JP2006522122A - Transmembrane antibody-induced apoptosis inhibition - Google Patents

Abstract

Cellular suicide (apoptosis) is associated with etiology, for example it is a major cause of neuronal loss in Alzheimer's disease. Caspase-3 is highly related to the apoptotic pathway. In an effort to inhibit live cell apoptosis, antibodies against caspase enzymes have been generated using superantibody (SAT) -transmembrane technology. The advantage of using transmembrane antibodies as apoptosis inhibitors is their specific target recognition in cells and their low toxicity compared to conventional apoptosis inhibitors. MTS transit peptide modified monoclonal anti-caspase-3 antibody has been shown to reduce actinomycin D-induced apoptosis and spectrin cleavage in living cells. These results indicate that antibodies conjugated with membrane transport peptides have therapeutic potential to inhibit apoptosis in various diseases.

Description

  This application is filed in US application no. X is a continuation-in-part application of X, which is a national phase international application PCT / US02 / 16651 filed on May 29, 2002, which is a US patent application filed on May 29, 2001. No. 09 / 865,281, which is a continuation-in-part of U.S. patent application Ser. No. 09 / 070,907, filed May 4, 1998, currently U.S. Pat. No. 6,238. 667. This application also claims the benefits of US Provisional Application No. 60 / 451,980, filed March 5, 2003. The disclosures of each of the aforementioned patents and patent applications are incorporated herein by reference.

  The present invention relates to fusion proteins comprising fully biologically active peptides and antibodies, or fragments thereof. Specifically, the fusion protein of the present invention combines biological activities such as antibody molecular recognition and immunostimulatory activity, membrane transport activity, and homophilic activity. The present invention further provides loop-forming sequences or other for binding the antibody and limiting the conformational flexibility of the biologically active peptide and increasing affinity for its biological target. Concerning a fusion protein comprising a biologically active peptide sequence located on the side of the sequence providing the conformation. The invention also relates to the use of antibodies and conjugates thereof in the inhibition of programmed cell death, ie apoptosis.

  Antibodies are praised as “magic bullets” to fight disease. However, the expectations made with respect to antibodies are by no means fully realized. This is partly due to the fact that antibodies are only a small part of immune defense and that T cells provide other strategies in immune defense. However, antibodies are an ideal target and delivery device. Antibodies are compatible with long-term survival in blood, have sites that aid blood vessel and tissue penetration, and are functionally related to several defense mechanisms of innate immunity. One such mechanism is the complement system, which helps destroy pathogens and is related to the regulation of immune responses. For example, complement fragment C3d binds to the CR2 receptor on B cells, which is also the binding site for Epstein-Barr virus. B cells are activated by the binding of Epstein-Barr virus and CR2. Accumulated evidence indicates that the CR2 receptor (CD19 / Cd20 / CD81 complex) has an immunostimulatory role and is activated by C3d.

  Monoclonal antibodies have been developed for many therapeutic applications. For example, diseases currently targeted by monoclonal antibodies include heart disease, cancer, neurological abnormalities and autoimmune diseases. Almost all these current therapeutic uses rely on the inherent therapeutic efficacy of certain monoclonal antibodies, drugs HERCEPTIN and RITUXAN. Since most monoclonal antibodies do not exhibit such intrinsic therapeutic activity, a variety of different toxicants, such as protein toxins or subunits thereof, drugs currently used in cancer chemotherapy treatment, unacceptably high toxicity Development is focused on adding therapeutic properties by combining drugs, or radioisotopes, for which clinical development has not progressed.

  In order for such a conjugate to be effective, a monoclonal antibody that delivers such a toxic substance must be able to bind to its target antigen and internalize in the cell to carry the toxic substance inside. Internally, toxic substances may be effective in damaging DNA of target cells or inhibiting protein synthesis or other metabolic functions. There are only a few antibodies that inherently exhibit this property and the ability to generate very strong immune complexes. Thus, although screening assays have been developed to test such antibodies, few antibodies have been identified that combine this property with the appropriate target specificity.

  Other approaches exist for penetrating the ability to internalize into antibodies. Complete protein toxins that combine active and cell binding subunits are effective in increasing internalization upon binding to antibodies, but reduce antibody selectivity and thereby potential toxicity There are many. Lipophilic drugs have also been used for increased internalization and intracellular delivery in a bound form, but reduce the selectivity of the conjugate as well as toxins. Other methods have been used to increase permeability, or microinjection allows better entry into the cell. Both of these methods have significant drawbacks. Increased cell permeability, for example by saponins, bacterial toxins, calcium phosphate, electroporation, etc., can actually be used only for ex vivo methods, and these methods cause damage to the cells. Microinjection requires a highly skilled technician (and therefore its use is limited to laboratory settings), and microinjection physically disturbs cells and has only limited applications. This is because microinjection cannot be used to treat, for example, a cell mass or complete tissue, and a large number of cells cannot actually be injected.

  Other examples of how antibodies can be used to increase the immune response are the work of Zanetti and Bona (Zanetti, M., Nature, 355: 466-477, 1992; Zaghouani H .; Anderson SA. , Superbeer KE, Daian C. Kennedy RC, Mayer L. and Bona CA, Proc. Nat. Acad. Science USA, 92: 631-635, 1995). These authors used molecular biology methods to replace the CDR3 sequences of Ig heavy chains with sequences resembling T cell and B cell antigens (epitopes), specific for those with these modified antibodies inserted It induces a strong immune response.

  The biological properties of antibodies can increase the overall avidity of the antigen and the ability to penetrate cell and nuclear membranes. Increasing antigen binding by increasing antibody valency as in pentamer IgM antibodies. Valency and avidity are also increased in some antibodies that are self-binding or isomorphic (Kang, CY, Cheng, HL, Rudikoff, S. and Kohler, H., J. Exp). Med.165: 1332, 1987; Xiyun, A.N., Evans, SV, Kaminki, M.J., Fillies, SFD, Resifeld, RA, Newton, A. N. and Chapman, P.B., J. Immunol.157: 1582-1588, 1996). Peptides in the heavy chain variable region that inhibited self-binding were identified (Kang, CY Brunck, TK, Kieber-Emmons, T., Blockock, JE, and Kohler, H.,). Science, 240: 1034-1036, 1988). Self-binding peptide sequences are inserted into the antibody to provide self-binding properties and increase valency and overall avidity for the antigen.

  Similarly, addition of a signal peptide to the antibody facilitates membrane trafficking, as demonstrated by Rojas et al., Nature Biotechnology, 16: 370-375 (1998). Rojas et al. Produced a fusion protein containing a 12-mer peptide, indicating that this protein has cell membrane permeability.

  Signal peptide sequences representing common hydrophobic motifs mediate the migration of most endocrine proteins across the mammalian endoplasmic reticulum (ER) and prokaryotic plasma membrane via putative protein channels. The main model shows that proteins are transported across the membrane via hydrophilic protein channels formed by several membrane proteins. In eukaryotes, newly synthesized proteins in the cytoplasm are directed to the ER membrane by signal sequences generally recognized by signal recognition particles (SRP) and their ER membrane receptors. This targeting step is followed by the actual movement of the protein from the cell through the ER membrane and through putative protein channels. Signal peptides can also interact strongly with lipids, supporting the proposal that transport of some secreted proteins across the cell membrane may occur directly through the lipid bilayer in the absence of protein channels. The Such signal peptides can be used to increase the internalization of antibodies or other biologically active molecules into cells, which are subject to several patents (US Pat. No. 5,807,746). No. 6,043,339 and 6,238,667).

  Antibodies have been used as delivery devices for some biologically active molecules such as toxins, drugs and cytokines. Antibody fragments, Fab or scFv are often preferred for better tissue penetration and low “viscosity”.

  There are two practical ways to link antibody molecules with molecules such as peptides. One method is to use chemical cross-linking, such as the affinity cross-linking method described in US 09 / 070,907. Another method is to design a fusion gene comprising DNA encoding the antibody and peptide, and to express the fusion gene, which is the subject of this application.

  Antibody fusion proteins are typically engineered with entire large protein genes or domains that confer the biological function of such proteins. Previous small peptide-antibody fusion proteins were typically made primarily to facilitate antibody purification or characterization.

  Methods for making fusion proteins are described, for example, in the following US patents, the related disclosures of which are incorporated herein by reference: US Pat. No. 5,563,046 to Mascarenhas et al .; Fell Jr. US Pat. No. 5,645,835 to Murphy US Pat. No. 5,668,225 to Murphy US Pat. No. 5,698,679 to Nemaze US Pat. No. 5,763,733 to Whitlow et al. US Pat. No. 5,811,265 to Quertermous et al. US Pat. No. 5,908,626 to Chang et al. US Pat. No. 5,969,109 to Bona et al. US Pat. US Pat. No. 6,008,319; US Pat. No. 6,117,656 to Seed; US Pat. No. 6,121,424 to Whitlow et al .; US Pat. No. 6,132,992 to Ledbetter et al. US Pat. No. 6,207,804 to others; and US Pat. No. 6,224,870 to Segal. Methods for making Ig fusion proteins are described, for example, in “Antibody Engineering”, 2nd edition: Carl A. et al. K. Borrebaeck, Oxford University Press, 1995) and “Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, 1989, disclosed in these Are incorporated herein by reference.

