# Patent

## Title
Immunotherapeutic molecules and uses

## Abstract

The invention provides molecule comprising: (i) a targeting moiety capable of directly or indirectly targeting to unwanted cells, and (ii) a further moiety that has a masked immune cell binding region so as to prevent binding of the further moiety to an immune cell, wherein the masked immune cell binding region is capable of being selectively unmasked when the molecule is in the vicinity of the unwanted cells so as to allow binding of the further moiety to an immune cell.

## Background
The present invention relates to immunotherapeutic molecules. In particular, it relates to immunotherapeutic molecules that can be used to prevent or treat a condition characterised by the presence of unwanted cells, such as tumours or other disease causing cells.
Immunotherapeutic strategies for targeting malignant disease are an active area of translational clinical research, and have been for several decades. The current models dictate that cancer represents either a functional or constitutional immunodeficiency which can be treated with immunotherapeutic manipulation of the host. These efforts can be broadly classified into two groups. The first serves to augment or support endogenous anti-tumour immunity through measures such as vaccination, cytokine support (IL-2, IFNγ) or reducing immunosuppressant environment (ipilimumab) whilst the second seeks to restore an absolute deficiency with components of a functional immune response (passive immunotherapy with antibodies, TCR transfer, Stem Cell Transplantation and adoptive immunotherapy). These approaches are unified by the argument that a highly effective functional anti-tumour immune response is indeed possible. Although irrefutable evidence exists for an effective anti-tumour immune response in some cases, this central pillar of tumour immunology is overwhelmingly countered by the current clinical reality that despite great efforts, no effective immunotherapeutics are available for the majority of patients with cancer. Almost all cancer vaccination trials have provided negative results, with those providing positive data most frequently demonstrating a small effect. The reality is that therapeutic antibodies offer very modest clinical benefit in the area of oncology. Thus, there remains a demand for more effective immunotherapeutic agents.
Bispecific antibodies unify two antigen binding sites of different specificity into a single construct giving them the ability to bring together two discrete antigens with exquisite specificity. The first bispecific antibodies resembled natural immunoglobulin G (IgG) molecules in which the two arms were equipped with distinct binding specificities. The concept of using these bispecific antibodies to engage cytotoxic T cells for cancer cell lysis was originally shown by Staerz and colleagues in 1985 (Nature 1985, 314: 628). Since then, different kinds of constructs have been made and developed for the same purpose. Recent focus has been on creating antibody constructs by joining two single chain Fv regions (scFv antibody fragments) while omitting the Fc portion present in full immunoglobulins. Each scFv unit in such constructs (so-called bispecific T cell engagers or BiTE antibodies) is made up of one variable domain from each of the heavy (VH) and light (VL) antibody chains, joined with one another via a synthetic polypeptide linker. The resulting bispecific single chain antibody is therefore a species containing two VH/VL pairs of different specificity on a single polypeptide chain of approximately 55 kDa.
Promising experimental results have emerged from the use of BiTE antibodies. Kufer and colleagues have demonstrated that CD3/target antigen-bispecific antibodies of this design have an exceptionally high potency, and can engage CD8+ and CD4+ T cells for redirected lysis of cancer cells at very low effector to target ratios (Mack, PNAS (1995), 92:7021-5). The antibodies have shown potential in treating tumours (Mack, J Immunol (1997), 158:3965-70; Kufer, Canc Immunol Immunother (1997), 45:193-7; and Loffler, Blood (2000), 95: 2098-103) and also in treating non-tumour diseases (Bruhl, J Immunol (2001), 166:2420-6). The antibodies rely on one scFv unit being capable of activating T cells, by specifically binding to an antigen on the T cells, and the other scFv unit specifically binding to an antigen on a target cell intended for destruction. In this way, the antibodies are able to activate and redirect the immune system's cytotoxic potential for the destruction of pathological cells.
Two BiTE antibodies are currently being tested in clinical trials. Blinatumomab (also known as MT103) is bispecific for CD3 on T cells and CD19 on B cells, and is being tested for the treatment of non-Hodgkin's lymphoma and acute lymphoblastic leukaemia. MT110 is bispecific for CD3 and epithelial cell adhesion molecule (EpCAM), and is being tested for the treatment of lung and gastrointestinal cancer patients.
However, a limitation of BiTE antibodies and similar bispecific therapeutic antibodies is that they can activate T cells when binding to any target that expresses the targeting antigen. For instance, if the BiTE or bispecific antibody was designed to target the EGF receptor, which is expressed at high levels in many epithelial cancers but also expressed at moderate levels in healthy tissues, both healthy and cancers tissue are targeted. Moreover, the agent becomes active when bound to any surface and thus non-specific binding to surfaces in a non-antigen binding manner can lead to T-cell activation and off-target effects. For these reasons, all BiTE antibodies in development are given at very low concentrations.
