GB2517190A - Intracellular immunity - Google Patents

Abstract

A conjugate of an agent (which may be cytotoxic) and a ligand (which may be an antibody) specific for an antigenic determinant of a virus for delivering the agent to the cytoplasm of a cell. The ligand and agent may be joined by a labile or non-labile linker. The conjugate may be used for killing virally infected cells.

Description

I

Intracellular Immunity The present invention relates to methods for delivering conjugates into cells. In particular, the invention employs the newly-discovered ability of antibodies to enter cells when bound to pathogens.

Viruses and their hosts have been co-evolving for millions of years and this has given rise to a complex system of immunity traditionally divided into innate and adaptive responses.

Innate immunity comprises germ-line encoded receptors and effector mechanisms that recognise pathogen-associated molecular patterns, or PAMPs. The advantage of innate immunity is that it is fast and generic; however viruses are adept at avoiding recognition by inhibiting innate immunity or by changing their molecular patterns. In contrast, adaptive immunity can cure' a host of infection and provide protection against future infection. Unlike the PAMP receptors of innate immunity, adaptive immunity uses proteins such as antibodies to target pathogens. Antibodies are unique in the human body in that they evolve during the lifetime of an individual and can continue to target evolving pathogens. The weakness of adaptive immunity is that it can take 1-2 weeks to reach full effectiveness. Furthermore, the dogma of antibody immunity for the last 100 years has been that antibodies only provide extracellular protection. It is thought that once a virus has entered the cytosol of a cell, antibodies are unable to prevent its infection.

Intracellular antibodies have been developed; for example, see Moutel S, Perez F., Med Sci (Paris). 2009 Dec; 25(12):1173-6; Stocks M., Curr Opin Chem Biol. 2005 Aug;9(4):359-65. However, results using intracellular antibodies, or intrabodies, have been mixed. In general, attempts to develop intracellular antibodies have focussed on single chain antibody fragments, such as scFvs and single domain antibodies, such as VHH antibodies and dAbs.

Antibodies and immune sera have long been used for the treatment of pathogenic infections. Fore example, horse antiserum was used in the 1890s to treat tetanus and diphtheria. However, antisera are seen as foreign by the human immune system, which reacts by producing antibodies against them, especially on repeat doses. During most of the 201h C, the adverse effect of animal antibodies prompted the use of human antiserum from donors who had recovered from disease, typically for prophylaxis of respiratory and hepatitis B infections. Following a reduction in the popularity of antibody therapy due to problems with toxicity, humanised and human antibodies have eliminated such concerns, and led to a return of such therapeutic approaches. See Casadevall et al., Nature Reviews Microbiology 2, 695-703 (September 2004), for a review. Diseases which have been targeted using antibody therapy include anthrax, whooping cough, tetanus, botulism, cryptococcosis, cryptosporidiosis, enterovirus gastrointestinal-tract infections, group a streptococcal infections, necrotizing fasciitis, hepatitis B, measles, tuberculosis, meningitis, aplastic anaemia, rabies, RSV infection, pneumonia, shingles, chickenpox and pneumonia due to VZV, and smallpox. Despite these developments, however, antibody therapy is considered only when no other suitable therapies are available, requiring high doses of antibody and producing unpredictable results.

The effectiveness of antibodies against pathogens is understood to be at least partly dependent on the Ec portion of the antibody, which is responsible for mediating the effects of complement. Therefore, antibody fragments have not been generally proposed for antiviral therapy, despite their advantages of small size and lower cost of production.

Antiviral conjugates, comprising cytotoxic agents attached to antibodies, are known.

However, such immunoconjugates have been targeted to viral epitopes that are displayed on the surface of infected cells; the strategy is for the immunoconjugate to target infected cells, not viruses.

The targeting of a conjugate affects the compartment which the conjugate enters on delivery to the cell, and thus the nature of the conjugate itself. For example, the approved ADC Mylotarg® which is targeted to the CD33 receptor uses an acyl hydrazine linker to link the antibody to the drug; this linker is hydrolysed in the lysosome of CD33 positive target cells. Viruses, however, enter pathways though a variety of different routes, and the processing agents targeted using viruses cannot be assumed to be identical to that of ADCs which target cell-surface receptors.

W02007/084692 describes the use of antibodies to attach therapeutic agents to viruses, in such a manner that "the therapeutic agent(s) comprised in the immunoconjugate may destroy the bound pathogen as well as the infected host cells as the pathogen enters the host cells, thereby effectively preventing the replication and further transmission of the pathogen". The language used in this document, "as the pathogen enters the host cells", reflects an understanding of antibody immunity that antibodies do not routinely exist in the intracellular environment and therefore that the method described would only be effective during the infection event itself. W02007/088692 does not disclose that antibodies can be taken up into the cell, and therefore that therapeutic agents can be delivered to an intracellular compartment.

US 5,521291 describes a method for delivering nucleic acids to cells using a virus as a deliveiy vehicle. The nucleic acids aie complexed with polyanions, and optionally with antibodies to attach them to the virus. However, this document does not demonstrate antibody delivery into a cellular compartment; the reporter gene expression indicates only S that nucleic acid is taken up into the cell. Nucleic acid transfection using polyanions is well known.

Recently, we described how antibodies bound to viruses are taken up into cells, and direct the viruses into the proteosomal degradation pathway by means of the polypeptide TRIM21 (doi: 10.1073/pnas.1014074107). Since viruses enter cells by a variety of mechanisms, including phagocytosis, clathrin and non-clathrin mediated pinocytosis and maciopinocytosis, the fate of antibody/virus complexes could vary substantially when the virus enters the cell. We have investigated whether viruses can be used to deliver a therapeutic payload, such as a cytotoxic compound, into a cell by attaching the payload to an antibody.

Summary of the Invention

Antibodies are extracellular proteins, as are all known mammalian IgO receptors (with the exception of FcRn, which is intracellular but not cytosolic). We recently described how antibodies entel cells bound to viluses, and diiect the vilus to the proteasome by means of the receptor TRIM 21. We have now shown that antibodies complexed to agents can be delivered by virus into the intracellular environment. This approach provides a new mechanism for deliveiy of agents to cells, and foi antiviral therapy. Moieovei, the invention provides means to deliver agents to specific subcellular compartments.

In a first aspect of the invention, therefore, there is provided the use an agent and a ligand specific for an antigenic determinant of a virus in a conjugate for delivering the cytotoxic agent to the cytoplasm of a cell, wherein said vilus infects the cell through pinocytosis.

In the piioi art, antiviral immunoconjugates have been diiected at viral proteins displayed on the cell surface; the strategy was to bind the immunoconjugate to the cell, and cause internalization of a cytotoxic or antiviral compound through internalization of the viral protein. In contrast, in the present invention, the virus carries the conjugate with it directly into the cell.

Accordingly, conjugates can be directed against antigens that are not displayed on the cell surface after viral infection. Unlike such conjugates, the conjugates according to the invention are taken up through pinocytosis of the virus by the cell. In one embodiment, the conjugate further includes the virus which comprises the antigenic determinant.

Viruses enter cells through a variety of pathways. Generally, viruses enter cells thorugh pinocytotic mechanisms; for example, viruses can enter cells though macropinocytosis or clathrin-mediated uptake. In different circumstances, viruses can use mechanisms such as plasma membrane fusion and receptor-mediated endocytosis. Some viruses can use more than one uptake mechanism. The selection of uptake mechanisms can depend on the cell type which is infected, or the conditions under which infection takes place.

In embodiments, the virus is taken up into the cell by macropinocytosis.

The conjugates of the invention are delivered into an intracellular compartment. Where the virus enters the cell through macropinocytosis, the conjugate will enter the macropinosome. Viruses which enter the cell through macropinocytosis include vaccinia virus, HIV-1, coxsackie B virus, HSV-1, ASFV, HPV-16, adenovirus 3, echovirus 1, ebola virus and KHSV.

In other embodiments the virus enters the cell through clathrin-mediated endocytosis. In this embodiment, viruses are taken up into clathrin-coated pits and pass into the endosome. Viruses which use clathrin-mediated endocytosis include SFV, VSV, HIV-1, Flavivirus, Bunya virus, influenza virus, adenovirus 2 and adenovirus 5.

In still further embodiments, the virus infects the cell by membrane fusion. Enveloped viruses can use this mode of infection, in which the viral envelop-e fuses with the plasma membrane, and the contents of the virus are introduced into the cell. Viruses which can employ this strategy include RSV, parainfluenza virus, metapneumovirus, HSV-2, coronavirus, measles virus and HIV.