Proteins such as cytokines, fusion proteins with immunoglobulins mainly containing active domains such as toxins and enzymes, immunoglobulin target domains containing CDRs (complementarity determining regions), and antigen binding but not directly involved Fusion proteins, including fusion proteins with other variable regions and domains that can provide high binding affinity through subsequent interactions, are described, for example, in the following publications incorporated herein by reference: Has been:
Guo L; Wang J; Qian S; Yan X; Chen R; Meng G, “Construction and structural modeling of the single-chain Fv-asparaginase fusion protein resistant to proteolysis. asparaginase fusion protein resistant to protease) "Biotechnol. Bioeng. 20 November 2000; 70 (4): 456-63;
Muller BH; Chevrier D; Boulain JC; Guesdon JL “Recombinant single-chain Fenfragmentation fragment” for a one-step immunodetection method of molecular hybridization. -Step immunodetection in molecular hybridization) "J. Immunol Methods July 30, 1999; 227 (1-2): 177-85;
Griep RA; van Twisk C; Kerschbaumer RJ; Harper K; Torrance L; Himmler G; van der Wolf JM; Schotts' pSKAP / S: expression vector for generating a single chain Fv alkaline phosphatase fusion protein (pSn: AP vector for the production of single-chain Fv alkalline phosphate fusion proteins) "Protein Expr. Purif. 16 June 1999 (1): 63-9;
Valera DA; Panoskaltsis-Mortari A; 1C; Ramakrishnan S; Eide CR; Kreitman RJ; Nichols PJ; Pennell C; Blazar BR "DT390 anti-CD3sFv, a Tv receptor-targeted CD3ε-specific F3 Anti-graft-versus-host disease effect of DT390-anti-CD3sFv, a-single-chain Fv fusion immunotoxicity pristine3 September 15, 1996; 8 (6): 2342-53;
Gupta S; Eastman J; Silski C; Ferkol T; Davis PB “Single-chain Fv: ligand in receptor-mediated gene delivery” Gene1 4 months (8): 586-92; and Goel A; Colcher D; Koo JS; Boot BJ; Pavlinkova G; Batra “The relative position of the hexahistidine tag affects the binding of the tumor-associated single-chain Fv construct. (Relative position of the hexhistagine tag effects binding properties of a tu-associated . Le-chain Fv construct) "Biochim Biophys Acta 9 January 2000; 1523 (1): 13-20.

  Fusion proteins designed to have biological activity can be constructed using chain peptide sequences derived from proteins with full biological activity. However, such peptides typically have a lower affinity than the complete protein. Since the incorporation of peptides into a fusion protein is not as cumbersome as the incorporation of a fully functional protein, a fusion protein comprising a peptide with a binding affinity that is as good as a full-length protein is needed.

  The invention also relates to the use of antibodies and fragments thereof in inhibiting apoptosis. Cell suicide (apoptosis) is a mechanism beneficially used by living organisms during cell differentiation, organogenesis and removal of damaged cells. However, apoptosis may be associated with some form of pathogenesis. For example, apoptosis is a major cause of loss of neurons in Alzheimer's disease and loss of tissue during myocardial infarction. Furthermore, T lymphocytes from HIV-1 infected individuals undergo spontaneous apoptosis in the absence of stimulation compared to uninfected T cells cultured under the same conditions. “Spontaneous apoptosis” of CD4 + and CD8 + cells has been shown to be accelerated by in vitro addition of HIV-1-related, anti-idiotypic antibodies.

  Caspase enzymes, such as caspase-3, are highly related to the apoptotic pathway. In an attempt to inhibit apoptosis, several substances and methods for inhibiting caspase action have been proposed. For example, US Pat. No. 6,566,338 (Weber et al.) Treats, ameliorates and prevents non-cancer cell death during chemotherapy and radiation therapy, and treats the side effects of cancer chemotherapy and radiation therapy. And to improve the use of caspase inhibitors in general. US Pat. No. 6,596,693 (Keana et al.) Reports that some dipeptides may be potent inhibitors of apoptosis. US Pat. Nos. 6,689,784 (Bebbington et al.) And 6,620,782 (Cai et al.) Propose a class of carbamates and substituted 2-aminobenzamides, respectively, as inhibitors of apoptosis. In addition, US Pat. No. 6,426,413 (Wannamaker et al.) Is a representative proposal for a class of caspase inhibitors called interleukin-1β-convertase inhibitors. In addition, US Pat. No. 6,228,603 (Reed et al.) Proposes a screening assay to identify substances that alter the specific binding of an inhibitor of apoptosis to a caspase such as caspase-3 or caspase-7. ing.

  Yet another novel approach for inhibiting caspase enzymes involves the use of so-called “superantibody technology (SAT)”. See, for example, WO 02/097041, entitled “Fusion Proteins of Biologically Active Peptides and Antibodies” (Immpheron, Inc. and Innexus Corporation). Simultaneous transfer). One proposed application of SAT is to inhibit apoptosis of living cells using antibodies against caspase enzymes. For example, one aspect of the invention contemplates intracellular delivery of antibodies or antibody fragments that are immunospecific for enzymes involved in apoptosis. Some anticipated advantages of transmembrane antibodies as apoptosis inhibitors are their specific target recognition in cells and their low toxicity compared to conventional apoptosis inhibitors.

  The object of the present invention is to provide such membrane permeable antibodies for therapeutic effect.

  The present invention provides a fusion protein comprising an antibody domain and a peptide domain, wherein the biological activity of the peptide domain is selected from the group consisting of immunostimulation, membrane transport and homophilic activity. The peptide is covalently bound to a site on the antibody so that the incorporated peptide does not reduce the antigen recognition of the antibody. In the present invention, this is the step of creating a fusion gene comprising a nucleic acid sequence encoding an antibody and a nucleic acid sequence encoding a peptide, wherein the nucleic acid sequence encoding the peptide is within the nucleic acid sequence encoding the antibody. The resulting fusion protein comprises an antibody and a peptide, wherein the peptide is located at a site that binds to the antibody at a site that does not impair the antigen binding of the antibody, and a fusion gene is expressed to express the fusion protein Is carried out by a method comprising the steps of: Specifically, the fusion protein is a step of providing a gene encoding an antibody, the gene being mutated to contain a restriction site, the restriction site being from any part of the gene that encodes the antigen binding site of the antibody. A DNA sequence coding for a peptide having a biological activity selected from the group consisting of immunostimulatory, membrane transport and allophilic activities is inserted into the restriction site of the gene encoding the antibody. A fusion gene, wherein a DNA sequence encoding a peptide is inserted in-frame with a gene encoding an antibody, and the fusion gene is expressed to produce a fusion protein Can be produced by the following steps.

  To increase the biological activity of the peptide, the peptide can be placed on the side of a sequence that provides a loop-forming sequence or conformation.

  The present invention also provides compositions and pharmaceutical compositions comprising a peptide-antibody fusion protein having biological activity selected from the group consisting of immunostimulation, membrane transport and homophilic activity.

  The present invention for making biologically active peptide-antibody fusion proteins includes peptides that include self-binding, stimulate lymphocytes, and allow transport across biofilms.

  Another aspect of the present invention relates to novel compounds and methods for modulating cellular function in normal or infected cells. In particular, such compounds and methods involve the use of antibodies or antibody fragments thereof conjugated to membrane transport peptides. The antibody or fragment thereof preferably comprises (a) intracellular signal proteins such as caspases, kinases, and phosphatases, (b) immature viral proteins prior to construction in the cells, (c) cell surface antigens or intracellular Immunospecific for protein targets such as tumor antigens, (d) nuclear or nucleolar proteins involved in the regulation of DNA synthesis and gene expression, or (e) cytoskeletal proteins involved in cell proliferation or cell division arrest That is, the antibody or fragment thereof recognizes them and binds specifically with high affinity. Polyclonal or monoclonal antibodies can be used.

  In a preferred embodiment of the invention, the aforementioned compound is effective to inhibit apoptosis and comprises an anti-caspase antibody or fragment thereof conjugated to a membrane transport peptide. A particularly preferred antibody is an anti-caspase-3 antibody.

  In a second preferred embodiment of the present invention, said membrane transport peptide is a translocation sequence (MTS) peptide such as Kaposi's sarcoma fibroblast growth factor, HIV-1 TAT peptide, Antennapedia homeodomain-derived peptide, herpes virus The protein VP22, or a peptide endogenous to the transport peptide. Particularly preferred MTS peptides include the amino acid residue sequence AAVLLPVLLAAP (SEQ ID NO: 9), the peptide sequence KGEGAAVLL PVLLAAPG (SEQ ID NO: 8), and the like.

  A pharmaceutical composition that is effective in inhibiting apoptosis in human cells and is therefore shown to be effective in treating human diseases, an anti-caspase antibody conjugated to a membrane transport peptide, such as an MTS peptide or the like Also contemplated are pharmaceutical compositions comprising the fragments. The antibody-peptide conjugates of the invention can cause internalization of the antibody or antibody fragment into cells.

  In another aspect of the invention, a method for treating or preventing a disease comprises a patient in need of a pharmacologically effective amount of a pharmaceutical composition comprising an anti-caspase antibody or fragment thereof conjugated to a membrane transport peptide or fragment thereof. Administration. Shown in detail are modified anti-caspase antibodies conjugated with membrane transport peptides that reduce chemically induced apoptosis. These results suggest that such antibodies have therapeutic potential for inhibiting apoptosis in various diseases such as Alzheimer's disease, Huntington's disease, or Parkinson's disease.

  The foregoing and other objects of the invention will become readily apparent to those skilled in the art from the following detailed description and drawings, which show and describe only preferred embodiments of the invention. As will be readily appreciated, the invention is capable of modifications within the skill of the relevant art without departing from the spirit and scope of the invention.

  The present invention describes a method of making a peptide-antibody fusion protein having a biological activity selected from the group consisting of immunostimulation, membrane transport and homophilic activity.

