JP6114936B2 - Compositions and methods of use of immunotoxins containing lampyrnase (RAP) exhibiting potent cytotoxic activity - Google Patents

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is related to US patent application Ser. No. 12 / 754,740 filed Apr. 6, 2010, No. 12 / 754,140 filed Apr. 5, 2010; 2010. No. 12 / 752,649 filed on April 1, No. 12 / 731,781 filed on March 25, 2010, No. 12 / 644,146 filed on December 22, 2009 And US Provisional Patent Application No. 61 / 238,473 filed on August 31, 2009, 61 / 266,305 filed on December 3, 2009, on March 24, 2010. Claims priority of 61 / 316,996 filed and 61 / 323,960 filed April 14, 2010. Each priority application is incorporated herein by reference in its entirety.
Financial assistance This study was supported in part by grant 2R44CA108083-02A2 from the National Cancer Institute and National Institutes of Health. The federal government may have certain rights in the invention.

  While the present invention relates to compositions and methods of use of toxin-antibody constructs (immunotoxins), preferably comprising lampirnase (Rap), those skilled in the art will recognize a wide variety of toxins and other cytotoxic agents in the art. It will be understood that any such toxin or cytotoxic agent can be used in the claimed compositions and methods. In other preferred embodiments, the construct comprises an anti-tumor antibody such as anti-EGP-1 (anti-Trop-2), anti-CD74, anti-CD22, or anti-CD20. However, the present compositions and methods are not so limited, and antibodies or antibody fragments may be cancer cells, B cells, T cells, autoimmune disease cells, pathogens, or antibodies thereto known in the art. It can bind to antigens associated with any target tissue, such as any other disease-related target cell.

  In a more preferred embodiment, the immunotoxin is preferably a dock-and-lock (DNL) construct comprising four copies of lumpirnase linked to an antibody or antibody fragment. Even more preferably, the toxin or other cytotoxic agent is a fusion protein, each of which comprises a DDD (dimerization and docking domain) moiety, and the antibody or antibody fragment comprises two AD (anchor domain) moieties. A fusion protein comprising The DDD moiety spontaneously forms a dimer that binds to the AD moiety, resulting in a DNL construct that contains four copies of a cytotoxin bound to one antibody or antibody fragment. The resulting immunotoxins exhibit very potent cytotoxic activity and can be administered to a subject with a disease to kill disease-related cells. The immunotoxin exhibits greater potency against the target cell than the parent antibody alone, cytotoxin alone, a combination of unbound antibody and cytotoxin, or a cytotoxin conjugated to a control antibody.

Ribonucleases, especially Rap (Lee, Exp Opin Biol Ther 2008; 8: 813-27) and its more basic variants, amphinase (Ardelt et al., Curr Pharm
Biotechnol 2008: 9: 215-25) is a potential anti-tumor agent (Lee and Raines,
Biodrugs 2008; 22: 53-8). Rap is a 104 amino acid single-stranded ribonuclease isolated for the first time from a Rana pipiens oocyte. Rap exhibits cytostatic and cytotoxic effects on various tumor cell lines in vitro and antitumor activity in vivo. Amphibian ribonucleases enter cells by receptor-dependent endocytosis and, when internalized into the cytosol, selectively degrade tRNA, resulting in inhibition of protein synthesis and induction of apoptosis.

Rap has completed a randomized phase IIIb clinical trial comparing the efficacy of Rap + doxorubicin with that of doxorubicin alone in patients with unresectable malignant mesothelioma, and according to an interim analysis, this combination of MST Has been shown to be 12 months, while monotherapy MST was 10 months (Mutti and Gaudino, Oncol Rev 2008; 2: 61-5). Rap can be administered repeatedly to patients, has no adverse immune response, and reversible nephrotoxicity has been reported to be dose limiting (Mikulski et al., J Clin Oncol 2002; 20: 274-81;
Int J Oncol 1993; 3: 57-64).

  Rap and other toxins or cytotoxins can be coupled to antibodies or antibody fragments for targeted delivery to selected disease-related cells, such as cancer cells or autoimmune disease cells. An exemplary tumor associated antigen is EGP-1, also known as Trop-2.

  Trop-2 is a type I transmembrane protein, human cells (Fornaro et al., Int J Cancer 1995; 62: 610-8) and mouse cells (Sewedy et al., Int J Cancer 1998; 75: 324 -30) has been cloned from both. In addition to its role as a tumor-associated calcium signaling agent (Ripani et al., Int J Cancer 1998; 76: 671-6), expression of human Trop-2 is required for tumorigenesis and invasiveness of colon cancer cells Which could be effectively reduced by a polyclonal antibody against the extracellular domain of Trop-2 (Wang et al., Mol Cancer Ther 2008; 7: 280-5).

Increased interest in Trop-2 as a therapeutic target for solid cancer (Cubas et al., Biochim Biophys
Acta 2009; 1796: 309-14) is a breast cancer (Huang et al., Clin Cancer
Res 2005; 11: 4357-64), colorectal cancer (Ohmachi et al., Clin Cancer
Res 2006; 12: 3057-63; Fang et al., Int J Colorectal Dis
2009; 24: 875-84) and oral squamous cell carcinoma (Fong et al., Modern Pathol)
2008; 21: 186-91) is further proved by a report that revealed the clinical significance of Trop-2 overexpression. Of particular note is the recent evidence that prostate basal cells expressing high levels of Trop-2 are abundant for in vitro and in vivo stem-like activity (Goldstein et al., Proc Natl Acad Sci USA 2008; 105: 20882-7).

The murine anti-Trop-2 mAb, mRS7, was generated by hybridoma technology using a crude membrane preparation derived from surgically removed human primary lung squamous cell carcinoma as an immunogen (Stein et al., Cancer Res 1990; 50: 1330-6). By immunoperoxidase staining of frozen tissue sections, antigens defined by mRS7 were present in lung, stomach, bladder, breast, ovary, uterus, and prostate tumors, and were not responsive to most normal human tissues (Stein et al., Int J Cancer 1993; 55: 938-46). The antigen recognized by mRS7 is later shown to be a 46-48 kDa glycoprotein and is named epithelial glycoprotein-1 or EGP-1 (Stein
et al., Int J Cancer 1994; 8: 98-102), literature (Ripani et al.,
Int J Cancer 1998; 76: 671-6) is also called Trop-2. Upon binding to target cells, mRS7 is rapidly internalized within 2 hours (Stein et al., Int J Cancer 1993; 55: 938-46).

Radioisotope-labeled mRS7 has been shown to effectively target and treat nude mouse cancer xenografts in several early studies (Stein et al., Antibody Immunoconj Radiopharm 1991; 4: 703- 12; Stein et al., Cancer 1994; 73: 816-23; Shih et
al., Cancer Res 1995; 55: 5857s-63s; Stein et al., J Nucl
Med 2001; 42: 967-74; Stein et al., Crit Rev Oncol
Hematol 2001; 39: 173-80). However, the field of immunoconjugates of RS7 or other disease-targeting antibodies (“immunotoxins”) that can be coupled with Rap or other cytotoxins to provide more effective agents for disease treatment include There is a need.

  The present invention relates to compositions and methods of use of immunotoxins comprising lampirnase (Rap) or other toxins conjugated to disease targeted antibodies or antigen binding antibody fragments. In certain preferred embodiments, the immunotoxin comprises 2L-Rap (Q) -hRS7 or 2L-Rap-hRS7 comprising two copies of Rap attached to the N-terminus of a humanized anti-Trop-2 antibody (hRS7) and The structure illustrated in FIG. 1 may be called. However, those skilled in the art will appreciate that immunotoxins are not so limited and antibodies against other tumor-related or disease-related antigens known in the art can be used. Such immunotoxins exhibit potent cytotoxicity and improved pharmacokinetics while minimizing the toxic side effects of Rap.

  In an alternative embodiment, the subject immunotoxins can be made using dock-and-lock (DNL) technology, and antibodies or antigen-binding antibody fragments of Rap or other toxins or cytotoxins are used. A conjugate may be included. As used herein below, the term “immunotoxin” may refer to an immunotoxin made by DNL technology, or the immunotoxin illustrated in FIG. In a preferred embodiment, the DNL construct comprises Rap conjugated to an anti-Trop-2 antibody such as hRS7. However, those of ordinary skill in the art will not be so limited in DNL constructs, and the subject DNL construct may be any conjugated to lampyrnase or other toxins or cytotoxins known in the art. It will be appreciated that antibodies or fragments thereof against disease associated antigens may be included.

  In certain embodiments, the immunotoxin comprises heavy chain CDR sequence CDR1 (NYGMN, SEQ ID NO: 1), CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO: 2), and CDR3 (GGGFSYSYWYFDV, SEQ ID NO: 3), and light chain CDR sequence CDR1 (KASQDVSIVAVA , SEQ ID NO: 4), CDR2 (SASYRYT, SEQ ID NO: 5), and CDR3 (QQHYITPLT, SEQ ID NO: 6), and humanized anti-Tropics such as hRS7 antibody conjugated to human antibody framework (FR) and constant region sequences -2 antibodies or fragments thereof (see, eg, US Pat. No. 7,238,785, column 34, line 6 to column 44, line 37, incorporated herein by reference).

  In other specific embodiments, the immunotoxin comprises a light chain variable region CDR1 (RASSSVSYIH, SEQ ID NO: 7), CDR2 (ATSNLAS, SEQ ID NO: 8), and CDR3 (QQWTSPPT, SEQ ID NO: 9), and a heavy chain variable region CDR1. A humanized anti-CD20 antibody such as veltuzumab or a fragment thereof comprising (SYNMH, SEQ ID NO: 10), CDR2 (AIYPGNGDTSYNQKFKG, SEQ ID NO: 11), and CDR3 (STYYGDWYFDV (SEQ ID NO: 95) or VVYYSNSYWYFDV, SEQ ID NO: 12) (See, eg, US Pat. No. 7,435,803, column 38, line 15 to column 46, line 52, incorporated herein by reference).

  In a more specific embodiment, the immunotoxin may comprise a lumpirnase (Rap) amino acid sequence known in the art (see, eg, NCBI protein database accession number 1PU3_A, and Gorbatyuk et al., J Biol Chem). 279: 5772-80, 2004).

  In various embodiments, the immunotoxin may comprise one or more antibodies or fragments thereof that bind to an antigen other than Trop-2 or CD20. In preferred embodiments, the antigen (s) may be selected from the group consisting of: carbonic anhydrase IX, CCCL19, CCCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8. CD11A, CD14, CD15, CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54 CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, AFP, PSMA, CEACAM5, CEA CAM-6, B7, ED-B of fibronectin, factor H, FHL-1, Flt-3, folate receptor, GROB, HMGB-1, hypoxia-inducible factor (HIF), HM1.24, insulin-like growth factor 1 (ILGF-1), IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6 , IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2 , MUC3, MUC4, MUC5, PAM4 antigen, NCA-95, NCA-90, Ia, HM1.24, EGP-1, EGP-2, HLA-DR, tenascin, Le (y), RANTES, T101, TAC, Tn Antigen, Thomson- Friedenreich antigen, tumor necrosis antigen, TNF-α, TRAIL receptors (R1 and R2), VEGFR, EGFR, PlGF, complement factors C3, C3a, C3b, C5a, C5, and oncogene products.

  Exemplary antibodies that can be used include, but are not limited to: hR1 (US patent application Ser. No. 12 / 722,645 filed in anti-IGF-1R, 3/12/10). HPAM4 (anti-mucin, US Pat. No. 7,282,567), hA20 (anti-CD20, US Pat. No. 7,251,164), hA19 (anti-CD19, US Pat. No. 7,109,304) , HIMMU31 (anti-AFP, US Pat. No. 7,300,655), hLL1 (anti-CD74, US Pat. No. 7,312,318), hLL2 (anti-CD22, US Pat. No. 7,074,403), hMu -9 (anti-CSAp, US Pat. No. 7,387,773), hL243 (anti-HLA-DR, US Pat. No. 7,612,180), hMN-14 (anti-CEACAM5, US special No. 6,676,924), hMN-15 (anti-CEACAM6, US Pat. No. 7,541,440), hRS7 (anti-EGP-1, US Pat. No. 7,238,785), and hMN-3 (Anti-CEACAM6, US Patent Application No. 7,541,440), the example portion of each cited patent or application is incorporated herein by reference. One skilled in the art will appreciate that this list is not limiting and that any known antibody can be used, as discussed in more detail below.

  Exemplary toxins that can be incorporated into immunotoxins include, but are not limited to, bacterial toxins, plant toxins, ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), DNase I, staphylococcal enterotoxin A, pokeweed Antiviral proteins, gelonin, diphtheria toxin, Pseudomonas exotoxin, Pseudomonas endotoxin, Lampyrinase (Rap), and Rap (N69Q) are included. The sequence of each of the listed toxins is known in the art (see, eg, the NCBI database), and clones encoding many exemplary toxins are available from Invitrogen, the American Cultured Cell Line Conservation Agency, and the It is commercially available from other sources known in the art.

  Various embodiments include, but are not limited to, non-Hodgkin lymphoma, B-cell acute and chronic lymphocytic leukemia, Burkitt lymphoma, Hodgkin lymphoma, hairy cell leukemia, acute and chronic myelogenous leukemia, T-cell lymphoma and leukemia, multiple Of using the subject immunotoxins to treat or diagnose diseases including multiple myeloma, glioma, Waldenstrom macroglobulinemia, carcinoma, melanoma, sarcoma, glioma, and skin cancer . Carcinomas from oral, gastrointestinal tract, colon, stomach, pulmonary duct, lung, breast, ovary, prostate, uterus, endometrium, cervix, bladder, pancreas, bone, liver, gallbladder, kidney, skin, and testicular carcinoma It may be selected from the group consisting of In addition, the subject immunotoxins can be used to treat the following: autoimmune diseases such as acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, sydnam chorea Disease, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polygonal syndrome, bullous pemphigoid, diabetes mellitus, Henoch-Schonlein purpura, post-streptococcal nephritis, erythema nodosum, Takayasu Arteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture syndrome, obstructive thrombotic blood vessels Inflammation, Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, thyroid poisoning, scleroderma, chronic active hepatitis, polymyositis / dermatomyositis, polychondritis, pemphigus vulgaris , Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, spinal cord fistula, giant cell arteritis / polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis, or fibrotic lung Cystitis. In certain embodiments, the subject antibodies can be used to treat leukemias such as chronic lymphocytic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, or acute myeloid leukemia.

  In one embodiment, the pharmaceutical composition of the invention can be used to treat a subject having a metabolic disease such as amyloidosis or a neurodegenerative disease such as Alzheimer's disease. In addition, the pharmaceutical composition of the present invention can be used to treat a subject having an immune dysregulation disorder.

The compositions of the present invention are also useful for the treatment of infectious diseases, where the immunoglobulin component of the immunotoxin specifically binds to the disease-causing microorganism. Within the scope of the present invention, disease-causing microorganisms include pathogenic bacteria, viruses, fungi, and various parasites, and antibodies target these microorganisms, their products or antigens associated with their lesions. can do. Examples of microorganisms include but are not limited to: Streptococcus agalactiae, Legionella pneumophila (Legionella)
pneumophilia), Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhea (Neisseria)
gonorrhoeae), Neisseria meningitidis, Pneumococcus, Hemophilis influenza B
influenzae B), Treponema pallidum, Lyme disease spirochetes, Pseudomonas
aeruginosa), Neisseria gonorrhoeae (Mycobacterium leprae), Bovine abortion bacteria (Brucella abortus), Mycobacterium tuberculosis (Mycobacterium)
tuberculosis), tetanus toxin, type 1, type 2, type 3, HIV, type A, type B, type C, hepatitis D, rabies virus, influenza virus, cytomegalovirus, herpes simplex I and II, human serum parvo-like virus , Papilloma virus, polyoma virus, RS virus, varicella-zoster virus, hepatitis B virus, papilloma virus, measles virus, adenovirus, human T-cell leukemia virus, Epstein-Barr virus, mouse leukemia virus, mumps virus Vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, sputum virus, bluetongue virus, Sendai virus, feline leukemia virus, reovirus, poliovirus, simian virus 40, mouse mammary tumor virus, dengue virus , Rubella virus, protozoa, Plasmodium falciparum (Plasmodium falciparum), vivax malaria parasite (Plasmodium
vivax), Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruise
cruzi), Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosoma
mansoni), Schistosoma japanicum, Babesia bovis, Elmeria tenella, Onchocerca volvulus, tropical Leishmania
tropica, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus ), Mesocestoides (Mesocestoides)
corti), Mycoplasma arthritidis, M. et al. M. hyorhinis, M.H. M. orale, M. Arginini, Acholeplasma
laidlawii), M.M. M. salivarium, M. M. pneumoniae. Monoclonal antibodies that bind to these pathogenic microorganisms are well known in the art.

The following drawings form part of the present specification and are included to further demonstrate certain embodiments of the present invention. Embodiments may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
It is a figure which shows the molecular design and size of (Q) -hRS7. A schematic structure of 2L-Rap-X, X may be IgG, and Rap may be Rap (Q). It is a figure which shows the cell binding curve of PC-3 (A), Calu-3 (B), and 22Rv1 (C) obtained from ELISA using a luminol substrate. Average fluorescence units were plotted against concentration and the resulting data was analyzed with Prism software to obtain _KD values. (A) shows that (Q) -hRS7 and rRap have equivalent RNase activity, and (B) plots the initial rate of rRap (left) and (Q) -hRS7 (right) activity against the concentration of yeast tRNA. FIG. 5 is a diagram showing representative data of an IVTT assay in which kcat / km is determined. Revealed by the MTS assay shown for ME-180 (A) and T-47D (B) and the colony formation assay shown for (C) DU-145 and (D) PC-3 (Q) − It is a figure which shows the in vitro cytotoxicity of hRS7. The data of (A) and (B) were analyzed with Prism software to obtain EC50 values. (A) Inhibits tumor growth and (B) Increases MST, showing the therapeutic efficacy of (Q) -hRS7 demonstrated in a Calu-3 human xenograft model. Nude mice were inoculated subcutaneously with 1 × 10 ⁷ Calu-3 cells. When the tumor reached approximately 0.15 cm ³ , the mice were treated with either a single intravenous dose of 50 μg or two injections of 25 μg administered at 7 day intervals. Control animals received saline. It is a figure which shows the RNase activity by an in vitro transcription | transfer translation assay. FIG. 6 shows a competitive binding assay demonstrating that both hLL1 and rap-hLL1 fusion proteins have the same affinity for WP, hLL1 anti-idiotype antibodies. FIG. 1 shows in vitro cytotoxicity of fusion proteins in Daudi cells: (A) cytotoxicity measured by MTS assay; (B) cytotoxicity measured by BRdU assay. FIG. 2 shows in vitro cytotoxicity of fusion proteins in MC / CAR cells by MTS assay. It is a figure which shows the blood clearance of 2L-Rap-hLL1-γ4P in naive SCID mice. Naive SCID mice were co-injected intravenously with ⁸⁸ Y-DTPA-hLL1 (◯) and ¹¹¹ In-DTPA-2L-Rap-hLL1-γ4P (□). At selected time points after administration, the mice were bled by cardiac puncture and the radioactivity of the blood samples was counted. Data are the mean of injected doses in blood ± S. D. (N = 3). FIG. 2 shows treatment of high grade minimal Daudi lymphoma with 2L-Rap-hLL1-γ4P or component proteins. SCID mice (8-10 mice / group) were inoculated intravenously with 1.5 × 10 ⁷ Daudi cells. One day later, the mice were treated with a single bolus injection of the indicated dose of 2L-Rap-hLL1-γ4P. Control groups were injected with component protein equal to 50 μg immunotoxin or PBS alone. FIG. 5 shows RNase activity measured by in vitro transcription / translation assay. The concentrations of rRap (■), 2L-Rap-hLL1-γ4P (▲), and hLL1-γ4P (♦) were plotted against relative luminescence units (RLU). FIG. 5 shows in vitro cytotoxicity of DNL-Rap immunotoxin constructs either treated sequentially with immunotoxin or washed after 1 hour of treatment. FIG. 5 shows in vitro cytotoxicity of DNL-Rap immunotoxin constructs in all cell lines.

Definitions Unless otherwise indicated, “a” or “an” means “one or more”.

  As used herein, the terms “and” and “or” can be used to mean either connected or disjunctive. That is, both terms should be understood to be the same as “and / or” unless stated otherwise.

  A “therapeutic agent” is an atom, molecule, or compound that is useful in the treatment of a disease. Examples of therapeutic agents include antibodies, antibody fragments, peptides, drugs, toxins, enzymes, nucleases, hormones, immunomodulators, antisense oligonucleotides, small interfering RNA (siRNA), chelating agents, boron compounds, photoactive effects Agents, dyes, and radioisotopes are included.

  A “diagnostic agent” is an atom, molecule, or compound that is useful in diagnosing a disease. Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (including biotin-streptavidin complexes, etc.), contrast agents, fluorescent compounds or molecules, and accelerators for magnetic resonance imaging (MRI) ( For example, paramagnetic ions) are included.

  An “antibody” as used herein is a full-length (ie, naturally occurring or formed by a normal immunoglobulin gene fragment recombination process) immunoglobulin molecule (eg, an IgG antibody), or Refers to the immunologically active (ie specifically binding) portion of an immunoglobulin molecule, such as an antibody fragment. “Antibodies” include monoclonal, polyclonal, bispecific, multispecific, mouse, chimeric, humanized, and human antibodies.

A “naked antibody” is an antibody or antigen-binding fragment thereof that is not conjugated to a therapeutic or diagnostic agent. The Fc portion of a fully naked antibody can provide complement binding and effector functions such as ADCC (eg Markrides, Pharmacol Rev 50: 59-87,
See 1998). Other mechanisms by which naked antibodies induce cell death may include apoptosis. (Vaswani
and Hamilton, Ann Allergy Asthma
Immunol 81: 105-119, 1998).

“Antibody fragments” are portions of a complete antibody such as F (ab ′) ₂ , F (ab) _2, Fab ′, Fab, Fv, sFv, scFv, and dAb. Regardless of structure, an antibody fragment binds with the same antigen that the full-length antibody recognizes. For example, to an antibody fragment, an isolated fragment composed of a variable region such as an “Fv” fragment composed of a variable region of a heavy chain and a light chain, or a light chain and a heavy chain variable region connected by a peptide linker Recombinant single chain polypeptide molecules (“scFv proteins”) are included. “Single-chain antibodies” are often abbreviated as “scFv” and are composed of polypeptide chains that include both V _H and V _L domains that interact to form an antigen-binding site. The _VH and _VL domains are usually joined by a peptide of 1 to 25 amino acid residues. Antibody fragments also include bispecific antibodies, trispecific antibodies, and single domain antibodies (dAbs).

  An antibody or immunotoxin preparation or composition described herein is said to be administered in a “therapeutically effective amount” if the amount administered is physiologically effective. An agent is physiologically effective if its presence results in a detectable change in the physiology of the recipient subject. In certain embodiments, an antibody preparation is physiologically effective if its presence causes an anti-neoplastic response or alleviates signs and symptoms of an autoimmune disease state. A physiologically effective effect may also be the induction of a humoral and / or cellular immune response in the recipient subject that leads to inhibition or death of the target cell.

Antibodies and Antibody Fragments Techniques for preparing monoclonal antibodies against virtually any target antigen are well known in the art. For example, Kohler and Milstein, Nature 256: 495 (1975), and Coligan et al. (Eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1,
See pages 2.5.1-2.6.7 (John Wiley & Sons 1991). Briefly, monoclonal antibodies are injected into a composition containing an antigen into a mouse, the spleen is removed to obtain B lymphocytes, the B lymphocytes are fused with myeloma cells to produce a hybridoma, and the hybridoma is cloned. It can be obtained by selecting a positive clone producing an antibody against the antigen, culturing a clone producing the antibody against the antigen, and isolating the antibody from the hybridoma culture.