In other embodiments, the virus infects the cell by receptor-mediated endocytosis. Viruse which can employ this mechanism include HSV-2, CMV, HPV-18, coronavirus, HBV, EBV, BK virus and Rhinovirus.

In embodiments, the virus is not the therapeutic target of the conjugate. A virus can be administered deliberately to target the cells of a patient which are infected by another pathogen or disease, thereby causing said cells to be neutralized by the agent. The conjugate and the virus are administered separately to the patient.

In embodiments! the virus is the therapeutic target of the conjugate. The conjugate is administered to a patient that is infected with the virus. In ceitain embodiments, the conjugate is administered prophylactically to a patient not infected with the virus.

The antibody/virus/agent complex can be assembled in the subject organism. In S embodiments, however, the complex can be preassembled prior to administration. The agent acts within the cell, once internalized together with the antibody and the virus.

Accordingly, the invention provides a complex which comprises a conjugate as described above and a virus. In some embodiments, the complex is intracellular. In this context, "intracellular" means that the complex is located inside the cell. It may be located, for example, in the cytoplasm or in an endosorne, or in the nucleus.

In embodiments, the complex does not comprise a polyanion. For example, the complex does not comprise an agent which is a nucleic acid, and a polyanion.

In the context of the present invention, the term "ligand" is used to refer to either half of a binding pair. For example, the binding pair can be biotin/streptavidin, and the virus can be biotinylated before exposeure to a streptavidin complex.

Wheie the ligand is an immunoglobulin, it can be any immunoglobulin molecule, for example an immunoglobulin molecule selected from the group consisting of an lgG, IgA.

1gM, I9E, lgD, F(ab')2, Fab, Fv, scFv, dAb, VHH, IgNAR, a TCR, and multivalent combinations thereof. Multivalent antibodies include, for instance, bivalent antibodies and antibody fiagments, bispecific antibodies and antibody fragments, trivalent veisions thereof, and proprietary formats such as diabodies. Single domain antibodies, such as dAbs and VHH antibodies, aie particularly suitable for combining to form multivalent and/or multispecific molecules.

Where the ligand is an antibody, the antibody molecule comprises at least one of a VH domain and a VL domain, or the equivalent thereof.

The agent can be a cytotoxic compound or payload which ii is desired to deliver into the cell. Examples can include proteins, nucleic acids and small molecules, as long as they can be conjugated to an antibody. Suitable toxins are known in the art, including bacterial toxins -such as diphtheiia toxin; plant toxins -such as ricin; small molecule toxins -such as geldanamycin, macrocyclic depsipeptides and calicheamicin. The toxins may effect their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, topoisomelase inhibition oi ribosome inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands, suggesting that smaller antibody fragments may be indicated in some cases.

In one embodiment, the agent may be an antiviral compound. Examples of antiviral agents which can be used include nucleoside analogues, such as 5-fluorouracil, sorivudine, zidovudine, didanosine, zalcitabine, aciclovir, ganciclovir, vidarabine and ribavirin.

Cytotoxic agents and antiviral compounds can be selected according to the ability of the virus to infect a cell more or less rapidly. Thus, a more rapidly-acting cytotoxic or antiviral agent can be combined with a more rapidly-infecting virus, to achieve optimal cell killing.

The complex according to the invention is assembled in an extracellular environment. In other words, the complex is assembled outside of the cell and carried in to the cell as a result of uptake of the virus as it infects the cell.

The ligand, for example the antibody, is preferably a non-neutralizing or non-blocking ligand. Neutralizing antibodies (Nabs) are capable of preventing viral infection by blocking the process of infection, usually by physically blocking viral entry into the cell.

Often, neutralizing antibodies are directed to viral envelope glycoproteins, or other viral surface markers. These markers are responsible for interacting with the cellular ligands which allow the virus to be taken up into the cell. Neutralizing antibodies are often preferred for antiviral therapy, because of their effectiveness against viruses; however, they are not indicated in the present invention. With a few exceptions, neutralizing antibodies are not take up into the cell, and thus do not permit entry of the virus into the cell. In the present invention, it is essential that the virus is taken up into the cell.

In a further aspect, there are provided conjugates comprising a ligand for an antigenic determinant of a virus and a cytotoxic agent, including a linker which releases the agent in an appropriate endosomal compartment as required by the biology of the appropriate viral uptake mechanism.

Thus, the invention provides a conjugate comprising a ligand specific for an antigenic determinant of a virus and a cytotoxic agent, linked together with a labile linker, wherein (a) the virus follows an infection cycle wherein it is released from the endosomal compartment of a cell in response to a signal; and (b) the labile linker is cleaved contemporaneously in the infection cycle.

Preferably, the linker is cleaved in response to the same signal as that which leads to release of the virus from the endosome.

For example, the linker can be cleaved in response to a change in pH. Acidification of endosomal pH is a trigger for some viruses to exit the endosomal compartment. For example, the virus is a pH-dependent virus which exits the relevant endosomal compartment in response to acidification of the environment. Viruses which exit the endosome early in response to acidification include Adenovirus. Acid labile linkers, such as hydrazone likers, are able to release the cytotoxic agent from the complex as the endosome environment acidifies. Suitable hydrazine linkers include (6-maleidimidocapropyl)hydrazone and 4-(4'-acetylphenoxy)butanoic acid linkers.

Vaccinia virus exits within 15 minutes of infection, in response to acidic pH. In one embodiment, there is provided a conjugate comprising a ligand specific for an antigenic determinant of a vaccinia virus and a cytotoxic agent, linked together with an acid labile linker.

The linker can moreover be cleaved in response to reducing agents. Alternative linkers include linkers cleavable as a result of reduction, such as disulphide-based linkers, which release the bound agent as the concentration of glutathione increases in the intracellular compartment. Glutathione is present in concentrations up to 1000 times higher in the cytoplasm compared with blood plasma, which leads to linker dissociation and agent release once the conjugate is internalized by the cell. Disulphide linkers include 4-mercaptopentanoate linkers and Immunogen® DM1 and DM4 toxin linkers.

In one embodiment, there is provided a conjugate comprising a ligand specific for an antigenic determinant of adenovirus and a cytotoxic agent, linked together with a disulphide linker.

Further linkers useful in embodiments of the present invention include enzyme labile linkers. Lysysomal proteases, including cathepsin and plasmin, can be exploited to release cytotoxic agents from conjugate at the appropriate time. Examples include the maleimido-contaiing peptide linkers, such as those conjugated by Seattle Genetics to auristatin E and F (MMAE and MMAF). Other examples include valine-citrulline linkers, which are hydrolysed by cathepsin B, and linkers cleavable by esterases or glycosidases such as beta-glucoronidase.

In one embodiment, there is provided a conjugate comprising a ligand specific tor an antigenic determinant of a rotavirus virus and a cytotoxic agent, linked together with a cathepsin labile linker.

In embodiments, the linker can be a non-cleavable linker. Examples include thioether linkers, such as succinimide linkers (SMCC) as used in the approved ADC Kadcyla®.

Non-cleavable linkers can be exploited for viruses which do not exit the endosome, or only exit at the late lysosomal stage. In such cases, the cytotoxic agent remains in the endosome until the virus and any attached antibody is degraded. In enbodiments, non-labile linkers are used to attach a virus selected from HSV-1, RSV and Adv5 to a cytotoxic agent such as auristatin.

Brief Description of the Figure

Figure 1: HeLa cells infected with adenovirus complexed with anti-adenovirus antibody HeLa cell infected with human adenovirus pre-incubated with AlexaFluor488-conjugated human serum lgG (antibody conjugate), permeabilized and stained with AlexaFluor568-conjugated anti-human lgG antibody to confirm the localization of the antiviral antibody conjugate. Scale bars, 10 pm.

A & B: The two dyes co-localise, indicating that the virus/antibody complex is taken up into the cell.

Figure 2: Infection of cells Bar chart showing the percentage of virus infected cells when exposed to antiviral antibody (black bars) or taxol-conjugated antiviral antibody (striped bars). Addition of taxol-conjugated antibody leads to markedly decreased infection levels at 1 day post-infection (p.i.). After 2 days p.i., there are no measurable infected cells in the presence of of taxol-conjugated antibody.

Figure 3: Inhibition of HSV-1 Infectivity Taxol labelled APi inhibits HSV-1 infectivity and plaque forming in HeLa cells. (a-c) APi-taxol antibody conjugate (10 or 40 pg/mI) or AP7-DMSO was added to HSV-1 and virus title determined by plaque assay on HeLa Cells. Both plaque number (a&b) and size (c&d) were determined. (d) AP7-taxol antibody conjugate and HSV-1 plaqued on Vero cells.