Accordingly, although bispecific antibody constructs have great therapeutic value in redirecting the body's own immune system to achieve the eradication or neutralisation of unwanted cells, the activation of such redirection requires that it be tightly controlled so that the cytotoxic potential is recruited and applied only in the direction intended. Thus, there is a strong need for further immunotherapeutic agents that overcome limitations described above.
The present inventors have now devised a means for recruiting and activating immune cells (eg T cells) to target unwanted cells, wherein the immune cells are only activated when in the vicinity of the targeted cells. As will become apparent below, such selective activation of immune cells is based on selective unmasking of an immune cell binding region in the vicinity of unwanted target cells by an agent that resides in the vicinity of the unwanted cells. In this way, the technology is more specific for the unwanted cell (eg more cancer specific) by virtue of the immune cell binding region only being unmasked in the vicinity of the unwanted cell (eg in the presence of a cancer associated protease). This dramatically expands the therapeutic window of bispecific antibodies and so facilitates larger doses of drug to be given leading to greater target effect.
Accordingly, a first aspect of the invention provides a molecule comprising: (i) a targeting moiety capable of directly or indirectly targeting to unwanted cells, and (ii) a further moiety that has a masked immune cell binding region so as to prevent binding of the further moiety to an immune cell, wherein the masked immune cell binding region is capable of being selectively unmasked when the molecule is in the vicinity of the unwanted cells so as to allow binding of the further moiety to an immune cell. The targeting moiety functions to bring the further moiety into the vicinity of the unwanted cells, for example in the same way as one of the antigen specificities of a BiTE antibody targets the construct to a cancer cell. The selective unmasking of the immune cell binding region in the vicinity of the unwanted cells ensures that the further moiety can only engage immune cells when in the vicinity of the unwanted cells.
The individual components of the molecule are described in detail below.
Targeting Moiety
By ‘targeting moiety’, we include the meaning of any moiety that is capable of targeting to the unwanted cells. Preferably, the targeting moiety is capable of targeting selectively to the unwanted cells. For example, it is preferred if the targeting moiety targets unwanted cells to a greater extent than it does normal cells, and most preferably targets only unwanted cells.
It will be appreciated that binding of the targeting moiety to normal cells may be tolerated if they can be functionally replaced by other therapeutic means or if they are not essential to life. Thus, a targeting moiety that targets to a cancer cell as well as, for example, an endocrine tissue or organ is not precluded. In this case, the targeting moiety acts to redirect an immune response to both unwanted cells and to other cells that can be functionally replaced by therapeutic means. In a life-saving situation for example, the tissue or organ may be sacrificed provided its function was either not essential to life, for instance in the case of the testes, prostate or pancreas, or could be supplied by hormone replacement therapy. Such considerations would apply to the thyroid gland, parathyroids, adrenal cortex and ovaries, for example.
It follows that the targeting moiety may be a moiety that is capable of targeting selectively to unwanted cells as opposed to wanted cells, wherein the unwanted cells may include cells whose presence in a host is undesired and optionally cells whose presence in the host is desired but whose presence can be functionally replaced by therapeutic means.
It is also appreciated that selective unmasking of the immune cell binding regionconfers specificity on where the immune cell binding region is unmasked, and so binding of the targeting moiety to normal cells, in the vicinity of which the immune cell binding region is not unmasked, may also be tolerated.
Most preferably, however, the targeting moiety targets selectively to unwanted cells as opposed to any other cells.
In one embodiment, the targeting moiety is capable of directly targeting to unwanted cells. However, the targeting moiety may be one that indirectly targets to unwanted cells, for example by targeting to another moiety that is localised in the vicinity of the unwanted cells (e.g. by association with the unwanted cells), as described below.
Conveniently, the targeting moiety is a specific binding partner of an entity expressed by or associated with the unwanted cell. Typically, the expressed entity is expressed selectively on the unwanted cell. For example, the abundance of the expressed entity is typically 10 or 100 or 500 or 1000 or 5000 or 10000 higher on the unwanted cell than on other cells within the body to be treated. However, as mentioned above, the selective unmasking of the immune cell binding region in the vicinity of the unwanted cells provides additional specificity on where the immune cell binding region is unmasked and so the binding partner may bind an entity that is similarly or even underexpressed on unwanted cells relative to other cells within the body.