MAbs can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with protein A sepharose, size exclusion chromatography, and ion exchange chromatography. See, for example, Coligan, pages 2.7.1 to 2.7.12, and 2.9.1 to 2.9.3. Baines et al., “Purification of
Immunoglobulin G (IgG), ”in METHODS IN
See MOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

  After first eliciting an antibody against the immunogen, the antibody can be sequenced and then prepared by recombinant techniques. Humanization and chimerization of mouse antibodies and antibody fragments are well known to those skilled in the art. The use of antibody components derived from humanized, chimeric, or human antibodies eliminates potential problems associated with the immunogenicity of mouse constant regions.

Chimeric antibody A chimeric antibody is a recombinant protein in which the variable region of a human antibody is replaced with the variable region of a mouse antibody, including, for example, the complementarity determining region (CDR) of a mouse antibody. Chimeric antibodies exhibit reduced immunogenicity and increased stability when administered to a subject. General techniques for cloning mouse immunoglobulin variable domains are described, for example, in Orlando et al., Proc. Nat'l Acad. Sci. USA
86: 3833 (1989). Techniques for constructing chimeric antibodies are well known to those skilled in the art. As an example, in Leung et al., Hybridoma 13: 469 (1994), DNA sequences encoding the V _κ and V _H domains of mouse LL2, anti-CD22 monoclonal antibody are combined with the respective human κ and IgG ₁ constant region domains. An LL2 chimera was produced.

Humanized antibodies Techniques for producing humanized MAbs are well known in the art (eg, Jones et al., Nature 321: 522 (1986), Riechmann
et al., Nature 332: 323 (1988), Verhoeyen et al.,
Science 239: 1534 (1988), Carter et al., Proc. Nat'l Acad. Sci. USA
89: 4285 (1992), Sandhu, Crit. Rev. Biotech. 12: 437
(1992) and Singer et al., J. Immun. 150: 2844 (1993)). A chimeric or mouse monoclonal antibody can be humanized by grafting mouse CDRs derived from the heavy and light chain variable chains of mouse immunoglobulin into the corresponding variable domains of a human antibody. The mouse framework region (FR) of the chimeric monoclonal antibody is also replaced with a human FR sequence. Simply transplanting mouse CDRs into human FRs often results in reduced or even loss of antibody affinity, and additional modifications are necessary to restore the original affinity of the mouse antibody. May be necessary. This can be accomplished by substituting one or more human residues of the FR region with their mouse counterparts to obtain antibodies with good binding affinity for that epitope. For example, Tempest et al., Biotechnology 9: 266 (1991) and Verhoeyen
et al., Science 239: 1534 (1988). In general, their FR counterparts differ from their murine counterparts, with human FR amino acid residues located close to or in contact with one or more CDR amino acid residues.

Methods for producing fully human antibodies using either human antibodies combinatorial techniques or transgenic animals transformed with human immunoglobulin loci are known in the art (eg, Mancini et al., 2004). 27: 315-28; Conrad and Scheller, 2005, Comb. Chem. High Throughput Screen. 8: 117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol. 3: 544-50). Fully human antibodies can also be constructed by gene or chromosomal transfection methods as well as phage display techniques, all of which are known in the art. See, for example, McCafferty et al., Nature 348: 552-553 (1990). Such fully human antibodies exhibit even fewer side effects than chimeric or humanized antibodies and are expected to function in vivo essentially as endogenous human antibodies. In certain embodiments, the claimed methods and procedures may use human antibodies produced by such techniques.

  In one alternative, human antibodies may be produced using phage display technology (eg, Dantas-Barbosa et al., 2005, Genet. Mol. Res. 4: 126-40). Human antibodies can be produced from normal humans or humans that exhibit certain disease states such as cancer (Dantas-Barbosa et al., 2005). An advantage of constructing human antibodies from diseased individuals is that the circulating antibody repertoire may be biased towards antibodies against disease-related antigens.

In one non-limiting example of this method, Dantas-Barbosa et al. (2005) constructed a phage display library of human Fab antibody fragments from osteosarcoma patients. In general, total RNA was obtained from circulating blood lymphocytes (Id.). Recombinant Fab was cloned from μ, γ, and κ chain antibody repertoires and inserted into a phage display library (Id.). RNA was converted to cDNA and used to construct a Fab cDNA library using specific primers for heavy and light chain immunoglobulin sequences (Marks et al., 1991, J. Mol. Biol 222: 581-97). Library construction was performed by Andris-Widhopf et al. (2000, In: Phage Display Laboratory).
Manual, Barbas et al. (Eds), 1 ^st edition, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor,
NY pp. 9.1 to 9.22). The final Fab fragment was digested with restriction endonucleases and inserted into the bacteriophage genome to create a phage display library. Such libraries can be screened by standard phage display methods known in the art (eg, Pasqualini and Ruoslahti, 1996, Nature 380: 364-366; Pasqualini, 1999, The Quart. J. Nucl Med. 43: 159-162).

Phage display can be performed in a variety of formats, and a review of them can be found, for example, in Johnson and Chiswell, Current Opinion in Structural Biology 3: 5564-571.
(1993). Human antibodies can also be produced by in vitro activated B cells. See US Pat. Nos. 5,567,610 and 5,229,275, which are hereby incorporated by reference in their entirety. One skilled in the art will appreciate that these techniques are exemplary and that any known method for producing and screening human antibodies or antibody fragments can be used.

In another alternative, transgenic animals genetically engineered to produce human antibodies can be used to produce antibodies against essentially any immunogenic target using standard immunization protocols. Methods for obtaining human antibodies from transgenic mice are described in Green et al., Nature Genet. 7:13 (1994), Lonberg.
et al., Nature 368: 856 (1994), and Taylor et al., Int. Immun.
6: 579 (1994). A non-limiting example of such a system is XenoMouse® from Abgenix (Fremont, CA) (eg, Green et al., 1999, J. Immunol. Methods 231: 11-23). ). In XenoMouse® and similar animals, the mouse antibody gene is inactivated and replaced with a functional human antibody gene, but the rest of the mouse immune system remains intact.

  XenoMouse® has been transformed with a germline YAC (yeast artificial chromosome) that contained a portion of the human IgH and Igkappa loci, including most variable region sequences, along with accessory genes and regulatory sequences. It was. The human variable region repertoire can be used to generate antibody-producing B cells that can be processed into known hybridomas. XenoMouse® immunized with the target antigen will produce a human antibody with a normal immune response, and the antibody can be recovered and / or produced by standard techniques discussed above. Different strains of XenoMouse® are available, each of which is capable of producing different classes of antibodies. Transgenically produced human antibodies have been shown to have therapeutic potential while retaining the pharmacokinetic properties of normal human antibodies (Green et al., 1999). One skilled in the art will be aware that the claimed compositions and methods are not limited to the use of the XenoMouse® system, but may use any transgenic animal that has been genetically engineered to produce human antibodies. You will understand what you can do.

Antibody Fragments Antibody fragments that recognize specific epitopes can be produced by known techniques. Antibody fragments are the antigen-binding portions of an antibody, such as F (ab ′) ₂ , Fab ′, F (ab) ₂ , Fab, Fv, and sFv. F (ab ′) ₂ fragments can be produced by pepsin digestion of antibody molecules, and Fab ′ fragments can be generated by reducing disulfide bridges of F (ab ′) ₂ fragments. Alternatively, Fab ′ expression libraries can be constructed (Huse et al., 1989, Science, 246: 1274-1281) to allow rapid and easy identification of monoclonal Fab ′ fragments with the desired specificity. it can. F (ab) ₂ fragments can be generated by papain digestion of antibodies.

Single chain Fv molecules (scFv) comprise a VL domain and a VH domain. The VL and VH domains bind to form a target binding site. These two domains are further covalently linked by a peptide linker (L). Methods for making scFv molecules and designing suitable peptide linkers are described in US Pat. No. 4,704,692, US Pat. No. 4,946,778, R. Raag and M. Whitlow, “Single Chain”.
Fvs. ”FASEB Vol 9: 73-80 (1995) and RE Bird and BW Walker,“ Single Chain
Antibody Variable Regions, ”TIBTECH, Vol 9: 132-137
(1991).

  Also, techniques for producing single domain antibodies (DAB) are described in, eg, Cossins et al. (2006, Prot Express Purif 51: 253-259), which is incorporated herein by reference. Known in the technical field.

Antibody fragments can be prepared by proteolysis of the full-length antibody or by expressing the DNA encoding the fragment in E. coli or another host. Antibody fragments can be obtained by digesting full length antibodies with pepsin or papain by conventional methods. These methods are described, for example, by Goldenberg, US Pat. Nos. 4,036,945, and 4,331,647, and references contained therein. Nisonoff et al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119 (1959), Edelman
et al., in METHODS IN ENZYMOLOGY VOL. 1, page 422 (Academic Press 1967), and Coligan 2.8-1.2.8.10 and 2.10. See pages ~ 2.10.4.

Known Antibodies The antibodies used can be obtained commercially from a wide variety of known sources. For example, various antibody-secreting hybridoma lines are available from the American Cultured Cell Line Conservation Agency (ATCC, Manassas, VA). Numerous antibodies against various disease targets, including but not limited to tumor-associated antigens, have been deposited with the ATCC and / or variable region sequences have been published and used in the claimed methods and compositions Is available for See, for example, U.S. Patent Nos. 7,312,318; 7,282,567; 7,151,164; 7,074,403; 7,060,802; No. 7,056,509; No. 7,049,060; No. 7,045,132; No. 7,041,803; No. 7,041,802; No. 7,041,293; No. 7,037,498; No. 7,012,133; No. 7,001,598; No. 6,998,468; No. 6,994,976; No. 6,994 No. 6,989,241; 6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813 No. 6,956,107; No. 6,951,924; No. 6, No. 49,244; No. 6,946,129; No. 6,943,020; No. 6,939,547; No. 6,921,645; No. 6,921,645; No. 533; No. 6,919,433; No. 6,919,078; No. 6,916,475; No. 6,905,681; No. 6,899,879; No. 6,893,625 6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226; 838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450; No. 6,764,688; No. 6,764,681; No. 6,764,679; No. 6,743,898; No. 6,733,981; No. 6,730, No. 307; No. 6,720,15; No. 6,716,966; No. 6,709,653; No. 6,693,176; No. 6,692,908; No. 6,689,607 6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852; 6,635,482 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618; 6,545,130; No. 544,749; No. 6,534,058; No. 6,528,625; No. 6,528,269; No. 6,521,227; No. 6,518,404; No. 665; No. 6,491,915; No. 6,488,930; No. 6,482,598; No. 6,482,408; No. 6,479,247; No. 6,468,531 6,468,529; 6,465,173; 6,461,8 No. 3, No. 6,458,356; No. 6,455,044; No. 6,455,040, No. 6,451,310; No. 6,444,206, No. 6,441,143 6,432,404; 6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; No. 376,654; No. 6,372,215; No. 6,359,126; No. 6,355,481; No. 6,355,444; No. 6,355,245; No. 244; No. 6,346,246; No. 6,344,198; No. 6,34 No. 6,340,459; No. 6,331,175; No. 6,306,393; No. 6,254,868; No. 6,187,287; No. 6,183,744 No. 6,129,914; No. 6,120,767; No. 6,096,289; No. 6,077,499; No. 5,922,302; No. 5,874,540; No. 5,814,440; No. 5,798,229; No. 5,789,554; No. 5,776,456; No. 5,736,119; No. 5,716,595; 6,577,136; 5,587,459; 5,443,953, 5,525,338, each of which is an example part of which is incorporated herein by reference. These are exemplary only and a wide variety of other antibodies and their hybridomas are known in the art. One skilled in the art can obtain antibody sequences or antibody-secreting hybridomas for almost any disease-related antigen by simply searching the ATCC, NCBI, and / or USPTO database for antibodies to the selected disease-related target of interest. You will understand that you can. The antigen-binding domain of the cloned antibody is amplified, cleaved, ligated into an expression vector, transfected into a suitable host cell, and used for protein production using standard techniques well known in the art. be able to.

Immunoconjugates In certain embodiments, the antibody or fragment thereof may be conjugated to one or more therapeutic or diagnostic agents. The therapeutic agents need not be the same and can be different, for example, a drug and a radioisotope. For example, ¹³¹ I can be incorporated into the tyrosine of an antibody or fusion protein and the drug can be linked to the epsilon amino group of a lysine residue. In addition, therapeutic agents and diagnostic agents can be bound to, for example, reduced SH groups and / or sugar side chains. Numerous methods for making covalent or non-covalent conjugates of therapeutic or diagnostic agents and antibodies or fusion proteins are known in the art and any such known method is used. be able to.

The therapeutic or diagnostic agent can be bound to the hinge region of the reduced antibody component by formation of a disulfide bond. Alternatively, such agents can be coupled using heterobifunctional crosslinkers such as N-succinyl 3- (2-pyridyldithio) propionate (SPDP). Yu et al., Int. J. Cancer 56: 244 (1994). The general techniques for forming such bonds are well known in the art. For example, Wong, CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press
1991); Upeslacis et al., “Modification of Antibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al.
(eds.), pages 187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterization of Synthetic Peptide-Derived
Antibodies, ”in MONOCLONAL ANTIBODIES: PRODUCTION,
ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (Eds.), Pages 60-84
See (Cambridge University Press 1995). Alternatively, the therapeutic or diagnostic agent can be attached via the sugar moiety of the Fc portion of the antibody. Sugar groups may be used to increase the loading of the same agent attached to the thiol group, or sugar moieties may be used to attach different therapeutic or diagnostic agents.

Methods for conjugating peptides to antibody components via an antibody sugar moiety are well known to those skilled in the art. For example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih, which is incorporated herein by reference.
et al., Int. J. Cancer 46: 1101 (1990); and Shih et al., US Pat. No. 5,057,313. A common method involves reacting an antibody component having an oxidized sugar moiety with a carrier polymer having at least one free amine function. This reaction initially results in a Schiff base (imine) linkage, which is stabilized by reducing the secondary amine to form the final conjugate.

If the antibody used as the antibody component of the immune complex is an antibody fragment, the Fc region may not be present. However, it is possible to introduce a sugar moiety into the light chain variable region of a full-length antibody or antibody fragment. For example, Leung et al., J. Immunol. 154: 5919 (1995); Hansen, which are incorporated herein by reference in their entirety.
See et al., US Pat. No. 5,443,953 (1995), Leung et al., US Pat. No. 6,254,868. The engineered sugar moiety is used to attach a therapeutic or diagnostic agent.

  In some embodiments, chelating agents can be attached to antibodies, antibody fragments, or fusion proteins and used to chelate therapeutic or diagnostic agents such as radionuclides. Exemplary chelating agents include, but are not limited to, DTPA (such as Mx-DTPA), DOTA, TETA, NETA, or NOTA. Methods of conjugating and using chelating agents to bind metals or other ligands to proteins are well known in the art (eg, US Pat. No. 7,563, the Example portion of which is incorporated herein by reference). , 433).

  In certain embodiments, a radioactive metal or paramagnetic ion can be bound to a protein or peptide by reacting with a reagent having a long tail that can bind a plurality of chelating groups for binding the ion to it. . Such tails are useful, for example, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoxim, and for this purpose It may be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chain having a pendant group that can be attached to a chelating group, such as a group as is known.

The chelating agent may be attached directly to the antibody or peptide, for example, as disclosed in US Pat. No. 4,824,659, which is incorporated herein by reference in its entirety. Particularly useful metal chelating agent combinations include ¹²⁵ I, ¹³¹ I, ¹²³ I, ¹²⁴ I, ⁶² Cu, ⁶⁴ Cu, ¹⁸ F, ¹¹¹ In, ⁶⁷ Ga, ⁶⁸ Ga, ^99m Tc, ^{94 m for radioimaging.} Included are 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs used with diagnostic isotopes in the general energy range of 60-4,000 keV, such as Tc, ¹¹ C, ¹³ N, ¹⁵ O, ⁷⁶ Br. The same chelating agent is useful for MRI when complexed with non-radioactive metals such as manganese, iron, and gadolinium. Macrocyclic chelators such as NOTA, DOTA, and TETA are used with various metals and radioactive metals, respectively, most specifically with gallium, yttrium, and copper radionuclides. Such metal chelator complexes can be very stabilized by matching the ring size to the target metal. Other ring-type chelating agents such as macrocyclic polyethers that are of interest to bind stably to nuclides such as ²²³ Ra for RAIT are included.

More recently, ¹⁸ F labeling methods used in PET scanning technology have been disclosed, for example by reacting F-18 with metals such as aluminum or other atoms. ^{The 18} F-Al conjugate can be bound directly to the antibody or complexed with a chelating group such as DOTA, NOTA, or NETA that is used to label constructs that can be targeted by pre-targeting methods. Also good. Such F-18 labeling technology is disclosed in US Pat. No. 7,563,433, an example portion of which is incorporated herein by reference.

Immunotoxins containing lampirnase (Rap) Binding or fusion of a tumor-targeting antibody or antibody fragment to Rap can be accomplished by first combining LL2-onconase (Newton et al., Blood 2001; 97: 528-35), Rap and mouse anti-tumor. For chemical conjugates containing the CD22 monoclonal antibody (mAb), a fusion protein comprising 2L-Rap-hLL1-γ4P (see, eg, Example 2 below), Rap and a humanized anti-CD74 mAb (Stein et al., As demonstrated for Blood 2004; 104: 3705-11), it is a promising technique to enhance its efficacy.

  The method used to generate 2L-Rap-hLL1-γ4P allows us to develop a series of structurally similar immunotoxins, commonly referred to as 2L-Rap-X. It was. All of them are composed of two Rap molecules, each connected to the N-terminus of one L chain of the antibody of interest (X) via a flexible linker. We have shown that the non-glycosylated form of Rap designated Rap (Q) and Rap to indicate that the potential glycosylation site of Asn69 has been changed to Gln (or Q, single letter code). Has also generated another series of immunotoxins of the same design called 2LRap (Q) -X. For both of a series of substances, we made IgG as either IgG1 (γ1) or IgG4 (γ4) to prevent the formation of IgG4 half molecules (Aalberse and Schuurman, Immunology 2002; 105 : 9-19), the present inventors converted the serine residue (S228) of the hinge region of IgG4 to proline (γ4P). The schematic structure of 2L-Rap-X or 52L-Rap (Q) -X is shown in FIG. This design is determined by the need for an N-terminal pyroglutamic acid residue of Rap for this RNase to be fully functional (Liao et al., Nucleic Acids Res 2003; 31: 5247- 55).

  To explore the utility of mRS7 as a potential therapeutic agent for solid tumors expressing Trop-2, humanized RS7 (hRS7) was created by transplanting the complementarity-determining region of mRS7 into the human IgG1 framework (Qu et al., Methods 2005; 36: 84-95) and fused with Rap (Q) to give 2L-Rap (Q) -hRS7, hereinafter abbreviated as (Q) -hRS7 .

  In the studies described in the examples below, we have found that (Q) -hRS7 can be (i) produced in a mammalian host and purified to homogeneity, (ii) the binding specificity of hRS7. And (iii) inhibits the growth of various Trop-2 expressing cancer cell lines in vitro at nanomolar concentrations, and (iv) in vivo human lung tumor xenografts Shown to inhibit growth indicates that N-terminal fusion of Rap or Rap (Q) to tumor-targeted mAbs is an effective and versatile approach to generate new immunotoxins.

  One skilled in the art will recognize that suitable cytotoxic RNase moieties for use in the present invention include polypeptides having the natural lampyrnase structure and all its enzyme activity variants. These molecules advantageously have an N-terminal pyroglutamate residue believed to be essential for RNase activity and are not substantially inhibited by mammalian RNase inhibitors. Nucleic acids encoding native cytotoxic RNases can be prepared by cloning and restriction of appropriate sequences or by using DNA amplification by polymerase chain reaction (PCR). The amino acid sequence of the leopard lamprumpnase can be obtained from Ardelt et al., J. Biol. Chem., 256: 245 (1991). Gene synthesis can be performed by a method similar to the batch V gene construction method used for hLL2 humanization. (Leung et al., Mol. Immunol., 32: 1413, 1995). Methods for producing cytotoxic RNase variants are known in the art and are within ordinary skill.

  Alternatively, a nucleic acid encoding a cytotoxic RNase or variant thereof can be synthesized in vitro. Chemical synthesis produces a single stranded oligonucleotide. This can be converted to double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using a short primer and single strand as a template. Chemical synthesis is best suited for sequences of about 100 bases, and longer sequences can be obtained by ligating shorter sequences. In Example 2 below, one exemplary method for obtaining a cytotoxic RNase gene is provided.

Dock and Lock (DNL) Method In the “Dock and Lock” (DNL) method, the regulatory (R) subunit of cAMP-dependent protein kinase (PKA) and the anchor domain (AD) of A kinase anchor protein (AKAP) Specific protein-protein interactions that occur in between (Baillie et al., FEBS
Letters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol.
Cell Biol. 2004; 5: 959). PKA plays a central role in one of the most studied signaling pathways caused by the binding of the second messenger cAMP to the R subunit and was first isolated from rabbit skeletal muscle in 1968 (Walsh et al., J.
Biol. Chem. 1968; 243: 3763). The structure of the holoenzyme is composed of two catalytic subunits held in an inactive form by the R subunit (Taylor, J. Biol. Chem. 1989; 264: 8443). PKA isozymes are found in two types of R subunits (RI and RII), each type having an α and β isoform (Scott, Pharmacol. Ther. 1991; 50: 123). The R subunit has been isolated only as a stable dimer and the dimerization domain has been shown to be composed of the first 44 amino terminal residues (Newlon et al., Nat .
Struct. Biol. 1999; 6: 222). The binding of cAMP to the R subunit leads to the release of the active catalytic subunit of broad spectrum serine / threonine kinase activity, which is oriented towards the selected substrate by compartmentalization of PKA by docking with AKAP. (Scott et al., J.
Biol. Chem. 1990; 265; 21561).

Since the first AKAP, microtubule-binding protein-2, was characterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA.
1984; 81: 6723), more than 50 AKAPs localized at various intracellular sites including the plasma membrane, actin cytoskeleton, nucleus, mitochondria, and endoplasmic reticulum are diverse in species ranging from yeast to humans. It is specified by structure (Wong and Scott, Nat. Rev. Mol. Cell Biol. 2004; 5: 959). The AKAP AD for PKA is an amphipathic helix of 14-18 residues (Carr et al., J.
Biol. Chem. 1991; 266: 14188). The amino acid sequence of AD is very different in individual AKAPs, and binding affinities for RII dimers ranging from 2 to 90 nM have been reported (Alto et al., Proc.
Natl. Acad. Sci. USA. 2003; 100: 4445). Interestingly, AKAP binds only to the dimeric R subunit. In the case of human RIIα, AD binds to a hydrophobic surface formed by 23 amino terminal residues (Colledge and Scott, Trends Cell Biol. 1999; 6: 216). Thus, the dimerization domain and AKAP binding domain of human RIIα are both located within the same N-terminal 44 amino acid sequence (Newlon et al., Nat.
Struct. Biol. 1999; 6: 222; Newlon et al., EMBO J. 2001; 20: 1651), referred to herein as DDD.

Human RIIα DDD and AKAP AD as linker modules
The inventors docked with any combination of substances, hereinafter referred to as A and B, to form a non-covalent complex, leaving cysteine residues in both DDD and AD at strategic positions that facilitate the formation of disulfide bonds. We have developed a platform technology that uses human PKA RIIα DDD and AKAP protein AD as an excellent pair of linker modules that can be further locked into a stable tethered structure by introducing groups. The general method of the “dock and lock” method is as follows. Substance A is constructed by linking the DDD sequence to the precursor of A, resulting in a first component, hereinafter referred to as a. DDD sequence, since that would give rise to spontaneous formation of a dimer, thus A will be composed of a _2. Substance B is constructed by linking the AD sequence to the precursor of B, resulting in a second component, hereinafter referred to as b. dimeric motif of DDD contained in a ₂ generates a docking site for binding to AD sequence contained in b, thus facilitating easy attachment of a ₂ and b, consists of a 2 _b A trimeric complex. This binding event is irreversible by a subsequent reaction that covalently anchors the two substances via disulfide bridges, which causes the first binding interaction to access a reactive thiol group located in both DDD and AD. (Chmura et al., Proc.
Natl. Acad. Sci. USA. 2001; 98: 8480), which should be ligated site-specifically, and thus occurs very efficiently based on the principle of effective local concentration. In certain embodiments, the a ₂ subunit may contain two identical effector moieties, such as antibodies, antibody fragments, or cytotoxins, each linked to the same DDD sequence. The trimer a ₂ b complex may comprise two copies of a first effector portion and one copy of a second effector portion.