  In particular, the present invention provides a biological activity selected from the group consisting of immunostimulation, membrane transport and homophilic activity, wherein the peptide is located at a site in the antibody so that the incorporated peptide does not impair the antigen recognition of the antibody. A fusion protein comprising a peptide having an antibody and an antibody is provided. In the present invention, this is a step of producing a fusion gene comprising a nucleic acid sequence encoding an antibody and a nucleic acid sequence encoding a peptide, wherein the nucleic acid sequence encoding the peptide is within the nucleic acid sequence encoding the antibody. The resulting fusion protein contains an antibody and a peptide, the peptide is located at the site where it binds to the antibody at a site that does not impair the antigen binding of the antibody, and the fusion gene is expressed to produce the fusion protein The method includes the steps of: Specifically, the fusion protein is a step of providing a gene encoding an antibody, the gene being mutated to contain a restriction site, the restriction site being from any part of the gene that encodes the antigen binding site of the antibody. A DNA sequence coding for a peptide having a biological activity selected from the group consisting of immunostimulatory, membrane transport and allophilic activities is inserted into the restriction site of the gene encoding the antibody. A step of inserting a DNA sequence encoding a peptide in frame with a gene encoding an antibody, and expressing the fusion gene to generate a fusion protein. It can be produced by steps.

  In other embodiments of the invention, the biologically active peptide can be linked to the C-terminus of the antibody. In other embodiments of the invention, the peptide can be placed on the side of a sequence that provides a loop-forming sequence or conformation to increase the biological activity of the peptide.

  As used herein, the term “targeting moiety” refers to any natural or synthetic protein molecule that contains an antigen binding site. The term refers to a CDR region, ScFv, Fab, F (ab) ′ 2, or a modified antibody mimetic or single chain domain, such as a full length immunoglobulin molecule or any functional fragment, a variable domain fragment of a full length immunoglobulin molecule. Includes a binding moiety. The individual target moieties are selected according to the desired target, such as a cellular receptor on the membrane structure, such as a protein, glycoprotein, polysaccharide or carbohydrate. A targeting moiety can be selected to bind to a cellular receptor on normal or tumor cells.

  Similarly, peptides having biological activity are selected according to the desired function of the fusion protein, or in other words, according to the desired result after the target moiety has bound to a target such as a normal cell or tumor cell. Biological activities that may be desirable include immunostimulation, membrane trafficking, and homophilic activity.

The loop-forming sequence or the conformation constraining sequence may be any amino acid sequence that, when placed on either side of a biologically active peptide, reduces the conformational flexibility of the peptide. An example is a sequence comprising amino acid residues, such as a cysteine pair, that can be bridged to form a loop. A specific example of a protein that constrains conformation is thioredoxin. Examples of conformationally constraining or loop-forming portions include, for example, the following US patents: US Pat. Nos. 6,242,163 and 6,004,746 to Brent, US Pat. No. 6,258,550. No. 6,147,189; No. 6,111,069; No. 6,100,044; No. 6,084,066; No. 5,952,465; No. 5,948,887; No. 5,928,896 to Brent et al., US Pat. Nos. 6,200,759 and 5,925,523 to Dove et al., And in the following publications:
Fairy DP; West ML; Wong AK “Tours protein surface mimetics” Curr Med Chem Feb 1998; 5 (1): 29-62;
Valero ML; Camarero JA; Haack T; Mateu MG; Domingo E; Giralt E; Andrew D “Circular peptide model resembling the prototype of the viral antigenic site: discovery of a balance between stiffness and flexibility (native-like cyclic peptide of a viral antigenic site: finding a balance between rigidity and flexibility) J Mol Recognition 2000 January-February 2000; 13 (1): 5-13;
Guraura TL; Narasihamurty S; Payan DG; “A novel artificial loop scaffold bioconstant strain.” July 2000; 7 (7): 515-27;
Venkatesh N; im SH; Balass M; Fuchs S; Katchalski-Katzir E "Prevention of passively immunized myasthenia gravis by passively transmitted experimental autoimmune myasthenia gravis. gravis by a phase library-derived cyclic peptide) "Proc Natl Acad Sci USA January 18, 2000; 97 (2): 761-6;
Stott K; Blackburn JM; Butler PJ; Perutz M “Incorporation of glutamine regenerative sensitizing sensitization rations: a protein is oligomerized by the incorporation of glutamine repeats.” July 3, 1995;
All of the aforementioned publications are incorporated herein by reference.

Conformational constraining sequences can also include sequences that form α-helices or β-pleated sheets. For example, see the following publications incorporated herein by reference:
Lee KH; Benson DR; Kuczera K “Transitions from alpha to pi symbiotic sym metric symmetries in the dynamic simulation of molecules of synthetic peptides” 14 November; 39 (45): 13737-47;
Dettin M; Roncon R; Simonetti M; Torinene S; Falcigno L; Paulillo L; Di Bello C “Synthesis, characterization and conformational analysis of gp120-derived synthetic peptides that specifically increase HIV-1 infectivity (Synthesis) , Characterization and conformational analysis of gp120-derived synthetic peptides that specifically enhanced HIV-1 infectivity.)-1 Jpept, Sci 1997;
Chavali GB; Nagpal S; Majudar SS; Singh O; Salunke DM “The helix-loop-helix motif in the GnRH-binding peptide is important for negative regulation of prolactin secretion (Helix-loop-helix motid in nipRips in ncId critical for negative regulation of production section) "J Mol Biol. 1997/10/10; 272 (5): 731-40; and Micheli R; Myszka D; Mao LI; Sathe G; Chaiken I “The coiled coil stem loop miniplot miniprote as a scaffold shown. as a presentation scaffold) "Drug Des Discov. 1996 April; 13 (3-4): 95-105.

  Expression of Ig fusion protein. Ig fusion proteins have the advantage of conjugating molecules that contribute to unique properties with antibodies that combine specificity and / or antibody effector function. The ability to generate this protein family was first demonstrated when c-myc was replaced with Fc of an antibody molecule (Neuberger MS, Williams GT and Fox RO, Nature, 125: 604, 1984). Many examples now exist. Ab fusion proteins can be obtained in several different ways. In one approach, a non-Ig sequence is replaced with a variable region; molecules with substituted V regions provide specificity to target antibodies that contribute properties such as effector function, and improved pharmacokinetics. Examples are IL-2 and CD4. Alternatively, the non-Ig sequence can be replaced with or combined with a constant region. The resulting molecule retains the binding specificity of the original antibody, but gains properties from the protein it binds to. Depending on the location of the substitution, different antibody-related effector functions and biological properties will be retained. For example, “Antibody Engineering”, 2nd edition: Carl A. et al. K. See Borrebaeck, Oxford University Press, 1995).

  A vector for constructing IgG fusion proteins. A series of vectors has now been generated, which allows for the fusion of proteins at different positions within the antibody molecule, thus facilitating the construction of fusion proteins with different properties. These vectors can be used to generate fusion protein families with molecules that differ in molecular weight, valence electrons, and have different subsets of antibody molecule functionality.

  As a specific example of a method that facilitates the construction of a fusion gene, a site-directed mutagenesis method was used to create a unique restriction enzyme site in the human IgG3 heavy chain gene. In this particular example, restriction sites were generated at the 3 'end of the CH1 exon, immediately after the hinge at the 5' end of the CH2 exon, and at the 3 'end of the CH3 exon. The restriction sites generated in this way are: SnaBI at the end of CH1 by replacing TtgGTg with TacGTa, PvuII at the first part of CH2 by replacing CAcCTG with CAgCTG, and CH3 by replacing AATgag with AATatt. SspI at the end of These manipulations provided unique blunt end cloning sites at these positions. In both cases, the restriction site was located so that Ig contributes to the first base of the codon after cleavage. Human IgG3 with an extended hinge region of 62 amino acids is selected for use as an immunoglobulin. When present, this hinge region should provide space and flexibility, thereby facilitating simultaneous antigen and receptor binding. An EcoRI site was also introduced at the 3 'end of the IgG3 gene to give a 3' cloning site and a poly A addition signal. Although originally designed for use with growth factors, these restriction sites can be used to place any new sequence at a well-defined position in the antibody. In addition, the variable regions can be easily altered using these cloning cassettes. Similar techniques can be used to create appropriate restriction sites in other antibody genes.

  Generation of fusion genes. As a first step in the production of the fusion protein, a blunt end restriction site must be introduced at the desired location, at the 5 'end of the gene to be fused. In order to maintain the correct reading frame, the site must be located so that after cleavage it contributes 2 bases to the codon. When the goal is to create a fusion protein with the complete molecule, restriction sites are usually introduced at any post-translational processing position, such as a position after the leader sequence. Alternatively, if the goal is to use only a portion of the protein, a blunt end site can be introduced at any position in the gene, but care must always be taken to maintain the correct reading frame. Furthermore, if there is post-translational processing at the carboxyl terminus of the fusion protein, it is often desirable to introduce a stop codon at this processing site.

  A significant concern during the production of the fusion protein is to preserve the biological activity of all elements. Generation of a fusion protein with an antibody is facilitated by the domain structure of the antibody, and all cloning sites are located immediately after the complete domain. This configuration should ensure correct folding of the immunoglobulin. The folding of the binding protein depends on its structure and its fusion location. Whenever structural information is available, it is desirable to generate the fusion at a location that preserves the structural integrity of the binding protein.

  In order to produce a sufficient amount of protein for functional analysis, it is desirable to secrete the protein into the medium. In the examples reported to date, the fusion protein to be constructed is constructed and secreted, but this is still a concern when designing other fusion proteins.