Figure 4: Blocking antibodies inhibit delivery of taxol Taxol labelled LP2 fails to inhibit HSV-1 infectivity in HeLa cells. LP2-taxol antibody conjugate (10, 2 or 0.4 pg/mI) or LP2-DMSO was added to HSV-1 and virus titre determined on HeLa Cells. LP2 is more effective without the Taxol.

Detailed Description of the Invention

Unless otherwise stated, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. Methods! devices, and materials suitable for such uses are now described. All publications cited herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing the methodologies, reagents, and tools reported in the publications that might be used in connection with the invention.

The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, cell biology, genetics, immunology and pharmacology, known to those of skill of the art. Such techniques are explained fully in the literature. See, e.g. , Gennaro, A. R., ed. (1990) Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.; Hardman, J. G., Limbird, L. E., and Gilman, A. G., eds. (2001) The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill Co.; Colowick, S. et al., eds., Methods In Enzymology, Academic Press, Inc.; Weir, D. M. , and Blackwell, C. C., eds. (1986) Handbook of Experimental Immunology, Vols. l-IV, Blackwell Scientific Publications; Maniatis, T. et al., eds. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Vols. I-Ill, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al., eds. (1999) Short Protocols in Molecular Biology, 4th edition, John Wiley & Sons; Ream et al., eds. (1998) Molecular Biology Techniques: An Intensive Laboratory Course, Academic Press; Newton, C. R., and Graham, A., eds. (1997) PCR (Introduction to Biotechniques Series), 2nd ed., Springer Verlag.

An antigen, in the context of the present invention, is a molecule which can be recognised by a ligand and which possesses an epitope specific for a virus. The antigen targeted by the ligands according to the present invention is not exposed on the surface of a cell infected by a virus. Methods for determining whether viral antigens are exposed on infected cell surfaces are well known; for example, see Pasternak, G. 1967, Nature.

214:1364-1365; Peters, C.J., and AN. Theofilopoulos. 1977, J Immunol. 119:1089-1096.

An antigenic determinant is an antigen which is specific for a particular pathogen in the circumstances, such that ligands which bind to the antigenic determinant can be considered specific for the pathogen.

Viruses may be enveloped or non-enveloped. In one embodiment, the virus is a non-enveloped virus.

A ligand which binds directly to an antigen is a ligand which is capable of binding specifically to an antigen under physiological conditions. As used herein, the term "ligand" can refer to either part of a specific binding pair; for instance, it can refer to the antibody or the antigen in an antibody-antigen pair. Antibodies are preferred ligands, and may be complete antibodies or antibody fragments as are known in the art, comprising for example lgG, IgA, 1gM, IgE, lgD, F(ab')2, Fab, Fv, scFv, dAb, VHH, I9NAR, a modified TCR, and multivalent combinations thereof. Ligands may also be binding molecules based on alternative non-immunoglobulin scaffolds, peptide aptamers, nucleic acid aptamers, structured polypeptides comprising polypeptide loops subtended on a non-peptide backbone, natural receptors or domains thereof.

A ligand which binds indirectly to an antigen is a ligand which binds to the antigen via a second ligand. For instance, it is a ligand which binds to an antibody. The ligand binds the antibody in a manner independent of the binding specificity of the antibody; for instance, it can bind the Ec region. In one embodiment, the ligand is selected from the group comprising Protein G, protein A, Protein L, the PRYSPRY domain of TRIM21, an anti-immunoglobulin antibody, and peptides which specifically recognise antibodies, for example in the Fc region.

The term "immunoglobulin" refers to a family of polypeptides that retain the immunoglobulin fold characteristic of antibody molecules, which contains two beta sheets and, usually, a conserved disulphide bond. Members of the immunoglobulin superfamily are involved in many aspects of cellular and non-cellular interactions in vivo, including widespread roles in the immune system (for example, antibodies, T-cell receptor molecules and the like), involvement in cell adhesion (for example the ICAM molecules) and intracellular signalling (for example, receptor molecules, such as the PDGF receptor).

The present invention is applicable to all immunoglobulin superfamily molecules which possess binding domains. Preferably, the present invention relates to antibodies.

An antigen is specific to a pathogen if targeting the antigen results in substantially exclusive targeting of the pathogen under physiological conditions.

The variable domains of the heavy and light chains of immunoglobulins, and the equivalents in other proteins such as the alpha and beta chains of T-cell receptors, are responsible for determining antigen binding specificity. VH and VL domains are capable of binding antigen independently, as in VH and V[ dAbs. References to VH and V[ domains include modified versions of VH and VL domains, whether synthetic or naturally occurring.

For example, naturally occurring VH variants include camelid VHH domains, and the heavy chain immunoglobulins IgNAR of cartilaginous fish.

Antibodies target pathogens before they infect cells. We show herein that upon infection these antibodies remain bound to pathogens and are able to transport linked agents into the cell.

1. Ligands Any ligand which can bind to a pathogen-associated antigen under physiological conditions, and be internalized by a cell, is suitable for use in the present invention. The natural immune system uses antibodies as ligands for pathogens, and antibodies or antibody fragments are ideal for use in the present invention. Other possibilities include binding domains from other receptors, as well as engineered peptides and nucleic acids.

Ia. Antibodies References herein to antigen-or pathogen-specific antibodies, antigen-or pathogen-binding antibodies and antibodies specific for an antigen or pathogen are coterminous and refer to antibodies, or binding fragments derived from antibodies, which bind to antigens which are present on a pathogen in a specific manner and substantially do not cross-react with other molecules present in the circulation or the tissues.

An "antibody" as used herein includes but is not limited to, polyclonal, monoclonal, recombinant, chimeric, complementarity determining region (CDR)-grafted, single chain, bi-specific, Fab fragments and fragments produced by a Fab expression library. Such fragments include fragments of whole antibodies which retain their binding activity for the desired antigen, Fv, F(ab'), F(ab')2 fragments, and F(v) or VH antibody fragments as well as fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody. Furthermore, the antibodies and fragments thereof may be human or humanized antibodies, as described in further detail below.

Antibodies and fragments also encompass antibody variants and fragments thereof.

S Variants include peptides and polypeptides comprising one or more amino acid sequence substitutions, deletions, and/or additions that have the same or substantially the same affinity and specificity of epitope binding as the antigen-specific antibody or fragments thereof.

The deletions, insertions or substitutions of amino acid residues may produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

Conservative substitutions may be made, for example according to the Table below.

Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other: ALIPHATIC Non-polar G A P

ILV

Polar -uncharged C S T M

NQ

Polar -charged D F

KR

AROMATIC HEWY

Homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur Le. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids -such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as 0), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.

Thus, variants may include peptides and polypeptides comprising one or more amino acid sequence substitutions, deletions, and/or additions to the antigen specific antibodies and fragments thereof wherein such substitutions, deletions and/or additions do not cause substantial changes in affinity and specificity of epitope binding. Variants of the antibodies or fragments thereof may have changes in light and/or heavy chain amino acid sequences that are naturally occurring or are introduced by in vitro engineering of native sequences using recombinant DNA techniques. Naturally occurring variants include "somatic" variants which are generated in vivo in the corresponding germ line nucleotide sequences during the generation of an antibody response to a foreign antigen.

Variants of antibodies and binding fragments may also be prepared by mutagenesis techniques. For example, amino acid changes may be introduced at random throughout an antibody coding region and the resulting variants may be screened for binding affinity for the target antigen, or for another property. Alternatively, amino acid changes may be introduced into selected regions of the antibody, such as in the light and/or heavy chain CDRs, and/or in the framework regions, and the resulting antibodies may be screened for binding to the target antigen or some other activity. Amino acid changes encompass one or more amino acid substitutions in a CDR, ranging from a single amino acid difference to the introduction of multiple permutations of amino acids within a given CDR. Also encompassed are variants generated by insertion of amino acids to increase the size of a CDR.

The antigen-binding antibodies and fragments thereof may be humanized or human engineered antibodies. As used herein, a humanized antibody", or antigen binding fragment thereof, is a recombinant polypeptide that comprises a portion of an antigen binding site from a non-human antibody and a portion of the framework and/or constant regions of a human antibody. A human engineered antibody or antibody fragment is a non-human (e.g., mouse) antibody that has been engineered by modifying (e.g., deleting, inserting, or substituting) amino acids at specific positions so as to reduce or eliminate any detectable immunogenicity of the modified antibody in a human.