  By coupling DDD and AD away from the precursor functional groups, it is expected that such site-specific ligation will preserve the original activity of the precursor. This approach is modular in nature and can potentially be applied to site-specifically bind a wide variety of substances. The DNL method is disclosed in US Pat. Nos. 7,550,143, 7,521,056, 76,534,866, 7,527,787, and 7,666,400. And each example portion is incorporated herein by reference. In a preferred embodiment, the DNL complex may comprise a trimeric structure, but in alternative embodiments, other chemistries such as four copies of the toxin moiety and one copy of the antibody or antibody fragment. Stoichiometry is possible.

In preferred embodiments, the effector moiety is a protein or peptide, more preferably an antibody, antibody fragment, or toxin, which can be attached to a DDD or AD moiety to form a fusion protein or peptide. Various methods are known for producing fusion proteins, including nucleic acid synthesis, hybridization, and / or amplification that produces a synthetic double stranded nucleic acid encoding a fusion protein of interest. Such double stranded nucleic acids can be inserted into expression vectors for production of fusion proteins by standard molecular biology techniques (eg, Sambrook et al., Molecular Cloning, A
referring to the ^{laboratory manual, 2 nd Ed, 1989} ). In such preferred embodiments, the AD and / or DDD moiety may be linked to either the N-terminus or C-terminus of the effector protein or peptide. However, those of ordinary skill in the art may have different binding sites for the AD or DDD moiety to the effector moiety, depending on the chemical nature of the effector moiety and part (s) of the effector moiety involved in its physiological activity. You will understand that there is. In the most preferred embodiment, the binding of the AD or DDD moiety to the antibody or antibody fragment occurs at the C-terminus of the heavy chain subunit at the opposite end of the antigen binding site of the molecule. However, as discussed below, N-terminal binding to an antibody or antibody fragment can also be used while retaining antigen binding activity. Site-specific attachment of various effector moieties can be performed using techniques known in the art, such as the use of bivalent cross-linking reagents and / or other chemical conjugation techniques.

DDD and AD Sequence Variants In one embodiment, the AD and DDD sequences incorporated into the immunotoxin DNL construct comprise the following DDD1 and AD1 amino acid sequences. In a more preferred embodiment, the AD and DDD sequences comprise the DDD2 and AD2 amino acid sequences that are designed to facilitate disulfide bond formation between the DDD and AD moieties.
DDD1
SHIQIPPGLTELLQGYTVEVLRRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 13)
DDD2
CGHIQIPPGLTELLQGYTVEVLRRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 14)
AD1
QIEYLAKQIVDNAIQQA (SEQ ID NO: 15)
AD2
CGQIEYLAKQIVDNAIQQAGC (SEQ ID NO: 16)

One skilled in the art will appreciate that DDD1 and DDD2 contain the human RIIα form DDD sequence of protein kinase A. However, in an alternative embodiment, the DDD and AD moieties may also be based on the human RIα-type DDD sequence of protein kinase A and the corresponding AKAP sequence, as exemplified in DDD3, DDD3C, and AD3 below. Good.
DDD3
SLRECELYVQKHNIQALLKDSIVQLCTARPERPFMAFLREEFERLEKEEEAK (SEQ ID NO: 17)
DDD3C
MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREEFERLEKEEEAK (SEQ ID NO: 18)
AD3
CGFEELAWKIAKMIWSDVFQQGC (SEQ ID NO: 19)

Still other alternative DDD moieties based on the known human RIβ and RIIβ amino acid sequences can be designed and used (see, eg, NCBI accession numbers NP_001158233 and NP_002727, see sequences below).
Human PKA RIbeta amino acid sequence MASPPACPSE EDESLKGCEL YVQLHGIQQV LKDCIVHLCI SKPERPMKFL REHFEKLEKE ENRQILARQK SNSQSDSHDE EVSPTPPNPV VKARRRRGGV SAEVYTEEDA VSYVRKVIPK DYKTMTALAK AISKNVLFAH LDDNERSDIF DAMFPVTHIA GETVIQQGNE GDNFYVVDQG EVDVYVNGEW VTNISEGGSF GELALIYGTP RAATVKAKTD LKLWGIDRDS YRRILMGSTL RKRKMYEEFL SKVSILESLE KWERLTVADA LEPVQFEDGE KIVVQGEPGD DFYIITEGTA SVLQRRSPN EYVEVGRLGP SDYFGEIALL LNRPRAATVV ARGPLKCVKL DRPRFERVLG PCSEILKRNI QRYNSFISLT V (SEQ ID NO: 20)
Human PKA RIIbeta amino acid sequence MSIEIPAGLT ELLQGFTVEV LRHQPADLLE FALQHFTRLQ QENERKGTAR FGHEGRTWGD LGAAAGGGTP SKGVNFAEEP MQSDSEDGEE EEAAPADAGA FNAPVINRFT RRASVCAEAY NPDEEEDDAE SRIIHPKTDD QRNRLQEACK DILLFKNLDP EQMSQVLDAM FEKLVKDGEH VIDQGDDGDN FYVIDRGTFD IYVKCDGVGR CVGNYDNRGS FGELALMYNT PRAATITATS PGALWGLDRV TFRRIIVKNN AKKRKMYESF IESLPFLKSL EFSERLKVVD VIGTKVYNDG EQIIAQGD A DSFFIVESGE VKITMKRKGK SEVEENGAVE IARCSRGQYF GELALVTNKP RAASAHAIGT VKCLAMDVQA FERLLGPCME IMKRNIATYE EQLVALFGTN MDIVEPTA (SEQ ID NO: 21)

In other alternative embodiments, various sequence variants of the AD and / or DDD portion can be used to construct an immunotoxin DNL construct. The structure-function relationship of AD and DDD domains has been the subject of research. (Eg, Burns-Hamuro et al., 2005, Protein Sci 14: 2982-92; Carr et al., 2001, J Biol Chem 276: 17332-38; Alto
et al., 2003, Proc Natl Acad Sci USA 100: 4445-50; Hundsrucker
et al., 2006, Biochem J 396: 297-306; Stokka et al.,
2006, Biochem J 400: 493-99; Gold et al., 2006, Mol Cell
24: 383-95; see Kinderman et al., 2006, Mol Cell 24: 397-408, the full text of each of which is incorporated herein by reference. )

For example, in Kinderman et al. (2006), the crystal structure of AD-DDD binding interaction has been examined, and human DDD sequences are either dimerized or AKAP-bound, as indicated by the underlined sequences below. It was concluded that it contains a number of important conserved amino acid residues. (See FIG. 1 of Kinderman et al., 2006, which is incorporated herein by reference.) When designing a sequence variant of a DDD sequence, one of ordinary skill in the art would not change any of the underlined residues. It will be appreciated that conservative amino acid substitutions of residues that are desirable to avoid but that are not as important for dimerization and AKAP binding may have been made. Thus, an alternative DDD sequence that may be used to construct a DNL construct is shown in SEQ ID NO: 22, where “X” represents a conservative amino acid substitution. Conservative amino acid substitutions, which are discussed in more detail below, include, for example, substitution of glutamic acid residues with aspartic acid residues or substitution of isoleucine residues with leucine or valine residues. Such conservative amino acid substitutions are well known in the art.
Human DDD sequence from protein kinase A SH I Q I PPG L TE LL QG Y T V E VL RQQPPD LV E F A V E YF TR L REARA ( SEQ ID NO: 13)
XX I X I XXX L XX LL XX Y X V X VL XXXXXXX LV X F X V X YF XX L XXXX (SEQ ID NO: 22)

Alto et al. (2003) performed bioinformatics analysis of AD sequences of various AKAP proteins and called AKAP-IS shown below, an RII-selective AD sequence with a binding constant for DDD of 0.4 nM. Designed. The AKAP-IS sequence was designed as a peptide antagonist of AKAP binding to PKA. Residues of the AKAP-IS sequence where substitution tends to reduce binding to DDD are underlined in the sequence below. Accordingly, one skilled in the art will appreciate that a variant capable of functioning in a DNL construct is represented by SEQ ID NO: 23, where “X” is a conservative amino acid substitution.
AKAP-IS sequence QIEYL A KQ IV DN AI QQA (SEQ ID NO: 15)
XXXXXX A XX IV XX AI XXX (SEQ ID NO: 23)

Similarly, Gold (2006) uses SuperAKAP-, shown below, which shows selectivity for the RKA isoform of PKA, which is 5 orders of magnitude higher than the RI isoform using crystallography and peptide screening. IS sequences were developed. Underlined residues indicate the position of the amino acid substitution that increases binding to the DDD portion of RIIα relative to the AKAP-IS sequence. In this sequence, the N-terminal Q residue is numbered as residue number 4 and the C-terminal A residue is numbered as residue number 20. Residues that could be substituted to affect affinity for RIIα were residues 8, 11, 15, 16, 18, 19, and 20 (Gold et al., 2006). In an alternative embodiment, it is contemplated that the SuperAKAP-IS sequence may be used in place of the sequence of the AKAP-IS AD moiety to prepare a cytotoxic DNL construct. Other alternative sequences that can be used in place of the AKAP-IS AD sequence are shown below. Substitutions relative to the AKAP-IS sequence are underlined. It is also expected that, like the AKAP-IS sequence, the AD moiety may contain additional N-terminal residues cysteine and glycine, and C-terminal residues glycine and cysteine.
SuperAKA-IS
QIEY V AKQIVD Y AI H QA (SEQ ID NO: 24)
Alternative AKAP sequence QIEY K AKQIVD H AI H QA (SEQ ID NO: 25)
QIEY H AKQIVD H AI H QA (SEQ ID NO: 26)
QIEY V AKQIVD H AI H QA (SEQ ID NO: 27)

In FIG. 2 of Gold et al., Additional DDD binding sequences derived from the various AKAP proteins shown below were disclosed.
RII specific AKAP
AKAP-KL
PLAYQAGLLVQNAIQQAI (SEQ ID NO: 28)
AKAP79
LLIETSSLSSLKNAIQLSI (SEQ ID NO: 29)
AKAP-Lbc
LIEEAASRIVAVIEQVK (SEQ ID NO: 30)
RI-specific AKAP
AKAPce
ALYQFADRFSELVISEAL (SEQ ID NO: 31)
RIAD
LEQVANQLADQIIKEAT (SEQ ID NO: 32)
PV38
FEELAWKIAKMIWSDVF (SEQ ID NO: 33)
Bispecific AKAP
AKAP7
ELVRLSKRLVENAVLKAV (SEQ ID NO: 34)
MAP2D
TAEEEVSARIVQVVTAEAV (SEQ ID NO: 35)
DAKAP1
QIKQAAFQLISQVILEAT (SEQ ID NO: 36)
DAKAP2
LAWKIAKMIVSDVMQQ (SEQ ID NO: 37)

Also, Stokka et al. (2006) developed a peptide competitor for AKAP binding to PKA, shown below. This peptide antagonist has been called Ht31, RIAD, and PV-38. The Ht-31 peptide showed greater affinity for the RII isoform of PKA, and RIAD and PV-38 showed higher affinity for RI.
Ht31
DLIEAASRIVDAVIEQVKAAGY (SEQ ID NO: 38)
RIAD
LEQYANQLADQIIKATE (SEQ ID NO: 39)
PV-38
FEELAWKIAKMIWSDVFQQC (SEQ ID NO: 40)

Hundsrucker et al. (2006) developed yet another peptide competitor for AKAP binding to PKA with a binding constant as low as 0.4 nM against PKA RII type DDD. The sequences of various AKAP antagonist peptides are provided in Table 1 of Hundsucker et al. Reproduced below.

Residues that are highly conserved in the AD domains of various AKAP proteins are shown below by underlining with reference to the AKAP-IS sequence below. These residues are the same as those observed by Alto et al. (2003) with the addition of a C-terminal alanine residue. (See FIG. 4 of Hundsrucker et al. (2006), incorporated herein by reference.) The sequences of peptide antagonists with particularly high affinity for the RII DDD sequence are AKAP-IS, AKAP7δ-wt-pep. , AKAP7δ-L304T-pep, and AKAP7δ-L308D-pep.
AKAP-IS
QIEYL A KQ IV DN AI QQ A (SEQ ID NO: 15)

Carr et al. (2001) examined the degree of sequence homology between AKAP-binding DDD sequences that differ from human and non-human proteins, and identified the most highly conserved DDD sequences in various DDD parts. Residues were identified. They are shown below by underlining with reference to the human PKA RIIα DDD sequence. Residues that were particularly conserved are further shown in italics. These residues overlap, but are not identical to, that Kinderman et al. (2006) suggested to be important for binding to the AKAP protein. Thus, a possible DDD sequence where “X” represents a conservative amino acid substitution is shown in SEQ ID NO: 59.
S HI Q IP P GL T ELLQGYT V EVLR Q QP P DLVEFA VE YF TR L R E A R A ( SEQ ID NO: 13)
X HI X IP X GL X ELLQGYT X EVLR X QP X DLVEFA XX YF XX L X E X R X (SEQ ID NO: 59)

  Those skilled in the art generally prefer that those amino acid residues that are highly conserved in DDD and AD sequences derived from various proteins still remain unchanged when making amino acid substitutions. However, it will be appreciated that residues that are less highly conserved may be more easily changed to generate sequence variants of the AD and / or DDD sequences described herein.

In addition to sequence variants of DDD and / or AD moieties, in certain embodiments, it may be preferable to introduce sequence mutations in the antibody moiety or linker peptide sequence that links the antibody to the AD sequence. As one illustrative example, three possible variants of the fusion protein sequence are shown below.
(L)
QKSLSLPSGLGGGGGSGGGCG (SEQ ID NO: 60)
(A)
QKSLSLSPGAGSGGGGSGGCG (SEQ ID NO: 61)
(-)
QKSLSLSPGGSGGGGGSGGCG (SEQ ID NO: 62)

Amino Acid Substitution In certain embodiments, the disclosed methods and compositions may involve the production and use of proteins or peptides having one or more substituted amino acid residues. The structural, physical, and / or therapeutic characteristics of natural, chimeric, humanized, or human antibodies, or AD or DDD sequences can be optimized by substitution of one or more amino acid residues. For example, it is known in the art that the functional characteristics of a humanized antibody can be improved by substituting a limited number of human framework region (FR) amino acids with the corresponding FR amino acids of the parent mouse antibody. It is well known. This is especially true when framework region amino acid residues are close to CDR residues.

  In other cases, the binding affinity for a target antigen, the rate of dissociation or off of the antibody from the target antigen, or further induction of CDC (complement dependent cytotoxicity) or ADCC (antibody dependent cytotoxicity) by the antibody. The therapeutic properties of the antibody, such as efficacy, can be optimized by a limited number of amino acid substitutions.

  In alternative embodiments, the DDD and / or AD sequences used to create the subject DNL construct may be further optimized, for example, to increase DDD-AD binding affinity. Possible sequence variations in DDD or AD sequences are discussed above.

  One skilled in the art will recognize that in general, amino acid substitutions typically involve replacing one amino acid with another with relatively similar properties (ie, conservative amino acid substitutions). Let's go. The effects of amino acid substitutions on various amino acid properties and protein structure and function are the subject of extensive research and knowledge in the art.

  For example, the hydrophobic and hydrophilic index of amino acids may be considered (Kyte & Doolittle, 1982, J. Mol. Biol., 157: 105-132). The relative hydrophobic and hydrophilic character of the amino acid contributes to the secondary structure of the resulting protein and then defines the interaction of the protein with other molecules. Each amino acid has been assigned its hydrophobicity index based on hydrophobicity and charge characteristics (Kyte & Doolittle, 1982), which are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4) Threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). When performing conservative substitution, it is preferable to use an amino acid whose hydrophobicity index is within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

  Amino acid substitutions may also take into account the hydrophilicity of amino acid residues (eg, US Pat. No. 4,554,101). Amino acid residues have been assigned hydrophilicity values: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0); glutamic acid (+3.0); serine (+0.3) Asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5. +-. 1); alanine (-0.5); histidine Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (- 2.3); phenylalanine (-2.5); tryptophan (-3.4). The amino acid is preferably substituted with another amino acid having similar hydrophilicity.

Other considerations include the size of amino acid side chains. For example, it will generally not be desirable to replace amino acids with small side chains such as glycine or serine with amino acids with bulky side chains, such as tryptophan or tyrosine. The effect of various amino acid residues on protein secondary structure should also be considered. Empirical studies have determined the influence of various amino acid residues on the propensity of protein domains to adopt an alpha helix, beta sheet, or reverse turn secondary structure and are known in the art (eg, Chou
& Fasman, 1974,
Biochemistry, 13: 222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979, Biophys.
J., 26: 367-384).

  Based on such considerations and extensive empirical studies, conservative amino acid substitution tables have been generated and are known in the art. For example, arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine. Or: Ala (A) leu, ile, val; Arg (R) gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glut; Cys (C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H) asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met, ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F) leu, val, ile, ala, tyr; Pro ( Ser (S), thr; Thr (T) ser; Trp (W) phe, tyr; Tyr (Y) trp, phe, thr, se ; Val (V) ile, leu, met, phe, is ala.

  Other considerations for amino acid substitutions include whether the residue is located within the protein or is exposed to a solvent. For internal residues, conservative substitutions may include: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala and Gly; Ile and Val; Val and Leu; Ile; Leu and Met; Phe and Tyr; Tyr and Trp (see, for example, the PROWL website at rockefeller.edu). In the case of solvent-exposed residues, conservative substitutions would include: Asp and Asn; Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala and Gly; And Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu; Leu and Ile; Ile and Val; Phe and Tyr (same as above). Various matrices such as PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlan matrix, Doottle matrix, Henikoff matrix, Miyata matrix, Fitch matrix, Jones matrix, Rao matrix, Levin matrix, and Risler matrix can be selected for amino acid substitution It is made to support (Id.).

  In determining amino acid substitution, formation of an ionic bond (salt bridge) between a positively charged residue (eg, His, Arg, Lys) and a negatively charged residue (eg, Asp, Glu), or a neighboring cysteine The presence of intermolecular or intramolecular bonds, such as the formation of disulfide bonds between residues, may be considered.

  Methods for substituting any amino acid in the encoded protein sequence with any other amino acid are well known, for example, by the technique of site-directed mutagenesis or by synthesizing and constructing an oligonucleotide encoding an amino acid substitution, It is a matter of routine experimentation by those skilled in the art by splicing into expression vector constructs.

Therapeutic agents In alternative embodiments, treatments such as cytotoxic agents, anti-angiogenic agents, pro-apoptotic agents, antibiotics, hormones, hormone antagonists, chemokines, drugs, prodrugs, toxins, enzymes, or other agents The drug can be used either conjugated to the subject immunotoxin or administered separately before, simultaneously with, or after the immunotoxin. The drug used is a pharmaceutical selected from the group consisting of anti-mitotic agents, anti-kinase agents, alkylating agents, antimetabolites, antibiotics, alkaloids, anti-angiogenic agents, pro-apoptotic agents, and combinations thereof It may have characteristics.

  Useful exemplary drugs include: 5-fluorouracil, apridin, azaribine, anastrozole, anthracycline, bendamustine, bleomycin, bortezomib, bryostatin 1, busulfan, calithiamycin ( calicheamycin), camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celeb, chlorambucil, cisplatin (CDDP), Cox-2 inhibitor, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecan , Cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX, 2-pyrrolinodoxorubicine), Ano-morpholino doxorubicin, doxorubi single clonide, epirubi single clonide, estramustine, epipodophyllotoxin, estrogen receptor binding agent, etoposide (VP16), etoposide glucuronide, etoposide phosphate, floxuridine (FUdR), 3 ', 5 '-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl-protein transferase inhibitor, gemcitabine, hydroxyurea, idarubicin, ifosfamide, L-asparaginase, lenolidamide, leucovorin, lomustine, mechlorethamine , Mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mitramycin, mitomycin, mitotane, nabe Bottle, nitrosurea, plicomycin, procarbazine, paclitaxel, pentostatin, PSI-341, raloxifene, semustine, streptozocin, tamoxifen, taxol, temazolomide (aqueous form of DTIC), transplatinum ( transplatinum), thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vinorelbine, vinblastine, vincristine, and vinca alkaloids.

  Toxins used may include: ricin, abrin, alpha toxin, saporin, ribonuclease (RNase) such as onconase, DNase I, staphylococcal enterotoxin A, pokeweed antiviral protein, gelonin , Diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.

  Chemokines that may be used may include RANTES, MCAF, MIP1-alpha, MIP1-beta, and IP-10.

  In certain embodiments, angiostatin, baculostatin, canstatin, maspin, anti-VEGF antibody, anti-PlGF peptide and antibody, anti-vascular growth factor antibody, anti-Flk-1 antibody, anti-Flt-1 antibody and Peptide, anti-Kras antibody, anti-cMET antibody, anti-MIF (macrophage migration inhibitory factor) antibody, laminin peptide, fibronectin peptide, plasminogen activator inhibitor, tissue metalloproteinase inhibitor, interferon, interleukin 12, IP-10 , Gro-β, thrombospondin, 2-methoxyestradiol, proliferin-related protein, carboxiamidotriazole, CM101, marimastat, pentosan polysulfate, angiopoietin-2, inter Ferron-alpha, herbimycin A, PNU145156E, 16K prolactin fragment, linimide (roquinimex), thalidomide, pentoxyphyllin, genistein, TNP-470, endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin, Anti-angiogenic agents such as AGM-1470, platelet factor 4 or minocycline can be used.

  The immunomodulator used may be selected from cytokines, stem cell growth factor, lymphotoxin, hematopoietic factor, colony stimulating factor (CSF), interferon (IFN), erythropoietin, thrombopoietin, and combinations thereof . Particularly useful are lymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors such as interleukin (IL), colonies such as granulocyte colony stimulating factor (G-CSF) or granulocyte macrophage colony stimulating factor (GM-CSF). Stimulating factors, interferons such as interferon-α, -β, or -γ, and stem cell growth factors such as those termed “factor S1”. Cytokines include: growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; follicle stimulating hormone ( Glycoprotein hormones such as FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; prostaglandin, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor -α and -β; Mueller inhibitor; mouse gonadotropin binding peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor such as NGF-β; platelet growth factor; TGF-α and Transformation growth such as TGF-β Insulin-like growth factor-I and -II; Erythropoietin (EPO); Osteoinductive factor; Interferon such as interferon-α, -β, and -γ; Macrophage-CSF (M-CSF) Colony stimulating factor (CSF) such as: IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL -10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25 and other interleukins (IL), LIF, kit-ligand or FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor, and LT.