  A method for designing a fusion gene comprising a biologically active peptide as part of a heavy or light chain gene is described in the established antibody engineering protocol “Antibody Engineering”, 2nd. Edition: Carl A. K. Borrebaeck, Oxford University Press, 1995, Chapter 9, pages 267-293). The peptide can be fused to the N-terminal residue or C-terminal residue of the H or L chain. Expression of such fusion genes is typically performed in mammalian cell systems, although other expression systems can be used, such as bacterial or yeast expression systems.

  The peptides of the present invention have a biological activity selected from the group consisting of immunostimulation, membrane transport and homophilic activity. Examples include immunostimulatory or immunomodulatory activity. The peptide may be, for example, a binding site derived from a cytokine hormone, ligand, or native ligand of a cellular receptor. In a preferred embodiment, the peptide is derived from C3d region 1217-1232 and ranges from about 10 to about 16-mer. In another embodiment, the peptide is a 16-mer peptide derived from C3d region 1217-1232.

  The peptide can bind to an antibody that is a full-length immunoglobulin molecule or a variable domain fragment of an antibody. As used herein, the term “antibody” generally refers to a heavy or light chain immunoglobulin molecule comprising an antigen binding site, or any functional combination or fragment thereof. The antibodies are preferably specific for cell receptors on membrane structures such as proteins, glycoproteins, polysaccharides or carbohydrates, and on normal or tumor cells.

  The use of a peptide derived from the ligand site of C3d as an immunostimulatory element to be incorporated into the antibody has unexpected utility as a molecular adjuvant. D. By Fearon et al. (Dempsey, P.W., Allison, M.E.D., Akkaraju, S., Goodnow, C.C. and Fearon, D.T., Science, 271: 348, 1996), C3d It has been used as a molecular adjuvant as part of a complete fusion protein with hen egg lysozyme (HEL). These authors have shown that the HEL-C3d fusion protein is 10,000 times more immunogenic than free HEL (see International Patent Publication No. WO 96/17625).

  In our latest animal experiments, a similar increase in immunogenicity has been observed using idiotype vaccines chemically cross-linked using peptides derived from C3d fragments (see examples below). Combining C3d peptides with idiotype and anti-idiotype vaccines increases the immunogenicity of these vaccines and has been approved for human use by the FDA in combination with powerful adjuvants such as Freund's adjuvant. This is considered to be an alternative to the need for binding of carrier molecules such as KLH.

  In other embodiments, the peptide may be derived from a human or non-human C3d region that is homologous to the human C3d residue at positions 1217-1232 and ranges from about 10 to about 16 mer. Other applications of peptides with affinity cross-linked biological activity for antibody vaccines include cytokine-derived active peptides. For example, nanopeptides derived from IL1-β cytokines have been described (Antoni et al., J. Immunol, 137: 3201-04, 1986) and have immunostimulatory effects that do not induce undesirable side effects. Other examples of active peptides that can be inserted into antibodies according to the present invention include signal peptides and peptides derived from antibody self-binding sites.

  Various peptides are known that have biological activity, such as cytokine hormones, ligands, or binding sites derived from the natural ligands of cellular receptors.

  Examples 1-3 below relate to C3d / antibody complexes made by affinity cross-linking, but show the impact on the immune response provided by the C3d peptide that provided them and bound to the antibody.

  Improvement of anti-idiotype vaccine. 3H1 is a murine anti-idiotype antibody (Bhattacharya-Chatterjee et al., J. Immunol., 145: 2758-65, 1990), which mimics carcinoembryonic antigen (CEA). 3H1 induces anti-CEA antibodies in animals when used as a KLH-conjugated vaccine in Freund's complete adjuvant. 3H1 has also been tested in clinical phase I trials, where 3H1 induces antibodies that bind to CEA in approximately half of treated cancer patients. However, due in part to low immunogenicity, no clinical response was observed in this study (Foon et al., J. Clin. Invst., 96: 334-342, 1995).

  3H1 mAb was affinity cross-linked with a 13-mer peptide (SEQ ID NO: 1) derived from C3d region 1217-1232. The amino acid sequence is derived from the Cd3 peptide and has the following sequence: KNRWEDPGKQLYNVEA (SEQ ID NO: 1).

  BALB / c mice were immunized twice intramuscularly with 25 μg of C3d-3H1 dissolved in phosphate-saline solution. Seven days after the last immunization, mice were bled and sera were tested for binding to 8019 (Ab1 idiotype) and CEA expressing tumor line LSI74T. As measured by FACS, sera from C3d-3H1 immunized mice bound to LS174T tumor cells, whereas control sera (normal mouse sera) showed only background fluorescence. Sera from mice immunized with C3d-3H1 were used in FACS of LS174T cells in a sandwich assay developed with FITC-conjugated goat anti-mouse IgG. The control was normal mouse serum. The number of cells analyzed was plotted against the relative fluorescence intensity of log 10 units.

  In addition, sera from mice immunized three times with 3H1 (25 micrograms in saline) or 3H1-C3d-peptide (affinity cross-linking, 25 micrograms in saline) were also tested for Ab3 responsiveness. Mice were bled and sera were tested for binding of 3H1 to F (ab) in an ELISA. By measuring the binding of 3H1F (ab) with a dilution of mouse serum, naked 3H1 did not induce Ab3 antibody, but 3H1-peptide induced, and affinity-crosslinked 3H1 increased immunogenicity. showed that.

  Other C3d peptides that can be used in practicing the present invention include Lambris et al., “The third component of complement, C3 phylogeny, incorporated herein by reference in its entirety. Phylogeny of the component of complement, C3) ", Erfi, A ed. “New Aspects of Complement Structure and Function”, Austin, R .; D. Landes Co. 1994 p. There are peptides reviewed in 15-34.

  Enhancement of mouse tumor idiotype vaccine (38C13). 38C13 is an idiotype expressed by the 38C13 B-lymphoma tumor cell line. The Levy group has developed this idiotype tumor vaccine model and has shown that pre-immunization with KLH-conjugated 38C13Id can protect against challenge by 38C13 tumor cells in mice ( Kaminski, MS, Kitamura, K., Maloney, DG, and Levy, R., J. Lnuniol., 138: 1289, 1987). Levy and colleagues (Tao, MH, and Levy, R., Nature, 362: 755-758, 1993) have developed a fusion protein (CSF-) produced from a chimeric gene and expressed in fermentation of mammalian cell cultures. Induction of tumor protection using 38C13) has also been reported. The 38C13Id protein was affinity cross-linked with a 16-mer azide-peptide derived from the C3D region 1217-1232.

  Ten mice were immunized three times intraperitoneally with 50 ug of C3d-38C13 conjugate dissolved in phosphate-saline solution. After the third vaccination, mice were challenged with 38C13 tumor cells. The control group included mice vaccinated with 38C13-KLH (adjuvant) dissolved in QS-21 considered as the “gold standard” in this tumor model, and mice injected with QS-21 alone. Seven of 10 mice vaccinated with the C3d-38C13 conjugate survived until day 35 after tumor challenge, as did mice vaccinated with KLH-38C13 dissolved in QS-21. All control mice were injected with QS-21 only and died by day 22.

  C3H mice were immunized three times with 38C13-KLH dissolved in QS-21 or 38C13-C3d peptide without QS-21 (50 μg, intraperitoneally). Control mice were injected with QS-21 only. Immunized and control mice were then challenged with 38C13 tumor cells and examined for survival.

  The results described in Examples 1-3 show that the immune response to tumors is significantly increased and protected against tumor challenge by cross-linking by affinity of immunostimulatory peptides and tumor anti-idiotype and idiotype vaccine antibodies Show that you can. Vaccination protocols using C3d cross-linked vaccines do not include any adjuvant, such as Freund's adjuvant, or KLH conjugate, both of which have not been approved by the FDA for use in humans.

  Some of the procedures used in the previous examples are known and the active binding peptide of C3d (complement fragment) is described by Lambris et al. (PNAS, 82: 4235-39, 1985), which The entirety of which is incorporated herein by reference.

  The following additional examples are given to demonstrate a general technique for making fusion proteins and to identify specific peptides with biological activity selected from the group consisting of immunostimulation, membrane transport and homophilic activity Show.

  A fusion non-Ig protein comprising a membrane transfer peptide (MTS-peptide). For example, Rojas, M, Donahue, JP, Tan, T .; And Lin, YZ. Nature Biotech. 16: 370, “Construction of the glutathione S-transferase-MTS peptide (GST-MTS) expression plasmids,” 1998. See, “Establishment of the glutathione S-transferase-MTS peptide (GST-MTS) expression plasmid”.

Two different GST-MTS expression plasmids were constructed to be able to generate target proteins or protein domains with hydrophobic MTS as the amino-terminal or carboxyl-terminal extension, depending on the biological application. To construct plasmids pGEX-3X-MTSI and pGEX3X-MTS2, the following complementary oligonucleotides were synthesized:

  After annealing, double stranded MTSI and MTS2 oligonucleotides were ligated in pGEX-3X digested with BamHI (Smith, DB and Johnson, KS, “Fusion with Glutathione S-Transferase”). "Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transfer", Gene, 67:31. DNA sequence analysis confirmed that the MTS coding sequence was correct and in-frame with the GST coding sequence in each plasmid.