Humanized antibodies include chimeric antibodies and CDR-grafted antibodies. Chimeric antibodies are antibodies that include a non-human antibody variable region linked to a human constant region. Thus, in chimeric antibodies, the variable region is mostly non-human, and the constant region is human. Chimeric antibodies and methods for making them are described in, for example, Proc. NatL Aced. ScL USA, 81: 6841-6855 (1984).

Although, they can be less immunogenic than a mouse monoclonal antibody, administrations of chimeric antibodies have been associated with human immune responses (HAMA) to the non-human portion of the antibodies.

CDR-grafted antibodies are antibodies that include the CDRs from a non-human "donor" antibody linked to the framework region from a human "recipient" antibody. Methods that can be used to produce humanized antibodies also are described in, for example, US 5,721,367 and 6,180,377.

"Veneered antibodies" are non-human or humanized (e.g., chimeric or CDR-grafted antibodies) antibodies that have been engineered to replace certain solvent-exposed amino acid residues so as to reduce their immunogenicity or enhance their function.

Veneering of a chimeric antibody may comprise identifying solvent-exposed residues in the non-human framework region of a chimeric antibody and replacing at least one of them with the corresponding surface residues from a human framework region.

Veneering can be accomplished by any suitable engineering technique.

Further details on antibodies, humanized antibodies, human engineered antibodies, and methods for their preparation can be found in Antibody Engineering, Springer, New York, NY, 2001.

Examples of humanized or human engineered antibodies are lgG, 1gM, IgE, IgA, and lgD antibodies. The antibodies may be of any class (lgG, IgA, 1gM, IgE, lgD, etc.) or isotype and can comprise a kappa or lambda light chain. For example, a human antibody may comprise an lgG heavy chain or defined fragment, such as at least one of isotypes, IgGi, lgG2, lgG3 or lgG4. As a further example, the antibodies or fragments thereof can comprise an lgGl heavy chain and a kappa or lambda light chain.

The antigen specific antibodies and fragments thereof may be human antibodies -such as antibodies which bind the antigen and are encoded by nucleic acid sequences which may be naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequence, and fragments, synthetic variants, derivatives and fusions thereof. Such antibodies may be produced by any method known in the art, such as through the use of transgenic mammals (such as transgenic mice) in which the native immunoglobulins have been replaced with human V-genes in the mammal chromosome.

Human antibodies to target a desired antigen can also be produced using transgenic animals that have no endogenous immunoglobulin production and are engineered to contain human immunoglobulin loci, as described in WO 98/24893 and WO 91/00906.

Human antibodies may also be generated through the in vitro screening of antibody display libraries (J1 MoL Blot (1991) 227: 381). Various antibody-containing phage display libraries have been described and may be readily prepared. Libraries may contain a diversity of human antibody sequences, such as human Fab, Fv, and scFv fragments, that may be screened against an appropriate target. Phage display libraries may comprise peptides or proteins other than antibodies which may be screened to identify agents capable of selective binding to the desired antigen.

Phage-display processes mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to an antigen of choice. One such method is described in WO 99/10494. Antigen-specific antibodies can be isolated by screening of a recombinant combinatorial antibody library, preferably a scFv phage display library, prepared using human VL and VH cDNAs prepared from mRNA derived from human lymphocytes.

Methodologies for preparing and screening such libraries are known in the art. There are commercially available kits for generating phage display libraries.

As used herein, the term "antibody fragments" refers to portions of an intact full length antibody -such as an antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); multispecific antibody fragments such as bispecific, trispecific, and multispecific antibodies (e.g., diabodies, triabodies, tetrabodies); binding-domain immunoglobulin fusion proteins; camelized antibodies; minibodies; chelating recombinant antibodies; tribodies or bibodies; intrabodies; nanobodies; small modular immunopharmaceuticals (SMIP), VHH containing antibodies; and any other polypeptides formed from antibody fragments.

The antigen binding antibodies and fragments encompass single-chain antibody fragments (scFv) that bind to the desired antigen. An scFv comprises an antibody heavy chain variable region (VH) operably linked to an antibody light chain variable region (V[) wherein the heavy chain variable region and the light chain variable region, together or individually, form a binding site that binds to the antigen. An scFv may complise a VH region at the amino-terminal end and a V[ region at the carboxy-terminal end.

Alternatively, scFv may comprise a V[ legion at the amino-teiminal end and a VH region at the carboxy-terminal end. Furthermore, although the two domains of the Fv fragment, V[ and VH, are coded for by separate genes, they can be joined, using iecombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv). An scFv may optionally furthei comprise a polypeptide linkei between the heavy chain variable legion and the light chain valiable legion.

The antigen binding antibodies and fragments thereof also encompass immunoadhesins.

One or moie CDRs may be incorporated into a molecule eithei covalently or noncovalently to make it an immunoadhesin. An immunoadhesin may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRs permit the immunoadhesin to specifically bind to the desired antigen.

The antigen binding antibodies and fragments thereof also encompass antibody mimics comprising one oi more antigen binding portions built on an organic or molecular scaffold (such as a protein or carbohydrate scaffold). Proteins having relatively defined three-dimensional structuies, commonly ieferred to as protein scaffolds, may be used as reagents for the design of antibody mimics. These scaffolds typically contain one or more regions which are amenable to specific or random sequence variation, and such sequence randomization is often carried out to produce libraries of proteins from which desired products may be selected. For example, an antibody mimic can comprise a chimeric non-immunoglobulin binding polypeptide having an irnmunoglobulin-like domain containing scaffold having two or more solvent exposed loops containing a different CDR from a paient antibody inserted into each of the loops and exhibiting selective binding activity toward a ligand bound by the parent antibody. Non-immunoglobulin protein scaffolds have been proposed for obtaining proteins with novel binding properties.

Antigen specific antibodies or antibody fragments theieof typically bind to the desired antigen with high affinity (e.g., as determined with BlAcore), such as for example with an equilibrium binding dissociation constant (KD) for the antigen of about l5nM or less, 10 nM or less, about 5 nM or less, about 1 nM or less, about 500 pM or less, about 250 pM or less, about 100 pM or less, about 50 pM or less, or about 25 pM or less, about 10 pM or less, about 5 pM or less, about 3 pM or less about 1 pM or less, about 0.75 pM or less, or about 0.5 pM or less.

lb Peptide Ligands Peptides, such as peptide aptamers, can be selected from peptide libraries by screening procedures. In practice, any vector system suitable for expressing short nucleic acid sequences in a eukaryotic cell can be used to express libraries of peptides. In a preferred embodiment, high-titer retroviral packaging systems can be used to produce peptide aptamer libraries. Various vectors, as well as amphotropic and ecotropic packaging cell lines, exist that can be used for production of high titers of retroviruses that infect mouse or human cells. These delivery and expression systems can be readily adapted for efficient infection of any mammalian cell type, and can be used to infect tens of millions of cells in one experiment. Aptamer libraries comprising nucleic acid sequences encoding random combinations of a small number of amino acid residues, e.g., 5, 6, 7, 8, 9, 10 or more, but preferably less than 100, more preferably less than 50, and most preferably less than 20, can be expressed in retrovirally infected cells as free entities, or depending on the target of a given screen, as fusions to a heterologous protein, such as a protein that can act as a specific protein scaffold (for promoting, e.g., expressibility, intracellular or intracellular localization, stability, secretability, isolatablitiy, or detectability of the peptide aptamer. Libraries of random peptide aptamers when composed of, for example 7 amino acids, encode molecules large enough to represent significant and specific structural information, and with 10' or more possible combinations is within a range of cell numbers that can be tested.

Preferably, the aptamers are generated using sequence information from the target antigen.

In identifying an aptamer, for example, a population of cells is infected with a gene construct expressing members of an aptamer library, and the ability of aptamers to bind to an antigen is assessed, for instance on a BlAcore platform. Coding sequences of aptamers selected in the first round of screening can be amplified by PCR, re-cloned, and re-introduced into naïve cells. Selection using the same or a different system can then be repeated in order to validate individual aptamers within the original pool.

Aptamer coding sequences within cells identified in subsequent rounds of selection can be iteratively amplified and subcloned and the sequences of active aptamers can then be determined by DNA sequencing using standard techniques.

Ic Structured polypeptides Polypeptides tethered to a synthetic molecular structure are known in the art (Kemp, D. S. and McNamara, P. E., J. Org. Chem, 1985; Timmerman, P. et al., ChemBioChem, 2005). Meloen and co-workers had used tris(bromomethyl)benzene and related molecules for rapid and quantitative cyclisation of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces (Timmerman, P. et al., ChemBioChem, 2005). Methods for the generation of candidate drug compounds wherein said compounds are generated by linking cysteine containing polypeptides to a molecular scaffold as for example tris(bromomethybenzene are disclosed in WO 2004/077062 and WO 2006/078161.