Radionuclides used include, but are not limited to: ¹¹¹ In, ¹⁷⁷ Lu, ²¹² Bi, ²¹³ Bi, ²¹¹ At, ⁶² Cu, ⁶⁷ Cu, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I , ³² P, ³³ P, ⁴⁷ Sc, ¹¹¹ Ag, ⁶⁷ Ga, ¹⁴² Pr, ¹⁵³ Sm, ¹⁶¹ Tb, ¹⁶⁶ Dy, ¹⁶⁶ Ho, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁸⁹ Re, ²¹² Pb, ²²³ Ra, ²²⁵ Ac, ⁵⁹ Fe, ⁷⁵ Se, ⁷⁷ As, ⁸⁹ Sr, ⁹⁹ Mo, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹⁴³ Pr, ¹⁴⁹ pm, ¹⁶⁹ Er, ¹⁹⁴ Ir, ¹⁹⁸ Au, ¹⁹⁹ Au, and ²¹¹ Pb. Preferably, the therapeutic radionuclide is an alpha emitter in the range of 20 to 6,000 keV for Auger emitters, preferably in the range of 60 to 200 keV, and 100 to 2,500 keV for beta emitters. The case has a decay energy of 4,000 to 6,000 keV. Useful beta particle radionuclides preferably have a maximum decay energy of 20 to 5,000 keV, more preferably 100 to 4,000 keV, and most preferably 500 to 2,500 keV. Also preferred are radionuclides that substantially decay with Auger radiation particles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161, Os-189m, and Ir-192. It is. Useful beta-particle radionuclides preferably have decay energy of less than 1,000 keV, more preferably less than 100 keV, and most preferably less than 70 keV. Also preferred are radionuclides that substantially decay with the production of alpha particles. Such radionuclides include, but are not limited to, Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac. -225, Fr-221, At-217, Bi-213, and Fm-255. The decay energy of useful alpha particle radionuclides is preferably 2,000 to 10,000 keV, more preferably 3,000 to 8,000 keV, and most preferably 4,000 to 7,000 keV. Additional potential radioisotopes used include: ¹¹ C, ¹³ N, ¹⁵ O, ⁷⁵ Br, ¹⁹⁸ Au, ²²⁴ Ac, ¹²⁶ I, ¹³³ I, ⁷⁷ Br, ^113m In , ⁹⁵ Ru, ⁹⁷ Ru, ¹⁰³ Ru, ¹⁰⁵ Ru, ¹⁰⁷ Hg, ²⁰³ Hg, ^{121 m} Te, ^{122 m} Te, ^{125 m} Te, ¹⁶⁵ Tm, ¹⁶⁷ Tm, ¹⁶⁸ Tm, ¹⁹⁷ Pt, ¹⁰⁹ Pd, ¹⁰⁵ Rh, ¹⁴² Pr, ¹⁴ Pr, ¹⁶¹ Tb, ¹⁶⁶ Ho, ¹⁹⁹ Au, ⁵⁷ Co, ⁵⁸ Co, ⁵¹ Cr, ⁵⁹ Fe, ⁷⁵ Se, ²⁰¹ Tl, ²²⁵ Ac, ⁷⁶ Br, and ¹⁶⁹ Yb. Some useful diagnostic nuclides include ¹⁸ F, ⁵² Fe, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁸⁹ Zr, ⁹⁴ Tc, ^{94 m} Tc, ^{99 m} Tc, or ¹¹¹ In May be included.

The therapeutic agent may include a photoactive agent or dye. Fluorescent compositions such as fluorescent dyes and other chromogens or dyes such as porphyrins sensitive to visible light have been used to detect and treat lesions by directing light suitable for the lesion. In therapy, this is called photoradiation therapy, phototherapy, or photodynamic therapy. Jori et al. (Eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER
DISEASES (Libreria Progetto 1985); van den Bergh, Chem. Britain
(1986), 22: 430. In addition, monoclonal antibodies are conjugated with photoactivatable dyes to achieve phototherapy. Mew et al., J. Immunol. (1983), 130: 1473; ibid. Cancer Res. (1985), 45: 4380; Oseroff et al., Proc. Natl. Acad. Sci. USA (1986), 83: 8744; ibid., Photochem. Photobiol. (1987), 46:83; Hasan et al.,
Prog. Clin. Biol. Res. (1989), 288: 471; Tatsuta et al.,
Lasers Surg. Med. (1989), 9: 422; Pelegrin et al., Cancer
(1991), 67: 2529.

  Other useful therapeutic agents may preferably include oligonucleotides directed against oncogenes and oncogene products such as bcl-2 or p53, particularly antisense oligonucleotides. A preferred form of therapeutic oligonucleotide is siRNA.

Diagnostic Agent The diagnostic agent is preferably selected from the group consisting of radionuclides, radiocontrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents, and photoactive agents. Such diagnostic agents are well known and any such known diagnostic agent can be used. Non-limiting examples of diagnostic agents may include radionuclides such as the following: ¹¹⁰ In, ¹¹¹ In, ¹⁷⁷ Lu, ¹⁸ F, ⁵² Fe, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁹⁰ Y, ⁸⁹ Zr, ^{94 m} Tc, ⁹⁴ Tc, ^{99 m} Tc, ¹²⁰ I, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ^{154 to 158} Gd, ³² P, ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸⁶ Re, ¹⁸⁸ Re, ⁵¹ Mn, ^{52 m} Mn, ⁵⁵ Co, ⁷² As, ⁷⁵ Br, ⁷⁶ Br, ^{82 m} Rb, ⁸³ Sr, or other gamma-, beta-, or positron-emitters . The paramagnetic ions used may include the following: chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), Copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), or erbium (III) . The metal contrast agent may contain lanthanum (III), gold (III), lead (II), or bismuth (III). The ultrasound contrast agent may contain liposomes such as gas-containing liposomes. The radiopaque diagnostic agent may be selected from compounds, barium compounds, gallium compounds, and thallium compounds. A wide variety of fluorescent labels are known in the art and include, but are not limited to, fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine. It is. The chemiluminescent label used may include luminol, isoluminol, aromatic acridinium ester, imidazole, acridinium salt, or oxalate ester.

Methods of Therapeutic Treatment Various embodiments are methods of treating cancer in a subject, such as a mammal, including a home or a companion pet, such as humans, dogs and cats, and comprising a therapeutically effective amount of a cytotoxic immune complex. It relates to a method comprising administering a body to a subject.

  In one embodiment, immune diseases that can be treated with the subject immunotoxins include the following: joint diseases such as ankylosing spondylitis, juvenile rheumatoid arthritis, rheumatoid arthritis; multiple sclerosis Neurologic diseases such as diabetes and myasthenia gravis; pancreatic diseases such as diabetes, especially juvenile diabetes; gastrointestinal diseases such as chronic active hepatitis, celiac disease, ulcerative colitis, Crohn's disease, and pernicious anemia; psoriasis or scleroderma Skin diseases such as asthma; allergic diseases such as asthma, and transplant-related conditions such as graft-versus-host disease and allograft rejection.

Administration of cytotoxic immune complexes can be supplemented by simultaneous or sequential administration of a therapeutically effective amount of another antibody that binds or reacts with another antigen on the surface of the target cell. Preferred additional MAbs include at least one humanized, chimeric, or human MAb selected from the group consisting of MAbs that react with: CD4, CD5, CD8, CD14, CD15, CD16, CD19, IGF -1R, CD20, CD21, CD22, CD23, CD25, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD70, CD74, CD79a, CD80, CD95, CD126, CD133, CD138 , CD154, CEACAM5, CEACAM6, B7, AFP, PSMA, EGP-1, EGP-2, carbonic anhydrase IX, PAM4 antigen, MUC1, MUC2, MUC3, MUC4, MUC5, Ia, MIF, HM1.24, HLA-D R, tenascin, Flt-3, VEGFR, PlGF, ILGF, IL-6, IL-25, tenascin, TRAIL-R1, TRAIL-R2, complement factor C5, oncogene product, or combinations thereof. The various antibodies used are known to those skilled in the art, such as anti-CD19, anti-CD20, and anti-CD22 antibodies. For example, see: Ghetie et al., Cancer Res. 48: 2610 (1988); Hekman
et al., Cancer Immunol. Immunother. 32: 364 (1991); Longo,
Curr. Opin. Oncol. 8: 353 (1996), US Pat. No. 5,798,554; 6,187,287; 6,306,393; 6,676,924; No. 109,304; No. 7,151,164; No. 7,230,084; No. 7,230,085; No. 7,238,785; No. 7,238,786; No. 7,282 No. 567; No. 7,300,655; No. 7,312,318; No. 7,501,498; No. 7,612,180; No. 7,670,804; No. 20070172920; 20060193865; and 20080138333, each example portion of which is incorporated herein by reference.

Immunotoxin therapy can be further supplemented with either simultaneous or sequential administration of at least one therapeutic agent. For example, “CVB” (1.5 g / m ² cyclophosphamide, 200-400 mg / m ² etoposide, and 150-200 mg / m ² karmastine) is used to treat non-Hodgkin lymphoma. This is a medication plan. Patti et al., Eur. J. Haematol. 51: 18 (1993). Other suitable combination chemotherapy regimens are well known to those of skill in the art. For example, Freedman et al., "Non-Hodgkin's
Lymphomas, "in CANCER MEDICINE, VOLUME 2, 3rd Edition, Holland et al.
(eds.), pages 2028-2068 (Lea & Febiger 1993). Illustratively, first generation chemotherapy regimens for treating moderate-grade non-Hodgkin's lymphoma (NHL) include C-MOPP (cyclophosphamide, vincristine, procarbazine, and prednisone) and CHOP (cyclophosphamis). Doxorubicin, vincristine, and prednisone). A useful second generation chemotherapy regimen is m-BACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone, and leucovorin) and a suitable third generation regimen is MACOP-B (Methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin, and leucovorin). Additional useful drugs include phenylbutyrate, bendamustine, and bryostatin-1.

The subject immunotoxins can be formulated by known methods of preparing pharmaceutically useful compositions, whereby the immunotoxin is mixed with a mixture having pharmaceutically suitable excipients. Sterile phosphate buffered saline is an example of a pharmaceutically suitable excipient. Other suitable excipients are well known to those skilled in the art. For example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS,
5th Edition (Lea & Febiger 1990), and Gennaro (ed.),
REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack
Publishing Company 1990), and their revisions.

  The subject immunotoxins can be formulated for intravenous administration, eg, by bolus injection or continuous infusion. Preferably, the immunotoxin is infused over a period of less than about 4 hours, more preferably less than about 3 hours. For example, the first 25-50 mg can be infused within 30 minutes, preferably even within 15 minutes, and the remainder can be infused over the next 2-3 hours. Injectable formulations may be provided in unit dosage form, eg, in ampoules or multi-dose containers, with the addition of a preservative. The composition can take the form of a suspension, solution, emulsion in an oily or aqueous medium, etc., and contains a formulation agent such as a suspending agent, a stabilizing agent, and / or a dispersing agent. Can do. Alternatively, the active ingredient may be in powder form for constitution with a suitable medium prior to use, such as pyrogen-free sterilized water.

Additional pharmaceutical methods can be used to control the duration of action of the immunotoxin. Controlled release preparations can be prepared by using polymers that are complexed or adsorbed with immunotoxins. For example, biocompatible polymers include matrices of poly (ethylene-vinyl acetate copolymer) and polyanhydride copolymers of stearic acid dimer and sebacic acid. Sherwood et al., Bio / Technology 10: 1446 (1992). The release rate from such a matrix depends on the molecular weight of the immunotoxin, the amount of immunotoxin within the matrix, and the size of the dispersed particles. Saltzman et al.,
Biophys. J. 55: 163 (1989); Sherwood et al., Supra. Other solid dosage forms include Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS,
5th Edition (Lea & Febiger 1990), and Gennaro (ed.),
REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack
Publishing Company 1990), and its revised version.

  The immunotoxin can also be administered subcutaneously to the mammal or even by other parenteral routes. Further, administration may be by continuous infusion or by single or multiple boluses. Preferably, the immunotoxin is infused over a period of less than about 4 hours, more preferably less than about 3 hours.

More generally, the dose of immunotoxin administered to a human will vary depending on factors such as the patient's age, weight, height, sex, general medical condition, and previous medical history. While it may be desirable to provide the recipient with a dose of immunotoxin ranging from about 1 mg / kg to 25 mg / kg as a single intravenous infusion, depending on the circumstances, lower or higher doses may be administered. May be. Dose of 1 to 20 mg / kg, for example, in the case of 70kg patient is 70～1,400Mg, or if 1.7m patient is 41~824mg / ^{m 2.} Administration may be repeated as needed, for example, once a week for 4-10 weeks, once a week for 8 weeks, or once a week for 4 weeks. Also, administration may be less frequent, such as biweekly for several months, or monthly or quarterly for months, as required for maintenance therapy.

Alternatively, the immunotoxin may be administered as a dose every 2 weeks or every 3 weeks and repeated for a total of at least 3 doses. Alternatively, the construct may be administered twice a week for 4-6 weeks. When the dose is reduced to approximately 200-300 mg / m ² (340 mg per dose for 1.7 m patients, or 4.9 mg / kg for 70 kg patients) once a week for 4-10 weeks It may be administered, or even twice a week for 4-10 weeks. Alternatively, the dosing schedule may be small, ie every other week or every 3 weeks for 2-3 months. However, it has been found that even higher doses, such as 20 mg / kg once a week or once every 2-3 weeks, can be administered by repeating the dosing cycle with slow intravenous infusion. The dosing schedule can optionally be repeated at other intervals, and the dose can be administered by various parenteral routes, with the dose and schedule appropriately adjusted.

  In a preferred embodiment, the immunotoxin is useful for the treatment of cancer. Examples of cancer include, but are not limited to, carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia, myeloma, or lymphoid malignancy. More specific examples of such cancers are noted below and include: squamous cell carcinoma (eg, epithelial squamous cell carcinoma), Ewing sarcoma, Wilms tumor, astrocytoma, Lung cancer including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of lung and squamous cell carcinoma of lung, peritoneal cancer, hepatocellular carcinoma, stomach cancer including stomach cancer, gastric cancer, pancreatic cancer, glioblastoma multiforme Cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, hepatocellular carcinoma, neuroendocrine tumor, medullary thyroid cancer, differentiated thyroid cancer, breast cancer, ovarian cancer, colon cancer, rectal cancer, endometrial cancer or uterus Cancer, salivary gland cancer, kidney or kidney cancer, prostate cancer, vulvar cancer, anal cancer, penile cancer, and head and neck cancer. The term “cancer” refers to primary malignant cells or tumors (eg, cells that have not migrated to a site in the subject other than the original malignant disease or tumor site), and secondary malignant cells or Tumors (eg, those that result from metastasis, malignant cells, or migration of tumor cells to a secondary site different from the original tumor site). Cancers that can be subjected to the therapeutic method of the present invention involve cells that express, overexpress, or abnormally express IGF-1R.

  Other examples of cancer or malignancies include, but are not limited to: acute childhood lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, adrenal gland Cortical cancer, adult (primary) hepatocellular carcinoma, adult (primary) liver cancer, adult acute lymphoblastic leukemia, adult acute myeloid leukemia, adult Hodgkin lymphoma, adult lymphocytic leukemia, adult non-Hodgkin lymphoma, adult primary liver cancer, Adult soft tissue sarcoma, AIDS-related lymphoma, AIDS-related malignancy, anal cancer, astrocytoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, renal pelvis and ureter cancer, central nervous system (Primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, childhood (primary) hepatocellular carcinoma, childhood (primary) liver cancer, childhood acute lymphoblast Spherical leukemia, infant Acute myeloid leukemia, childhood brain stem glioma, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, childhood extracranial germ cell tumor, childhood Hodgkin's disease, childhood Hodgkin lymphoma, childhood hypothalamus and Optic glioma, childhood lymphoblastic leukemia, childhood medulloblastoma, childhood non-Hodgkin lymphoma, childhood pineal gland and tent upper undifferentiated neuroectodermal tumor, childhood primary liver cancer, childhood Rhabdomyosarcoma, childhood soft tissue sarcoma, childhood visual and hypothalamic glioma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, cutaneous T-cell lymphoma, endocrine pancreatic islet cell carcinoma, endometrial cancer , Ventricular ependymoma, epithelial cancer, esophageal cancer, Ewing sarcoma and related tumors, exocrine pancreatic cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, female breast cancer, Gaucher disease, gallbladder Cancer, gastric cancer, gastrointestinal carcinoid Tumor, gastrointestinal tumor, germ cell tumor, gestational choriocarcinoma, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin lymphoma, hypergammaglobulinemia, hypopharyngeal cancer, intestinal cancer, intraocular melanoma, islet cell carcinoma , Islet cell pancreatic cancer, Kaposi sarcoma, kidney cancer, laryngeal cancer, lip and oral cancer, liver cancer, lung cancer, lymphoproliferative disorder, macroglobulinemia, male breast cancer, malignant mesothelioma, malignant thymoma, medulloblast Melanoma, mesothelioma, metastatic latent primary squamous neck cancer, metastatic primary squamous neck cancer, metastatic squamous neck cancer, multiple myeloma, multiple myeloma / plasma cell neoplasm, Myelodysplastic syndrome, myeloid leukemia, myeloid leukemia, myeloproliferative disorder, nasal and sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, latent primary Metastatic squamous neck cancer, oropharynx Cancer, bone / malignant fibrous sarcoma, osteosarcoma / malignant fibrous histiocytoma, osteosarcoma / malignant fibrous histiocytosis of bone, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low-grade potential tumor, pancreatic cancer , Paraproteinemia, polycythemia vera, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, primary central nervous system lymphoma, primary liver cancer, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and urine Duct cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoidosis sarcoma, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cervical cancer, gastric cancer, extratendiary primary extraneural nerve Germ and pineal tumors, T cell lymphoma, testicular cancer, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional renal pelvis and ureteral cancer, chorionic tumor, ureteral and renal pelvic cell carcinoma, urethra Cancer, uterine cancer, uterine sarcoma, vaginal cancer, visual tract and hypothalamic glioma Vulvar cancer, Waldenstrom's macroglobulinemia, Wilms tumor, and any other hyperproliferative diseases other than tumorigenesis located organ system listed above.

The methods and compositions described and claimed herein are used to treat malignant or pre-malignant conditions, including neoplastic conditions or including but not limited to those disorders described above Progression to a malignant state can be prevented. Such use is a condition that is known or suspected to precede tumorigenesis or progression to cancer, particularly hyperplasia, dysplasia, or most specifically non-new composed of dysplasia. Applies to conditions in which biological cell growth occurs (for a review of such proliferative conditions see Robbins and Angell, Basic Pathology, 2d Ed., WB Saunders Co., Philadelphia, pp. 68-79
(1976)).

  Dysplasia is often a precursor to cancer and is found primarily in the epithelium. Dysplasia is the most disordered form of non-neoplastic cell growth with a loss of individual cell uniformity and cellular orientation. Dysplasia is characterized by the occurrence of chronic irritation or inflammation. Dysplasia disorders that can be treated include, but are not limited to: non-sweat ectodermal dysplasia, anteroposterior dysplasia, asphyxia thoracic dysplasia, atrial finger dysplasia, bronchopulmonary abnormality Dysplasia, telencephalon dysplasia, cervical dysplasia, cartilage ectodermal dysplasia, clavicle skull dysplasia, congenital ectodermal dysplasia, skull trunk dysplasia, skull carpal tarsal bone dysplasia, skull base Dysplasia, dentin dysplasia, diaphyseal dysplasia, ectodermal dysplasia, enamel dysplasia, cerebral ocular dysplasia, dysplasia epiphysialis hemimelia, multiple epiphyseal dysplasia, punctate bone Abnormal dysplasia, epithelial dysplasia, facial digital genital dysplasia, familial fibrous mandibular dysplasia, familial white mandible dysplasia, fibromuscular dysplasia, fibrous bone dysplasia, flowering bone dysplasia, Hereditary renal retinal dysplasia, sweating Embryonic dysplasia, hypohidrotic ectodermal dysplasia, lymphocytopenic thymic dysplasia, mammary dysplasia, mandibular facial dysplasia, metaphyseal dysplasia, Mondini inner ear dysplasia, single bone fibrosis dysplasia , Mucosal epithelial dysplasia, multiple epiphyseal dysplasia, ocular ear vertebral dysplasia, ocular finger dysplasia, ocular spinal dysplasia, odontogenic dysplasia, ophthalmomandibular dysplasia, apical cementum dysplasia , Dysplasia of multiple bones, hypochondral dysplasia vertebral tip dysplasia, retinal dysplasia, septal vision dysplasia, vertebral tip dysplasia, and ventricular rib dysplasia.

  Additional preneoplastic diseases that can be treated include, but are not limited to: benign proliferative dysfunction disorders (eg, benign tumors, fibrous cystic conditions, tissue hypertrophy, intestinal polyps) Or adenoma and esophageal dysplasia), vitiligo, keratosis, Bowen's disease, farmer skin, sun cheilitis, and sun keratosis.

  In preferred embodiments, the methods of the invention are used to inhibit the growth, progression, and / or metastasis of cancer, particularly those listed above.

  Additional hyperproliferative diseases, disorders, and / or conditions include, but are not limited to, progression and / or metastasis of malignant diseases and related disorders such as: leukemia (acute leukemia (eg, Acute lymphocytic leukemia, acute myeloid leukemia (myeloblasts, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemia (eg, chronic myelocytic (granulocytic) leukemia and chronic Lymphocytic leukemia)), true red blood cell increase, lymphoma (eg, Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and but not limited to: Solid tumors including sarcomas and carcinomas such as: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synovium Mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell cancer, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, nipple Carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary cancer, bronchial cancer, renal cell cancer, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonic cancer, Wilms tumor, cervical cancer, testicular cancer, lung cancer, Small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ventricular ependymoma, pineal tumor, emangioblastoma, acoustic neuroma, Oligodenoma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

Expression Vectors Still other embodiments may relate to a DNA sequence comprising a nucleic acid encoding a constituent fusion protein of an immunotoxin, such as an antibody, antibody fragment, toxin, or DNL construct. A fusion protein may comprise, for example, an antibody or fragment or toxin linked to an AD or DDD moiety.

  Various embodiments relate to an expression vector comprising a coding DNA sequence. The vector may contain sequences encoding the light and heavy chain constant regions and the hinge region of a human immunoglobulin to which chimeric, humanized, or human variable region sequences may be linked. The vector can further contain a promoter, enhancer, and signal or leader sequence that expresses the encoded protein (s) in the selected host cell. Particularly useful vectors are pdHL2 or GS. More preferably, the light and heavy chain constant regions and the hinge region optionally have at least one amino acid at the allotype position changed to that found in a different IgG1 allotype, optionally based on the EU numbering system. EU heavy chain amino acid 253 may be derived from human EU myeloma immunoglobulin, which may be substituted with alanine. See Edelman et al., Proc. Natl. Acad. Sci USA 63: 78-85 (1969). In other embodiments, the IgG1 sequence may be converted to an IgG4 sequence.

  Those skilled in the art understand that methods for genetically manipulating expression constructs and inserting into engineered cells to express engineered proteins are well known in the art and are a matter of routine experimentation. will do. Methods for expressing host cells and cloned antibodies or fragments are described, for example, in US Pat. Nos. 7,531,327 and 7,537,930, each of which is incorporated herein by reference. Embedded in the book.

Kits Various embodiments may relate to kits containing components suitable for treating or diagnosing diseased tissue in a patient. Exemplary kits may contain one or more immunotoxins described herein. If the composition containing the administration component is not formulated for delivery via the gastrointestinal tract, such as by oral delivery, a device is included that can deliver the kit component by some other route. Also good. One type of device for applications such as parenteral delivery is a syringe used to inject the composition into the body of a subject. An inhaler can also be used. In certain embodiments, the therapeutic agent can be provided in the form of a pre-filled syringe or pen-type auto-injector containing a sterile liquid formulation or lyophilized preparation.

  The kit components may be packaged together or separated into two or more containers. In some embodiments, the container may be a vial containing a sterile lyophilized formulation of the composition that is suitable for reconstitution. The kit may also contain one or more buffers suitable for reconstitution and / or dilution of other reagents. Other containers that can be used include, but are not limited to, pouches, trays, boxes, or tubes. The kit components may be sterile packaged and maintained in a container. Another element that may be included is instructions for the person using the kit.

Examples The following examples are provided for the purpose of illustration and are not intended to limit the scope of the claims of the invention.

Example 1. Lampyrinase (frog RNase) targeted with humanized internalizing anti-Trop-2 antibody exhibits potent cytotoxicity against a variety of human epithelial cancer cells. Novel immunotoxins containing recombinantly tethered amphibian ribonucleases have been demonstrated in vitro against a variety of human epithelial cancer cell lines such as cervical cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, and prostate cancer. As well as in vivo, it has been shown to exhibit a broad and potent antiproliferative activity against human lung cancer xenografts.