Construction of GST-Grb2SH2, GST-Grb2SH2-MTS, and GST-StatlSH2-MTS expression plasmids. Human Grb2SH2 domain (amino acid residues 54-164) (Lowenstein, EJ, Daly, RJ, Batzer, AG, U, W., Margollis, B., Lammers, R et al., “SH2 And SH3 domain-containing protein Grb2 binds to receptor tyrosine kinases and signals ras (The SH2 and SH3 domain-containing protein Grb2 links receptor tyrosine kinases to rassign, 19, 43, StatlSH2 domain (residues 567-716) (Schindler, C., Fu, XY, Impnota, T., Abersold, R., and Darne) ll, JE Jr., Proc. Natl Acad. Sci USA 89: 7836, 1992) was synthesized by PCR from a Grb2 cDNA clone or a Statl cDNA clone. The primers used for PCR, each containing a BamHI site at their 5 ′ end, were:

  The PCR product was digested with BamHI and ligated in pGEX-3X or pGEX-3XMTS2 digested with BamHI. DNA sequence analysis of the vector / insert junction confirmed that the translation reading frames of GST-Grb2SH2, GST-Grb2SH2-MTS, and GST-Stat1SH2-MTS were maintained in the respective expression plasmids.

Expression of MTS fusion protein Expression and purification of GST fusion protein. E. coli strain DHSor containing the appropriate expression plasmid 74 was grown in LB broth containing 100 μg / ml ampicillin at 37 ° C. Expression of the GST fusion protein was induced by adding isopropyl, BD-thiogalactoside (0.5 mM final concentration) and incubation at 37 ° C. was continued for 2-3 hours. The GST fusion protein was purified from bacterial cell lysates by glutathione-agarose affinity chromatography (Smith, DB and Johnson, KS Gene, 67:31, 1988), but after sonication Prior to mixing with glutathione-agarose beads, cell lysates were removed by centrifugation at 2000 rpm for 5 minutes. The protein preparation was concentrated by ultrafiltration using a PMIO membrane (Amicon, Beverly, Mass.) And stored at 4 ° C for immediate use or at -70 ° C for long-term storage. Protein concentration was measured by a spectrophotometer at 280 nm. Immediately prior to their use in biological assays, protein concentrations were confirmed by SDS-PAGE using Coomassie brilliant blue staining intensity compared to known concentrations of wild-type GST. In order to confirm the amino acid content of MTS in the GST-MTS protein, a protease factor almost as described in (Smith, DB and Johnson, KS, Gene 67:31, 1988). The MTS peptide was cleaved from glutathione-agarose-bound GST-MTSI using Xa. The cleaved MTS-containing peptide was purified by C reverse phase HPLC and characterized by mass spectrometry as described in (Smith, DB and Johnson, KS, Gene 67:31, 1988). did. The cleaved MTS-containing peptide was purified by _C18 reverse phase HPLC (Lin, Y-Z., Yao, S., Veach, RA, Torgerson, R., and Hawiger, J., JBiol. Chem). 270: 14255, 1995) was characterized by mass spectrometry.

C3d-HEL fusion protein (Dempsey et al., Science, 271: 348, 1996). HEL, C3d (H. Domdey et al., Pro. Natl Acad Sci USA, 79: 7619, 1982) doq pre-pro-insulin signal sequence (ME Taylor and K. Drickkamer, Biochem. J., 274, 575, 1991), and complementary DNA encoding the (G4S) ₂ linking group was amplified by polymerase chain reaction. The epitope tag and stop codon were encoded by the oligonucleotide linking group. The fusion protein cassette is tandem: doq pre-pro-insulin signal sequence, HEL, and 1 to 3 copies of C3d linked by (G ₄ S) _{2 in} pSG5 (Stratagene Cloning Systems, La Jolla, Calif.). Built with. The HEL-C3d3 cassette was subcloned into the A71d vector. Plasmid pSG. HEL, pSG. HEL. C3d, and pSG. HEL. C3d2 was cotransfected into L cells and A71d with pSV2-neo. HEL. C3d3 was transiently expressed in COS cells. The recombinant protein was purified by affinity chromatography on the YL1 / 2 antibody (H. Skinner et al., J. Biol. Chem., 66: 14163, 1991) and fractionation on Sephacryl S-200 (Pharmacia). .

  Fusion tails are useful on a laboratory scale and have the ability to enhance recovery using economical recovery methods, which are easily scaled up for industrial downstream processes. The fusion tail can be used to facilitate secretion of the target protein and can provide useful assay tags based on enzyme activity or antibody binding. Many fusion tails have not been compromised by the biological activity of the target protein and in some cases have been shown to stabilize it. Nevertheless, for the purification of the authentic protein, a site that is specifically cleaved is often included, and the tail can be removed after recovery.

Fusion tail for recovering and purifying the recombinant protein. (See, for example, Ford C., Suominen I., Glatz C., Protein Expr. Purif. 2-3: 95-107, 1991). The fusion proteins of the invention can also include a fusion tail that has been developed to facilitate efficient recovery and purification of recombinant proteins from crude cell extracts or culture media. In these systems, the target protein is genetically modified to include a C-terminal or N-terminal polypeptide tail, which provides a biochemical basis for specificity in recovery and purification. Tail with various characteristics have been used:
(1) a complete enzyme with affinity for an immobilized substrate or inhibitor;
(2) a peptide binding protein having affinity for immunoglobulin G or albumin;
(3) a carbohydrate binding protein or domain;
(4) a biotin-binding domain for in vivo biotinylation to promote the affinity between the fusion protein and avidin or streptavidin;
(5) an antigenic epitope having an affinity for the immobilized monoclonal antibody;
(6) poly (His) residues for recovery by affinity chromatography of fixed metals; and (7) other poly (amino acids) with binding specificity based on the nature of the amino acid side chain.

  Fusion tails are useful on a laboratory scale and have the ability to enhance recovery using economical recovery methods, which are easily scaled up for industrial downstream processes. The fusion tail can be used to facilitate secretion of the target protein and can provide useful assay tags based on enzyme activity or antibody binding. Many fusion tails have not been compromised by the biological activity of the target protein and in some cases have been shown to stabilize it. Nevertheless, for the purification of the authentic protein, a site that is specifically cleaved is often included, and the tail can be removed after recovery.

  The present invention describes the production of antibody-peptide fusion proteins that enhance the biological and immune activity of the antibody without changing the specificity of the antibody for the corresponding antigen. This genetically modified fusion protein is similar to the chemically modified chimeric antibody described in patent application 09 / 070,907. In particular, the present invention provides for the production of an antibody fusion protein comprising a complete or partial autotrophic 24-mer peptide, a membrane transport peptide (MTS) or a C3d peptide, all as previously described.

  The present invention relates to a peptide having a biological activity selected from the group consisting of (1) an antibody and (2) immunostimulation, membrane transport, and homophilic activity, wherein the antibody does not harm the antigen binding of the antibody. Also provided are compositions and pharmaceutical compositions comprising a fusion protein composed of a peptide linked by a peptide bond.

  Any antibody can be used in the peptide / antibody complex of the invention. A preferred antibody is an anti-idiotype antibody. For example, anti-idiotype antibody 3H1 can be used ("Anti-idiotype Antibody Vaccine (3H1) that Mimics the Carcinoembrym that mimics fetal cancer antigen (CEA) as an adjuvant treatment." Antigen (CEA) as an Adjuvant Treatment) ", Foon et al., Cancer Weekly, Jun. 24, 1996). Other anti-idiotype antibodies that can be used in the present invention include, for example, anti-idiotype antibodies to Chlamydia glycolipid extra antigen (US Pat. No. 5,656,271); anti-therapeutic agents for the treatment of melanoma and small cell carcinoma Idiotype antibody 1A7 (US Pat. No. 5,612,030); anti-idiotype antibody MK2-23 anti-melanoma antibody (US Pat. No. 5,493,009); anti-idiotype gonococcal antibody (US Pat. No. 5,476) , 784) Pseudomonas aeruginosa anti-idiotype antibody (US Pat. No. 5,233,024); antibody against surface Ig of human B cell tumor (US Pat. No. 4,513,088); and monoclonal antibody BR96 (US patent) No. 5,491,088). Any restrictions on peptide length are actual restrictions on peptide synthesis, but not on the practice of the method of the invention.

  Further, (Kang, CY Brunck, TK, Kiever-Emmons, T., Blicker, JE and Kohler, H., “the entire contents of which are incorporated herein by reference.” Inhibition of self-binding proteins (autoantibodies) by VH-derived peptides (Inhibition of self-binding proteins (auto-antibodies) by a VH-derived peptide) ", Science, 240: 1034-1036, 1988). Self-binding peptides such as can be used in the methods of the invention.

  Furthermore, Rojas et al., “Genetic Engineering of Proteins with Cellular Permeability”, Nature Biotechnology 75, 37:37, the entirety of which is incorporated herein by reference. (1988) and “Calbiochem Signal Transduction Catalog” 1997/98, signal peptides such as those disclosed in 1997/98 can be used in the methods of the present invention.

  In accordance with the disclosure of US Pat. No. 5,523,208 (incorporated herein by reference in its entirety), it has reverse hydrotherapeutic properties and has mutual affinity and homophilic (self) binding within the peptide. The peptides shown can be designed.

The present invention contemplates novel compounds and methods for modulating cell function in normal or infected cells. Such compounds include antibodies or fragments thereof that can be internalized into cells by the cell osmotic action of the peptides bound thereto. Such peptides are referred to herein as “membrane transport peptides” and the like. Known membrane transport peptides, or active fragments thereof, can be used as binding peptides. Such antibodies or fragments thereof include: (a) intracellular signal proteins such as caspases, kinases, and phosphatases; (b) immature virion proteins prior to construction in cells; (c) cell surface antigens or cells Immunospecific for protein targets such as internal tumor antigens, (d) nuclear or nucleolar proteins involved in the regulation of DNA synthesis and gene expression, or (e) cytoskeletal proteins involved in cell proliferation or cell division arrest is there. Polyclonal or monoclonal antibodies can be used. Such antibodies or fragments thereof preferably bind to their antigenic determinants with an affinity of 10 ⁻⁹ M or higher.