W02004/077062 discloses a method of selecting a candidate drug compound. In particular, this document discloses various scaffold molecules comprising first and second reactive groups, and contacting said scaffold with a further molecule to form at least two linkages between the scaffold and the further molecule in a coupling reaction.

W02006/078161 discloses binding compounds, immunogenic compounds and peptidomimetics. This document discloses the artificial synthesis of various collections of peptides taken from existing proteins. These peptides are then combined with a constant synthetic peptide having some amino acid changes introduced in order to produce combinatorial libraries. By introducing this diversity via the chemical linkage to separate peptides featuring various amino acid changes, an increased opportunity to find the desired binding activity is provided. Figure 7 of this document shows a schematic representation of the synthesis of various loop peptide constructs.

International patent application WO2009098450 describes the use of biological selection technology, such as phage display, to select peptides tethered to synthetic molecular structures. In this approach, peptides are expressed on phage, and then reacted under suitable conditions with molecular scaffolds, such that a structurally constrained peptide is displayed on the surface of the phage.

Such structured peptides can be designed to bind to any desired antigen, and can be coupled to an agent in order to direct the complex to the cell.

Id Indirect ligands Indirect ligands bind to the antigen via a second ligand, which recognises the antigen specifically. For example, the second ligand is an antibody which is specific to the antigen. Ligands described in sections la-ic above may be prepared which are specific S for immunoglobulins, but which bind thereto in a manner which is not dependent on the binding specificity of the target immunoglobulin. For instance, anti-Fc antibodies, peptides and structured peptides may be prepared. Antibody-binding peptides such as Protein A, Protein G and Protein L can be used.

2. Antibody conjugates Methods for attaching a drug or other small molecule pharmaceutical to an antibody fragment are well-known. various peptide conjugation chemistries are established in the art and include bifunctional chemical linkers such as N-succinimidyl (4-iodoacetyl)- aminobenzoate; sulfosuccinimidyl (4-iodoacety-aminobenzoate; 4-succinimidyl- oxycarbonyl-[alpha]-(2-pyridyldithio) toluene; sulfosuccinimidyl-6-[alpha]-methyl- [alpha]-(pyridyldithiol)-toluamido]hexanoate; N-succininiidyl-3-(-2-pyridyldithio)-proprionate; succinimidyl-6 -[3(-(-2-pyridyldithio)-proprionamido] hexanoate; sulfosuccinimidyl-6-[3(-(-2-pyridyldithio)-propionamido] hexanoate; 3-(2-pyridyldithio)-propionyl hydrazide, ElIman's reagent, dichlorotriazinic acid, S-(2-thiopyridyl)-L-cysteine, and the like. Further bifunctional linking molecules are disclosed in U.S. Pat. Nos. 5,349,066; 5,618,528; 4,569,789; 4,952,394; and 5,137,877, as well as Corson et al., ACS Cemical Biology 3, 11, pp677-692, 2008.

Polypeptide agents and polypeptide ligands, including antibodies, may be conjugated via functional or reactive groups on one (or both) polypeptide(s). These are typically formed from the side chains of particular amino acids found in the polypeptide polymer. Such reactive groups may be a cysteine side chain, a lysine side chain, or an N-terminal amine group or any other suitable reactive group.

Reactive groups are capable of forming covalent bonds to the ligand to be attached.

Functional groups are specific groups of atoms within either natural or non-natural amino acids which form the functional groups.

Suitable functional groups of natural amino acids are the thiol group of cysteine, the amino group of lysine, the carboxyl group of aspartate or glutamate, the guanidinium group of arginine, the phenolic group of tyrosine or the hydroxyl group of serine. Non-natural amino acids can provide a wide range of functional groups including an azide, a keto-carbonyl, an alkyne, a vinyl, or an aryl halide group. The amino and carboxyl group of the termini of the polypeptide can also serve as functional groups to form covalent bonds to a desired ligand.

S Alternatives to thiol-mediated conjugations can be used to attach a ligand to a polypeptide via covalent interactions. These methods may be used instead of (or in combination with) the thiol mediated methods by producing polypeptides bearing unnatural amino acids with the requisite chemical functional groups, in combination small molecules that bear the complementary functional group, or by incorporating the unnatural amino acids into a chemically or recombinantly synthesised polypeptide when the molecule is being made after the selection/isolation phase.

The unnatural amino acids incorporated into peptides and proteins on phage may include 1) a ketone functional group (as found in para or meta acetyl-phenylalanine) that can be specifically reacted with hydrazines, hydroxylamines and their derivatives (Addition of the keto functional group to the genetic code of Escherichia coli. Wang L, Zhang Z, Brock A, Schultz PG. Proc NatI Acad Sci US A. 2003 Jan 7;100(1):56-61; Bioorg Med Chem Lett.

2006 Oct 15;16(20):5356-9. Genetic introduction of a diketone-containing amino acid into proteins. Zeng H, Xie U, Schultz PG), 2) azides (as found in p-azido-phenylalanine) that can be reacted with alkynes via copper catalysed "click chemistry" or strain promoted (3+2) cyloadditions to form the corresponding triazoles (Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli. Chin JW, Santoro SW, Martin AB, King DS, Wang L, Schultz PG. U Am Chem Soc. 2002 Aug 7;124(31):9026-7; Adding amino acids with novel reactivity to the genetic code of Saccharomyces cerevisiae.

Deiters A, Cropp TA, Mukherji M, Chin JW, Anderson JC, Schultz PG. U Am Chem Soc. 2003 Oct 1 125(39):1 1782-3), or azides that can be reacted with aryl phosphines, via a Staudinger ligation (Selective Staudinger modification of proteins containing p- azidophenylalanine. Tsao ML, Tian F, Schultz PG. Chembiochem. 2005 Dec;6(12):2147- 9), to form the corresponding amides, 4) Alkynes that can be reacted with azides to form the corresponding triazole (In vivo incorporation of an alkyne into proteins in Escherichia coli. Deiters A, Schultz PG. Bioorg Med Chem Lett. 2005 Mar 1;15(5):1521-4), 5) Boronic acids (boronates) than can be specifically reacted with compounds containing more than one appropriately spaced hydroxyl group or undergo palladium mediated coupling with halogenated compounds (Angew Chem Int Ed EngI. 2008;47(43):8220-3. A genetically encoded boronate-containing amino acid., Brustad E, Bushey ML, Lee JW, Groff D, Liu W, Schultz PG), 6) Metal chelating amino acids, including those bearing bipyridyls, that can specifically co-ordinate a metal ion (Angew Chem Int Ed EngI. 2007;46(48):9239-42.

A genetically encoded bidentate, metal-binding amino acid. Xie J, Liu W, Schultz FG).

Unnatural amino acids may be incorporated into proteins and peptides by transforming E. coli with plasmids or combinations of plasmids bearing: 1) the orthogonal aminoacyl-S tRNA synthetase and tRNA that direct the incorporation of the unnatural amino acid in response to a codon, 2) The phage DNA or phagemid plasmid altered to contain the selected codon at the site of unnatural amino acid incorporation (Proc NatI Acad Sci U S A. 2008 Nov 18;105(46):17688-93. Protein evolution with an expanded genetic code. Liu CC, Mack AV, Tsao ML, Mills JH, Lee HS, Choe H, Farzan M, Schultz PG, Smider VV; A phage display system with unnatural amino acids. Tian F, Tsao ML, Schultz PG. J Am Chem Soc. 2004 Dec 15;126(49):15962-3). The orthogonal aminoacyl-tRNA synthetase and tRNA may be derived from the Methancoccus janaschii tyrosyl pair or a synthetase (Addition of a photocrosslinking amino acid to the genetic code of Escherichiacoli. Chin JW, Martin AB, King DS, Wang L, Schultz PG. Proc NatI Acad Sci U S A. 2002 Aug 20;99(17):11020-4) and tRNA pair that naturally incorporates pyrrolysine (Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N(epsilon)-(o-azidobenzyloxycarbonyl) lysine for site-specific protein modification. Yanagisawa T, lshii R, Fukunaga R, Kobayashi T, Sakamoto K, Yokoyama S. Chem Biol. 2008 Nov 24;15(11):1187-97; Genetically encoding N(epsilon)-acetyllysine in recombinant proteins.