Summary We hereby describe Rap (Q), a mutant form of Rap from which the putative N-glycosylation site has been removed, and hRS7, a cell surface glycoprotein that is overexpressed in various epithelial cancers. The generation of a novel IgG-based immunotoxin called 2L-Rap (Q) -hRS7 containing an internalizing humanized antibody against certain Trop-2 is described. Various tests including size-exclusion HPLC, SDS-PAGE, flow cytometry, RNase activity, internalization, cell viability, and colony formation can be used to determine the purity, molecular integrity, and several different Trop-2 of this immunotoxin. It has been demonstrated that the binding affinity of hRS7 to the expression cell lines is comparable and the potency of inhibiting the growth of these cell lines at nanomolar concentrations. In addition, 2L-Rap (Q) -hRS7 suppresses tumor growth in a nude mouse prevention model carrying Calu-3 human non-small cell lung cancer xenografts, with a median survival (MST) of 55 to 96 days. (P <0.01). These results indicate the excellent efficacy of 2L-Rap (Q) -hRS7 as a therapeutic agent for various Trop-2 expressing cancers such as cervical cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, and prostate cancer. Demonstrate.

Materials and Methods Cell lines and cell cultures. Cervical cancer system (ME-180), breast cancer system (T-47D, MDA-MB-468, SK-BR-3), prostate cancer system (DU-145, PC-3, 22Rv1), lung adenocarcinoma system ( Calu-3), pancreatic cancer lines (Capan-1, BxPC-3, and AsPc-1), and ovarian cancer lines (SK-OV-3) were obtained from the American Cultured Cell Line Conservation Agency (Manassas, VA) And cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 200 units / mL penicillin, and 100 μg / mL streptomycin at 37 ° C. with 5% CO ₂ . .

Antibodies and reagents. Miratuzumab (hLL1, anti-CD74), hRS7, recombinant lampirnase (rRap), and mouse anti-Rap IgG were obtained from Immunomedics. Fluorescein isothiocyanate (FITC) conjugated, phycoerythrin (PE) conjugated, or horseradish peroxidase (HRP) conjugated goat anti-human (GAH) or goat anti-mouse (GAM) IgG Fc specific antibodies were purchased from Jackson ImmunoResearch Labs ( Purchased from West Grove, Pennsylvania. GAH IgG conjugated to Alexa fluor 488, human transferrin conjugated to Alexa fluor 568, and Hoechst 33258 were obtained from Molecular Probes (Invitrogen, Carlsbad, CA). All restriction enzymes are available from New England Biolabs (
From Beverly, Massachusetts).

  Vector construction. Construction of plasmid pdHL2-Rap-L-hLL1-γ4P for 2L-Rap-hLL1-γ4P expression is as described in Example 2 below. The expression vector pdHL2-Rap (Q) -L-hLL1-γ4P is derived from pdHL2-Rap-L-hLL1-γ4P by replacing Rap with Rap (Q), and a plasmid for expressing (Q) -hRS7 pdHL2-Rap (Q) L-hRS7-γ1 was constructed by subcloning the Rap (Q) gene derived from pdHL2-Rap (Q) -L-hLL1-γ4P into the pdHL2-hRS7-γ1 vector. Briefly, an EcoRV restriction site was introduced into the N-terminal (5 ') side of the hRS7 VL gene by PCR using suitable primers. The XbaI-EcoRV fragment of pdHL2-Rap (Q) -L-hLL1-γ4P containing the leader peptide-Rap-linker was ligated with the EcoRV-BamHI fragment containing the hRS7 VL gene generated by PCR and intermediate The vector was pBS-Rap (Q) -L-hRS7. The Xba-BamHI fragment of pdHL2-hRS7-γ1 was replaced with the Xba-BamHI fragment of pBS-Rap (Q) -L-hRS7.

  Transfection and selection. The pdHL2-Rap (Q) -L-hRS7-γ1 vector (30 μg) was linearized with SalI and transfected into Sp2 / 0 cells by electroporation, which was treated with 10% low IgG FBS, 100 units / mL penicillin, And 100 μg / mL streptomycin, 2 mM L-glutamine, 1 μM sodium pyruvate, 100 μM essential amino acid, and 0.05 μM methotrexate (MTX) in complete hybridoma serum-free medium. Culture supernatants from viable cell wells were analyzed for fusion protein expression by ELISA using HRP-conjugated GAH IgG Fc-specific antibodies. Positive clones were expanded and frozen for future use.

  Expression and purification. Cells were grown in rotating bottles for final culture (viability 10-20%). The supernatant was filtered and applied to a protein A column that had already been equilibrated with a pH 8.5 buffer containing 20 mM Tris-HCl and 100 mM NaCl. After loading, the column was washed with 100 mM sodium citrate buffer (pH 7.0) and eluted with 100 mM sodium citrate buffer (pH 3.5) to obtain a fusion protein. The peak containing the product was adjusted to pH 7.0 using 3M Tris-HCl, pH 8.5 and dialyzed against 40 mM phosphate buffered saline (PBS). After concentration, the product was filtered through a 0.22 μm filter and stored at 2-8 ° C.

  Size exclusion HPLC and SDS-PAGE analysis. The purity and molecular integrity of (Q) -hRS7 was determined by size exclusion HPLC using a Zorbax column purchased from BioRad (Hercules, CA) and 4-20% Tris-glycine gel (PAGEr®). GOLD precast gel (Cambrex, Rockland, Maine) was used and evaluated by SDS-PAGE under reducing conditions.

  In vitro transcription and translation (IVTT) assay. RNase activity was determined by measuring the activity of de novo synthesized luciferase using the TNT® Quick Coupled Transcription / Translation System (Promega, Madison, Wis.) According to the manufacturer's instructions. Determined in a cell-free system. Briefly, various test samples at concentrations ranging from 10 pM to 100 nM in 2 μL are added to 8 μL TNT® Quick Master Mix containing methionine and luciferase control DNA, and a 96-well round bottom plate is added. And then incubated at 30 ° C. for 2 hours, then 2 μL was removed and analyzed in a black 96 well flat bottom plate using 50 μL Bright-Glo substrate. Plates were measured with an Envision chemiluminescence reader. Relative luciferase units (RLU) were plotted against the concentration of the test sample.

  Yeast tRNA degradation assay. In addition, RNase activity was determined by measuring the amount of perchlorate-soluble nucleotide formed using yeast tRNA (Invitrogen) as a substrate (Newton et al, Blood 2001; 97: 528-35). . Each sample was placed in a final volume of 100 μL with 5 nM (Q) -hRS7 or rRap; 10 mM HEPES, pH 6.0; 200 μg / mL human serum albumin; and 100 μg / mL (3.09 μM) to 600 μg / mL (18. It was prepared in a 1.5 mL RNase-free Eppendorf tube using RNase-free water (Ambion, Austin, TX) to contain a predetermined concentration of tRNA in the range of 54 μM). The enzymatic reaction was performed at 37 ° C. for 2 hours and terminated by adding 233 μL of 3.4% ice-cold perchloric acid to each sample on ice. After 10 minutes, the sample was centrifuged at 12,000 rpm for 10 minutes using a microcentrifuge in a cold room. A certain amount was taken from the supernatant of each sample, diluted 10-fold with water, and its optical density (OD) at 260 nm was measured against water as a blank. The initial rate of each substrate concentration was calculated by dividing the corresponding OD value by the reaction time (7200 seconds) and plotted against the substrate concentration to determine kcat / Km. According to the Michaelis-Menten equation, under the condition of Km >> [S], the slope of the resulting least-squares line is the total enzyme concentration (5 nM for rRap, for (Q) -hRS7 Should be equal to 10 nM).

Cell binding measurement. Using an ELISA-based method, binding of (Q) -hRS7 to selected cell lines was evaluated as follows. Cells were seeded in black 96 well flat bottom plates (1 × 10 ⁵ cells / well; 100 μL / well) and incubated overnight at 37 ° C. in a 5% CO ₂ humidified incubator. The next day, the plates containing the cells were removed from the incubator and the media was removed from the wells by flicking and then tapped on a paper towel to dry. Each well was then filled with 50 μL of fresh growth medium. Serial serial 1: 4 dilutions of (Q) -hRS7 (200 nM to 1.9 × 10 ⁻⁴ nM) were made in assay medium (RPMI 1640; 10% FBS complete medium) and added in triplicate to the corresponding wells (50 μL). / Well) (final concentration 100 nM to 0.95 × 10 ⁻⁴ nM). After a 1.5 hour incubation at 4 ° C., the plate was centrifuged at 600 × g for 2 minutes, the medium removed and blotted dry with a paper towel and washed by adding 150 μL ice-cold medium to each well. And then centrifuged at 600 × g for 2 minutes. The medium was removed and the plates were blotted dry. HRP-conjugated GAH antibody was used at a 1: 20,000 dilution and then added to all wells (100 μL / well). For background controls, only a set of cells plus secondary antibody was placed in a set of wells. Plates were incubated for 1 hour at 4 ° C. The plates were then centrifuged and blot dried. Thereafter, the cells were washed twice with ice-cold medium, followed by a third wash with ice-cold PBS. Centrifugation, media removal, and plate blot procedures were repeated after each wash.

After the last wash step, LumiGLO (KPL, Gaithersburg, MD) was added to all wells (100 μL / well) and the plate luminescence was measured using an Envision plate reader. . Data was analyzed using GraphPad Prism software to determine the apparent affinity, the concentration corresponding to half-maximum saturation. In each experiment, hRS7 and hLL1 were included as positive and isotype controls, respectively. Alternatively, binding of (Q) -hRS7 to human cancer cell lines using Guava PCA (Guava Technologies, Inc., Hayward, Calif.) Using manufacturer's reagents, protocols, and software. Determined by cytometry. In parallel, similar studies were performed for each cell line using hRS7 and hLL1. Briefly, various lines to be analyzed of approximately 5 × 10 ⁵ cells were obtained and resuspended in PBS / 1% BSA (bovine serum albumin). Cells were centrifuged, resuspended in 100 μL PBS / 1% BSA containing 10 μg / mL (Q) -hRS7, hRS7, or hLL1 and incubated for 45 minutes at 4 ° C. After washing twice with PBS / 1% BSA and centrifuging after each wash, the cells were resuspended in 50 μL of FITC-conjugated GAH Fc specific antibody (1:25 dilution) and incubated at 4 ° C. for 30 minutes. . Cells were washed twice with PBS / 1% BSA, resuspended in 0.5 mL PBS / 1% BSA and then analyzed by flow cytometry. To separate dead and live cells, 1 μg / mL propidium iodide was added. 10,000 cells were acquired for each analysis.

Cell proliferation assay. Tumor cells were seeded in 96-well plates (1 × 10 ⁴ cells per well) and incubated with 0.01-100 nM test substance for 72 hours. Subsequently, a soluble tetrazolium salt, MTS [3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium] was obtained from the manufacturer. The number of viable cells was determined using according to the protocol. Data from the dose response curve was analyzed using GraphPad Prism software to obtain EC50 values (concentration at which 50% inhibition occurs).

Colony formation assay. Tumor cells were trypsinized and seeded in 60 mm dishes (1 × 10 ³ cells). Cells were treated with each test substance to form colonies. Fresh medium containing the test substance was added every 4 days and after 2 weeks of incubation, colonies were fixed with 4% formaldehyde and Giemsa stained. Colonies exceeding 50 cells were counted with a microscope.

  Internalization studies with a fluorescence microscope. ME-180 cells were seeded into 8-well Lab-Tek ™ II chamber slides (Nalge Nunc International, Naperville, IL) (2000 cells in 500 μL per well), (Q) -hRS7 (10 μg / mL) Alternatively, it was incubated with hRS7 (6 μg / mL) at 37 ° C. for 16 hours. All subsequent steps were performed at room temperature. After washing twice with PBS / 2% BSA or twice with PBS / 2% BSA and then twice with 0.1M glycine, pH 2.5 (500 μL, 2 minutes), the cells were treated with 4% Fix with formalin for 15 minutes, wash twice with PBS, then probe with mouse anti-Rap mAb, then probe with PE-conjugated GAM Fc-specific antibody or directly with FITC-conjugated GAH Fc-specific antibody ( Q) The location of -hRS7 or hRS7 was revealed using a fluorescence microscope.

  A second study addressing the intracellular location of (Q) -hRS7 was performed as follows. Alexa fluor 568-conjugated human transferrin (hTf) was added to MDA-MB-468 human breast cancer cells (3000 cells in 500 μL per well) seeded in 8-well chamber slides to (Q) -hRS7 (10 μg / mL) or hRS7 ( 6 μg / mL). After 2 hours incubation at 37 ° C., cells were washed and fixed as described above and then treated with Alexa Fluor 488-conjugated GAH IgG for 15 minutes at room temperature. After washing twice with PBS, the cells were treated with Hoechst 33258 for 15 minutes at room temperature, washed and examined with a fluorescence microscope.

  In vivo toxicity. Naive BALB / c mice (female, 7 weeks old, Taconic Farms, Germantown, NY) were intravenously administered various doses of (Q) -hRS7 ranging from 25-400 μg per mouse. Injections were monitored daily for visible toxicity signs and body weight changes. The maximum tolerated dose (MTD) was defined as the maximum dose at which death did not occur and weight loss was 20% or less of pre-treatment animal body weight (approximately 20 g). Animals that experienced toxic effects were euthanized.

Therapeutic efficacy in tumor-bearing mice. Approximately 20 g of female NCr homozygous athymic nu / nu mice (5 weeks old when received from Taconic Farms) were inoculated subcutaneously with 1 × 10 ⁷ Calu-3 human NSCLC cells and the length of the tumor X Tumor growth was monitored by measuring caliper width. Tumor volume was calculated as (L × W2) / 2. When tumors reached a size of approximately 0.15 cm ³ , animals were divided into 5 treatment groups per group. Treatment consisted of either a single intravenous injection of 50 μg (Q) -hRS7 or two injections of 25 μg administered at 7 day intervals. The control group received saline. Animals were monitored daily for signs of toxicity and were considered humanely euthanized and died due to disease progression if the tumor reached a size greater than 2.0 cm ³ or became ulcerated. In addition, mice were euthanized if they lost more than 20% of their initial body weight or were otherwise moribund. Survival data were analyzed using Kaplan-Meier plots (log rank analysis) using GraphPad Prism software. Differences were considered statistically significant when P <0.05.

Results Purity and molecular integrity. (Q) -hRS7 was shown by size exclusion HPLC to be composed of a single peak (not shown) whose observed residence time (7.8 minutes) showed a larger molecular size than IgG. The purity of (Q) -hRS7 is only about two bands in reduced SDS-PAGE, one is about 50 kDa attributed to the heavy chain of hRS7, and the other is about 37 KDa attributed to the Rap (Q) fusion light chain, Was supported by the observation (not shown).

Combined analysis. The reactivity of the Trop-2-expressing cell line with (Q) -hRS7 was first evaluated by ELISA, and in the case of PC-3 (FIG. 2A) and Calu-3 (FIG. 2B), both were approximately twice that of hRS7. It has been demonstrated to yield a high apparent dissociation constant (K _D ) (0.28 nM vs. 0.14 nM). In the case of Trop-2 negative 22Rv1, no binding was observed (FIG. 2C). Subsequently, flow cytometry studies were performed on a total of 10 Trop-2 expressing cell lines. The result (not shown) shows that there was virtually no difference between the binding characteristics of (Q) -hRS7 and hRS7.

RNase activity. In the IVTT assay, inhibition of protein synthesis due to mRNA degradation by RNase is measured. As shown in FIG. 3A, (Q) -hRS7 and rRap show comparable RNase activity in this cell-free assay, but no enzyme activity was observed with hRS7. Using yeast tRNA as a substrate, we found that kcat / Km (10 ⁹ M ⁻¹ s ⁻¹ ) of rRap and (Q) -hRS7 was 4.10 (± 0.42) and 1 respectively. .98. Thus, the catalytic efficiency of (Q) -hRS7 based on the concentration of Rap is about 50% of rRap, similar to the report that the catalytic efficiency of LL2-onconase is 40% compared to native Rap. (Newton et al., Blood 2001; 97: 528-35). A plot of initial rate versus tRNA concentration from a representative set of experiments is shown in FIG. 3B.

  In vitro cytotoxicity. Based on the results of the MTS assay, (Q) -hRS7 is most potent against ME-180 (FIG. 4A), T-47D (FIG. 4B), MDA-MB-468, and Calu-3, with EC50 The values are 1.5, 2.0, 3.8, and 8.5 nM, respectively. For cell lines that show less than 50% growth inhibition with 100 nM (Q) -hRS7 in the MTS assay, we also performed a colony formation assay where (Q) -hRS7 was 10 or 100 nM and DU -145, PC-3, MCF7, SK-BR-3, BxPC-3, Capan-1, and SK-OV-3 were confirmed to be cytotoxic (not shown). Representative results are shown for DU-145 (FIG. 4C) and PC-3 (FIG. 4D). In both assays, hRS7, rRap, and the combination of hRS7 and rRap showed very little, if any, toxicity at 100 nM in all cell lines evaluated. Trop-2 negative AsPC-1 was resistant to (Q) -hRS7 in both assays.

  Internalization and intracellular location. Internalization of (Q) -hRS7 into ME-180 cells was achieved for samples fixed after washing with PBS / 0.2% BSA or low pH glycine buffer to remove membrane bound proteins. It was clearly obvious (hidden). When the distribution pattern of intracellular (Q) -hRS7 in ME-180 was detected with mouse anti-Rap IgG directly by FITC-conjugated GAH or indirectly by PE-conjugated GAM, the distribution pattern was almost the same. It was suggested that (Q) -hRS7 remained intact after entering these cells (not shown). The intracellular location of (Q) -hRS7 was further explored in MDA-MB-468 cells using fluorescently labeled hTf as a marker for recycled endosomes and Hoechst 33258 staining nuclei. It was clear from these results that (Q) -hRS7 and hTf occupy the same intracellular location as MDA-MB-468 when examined after incubation at 37 ° C. for 2 hours (not shown). In both cell lines, hRS7 showed internalization characteristics similar to (Q) -hRS7 except that it was not visualized by PE-GAM / anti-Rap as expected (data not shown).

  MTD in mice. We determined the MTD of (Q) -hRS7 in normal BALB / c mice given a single intravenous injection of 50 μg-100 μg. Other 2L-Rap-X fusion proteins or 2L-Rap (Q) -X fusion proteins that have been produced to date exhibit similar MTD ranges. In addition, we provided (Q) -hRS7 MTD when injected multiple times in naïve SCID mice to give 80 μg (q5d × 4) by giving 20 μg four times every 5 days. It was determined.

  Therapeutic efficacy in tumor-bearing mice. As shown in FIG. 5A, treatment with (Q) -hRS7 (either a single dose of 50 μg or two doses of 25 μg given at 5-day intervals) compared to an untreated control Significantly inhibited the growth of Calu-3 xenografts (P <0.019) and the median survival increased from 55 days to 96 days (P <0.01; FIG. 5B).

DISCUSSION Clinical trials for cancer treatment have been completed or many are underway (Pastan et al., Nat Rev Cancer 2006; 6: 559-65; Pastan
et al., Annu Rev Med 2007; 58: 221-37; Kreitman, BioDrugs
2009; 23: 1-13) compared to immunotoxins made with plant or bacterial toxins (Kreitman, AAPS)
J 2006; 8: E532-51), an antibody-targeted RNase referred to as immune RNase (ImmunoRNase) (De Lorenzo et al., FEBS Lett 2002; 516: 208-12; Cancer Res
2004; 64: 4870-4) has relatively few advantages, mostly developed for the treatment of hematological malignancies, and the targeting component is conferred by several forms of scFv ( Schirrmann et al., Exp Opin Biol Ther 2009; 9: 79-95). To date, immune RNase has never been evaluated in patients with cancer.

  Two obstacles that have been shown in clinical development of other plant or microbial immunotoxins are undesirable toxicity and immunogenicity. Normal tissue toxicity observed with these immunotoxins includes vascular leakage syndrome (VLS), hemolytic uremic syndrome (HUS), and hepatotoxicity (Kreitman, BioDrugs 2009; 23: 1-13). The structural motif (x) D (y) identified to be involved in the binding of ricin A chain or IL-2 to endothelial cells is not present in the native sequence of Rap (Q) and hRS7 is a human endothelial cell Does not cross-react with. Therefore, the present inventors consider that (Q) -hRS7 is unlikely to cause VLS. The large size of (Q) -hRS7 (approximately 180 kDa) raises the potential concern that tumor penetration is not so rapid (Yokota et al., Cancer Res 1992; 52: 3402-8), but the kidney Should prevent that clearance and mitigate the risk of HUS. Regarding hepatotoxicity, we note the following: BL22, a recombinant anti-CD22 immunotoxin composed of a disulfide stabilized Fv of RFB4 fused to PE38, and LMB-2 (anti-Tac (Fv Similar immunotoxins such as) -PE38) showed very low MTD in mice due to nonspecific hepatotoxicity, but BL22 is safe in clinical trials of patients with hairy cell leukemia It has been reported to be effective (Kreitman et al., N Engl J Med 2001; 345: 241-7). Therefore, the dose-limiting hepatotoxicity commonly observed in mice may rarely appear in humans (Kreitman, BioDrugs 2009; 23: 1-13). On the other hand, immunogenicity is a more common problem. Most genetically engineered immunotoxins that are being evaluated in cancer patients induce a strong humoral immune response, thereby reducing serum half-life and preventing further administration. Several approaches to reduce the immune response have been tested in laboratory animals, deoxyspargarine (Pai et al., Cancer Res 1990; 50: 7750-3) and CTLA4Ig (Sieall et al., J Immunol 1997; 159: 5168-73) and several clinical trials of these and other immunosuppressive agents in combination with immunotoxins have been proposed (Frankel, Clin Cancer Res 2004; 10: 13-5 ). (Q) -hRS7 may not be very immunogenic because it involves fusing a humanized antibody to a toxin that is thought to induce little antibody response in the patient (Mikulski et al., J Clin Oncol 2002; 20: 274-81).

The cytotoxicity of the immunotoxin requires that it enters the target cell and then moves to the cytosol. The intracellular pathway after internalization is determined by rampyrnase (Rodriguez et al., J Cell Sci 2007; 120: 1405-11; Haigis and Raines, J
Cell Sci 2003; 116: 313-24; Wu et al., J Biol Chem 1995; 270: 17476-81) and other RNases (Wu et al., J Biol Chem 1995; 270: 17476-81; Leich et al ., J Biol Chem
2007; 282: 27640-6; Bracale et al., Biochem J 2002; 362: 553-60), as well as human anti-ErbB2 scFv ((De Lorenzo et al., Cancer Res 2004; 64: 4870-4; FEBS Lett
2007; 581: 296-300) or an immune RNase comprising human pancreatic RNase fused to either human anti-CD30 scFv-Fc (Menzel et al., Blood 2008; 111: 3830-7). Still not fully understood. Our internalization experiment showed that (Q) -hRS7 was co-localized with hTf when examined 2 hours after addition to MDA-MB-468, and was proposed for lampyrnase. (Haigis and Raines, J Cell Sci 2003; 116: 313-24), suggesting that (Q) -hRS7 can exit the endosome and enter the cytosol directly. The close similarity of the fluorescence images observed with ME-180 for intracellular (Q) -hRS7 between anti-Rap and anti-human Fc is resistant to degradation by proteases during the endocytosis process. The indicated (Q) -hRS7 ability is further suggested.

The in vitro potency of (Q) -hRS7 was found to vary depending on the Trop-2-expressing cell line, as measured by a 3-day MTS assay, which is partly different in the intracellular pathway. The cytotoxicity of (Q) -hRS7 was clearly demonstrated at 10 nM for all cell lines using a 14-day colony formation assay. In addition to its potent cytotoxicity in vitro against a variety of cancer cell lines, (Q) -hRS7 has been shown to be effective in inhibiting the growth of Calu-3 human lung cancer xenografts in nude mice. Therefore, the antitumor activity and stability of (Q) -hRS7 in vivo is verified and it is appropriate to add Trop-2 to the current list of solid tumor antigens targeted by immunotoxins Was confirmed (Kreitman, AAPS J
2006; 8: E532-51; Pastan et al., Nat Rev Cancer 2006;
Pastan et al., Annu Rev Med 2007; 58: 221-37; Schirrmann
et al., Exp Opin Biol Ther 2009; 9: 79-95).

