excitation and 530nm detection.

In the experiment, 100. mu.l of DHL-4B-cells (1X 10)⁶Individual cells/ml) were plated in black 96-well flat-bottomed tissue culture plates and incubated in a standard tissue culture incubator at 37 ℃ with 5% CO₂Next, the cells were treated with 1. mu.g/ml of CD20 × CD20 scorpion, rituximab, IgG control antibody, or 5. mu.M staurosporine for 24 or 48 hours. (staurosporine is a small molecule broad-spectrum protein kinase inhibitor known in the art to be a potent inducer of classical apoptosis in a variety of cell types). After 24 or 48 hours, 100 μ Ι of 100 fold dilution substrate was added to each well, gently mixed on a plate shaker (300rpm) for one minute and incubated at room temperature for two hours. Fluorescence was measured using 485nM excitation and 527nM emission filter (Fluoroskan Ascent FL, Thermo Labsystems). The graph shown in figure 57 shows the mean fluorescence intensity +/-standard deviation of three treatments after 24 hours and 48 hours (only 24 hours for staurosporine). The results demonstrate that scorpion-like molecules that bind CD20 are not involved in cysts The aspartase 3/7 activated apoptotic pathway kills B-cells directly.

The results obtained in the Apo-1 assay were validated by western blot analysis designed to detect either pro-caspase cleavage leading to caspase activation or to detect cleavage of PARP (poly (ADP-ribose) polymerase), a protein known to be cleaved by activation of caspase 3. DHL-4B-cells were exposed to CD 20-binding scorpion or control for 4, 24, or 48 hours, and cell lysates were separated by SDS-PAGE and analyzed by western blotting using conventional techniques. The results shown in figure 58 are in the form of a collection of western blots. The bottom three western blots were made using anti-caspase antibodies to detect changes in molecular weight of caspases reflecting proteolytic activation. For caspases 3, 7 and 9, there was no evidence of caspase activation by any CD 20-binding molecule. Staurosporine served as a positive control for the assay and induced pro-caspase cleavage of the active caspases for each of caspases 3, 7 and 9. The fourth western blot, shown in figure 58, shows that PARP, a known substrate for activating caspase 3, is not cleaved, consistent with the inability of scorpion-like molecules that bind CD20 to activate caspase 3. The results of all the experiments were consistent, suggesting that caspase 3 activation is not an important feature of direct cell killing of DHL-4B-cells induced by scorpion binding to CD 20.

In addition, time-series studies were aimed at determining the effect of CD 20-binding proteins (including CD20 × CD20 scorpion) on caspase 3. DoHH2 or Su-DHL-6B-cells were conjugated with 10nM of CD20 binding protein (S0129 scorpion, 2Lm20-4SMIP or) +/-soluble CD16 Ig (40nM), soluble CD16 Ig alone, or vehicle incubated together. Cells were cultured at 3X 10⁵Pieces/well/300 microliter were cultured in complete RPMI with 10% FBS and harvested at 4 hours, 24 hours or 72 hours. Samples at the 72 hour time point were plated in 500 μ l of test agent. Cells were washed with PBSAnd then using BD Pharmingen caspase 3 (active form), mAB apoptosis kit: FITC (Cat No. 55048, BDPharmingen, San Jose, Calif.) was stained with intracellular active caspase-3. Briefly, after 2 more washes with cold PBS, cells were suspended in cold cytofix/cytoperm solution and incubated on ice for 20 minutes. The cells were then washed by centrifugation, aspirated, and washed twice with Perm/Wash buffer at room temperature. The samples were then stained with 20. mu.l FITC-anti-caspase 3 in 100. mu.l Perm-Wash buffer for 30 min at room temperature in the dark. The sample was then washed twice with Perm-Wash buffer and resuspended in 500. mu.l of Perm-Wash buffer. The washed cells were then transferred to FACs tubes and run on FACs Calibur (BD Biosciences, San Jose, Calif.) and analyzed with Cell Quest software (BD Biosciences, San Jose, Calif.). The results are shown in table 16.

TABLE 16

The results of all the experiments are consistent, demonstrating limited activation of caspase 3 in the absence of CD16, which does not imply an important feature of caspase 3 activation as a direct cell killing effect induced by scorpion molecules binding to CD 20.

DNA fragmentation

Induction of the classical apoptotic signaling pathway ultimately leads to condensation and fragmentation degradation of chromosomal DNA. To determine whether a scorpion binding CD20 killed B-cells directly via a classical apoptotic mechanism, the status of B-cell chromosomal DNA was examined after exposing the cells to a scorpion binding CD20 or a control. Initially, DHL-4B-cells were treated ex vivo with CD20 binding molecules (i.e., monospecific CD20 xcd 20(2Lm20-4 × 2Lm20-4) scorpion, CD20 xcd 20(011 × 2Lm20-4) scorpion, or rituximab) or with controls for 4 hours, 24 hours, or 48 hours. Subsequently, the cells are lysed and the chromosomal DNA is purified using conventional techniques. The chromosomal DNA was then fractionated by gel electrophoresis. The gel electrophoresis pattern shown in fig. 59 shows the lack of DNA fragmentation, demonstrating that cell death caused by scorpion binding to CD20 is not mediated by the classical apoptotic pathway. Staurosporine treated cells were used as positive controls in the assay.

Phosphorylation of SYK

SYK is a phospho-regulatory protein with several phosphorylation sites, which acts as a transcriptional repressor. SYK is located in the nucleus, but can be rapidly relocated to the cell membrane after activation. For activation, SYK must retain its nuclear localization sequence. Activated SYK has the effect of inhibiting breast cancer tumors and SYK is activated by pro-apoptotic signals such as ionizing radiation, BCR ligation and MHC class II cross-linking. In addition, SYK has been shown to affect PLC-. gamma.and Ca⁺⁺And (4) a passage. Via these observations, the ability of scorpion molecules binding to CD20 to affect SYK was investigated.

DHL-6B-cells were exposed to bispecific CD20 xcd 37 scorpion for 0, 5, 7, or 15 hours and the cells were lysed. Immunoprecipitates were separated by gel electrophoresis by immunoprecipitation of the lysates with an anti-phosphotyrosine antibody or with an anti-SYK antibody, and the results are shown in fig. 60. As is evident from examination of figure 60, the bispecific CD20 xcd 37 scorpion failed to induce phosphorylation of SYK, thereby failing to activate it. Consistent with the above-described studies of caspase activation and chromosomal DNA fragmentation, scorpion molecules that bind CD20 do not appear to directly kill B-cells using classical apoptotic pathways (such as the caspase-dependent pathway). While not wishing to be bound by theory, it is expected that the scorpion molecule that binds CD20 kills B-cells directly via the caspase-independent and SYK-independent pathways, which do not have significant chromosomal DNA fragmentation characteristics, at least during the same time period that fragmentation occurs during caspase-dependent apoptosis.

Example 19

Scorpion-shaped molecule application

a. In vivo Activity of Scorpion-like molecules

The activity of scorpion molecules was also assessed using a mouse model. Measurement of the in vivo activity of scorpion involves administration of 10-300 μ g of scorpion and subsequent time-sequential determination of the serum concentration of this scorpion. The results of the study are shown in figure 40, which is a plot of serum concentration for each of the two bispecific scorpion molecules (i.e., S0033, CD20 × CD27 scorpion and CD20 × CD37 scorpion) studied for three weeks of pharmacokinetics in mice. The data in figure 40 show that it takes at least 500 hours after administration before the serum level of each of the two bispecific scorpion drops back to baseline levels. Thus, scorpion molecules exhibit serum stability and a reproducible, long-lasting half-life in circulation in vivo.

The in vivo efficacy of scorpion molecules was also assessed. An invasive Ramos xenograft model was used in parallel experiments with SMIP versus historical immunoglobulin controls. The survival curves provided in figure 41 reveal that administration of 10 μ g of bispecific scorpion had a negligible effect on survival, but administration of 100-300 μ g had a significant positive effect on survival of mice bearing Ramos xenografts.

b. Combination therapy

It is expected that scorpion molecules will be useful in preventing, treating, or ameliorating symptoms of a variety of conditions affecting humans, other mammals, and other organisms. For example, scorpion molecules that bind CD20 are expected to be useful in the treatment or prevention of a variety of diseases associated with B-cell excess or abnormality. Virtually any disease susceptible to treatment (involving depletion of B-cells) can be treated with scorpion molecules that bind CD 20. In addition, scorpion molecules (e.g., scorpion molecules that bind CD 20) may be used in combination therapy with other therapeutic agents. To demonstrate the feasibility of various combination therapies, a monospecific CD20 XCD 20 scorpion (S0129) was administered in combination with either daunorubicin (doxorubicin), vincristine (vincristine) or rapamycin (rapamycin) to Su-DHL-6B-cells. Erythromycin hydroxydocusate is a topoisomerase II poison that interferes with DNA biochemical mechanisms and belongs to a class of drugs directed to anticancer therapy. Rapamycin (Sirolimus) is a macrolide antibiotic that inhibits initiation of protein synthesis and suppresses the immune system, is suitable for organ transplantation and can be used as an antiproliferative agent in conjunction with coronary intravascular stents to inhibit or prevent restenosis. Vincristine is a vinca alkaloid that inhibits tubule formation and has been used to treat cancer.

The experimental results shown in fig. 61 are combination index values shown as ranges of the degrees of effect for each combination. The monospecific CD20 XCD 20 scorpion S0129 has different interaction with various drugs, but has different interaction with various drugsThe curve form of (RTXN) is similar. The effect seen with high concentrations of erythromycin hydroxydaxole may reflect a shift towards monovalent binding. The data demonstrate that scorpion binding CD20 can be used in combination with a variety of other therapeutic agents and that such combinations will be apparent to those skilled in the art upon review of the present disclosure.

Variations on the structural theme of multivalent binding molecules or scorpion-like molecules with effector functions will be apparent to those skilled in the art upon review of this disclosure, and such altered structures fall within the scope of the present invention.