Neumann H, Peak-Chew SY, Chin JW. Nat Chem Biol. 2008 Apr;4(4):232-4. Epub 2008 Feb 17). The codon for incorporation may be the amber codon (UAG) another stop codon (UGA, or UAA), alternatively it may be a four base codon. The aminoacyl-tRNA synthetase and tRNA may be produced from existing vectors, including the pBK series of vectors, pSUP (Efficient incorporation of unnatural amino acids into proteins in Escherichia coli. Ryu Y, Schultz PG.Nat Methods. 2006 Apr;3(4):263-5) vectors and pDULE vectors (Nat Methods. 2005 May;2(5):377-84. Photo-cross-linking interacting proteins with a genetically encoded benzophenone. Farrell IS, Toroney R, Hazen JL, MehI RA, Chin JW). The E.coli strain used will express the F' pilus (generally via a tra operon). When amber suppression is used the F. coli strain will not itself contain an active amber suppressor tRNA gene. The amino acid will be added to the growth media, preferably at a final concentration of 1mM or greater. Efficiency of amino acid incorporation may be enhanced by using an expression construct with an orthogonal ribosome binding site and translating the gene with ribo-X(Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion. Wang K, Neumann H, Peak- Chew SY, Chin JW. Nat Biotechnol. 2007 Jul;25(7):770-7). This may allow efficient multi-site incorporation of the unnatural amino acid providing multiple sites of attachment to the ligand.

Such methods are useful to attach agents to antibodies and other ligands, including non-peptide ligands.

Techniques for conjugating antibodies to drugs and other compounds are also described in Caitei & Senter, Cancer Journal: May/June 2008 -Volume 14 -Issue 3 -pp 154-169; Ducry and Stump, Bioconjugate Chem., 2010,21(1), pp 5-13.

Alternatively! bispecific antibodies may be used. For example! bispecific domain antibodies are known in the alt, and are useful for taigeting both a desired antigen and an agent.

The half-life of antibody conjugates in the serum is dependent no a number of factors, but smaller antibody fragments tend to be eliminated quickly from the circulation.

Accordingly, smaller constructs, for example comprising a domain antibody and a small peptide toxin, are advantageously coupled to a polypeptide which increases serum half-life. For example, they can be coupled to HSA. Preferably, the bond to HSA is labile, for example having a defined half life, such that the construct is released from the HSA when bound to a cell, and is internalised without the HSA. A useful approach is to use a multispecific ligand construct, such that the ligand also binds HSA, maintaining it in circulation. The affinity of the ligand for HSA can be tailored such that the ligand can be internalised by the cell as appropriate.

3. Agents As used herein, the term "agent" is used to refei to any compound which can be deliveied into a cell on a virus. Many possibilities will be appaient to those skilled in the alt, including delivering therapeutic proteins to cell, replacement or supplementation of defective proteins to correct defects and delivery of small molecule drugs to cells. The system according to the invention can use viruses to deliver agents directly to the cytosol.

Using specific viruses, it is also possible to target agents to specific cells. Cell-specific viruses are known, and/or can be engineered. For example, CD-40 targeted adenovirus vectois have been sued to delivei nucleic acids to dendritic cells. Moreovei, viruses can be modified to reduce their pathogenicity, for example by making them replication detective or otherwise attenuating them. For a review of the use ot viruses to deliver therapeutic agents, see Youg et al., J Pathol. 2006 Jan; 208(2): 299-318.

Agents can also be cytotoxic, cytostatic or antiviral drugs. In one embodiment! the drug is a cytotoxic agent that inhibits or prevents the function of cells and/or causes destruction of cells. Examples of cytotoxic agents include radioactive isotopes, chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including synthetic analogues and derivatives thereof. The cytotoxic agent may be selected from the group consisting of an auristatin, a DNA minor groove binding agent, a DNA minor groove alkylating agent, an enediyne, a lexitropsin, irinotecan derivatives, a calichaemycin, a microtubule inhibitor, a nucleoside analogue, a toxin such as ricin, a radionuclide, an interleukin, a duocarmycin, a taxane, a puromycin, a dolastatin, a maytansinoid, a ymca alkaloid, pyrrolobenzodiazepines (PBD5), tubulysins, or a combination of two or more thereof.

Toxins which are effective in non-dividing cells are important where the therapeutic target is not cancer. Examples include ribosome inhibitors, for example toxins such as vero toxin and shiga toxin, drugs such as Saporin, and other metabolic blockers.

Radionuclides include Radium 223. Radium 226. Thorium 232, Thorium 227, Astatine 211, Bismuth 213 and Actinium 225, as well as isotopes of phosphorous, copper, strontium, yttrium, silver, iodine, samarium, holmium, rhenium, lutetium, polonium, and the like. For example, the radionuclide can be selected from comprising a metal ion selected from the group consisting of 68Ga, 64Cu 67Cu, 212Pb, 203Pb, 212Bi, 213Bi, 89Zr, 186Re, Q9mTc, 90Y, 86Y, 111Lu, "In and 188Re. See Neves et al., Journal of Radioanalytical and Nuclear Chemistry, Vol. 266, No. 3 (2005) 377-384.

Exeuiplary of metal chelator groups for conjugates comprising radionuclides include those selected from the group consisting of DOTA, DTPA, EDTA, cyclohexyl DTPA, hydroxamic acid chelators, deferrioxamine, hynic, thiosemicarbazone, TETA, NOTA, Mag3, and N2S2.

Approaches for attaching multiple drugs to antibodies are known in the art, such as the fleximer technology developed by Mersana: Papisov et al. Biomacromolecules. 2005 Sep-Oct;6(5):2659-70.

In one embodiment, the agent may be taxol.

Agent loading on the conjugate may range from 1 to 2 or more agents per ligand.

Accordingly, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more agent moieties may be covalently attached to the ligand. for example via a linker. Thus, compositions of conjugates may include collections of ligands conjugated with one or more different agents. The number of agents per ligand in preparations of conjugates may be characterized by conventional means -such as mass spectroscopy, ELISA assay, electrophoresis, and HPLC.

4. Administration of Compounds Generally, the immnoconjugates according to the invention will be utilised in purified form together with pharmacologically appropriate carriers. Typically, these carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, any including saline and/or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically-acceptable adjuvants, if necessary to keep a polypeptide complex in suspension, may be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition).

The conjugates of the present invention may be used as separately administered compositions or in conjunction with other agents. These can include further antibodies, antibody fragments and conjugates, and various immunotherapeutic drugs, such as cylcosporine, methotrexate, adriamycin or cisplatinum, and immunotoxins.

Pharmaceutical compositions can include "cocktails" of various cytotoxic or other agents in conjunction with the selected antibodies, receptors or binding proteins thereof of the present invention, or even combinations of selected polypeptides according to the present invention having different specificities, such as polypeptides selected using different target ligands, whether or not they are pooled prior to administration.

The route of administration of pharmaceutical compositions according to the invention may be any of those commonly known to those of ordinary skill in the art. For therapy, including without limitation immunotherapy, the selected antibodies, receptors or binding proteins thereof of the invention can be administered to any patient in accordance with standard techniques. The administration can be by any appropriate mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or also, appropriately, by direct infusion with a catheter. The dosage and frequency of administration will depend on the age, sex and condition of the patient.

concurrent administration of other drugs, counterindications and other parameters to be taken into account by the clinician.

The compounds of this invention can be lyophilised for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective and art-known lyophilisation and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of activity loss and that use levels may have to be adjusted upward to compensate.

The compositions containing the present peptide ligands or a cocktail thereof can be administered for prophylactic and/or therapeutic treatments. In certain therapeutic applications, an adequate amount to accomplish at least partial inhibition, suppression, modulation, killing, or some other measurable parameter, of a population of selected cells is defined as a "therapeutically-effective dose". Amounts needed to achieve this dosage will depend upon the severity of the disease and the general state of the patient's own immune system, but generally range from 0.005 to 5.0 mg of selected peptide ligand per kilogram of body weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonly used. For prophylactic applications, compositions containing the present peptide ligands or cocktails thereof may also be administered in similar or slightly lower dosages.

A composition containing a compound according to the present invention may be utilised in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing or removal of a select target cell population in a mammal. In addition, the selected repertoires of polypeptides described herein may be used extracorporeally or in vitro selectively to kill, deplete or otherwise effectively remove a target cell population from a heterogeneous collection of cells. Blood from a mammal may be combined extracorporeally with the selected peptide ligands whereby the undesired cells are killed or otherwise removed from the blood for return to the mammal in accordance with standard techniques.