  In conclusion, we show that amphibian RNase recombinantly fused to humanized anti-Trop-2 antibody exhibits selective and potent cytotoxicity against various epithelial cancers both in vitro and in vivo. Proved that.

Example 2.2 Expression and Characterization of L-rap-hLL1-γ4P As used below, rap stands for lampirnase.

Construction of pdHL-IgG4P variants:
B13-24 cells containing the IgG4 gene were purchased from ATCC (ATCC number CRL-11397) and genomic DNA was isolated. Cells were washed with PBS, resuspended in digestion buffer (100 mM NaCl, 10 mM Tris-HCl pH 8.0, 25 mM EDTA, 0.5% SDS, 0.1 mg / ml proteinase K) and incubated at 50 ° C. for 18 hours. did. Samples were extracted with an equal volume of phenol / chloroform / isoamyl alcohol and precipitated with 7.5M NH ₄ Ac / 100% EtOH. Genomic DNA was collected by centrifugation and dissolved in TE buffer. The IgG4 gene was amplified by PCR using genomic DNA as a template.

  The amplified PCR product was cloned into a TOPO-TA sequencing vector (Invitrogen) and confirmed by DNA sequencing. Replacing the SacII-EagI fragment containing the heavy chain constant region of IgG1 in pdHL-hLL2 with SacII-EagI of the TOPO-TA-IgG4 plasmid to generate the pdHL2-hLL2-IgG4 (pdHL2-hLL2-γ4) vector did.

In order to avoid the formation of an IgG ₄ -proline mutant half molecule, a Ser228Pro mutation was introduced into the hinge region of IgG4. Mutations hinge region 56bp fragment (PstI-StuI) were synthesized, annealed, and replaced with PstI-StuI fragment of IgG _4. This construction resulted in the final vector pdHL2-hLL2-γ4P.

Construction of pdHL2-hLL1-γ4P The XbaI-HindIII fragment of pdHL2-hLL2-γ4P was replaced with the Xba-HindIII fragment of pdHL2-hLL1 containing the Vk and VH regions to generate the hLL1-γ4P construct.

Construction of pdHL2-2L-rap-hLL1-γ4P A flexible linker containing three copies of a monomer of glycine 4-serine 1 was used to attach the C-terminus of Rap to the N-terminus of hLL1 Vk. . One rap molecule was attached to the N-terminus of each light chain. The DNA of this molecule was constructed by PCR. The Xba-BamHI fragment of pdHL2-hLL1-γ4P is replaced with the Xba-BamHI (Xba-leader-rap-linker-Vk-BamHI) fragment of pBS-2L-rap-hLL1 to give the final vector pdHL2-2L-rap- hLL1-γ4P was completed.

Transfection Vector DNA (30 μg) was linearized with SalI enzyme and electroporated (450 V) by NSO (4 × 10 ⁶ cells / mL) or Sp2 / 0-Ag14 (5 × 10 ⁶ cells / mL) myeloma cells. Was transfected. Low IgG FBS (10%), penicillin (100 units / mL), streptomycin (100 μg / mL), L-glutamine (2 mM), sodium pyruvate (1 mM), non-essential amino acids (100 μM), and methotrexate (0.1 μM) The cells were grown in complete hybridoma SFM medium supplemented with Positive clones were screened by ELISA. Briefly, plates were coated with 5 μg / mL 50 μl anti-rap antibody in PBS medium and incubated overnight at 4 ° C. The plate was washed with PBS and blocked with 2% BSA, and then cell culture supernatant was added. HRP-conjugated goat anti-human IgG. sub. Four antibodies were used for detection and OPD was used as a substrate for color development. Plates were measured at 490 nm. Positive clones were expanded and frozen for future use. Clone C6 was identified as the best producer and was used for further development.

Expressed and purified cells were grown in two rotating bottles each with 500 ml of media to final culture (10-20% viability) and the cells were removed by centrifugation. The culture supernatant was filtered and applied to a protein A column equilibrated with 20 mM Tris-HCl / 100 mM NaCl buffer (pH 8.5). After loading, the column was washed with 100 mM sodium citrate buffer (pH 7.0) and eluted with 100 mM sodium citrate buffer (pH 3.5) to obtain a fusion protein. The peak containing the product was adjusted to pH 7.0 using 3M Tris-HCl, pH 8.0 and dialyzed against 10 mM PBS buffer. After concentration, the product was filtered through a 0.22 μm filter and stored at 2-8 ° C. 16 mg was recovered from 1 L culture after purification.

Characterization of 2L-rap-hLL1-γ4P HPLC: Protein purity and concentration were investigated by HPLC. A sharp single peak was observed at 7.7 minutes (not shown) and this residence time indicated that this molecule was larger than IgG.

  SDS-PAGE: SDS-PAGE was performed under reducing conditions using 4-20% Tris-glycine gel. Bands associated with the expected heavy chain of about 50 kD and two bands of molecular weights of about 37 and 39 kD, both larger than the light chain of hLL1 (about 25 kD), were observed (not shown). The presence of two light chains has been shown to be due to rap glycosylation of the fusion protein (see below).

  Mass spectrometry: Mass spectrometry was performed at the Scriptus Laboratories, California by the MALDI-TOF method. Two samples were sent for analysis. One was in the native state (1.6 mg / mL in 10 mM PBS) and the other was in the reduced state (1.6 mg / mL in 1 mM HEPES / 10 mM DTT, pH 7.5 buffer). The natural sample shows one major peak with a mass of 177150, in good agreement with the MW of one IgG + 2 raps (not shown). The reduced sample showed three major peaks of 50560 (corresponding to heavy chain), 38526, and 36700 (corresponding to two light chains containing rap) (not shown).

  Western blotting: Western blotting was performed to confirm the presence of rap in the purified protein. Samples from SDS-PAGE gels under reducing conditions were electrically transferred to PVDF membrane. After blocking with 5% BSA, mouse anti-rap antibody was added at 1: 10,000 dilution or 10 ng / ml and incubated for 1 hour. After washing, HRP-conjugated goat anti-mouse Fc antibody was added and incubated for 1 hour. After 6 washes, LumiGlo ™ (from Kirkegaard & Perry Laboratories) substrate was added and the Kodak film was developed. Both bands corresponding to the fused light chain were detected in the film, confirming the presence of rap in both light chains (not shown).

  Treatment with N-glycosidase: Because rap has potential N-glycosylation sites, Asn-X-Thr / Ser, Asn69-Val70-Thr71, the observation that the two light chains have molecular weights different by 2 kD is It is possible that rap is the result of uneven glycosylation. To investigate this possibility, the rap-hLL1 antibody was incubated with N-glycosidase (New England Biolabs) under denaturing conditions according to the supplier's recommendations. After N-glycosidase treatment, the two bands corresponding to the two light chains converge to one (the band that migrates more rapidly), and thus the unequal distribution of sugar chains results in two bands on SDS-PAGE. It was confirmed that the reason was observed (hidden). Further support is provided by the fact that only one Rap fusion light chain was observed when Rap (N69Q), a Rap variant with the glycosylation site removed, was used in place of the rap of the recombinant construct. (Data not shown).

Activity of rap: RNase activity was tested by TNT® Quick Coupled Translation / Translation System (Promega) using the Bright-Glo ™ Luciferase Reporter Assay System (Promega) according to the supplier's recommendations. did. The principle of this assay was to measure protein synthesis inhibition (mRNA degradation) as a result of RNase activity using a luciferase reporter system. Samples were chemistry of hLL2-rap expressed as different dilutions, free rap (0.001-2.5 nM), hLL1-rap (0.01-20 nM), or PK1-LL2-One and PKII-LL2-One. Prepared with conjugate (0.01-20 nM). Each sample (5 μL) was mixed with 20 μl TNT master mix and incubated in a 96-well plate for 2 hours at 30 ° C., then 1 μl was removed and analyzed with 50 μl Bright-Glo ™ substrate. Results are shown in FIG. 6 using Excel or Prism Pad software. The EC ₅₀ value was about 300 pM for the rap-hLL1 and hLL2-One chemical conjugates and 30 pM for the free rap.

WP competitive binding. WP is an anti-idiotype antibody of hLL1. The affinity of the rap-hLL1 antibody compared to the hLL1 antibody for WP was assessed by a competitive binding assay. Briefly, 96 well plates were coated with 50 μl of 5 μg / mL WP and incubated overnight at 4 ° C. Three different types of protein samples, hLL1, rap-hLL1, or hA20 mixed with an equal volume of 2 × HRP-conjugated mLL1 antibody (final dilution is 1/20000) (final concentration is In the range of 0.49 to 1000 nM). 50 μL of protein sample mixed with HRP-conjugated mLL1 as described above was added to each well and incubated for 1 hour. After washing, OPD substrate containing H ₂ O ₂ was added and the plate was measured at 490 nm. Protein concentration versus absorbance was plotted using Excel or Prism Pad graph software as shown in FIG. hA20 (humanized anti-CD20 antibody) was used as a negative control. From FIG. 7, it is clear that rap-hLL1 shows similar binding affinity as hLL1, and the negative control, hA20, shows no affinity. Similar results were obtained using Raji cells as a source of antigen.

In vitro cytotoxicity. In vitro cytotoxicity was determined in B cell lymphoma cell lines (Daudi) and multiple myeloma cell lines (MC / CAR). Cells (10,000 in 0.1 ml) were seeded in each well of a 96 well plate. After 24 hours, free hLL1, free rap, or rap-hLL1 (10 μl) was added to the appropriate wells and the cells were incubated in an incubator for 3 days at 37 ° C. Cell proliferation was determined using MTS tetrazolium dye reduction assay or BrDU colorimetric assay. Results were expressed as EC ₅₀ obtained graphically using Prism Pad software. It is clear from FIGS. 8-9 that rap-hLL1 was sensitive to both the B cell lymphoma cell line (Daudi) and the multiple myeloma cell line (MC / CAR). rap-hLL1 was significantly more potent (cytotoxic) on Daudi cells compared to MC / CAR cells as reflected in EC ₅₀ values (FIGS. 8 and 9). In the case of MC / CAR cells, EC ₅₀ values were not achieved at the concentrations tested. At the highest concentration (56 nM), cell viability was 57%. hLL1 or free rap itself did not show cytotoxicity in any cell line.

Pharmacokinetics and biodistribution methods. hLL1 or 2L-Rap-hLL1-γ4P was prepared as described in Sharkey et al. (Int J Cancer. 1990; 46: 79-85) using 2- (4-isothiocyanatobezyl) DTPA (Macrocyclics, Dallas, Texas) using conjugated to diethylenetriaminepentaacetic acid (DTPA) to give DTPA-hLL1 or DTPA-2L-Rap-hLL1-γ4P, which was used to study pharmacokinetics and biodistribution, respectively. Labeled with ⁸⁸ Y chloride (Los Alamos National Laboratory (Los Alamos, New Mexico)) or ¹¹¹ In chloride (Perkin Elmer Life Sciences, Boston, Mass.) Naive female SCID mice (8 weeks old, 18-22 g) Supplemented with unlabeled DTPA conjugate of hLL1 or 2L-Rap-hLL1-γ4P Has been a mixture of ^{111 In-DTPA-2L-Rap} -hLL1-γ4P of ⁸⁸ Y-DTPA-hLL1 and 0.02mCi of 0.001MCi, each animal of the total dose of 10μg each hLL1 and 2L-Rap-hLL1 Intravenous injection to receive γ4P At selected time points after administration (1, 2, 4, 16, 48, 72, 168 hours), groups of 5 mice were anesthetized and blood samples were was collected by cardiac puncture. removed major tissue, weighed, was placed in a container. blood samples and tissue ¹¹¹ an in (channels 120-480), and ⁸⁸ Y (channels 600 to 2000), counted in a calibrated gamma counter A cross curve was generated to correct the backscattering of ⁸⁸ Y energy and fit to the ¹¹¹ In counting window.

  In vivo toxicity. Naive SCID or BALB / c mice were intravenously injected with various doses of 2L-Rap-hLL1-γ4P ranging from 25-400 μg / mouse and monitored daily for visible signs of toxicity and weight loss. The maximum tolerated dose (MTD) was defined as the maximum dose at which no death occurred and the weight loss was less than 20% of the pre-treatment animal weight (approximately 20 g). Animals that experienced toxic effects were sacrificed, collected, and subjected to histopathological analysis. In naive SCID mice, a single intravenous dose of 100, 150, 200, 250, 300, or 400 μg of 2L-Rap-hLL1-γ4P resulted in severe animal weight loss and death, although 25 Or, at a dose of 50 μg, all mice survived (not shown). In the case of BALB / c mice, all mice survived with a single intravenous administration of 30 or 50 μg of 2L-Rap-hLL1-γ4P, but did not survive at 100 or 200 μg (not shown). In another experiment, administration of 75 μg of 2L-Rap-hLL1-γ4P was found to be toxic to SCID mice (data not shown). Therefore, the MTD of 2L-Rap-hLL1-γ4P given as a single bolus injection is 50-75 μg in SCID mice and 50-100 μg in BALB / c mice. Gross pathological examination of dead or sacrificed mice showed severe hepatotoxicity and splenic toxicity. The liver was pale white and the spleen was wrinkled and smaller than normal size. Histopathological examination revealed hepatic necrosis and splenic necrosis. Representative mouse serum samples showed elevated levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin, suggesting that these high doses are significantly hepatotoxic.

Data analysis. For in vitro cytotoxicity studies, dose response curves were generated from triplicate averages and using 50% inhibition concentration (IC ₅₀ ) values using GraphPad Prism software (Advanced Graphics Software, Encinitas, Calif.). Obtained. Pharmacokinetic data was analyzed using the standard algorithm of the non-compartmental analysis program WinNonlin, version 4.1 (Pharsight, Mountain View, CA). This program calculates the area under the curve (AUC) using the linear trapezoidal method with linear interpolation. The disappearance rate constant (k _β ) was calculated from the terminal phase half-life (t _{1 / 2β} ) assuming first order kinetics. Survival data was analyzed with GraphPad Prism software using Kaplan-Meier plot (log rank analysis). Differences were considered statistically significant when P <0.05.

Pharmacokinetic and biodistribution data. The pharmacokinetics and biodistribution of radioisotope labeled hLL1 and 2L-Rap-hLL1-γ4P were determined in naïve SCID mice. hLL1 and 2L-Rap-hLL1-γ4P were conjugated with DTPA and the ⁸⁸ Y and ¹¹¹ In labels were followed, respectively. As shown in FIG. 10, ¹¹¹ In-labeled 2L-Rap-hLL1-γ4P is characterized by ⁸⁸ Y-labeled, characterized by an early rapid redistribution (α) phase and a later slower disappearance (β) phase. Biphasic blood clearance similar to hLL1 is shown. A slightly shorter half-life was observed with 2L-Rap-hLL1-γ4P (5.1 hours) compared to hLL1 (4 hours). Data points over 5 hours were used to calculate t _{1 / 2β} , k _β , AUC, mean residence time (MRT), apparent volume of distribution (V _d ), and clearance rate (Cl). The values for these parameters are shown in Table 2. Incorporation of ¹¹¹ In-labeled 2L-Rap-hLL1-γ4P into tissues was similar to that of ⁸⁸ Y-labeled hLL1 (data not shown).

Therapeutic efficacy in tumor-bearing mice Therapeutic efficacy in tumor-bearing mice: Female SCID mice (8 weeks old, 18-22 g), 8-9 mice per group were intravenously injected with 1.5 × 10 ⁷ Daudi cells Treated one day later. Mice were examined daily for hindlimb paralysis and weighed once a week. Animals were euthanized when they developed hind limb paralysis or when 20% of their pre-treatment weight was lost. Each set of treatment experiments was terminated after 180 days.

  As shown in FIG. 11, all untreated mice (PBS group) died within 30 days and the median survival (MST) was 28 days. The control group that received 50 μg of a mixture of hLL1-γ4P (43.2 μg) and Rap (6.6 μg), which is a component protein composition of 2L-Rap-hLL1-γ4P, had a MST of 70 days (with PBS group) Compared to P <0.0001). In contrast, all mice that received a single injection of either 5 or 15 μg of 2L-Rap-hLL1-γ4P survived more than 100 days (MST> 180 days; P compared to the component treatment group). = 0.0005) Only one mouse was lost from each group at the end of the study. When the study ended 180 days, 90% of the mice that received a single injection of 5, 15, 30, 40, or 50 μg of 2L-Rap-hLL1-γ4P had been cured. Of note is that the MST of mice receiving a single injection of 1 μg was 92 days, showing an increase of 230% compared to 28 days in the untreated group (P <0.0001). .

Synthesis of PCR Amplified DNA Encoding Cytotoxic RNase 139-mer DNA nucleotides of the sense strand sequence encoding the N-terminal sequence (46 amino acids) of recombinant cytotoxic RNase, ONCO--N:
5'-TGG CTA ACG TTT CAG AAG AAA ACAT CAT ATC ACG AAT ACA CGA GAT GTA GAC TGG GAC AAT ATA ATG TCT ACG AAT CTG TTT CAC TGT AAG -3 ′ (SEQ ID NO: 88)
Was synthesized with an automatic DNA synthesizer and used as a template for PCR amplification using the following primers.
ONNBACK
5′-AAG CTT CAT ATG CAG GAT TGG CTA ACG TTT CAG AAG AAA-3 ′ (SEQ ID NO: 89)
ONNFOR
5′-CTT ACT CGC GAT AAT GCC TTT ACA GAT AGC CTT TAC AGG CTC TG-3 ′ (SEQ ID NO: 90)

  The resulting double stranded PCR product contains a cDNA sequence encoding the 54 amino acid residues of the N-terminal half of cytotoxic RNase. ONNBACK contains restriction sites HindIII and NdeI to facilitate either subcloning into a staging vector or in-frame ligation (NdeI site) into a bacterial expression vector. A NruI site is incorporated into the ONNFOR primer to facilitate in-frame ligation with the cDNA encoding the C-terminal half of cytotoxic RNase.

Similarly, a 137-mer DNA nucleotide of the sense strand sequence encoding the C-terminal sequence (46 amino acids) of recombinant cytotoxic RNase, ONCO--C:
5'-TGC TGA CTA CTT CCG AGT TCT ATC TGT CCG ATT GCA ATG TGA CTT CAC GGC CCT GCA AAT ATA AGC TGA AGA AAA GCA CTA ACA AAT TTT GCG TAA CTT GCG AGA ACC AGG CTC CTG TAC ATT TCG TTG GAG TCG GG- 3 ′ (SEQ ID NO: 91)
Was synthesized and PCR amplified with the following primers.
ONCBACK
5′-ATT ATC GCG AGT AAG AAC GTG CTG ACT ACT TCC GAG TTC TAT-3 ′ (SEQ ID NO: 92)
ONCFOR
5′-TTA GGA TCC TTA GCA GCT CCC GAC TCC AAC GAA ATG TAC-3 ′ (SEQ ID NO: 93)

  The final double-stranded PCR product contained a cDNA sequence encoding 51 amino acids of the remaining C-terminal half of cytotoxic RNase. An NruI site was incorporated into ONCBACK that allowed in-frame ligation with the N-terminal half of the PCR amplified DNA. A stop codon and BamHI restriction site for subcloning into staging or expression vectors were included in the ONCFOR sequence.

  PCR amplified DNA encoding the N-terminal half and C-terminal half of cytotoxic RNase was treated with appropriate restriction enzymes, ligated at the NruI site, and subcloned into a staging vector, eg, pBluescript from Stratagene. The ligated sequence encodes a 105 amino acid polypeptide with an N-terminal Met.

Cloning of LL2 and MN-14 V region sequences and humanization of LL2 and MN-14 The V region sequences of hLL2 and hMN-14 have been published. Leung et al., Mol.
Immunol., 32: 1413 (1995); US
Pat. No. 5,874,540. The VK and VH sequences of LL2 and MN-14 were PCR amplified using published methods and primers. Sequence analysis of the PCR amplified DNA showed that they encoded proteins typical of antibody VK and VH domains. Chimeric antibodies constructed based on the PCR amplified LL2 and MN-14 sequences showed immunoreactivity equivalent to their parent antibodies, confirming the reliability of the resulting sequences.

  Sequence analysis of the LL2 antibody revealed the presence of an N-linked glycosylation site added to the framework 1 region VK. Mutational studies have shown that glycosylation at sites added to VK is not necessary to maintain antibody immunoreactivity (not shown). The REI framework sequence, which does not contain the FR-1 glycosylation site, was used as a scaffold for grafting light chain CDRs and as EU / NEWM for grafting LL2 heavy chain CDRs. The immunoreactivity of humanized LL2 (hLL2) was shown to be equivalent to that of mouse and chimeric LL2 (not shown). The internalization rate of LL2 was not affected by antibody chimerization or humanization (not shown).

Construction of Gene Encoding Humanized LL2 and Cytotoxic RNase Fusion Protein The VLL and VK sequences of hLL2 were used as a template to construct the hLL2-scFv gene by standard PCR procedures. -The Met start codon at position 1 was incorporated into the N-terminus of the VL gene that had been linked to the VH domain via a 16 amino acid linker. To facilitate purification of the fusion protein by metal chelate chromatography, a tail composed of 6 histidyl residues was included at the carboxyl terminus of the VH chain.

The lumpirnase-hLL2scFv immunotoxin fusion protein gene was constructed in a similar manner by restriction digestion and ligation methods. When expressed, the cDNA sequence encoded a fusion protein with lampirnase linked via a short linker to the N-terminus of the LL2 VL sequence. There are various linkers that can be inserted between the cytotoxic RNase C-terminus and the VL domain N-terminus. A preferred linker is the amino acid sequence TRHRQPRGW (SEQ ID NO: 94) derived from the C-terminal positions 273 to 281 of Pseudomonas exotoxin (PE). This sequence has been shown to be a recognition site for subtilisin to intracellularly cleave PE into active fragments, with cleavage occurring between the G and W residues of this sequence.
Chiron et al., J. Biol. Chem., 269: 18167
(1994). Incorporation of this sequence facilitates release of active cytotoxic RNase after the fusion immunotoxin is internalized. Alternatively, a 13 amino acid residue spacer composed of amino acid residues 48-60 of the B fragment of staphylococcal prontain A used in the construction of the EDN-scFv fusion was used instead to form cytotoxic RNase and Flexible coupling with scFv can be allowed. Tai et al., Biochemistry, 29: 8024 (1990) and Rybak et al., Tumor
Targeting, 1: 141 (1995).

Construction of Gene Encoding Humanized MN-14 and Lampyrinase Fusion Protein MN-14 scFv was generated by PCR amplification of cDNA derived from a humanized MN-14 transfectoma. The linker used for MN-14scFv was a 15 amino acid linker and the orientation was VL-linker VH. After confirming the DNA sequence, the single-stranded construct was subcloned into a eukaryotic expression vector and transfected into a suitable mammalian host cell for expression.

  Another single chain construct was also made. This was constructed with the opposite 5'-3 'heavy and light chain orientations, constructed in pCANTABE5E (Pharmacia Biotech, Piscataway, NJ) and expressed in phage. Specific binding of recombinant phage expressing this scFv was demonstrated by ELISA (not shown).

  The scFv sequence was used to construct a lumpirnase-MN-14 fusion protein in which lumpirnase was attached to the N-terminus of the VL sequence via a linker. A DNA fragment encoding lumpirnase was obtained as discussed above. A 23 amino acid linker was used between the lumpirnase sequence and the scFv. Kurucz et al. (1995).

Dock-and-lock Example 3 Basic Strategy for Producing Modular Fab Subunits Fab modules can be produced as fusion proteins containing either DDD or AD sequences. Independent transgenic cell lines were developed for each fusion protein. Once produced, the module may be purified as necessary or maintained in cell culture supernatant. Once produced, any DDD ₂ module can be combined with any AD module to generate a DNL construct.