Sequence listing

<110> Thompson et al

<120> Single-chain multivalent binding proteins with Effector function

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<400>10

<210>11

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<223> Race simplified anchor primer

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<223> n is a, c, g or t

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<400>11

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<223> T7 primer

<400>12

<210>13

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<223> M13 reverse primer

<400>13

<210>14

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<213> Intelligent people

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<223> human IgG1 hinge domain

<400>14

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<223>hVK3L-F3H3

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<223>hVK3LF12HVL

<400>17

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<223>2H7VHNheF

<400>18

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<223>G4SNheR

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<223>015VHXhoR

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<223>2E12VLXbaR

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<223>2E12VLR1F

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<223>2e12VHdXbaF1

<400>31

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<223>IgBsrG1F

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<223>T7

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<400>40

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<400>41

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<223>CH3seqF1

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<400>45

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<223>L1-11R

<400>46

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<223>L3R

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<223>L4R

<400>49

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<223>L5R

<400>50

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<211>23

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<223>IgBsrG1F

<400>51

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<223>L-CPPCPR

<400>52

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<223>G281-LH-LPinF

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<220>

<223> Synthesis of primers

<220>

<223>G281-HL-LNheF

<400>63

<210>64

<211>37

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>G281-HL-LXhoR

<400>64

<210>65

<211>42

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>G281-HL-LXhoR

<400>65

<210>66

<211>37

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>G281-HL-EcoF

<400>66

<210>67

<211>40

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-LH-LF1

<400>67

<210>68

<211>50

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-LF2

<400>68

<210>69

<211>52

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-LF3

<400>69

<210>70

<211>50

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-LF4

<400>70

<210>71

<211>50

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-LF5

<400>71

<210>72

<211>50

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-LF6

<400>72

<210>73

<211>52

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-LF7

<400>73

<210>74

<211>52

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-LR7

<400>74

<210>75

<211>50

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-LR6

<400>75

<210>76

<211>51

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-LR5

<400>76

<210>77

<211>52

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-LR4

<400>77

<210>78

<211>46

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-LR3

<400>78

<210>79

<211>50

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-LR2

<400>79

<210>80

<211>42

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-LH-LR1

<400>80

<210>81

<211>54

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-LH-HF1

<400>81

<210>82

<211>50

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-HF2

<400>82

<210>83

<211>50

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-HF3

<400>83

<210>84

<211>53

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-HF4

<400>84

<210>85

<211>52

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-HF5

<400>85

<210>86

<211>52

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-HF6

<400>86

<210>87

<211>52

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-HR6

<400>87

<210>88

<211>52

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-HR5

<400>88

<210>89

<211>55

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-HR4

<400>89

<210>90

<211>52

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-HR3

<400>90

<210>91

<211>52

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-HR2

<400>91

<210>92

<211>52

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-LH-HR1

<400>92

<210>93

<211>42

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-HL-HF1

<400>93

<210>94

<211>55

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-HL-HR1

<400>94

<210>95

<211>52

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-HL-HRO

<400>95

<210>96

<211>49

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-HL-LF1

<400>96

<210>97

<211>34

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-HL-LR3Xho

<400>97

<210>98

<211>37

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-HL-LR3Xba

<400>98

<210>99

<211>43

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-HL-HF1R1

<400>99

<210>100

<211>41

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-LH-LF1R1

<400>100

<210>101

<211>54

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>194-LH-HR1Xba

<400>101

<210>102

<211>725

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>G28-1VLVH(DNA)

<400>102

<210>103

<211>239

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>G-28-1VLVH(AA)

<220>

<221>misc_feature

<222>(1)..(107)

<223>VL

<220>

<221>misc_feature

<222>(108)..(124)

<223> linker

<220>

<221>misc_feature

<222>(125)..(239)

<223>VH

<400>103

<210>104

<211>767

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>G28-1 VHVL(DNA)

<400>104

<210>105

<211>253

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>G28-1 VHVL(AA)

<220>

<221>misc_feature

<222>(1)..(121)

<223>VH

<220>

<221>misc_feature

<222>(122)..(144)

<223> linker

<220>

<221>misc_feature

<222>(145)..(253)

<223>VL

<400>105

<210>106

<211>749

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>G19-4 VLVH(DNA)

<400>106

<210>107

<211>247

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>G-19-4 VLVH(AA)

<220>

<221>misc_feature

<222>(1)..(108)

<223>VL

<220>

<221>misc_feature

<222>(109)..(125)

<223> linker

<220>

<221>misc_feature

<222>(126)..(247)

<223>VH

<400>107

<210>108

<211>752

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>G19-4 VHVL(DNA)

<400>108

<210>109

<211>248

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>G19-4 VHVL(AA)

<220>

<221>misc_feature

<222>(1)..(122)

<223>VH

<220>

<221>misc_feature

<222>(123)..(139)

<223> linker

<220>

<221>misc_feature

<222>(140)..(248)

<223>VL

<400>109

<210>110

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<221>misc_feature

<223>ssc(s)-hIgG1(DNA)

<400>110

<210>111

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<221>misc_feature

<223>ssc(s)-hIgG1(AA)

<400>111

<210>112

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<221>misc_feature

<223>scc(s)-hIgG1(DNA)

<400>112

<210>113

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<221>misc_feature

<223>scc(s)-hIgG1(AA)

<400>113

<210>114

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<221>misc_feature

<223>css(s)-hIgG1(DNA)

<400>114

<210>115

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<221>misc_feature

<223>css(s)-hIgG1(AA)

<400>115

<210>116

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221>misc_feature

<223>scs(s)-hIgG1(DNA)

<400>116

<210>117

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<221>misc_feature

<223>scs(s)-hIgG1(AA)

<400>117

<210>118

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<400>118

<210>119

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>119

<210>120

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<400>120

<210>121

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>121

<210>122

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<400>122

<210>123

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>123

<210>124

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<221>misc_feature

<223>csc(p)-hIgG1(DNA)

<400>124

<210>125

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<221>misc_feature

<223>csc(p)-hIgG1(AA)

<400>125

<210>126

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<221>misc_feature

<223>ssc(p)-hIgG1(DNA)

<400>126

<210>127

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<221>misc_feature

<223>ssc(p)-hIgG1(AA)

<400>127

<210>128

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<221>misc_feature

<223>scc(p)-hIgG1(DNA)

<400>128

<210>129

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<221>misc_feature

<223>scc(p)-hIgG1(AA)

<400>129

<210>130

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<221>misc_feature

<223>css(p)-hIgG1(DNA)

<400>130

<210>131

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<221>misc_feature

<223>css(p)-hIgG1(AA)

<400>131

<210>132

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<221>misc_feature

<223>ssc(p)-hIgG1(DNA)

<400>132

<210>133

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<221>misc_feature

<223>ssc(p)-hIgG1(DNA)

<400>133

<210>134

<211>18

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223>ssc(p)(DNA)

<220>

<223> Synthesis of primers

<400>134

<210>135

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>135

<210>136

<211>7

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<400>136

<210>137

<211>2

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>137

<210>138

<211>7

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<400>138

<210>139

<211>2

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>139

<210>140

<211>654

<212>DNA

<213> Intelligent people

<220>

<223> hIgG1 wild type

<400>140

<210>141

<211>217

<212>PRT

<213> Intelligent people

<220>

<223> hIgG1 wild type

<400>141

<210>142

<211>654

<212>DNA

<213> Intelligent people

<220>

<223>hIgG1(P238S)

<400>142

<210>143

<211>217

<212>PRT

<213> Intelligent people

<220>

<223>hIgG1(P238S)

<400>143

<210>144

<211>654

<212>DNA

<213> Intelligent people

<220>

<223>hIgG1(P331S)

<400>144

<210>145

<211>217

<212>PRT

<213> Intelligent people

<220>

<223>hIgG1(P331S)

<400>145

<210>146

<211>654

<212>DNA

<213> Intelligent people

<220>

<223>hIgG1(P238S/P331S)

<400>146

<210>147

<211>217

<212>PRT

<213> Intelligent people

<220>

<223>hIgG1(P238S/P331S)

<400>147

<210>148

<211>60

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>STD1(DNA)

<400>148

<210>149

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223>STD1(AA)

<400>149

<210>150

<211>114

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223>STD2(DNA)

<400>150

<210>151

<211>38

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223>STD2(AA)

<400>151

<210>152

<211>6

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223> linker H1(PN)

<400>152

<210>153

<211>2

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H1(AA)

<400>153

<210>154

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223> linker H2(PN)

<400>154

<210>155

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223> linker H2(AA)

<400>155

<210>156

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223> linker H3(PN)

<400>156

<210>157

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H3(AA)

<400>157

<210>158

<211>39

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223> linker H4(PN)

<400>158

<210>159

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H4(AA)

<400>159

<210>160

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223> linker H5(PN)

<400>160

<210>161

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H5(AA)

<400>161

<210>162

<211>54

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223> linker H6(PN)

<400>162

<210>163

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H6(AA)

<400>163

<210>164

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223> linker H7(PN)

<400>164

<210>165

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H7(AA)

<400>165

<210>166

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223> linker (G4S)3

<400>166

<210>167

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker (G4S)3

<400>167

<210>168

<211>60

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of primers

<220>

<223> linker (G4S)4

<400>168

<210>169

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker (G4S)4

<400>169

<210>170

<211>2337

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>2H7sssIgG1-STD1-2e12HL(DNA)

<400>170

<210>171

<211>772

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>2H7sssIgG1-STD1-2e12HL(AA)

<220>

<221>misc_feature

<222>(1)..(22)

<223> leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

<223> linker

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(281)

<223> hinge

<220>

<221>misc_feature

<222>(500)..(519)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(520)..(640)

<223>VH2

<220>

<221>misc_feature

<222>(641)..(660)

<223> linker 2

<220>

<221>misc_feature

<222>(661)..(772)

<223>VL2

<400>171

<210>172

<211>772

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>2H7sssIgG1(P238S/P331S) -STD1-2e12IIL (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

<223> leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

<223> linker

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

<223> hinge

<220>

<221>misc_feature

<222>(500)..(519)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(520)..(640)

<223>VH2

<220>

<221>misc_feature

<222>(641)..(660)

<223> linker 2

<220>

<221>misc_feature

<222>(661)..(772)

<223>VL2

<400>172

<210>173

<211>2322

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>2H7sssIgG1-STD1-2e12LH(DNA)

<400>173

<210>174

<211>767

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>2H7sssIgG1-STD1-2e12LH (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

<223> leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

<223> linker

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

<223> hinge

<220>

<221>misc_feature

<222>(500)..(519)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(520)..(631)

<223>VL2

<220>

<221>misc_feature

<222>(632)..(646)

<223> linker 2

<220>

<221>misc_feature

<222>(647)..(767)

<223>VH2

<400>174

<210>175

<211>767

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>2H7sssIgG1(P238S/P331S) -STD1-2e12LH (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

<223> leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

<223> linker

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

<223> hinge

<220>

<221>misc_feature

<222>(500)..(519)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(520)..(631)

<223>VL2

<220>

<221>misc_feature

<222>(632)..(646)

<223> linker 2

<220>

<221>misc_feature

<222>(647)..(767)

<223>VH2

<400>175

<210>176

<211>2376

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>2H7sssIgG1-STD2-2e12LH(DNA)

<400>176

<210>177

<211>785

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>2H7sssIgG1-STD2-2e12LH (w/2e12 leader sequence)

<220>

<221>misc_feature

<222>(1)..(22)

<223> leader sequence

<220>

<221>misc_feature

<222>(23)..(129)

<223>VL

<220>

<221>misc_feature

<222>(130)..(144)

<223> linker

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

<223> hinge

<220>

<221>misc_feature

<222>(500)..(537)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(538)..(649)

<223>VL2

<220>

<221>misc_feature

<222>(650)..(664)

<223> linker 2

<220>

<221>misc_feature

<222>(665)..(785)

<223>VH2

<400>177

<210>178

<211>785

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>2H7sssIgG1(P238S/P331S) -STD2-2e12LH (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

<223> leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

<223> linker

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(281)

<223> hinge

<220>

<221>misc_feature

<222>(500)..(537)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(538)..(649)