By “binding partner” we include the meaning of a molecule that binds to an entity expressed by a particular cell. Preferably, the binding partner binds selectively to that entity. For example, it is preferred if the binding partner has a Kd value (dissociation constant) which is at least five or ten times lower (i.e. higher affinity) than for at least one other entity expressed by another cell (e.g. a normal cell type), and preferably more than 100 or 500 times lower. More preferably, the binding partner of that entity has a Kd value more than 1000 or 5000 times lower than for at least one other entity expressed by another cell (e.g. normal cell type). Kd values can be determined readily using methods well known in the art. However, as discussed above, it is appreciated that the binding partner may bind selectively to an entity expressed by an unwanted cell and by a normal cell provided that the normal cell may be functionally replaced or else is not essential to life. For example, in lymphoma, anti-CD20 (which targets all B cells) is very effective and kills all B cells, healthy and malignant. However, this can be tolerated as B cells are not critical for health. Further, in the case of melanoma, lymphoma, prostate cancer, thyroid, testicular or ovarian cancer, targeting healthy counterpart tissue may also be tolerated.
Typically, the binding partner is one that binds to an entity that is present or accessible to the binding partner in significantly greater concentrations in or on unwanted cells than in any normal cells of the host. Thus, the binding partner may bind to a surface molecule or antigen on the unwanted cell that is expressed in considerably higher amounts than on normal cells. Similarly, the binding partner may bind to an entity that has been secreted into the extracellular fluid by the unwanted cells to a greater extent than by normal cells. For example, the binding partner may bind to a tumour associated antigen which is expressed on the cell membrane or which has been secreted into tumour extracellular fluid.
The targeting moiety may be a polypeptide or a peptide. In a particularly preferred embodiment, the targeting moiety is an antibody that binds to an antigen expressed by the unwanted cell, for example a tumour associated antigen.
Preferred antibody targets (with examples of unwanted cell types in parentheses) include: Her2/Neu (Epithelial malignancies); CD22 (B cells, autoimmune or malignant); EpCAM (CD326) (Epithelial malignancies); EGFR (epithelial malignancies); PMSA (Prostate Carcinoma); CD30 (B cell malignancies); CD20 (B cells, autoimmune, allergic or malignant); CD33 (Myeloid malignancies); membrane IgE (Allergic B cells); IgE Receptor (CD23) (Mast cells or B cells in allergic disease), CD80 (B cells, autoimmune, allergic or malignant); CD86 (B cells, autoimmune, allergic or malignant); CD2 (T cell or NK cell lymphomas); CA125 (multiple cancers including Ovarian carcinoma); Carbonic Anhydrase IX (multiple cancers including Renal Cell Carcinoma); CD70 (B cells, autoimmune, allergic or malignant); CD74 (B cells, autoimmune, allergic or malignant); CD56 (T cell or NK cell lymphomas); CD40 (B cells, autoimmune, allergic or malignant); CD19 (B cells, autoimmune, allergic or malignant); c-met/HGFR (Gastrointestinal tract and hepatic malignancies; TRAIL-R1 (multiple malignancies including ovarian and colorectal carcinoma); DR5 (multiple malignancies including ovarian and colorectal carcinoma); PD-1 (B cells, autoimmune, allergic or malignant); PDIL (Multiple malignancies including epithelial adenocarcinoma); IGF-1R (Most malignancies including epithelial adenocarcinoma); VEGF-R2 (The vasculature associated with the majority of malignancies including epithelial adenocarcinomas; Prostate stem cell antigen (PSCA) (Prostate Adenocarcinoma); MUC1 (Epithelial malignancies); CanAg (tumors such as carcinomas of the colon and pancreas); Mesothelin (many tumours including mesothelioma and ovarian and pancreatic adenocarcinoma); P-cadherin (Epithelial malignancies, including breast adenocarcinoma); Myostatin (GDF8) (many tumours including sarcoma and ovarian and pancreatic adenocarcinoma); Cripto (TDGF1) (Epithelial malignancies including colon, breast, lung, ovarian, and pancreatic cancers); ACVRL1/ALK1 (multiple malignancies including leukaemias and lymphomas); MUC5AC (Epithelial malignancies, including breast adenocarcinoma); CEACAM (Epithelial malignancies, including breast adenocarcinoma); CD137 (B cells or T cells, autoimmune, allergic or malignant); CXCR4 (B cells or T cells, autoimmune, allergic or malignant); Neuropilin 1 (Epithelial malignancies, including lung cancer); Glypicans (multiple cancers including liver, brain and breast cancers); HER3/EGFR (Epithelial malignancies); PDGFRa (Epithelial malignancies); EphA2 (multiple cancers including neuroblastoma, melanoma, breast cancer, and small cell lung carcinoma); and CD138 (Myeloma).
Particularly preferred targeting moiety antibodies include an anti-epidermal growth factor receptor antibody such as Cetuximab, an anti-Her2 antibody, an anti-CD20 antibody such as Rituximab, an anti-CD22 antibody such as Inotuzumab, an anti-CD70 antibody, an anti-CD33 antibody such as hp67.6 or Gemtuzumab, an anti-MUC1 antibody such as GP1.4 and SM3, an anti-CD40 antibody, an anti-CD74 antibody, an anti-P-cadherin antibody, an anti-EpCAM antibody, an anti-CD138 antibody, an anti-E-cadherin antibody, an anti-CEA antibody, and an anti-FGFR3 antibody.
Yet further selective targets useful for preventing or treating various conditions characterised by the presence of unwanted cells are provided below. For all of the examples below, therapeutic antibodies are already available or can be readily prepared by the skilled person.