5. Viral infection processes The invention can take advantage of a number of processes used by viruses to infect cells, thereby transporting the ligand/agent conjugate into the cell. Virus entry mechanism have been reviewed in a number of publications, including Marsh et al (Cell 124, 729-740, February 24, 2006); Grove and Marsh, (J. Cell Biol. Vol. 195 No. 7 1071- 1082, 2011). Macropinocytosis, reviewed by Kerr and Teasdale (Traffic 2009, 10:364- 371) and Mercer and Helenius (Curr. Opin. Microbiol. 15:490-499, 2012; also Nature Cell Biology, 11:510, 2009) is involved in the uptake of many different viruses, which exit from the macropinosome or lysosome at deifferent stages after infection. Clathrin-coated pits are described in Hewlett et al., (JOB 124:689-703, 1994).

Viruses exit the endosome compartments into which they are taken up at different times and by different methods. Adenovirus exits the endosome rapidly, within 15 minutes of infection in a process that is pH-dependent [Greber, U.F., et al., Stepwise dismantling of adenovirus 2 during entry into cells. Cell, 1993. 75(3): p. 477-86]. Other viruses, such as Rotavirus exit much later from the lysosome in a process that is acid-independent but Calcium and cathepsin dependent [Golden, J.W., et al., Cathepsin S supports acid-independent infection by some reoviruses. The Journal of biological chemistry, 2004.

279(10): p. 8547-57]. Other viruses are also pH-independent such as Ebola [Chandran, K., et al., Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection.

Science, 2005. 308(5728): p. 1643-5] and SARS coronavirus [Silvey, K.J., et al., Role of immunoglobulin A in protection against reovirus entry into Murine Peyer's patches.

Journal of virology, 2001. 75(22): p. 10870-9]. Dengue virus exits from late endosomal compartments (rather than early ones like adenovirus) in a process thought to be triggered by interaction with bis(monoacylglycero)phosphate, a lipid specific to late endosomes [Zaitseva, E., et al., Dengue virus ensures its fusion in late endosomes using compartment-specific lipids. PLoS pathogens, 2010. 6(10): p. e1001131]. Rhinovirus may form size-selective pore rather than rupturing the endosome [Brabec, M., et al., Opening of size-selective pores in endosomes during human rhinovirus serotype 2 in vivo uncoating monitored by single-organelle flow analysis. Journal of virology, 2005. 79(2): p. 1008-16]. Entry of viruses also depends on cell type. For instance, HIV-1 transcytoses through polarized epithelial cells without infecting them [Bomsel, M., Transcytosis of infectious human immunodeficiency virus across a tight human epithelial cell line barrier.

Nature medicine, 1997. 3(1): p.42-i].

The invention is further described in the following examples.

Examples

Example 1: Antibodies carry agents into cells We have recently made two key discoveries concerning antibody immunity. First, we have shown that antibodies can routinely enter cells attached to viral particles. Second, that antibodies carried into cells on viruses are stable and remain active. We postulated that this might allow us to modify virus-binding antibodies with active molecules, either protein, peptide or small molecule and that these would get carded only into cells infected with virus. Antibodies could be modified with molecules that either specifically interfere with viral replication or kill the cell, thereby preventing infection.

We conjugated a monoclonal anti-adenovirus antibody to a small molecule fluorophore and showed that this modified antibody is carried into cells upon infection with adenovirus (Figure 1A&B). The modified antibody stays attached to the virus and is clearly present in the cell. To show that the antibody conjugate is stable and still covalently associated we stained cells after infection with a secondary antibody. The antibody-conjugate and secondary exactly co-localise. Finally, addition of modified antibody without virus resulted in no modified antibodies inside cells. Thus by replacing the fluorophore with a small molecule (or protein or peptide) that is cytotoxic instead of fluorescent we could kill those cells that are infected.

Method 1 5 Preparation of the Antibody Conjugate Alexa Fluor 488 5-SDP ester (Invitrogen) was conjugated to human serum lgG following supplier's guidelines. After the reaction, remaining free dye was removed by extensive dialysis against PBS. The preparation of antibody conjugate was analyzed by absorbance measurements, size-exclusion chromatography, and by both fluorescent imaging and Coomassie staining of an SDS-PAGE gel. The analytical chemistry confirmed efficient conjugation of Alexa 488 to human serum lgG and showed no evidence of contamination with free dye or with other non-conjugated proteins.

Selective Delivery of a Small Organic Molecule (the Alexa Dye) to Virus-infected Cells by Means of the Antibody Conjugate HeLa cells were grown on coverslips over night and then infected with human adenovirus that had been pre-incubated with AlexaFluor488-conjugated human serum lgG. 30 minutes post infection cells were fixed in formaldehyde. Some samples of cells were subsequently permeabilized and stained with an AlexaFluor56B-conjugated anti-human lgG antibody (Invitrogen) to confirm the intracellular localization of the AlexaFluor488-antibody conjugate. Coverslips were then mounted on microscopy slides using mounting medium containing DAPI. Confocal microscopic images were taken using a Zeiss 63x lens on a Jena LSM 710 microscope (Carl Zeiss Microlmaging). Images of several focal planes within cells were combined into a projection along the z-axis to allow visualization in one image of antibody conjugate-bound viruses residing in different focal planes within the same cell.

Example 2: Immunotoxin kills virus-infected cells The anti-adenovirus antibody 9C12 was fused to the cytotoxic agent Taxol through a reactive NHS ester. Cell infection experiments were performed by exposing cells to adenovirus in the presence of either 9C12 or Taxol-conjugated 9C12.

P OH Taxol

Exposing cells to adenovirus in the presence of unconjugated 9C12 antibody results in the production of virus-infected cells, see as the black bars in Figure 2.

Using the Taxol-conjugated immunotoxin led to substantial reduction in numbers of virus-infected cells both 1 and 2 days post-infection, as shown in the striped bars Figure 2. 2 days post-infection, virus-infected cells were substantially eliminated, except at very low antibody concentrations; at O.lpgIml, insufficient antibody is present to eliminate all infected cells. However, the number of infected cells is still reduced in comparison to the cells which are exposed to adenovirus in the presence of the unconjugated 9C12 antibody.

Method Taxol conjugation: lml of 0.8mg/mi 9C12 antibody was first dialysed in 0.1 M NaHCO3 (pH8.5), 0.2M NaCI at 4°C. A stock solution of Taxol was formed by dissolving 2.5mg Taxol in 50m1 DMSO.

Dilutions of this stock solution were made, such that lOpI would form 10, 20 and 100 molar excesses over the 9C12 antibody were produced. lOmI of the Taxol solutions were then mixed with the antibody, and incubated at room temperature on a rotary mixer for 3 hours. After this, the antibody was dialysed in PBS overnight at 4 °C. The conjugated antibody was analysed by LCMS, and compared with the chromatograms of unlabelled 9C12. Increases in mass of multiples of lkDa were counted as being conjugated with molecules of Taxol.

Infection Experiments: The Taxol-9C12 was serially diluted (eg.1 00, 10, 1.0, 0.1, 0.01 pg/mI concentrations. i OpI of these solutions were incubated with lOpI human Adenovirus (with a GEP reporter gene) for 30 minutes, before addition to 5.OxlOe4 cells (HeLa, or TRIM21 shRNA knock down -Dharm) in imI of DMEM media (supplemented with 10% Foetal Calf Serum, and 1% Penicillin/Streptomycin). Cells were incubated at 37°C, in a 5% C02 incubator before harvesting and resuspension in 250pl 4% paraformealdehyde. The samples were analysed by FACS, gating for GFP positive cells.

IS Example 3

Targeting of HSV-1 infected Cells and Inhibition of HSV-1 Infectivity AF7 is a non-neutralizing antibody that targets glycoprotein D on HSV-1. Therefore upon conjugation with a cytotoxic, any reduction in viral replication mediated by an APT conjugate has to derive from the activity of the attached drug. Use of lOyg/mI or 4Opg/ml AP7 conjugated to taxol was sufficient to significantly reduce infectivity (Figure la&b).

Importantly, use of lOpg/ml AP7-taxol conjugate gave a dramatic reduction in plaque size, showing that the drug is efficiently inhibiting viral spread. Taxol is a relatively slow acting cytotoxic and this is consistent with its greater effect on viral spread versus initial infection. AP7-DMSO showed no activity, confirming that the cytotoxic is mediating antiviral affects not the antibody itself. To further confirm that the effect we observe is due to the action of taxol inside the cell we compared use of the AP7-taxol conjugate in Hela cells to Vero cells. Unlike HeLa cells, which HSV-1 infects by endocytosis, Vero cells are infected by HSV-1 following fusion at the plasma membrane. Given that infection of Vero cells would therefore leave antibody conjugate behind on the outside of the cell we predicted that no antiviral effect would be observed. As can be seen in Figure 10, no reduction in plaque size was observed when Vero cells were infected with HSV-1 in the presence of AP7-taxol. This confirms our hypothesis that taxol delivered into the cell by an antiviral antibody can inhibit viral replication.