<223>VL2

<220>

<221>misc_feature

<222>(650)..(664)

<223> linker 2

<220>

<221>misc_feature

<222>(665)..(785)

<223>VH2

<400>178

<210>179

<211>2391

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>2H7sssIgG1-STD2-2e12HL(DNA)

<400>179

<210>180

<211>790

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>2H7sssIgG1-STD2-2e12HL (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

<223> leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

<223> linker

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

<223> hinge

<220>

<221>misc_feature

<222>(500)..(537)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(538)..(658)

<223>VH2

<220>

<221>misc_feature

<222>(659)..(678)

<223> linker 2

<220>

<221>misc_feature

<222>(679)..(790)

<223>VL2

<400>180

<210>181

<211>790

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>2H7sssIgG1(P238S/P331S) -STD2-2e12HL (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

<223> leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

<223> linker

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

<223> hinge

<220>

<221>misc_feature

<222>(500)..(537)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(538)..(658)

<223>VH2

<220>

<221>misc_feature

<222>(659)..(678)

<223> linker 2

<220>

<221>misc_feature

<222>(679)..(790)

<223>VL2

<400>181

<210>182

<211>2283

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>2H7sssIgG1-H1-2e12HL(DNA)

<400>182

<210>183

<211>754

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>2H7sssIgG1-H1-2e12HL (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

<223> leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

<223> linker

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(281)

<223> hinge

<220>

<221>misc_feature

<222>(500)..(501)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(502)..(622)

<223>VH2

<220>

<221>misc_feature

<222>(623)..(642)

<223> linker 2

<220>

<221>misc_feature

<222>(643)..(754)

<223>VL2

<400>183

<210>184

<211>2301

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>2H7sssIgG1-H2-2e12HL(DNA)

<400>184

<210>185

<211>760

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>2H7sssIgG1-H2-2e12HL (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

<223> leader sequence

<220>

<221>misc_feature

<222>(23)..(127)

<223>VL

<220>

<221>misc_feature

<222>(128)..(144)

<223> linker

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

<223> hinge

<220>

<221>misc_feature

<222>(500)..(507)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(508)..(628)

<223>VH2

<220>

<221>misc_feature

<222>(629)..(648)

<223> linker 2

<220>

<221>misc_feature

<222>(649)..(760)

<223>VL2

<400>185

<210>186

<211>2307

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>2H7sssIgG1-H3-2e12HL(DNA)

<400>186

<210>187

<211>762

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>2H7sssIgG1-H3-2e12HL (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

<223> leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

<223> linker

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

<223> hinge

<220>

<221>misc_feature

<222>(500)..(509)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(510)..(630)

<223>VH2

<220>

<221>misc_feature

<222>(631)..(650)

<223> linker 2

<220>

<221>misc_feature

<222>(651)..(762)

<223>VL2

<400>187

<210>188

<211>2316

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>2H7sssIgG1-H4-2e12HL(DNA)

<400>188

<210>189

<211>765

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>2H7sssIgG1-H4-2e12HL(AA)

<220>

<221>misc_feature

<222>(1)..(22)

<223> leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

<223> linker

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

<223> hinge

<220>

<221>misc_feature

<222>(500)..(512)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(513)..(633)

<223>VH2

<220>

<221>misc_feature

<222>(634)..(653)

<223> linker 2

<220>

<221>misc_feature

<222>(654)..(765)

<223>VL2

<400>189

<210>190

<211>2322

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>2H7sssIgG1-H5-2e12HL(DNA)

<400>190

<210>191

<211>767

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>2H7sssIgG1-H5-2e12HL (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

<223> leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

<223> linker

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

<223> hinge

<220>

<221>misc_feature

<222>(500)..(514)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(515)..(635)

<223>VH2

<220>

<221>misc_feature

<222>(636)..(655)

<223> linker 2

<220>

<221>misc_feature

<222>(656)..(767)

<223>VL2

<400>191

<210>192

<211>2331

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>2H7sssIgG1-H6-2e12HL(DNA)

<400>192

<210>193

<211>770

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>2H7sssIgG1-H6-2e12HL (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

<223> leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

<223> linker

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

<223> hinge

<220>

<221>misc_feature

<222>(500)..(517)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(518)..(638)

<223>VH2

<220>

<221>misc_feature

<222>(639)..(658)

<223> linker 2

<220>

<221>misc_feature

<222>(659)..(770)

<223>VL2

<400>193

<210>194

<211>2301

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>2H7ssc_IgG1-H7-2e12HL(DNA)

<400>194

<210>195

<211>760

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>2H7ssc _ IgG1-H7-2e12HL (w/2e12 linker) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

<223> leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

<223> linker

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

<223> hinge

<220>

<221>misc_feature

<222>(500)..(507)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(508)..(628)

<223>VH2

<220>

<221>misc_feature

<222>(629)..(648)

<223> linker 2

<220>

<221>misc_feature

<222>(649)..(760)

<223>VL2

<400>195

<210>196

<211>2283

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>2H7sssIgG1-H7-G194HL(DNA)

<400>196

<210>197

<211>754

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>2H7sssIgG1-H7-G194HL (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

<223> leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

<223> linker

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH1

<220>

<221>misc_feature

<222>(268)..(282)

<223> hinge

<220>

<221>misc_feature

<222>(500)..(507)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(508)..(629)

<223>VH2

<220>

<221>misc_feature

<222>(630)..(646)

<223> linker 2

<220>

<221>misc_feature

<222>(647)..(754)

<223>VL2

<400>197

<210>198

<211>2298

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>2H7sssIgG1-H7-G281HL(DNA)

<400>198

<210>199

<211>759

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>2H7sssIgG1-H7-G281HL (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

<223> leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL1

<220>

<221>misc_feature

<222>(129)..(144)

<223> linker

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH1

<220>

<221>misc_feature

<222>(268)..(282)

<223> hinge

<220>

<221>misc_feature

<222>(500)..(507)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(508)..(629)

<223>VH2

<220>

<221>misc_feature

<222>(630)..(651)

<223> linker 2

<220>

<221>misc_feature

<222>(652)..(759)

<223>VL2

<400>199

<210>200

<211>1533

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>2e12-sss-IgG1HL SMIP(DNA)

<400>200

<210>201

<211>510

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>2e12-sss-IgG1HL SMIP(AA)

<220>

<221>misc_feature

<222>(1)..(22)

<223> leader sequence

<220>

<221>misc_feature

<222>(24)..(144)

<223>VH

<220>

<221>misc_feature

<222>(145)..(164)

<223> linker

<220>

<221>misc_feature

<222>(165)..(276)

<223>VL

<220>

<221>misc_feature

<222>(279)..(293)

<223> hinge

<400>201

<210>202

<211>1518

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>2e12-sss-IgG1LH SMIP(DNA)

<400>202

<210>203

<211>505

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>2e12-sss-IgG1LH SMIP(AA)

<220>

<221>misc_feature

<222>(1)..(23)

<223> leader sequence

<220>

<221>misc_feature

<222>(24)..(135)

<223>VL

<220>

<221>misc_feature

<222>(136)..(150)

<223> linker

<220>

<221>misc_feature

<222>(151)..(271)

<223>VH

<220>

<221>misc_feature

<222>(274)..(288)

<223> hinge

<400>203

<210>204

<211>1498

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>G28-1LH SMIP(DNA)

<400>204

<210>205

<211>492

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>G28-1LH SMIP(AA)

<220>

<221>misc_feature

<222>(1)..(20)

<223> leader sequence

<220>

<221>misc_feature

<222>(21)..(127)

<223>VL

<220>

<221>misc_feature

<222>(128)..(144)

<223> linker

<220>

<221>misc_feature

<222>(145)..(260)

<223>VH

<220>

<221>misc_feature

<222>(261)..(275)

<223> hinge

<400>205

<210>206

<211>1522

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>G28-1HL SMIP(DNA)

<400>206

<210>207

<211>500

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>G28-1HL SMIP(AA)

<220>

<221>misc_feature

<222>(1)..(20)

<223> leader sequence

<220>

<221>misc_feature

<222>(21)..(136)

<223>VH

<220>

<221>misc_feature

<222>(137)..(158)

<223> linker

<220>

<221>misc_feature

<222>(159)..(266)

<223>VL

<220>

<221>misc_feature

<222>(268)..(283)

<223> hinge

<400>207

<210>208

<211>1522

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>G19-4LH SMIP(DNA)

<400>208

<210>209

<211>500

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>G19-4LH SMIP(AA)

<220>

<221>misc_feature

<222>(1)..(20)

<223> leader sequence

<220>

<221>misc_feature

<222>(21)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(145)

<223> linker

<220>

<221>misc_feature

<222>(146)..(267)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

<223> hinge

<400>209

<210>210

<211>1525

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>G19-4HL SMIP(DNA)

<400>210

<210>211

<211>501

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>G19-4HLSMIP(AA)

<220>

<221>misc_feature

<222>(1)..(20)

<223> leader sequence

<220>

<221>misc_feature

<222>(21)..(142)

<223>VH

<220>

<221>misc_feature

<222>(143)..(159)

<223> linker

<220>

<221>misc_feature

<222>(160)..(267)

<223>VL

<220>

<221>misc_feature

<222>(270)..(284)

<223> hinge

<400>211

<210>212

<211>2328

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>n2H7sssIgG1-STD1-2e12HL(DNA)

<400>212

<210>213

<211>769

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>n2H7sssIgG1-STD1-2e12HL(AA)

<220>

<221>misc_feature

<222>(1)..(20)

<223> leader sequence

<220>

<221>misc_feature

<222>(21)..(127)

<223>VL

<220>

<221>misc_feature

<222>(128)..(142)

<223> linker

<220>

<221>misc_feature

<222>(143)..(264)

<223>VH

<220>

<221>misc_feature

<222>(265)..(279)

<223> hinge

<220>

<221>misc_feature

<222>(497)..(516)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(517)..(637)

<223>VH2

<220>

<221>misc_feature

<222>(638)..(657)

<223> linker 2

<220>

<221>misc_feature

<222>(658)..(769)

<223>VL2

<400>213

<210>214

<211>2313

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>n2H7sssIgG1-STD2-2e12LH(DNA)

<400>214

<210>215

<211>764

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>n2H7sssIgG1-STD2-2e12LH(AA)

<220>

<221>misc_feature

<222>(1)..(20)

<223> leader sequence

<220>

<221>misc_feature

<222>(21)..(126)

<223>VL

<220>

<221>misc_feature

<222>(127)..(142)

<223> linker

<220>

<221>misc_feature

<222>(143)..(264)

<223>VH

<220>

<221>misc_feature

<222>(265)..(279)

<223> hinge

<220>

<221>misc_feature

<222>(497)..(516)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(517)..(628)

<223>VL2

<220>

<221>misc_feature

<222>(629)..(643)

<223> linker

<220>

<221>misc_feature

<222>(644)..(764)

<223>VH2

<400>215

<210>216

<211>2048

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>n2H7sssIgG1-H1-2e12HL(DNA)