Target
Unwanted cell







Activin A
Many types of carcinoma, lymphoma, sarcoma



and leukaemia


activin A, activin B and inhibin B
Many types of carcinoma, lymphoma, sarcoma



and leukaemia


Adenocarcinoma antigen
Many types of carcinoma,


AFP (alpha-fetoprotein)
Many types of carcinoma,


amyloid beta (Abeta)
Alzheimer's Disease


amyloid beta (Abeta) peptide Aβ40
Alzheimer's Disease


amyloid beta (Abeta) peptide soluble monomer
Alzheimer's Disease


amyloid beta (Abeta) peptides Aβ42 and Aβ40
Alzheimer's Disease


ANGPT2 (Ang2, angiopoietin 2)
Multiple carcinomas


N-glycolyl GM3 ganglioside (N-
Brain tumours


glycolylneuraminic acid (NeuGc, NGNA) GM3


gangliosides, NeuGcGM3) Mus musculus IgM-


kappa P3


BSG (basigin, Ok blood group, CD147)
Many types of carcinoma, lymphoma, sarcoma



and leukaemia


CA 72-4 (tumour associated glycoprotein 72,
Many types of carcinoma,


TAG-72, TAG, HMW mucin-like glycoprotein)


CA9 (carbonic anhydrase IX, CAIX, MN, G250)
Many types of carcinoma,


carcinoma associated antigen CTAA16.88
Many types of carcinoma,


(complex of cytokeratin polypeptides (35-


40 kDa))


CCL11 (chemokine (C-C motif) ligand 11,
Many types of carcinoma and


chemokine CC 11, eotaxin1)
lymphoma/leukaemia


CCL2 (chemokine (C-C motif) 2, chemokine CC
Many types of carcinoma and


2, monocyte chemoattractant protein-1, MCP-1,
lymphoma/leukaemia


monocyte chemotactic and activating factor,


MCAF, small inducible cytokine A2, SCYA2,


HC11)


CCR4 (chemokine (C-C motif) receptor 4,
Many types of carcinoma and


chemokine CC receptor 4, CCR-4, CKR4, k5-5,
lymphoma/leukaemia


CD194)


CD14
Many types of carcinoma and



lymphoma/leukaemia


CD15 (3-fucosyl-N-acetyl-lactosamine, Lewis x,
Many types of carcinoma and


stage-specific embryonic antigen 1, SSEA-1)
lymphoma/leukaemia


CD19 (B lymphocyte surface antigen B4, Leu-
Lymphoma and Acute lymphoblastic leukaemia