Particularly preferred compounds of the invention are those comprising an anti-caspase antibody conjugated to a membrane transport protein or peptide fragment thereof. A preferred membrane transport fragment is a membrane translocation sequence (MTS) peptide. Particularly preferred membrane transport peptides include the following:
(1) KGEGAAVLL PVLLAAPG (SEQ ID NO: 8), derived from Kaposi's sarcoma fibroblast growth factor [K-FGF] (Rojas et al., Nature Biotechnology, 16: 370-375 (1988).
(2) AAVLLPVLLAAP (SEQ ID NO: 9), truncated form of the aforementioned peptide, Lin et al. Biol. Chem. 271: 5305 (1996).
(3) See RQKIKIWFQNRRMKWKK (SEQ ID NO: 10), “Penetratin” from Homeodomain (Ant) of Antennapedia (Lindberg, M. et al., Eur. J. Biochem., 270 (14): 3055-3063 (2003) )
(4) RRMKWKK (SEQ ID NO: 11), penetratin C-terminal sequence, such as Fischer, P. et al. J. et al. Peptide Res. 55 (2): 163-172 (2000).
(5) TAT peptides, such as aa 47-57 and 48-60 from HIV-I TAT (eg, Schwarze, S. et al., Trends Pharmacol. Sci., 21:45, 2000; Li Y. et al., Biochem. Biophys. Res. Commun. 298 (3): 439-449, 2002; Hallbrink M. et al., Biochim. Biophys. Acta, 1515 (2): 101-109, 2001).
(6) Herpesvirus protein VP22 (Elliot, G. et al., Cell, 88: 223 (1997)).
(7) GWTLNSAGYLLLGKINLKALAALAKIL (SEQ ID NO: 12), “Transportan”, 27-mer peptide (Pouga, M. et al., FASEB J., 12:67 (1998); Lindberg, M. et al., Biochem., 40: 3141 ~ 3149, 2001).
(8) AGYLLGKINLKLALAALAKIL (SEQ ID NO: 13), transportan from which the N-terminal 6 residues have been deleted (see Somets, U., et al., Biochim. Biophys. Acta, 1467: 165-176, 2000).
(9) Lys-Leu-Ala-Leu (KLAL) (SEQ ID NO: 14), also called MAP (see also Hallblink M. et al., Blochim. Biophys. Acta, 1515 (2): 101-109, 2001).

  Also contemplated are pharmaceutical compositions effective for inhibiting apoptosis comprising an anti-caspase antibody conjugated to a membrane transport protein or fragment thereof as discussed herein. Such fusion proteins and methods for making them are disclosed in US patent application Ser. No. 09 / 865,281 (Kohler et al.), Which is incorporated herein by reference.

  Preferred immunoconjugates of the invention comprise a secondary antibody linked to an MTS sequence by one of several types of linkages, including antibody nucleotide or tryptophan site mediated, or antibody N-terminal linked carbohydrate mediated. As used herein, “secondary antibody” refers to an antibody or fragment thereof that binds specifically and with high affinity to a primary antibody. Secondary antibodies useful in the present invention include polyclonal or monoclonal antiglobulins directed against murine or human IgG, or T15 sequences that confer autotrophic properties to the antibody (Kang, CY, Brunck, TK, Kieber-Emmons, T et al. Inhibition of self-binding antibodies (self-body) by derived peptides (Inhibition of self-bindings (autobodies) by a VH-derived peptide), Science, 240: 1034-6, 1988) There are secondary antibodies that target the sequence.

  Pre-administer or pre-inject monoclonal antibody or immunoconjugate targeting cell surface antigen to allow sufficient time for target binding and removal from tissue and continue administration of secondary antibody covalently bound to MTS peptide Deliver by Primary antibodies can bind to inhibitors such as toxins, drugs, enzymes or isotopes, which can enhance the delivery of inhibitor molecules into cells. The secondary antibody bound to the MTS peptide recognizes and binds to the primary antibody and is internalized in the cell by MTS peptide activity. In this way, the primary immune complex is carried into the cell where its inhibitory action is enhanced.

  Such secondary conjugates can be used by mixing primary antibodies and secondary antibodies conjugated with MTS, then exposing the cells and testing for inhibition of cellular activity targeting the primary antibodies, The usefulness of monoclonal antibodies against internal targets can also be evaluated. In this rapid screen, many antibodies against intracellular targets can be screened for utility as antagonists or agonists. Thus, an active antibody can be directly conjugated to a membrane transport peptide such as MTS for in vivo use.

  Preferred embodiments of the invention include toxins, drugs bound via sulfhydryls, epsilon amino acids or carbohydrate residues by chemical or peptide linking groups or chelating compounds linked to tryptophan or nucleotide binding sites of secondary and primary antibodies. Alternatively, an MTS peptide coupled to an isotope is used.

  The present invention generally relates to delivery of antibody conjugates to the interior of cells in vivo. Such antibodies can potentially be neutralizing, antiviral antibodies, antiregulatory protein antibodies, or antitumor antibodies. For example, by administering to a living organism an antibody conjugate comprising an MTS peptide, and an antibody directed to an antigenic determinant on an immature virus or pathogen most expressed virus or other intracellular pathogen, Delivery can be performed. Such a conjugate has sufficient opportunity to bind with high affinity, destroy the viral construct and neutralize the virus before it has the opportunity to mature and infect other cells.

The present invention thus provides antiviral (anti-HIV) therapy as an example of a wide range of antibody therapies. Preferred antibodies in the present invention have the following preferred properties:
(1) The antibody of the present invention mainly binds to an antigen expressed in cells. This includes tumor associated antigen (TAA) and viral glycoproteins. The former includes TAAs such as CEA. Individual antigenic determinants can primarily bind to intracellular forms of proteins, while other antigenic determinants can be expressed primarily on the surface. Prior to the present invention, many useful therapeutic antibodies have been selected that have the ability to target intracellular antigens with respect to their reactivity with cell surface molecules, and the selection criteria are the main reactivity with intracellular antigens. Seems to contain.
(2) Intracellular targets include viral glycoproteins. For example, most monoclonal antibodies are produced against viruses that propagate in cells of multiple passages rather than viruses that propagate in cells of only a few passages; as a result, most monoclonal antibodies against viruses Reacts better with laboratory virus strains than new isolates. The proposed explanation for this binding difference is that most antibodies, like antibodies to HIV, are only a few passages of virus (and possibly new) due to sufficient glycosylation and viral glycoprotein folding. It reacts with an antigenic determinant that is hidden on and partially buried in the viral glycoprotein from the synthesized virus. This is because most antibodies are better with immature or incomplete virions with underglycosylated or incompletely glycosylated glycoproteins and / or immature or incomplete virions that are not fully assembled. It seems to mean that they should be combined. Thus, antibodies that are not considered useful for therapy due to limited reactivity with the original virus appear to be useful for targeting by contact with intracellular immature forms.
(3) The antibody of the present invention binds to a linear sequence of amino acids on TAA or viral glycoprotein rather than a conformation-dependent sequence. Such antibodies are more likely to bind to intracellular antigens early in synthesis and maturation, which may include immature virions or non-constructed, glycoprotein precursors present in the cell.
(4) These antibodies should bind to their antigenic determinants with an affinity of 10 ⁻⁹ M or higher.

  The MTS transit peptide modified monoclonal anti-caspase-3 antibody is shown herein by way of a specific example to reduce actinomycin D-induced apoptosis and spectrin cleavage in living cells. These results suggest that such antibodies have therapeutic potential to inhibit apoptosis in various diseases.

  Cell lines and antibodies. Human Jurkat T cell lymphomas were grown in RPMI 1640 supplemented with 10% fetal bovine serum and antibiotics (penicillin, streptomycin and amphetericin). Rabbit polyclonal anti-active caspase-3 antibody and anti-cleaved fodrine, αII spectrin, were obtained from Cell Signaling, Inc. (Beverly, MA). Rabbit monoclonal anti-active caspase-3 antibody was purchased from BD PharMingen (San Diego, Calif.). Rabbit anti-spectrin antibody was purchased from Cell Signaling (Beverly, MA). Mouse monoclonal antibody 3H1 (anti-CEA) was purified from cell culture supernatant by protein G affinity chromatography. Anti-mouse and anti-rabbit HRP-conjugated secondary antibodies are available from Santa Cruz Biotechnologies, Inc. Purchased from. The ApoAlert caspase-3 fluorescence assay kit was purchased from Clontech Laboratories, (Palo Alto, CA). Cell death detection ELISA was purchased from Roche Applied Science (Indianapolis, IN). Caspase inhibitors were purchased from Enzyme Systems Products (Livermore, CA).

Synthesis of antibody-peptide conjugates. MTS peptide (KGEGAAVLLPVLLAAPG) is a signal peptide membrane translocation sequence (1) and was synthesized by Genemed synthesis (San Francisco, Calif.). The antibody was dialyzed against PBS (pH 6.0) buffer and oxidized by adding 1/10 volume of 200 mmol / L NaIO ₄ and incubating at 4 ° C. for 30 minutes in the dark. Oxidation was stopped by adding glycerol to 30 mM and the sample was dialyzed against PBS (pH 6.0) buffer for 30 minutes at 4 ° C. More than 50-fold MTS peptide was used in the molecule to allow the antibody to bind by incubation for 1 hour at 37 ° C., and then the antibody-peptide was dialyzed against PBS (pH 7.4).