<400>216

<210>217

<211>751

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>n2H7sssIgG1-H1-2e12HL(AA)

<220>

<221>misc_feature

<222>(1)..(20)

<223> leader sequence

<220>

<221>misc_feature

<222>(21)..(126)

<223>VL

<220>

<221>misc_feature

<222>(127)..(142)

<223> linker

<220>

<221>misc_feature

<222>(143)..(264)

<223>VH

<220>

<221>misc_feature

<222>(265)..(279)

<223> hinge

<220>

<221>misc_feature

<222>(497)..(498)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(499)..(619)

<223>VH2

<220>

<221>misc_feature

<222>(620)..(639)

<223> linker 2

<220>

<221>misc_feature

<222>(640)..(751)

<223>VL2

<400>217

<210>218

<211>2292

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>n2H7sssIgG1-H2-2e12HL(DNA)

<400>218

<210>219

<211>757

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>n2H7sssIgG1-H2-2e12HL(AA)

<220>

<221>misc_feature

<222>(1)..(20)

<223> leader sequence

<220>

<221>misc_feature

<222>(21)..(126)

<223>VL

<220>

<221>misc_feature

<222>(127)..(142)

<223> linker

<220>

<221>misc_feature

<222>(143)..(264)

<223>VH

<220>

<221>misc_feature

<222>(265)..(279)

<223> hinge

<220>

<221>misc_feature

<222>(497)..(504)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(505)..(625)

<223>VH2

<220>

<221>misc_feature

<222>(626)..(645)

<223> linker 2

<220>

<221>misc_feature

<222>(646)..(757)

<223>VL2

<400>219

<210>220

<211>2298

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>n2H7sssIgG1-H3-2e12HL(DNA)

<400>220

<210>221

<211>759

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>n2H7sssIgG1-H3-2e12HL(AA)

<220>

<221>misc_feature

<222>(1)..(20)

<223> leader sequence

<220>

<221>misc_feature

<222>(21)..(126)

<223>VL

<220>

<221>misc_feature

<222>(127)..(142)

<223> linker

<220>

<221>misc_feature

<222>(143)..(264)

<223>VH

<220>

<221>misc_feature

<222>(265)..(279)

<223> hinge

<220>

<221>misc_feature

<222>(497)..(506)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(507)..(627)

<223>VH2

<220>

<221>misc_feature

<222>(628)..(647)

<223> linker 2

<220>

<221>misc_feature

<222>(648)..(759)

<223>VL2

<400>221

<210>222

<211>2307

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>n2H7sssIgG1-H4-2e12HL(DNA)

<400>222

<210>223

<211>762

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>n2H7sssIgG1-H4-2e12HL(AA)

<220>

<221>misc_feature

<222>(1)..(20)

<223> leader sequence

<220>

<221>misc_feature

<222>(21)..(126)

<223>VL

<220>

<221>misc_feature

<222>(127)..(142)

<223> linker

<220>

<221>misc_feature

<222>(143)..(264)

<223>VH

<220>

<221>misc_feature

<222>(265)..(279)

<223> hinge

<220>

<221>misc_feature

<222>(497)..(509)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(510)..(630)

<223>VH2

<220>

<221>misc_feature

<222>(631)..(650)

<223> linker 2

<220>

<221>misc_feature

<222>(651)..(762)

<223>VL2

<400>223

<210>224

<211>2313

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>n2H7sssIgG1-H5-2e12HL(DNA)

<400>224

<210>225

<211>764

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>n2H7sssIgG1-H5-2e12HL(AA)

<220>

<221>misc_feature

<222>(1)..(20)

<223> leader sequence

<220>

<221>misc_feature

<222>(21)..(126)

<223>VL

<220>

<221>misc_feature

<222>(127)..(142)

<223> linker

<220>

<221>misc_feature

<222>(143)..(264)

<223>VH

<220>

<221>misc_feature

<222>(265)..(279)

<223> hinge

<220>

<221>misc_feature

<222>(497)..(511)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(512)..(632)

<223>VH2

<220>

<221>misc_feature

<222>(633)..(652)

<223> linker 2

<220>

<221>misc_feature

<222>(653)..(764)

<223>VL2

<400>225

<210>226

<211>2322

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>n2H7sssIgG1-H6-2e12HL(DNA)

<400>226

<210>227

<211>767

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>n2H7sssIgG1-H6-2e12HL(AA)

<220>

<221>misc_feature

<222>(1)..(20)

<223> leader sequence

<220>

<221>misc_feature

<222>(21)..(126)

<223>VL

<220>

<221>misc_feature

<222>(127)..(142)

<223> linker

<220>

<221>misc_feature

<222>(143)..(264)

<223>VH

<220>

<221>misc_feature

<222>(265)..(279)

<223> hinge

<220>

<221>misc_feature

<222>(497)..(514)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(515)..(635)

<223>VH2

<220>

<221>misc_feature

<222>(636)..(655)

<223> linker 2

<220>

<221>misc_feature

<222>(656)..(767)

<223>VL2

<400>227

<210>228

<211>2337

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>2H7sssIgG1-H7-G281HL(DNA)

<400>228

<210>229

<211>772

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>2H7cscIgG1-STD1-2E12HL (w/2E12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

<223> leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

<223> linker

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

<223> hinge

<220>

<221>misc_feature

<222>(500)..(519)

<223> EFD-BD2 linker

<220>

<221>misc_feature

<222>(520)..(640)

<223>VH2

<220>

<221>misc_feature

<222>(641)..(660)

<223> linker 2

<220>

<221>misc_feature

<222>(661)..(772)

<223>VL2

<400>229

<210>230

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>scs(s)-hIgG1(DNA)

<400>230

<210>231

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>scs(s)-hIgG1(AA)

<400>231

<210>232

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223>ccc(s)(DNA)

<400>232

<210>233

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223>ccc(s)(AA)

<400>233

<210>234

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H8(PN)

<400>234

<210>235

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>235

<210>236

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H9(PN)

<400>236

<210>237

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H9(AA)

<400>237

<210>238

<400>238

000

<210>239

<400>239

000

<210>240

<211>51

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H11(PN)

<400>240

<210>241

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H11(AA)

<400>241

<210>242

<211>51

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H12(PN)

<400>242

<210>243

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H12(AA)

<400>243

<210>244

<211>51

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H13(PN)

<400>244

<210>245

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H13(AA)

<400>245

<210>246

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H15(PN)

<400>246

<210>247

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H15(AA)

<400>247

<210>248

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H16(PN)

<400>248

<210>249

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H16(AA)

<400>249

<210>250

<211>60

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H17(PN)

<400>250

<210>251

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H17(AA)

<400>251

<210>252

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H18(PN)

<400>252

<210>253

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H18(AA)

<400>253

<210>254

<211>60

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H19(PN)

<400>254

<210>255

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H19(AA)

<400>255

<210>256

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H20(PN)

<400>256

<210>257

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H20(AA)

<400>257

<210>258

<211>60

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223> linker H21(PN)

<400>258

<210>259

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H21(AA)

<400>259

<210>260

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<223> linker H22(PN)

<400>260

<210>261

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H22(AA)

<400>261

<210>262

<211>63

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H23(PN)

<400>262

<210>263

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H23(AA)

<400>263

<210>264

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H24(DNA)

<400>264

<210>265

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H24(AA)

<400>265

<210>266

<211>60

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H25(PN)

<400>266

<210>267

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H25(AA)

<400>267

<210>268

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H26(PN)

<400>268

<210>269

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H26(AA)

<400>269

<210>270

<211>60

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H27(PN)

<400>270

<210>271

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H27(AA)

<400>271

<210>272

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H28(PN)

<400>272

<210>273

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H28(AA)

<400>273

<210>274

<211>63

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H29(PN)

<400>274

<210>275

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H29(AA)

<400>275

<210>276

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H30(PN)

<400>276

<210>277

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H30(AA)

<400>277

<210>278

<211>63

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H31(PN)

<400>278

<210>279

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H31(AA)

<400>279

<210>280

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H32(PN)

<400>280

<210>281

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H32(AA)

<400>281

<210>282

<211>60

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H33(PN)

<400>282

<210>283

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H33(AA)

<400>283

<210>284

<400>284

<210>285

<400>285

<210>286

<400>286

<210>287

<400>287

<210>288

<211>39

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H36(PN)

<400>288

<210>289

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H36(AA)

<400>289

<210>290

<400>290

<210>291

<400>291

<210>292

<400>292

<210>293

<400>293

<210>294

<400>294

<210>295

<400>295

<210>296

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H40(PN)

<400>296

<210>297

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H40(AA)

<400>297

<210>298

<400>298

<210>299

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<221>misc_feature

<223> linker H41

<400>299

<210>300

<400>300

<210>301

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H42(AA)

<400>301

<210>302

<400>302

<210>303

<400>303

<210>304

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H44(PN)

<400>304

<210>305

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H44(AA)

<400>305

<210>306

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H45(PN)

<400>306

<210>307

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H45(AA)

<400>307

<210>308

<211>33

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<223> linker H46

<400>308

<210>309

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H46

<400>309

<210>310

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H47

<400>310

<210>311

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H48

<400>311

<210>312

<400>312

<210>313

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H50

<400>313

<210>314

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H51

<400>314

<210>315

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H52

<400>315

<210>316

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H53

<400>316

<210>317

<211>19

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H54

<400>317

<210>318

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H55

<400>318

<210>319

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H56

<400>319

<210>320

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H57

<400>320

<210>321

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H58

<400>321

<210>322

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H59

<400>322

<210>323

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H60

<400>323

<210>324

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H61

<400>324

<210>325

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H62

<400>325

<210>326

<211>19

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H63

<400>326

<210>327

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>327

<210>328

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H65

<400>328

<210>329

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H66

<400>329

<210>330

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H67

<400>330

<210>331

<211>118

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> linker H68

<400>331

<210>332

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> anti-CD-20 VL CDR1

<400>332

<210>333

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> anti-CD-20 VL CDR1

<400>333

<210>334

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> anti-CD-20 VL CDR3

<400>334

<210>335

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> anti-CD-20 VL CDR3

<400>335

<210>336

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> anti-CD-20 VH CDR2

<400>336

<210>337

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> anti-CD-20 VH CDR2

<400>337

<210>338

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> anti-CD 20 VH CDR3

<400>338

<210>339

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> anti-CD 20 VH CDR3

<400>339

<210>340

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> anti-CD 20 VH CDR3

<400>340

<210>341

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> anti-CD 20 VH CDR3

<400>341

<210>342

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> anti-CD 20 VH CDR3

<400>342

<210>343

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> anti-CD 20 VH CDR3

<400>343

<210>344

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> anti-CD-20 VH CDR3

<400>344

<210>345

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> anti-CD 20 VH CDR3

<400>345

<210>346

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> Scorpion linker core (2H7) sequence