The plasmid vector pdHL2 has been used to generate a large number of antibodies and antibody-based constructs. See Gillies et al., J Immunol Methods (1989), 125: 191-202; Losman et al., Cancer (Phila) (1997), 80: 2660-6. Bicistronic mammalian expression vectors direct the synthesis of IgG heavy and light chains. The vector sequence is almost identical to many different IgG-pdHL2 constructs, the only difference being in the variable domain (VH and VL) sequences. These IgG expression vectors can be converted to Fab-DDD or Fab-AD expression vectors using molecular biology tools known to those skilled in the art. To generate an expression vector for Fab-DDD, the coding sequence of the heavy chain hinge, CH2, and CH3 domains is the first 4 residues of the hinge, the 14 residue Gly-Ser linker, and the first of human RIIα. Of 44 residues (referred to as DDD1). To generate a Fab-AD expression vector, the sequence of the IgG hinge, CH2, and CH3 domains, the first 4 residues of the hinge, the 15 residue Gly-Ser linker, and bioinformatics and peptide array technology A sequence encoding a 17-residue synthetic AD called AKAP-IS (called AD1) that was generated using and shown to bind the RIIα dimer with very high affinity (0.4 nM). Replace with Alto,
et al. Proc. Natl. Acad. Sci., USA (2003), 100: 4445-50.

  In order to facilitate conversion of the IgG-pdHL2 vector into either a Fab-DDD1 or Fab-AD1 expression vector, two shuttle vectors were designed as described below.

Preparation of CH1 The CH1 domain was amplified by PCR using the pdHL2 plasmid vector as a template. The left PCR primer is composed of an upstream (5 ′) CH1 domain and a SacII restriction endonuclease site 5 ′ of the CH1 coding sequence. The right primer consists of a sequence encoding the first 4 residues of the hinge followed by a short linker, with the last two codons containing a BamHI restriction site.
5 'of CH1 Left primer
5 ′ GAACCCTCGCGGACAGTTAAG-3 ′ (SEQ ID NO: 63)
CH1 + G ₄ S-Bam Right (“G ₄ S” is disclosed as SEQ ID NO: 96)
5 ′ GGATCCTCCGCGCGCCGCAGCCTCTTAGGTTTCTTGTCCCACTTGGTGTTGCTGG-3 ′ (SEQ ID NO: 64)

  A 410 bp PCR unprimer was cloned into the pGemT PCR cloning vector (Promega) and clones were screened for T7 (5 ') oriented inserts.

Construction of (G ₄ S) ₂ DDD1 (“(G ₄ S) ₂ ” is disclosed as SEQ ID NO: 97) 11 residues of the linker peptide encode the preceding amino acid sequence of DDD1, and the first two codons A double stranded oligonucleotide designated (G ₄ S) ₂ DDD1 (“(G ₄ S) ₂ ” is disclosed as SEQ ID NO: 97), which contains a BamHI restriction site, is available from Sigma Genosys (Haveril, UK) Was synthesized. A stop codon and an EagI restriction site are added to the 3 ′ end. The encoded polypeptide sequence is shown below.
GSGGGGSGGGGG SHIQIPPGLTELLQGYTVEVLRRQQPDLVEFAVEYFTRLREARA (SEQ ID NO: 65)

Two oligonucleotides, designated RIIA1-44top and RIIA1-44bottom, which overlap 30 base pairs at their 3 ′ ends, were synthesized (Sigma Genosys) and combined to include the middle 154 bp of the 174 bp DDD1 sequence. Oligonucleotides were annealed and subjected to a primer extension reaction using Taq polymerase.
RIIA1-44 top
5'GTGGCGGGTCTGGCGGAGGGTGGCAGCCACATCCAGATCCGCCGGGGGCTCACGGAGCTGCTGCAGGGCTACACGGTGGAGGGTGCTGCGAGAG-3 '(SEQ ID NO: 66)
RIIA1-44 bottom
5 'GCGCGAGCTTCTCTCCAGGCGGGGTGAAGTACTCCCACTGCGAATTCGACGAGGTCAGGGCGGCTGCTGTCGCAGCACCTCCCACCGTTAGCCCCTG-3' (SEQ ID NO: 67)

After extending the primers, the duplex was amplified by PCR using the following primers:
G4S Bam-Left (“G4S” is disclosed as SEQ ID NO: 96)
5′-GGATCCGGAGGTGGCGGGTCTGGCGGAGGT-3 ′ (SEQ ID NO: 68)
1-44 stop Eag Right
5′-CGGCCGTCAAGCGCGAGCTTCTCTCAGGGCG-3 ′ (SEQ ID NO: 69)

  This unprimer was cloned into pGemT and screened for T7 (5 ') oriented inserts.

Construction of (G ₄ S) ₂ -AD1 (“(G ₄ S) ₂ ” is disclosed as SEQ ID NO: 97) 11 residues of the linker peptide encode the amino acid sequence of AD1 preceded by the first two A double stranded oligonucleotide designated as (G ₄ S) ₂ -AD1 (“(G ₄ S) ₂ ” is disclosed as SEQ ID NO: 97), in which the codon contains a BamHI restriction site (Sigma Genosys) . A stop codon and an EagI restriction site are added to the 3 ′ end. The encoded polypeptide sequence is shown below.
GSGGGGSGGGGS QIEYLAKQIVDNAIQQA (SEQ ID NO: 70)

Two complementary overlapping oligonucleotides called AKAP-IS Top and AKAP-IS Bottom were synthesized.
AKAP-IS Top
5 'GGATCCGGAGGTGGCGGGGTCTGGCGCAGCCAGATCGAGTACCTGGCCAAGCAGATCGTGACACAGCCCCATCCAGCAGCCCTG-3' (SEQ ID NO: 71)
AKAP-IS Bottom
5'CGGCCGTCAGGCCCTGCTGGATGGCGGTGTCCACGATCTGCTTGGCCAGGTACCTGATCTGGCTGCCACCTCCGCCCAGACCCCCCACCTCCCGGATCC-3 '(SEQ ID NO: 72)

The duplex was amplified by PCR using the following primers:
G4S Bam-Left (“G4S” is disclosed as SEQ ID NO: 96)
5′-GGATCCGGAGGTGGCGGGTCTGGCGGAGGT-3 ′ (SEQ ID NO: 73)
AKAP-IS stop Eagle Right
5′-CGGCCGTCAGGGCCTGCTGGATG-3 ′ (SEQ ID NO: 74)

  This unprimer was cloned into the pGemT vector and screened for T7 (5 ') oriented inserts.

Ligation of DDD1 and CH1 A 190 bp fragment encoding the DDD1 sequence was excised from pGemT using BamHI and NotI restriction enzymes and then ligated to the same site of CH1-pGemT to generate the shuttle vector CH1-DDD1-pGemT.

Ligation of AD1 and CH1 A 110 bp fragment containing the AD1 sequence was excised from pGemT using BamHI and NotI restriction enzymes and then ligated to the same site of CH1-pGemT to generate the shuttle vector CH1-AD1-pGemT.

Cloning of CH1-DDD1 or CH1-AD1 into a pdHL2-based vector This modular design allows either CH1-DDD1 or CH1-AD1 to be incorporated into any IgG construct in the pdHL2 vector. By removing the SacII / EagI restriction fragment (CH1-CH3) from pdHL2 and replacing it with the SacII / EagI fragment of CH1-DDD1 or CH1-AD1 excised from the respective pGemT shuttle vector, The entire domain is replaced with one of the above constructs.

N-terminal DDD domain The position of DDD or AD is not limited to the carboxyl terminus of CH1. A construct was made in which the DDD1 sequence was attached to the amino terminus of the VH domain.

Example 4: Construction of the DNL expression vector h679-Fd-AD1-pdHL2 h679-Fd-AD1-pdHL2 is an Fd CH1 domain via a flexible Gly / Ser peptide spacer composed of 14 amino acid residues. An expression vector for producing h679 Fab with AD1 linked to the carboxyl terminus. By replacing the SacII / EagI fragment with the CH1-AD1 fragment excised from the CH1-AD1-SV3 shuttle vector using SacII and EagI, a vector based on pdHL2 containing the variable domain of h679 was obtained. Converted to.

Construction of C-DDD1-Fd-hMN-14-pdHL2 C-DDD1-Fd-hMN-14-pdHL2 is obtained by binding DDD1 to hMN-14 Fab at the carboxyl terminus of CH1 via a flexible peptide spacer. An expression vector for producing the fusion protein C-DDD1-Fab-hMN-14. Digestion with SacII and EagI restriction endonuclease removes the CH1-CH3 domain and inserts a CH1-DDD1 fragment excised from the CH1-DDD1-SV3 shuttle vector using SacII and EagI to produce hMN-14 IgG The plasmid vector hMN14 (I) -pdHL2 used for this purpose was converted to C-DDD1-Fd-hMN-14-pdHL2.

Construction of N-DDD1-Fd-hMN-14-pdHL2 N-DDD1-Fd-hMN-14-pdHL2 is obtained by binding DDD1 to hMN-14 Fab at the amino terminus of VH via a flexible peptide spacer. An expression vector for producing a stable dimer containing two copies of the fusion protein N-DDD1-Fab-hMN-14. The expression vector was manipulated as follows. The DDD1 domain was amplified by PCR using the two primers shown below.
DDD1 Nco Left
5 ′ CCATGGGCAGCCACCATCCAGATCCCGCC-3 ′ (SEQ ID NO: 75)
DDD1-G ₄ S Bam Right (“G ₄ S” is disclosed as SEQ ID NO: 96)
5 ′ GGATCCGCCACCTCCCAGATCCTCCCGCCCCACGCGGAGCTTCTCTCAGGCGGGTG-3 ′ (SEQ ID NO: 76)

  As a result of PCR, the coding sequence of the NcoI restriction site and a part of the linker containing the BamHI restriction was added to the 5 'and 3' ends, respectively. A 170 bp PCR unprimer was cloned into the pGemT vector and clones were screened for T7 (5 ') oriented inserts. The 194 bp insert was excised from the pGemT vector using NcoI and SalI restriction enzymes and cloned into the SV3 shuttle vector prepared by digestion with those same enzymes to generate the intermediate vector DDD1-SV3.

The hMN-14 Fd sequence was amplified by PCR using the oligonucleotide primers shown below.
hMN-14VH left G4S Bam (“G4S” is disclosed as SEQ ID NO: 96)
5′-GGATCCGGCGGAGGTGCGCTCTGAGGTCCAACTGGTGGAGAGCGG-3 ′ (SEQ ID NO: 77)
CH1-C stop Eag
5′-CGGCCGTCAGCAGCTCTTAGGTTTCTTGTC-3 ′ (SEQ ID NO: 78)

  As a result of PCR, a BamHI restriction site and a coding sequence of a part of the linker were added to the 5 'end of the unprimer. A stop codon and an EagI restriction site were added at the 3 'end. A 1043 bp amp primer was cloned into pGemT. The hMN-14-Fd insert was ligated into the DDD1-SV3 vector prepared by excision from pGemT using BamHI and EagI restriction enzymes and subsequent digestion with those same enzymes to construct N-DDD1-hMN- 14Fd-SV3 was produced.

  N-DDD1-hMN-14 Fd sequence was excised using XhoI and EagI restriction enzymes and a 1.28 kb insert fragment was prepared by digesting C-hMN-14-pdHL2 using these same enzymes Ligated into fragments. The final expression vector is N-DDD1-Fd-hMN-14-pDHL2.

Example 5 FIG. Production and purification of h679-Fab-AD1 679 antibodies bind to the HSG target antigen and can be purified by affinity chromatography. The h679-Fd-AD1-pdHL2 vector was linearized by digestion with SalI restriction endonuclease and transfected into Sp / EEE myeloma cells by electroporation. The bicistronic expression vector directs the synthesis and secretion of both h679 kappa light chain and h679Fd-AD1, which together form h679Fab-AD1. After electroporation, cells were seeded in 96 well tissue culture plates and transfected clones were selected with 0.05 μM methotrexate (MTX). Clones were screened for protein expression by ELISA detected with HRP-conjugated goat anti-human Fab using microtiter plates coated with BSA-IMP-260 (HSG) conjugate. BIAcore analysis using an HSG (IMP-239) sensor chip was used to determine productivity by measuring the initial slope obtained from the injection of diluted media samples. The clone with the highest productivity showed an initial productivity of approximately 30 mg / L. A total of 230 mg of h679-Fab-AD1 was purified from a 4.5 liter rotating bottle culture by one-step IMP-291 affinity chromatography. The medium was concentrated approximately 10-fold by ultrafiltration before being loaded onto the IMP-291-affigel column. The column was washed with PBS until the baseline was reached, and h679-Fab-AD1 was eluted with 1M imidazole, 1 mM EDTA, 0.1M NaAc, pH 4.5. SE-HPLC analysis of the eluate showed a single sharp peak showing a residence time (9.63 min) consistent with the 50 kDa protein (not shown). In reduced SDS-PAGE analysis, only two bands representing the polypeptide component of h679-AD1 were evident (not shown).

Example 6 Production and purification of N-DDD1-Fab-hMN-14 and C-DDD1-Fab-hMN-14 C-DDD1-Fd-hMN-14-pdHL2 and N-DDD1-Fd-hMN-14-pdHL2 vectors were Sp2 / 0-derived myeloma cells were transfected by poration. C-DDD1-Fd-hMN-14-pdHL2 is a bicistronic expression vector, both hMN-14 kappa light chain and hMN-14 Fd-DDD1, which together form C-DDD1-hMN-14 Fab Directs synthesis and secretion. N-DDD1-hMN-14-pdHL2 is a bicistronic expression vector of hMN-14 kappa light chain and N-DDD1-Fd-hMN-14 that together form N-DDD1-Fab-hMN-14 Directs both synthesis and secretion. Each fusion protein forms a stable homodimer due to the interaction of the DDD1 domain.

  After electroporation, cells were seeded in 96 well tissue culture plates and transfected clones were selected with 0.05 μM methotrexate (MTX). Clones were screened for protein expression by ELISA detected with HRP-conjugated goat anti-human Fab using microtiter plates coated with rat anti-id monoclonal antibody against hMN-14. The initial productivity of -Fab-hMN14 Fab and N-DDD1-Fab-hMN14 Fab clones was 60 mg / L and 6 mg / L, respectively.

Affinity purification of N-DDD1-hMN-14 and C-DDD1-hMN-14 by AD1-Affigel DDD1-AD interaction was used to affinity purify DDD1-containing constructs. AD1-C is a synthetically engineered peptide composed of the AD1 sequence and the carboxyl terminal cysteine residue used to bind the peptide to Affigel after reacting the sulfhydryl group with chloroacetic anhydride. . DDD containing _{a 2} structure at neutral pH AD1-C-Affigel resin specifically bound, low pH (e.g., pH 2.5) can be eluted with.

  A total of 81 mg of C-DDD1-Fab-hMN-14 was purified from a 1.2 liter rotating bottle culture by one-step AD1-C affinity chromatography. The medium was concentrated approximately 10-fold by ultrafiltration before being loaded onto the AD1-C-affigel column. The column was washed with PBS until the baseline was reached and C-DDD1-Fab-hMN-14 was eluted with 0.1 M glycine, pH 2.5. SE-HPLC analysis of the eluate showed a single sharp peak showing a residence time (8.7 minutes) consistent with the 107 kDa protein (not shown). The purity was also confirmed by reduced SDS-PAGE showing only two bands of the expected molecular size in the two polypeptide components of C-DDD1-Fab-hMN-14 (not shown).

  A total of 10 mg of N-DDD1-hMN-14 was purified from a 1.2 liter rotating bottle culture by one-step AD1-C affinity chromatography. SE-HPLC analysis of the eluate showed a single protein peak with a residence time (8.77 min) consistent with the 107 kDa protein, similar to C-DDD1-Fab-hMN-14 (not shown). Reduced SDS-PAGE showed only two bands due to the polypeptide component of N-DDD1-Fab-hMN-14 (not shown).

  The binding activity of C-DDD1-Fab-hMN-14 was determined by SE-HPLC analysis of samples in which test substances were mixed with various amounts of WI2. A sample prepared by mixing WI2 Fab and C-DDD1-Fab-hMN-14 at a molar ratio of 0.75: 1 was unbound C-DDD1-Fab-hMN14 (8.71 min), one WI2 Fab. Showed three peaks due to C-DDD1-Fab-hMN-14 (7.95 min) bound to 2 and C-DDD1-Fab-hMN14 (7.37 min) bound to two WI2 Fabs (not shown) ). When a sample containing WI2 Fab and C-DDD1-Fab-hMN-14 at a molar ratio of 4 was analyzed, only a single peak at 7.36 minutes was observed (not shown). These results demonstrate that hMN14-Fab-DDD1 is a dimer and has two active binding sites. When this experiment was repeated with N-DDD1-Fab-hMN-14, very similar results were obtained.

  By competitive ELISA, C-DDD1-Fab-hMN-14 and N-DDD1-Fab-hMN-14 both have binding activity similar to hMN-14 IgG and are significantly stronger than monovalent hMN-14 Fab. (Not shown). The ELISA plate was coated with a fusion protein containing an epitope of CEA (A3B3) where hMN-14 is specific.

Example 7 forming a ₂ b basis of complex formation of a ₂ b complex, the C-DDD1-Fab-hMN- 14 ( as _{a 2)} and a mixture containing equimolar amounts h679-Fab-AD1 (as a b) Provided by SE-HPLC analysis. Analysis of such a sample is consistent with the formation of a new protein larger than either h679-Fab-AD1 (9.55 min) or C-DDD1-Fab-hMN-14 (8.73 min). A single peak with a residence time of minutes was observed (not shown). When hMN-14F (ab ′) ₂ was mixed with h679-Fab-AD1 or C-DDD1-Fab-hMN-14 was mixed with 679-Fab-NEM, no upfield shift was observed, It was demonstrated that the interaction is specifically mediated through the DDD1 and AD1 domains. Very similar results were obtained using h679-Fab-AD1 and N-DDD1-Fab-hMN-14 (not shown).

BIAcore was used to further demonstrate and characterize the specific interaction between DD1 and AD1 fusion proteins. First, either h679-Fab-AD1 or 679-Fab-NEM is bound to the surface of the high density HSG binding (IMP 239) sensor chip, and then C-DDD1-Fab-hMN-14 or hMN-14F (ab ') Experiments were performed by injecting ₂ afterwards. As expected, only the combination of h679-Fab-AD1 and C-DDD1-Fab-hMN-14 resulted in a further increase in response units when the latter was injected (not shown). Similar results were obtained using N-DDD1-Fab-hMN-14 and h679-Fab-AD1 (not shown).

Example 8 FIG. Affinity purification of either DDD or AD fusion proteins A universal affinity purification system can be developed by producing DDD or AD proteins that exhibit lower affinity docking. DDD formed by the RIα dimer binds to AKAP-IS (AD1) with a 500-fold weaker affinity (225 nM) compared to RIIα. Thus, an RIα dimer formed with the first 44 amino acid residues can be produced and attached to the resin to create an affinity matrix for purifying any AD1-containing fusion protein.

  Many low affinity (0.1 μM) AKAP anchor domains exist in nature. If necessary, highly predictable amino acid substitutions can be introduced to further reduce binding affinity. Low affinity AD can be produced either synthetically or biologically and can be conjugated to a resin used for affinity purification of any DDD1 fusion protein.

Example 9 Vector N-DDD2-Fd-hMN-14-pdHL2 for producing disulfide stabilized structures
N-DDD2-hMN-14-pdHL2 is an expression vector for producing N-DDD2-Fab-hMN-14 having a DDD2 dimerization and docking domain sequence added to the amino terminus of Fd. DDD2 is attached to the _VH domain via a 15 amino acid residue Gly / Ser peptide linker. DDD2 had a cysteine residue preceding the dimerization and docking sequence, which was identical to that of DDD1.

The expression vector was manipulated as follows. Two overlapping complementary oligonucleotides (DDD2 Top and DDD2 Bottom) containing residues 1-13 of DDD2 were made synthetically. Oligonucleotides were annealed and phosphorylated with T4 polynucleotide kinase (PNK), resulting in 5 ′ and 3 ′ end overhangs compatible with ligation with DNA digested with restriction endonucleases NcoI and PstI, respectively.
DDD2 Top
5'CATGTGCGGCCACCATCCAGATCCGCCCGGGGCTCACGGAGCTCTGCA-3 '(SEQ ID NO: 79)
DDD2 Bottom
5′GCAGCTCCGTGAGCCCCCGGCGGGATCTGGATGTGGCCGCA-3 ′ (SEQ ID NO: 80)

  Double-stranded DNA was ligated to the vector fragment, DDD1-hMN14 Fd-SV3, prepared by digesting with NcoI and PstI, to generate the intermediate construct DDD2-hMN14 Fd-SV3. The 1.28 kb insert fragment, which contained the DDD2-hMN14 Fd coding sequence, was excised from the intermediate construct using XhoI and EagI restriction endonucleases and digested with those same enzymes into the hMN14-pdHL2 vector DNA prepared. Ligated. The final expression vector is N-DDD2-Fd-hMN-14-pdHL2.

C-DDD2-Fd-hMN-14-pdHL2
C-DDD2-Fd-hMN-14-pdHL2 is a C-DDD2 having a DDD2 dimerization and docking domain sequence added to the carboxyl terminus of Fd via a 14 amino acid residue Gly / Ser peptide linker -An expression vector for producing Fab-hMN-14. The expression vector was manipulated as follows. Two overlapping complementary oligonucleotides containing a portion of the linker peptide (GGGGSGGGCG, SEQ ID NO: 81) and the coding sequence of residues 1-13 of DDD2 were made synthetically. Oligonucleotides were annealed, phosphorylated with T4 PNK, resulting in 5 ′ and 3 ′ end overhangs compatible with ligation with DNA digested with the restriction endonucleases BamHI and PstI, respectively.
G4S-DDD2 top (“G4S” is disclosed as SEQ ID NO: 96)
5'GATCCGGAGGTGGCGGGGTCTGGCGGAGGTTGCGGCCACATCCAGATCCCGCCGGGGGCTCACGGAGGCTGCTGCA-3 '(SEQ ID NO: 82)
G4S-DDD2 bottom ("G4S" is disclosed as SEQ ID NO: 96)
5′GCAGCTCCGTGAGCCCCGGCGGGATCTGGATGTGGCCGCAACCTCCCGCCAGCCCGCCCACCTCCG-3 ′ (SEQ ID NO: 83)

  The double-stranded DNA was ligated to the shuttle vector CH1-DDD1-pGemT prepared by digesting with BamHI and PstI to generate the shuttle vector CH1-DDD2-pGemT. The 507 bp fragment was ligated into the IgG expression vector hMN14 (I) -pdHL2 prepared by excising from CH1-DDD2-pGemT using SacII and EagI and digesting with SacII and EagI. The final expression construct is C-DDD2-Fd-hMN-14-pdHL2.

h679-Fd-AD2-pdHL2
h679-Fd-AD2-pdHL2 is expressed to produce h679-Fab-AD2 with an AD2 anchor domain sequence appended to the carboxyl terminus of the CH1 domain via a 14 amino acid residue Gly / Ser peptide linker It is a vector. AD2 has one cysteine residue in front of the anchor domain sequence of AD1 and another cysteine residue after.

The expression vector was manipulated as follows. Two overlapping complementary oligonucleotides (AD2Top and AD2Bottom) containing the coding sequences of part of AD2 and the linker peptide were made synthetically. Oligonucleotides were annealed, phosphorylated with T4 PNK, resulting in 5 ′ and 3 ′ end overhangs that were compatible with ligation with DNA digested with the restriction endonucleases BamHI and SpeI, respectively.
AD2 Top
5'GATCCGGAGGTGGCGGGGTCTGGGCGGATGCGGCAGATCGAGTACCTGGCCAAGCAGATCGTGGACAACGCCATCCAGCAGCGCGCGCTGCTGAA-3 '(SEQ ID NO: 84)
AD2 Bottom
5'TTCAGCAGCCGGCCTGCTGGATGGCGGTGTCCACGATCTGCTTGGCCAGGTACCTGATCTGGCCACATCCGCCAGCCCGCCCACCTCCG-3 '(SEQ ID NO: 85)

  Double-stranded DNA was ligated to shuttle vector CH1-AD1-pGemT prepared by digestion with BamHI and SpeI to generate shuttle vector CH1-AD2-pGemT. A 429 base pair fragment containing the CH1 and AD2 coding sequences was excised from the shuttle vector using SacII and EagI restriction enzymes and ligated into the h679-pdHL2 vector prepared by digestion with those same enzymes. The final expression vector is h679-Fd-AD2-pdHL2.