Effect of MTS-conjugated anti-active caspase-3 antibody on cell proliferation. 2.5 × 10 ⁵ Jurkat cells were seeded in a 96-well culture plate. After incubation with 0.5 μg MTS antibody conjugate for 6, 12, 18 and 24 hours, aliquots were removed and viable cells were counted using a dye exclusion test (trypan blue).

  Test of antibody internalization by ELISA. Jurkat cells grown in 1 ml medium were incubated with 2 μg naked or MTS antibody conjugate for 0, 1, 3, 6, 12 and 18 hours in 6-well culture plates (Costar, Cambridge, Mass.). The cells were spun down and the culture supernatant was transferred to a new tube and the cell pellet was washed twice with PBS (pH 7.4) before being homogenized by Pellet Pellet Motor (Kontes, Vineland, NJ) for 30 seconds. All cell homogenates and the same volume (10 μl) of culture supernatant were added to sheep anti-rabbit IgG coated ELISA plates (Falcon, Oxford, Calif.) And incubated for 2 hours at room temperature. After washing, HRP-labeled goat anti-rabbit light chain antibody was added and the antibody was made visible using o-phenylenediamine.

  DNA fragmentation. Jurkat cells were pretreated with antibody or caspase-3 inhibitor (DEVD-fmk) for 1 hour, centrifuged and incubated with fresh medium containing actinomycin D (1 μg / ml) for 4 hours. After treatment, Jurkat cells were collected and washed with PBS (pH 7.4), then in 700 μl HL buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.2% Triton X-100) And incubated at room temperature for 15 minutes. The crude DNA preparation was extracted twice with phenol: chloroform: isoamyl alcohol (25: 24: 1) and precipitated with 0.1 volume of 5M NaCl and 1 volume of isopropanol at −20 ° C. for 24 hours. The recovered DNA was dissolved in TE buffer (10 mM Tris, pH 8.0 and 1 mM EDTA). The same amount of DNA was resolved by electrophoresis on a 1.5% agarose gel and visualized by UV fluorescence after staining with ethidium bromide. DNA fragmentation was also detected by a cell death detection ELISA (Roche, Indianapolis, Ind.) And was performed with minor modifications according to the manufacturer's instructions: JB6 cells were grown in p100 plates and the cells were treated after treatment. Collected and 25 μl of complete cell lysate was applied to each sample well.

  Preparation of whole cell lysate. Jurkat cells were treated in the same manner as in the previous section. After treatment, Jurkat cells were collected and washed twice with PBS (pH 7.4) and then suspended in 300 μl CHAPS buffer (50 mM PIPES, pH 6.5, 2 mM EDTA, 0.1% CHAPS). I let you. Samples were sonicated for 10 seconds and centrifuged at 14,000 rpm for 15 minutes at 4 ° C. The supernatant was transferred to a new tube and called “whole cell lysate”.

  Caspase-3-like cleavage activity assay. Jurkat cells were treated in the same manner as in the previous section. Caspase-3 activity was analyzed using whole cell lysates of equal protein concentration and ApoAlert caspase-3 fluorescence assay kit according to the manufacturer's instructions. Fluorescence was measured using a spectral MAX GEMINI reader (Molecular Devices, Sunnyvale, CA).

  Western blot analysis. Jurkat cells were treated in the same manner as in the previous section. Ten μg of whole cell lysate was separated on a 10% SDS-PAGE gel to detect immunoreactive protein against truncated spectrin (1: 1000 dilution). Ponceau staining was used to examine the uniformity of protein transfer onto the nitrocellulose membrane. Membranes were washed with distilled water to remove excess staining solution and in Blotto (5% milk, 10 mm Tris-HCl [pH 8.0], 150 mM NaCl and 0.05% Tween 20) for 2 hours at room temperature. Blocked with. Prior to adding the secondary antibody, the membrane was washed twice with TBST (10 mM Tris-HCl, 150 mM NaCl and 0.05% Tween 20) and the membrane was then diluted with horseradish peroxidase at 1: 4000 dilution. Incubated with next antibody (Santa Cruz Biotechnology). The final wash step included 3 times with TBST (5 minutes each) and 2 times with TBS (10 mM Tris-HCl and 150 mM NaCl) (5 minutes each). The antibody band was made visible by an advanced chemiluminescence detection system (ECL, Amersham Pharmacia Biotech, Piscataway, NJ).

Results MTS-conjugated anti-active caspase-3 antibody shows little inhibition of cell proliferation. Initially tested was the potential toxicity of MTS antibody conjugates to cells. Cell viability assays showed that MTS antibody conjugates had little effect on cell proliferation (FIG. 1).

  MTS peptide promotes rapid penetration of anti-active caspase-3 antibody into living cells. Using a sandwich assay, an ELISA was designed to capture rabbit Ig. As can be seen in FIG. 2, the MTS conjugate rapidly facilitated internalization of the monoclonal anti-active caspase-3 antibody into living cells. Ig migration increased within 1 hour and reached a steady state after 18 hours. The antibody remaining in the culture decreased in 1 hour and appeared to reach equilibrium in 18 hours. Naked antibody internalization was delayed (at 3 hours) and was at a lower level compared to MTS-conjugated anti-caspase-3 antibody.

  Polyclonal MTS anti-active caspase-3 antibody inhibits DNA fragmentation. MTS-conjugated or naked polyclonal anti-caspase-3 antibody (1 μg / ml final concentration-equivalent to 1:64 dilution) was added to 6 ml cultured Jurkat cells and preincubated for 1 hour. The antibody was washed away by centrifugation, fresh medium containing only actinomycin D (1 μg / ml) and no antibody was added, and the cells were incubated for 4 hours. 5 ml of culture was collected for DNA fragmentation. Naked (unbound) anti-caspase-3 polyclonal antibody did not prevent DNA laddering by actinomycin D treatment. In contrast, MTS-conjugated anti-caspase-3 polyclonal antibody significantly inhibited DNA fragmentation (apoptosis) induced by actinomycin D (data not shown).

Monoclonal MTS anti-active caspase-3 antibody prevents DNA fragmentation. MTS-conjugated or naked monoclonal anti-caspase-3 antibody (1 μg / ml final concentration) was added to 6 ml cultured Jurkat cells and preincubated for 1 hour. The antibody was washed away by centrifugation, fresh medium containing only actinomycin D (1 μg / ml) and no antibody was added, and the cells were incubated for 4 hours. 5 ml cultures were collected for DNA fragmentation and the rest saved for cell death ELISA assay. While MTS-conjugated antibodies were observed to suppress DNA ladder formation, naked (unbound) anti-caspase-3 monoclonal antibodies did not prevent DNA laddering by actinomycin D treatment (data not shown). Cell death ELISA assay (FIG. 3) confirmed a significant decrease in cell apoptosis when cells were pretreated with MTS-conjugated antibody. Jurkat cells incubated with caspase-3 inhibitor (DEVD-fmk) maintained 100% viability and vehicle (DMSO) treated control cells maintained approximately 80% viability. In the naked anti-caspase-3 antibody treated group, only 36% of the cells were viable after 4 hours. However, as 70% of the cells were viable, treatment with MTS anti-caspase-3 binding antibody dramatically protected against actinomycin D-induced apoptosis (see Table 1).

  MTS-conjugated anti-active caspase-3 antibody suppresses caspase-3 activity. Jurkat cells were treated as before, murine anti-CEA antibody was modified and used as a control. As shown in FIG. 4, caspase-3-like cleavage activity is increased by actinomycin D treatment, MTS-conjugated monoclonal anti-active caspase-3 antibody reduces caspase-3-like cleavage activity, whereas MTS-3H1 antibody has no effect. Did not show. Cell death ELISA assay also confirmed that the MTS-conjugated monoclonal anti-caspase-3 antibody showed significantly lower DNA fragmentation (data not shown).

  MTS anti-active caspase-3 antibody inhibits spectrin cleavage. As a downstream target of caspase-3, the protein level of spectrin was examined. Two truncated fragments of spectrin were observed in actinomycin D treated Jurkat cells (data not shown). Neither 3H1 nor MTS-3H1 protected spectrin cleavage. Naked monoclonal anti-active caspase antibody has little effect on protection; whereas MTS-conjugated anti-active caspase-3 antibody, like caspase-3 inhibitor DEVD-fmk, completely cleaves 100 kDa and 75 kDa αII spectrin fragments. Suppressed. The 150 kDa cleavage band showed no obvious change in all antibody pretreated cell samples.

Conclusion The foregoing results indicate that anti-caspase-3 antibodies can significantly inhibit in vitro apoptosis related events such as caspase-3 activity, DNA fragmentation, and spectrin cleavage. Thus, anti-caspase-3 antibodies can be used to inhibit apoptosis in various diseases. In contrast to antibodies used therapeutically, as shown in rodent animal models, conventional peptide apoptosis inhibitors not only show strong inhibition but also have adverse side effects such as high toxicity. Therefore, transport membrane-bound antibodies have low toxicity compared to conventional apoptosis inhibitors. Transport membrane (MTS) -bound antibodies are therefore promising new candidates for treating diseases, including apoptosis of the central nervous system, particularly diseases such as Alzheimer's disease, Huntington's disease and Parkinson's disease.

  Compositions of the present invention comprise unit dosage forms, sterile solutions or suspensions, sterile oral solutions or suspensions, oral solutions or suspensions, oil-in-water or water-in-oil emulsions, etc., containing appropriate amounts of the active ingredients In pharmaceutical compositions for systemic administration to humans and animals. Topical application may be in the form of ointments, creams, lotions, jellies, sprays, irrigants and the like. The compositions of the present invention are useful in pharmaceutical compositions (% by weight) of the active ingredient and carrier or excipient of about 1-20%, and preferably about 5-15%.