<400>346

<210>347

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> spreading sequence

<400>347

<210>348

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> spreading sequence

<400>348

<210>349

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> spreading sequence

<400>349

<210>350

<211>19

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> spreading sequence

<400>350

<210>351

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> expanded scorpion linker

<400>351

<210>352

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> expanded scorpion linker

<400>352

<210>353

<211>22

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> expanded scorpion linker

<400>353

<210>354

<211>27

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> expanded scorpion linker

<400>354

<210>355

<211>51

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223>VL CDR1-X-VL CDR3-X-VH CDR2-X-VH CDR3

<220>

<221>misc_feature

<222>(11)..(11)

<223> Xaa ═ amino acid range between VL CDR1 and VL CDR3

<220>

<221>misc_feature

<222>(21)..(21)

<223> Xaa ═ the range of amino acids between VL CDR3 and VH CDR2

<220>

<221>misc_feature

<222>(39)..(39)

<223> Xaa ═ the range of amino acids between VH CDR2 and VH CDR3

<400>355

<210>356

<211>51

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223>VLCDR1-X-VLCDR3-X-VHCDR2-X-VHCDR3

<220>

<221>misc_feature

<222>(11)..(11)

<223> Xaa ═ amino acid range between VL CDR1 and VL CDR3

<220>

<221>misc_feature

<222>(21)..(21)

<223> Xaa ═ the range of amino acids between VL CDR3 and VH CDR2

<220>

<221>misc_feature

<222>(39)..(39)

<223> Xaa ═ the range of amino acids between VH CDR2 and VH CDR3

<400>356

<210>357

<211>51

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223>VLCDR1-X-VLCDR3-X-VHCDR2-X-VHCDR3

<220>

<221>misc_feature

<222>(11)..(11)

<223> Xaa ═ amino acid range between VL CDR1 and VL CDR3

<220>

<221>misc_feature

<222>(21)..(21)

<223> Xaa ═ the range of amino acids between VL CDR3 and VH CDR2

<220>

<221>misc_feature

<222>(39)..(39)

<223> Xaa ═ the range of amino acids between VH CDR2 and VH CDR3

<400>357

<210>358

<211>51

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223>VLCDR1-X-VLCDR3-X-VHCDR2-X-VHCDR3

<220>

<221>misc_feature

<222>(11)..(11)

<223> Xaa ═ amino acid range between VL CDR1 and VL CDR3

<220>

<221>misc_feature

<222>(21)..(21)

<223> Xaa ═ the range of amino acids between VL CDR3 and VH CDR2

<220>

<221>misc_feature

<222>(39)..(39)

<223> Xaa ═ the range of amino acids between VH CDR2 and VH CDR3

<400>358

<210>359

<211>51

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223>VLCDR1-X-VLCDR3-X-VHCDR2-X-VHCDR3

<220>

<221>misc_feature

<222>(11)..(11)

<223> Xaa ═ amino acid range between VL CDR1 and VL CDR3

<220>

<221>misc_feature

<222>(21)..(21)

<223> Xaa ═ the range of amino acids between VL CDR3 and VH CDR2

<220>

<221>misc_feature

<222>(39)..(39)

<223> Xaa ═ the range of amino acids between VH CDR2 and VH CDR3

<400>359

<210>360

<211>51

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223>VLCDR1-X-VLCDR3-X-VHCDR2-X-VHCDR3

<220>

<221>misc_feature

<222>(11)..(11)

<223> Xaa ═ amino acid range between VL CDR1 and VL CDR3

<220>

<221>misc_feature

<222>(21)..(21)

<223> Xaa ═ the range of amino acids between VL CDR3 and VH CDR2

<220>

<221>misc_feature

<222>(39)..(39)

<223> Xaa ═ the range of amino acids between VH CDR2 and VH CDR3

<400>360

<210>361

<211>51

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223>VLCDR1-X-VLCDR3-X-VHCDR2-X-VHCDR3

<220>

<221>misc_feature

<222>(11)..(11)

<223> Xaa ═ amino acid range between VL CDR1 and VL CDR3

<220>

<221>misc_feature

<222>(21)..(21)

<223> Xaa ═ the range of amino acids between VL CDR3 and VH CDR2

<220>

<221>misc_feature

<222>(39)..(39)

<223> Xaa ═ the range of amino acids between VH CDR2 and VH CDR3

<400>361

<210>362

<211>51

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223>VLCDR1-X-VLCDR3-X-VHCDR2-X-VHCDR3

<220>

<221>misc_feature

<222>(11)..(11)

<223> Xaa ═ amino acid range between VL CDR1 and VL CDR3

<220>

<221>misc_feature

<222>(21)..(21)

<223> Xaa ═ the range of amino acids between VL CDR3 and VH CDR2

<220>

<221>misc_feature

<222>(39)..(39)

<223> Xaa ═ the range of amino acids between VH CDR2 and VH CDR3

<400>362

<210>363

<211>51

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223>VLCDR1-X-VLCDR3-X-VHCDR2-X-VHCDR3

<220>

<221>misc_feature

<222>(11)..(11)

<223> Xaa ═ amino acid range between VL CDR1 and VL CDR3

<220>

<221>misc_feature

<222>(21)..(21)

<223> Xaa ═ the range of amino acids between VL CDR3 and VH CDR2

<220>

<221>misc_feature

<222>(39)..(39)

<223> Xaa ═ the range of amino acids between VH CDR2 and VH CDR3

<400>363

<210>364

<211>51

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223>VLCDR1-X-VLCDR3-X-VHCDR2-X-VHCDR3

<220>

<221>misc_feature

<222>(11)..(11)

<223> Xaa ═ amino acid range between VL CDR1 and VL CDR3

<220>

<221>misc_feature

<222>(21)..(21)

<223> Xaa ═ the range of amino acids between VL CDR3 and VH CDR2

<220>

<221>misc_feature

<222>(39)..(39)

<223> Xaa ═ the range of amino acids between VH CDR2 and VH CDR3

<400>364

<210>365

<211>51

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223>VLCDR1-X-VLCDR3-X-VHCDR2-X-VHCDR3

<220>

<221>misc_feature

<222>(11)..(11)

<223> Xaa ═ amino acid range between VL CDR1 and VL CDR3

<220>

<221>misc_feature

<222>(21)..(21)

<223> Xaa ═ the range of amino acids between VL CDR3 and VH CDR2

<220>

<221>misc_feature

<222>(39)..(39)

<223> Xaa ═ the range of amino acids between VH CDR2 and VH CDR3

<400>365

<210>366

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> partial CH3 sequence

<400>366

<210>367

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> partial CH3 sequence

<400>367

<210>368

<211>1

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> partial CH3 sequence

<400>368

<210>369

<211>3

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> partial CH3 sequence

<400>369

<210>370

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> partial CH3 sequence

<400>370

<210>371

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> partial CH3 sequence

<400>371

<210>372

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> scorpion-shaped connector

<400>372

<210>373

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> scorpion-shaped connector

<400>373

<210>374

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> scorpion-shaped connector

<400>374

<210>375

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> scorpion-shaped connector

<400>375

<210>376

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> scorpion-shaped connector

<400>376

<210>377

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> scorpion-shaped connector

<400>377

<210>378

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> anti-CD 20VL CDR1(TRU-015)

<400>378

<210>379

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<223> anti-CD 20VH CDR3(TRU-015)

<400>379

Claims (98)

1. A multivalent single chain binding protein having effector function, comprising:

a. a first binding domain derived from an immunoglobulin or immunoglobulin-like molecule;

b. a constant sub-region providing an effector function, the immunoglobulin constant sub-region being C-terminal to the first binding domain;

c. a scorpion linker at the C-terminus of the constant sub-region; and

d. a second binding domain derived from an immunoglobulin or immunoglobulin-like molecule and located C-terminal to the constant sub-region;

thus, the constant sub-region is positioned between the first binding domain and the second binding domain.

2. The protein of claim 1, wherein the first binding domain comprises a light chain variable region derived from a first immunoglobulin and a heavy chain variable region derived from a second immunoglobulin.

3. The protein of claim 2, wherein the first immunoglobulin and the second immunoglobulin are the same immunoglobulin.

4. The protein of claim 1, wherein the second binding domain comprises a light chain variable region derived from a first immunoglobulin and a heavy chain variable region derived from a second immunoglobulin.

5. The protein of claim 4, wherein the first immunoglobulin and the second immunoglobulin are the same immunoglobulin.

6. The protein of claim 1, wherein the first binding domain and the second binding domain recognize the same molecular target.

7. The protein of claim 1, wherein the first binding domain and the second binding domain recognize the same epitope.

8. The protein of claim 1, wherein the first binding domain and the second binding domain recognize different molecular targets on the same eukaryotic cell, prokaryotic cell, virus, vector, or object.

9. The protein of claim 1, wherein the first binding domain and the second binding domain recognize molecular targets associated with a eukaryotic cell, prokaryotic cell, virus, vector, or object that is physically distinct.

10. The protein of claim 1, wherein at least one of the first binding domain and the second binding domain recognizes at least one molecular target that is not associated with a eukaryotic cell, prokaryotic cell, virus, vector, or object.

11. The protein of claim 1, wherein each of the first binding domain, the second binding domain, and the constant sub-region is derived from a human immunoglobulin.

12. The protein of claim 1, wherein at least one of the first binding domain and the second binding domain recognizes a target on a cancer cell.

13. The protein of claim 11, wherein at least one of the first binding domain and the second binding domain recognizes a target selected from the group consisting of: tumor antigens, B-cell targets, TNF receptor superfamily members, Hedgehog family members, receptor tyrosine kinases, proteoglycan-related molecules, TGF- β superfamily members, Wnt-related molecules, receptor ligands, T-cell targets, dendritic cell targets, NK cell targets, monocyte/macrophage targets, and angiogenesis targets.

14. The protein of claim 13, wherein the tumor antigen is selected from the group consisting of: squamous cell carcinoma antigen 1, squamous cell carcinoma antigen 2, ovarian carcinoma antigen CA125, mucin 1, CTCL tumor antigen se1-1, CTCL tumor antigen se14-3, CTCL tumor antigen se20-4, CTCL tumor antigen se20-9, CTCL tumor antigen se33-1, CTCL tumor antigen se37-2, CTCL tumor antigen se57-1, CTCL tumor antigen se89-1, prostate specific membrane antigen, 5T4 carcinotrophoblast glycoprotein, Orf73 Kaposi's sarcoma-associated herpesvirus, MAGE-C1, MAGE-B1 antigen, MAGE-B2 antigen, MAGE-2 antigen, MAGE-4a antigen, MAGE-4B antigen, colon cancer antigen NY-CO-45, lung cancer antigen NY-LU-12 variant A, cancer-associated surface antigen, adenocarcinoma antigen ART1, paratumor-associated brain-testis cancer antigen, Nerve tumor ventral antigen 2, hepatocellular carcinoma antigen gene 520, tumor-associated antigen CO-029, tumor-associated antigen MAGE-X2, synovial sarcoma X breakpoint 2, squamous cell carcinoma antigen recognized by T cells, serologically defined colon cancer antigen 1, serologically defined breast cancer antigen NY-BR-15, serologically defined breast cancer antigen NY-BR-16, chromogranin A; parathyroid secretory protein 1, DUPAN-2, CA19-9, CA72-4, CA195 and L6.