Example 10 Generation of TF1 A large scale preparation of a trivalent DNL construct designated TF1 was performed as follows. First, N-DDD2-Fab-hMN-14 (purified with protein L) and h679-Fab-AD2 (purified with IMP-291) were added at approximately stoichiometric concentrations in 1 mM EDTA, PBS, pH 7.4. Mixed. Prior to the addition of TCEP, SE-HPLC showed no evidence of a ₂ b formation (not shown). Instead, there were peaks representing a ₄ (7.97 min; ₂₀₀ kDa), a ₂ (8.91 min; 100 kDa), and B (10.01 min; 50 kDa). Addition of 5 mM TCEP rapidly resulted in the formation of an a ₂ b complex indicated by a new peak at 8.43 min consistent with the 150 kDa protein (not shown). In this experiment, the peak due to h679-Fab-AD2 (9.72 min) was still evident so it was clear that B was present in excess but corresponds to either a ₂ or a ₄ No obvious peak was observed. After 1 hour of reduction, TCEP was removed by dialyzing overnight with several changes of PBS. The resulting solution was adjusted to 10% DMSO and kept at room temperature overnight.

When analyzed by SE-HPLC, the peak representing a ₂ b became sharper and it was observed that the residence time was slightly delayed by 0.1 min to 8.31 min (not shown). This indicates an increase in binding affinity based on our previous findings. The complex was further purified by IMP-291 affinity chromatography to remove kappa chain contaminants. As expected, excess h679-AD2 was simultaneously purified and then removed by preparative SE-HPLC.

TF1 is a very stable complex. When TF1 was tested for binding to the HSG (IMP-239) sensor chip, no apparent decrease in response was observed at the end of sample injection. In contrast, when a solution containing equimolar mixtures of both C-DDD1-Fab-hMN-14 and h679-Fab-AD1 was tested under similar conditions, the observed increase in response units was There was a detectable drop between and immediately after injection, indicating that the initially formed a ₂ b structure was unstable. Furthermore, subsequent injection of WI2 resulted in a substantial increase in the response unit of TF1, but for the C-DDD1 / AD1 mixture, the increase was not obvious.

The further increase in response units due to the binding of WI2 to TF1 immobilized on the sensor chip corresponds to two fully functional binding sites, with one subunit of N-DDD2-Fab-hMN-14 being It contributed to each of them. This was confirmed by the ability of TF1 to bind to the two Fab fragments of WI2 (not shown). When a mixture containing h679-AD2 and N-DDD1-hMN14 reduced and oxidized exactly as TF1 was analyzed by BIAcore, there was little further binding of WI2 (not shown) and stabilized with disulfides such as TF1. It has been shown that the a ₂ b complex produced can only be caused by the interaction of DDD2 and AD2.

Two improvements were made to the process that reduced process time and efficiency. First, using a slight molar excess of N-DDD2-Fab-hMN-14 present as a mixture of a ₄ / a ₂ structures, no free h679-Fab-AD2 remained and h679-Fab- Reacted with h679-Fab-AD2 so that any a ₄ / a ₂ structure not tethered to AD2 as well as the light chain would be removed by IMP-291 affinity chromatography. Second, hydrophobic interaction chromatography (HIC) was used instead of dialysis or diafiltration as a means of removing TCEP after reduction. This will not only shorten the process time, but may also add a virus removal step. N-DDD2-Fab-hMN-14 and 679-Fab-AD2 were mixed and reduced with 5 mM TCEP for 1 hour at room temperature. The solution was adjusted to 0.75M ammonium sulfate and then loaded onto a butyl FF HIC column. The column was washed with 0.75 M ammonium sulfate, 5 mM EDTA, PBS to remove TCEP. The reduced protein was eluted from the HIC column with PBS and adjusted to 10% DMSO. After overnight incubation at room temperature, highly purified TF1 was isolated by IMP-291 affinity chromatography (not shown). Additional purification steps such as gel filtration were not necessary.

Example 11 Generation of TF2 After successful generation of TF1, an analog called TF2 was obtained by reacting C-DDD2-Fab-hMN-14 with h679-Fab-AD2. A trial batch of TF2 was produced in a yield greater than 90% as follows. Protein L purified C-DDD2-Fab-hMN-14 (200 mg) was mixed with h679-Fab-AD2 (60 mg) in a molar ratio of 1.4: 1. The total protein concentration was 1.5 mg / ml in PBS containing 1 mM EDTA. Subsequent steps involving TCEP reduction, HIC chromatography, DMSO oxidation, and IMP-291 affinity chromatography were the same as described for TF1. Prior to the addition of TCEP, SE-HPLC showed no evidence of a ₂ b formation (not shown). Instead, there were peaks corresponding to a ₄ (8.40 min; 215 kDa), a ₂ (9.32 min; 107 kDa), and b (10.33 min; 50 kDa). Addition of 5 mM TCEP rapidly resulted in the formation of an a ₂ b complex indicated by a new peak at 8.77 minutes (not shown) consistent with the expected 157 kDa protein for the binary structure. TF2 was purified to near homogeneity by IMP-291 affinity chromatography (not shown). IMP-291 the unbound fraction by analyzing by _SE-HPLC, it was demonstrated that _a 4, a _2, and free kappa chains were removed from the product (not shown).

The functionality of TF2 was determined with BIACORE as described for TF1. TF2, C-DDD1-hMN-14 + h679-AD1 (used as a control sample for non-covalent a ₂ b complex) or C-DDD2-hMN-14 + h679-AD2 (as a control sample for non-reduced a ₂ and b components) Used) was diluted to 1 μg / ml (total protein) and passed through a sensor chip on which HSG had been immobilized. The TF2 response was approximately twice that of the two control samples, indicating that only the h679-Fab-AD component in the control sample would bind and remain on the sensor chip. Subsequent injection of WI2 IgG demonstrated that only TF2 has a DDD-Fab-hMN-14 component that is strongly bound to h679-Fab-AD as indicated by the additional signal response. . Also, the further increase in response units due to the binding of WI2 to TF2 immobilized on the sensor chip corresponds to two fully functional binding sites, and one sub-part of C-DDD2-Fab-hMN-14 Units contributed to each of them. This was confirmed by the ability of TF2 to bind to the two Fab fragments of WI2 (not shown).

  The relative CEA binding activity of TF2 was determined by competitive ELISA. Plates were coated with a fusion protein containing the A3B3 domain of CEA recognized by hMN-14 (0.5 μg / well). Serial dilutions of TF1, TF2, and hMN-14 IgG were made in quadruplicate and incubated in wells containing HRP-conjugated hMN-14 IgG (1 nM). The data show that TF2 binds to CEA with a binding activity that is at least similar to the binding activity of IgG and twice as strong as TF1 (not shown).

Example 12 Serum stability of TF1 and TF2 TF1 and TF2 were designed to be stable tethered structures that can be used in vivo where extensive dilutions occur in blood and tissues. The stability of TF2 in human serum was evaluated using BIACORE. TF2 was diluted to 0.1 mg / ml with fresh human serum collected from 4 donors and incubated for 7 days at 37 ° C. under 5% CO ₂ . Daily samples were diluted 1:25 and then analyzed by BIACORE using an IMP-239HSG sensor chip. WI2 IgG injection was used to quantify the amount of complete and fully active TF2. Serum samples were compared to control samples diluted directly from the stock solution. TF2 is highly stable in serum and retains 98% of its bispecific binding activity after 7 days (not shown). Similar results were obtained for TF1 in either human or mouse serum (not shown).

Example 13 Generation of C-H-AD2-IgG-pdHL2 Expression Vector The pdHL2 mammalian expression vector has been used to mediate the expression of numerous recombinant IgGs (Qu et al., Methods 2005, 36: 84-95 ). In order to facilitate the conversion of any IgG-pdHL2 vector to a C-H-AD2-IgG-pdHL2 vector, a plasmid shuttle vector was generated. The gene for Fc (CH2 and CH3 domains) was amplified using the pdHL2 vector as a template and the oligonucleotides Fc BglII Left and Fc Bam-EcoRI Right as primers.
Fc BglII Left
5′-AGATCTGGCGCACCTGACTACTCG-3 ′ (SEQ ID NO: 86)
Fc Bam-EcoRI Right
5′-GAATTCGGATCTCTTTACCCGGAGACAGGGAGAG-3 ′ (SEQ ID NO: 87)

  The unprimer was cloned into the pGemT PCR cloning vector. The Fc insert fragment was excised from pGemT using XbaI and BamHI restriction enzymes and ligated to the AD2-pdHL2 vector prepared by digesting h679-Fab-AD2-pdHL2 with XbaI and BamHI to generate the shuttle vector Fc-AD2 -pdHL2 was produced.

  To convert any IgG-pdHL2 expression vector into a C-H-AD2-IgG-pdHL2 expression vector, an 861 bp BsrGI / NdeI restriction fragment was excised from the former and a 952 bp BsrGI excised from the Fc-AD2-pdHL2 vector. Replaced with / NdeI restriction fragment. BsrGI cleaves the CH3 domain and NdeI cleaves downstream (3 ') of the expression cassette.

Example 14 Production of C-H-AD2-hLL2 IgG Epiratuzumab, or hLL2 IgG, is a humanized anti-human CD22 MAb. An expression vector for C-H-AD2-hLL2 IgG was generated from hLL2 IgG-pdHL2 as described above and used to transfect Sp2 / 0 myeloma cells by electroporation. After transfection, cells were seeded in 96-well plates and transgenic clones were selected in medium containing methotrexate. Clones were screened for C-H-AD2-hLL2 IgG productivity by sandwich ELISA detecting with peroxidase-conjugated anti-human IgG using 96-well microtiter plates coated with hLL2-specific anti-idiotype MAb. Clones were grown in a rotating bottle for protein production and C-H-AD2-hLL2 IgG was purified from spent media in one step using protein A affinity chromatography. SE-HPLC analysis separated two protein peaks (not shown). The residence time of the slower elution peak (8.63 minutes) was similar to hLL2 IgG. The residence time of the more rapidly eluted peak (7.75 minutes) was consistent with the approximately 300 kDa protein. Later, this peak was found to be a disulfide linked dimer of C-H-AD2-hLL2-IgG. This dimer is reduced to the monomer form during the DNL reaction. SDS-PAGE analysis showed that purified CH-AD2-hLL2-IgG was composed of both a monomeric form of the module and a dimeric form of the module linked by disulfide (non- display). The protein bands representing these two forms were evident in SDS-PAGE under non-reducing conditions, but under reducing conditions all forms consisted of two representing the component polypeptides (heavy chain-AD2 and kappa chain). Reduced to band (not shown). Other contaminant bands were not detected.

Example 15. Production of C-H-AD2-hA20 IgG hA20 IgG is a humanized anti-human CD20 MAb. An expression vector for C-H-AD2-hA20 IgG was generated from hA20 IgG-pDHL2 and used to transfect Sp2 / 0 myeloma cells by electroporation. After transfection, cells were seeded in 96-well plates and transgenic clones were selected in medium containing methotrexate. Clones were screened for C-H-AD2-hA20 IgG productivity by sandwich ELISA detecting with peroxidase-conjugated anti-human IgG using 96 well microtiter plates coated with hA20 specific anti-idiotype MAb. Clones were grown in a rotating bottle for protein production and C-H-AD2-hA20 IgG was purified from spent media in one step using protein A affinity chromatography. SE-HPLC and SDS-PAGE analysis gave results very similar to those obtained for C-H-AD2-hLL2 IgG (not shown).

Example 16 Production of AD and DDD-binding Fab and IgG fusion proteins derived from multiple antibodies Using the techniques described in the examples above, the following IgG or Fab fusion proteins were constructed and incorporated into DNL constructs. The fusion protein retained the antigen-binding characteristics of the parent antibody, and the DNL construct showed the antigen-binding activity of the incorporated antibody or antibody fragment.

Example 17. A DNL immunotoxin based on a ribonuclease containing four lampyrnases (Rap) conjugated to a B cell lymphoma targeted antibody We applied a dock-and-lock (DNL) method, each of which is a bivalent IgG A new class of immunotoxins was generated that contained four copies of Rap site-specifically bound to. We have produced recombinant Rap-DDD modules produced in E. coli in myeloma cells, CD20 (hA20, veltuzumab), CD22 (hLL2, epilatuzumab), or HLA-DR (hL243, IMMU-114). In combination with recombinant humanized IgG-AD modules targeting B cell lymphoma and leukemia to produce 20-Rap, 22-Rap, and C2-Rap, respectively. For each construct, Rap dimers were covalently tethered to the C-terminus of each heavy chain of the respective IgG. A control construct, 14-Rap, was similarly constructed using rabetuzumab (hMN-14) that binds to an antigen not expressed in B cell lymphoma / leukemia (CEACAM5).
RapDDD2
pQDWLTFQKKHIITNTRDVDCDNIMSTNLFHCKDKNTFIYSRPEPVKAICKGIIASKNVLLTSEFYLDSDCNVTSRPCKYKLKKSTHFCLGTCHLQHLVHH

  The deduced amino acid sequence of secreted Rap-DDD2 is shown above (SEQ ID NO: 94). Rap, underlined; linker, italic; DDD2, bold; pQ, amino terminal glutamine converted to pyroglutamic acid. Rap-DDD2 was produced as inclusion bodies in E. coli, which was purified by IMAC under denaturing conditions, refolded, then dialyzed against PBS, and then purified by Q-Sepharose anion exchange chromatography. By SDS-PAGE under reducing conditions, a protein band having Mr corresponding to Rap-DDD2 (18.6 kDa) was separated (not shown). The final yield of purified Rap-DDD2 was 10 mg per liter culture.

  A group of IgG-Rap conjugates was rapidly generated using the DNL method. The IgG-AD module was expressed in myeloma cells and purified from the culture supernatant using protein A affinity chromatography. A Rap-DDD2 module was produced and mixed with IgG-AD2 to form a DNL complex. Since the CH3-AD2-IgG module has two AD2 peptides, each capable of tethering a Rap dimer, the resulting IgG-Rap DNL construct has four Rap groups and one IgG including. IgG-Rap is formed almost quantitatively from the component module and purified to near homogeneity using protein A.

  Prior to the DNL reaction, CH3-AD2-IgG exists as both a monomer and a disulfide-linked dimer (not shown). Under non-reducing conditions, IgG-Rap is separated as a cluster of high molecular weight bands of the expected size between monomeric CH3-AD2-IgG size and dimeric CH3-AD2-IgG size (not shown) . Under reducing conditions that reduce conjugates to their component polypeptides, only the bands representing heavy chain-AD2 (HC-AD2), kappa light chain, and Rap-DDD2 are visible (not shown), so IgG-Rap And the consistency of the DNL method is shown.

  Between two peaks of CH3-AD2-IgG-hLL2 representing the monomeric form (7.55 min) and the dimeric form (8.00 min) by reverse phase HPLC analysis (not shown) of 22-Rap A single protein peak that eluted at 9.10 minutes was separated. The Rap-DDD2 module was isolated as a mixture of dimers and tetramers (reduced to dimers in DNL) eluted at 9.30 and 9.55 minutes, respectively (not shown).

  LC / MS analysis of 22-Rap was achieved by combining reverse phase HPLC using a C8 column with ESI-TOF mass spectrometry (not shown). In the spectrum of unmodified 22-Rap, in addition to several minor glycoforms, two major ones with either two G0F (G0F / G0F) or one G0F + 1 G1F (G0F / G1F) N-linked glycans Species are identified (hidden). Enzymatic deglycosylation resulted in a single deconvolution mass consistent with the calculated mass of 22-Rap (not shown). The spectra obtained after reduction with TCEP identified heavy chain-AD2 polypeptides modified with N-linked glycans of G0F or G1F structure, as well as additional trace forms (not shown). Each of the three subunit polypeptides, including 22-Rap, was identified in the deconvoluted spectrum of the reduced and deglycosylated sample (not shown). These results show that both Rap-DDD2 and HC-AD2 polypeptides have an amino-terminal glutamine that has been converted to pyroglutamic acid (pQ), and thus 22-Rap has its 8 modified with pQ. Make sure you have 6 of the 1 component polypeptides.

In vitro cytotoxicity was assessed in three NHL cell lines. Each cell line expresses CD20 at a significantly higher surface density compared to CD22, but the internalization rate of hLL2 (anti-CD22) is much faster than hA20 (anti-CD20). 14-Rap shares the same structure as 22-Rap and 20-Rap, but its antigen (CEACAM5) is not expressed in NHL cells. In FIG. 13, left panel, cells were treated sequentially with IgG-Rap as a single agent or with a combination of parental MAb + rRap. Both 20-Rap and 22-Rap killed each cell line at concentrations above 1 nM, indicating that their effects are cytotoxic rather than simply cytostatic. 20-Rap is the most potent IgG-Rap, suggesting that antigen density may be more important than internalization rate. Similar results were obtained with Daudi and Ramos, with 20-Rap (EC50 ~ 0.1 nM) 3-6 times more potent than 22-Rap. The rituximab resistant mantle cell lymphoma line, Jeko-1, shows an increase in CD20 but a decrease in CD22 compared to Daudi and Ramos. Importantly, 20-Rap showed very strong cytotoxicity in Jeko-1 (EC ₅₀ ca. 20 pM) and was 25 times more potent than 22-Rap.

  As shown in FIG. 13, right panel, as expected, washing the cells after 1 hour treatment significantly reduced the cytotoxicity of each agent (approximately 50 times). Again, 20-Rap was the most potent, suggesting that its slower internalization rate is not a limitation. 14-Rap shows increased cytotoxicity compared to rRap (in combination with MAb), suggesting that the four Rap structures of IgG-Rap can enhance its internalization. By washing after 1 hour incubation, 14-Rap cytotoxicity was reduced over targeted 22-Rap and 20-Rap.

  IgG-Rap was evaluated in all three cell lines (Figure 14). The relative antigen density was similar in the three lines, HLA-DR >> CD22 >> CD20. In these assays, none of the parental MAbs were cytotoxic, either alone or in combination with rRap. However, non-targeted 14-Rap showed some activity and was similar to the results in the NHL system. In each cell line, C2-Rap targeting the most abundant antigen (HLA-DR) showed the strongest response, approximately 50 times more potent than 22-Rap. In all lines with very low CD20 antigen density, 20-Rap showed little cytotoxicity and was similar to that of non-targeted 14-Rap. This is in contrast to the results of the NHL line which has a high CD20 density and was most responsive to 20-Rap. Thus, the efficacy of IgG-Rap correlates with the relative abundance of the targeted antigen.

CONCLUSION The DNL method provides a modular approach to efficiently tether multiple cytotoxins to targeted antibodies and may exhibit higher in vivo efficacy for improved pharmacokinetics and specific targeting Resulting in the expected new immunotoxins. LC / MS, RP-HPLC, and SDS-PAGE demonstrated the homogeneity and purity of IgG-Rap. Targeting Rap with MAb to cell surface antigen enhances its tumor-specific cytotoxicity. Both antigen density and internalization rate are important factors in the observed in vitro potency of IgG-Rap. In vitro results indicate that CD20-, CD22-, or HLA-DR-targeted IgG-Rap has potent biological activity to treat B-cell lymphoma and leukemia.

Example 18 High potency of Rap-anti-Trop-2 IgG DNL construct against carcinomas Using the same technique as described in Example 17, 4 copies of hRS7-IgG-Ad2 (anti-Trop-) bound to Rap-DDD2. An E1-Rap DNL construct containing 2) was generated, which showed potent in vitro growth inhibitory properties against various cancer cell lines (not shown). In breast (MDA-MB-468), cervix (ME-180), and pancreas (BxPC-3 and Capan-1) tumor lines, all of which express high levels of Trop-2, E1-Rap is highly to a potent, it showed an _{EC 50} in the range of less than nanomolar (5~890pM), it was 000 to 10,000 times higher than the combination of untargeted Rap or Rap and hRS7. In cell lines expressing slight levels of Trop-2, such as the three prostate cancer lines (PC-3, DU145, and LNCaP), E1-Rap was not as potent, but was still in the nanomolar range (1- EC ₅₀ of 890 nM). The cell binding data obtained with these solid cancer cell lines suggests that the sensitivity of the cell line to E1-Rap is thought to correlate with its Trop-2 expression on the cell surface. E1-Rap toxicity was not observed in the prostate cancer line 22Rv1, which does not bind to hRS7. These results indicate that E1-Rap as a new therapeutic agent for Trop-2 positive solid tumors including breast cancer, colon cancer, stomach cancer, lung cancer, ovarian cancer, endometrial cancer, cervical cancer, pancreatic cancer, and prostate cancer. Indications.

  All of the compositions and methods disclosed and claimed herein can be made and used without undue experimentation in light of the present disclosure. Although the present compositions and methods have been described in terms of preferred embodiments, those skilled in the art will understand the compositions and methods described herein without departing from the concept, spirit, and scope of the present invention. Obviously, mutations can be applied to the steps or sequence of steps of the method. More particularly, certain agents that are both chemically and physiologically related can be used in place of the agents described herein, and the same or similar results will be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims (8)

A DNL (dock and lock) construct,
a) four copies of the toxin each bound to a DDD (dimerization and docking domain) portion from protein kinase A (PKA);
b) an antibody or antigen-binding antibody fragment bound to an AD (anchor domain) portion derived from an AKAP protein and comprising two copies of said AD portion;
The DDD moiety has an amino acid sequence derived from human RIα, RIβ, RIIα, or RIIβ;
The antibody or antigen-binding antibody fragment is anti-Trop-2, anti-CD20, anti-CD22 or anti-HLA-DR;
Two copies of the DDD moiety form a dimer and bind to the AD moiety to form the DNL construct;
The toxin Ri Oh in ranpirnase,
A construct wherein the AD moiety is attached to the C-terminus of the heavy chain of the antibody or antibody fragment .   The DNL construct of claim 1, wherein the toxin and the antibody or antibody fragment are fusion proteins, each fusion protein comprising an AD or DDD moiety. The DDD portion is represented by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, first 44 amino acids of SEQ ID NO: 20, first 44 amino acids of SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 59 The DNL construct according to claim 1 or 2 , which has an amino acid sequence. The AD part is SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 30 SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 , SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, The DNL construct according to any one of claims 1 to 3 , which has the amino acid sequence of SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58. Said antibody or antibody _{fragment, IgG, F (ab ')} 2, F (ab) 2, Fab', Fab, and scF v is selected from either Ranaru group, to any one of claims 1-4 The DNL construct described. A pharmaceutical composition for treating a disease selected from the group consisting of cancer, immune dysfunction, and autoimmune disease, comprising a DNL construct according to any one of claims 1 to 5. Composition. The cancer is non-Hodgkin lymphoma, B cell lymphoma, B cell leukemia, T cell lymphoma, T cell leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, Burkitt lymphoma, Hodgkin lymphoma, hairy cell leukemia, acute bone marrow leukemia, chronic Myelogenous leukemia, multiple myeloma, glioma, Waldenstrom macroglobulinemia, carcinoma, melanoma, sarcoma, glioma, skin cancer, oral cancer, digestive organ cancer, colon cancer, stomach cancer, lung cancer, Selected from the group consisting of lung cancer, breast cancer, ovarian cancer, prostate cancer, uterine cancer, endometrial cancer, cervical cancer, bladder cancer, pancreatic cancer, bone cancer, liver cancer, gallbladder cancer, kidney cancer, and testicular cancer The pharmaceutical composition according to claim 6 . The autoimmune disease is acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, chondomia chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, multigonal Syndrome, bullous pemphigoid, diabetes mellitus, Henoch-Schönlein purpura, streptococcal post-nephritis, erythema nodosum, Takayasu arteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, Erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture syndrome, obstructive thrombotic vasculitis, Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, thyroid poisoning, scleroderma Disease, chronic active hepatitis, polymyositis / dermatomyositis, polychondritis, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, spinal cord fistula, giant cell 7. The pharmaceutical composition according to claim 6 , selected from the group consisting of arteritis / polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis, or fibrotic alveolitis.

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