The Neutralising Antibody LP2 is not effective The experiment performed with AP7 was also attempted using a neutralizing antibody (LP2) against HSV-1 fused to Taxol. This attempt was unsuccessful and the antibody was more effective by itself than as an ADC. We infer that this is because the conjugate did not enter the cell and only served to reduce the specific activity of the antibody. The results are shown in Figure 4.

Materials and methods Ce/Is Spontaneously immortalised human keratinocyte cell line HaCaT (Boukamp et al., 1988) and African green monkey kidney cell line Vero (ATCC) were grown at 37 °C incubator with 5% 002 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/mI penicillin, and 100 -g/ml streptomycin. Virus

Herpes simplex virus type 1 (HSV-1) strain KOS (Smith, 1964) was propagated in HaCaT cells.

Antibody Purified anti-glycoprotein D antibody APi was a gift from S. Bell at Division of Virology, Department of Pathology, University of Cambridge (Minson AC, Hodgman TC, Digard P, Hancock DO, Bell SE, Buckmaster EA. An analysis of the biological properties of monoclonal antibodies against glycoprotein D of herpes simplex virus and identification of amino acid substitutions that confer resistance to neutralization. J Gen Virol. 1986 67:1001-13.) Plaque assay and plaque size quantification HSV-1 titres were determined by plaque assay on Vero cell monolayer in six-well plates.

Ten-fold serial dilutions of virus samples were prepared with supplemented DMEM media and 0.4 ml of the virus dilutions were added to each well. The cells were incubated at 37 °C for one hour and overlaid with 2 ml of DMEM solution containing 0.6% carboxymethyl cellulose, 2% FOS and antibiotics. After incubation at 37 00 for 2 to 3 days the plaques were fixed in 3.7% formaldehyde for 20 mm and stained with 0.1% toluidine blue solution for 20 mm. Plaques were counted and the virus titre was calculated as plaque forming units per ml (PFUImI). Microscopic images of plaques were taken to calculate plaque size in pixels using lmagej software.

Virus purification HaCaT cells were incubated with virus with M.O.l. of 0.05 at 37 °C for 1 h and further incubated in DMEM supplemented as described above for 3 days. The culture medium was harvested and cellular debris was removed by centrifugation at 2,000 rpm for 20 mm at 4 °C. Virus particles were pelleted at 24,800 rpm for 2 h at 4 °C (Beckman 45Ti) and the pellets were resuspended in 2 ml of 1% FCS/PBS and layered onto a 30 ml of 5-15% Ficoll 400 continuous gradient. After centrifugation at 20,000 rpm for 90 mm at 4 CC (Beckman SW 28), the visible band approximately in the middle of the gradient was collected carefully and the virus was harvested by centrifugation as described above.

Virus pellets were resuspended in 1% FCS!PBS and stored at -70 °C. The titres were determined by plaque assay on Vero cells.

1 5 Antibody Taxof conjugation Purified AP7 antibody was incubated with Taxol or DMSO at 1:100 molecule concentration in 0.1 M NaHCO3, 0.2 M NaCI at RT for 4 h. The antibody was dialysed in PBS at 4 °C over night and the concentration was measured by Nanodrop.

Virus and antibody binding Purified viruses were diluted to 10,000 PFU/ml and incubated with APi (10 pg/mI) at 37 °C for 1 h with rotation.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described aspects and embodiments of the present invention will be apparent to those skilled in the art without departing from the scope of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are apparent to those skilled in the art are intended to be within the scope of the following claims.

Claims (31)

1. Claims 1. Use of an agent and a ligand specific for an antigenic determinant of a virus in a conjugate for delivering the cytotoxic agent to the cytoplasm of a cell, wherein said virus infects the cell through pinocytosis.
2. 2. Use according to claim 1, wherein said virus infects the cell through macropinocytosis.
3. 3. Use according to claim 2, wherein said antigenic determinant is a non-neutralising antigenic determinant.
4. 4. Use according to claim 2 or claim 3, wherein said cytotoxic agent and ligand are delivered to a macropinosome.
5. 5. Use according to any one of claims 2 to 4, wherein the virus is selected from the group consisting of vaccinia virus! HIV-1, coxsackie B virus, HSV-1, ASFV, HPV-16, adenovirus 3, echovirus 1, ebola virus, norovirus, parvovirus, RSV, parainfluenza, metapneumovirus, HSV-2, CMV and KHSV.
6. 6. Use according to claim 1, wherein said virus infects the cell through clathrin-mediated uptake.
7. 7. Use according to claim 6, wherein said cytotoxic agent and said ligand are delivered to the endosome.
8. 8. Use according to claim 6 or claim 7, wherein the virus is selected from the group consisting of SFV, VSV, HIV-1, Flavivirus, Bunya virus, influenza virus, adenovirus 2 and adenovirus 5.
9. 9. Use of an agent and a ligand specific for an antigenic determinant of a virus in a conjugate for delivering the cytotoxic agent to the cytoplasm of a cell, wherein said virus infects the cell through receptor-mediated endocytosis.
10. 10. Use according to claim 9, wherein said virus is selected from the group consisting of HSV-2, CMV, HPV-18, coronavirus, HBV, EBV, BK virus and Rhinovirus.
11. 11. Use of an agent and a ligand specific for an antigenic determinant of a virus in a conjugate for delivering the cytotoxic agent to the cytoplasm of a cell, wherein said virus infects the cell through plasma membrane fusion.
12. 12. Use according to claim 11, wherein said virus is selected from the group consisting of RSV, parainfluenza virus, metapneumovirus, HSV-2, coronavirus, measles virus and HIV.
13. 13. A conjugate comprising a ligand specific for an antigenic determinant of a virus S and an agent, linked together with a labile linker, wherein (a) the virus follows an infection cycle wherein it is released from the endosomal compartment of a cell in response to a signal; and (b) the labile linker is cleaved contemporaneously in the infection cycle.
14. 14. A conjugate according to claim 13, wherein the linker is cleaved in response to the same signal as that which leads to release of the virus from the endosome.
15. 15. A conjugate according to claim 13 or claim 14, wherein the signal is an acidification of pH.
16. 16. A conjugate according to claim 15, comprising a ligand specific for an antigenic determinant of a vaccinia virus and an agent, linked together with an acid labile linker.
17. 17. A conjugate according to claim 13 or claim 14, wherein the signal is an increase in the concentration of a reducing agent.
18. 18. A conjugate according to claim 17! comprising a ligand specific for an antigenic determinant of an adenovirus and an agent, linked together with a disulphide linker.
19. 19. A conjugate according to claim 13 or claim 14, wherein the signal is an increase in the concentration of an enzyme.
20. 20. A conjugate according to claim 19! comprising a ligand specific for an antigenic determinant of a rotavirus virus and an agent, linked together with a cathepsin labile linker.
21. 21. A conjugate according to any one of claim 13 to 20, further comprising a virus.
22. 22. A conjugate according to any one of claims 13 to 21, which conjugate is intracellular.
23. 23. Use or conjugate according to any preceding claim, wherein the ligand is an antibody.
24. 24. Use or conjugate according to claim 23, wherein the antibody is a non-neutralising antibody.
25. 25. A conjugate comprising a ligand specific for an antigenic determinant of a virus and an agent, linked together with a non-labile linker.
26. 26. A conjugate according to claim 25, wherein the virus is selected from HSV-1, RSV and Adv5.
27. 27. Use or conjugate according to any one of claims 1 to 22, wherein the ligand is a first half of a specific binding pair, and the virus is complexed with the second half of said specific binding pair.
28. 28. Use or conjugate according to claim 27, wherein the specific binding pair is biotin/streptavidin.
29. 29. Use or conjugate according to any preceding claim, wherein the agent is selected from a cytotoxic agent and an antiviral agent.
30. 30. Use or conjugate according to claim 29, wherein the cytotoxic agent is selected from the group consisting of an auristatin, a DNA minor groove binding agent, a DNA minor groove alkylating agent, an enediyne, a lexitropsin, a duocarmycin. a taxane, a puromycin, a dolastatin, a maytansinoid and a ymca alkaloid or a combination of two or more thereof.
31. 31. Use according to any one of claims 1 to 29, wherein the antiviral agent is a nucleoside analogue selected from the group consisting of 5-fluorouracil, sorivudine, zidovudine, didanosine, zalcitabine, aciclovir, ganciclovir, vidarabine and ribavirin.