15. The protein of claim 13, wherein the B cell target is selected from the group consisting of: CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a/b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138 and CDw 150.

16. The protein of claim 13, wherein the TNF receptor superfamily member is selected from the group consisting of: 4-1BB/TNFRSF9, NGF/TNFRSF 16, BAFFR/TNFRSF13C, osteoprotegerin/TNFRSF 11B, BCMA/TNFRSF 17, OX40/TNFRSF4, CD27/TNFRSF7, RANK/TNFRSF 11A, CD30/TNFRSF A, CD30, RELT/TNFRSF 19A, CD30, CD A, CD30/TNFRSF 5, TACI/TNFRSF 13A, CD30, DcR A, CD30/TNFRSF 6A, CD30, TNF RI/TNFRSF 1A, CD30/TNFRSF A, CD30, TNF RII/TNFRSF 1A, CD30, DcR A, CD30/TNFRSF A, CD30, TRAILRSF A, CD30/TNFRSF 10A, CD30, DR A, CD30/TNFRSF A, CD30, TRANFR A, CD30/TNFRSF A, CD30, TNFRRSF A, CD30/TNFRSF A, CD30, TNFRSF A, CD30/TNFRSF A, CD30, TNFRFSF A, CD 30/TNFRFSF A, CD30, TNFRFSF A, CD30/TNFRSF A, CD30, TNFRSF A, CD30/TNFRSF A, CD30, TNFRFSF A, CD30, TNFRSF A, CD30, TNFRFSF-A, CD30/TNFRSF A, CD30, TNFRFIF A, CD30, TNFRFSF-A, CD30, TNFRFIFLP-A, CD30, TNFRSF A, CD30/TNFRSF A, CD30, CD27 ligand/TNFSF 7, TL1A/TNFSF15, CD30 ligand/TNFSF 8, TNF-alpha/TNFSF 1A, CD40 ligand/TNFSF 5, TNF-beta/TNFSF 1B, EDA-A2, TRAIL/TNFSF10, Fas ligand/TNFSF 6, TRANCE/TNFSF11, GITR ligand/TNFSF 18, TWEAK/TNFSF12, and LIGHT/TNFSF 14.

17. The protein of claim 13, wherein the Hedgehog family member is selected from the group consisting of Patched and Smoothened.

18. The protein of claim 13, wherein the receptor tyrosine kinase is selected from the group consisting of: ax1, FGF R4, Clq R1/CD93, FGF R93, DDR 93, Flt-3, DDR 93, HGF R, Dtk, IGF-93, EGF R, IGF-II R, Eph, INSRR, EphA 93, insulin R/CD220, EphA 93, M-CSF R, EphA 93, Mer, EphA 93, MSP R/Ron, EphA 93, MuSK, EphA 93, PDGF R alpha, EphA 93, PDGF R beta, EphA 93, Ret, EphB 93, ROR 93, EphB 93, SCF R/c-kit, EphB 93, Tie-1, EphB 93, Tie-2, ErbB 93, TrkB 93, TrkC, VEGF C, Flt/FLR 93, Flt-93, Flt/FLR 93, VEGF R93, Flt-93, and Flt/FLR 93.

19. The protein of claim 13, wherein the proteoglycan-related molecule is selected from the group consisting of proteoglycans and modulators thereof.

20. The protein of claim 13, wherein the Transforming Growth Factor (TGF) - β superfamily member is selected from the group consisting of: activin RIA/ALK-2, GFR alpha-1, activin RIB/ALK-4, GFR alpha-2, activin RIIA, GFR alpha-3, activin RIIB, GFR alpha-4, ALK-1, MIS RII, ALK-7, Ret, BMPR-IA/ALK-3, TGF-beta RI/ALK-5, BMPR-IB/ALK-6, TGF-beta RII, BMPR-II, TGF-beta RIIb, endothelial factor/CD 105, and TGF-beta RIII.

21. The protein of claim 13, wherein the Wnt-related molecule is selected from the group consisting of: frizzled-1, Frizzled-8, Frizzled-2, Frizzled-9, Frizzled-3, sFRP-1, Frizzled-4, sFRP-2, Frizzled-5, sFRP-3, Frizzled-6, sFRP-4, Frizzled-7, MFRP, LRP5, LRP6, Wnt-1, Wnt-8a, Wnt-3a, Wnt-10b, Wnt-4, Wnt-11, Wnt-5a, Wnt-9a, and Wnt-7 a.

22. The protein of claim 13, wherein the receptor ligand is selected from the group consisting of: 4-1BB ligand/TNFSF 9, lymphotoxin, APRIL/TNFSF 13, lymphotoxin beta/TNFSF 3, BAFF/TNFSF13C, OX40 ligand/TNFSF 4, CD27 ligand/TNFSF 7, TL1A/TNFSF15, CD30 ligand/TNFSF 8, TNF-alpha/TNFSF 1A, CD40 ligand/TNFSF 5, TNF-beta/TNFSF 1 40, EDA-A40, TRAIL/TNFSF 40, Fas ligand/TNFSF 40, TRANCE/TNFSF 40, GITR ligand/TNFSF 18, TWEAK/TNFSF 40, LIGHT/TNFSF 40, amphiregulin, NRG 40 isoform GGF 40, beta cell growth factor, NRG 40 isoform SMDF, EGF, NRG 40-alpha/TNGF 40-alpha, NRIG, NRG 40-beta-regulatory TGF-alpha, EGF, TMF-1/TMF-1-beta-regulatory TGF-alpha, TMF 40, TMF-alpha-regulatory EGF, TMF-alpha, TMF-3, TMF-beta-regulatory EGF, TMF-alpha, TMF-alpha, IGF-II, insulin, activin A, activin B, activin AB, activin C, BMP-2, BMP-7, BMP-3, BMP-8, BMP-3B/GDF-10, BMP-9, BMP-4, BMP-15, BMP-5, Dpp protein (Decapentaplegic), BMP-6, GDF-1, GDF-8, GDF-3, GDF-9, GDF-5, GDF-11, GDF-6, GDF-15, GDF-7, Artemin (Artemin), Neurturin (Neurturin), GDNF, praseffin (Persephin), TGF-beta 2, TGF-beta 1, TGF-beta 3, LAP (TGF-beta 1), TGF-beta 5, latent TGF-beta 1, latent TGF-beta-bp 1, TGF-beta 1.2, Lefty, Nodal, MIS/AMH, acidic FGF, FGF-12, basic FGF, FGF-13, FGF-3, FGF-16, FGF-4, FGF-17, FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF-21, FGF-9, FGF-23, FGF-10, KGF/FGF-7, FGF-11, Neuropilin (Neuropilin) -1, PlGF, Neuropilin-2, PlGF-2, PDGF-A, VEGF, PDGF-B, VEGF-B, PDGF-C, VEGF-C, PDGF-D, VEGF-D, and PDGF-AB.

23. The protein of claim 13, wherein the T-cell target is selected from the group consisting of: 2B/SLAMF, IL-2 Ra, 4-1BB/TNFRSF, IL-2 Rbeta, ALCAM, B-1/CD, IL-4-H, BLAME/SLAMF, BTLA, IL-6, IL-7 Ra, CCR, CXCR/IL-8 RA, CCR, IL-10 Ra, CCR, IL-10 Rbeta, CCR, IL-12 Rbeta 1, CCR, IL-12 Rbeta 2, CD, IL-13 Ralpha 1, IL-13, CD, ILT/CD 85, integrin alpha 4/CD49, CD, integrin alpha E/CD103, CD, integrin alpha M/CD11, CD, integrin alpha X/CD11, integrin beta 2/CD, KIR/CD158, CD27/TNFRSF7, KIR2DL1, CD28, KIR2DL3, CD30/TNFRSF8, KIR2DL4/CD158d, CD31/PECAM-1, KIR2DS4, CD40 ligand/TNFSF 5, LAG-3, CD43, LAIR1, CD45, LAIR2, CD83, leukotriene B4R 83, CD 83/SLAMF 83, NCAM-L83, CD83, NKG2 83, CD229/SLAMF 83, NKG2 83, CD2 83-10/SLAMF 83, CD 364, CD83, NTB-A/SLAMF 83, common gamma chain/IL-2R gamma, FasFasP, CRASF 1/SLCF 1-CX-1, CTCXCR 3, CTCXCR 1/TAMC-1, CTMA-11, CTSC-CR 3, CTCXCR 3, CTLA-11, CTLA-L83, CTLA-3, CTLA-X-3, CTLA-L83, CTSC-3, CTLA-L83, CTLA-3, CTLA-L83, CTLA-3, CTLA-, Fas ligand/TNFSF 6, TIM-4, Fc gamma RIII/CD16, TIM-6, GITR/TNFRSF18, TNF RI/TNFRSF1A, granulysin, TNF RII/TNFRSF 1B, HVEM/TNFRSF14, TRAIL R1/TNFRSF10A, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM-2/CD102, TRAIL R3/TNFRSF10C, IFN-gamma R1, TRAIL R4/TNFRSF10D, IFN-gamma R2, TSLP, IL-1RI, and TSLP R.

24. The protein of claim 13, wherein the NK cell target is selected from the group consisting of: 2B4/SLAMF4, KIR2DS4, CD155/PVR, KIR3DL1, CD94, LMIR1/CD300A, CD69, LMIR2/CD300c, CRACC/SLAMF7, LMIR3/CD300LF, DNAM-1, LMIR5/CD300LB, Fc epsilon RII, LMIR6/CD300LE, Fc gamma RI/CD64, MICA, Fc gamma RIIB/CD32 LE, MICB, Fc gamma RIIC/CD32 LE, MURLT-1, Fc gamma RIIA/CD32 LE, nectin-2/CD 112, FcRIII/CD LE, FcRG 2 LE, FcRH LE/IRTA LE, NKG2 LE/IRTA LE, NKH LE/IRTA LE, RaKL LE/LE, RaNKG 2 NKP LE, RaLN LE/NKP LE, RaLN LE, IRH LE/NKP LE, IRCP LE, IRC-LE, and NKP-LE, RAL LE, and K LE, and its receptor alpha-LE, or its receptor family, ILT3/CD85k, TREM-1, ILT4/CD85d, TREM-2, ILT5/CD85a, TREM-3, KIR/CD158, TREML1/TLT-1, KIR2DL1, ULBP-1, KIR2DL3, ULBP-2, KIR2DL4/CD158d and ULBP-3.