  The parenteral solutions or suspensions described above can be administered transdermally, and if desired, further concentrated sustained release forms can be administered. The cross-linked peptides of the present invention can be administered intravenously, intramuscularly, intraperitoneally, or locally. Thus, active compounds can be incorporated into sustained release materials for transdermal administration. Pharmaceutical carriers recognized for the purposes of the present invention are carriers known in the art that do not adversely affect the substance, including the drug, host, or drug delivery device. The carrier can also contain adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, and the like, and permeability enhancers of the invention. Effective doses for mammals can vary depending on factors such as the age, weight, level of activity or condition of the subject being treated. Typically, an effective dose of a compound of the invention is about 10-500 mg, preferably 2-15 mg, when administered by suspension at least once a day. Administration can be repeated at appropriate intervals.

  The purpose of the foregoing description and examples is to illustrate some embodiments of the present invention without showing any limitation. It will be readily apparent to those skilled in the art that various modifications and variations of the composition and method of the present invention can be made within the scope of the appended claims without departing from the spirit or scope of the invention. I will. All patents and publications cited herein are incorporated by reference in their entirety.

It is a figure which shows the viability by detection of the Jurkat cell processed with the MTS anti-active caspase-3 antibody conjugate. 2.5 × 10 ⁵ Jurkat cells were seeded in a 96-well culture plate. After incubation with 0.5 μg MTS antibody for 6, 12, 18 and 24 hours, aliquots were removed and viable cells were counted using a dye exclusion test (trypan blue). It is a figure which shows the detection of the antibody internalization by sandwich ELISA. Sheep anti-rabbit antibody was coated on ELISA plates (400 ng / well). Cell homogenate and an equal volume of culture supernatant were added to sheep anti-rabbit IgG-coated ELISA plates (Falcon, Oxford, Calif.) And incubated for 2 hours at room temperature. After washing, an HRP-labeled goat anti-rabbit light chain antibody was added and the antibody was made visible by adding o-phenylene-diamine. Plot the ratio of internalized antibody to antibody in the culture. FIG. 5 shows the extent of DNA fragmentation as measured by cell death ELISA assay. MTS-conjugated or naked anti-caspase-3 antibody (2 μg / ml) was added to 6 ml of cultured Jurkat cells and preincubated for 1 hour. The antibody was washed by centrifugation, fresh medium containing actinomycin D (1 μg / ml) was added and incubated for 4 hours. Five ml cultures were collected for evaluation of DNA fragmentation by ladder electrophoresis; the rest were collected for an ELISA assay. AD = actinomycin D; naked antibody = caspase-3 antibody; MTS-Ab = MTS-conjugated anti-caspase-3 antibody; caspase-3 inhibitor = DEVD-fmk (100 μM). *, P <0.01 compared to control; #, p <0.01 compared to naked caspase-3 antibody. FIG. 3 shows a caspase-3-like cleavage activity assay. An equal volume of protein from the whole cell lysate was applied to this assay by using the ApoAlert caspase-3 fluorescence assay kit. *, P <0.01 compared to control; #, p <0.01 compared to naked caspase-3 antibody.

Claims (36)

  A compound effective for regulating the function of normal cells or infected cells, comprising an antibody or fragment thereof bound to a membrane transport peptide, wherein the antibody or fragment thereof comprises (a) a caspase, a kinase, and a phosphatase Intracellular signal proteins selected from the group, (b) immature viral proteins, (c) cell surface antigens or intracellular tumor antigens, (d) nuclear proteins or nucleoli involved in the regulation of DNA synthesis and gene expression A compound that is immunospecific for a protein or (e) a cytoskeletal protein involved in cell proliferation or cell division arrest.   2. The compound of claim 1, wherein the antibody is a monoclonal antibody.   2. The compound of claim 1 comprising an anti-caspase antibody or fragment thereof effective to inhibit apoptosis and conjugated to a membrane transport peptide.   4. The compound of claim 3, wherein the antibody is an anti-caspase-3 antibody.   2. The compound of claim 1, wherein the membrane transport peptide is a translocation sequence (MTS) peptide.   6. The compound of claim 5, wherein the MTS peptide is endogenous to Kaposi's sarcoma fibroblast growth factor, HIV-1 TAT peptide, antennapedia homeodomain-derived peptide, herpesvirus protein VP22, or transport peptide.   7. The compound of claim 6, wherein the MTS peptide comprises the amino acid residue sequence AAVLLPVLLAAP (SEQ ID NO: 9).   8. The compound of claim 7, wherein the MTS peptide comprises the amino acid residue sequence KGEGAAVLLPVLLAAPG (SEQ ID NO: 8).   The membrane transport peptide has low hydrophobicity compared to a second peptide comprising the amino acid residue sequence: KGEGAAVLL PVLLAAPG (SEQ ID NO: 8), and the membrane transport peptide is greatly internalized compared to the second peptide 2. The compound of claim 1, which provides enhancement and immunobinding power.   A pharmaceutical composition effective for inhibiting apoptosis in humans, comprising an anti-caspase antibody or fragment thereof bound to a membrane transport peptide.   The composition according to claim 10, wherein the antibody is a monoclonal antibody.   The composition according to claim 10, wherein the antibody is an anti-caspase-3 antibody.   11. The composition of claim 10, wherein the membrane transport peptide is a membrane translocation sequence (MTS) peptide.   11. The composition of claim 10, wherein the MTS peptide comprises the amino acid residue sequence AAVLLPVLLAAP (SEQ ID NO: 9).   15. The composition of claim 14, wherein the MTS peptide comprises the amino acid residue sequence KGEGAAVLLPVLLAAPG (SEQ ID NO: 8).   A method for treating or preventing a human disease, comprising administering to a patient in need thereof a pharmacologically effective amount of a composition comprising an anti-caspase antibody or fragment thereof conjugated to a membrane transport peptide. .   17. The method of claim 16, wherein the disease is Alzheimer's disease, Huntington's disease, or Parkinson's disease.   An immune complex comprising a membrane transport peptide or fragment thereof conjugated with a secondary antibody.   19. The immune complex according to claim 18, wherein the secondary antibody is a polyclonal or monoclonal immunoglobulin.   19. The immune complex according to claim 18, wherein the membrane transport peptide or fragment thereof is an MTS sequence.   19. The immune complex of claim 18, wherein the membrane transport peptide or fragment thereof is covalently linked to a tryptophan residue or nucleotide binding site of the secondary antibody.   19. The immune complex according to claim 18, wherein the secondary antibody is covalently bound to an inhibitor via a sulfhydryl, ε amino acid or carbohydrate group of the antibody. A method of treating or preventing a human disease comprising:
Pre-administering to a patient in need of a primary antibody immunospecific for a cell surface target;
Providing sufficient time for binding of the primary antibody to the target and removal from normal tissue, and a secondary antibody covalently linked to a membrane transport peptide or fragment thereof, wherein the primary antibody is immunospecific Administering a secondary antibody.   24. The method of claim 23, wherein the primary antibody is conjugated with a toxin, drug or radioisotope.   The toxin is (a) a holoprotein toxin selected from the group consisting of ricin, abrin, diphtheria and Pseudomonas exotoxin, (b) a complete protein toxin subunit, or (b) ricin A chain, abrin A chain, 25. The method of claim 24, which is a natural A chain toxin subunit selected from the group consisting of diphtheria toxin A chain, Pseudomonas exotoxin A and gelonin.   25. The chemotherapeutic agent of claim 24, wherein the agent is a chemotherapeutic agent suitable for treating a human disease or a chemotherapeutic agent having high efficacy but unacceptably high toxicity for use in humans. Method.   25. The method of claim 24, wherein the radioisotope is an alpha, beta or Auger radioisotope that is bound to the antibody via a chelate compound.   24. The method of claim 23, wherein the secondary antibody is covalently bound to the membrane transport peptide or fragment thereof by photoactivation.   24. The method of claim 23, wherein the secondary antibody is conjugated with a toxin, drug or radioisotope.   The toxin is (a) a holoprotein toxin selected from the group consisting of ricin, abrin, diphtheria and Pseudomonas exotoxin, (b) a complete protein toxin subunit, or (b) ricin A chain, abrin A chain, 30. The method of claim 29, which is a naturally occurring A chain toxin subunit selected from the group consisting of diphtheria toxin A chain, Pseudomonas exotoxin A and gelonin.   30. The chemotherapeutic agent of claim 29, wherein the agent is a chemotherapeutic agent suitable for treating a human disease or a chemotherapeutic agent having high efficacy but unacceptably high toxicity for use in humans. Method.   30. The method of claim 29, wherein the radioisotope is an alpha, beta or Auger radioisotope that is conjugated to the antibody via a chelate compound.   30. The method of claim 29, wherein the secondary antibody is covalently linked to an inhibitor via a sulfhydryl, ε amino acid or carbohydrate group of the antibody.   24. The method of claim 23, wherein the patient is suffering from cancer, HIV or other viral vector, or bacterial material. an in-vitro screening assay comprising:
Contacting a plurality of cells with a primary antibody that is immunospecific for a cellular receptor or an intracellular target, wherein the primary antibody is bound to a membrane transporting peptide or fragment thereof; and An assay comprising the step of assessing the possibility of antagonism or agonism of cellular activity by activation. an in-vitro screening assay comprising:
Contacting a plurality of cells with a primary antibody that is immunospecific for a cellular receptor or an intracellular target;
Mixing a secondary antibody bound to a membrane transport peptide or fragment thereof with the primary antibody, wherein the secondary antibody is immunospecific for the primary antibody, and cellular activity due to internalization of the primary antibody An assay comprising the step of assessing the possibility of antagonism or agonism.