25. The protein of claim 13, wherein the monocyte/macrophage target is selected from the group consisting of: B7-1/CD80, ILT4/CD85d, B7-H1, ILT5/CD85a, common beta chain, integrin alpha 4/CD49d, BLAME/SLAMF8, integrin alpha X/CD11C, CCL6/C10, integrin beta 2/CD18, CD155/PVR, integrin beta 3/CD 18, CD 18/PECAM-1, laticifin (Latexin), CD 18/SR-B18, leukotriene B4R 18, CD 18/TNFRSF 18, LIMPII/SR-B18, CD18, LMIR 18/CD 300 18/CD 18, CD 18/CD 18, EMIR 18/CD 18, EMAMF CD 18/CD 18, EMMC-18, CD 18/CD 18, EMMC-18, CD18, EMC-1/CD 18, EMC-1/PMC-18, EMC-1/, Osteopontin, Fc γ RIIB/CD32, PD-L, Fc γ RIIC/CD32, Siglec-3/CD, Fc γ RIIA/CD32, SIGNR/CD 209, Fc γ RIII/CD, SLAM, GM-CSF Ra, TCCR/WSX-1, ICAM-2/CD102, TLR, IFN- γ R, TLR- γ R, TREM-1, IL-1RII, TREM-2, ILT/CD 85, TREM-3, ILT/CD 85, TREML/TLT-1, 2B/SLAMF, IL-10 Ra, ALCAM, IL-10 Rbeta, aminopeptidase N/ANPEP, ILT/CD 85, common beta chain, ILT/CD 85, C1R/CD, ILT/CD 85, CCR/CD 85, ILT/CD 85, CCR integrin, CCR 4/CD49, CCR α 4/CD 11, α M/CD11, and CD11, CCR, integrin α X/CD11, CD155/PVR, integrin β 2/CD18, CD, integrin β 3/CD, CD/SR-B, LAIR, CD, leukotriene B4R, CD, LIMPII/SR-B, CD/SLAMF, LMIR/CD 300, CD163, LMIR/CD 300, coagulation factor III/tissue factor, LMIR/CD 300, CX3CR, CX3CL, LMIR/CD 300, CXCR, LRP-1, CXCR, M-CSF R, DEP-1/CD148, MD-1, DNAM-1, MD-2, EMMPIN/CD 147, MMR, endothelial factor/CD 105, NCAM-L, Fc γ/CD, PSGL-1, Fc γ RIII/CD, RP105, G-CSF R, L-selectin, GM-R, CD 3/lec-3, CD, HVEM/TNFRSF14, SLAM, ICAM-1/CD54, TCCR/WSX-1, ICAM-2/CD102, TREM-1, IL-6R, TREM-2, CXCR1/IL-8RA, TREM-3, and TRML 1/TLT-1.

26. The protein of claim 13, wherein the dendritic cell target is selected from the group consisting of: CD36/SR-B3, LOX-1/SR-E1, CD68, MARCO, CD163, SR-AI/MSR, CD5L, SREC-I, CL-P1/COLEC12, SREC-II, LIMPII/SR-B2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-1BB ligand/TNFSF 9, IL-12/IL-23P40, 4-amino-1, 8-naphthalimide, ILT 40/CD 85 40, CCL 40/6 Ckine, ILT 40/CD 85 40, 8-oxy (oxo) -dG, ILT 40/CD 85 40, 8D6 40, ILT 40/CD 85, A2B 40, alpha 4/CD 40, AAIR 40, LAB 40/CD 40, LACI/CD 40, LAMIR 40, LAR/CD 40, LAR 40, LAMIR 36, C1q R q/CD q, LMIR q/CD 300 q, CCR q, LMIR q/CD 300 q, CD q/TNFRSF q, MAG/Siglec-4a, CD q, MCAM, CD q, MD-1, CD q, MD-2, CD q, MDL-1/CLEC5 q, CD q/SLAMF q, MMR, CD q, NCAM-L q, CD2 q-10/SLAMF q, Signalin/GPNMB, Chem q, PD-L q, CLEC-1, RP105, CLEC-2, Siglec-2/CD q, CLEC/SLAMF q, Sig-3/CD q, DC-SIG 5, SIG-DC-299, CLEC-2/CD q, CLEC-DCDEC-3/CD q, CRA-DC-SIGC-3/CD q, SIGC-5, CLEC-DC-299, CLEC-6-DCDEC-3, CRA-3/DCC-q, CRA-7, CRA-3/DCC-3, SIGC-3, CRA-3, SIGC-3/DCC-3, SIGC-3, CRA-3, DEP-1/CD148, SIGNR4, DLEC, SLAM, EMMPRIN/CD147, TCCR/WSX-1, Fc γ RI/CD64, TLR3, Fc γ RIIB/CD32b, TREM-1, Fc γ RIIC/CD32c, TREM-2, Fc γ RIIA/CD32a, TREM-3, Fc γ RIII/CD16, TREML1/TLT-1, ICAM-2/CD102, and capsaicin R1(Vanilloid R1).

27. The protein of claim 13, wherein the angiogenesis target is selected from the group consisting of: angiopoietin (Angiopoietin) -1, Angiopoietin-2, Angiopoietin-3, Angiopoietin-7/CDT 6, Angiopoietin-4, Tie-1, Angiopoietin-1, Tie-2, Angiogenin (Angiogenin), iNOS, coagulation factor III/tissue factor, nNOS, CThAA/CCN 2, NOV/CCN3, DANCE, OSM, EDG-1, Plfr, EG-VEGF/PK1, Proliferin (Proliferin), Endostatin (Endostatin), ROBO4, Erythropoietin (Erythropoiin), thrombospondin-1, Kininostatin (Kininostatin), thrombospondin-2, MFG-E8, thrombospondin-4, Nitric Oxide (Nitric Oxide), VG5, VG 383, EpOS 3884, EphB 9638, EphAB 369636, EphAb-9, EphA-7, EphhhA-7, EphhhhhA-7, EphA-2, EphA-7, EphhhhA-7, EphA, EphB2, EphB6, EphB3, Ephrin-A1, Ephrin-A4, Ephrin-A2, Ephrin-A5, Ephrin-A3, Ephrin-B1, Ephrin-B3, Ephrin-B2, acidic FGF, FGF-12, basic FGF, FGF-13, FGF-3, FGF-16, FGF-4, FGF-17, FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF-21, FGF-9, FGF-23, FGF-10, KGF/FGF-7, FGF-11, FGF R1, FGF R4, FGF R2, FGF R5, FGF R3, neurotrophin-1, neurotrophin-2, brachial plate protein (Semaphorin)3A, brachial plate 6B 3, brachial plate 6C 3, brachial plate C6, brachial plate D3, brachial plate D6, brachial plate 3, brachial plate 6, brachial plate 3, and brachial plate 6, Bradrin 7A, MMP-11, MMP-1, MMP-12, MMP-2, MMP-13, MMP-3, MMP-14, MMP-7, MMP-15, MMP-8, MMP-16/MT3-MMP, MMP-9, MMP-24/MT5-MMP, MMP-10, MMP-25/MT6-MMP, TIMP-1, TIMP-3, TIMP-2, TIMP-4, ACE, IL-13 Ra 1, IL-13, C1q R1/CD 93, integrin alpha 4/CD49d, VE-Cadherin (Caerin), integrin beta 2/CD18, CD31/PECAM-1, KLF4, CD36/SR-B3, LYVE-1, CD151, MCAM, COLCL-P1/dhEC 12, fibronectin-2/CD 112, Coagulation factor III/tissue factor, E-selectin, D6, P-selectin, DC-SIGNR/CD299, SLAM, EMMPRIN/CD147, Tie-2, endoglin/CD 105, TNF RI/TNFRSF1A, EPCR, TNF RII/TNFRSF1B, erythropoietin R, TRAIL R1/TNFRSF10A, ESAM, TRAILR2/TNFRSF10B, FABP5, VCAM-1, ICAM-1/CD54, VEGF R2/Flk-1, ICAM-2/CD102, VEGF R3/Flt-4, IL-1RI, and VG 5Q.

28. The protein of claim 1, wherein at least one of the first binding domain and the second binding domain specifically binds to a target selected from the group consisting of: prostate specific membrane antigen (folate 1), epidermal growth factor receptor, receptors for advanced glycosylation end products, IL-17A, IL-17F, P19, Dickkopf-1, NOTCH1, NG2, IgHE, IL-22R, IL-21, amyloid beta oligomers, amyloid beta preproprotein, NOGO receptor (RTN4R), low density lipoprotein receptor-related protein 5, IL-4, myostatin, very late antigen 4, and IGF-1R.

29. The protein of claim 12, wherein the cancer cell is a transformed hematopoietic cell.

30. The protein of claim 29, wherein at least one of the first binding domain and the second binding domain recognizes a target selected from the group consisting of: a B-cell target, a T-cell target, a dendritic cell target, or an NK-cell target.

31. The protein of claim 29, wherein at least one of the first binding domain and the second binding domain recognizes a target selected from the group consisting of: CD5, CD10, CD11c, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD30, CD38, CD45, CD70, CD80, CD86, CD103, IRTA1, IRTA2, IRTA3, IRTA4, IRTA5, B-B2, B-B8 and B-cell antigen receptors.

32. The protein of claim 1, comprising a sequence selected from the group consisting of seq id no: SEQ ID NO: 2. 4, 6, 103, 105, 107, 109, 332, 333, 334, and 345.

33. The protein of claim 1, comprising a sequence selected from the group consisting of seq id no: SEQ ID NO: 355. 356, 357, 358, 359, 360, 361, 362, 363, 364, and 365.

34. The protein of claim 1, wherein the constant sub-region recognizes an effector cell FC receptor.

35. The protein of claim 1, wherein the constant sub-region recognizes an effector cell surface protein selected from the group consisting of: CD16, CD32a, CD32b, CD64, CD89, fcer 1, FcRn, and pIgR.

36. The protein of claim 1, wherein the constant sub-region comprises C_H2Domains and C_H3A domain.

37. The protein of claim 36, wherein the CH₃The domain is truncated and comprises a C-terminal sequence selected from the group consisting of: SEQ ID NO: 366. 367, 368, 369, 370 and 371.

38. The protein of claim 1, wherein the scorpion linker is derived from an immunoglobulin hinge.

39. The protein of claim 38, wherein the scorpion linker has a reduced but non-zero number of cysteines relative to the hinge from which the scorpion linker is derived, and wherein one cysteine corresponds to an N-terminal hinge cysteine of the immunoglobulin hinge.

40. The protein of claim 39, wherein the scorpion linker is derived from an IgG1 hinge region, and wherein the linker comprises 1 or 2 cysteines, further wherein the linker retains a cysteine corresponding to the N-terminal hinge cysteine of the IgG1 hinge region.

41. The protein of claim 1, wherein the scorpion linker is derived from the stem region of a C-lectin.

42. The protein of claim 1, wherein the scorpion linker comprises a sequence selected from the group consisting of SEQ ID NOs: 373. 374, 375, 376 and 377.

43. The protein of claim 1, wherein the scorpion linker comprises a sequence selected from the group consisting of SEQ ID NOs: 351. 352, 353 and 354.

44. The protein of claim 1, further comprising a linker of at least about 5 amino acids attached to the constant sub-region and to the first binding domain such that the linker is positioned between the constant sub-region and the first binding domain.

45. The protein of claim 1, wherein the first binding domain has a binding affinity of less than 10 to at least one of the second binding domains ^-9M。

46. The protein of claim 1, wherein the binding affinity of the first binding domain to at least one of the second binding domains is at least 10^-6M。

47. The protein of claim 1, wherein the effector function is selected from the group consisting of antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

48. The protein of claim 1, wherein the protein has an in vivo half-life of at least 28 hours in a human.

49. A pharmaceutical composition comprising the protein of claim 1 and a pharmaceutically acceptable adjuvant, carrier or excipient.

50. A nucleic acid encoding the protein of claim 1.

51. A vector comprising the nucleic acid of claim 50.

52. A host cell comprising the vector of claim 51.

53. A method of making the protein of claim 1, the method comprising:

a. introducing a nucleic acid encoding the protein of claim 1 into a host cell; and

b. incubating the host cell under conditions suitable for expression of the protein, thereby expressing the protein at a level of at least 1 mg/ml.

54. The method of claim 53, further comprising isolating the protein.

55. The method of claim 53, wherein the host cell is selected from the group consisting of: VERO cells, Hela cells, CHO cells, COS cells, W138 cells, BHK cells, HepG2 cells, 3T3 cells, RIN cells, MDCK cells, A549 cells, PC12 cells, K562 cells, HEK293 cells, N cells, Spodoptera frugiperda (Spodoptera frugiperda) cells, Saccharomyces cerevisiae (Saccharomyces cerevisiae) cells, Pichia pastoris (Pichia pastoris) cells, fungal cells, and bacterial cells.

56. A method of making a nucleic acid encoding the protein of claim 1, the method comprising:

a. covalently linking the 3 'end of a polynucleotide encoding a first binding domain derived from an immunoglobulin variable region to the 5' end of a polynucleotide encoding a constant sub-region;

b. covalently linking the 5 'end of the polynucleotide encoding the scorpion linker to the 3' end of the polynucleotide encoding the constant sub-region; and

c. covalently linking the 5 'end of a polynucleotide encoding a second binding domain derived from an immunoglobulin variable region to the 3' end of a polynucleotide encoding the scorpion linker;

thereby forming a nucleic acid encoding a multivalent binding protein with effector function.

57. The method of claim 56, wherein the polynucleotide encoding a first binding domain comprises a sequence selected from the group consisting of SEQ ID NOs: SEQ ID NO: 1 (anti-CD 20 variable region), SEQ ID NO: 3 (anti-CD 28 variable region, V)_L-V_HOrientation) and SEQ ID NO: 5 (anti-CD 28 variable region, V)_H-V_LOrientation).

58. The method of claim 56, further comprising inserting a linker polynucleotide encoding a peptide linker of at least 5 amino acids between the polynucleotide encoding a first binding domain and the polynucleotide encoding a constant sub-region.

59. The method of claim 56, further comprising inserting a linker polynucleotide encoding a peptide linker of at least 5 amino acids between the polynucleotide encoding a constant sub-region and the polynucleotide encoding a second binding domain.

60. A method of inducing damage to a target cell, the method comprising contacting the target cell with a therapeutically effective amount of the protein of claim 1.

61. The method of claim 60, wherein the target cell is contacted in vivo by administering the protein to an organism in need thereof.

62. A method of treating a cell proliferative disorder, the method comprising administering to an organism in need thereof a therapeutically effective amount of the protein of claim 1.

63. The method of claim 62, wherein the disorder is selected from the group consisting of cancer, an autoimmune disorder, Rous sarcoma virus infection, and inflammation.

64. The method of claim 62, wherein the protein is administered by in vivo expression of a nucleic acid encoding the protein of claim 1.

65. The method of claim 62, wherein the protein is administered by a route selected from the group consisting of: intravenous injection, intra-arterial injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, and direct tissue injection.

66. A method of ameliorating a symptom associated with a cell proliferative disorder, the method comprising administering to an organism in need thereof a therapeutically effective amount of a protein of claim 1.

67. The method of claim 66, wherein the symptom is selected from the group consisting of pain, heat, swelling, and joint stiffness.

68. A method of treating an infection associated with an infectious agent, the method comprising administering to a patient in need thereof a therapeutically effective amount of the protein of claim 1, wherein the protein comprises a binding domain that specifically binds to a target molecule of the infectious agent.

69. A method of ameliorating a symptom of an infection associated with an infectious agent, the method comprising administering to a patient in need thereof a therapeutically effective amount of the protein of claim 1, wherein the protein comprises a binding domain that specifically binds to a target molecule of the infectious agent.

70. A method of reducing the risk of infection due to an infectious agent, the method comprising administering to a patient at risk of infection a prophylactically effective amount of the protein of claim 1, wherein the protein comprises a binding domain that specifically binds to a target molecule of the infectious agent.

71. A kit comprising the protein of claim 1 and a set of instructions for administering the protein to treat a cell proliferative disorder or ameliorate a symptom of the cell proliferative disorder.

72. The multivalent single chain binding protein of claim 1, wherein at least one of the first binding domain and the second binding domain specifically binds an antigen selected from the group consisting of: CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and a major histocompatibility complex class II peptide.

73. The protein of claim 72, wherein one of the first and second binding domains specifically binds CD 20.

74. The protein according to claim 73 wherein the further binding domain specifically binds to an antigen selected from the group consisting of: CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and a major histocompatibility complex class II peptide.

75. The protein of claim 72, wherein one of the first and second binding domains specifically binds CD79 b.

76. The protein according to claim 75 wherein said further binding domain specifically binds to an antigen selected from the group consisting of: CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and a major histocompatibility complex class II peptide.

77. The protein of claim 72, wherein one of said first binding domain and said second binding domain specifically binds to a major histocompatibility complex class II peptide.

78. The protein according to claim 77, wherein the further binding domain specifically binds to an antigen selected from the group consisting of: CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and a major histocompatibility complex class II peptide.

79. The protein of claim 72, wherein one of the first and second binding domains specifically binds CD 22.

80. The protein according to claim 79 wherein the further binding domain specifically binds to an antigen selected from the group consisting of: CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and a major histocompatibility complex class II peptide.

81. The protein of claim 72, wherein the protein has a synergistic effect on target cell behavior relative to the sum of the effects of each binding domain, and wherein the protein comprises a pair of binding domains that specifically recognize a pair of antigens selected from the group consisting of: CD20-CD19, CD20-CD21, CD20-CD22, CD20-CD40, CD20-CD79a, CD20-CD79b and CD20-CD 81.

82. The protein of claim 72, wherein the protein has an additive effect on target cell behavior relative to the sum of the effects of each binding domain, and wherein the protein comprises a pair of binding domains that specifically recognizes a pair of antigens selected from the group consisting of: CD20-CD23, CD20-CD30, CD20-CD37, CD20-CD70, CD20-CD80, CD20-CD86, CD79b-CD37, CD79b-CD81, major histocompatibility complex class II peptide-CD 30 and major histocompatibility complex class II peptide-CD 72.

83. The protein of claim 72, wherein the protein has an inhibitory effect on target cell behavior relative to the sum of the effects of each binding domain, and wherein the protein comprises a pair of binding domains that specifically recognize a pair of antigens selected from the group consisting of: CD 20-major histocompatibility complex class II peptide, CD79b-CD19, CD79b-CD20, CD79b-CD21, CD79b-CD22, CD79b-CD23, CD79b-CD30, CD79b-CD40, CD79b-CD70, CD79b-CD72, CD79b-CD79a, CD79b-CD80, CD79b-CD86, CD79 b-major histocompatibility complex class II peptide, major histocompatibility complex class II peptide-CD 19, major histocompatibility complex class II peptide-CD 20, major histocompatibility complex class II peptide-CD 21, major histocompatibility complex class II peptide-CD 22, major histocompatibility complex II peptide-CD 22, major histocompatibility complex 3679-CD 22, major histocompatibility complex II peptide-CD 46, Major histocompatibility complex class II peptide-CD 79b, major histocompatibility complex class II peptide-CD 80, major histocompatibility complex class II peptide-CD 81, major histocompatibility complex class II peptide-CD 86, CD22-CD19, CD22-CD40, CD22-CD79b, CD22-CD86 and CD 22-major histocompatibility complex class II peptide.

84. A method of identifying at least one of the binding domains of a multivalent binding molecule of claim 1, the method comprising:

(a) contacting an anti-isotype antibody with an antibody that specifically recognizes a first antigen and an antibody that specifically recognizes a second antigen;

(b) further contacting a target comprising at least one of the antigens with the composition of step (a); and

(c) measuring an activity of the target, wherein the activity is for identifying at least one of the binding domains of the multivalent binding molecule.

85. The method of claim 84, wherein the target is a diseased cell.

86. The method of claim 60, wherein the multivalent single chain binding protein induces a synergistic amount of damage to the target cell as compared to the sum of damage induced by a first antibody comprising the first binding domain but not the second binding domain and a second antibody comprising the second binding domain but not the first binding domain.

87. The method of claim 86, wherein the multivalent single chain binding protein comprises a pair of binding domains that specifically recognize a pair of antigens selected from the group consisting of: CD19/CD20, CD20/CD21, CD20/CD22, CD20/CD40, CD20/CD79a, CD20/CD79b, CD20/CD81, CD21/CD79b, CD37/CD79b, CD79b/CD81, CD19/CL II, CD20/CL II, CD30/CL II, CD37/CL II, CD72/CL II and CD79b/CL II.

88. The method of claim 60, wherein the multivalent single chain binding protein induces an inhibitory amount of damage to the target cell as compared to the sum of the damage induced by a first antibody comprising the first binding domain but not the second binding domain and a second antibody comprising the second binding domain but not the first binding domain.

89. The method of claim 88, wherein the multivalent single chain binding protein comprises a pair of binding domains that specifically recognize a pair of antigens selected from the group consisting of: CD20/CL II, CD21/CD79 21, CD79 21/CD 21, CD21/CLII, CD21/CL II, CD21/CD 21, CD21/CD 3679 a, CD21/CD 21, CD21/CD 3679 b, CD21/CD 21 and CD21/CD 21.

90. The method of claim 60, further comprising a plurality of multivalent single chain binding proteins.

91. The method of claim 90, wherein the binding domain of the first multivalent single chain binding protein and the binding domain of the second multivalent single chain binding protein induce a synergistic amount of damage to the target cell.

92. The method of claim 90, wherein the binding domain of the first multivalent single chain binding protein and the binding domain of the second multivalent single chain binding protein induce an inhibitory amount of damage to the target cell.

93. A composition comprising a plurality of multivalent single chain binding proteins of claim 1.

94. The composition of claim 93, wherein the binding domain of the first multivalent single chain binding protein and the binding domain of the second multivalent single chain binding protein are capable of inducing a synergistic amount of damage to the target cell.

95. The composition of claim 93, wherein the binding domain of the first multivalent single chain binding protein and the binding domain of the second multivalent single chain binding protein are capable of inducing an additive amount of damage to the target cell.

96. The composition of claim 93, wherein the binding domain of the first multivalent single chain binding protein and the binding domain of the second multivalent single chain binding protein are capable of inducing an inhibitory amount of damage to the target cell.

97. A pharmaceutical composition comprising the composition of claim 93 and a pharmaceutically acceptable carrier, diluent, or excipient.

98. A kit comprising the composition of claim 93 and a set of instructions for administering the composition to damage a target cell.