12)


CD2 (lymphocyte function-antigen 2, LFA-2)
T-cell and NK-cell lymphoma


CD200 (OX-2)
T-cell and NK-cell lymphoma


CD22 (sialic acid binding Ig-like lectin 2,
Lymphoma and Acute lymphoblastic leukaemia


SIGLEC2, SIGLEC-2, B-lymphocyte cell


adhesion molecule, BL-CAM, Leu-14


CD33 (sialic acid binding Ig-like lectin 3,
Myeloid leukaemia and Stem cells


SIGLEC3, SIGLEC-3, gpG7, p67)


CD38 (ADP-ribosyl cyclase 1, cyclic ADP-
Myeloid leukaemia and many types of


ribose hydrolase 1, cADPr hydrolase 1, T10)
carcinoma


CD40 (tumor necrosis factor receptor
Lymphoma and many types of carcinoma


superfamily member 5, TNFRSF5, p50)


CD40LG (CD40 ligand, CD40L, tumor necrosis
Lymphoma and many types of carcinoma


factor ligand superfamily member 5, TNFSF5,


tumor necrosis factor related activation protein,


TRAP, CD154)


CD44 (homing function and Indian blood group
Myeloid leukaemia and many types of


system, chondroitin sulfate proteoglycan 8,
carcinoma including cancer stem cells


CSPG8)


CD5 (T1, LEU-1)
T-cell lymphoma, T-cells and B-cell lymphomas



such as chronic lymphocytic leukaemia.


CD52
T-cell lymphoma, T-cells and B-cell



lymphomas. Autoimmune induced immune



cells may also be targeted.


CD6 (Tp120)
T-cell lymphoma, T-cells and B-cell lymphomas



such as chronic lymphocytic leukaemia.


CD70 (tumor necrosis factor superfamily
Lymphoma and many types of carcinoma


member 7, TNFSF7, CD27LG, CD27L)


CD74 (major histocompatibility class II invariant
Lymphoma and many types of carcinoma


chain, MH2)


CD80 (B7-1, CD28LG1)
Lymphoma and many types of carcinoma


CD86 (B7-2, CD28LG2)
Lymphoma and many types of carcinoma


CEA (anticarcinoembryonic antigen)
Many types of carcinoma,


CEACAM3 (carcinoembryonic antigen-related
Many types of carcinoma,


cell adhesion molecule 3, CGM1, CD66d)


CEACAM5 (carcinoembryonic antigen-related
Many types of carcinoma,


cell adhesion molecule 5, CEA, CD66e)


CEACAM8 (carcinoembryonic antigen-related
Many types of carcinoma,


cell adhesion molecule 8, NCA-95, nonspecific


cross- reacting antigen 95 kDa, granulocyte cell


antigen, CGM6, CD66b)


ClfA (Clumping factor A)
Many types of carcinoma,


complement C3b, C4b
Many types of unwanted cells.


CSF2 (colony stimulating factor 2 (granulocyte-
Myeloid diseases


macrophage), granulocyte-macrophage colony


stimulating factor, GM-CSF)


CSF2RA (colony-stimulating factor
Myeloid diseases


2(granulocyte-macrophage) receptor alpha


subunit, GM-CSF-R-alpha, CD116)


CSPG4 (chondroitin sulfate proteoglycan 4,
Many types of carcinoma, lymphoma, sarcoma


high molecular weight-melanoma-associated
and leukaemia


antigen, HMW-MAA)


CTLA4 (cytotoxic T lymphocyte-associated
Regulatory T-cells and unwanted immune cells.


antigen 4, CD152)


ED-B (fibronectin extra domain B)
Many types of carcinoma,


EGFR (epidermal growth factor receptor,
Many types of carcinoma, lymphoma, sarcoma


receptor tyrosine-protein kinase erbB-1,
and leukaemia


ERBB1, HER1, HER-1, ERBB)


EPCAM (epithelial cell adhesion molecule,
Many types of carcinoma, lymphoma, sarcoma


tumor-associated calcium signal transducer 1,
and leukaemia


TACSTD1, gastrointestinal tumor-associated


protein 2, GA733-2, epithelial glycoprotein 2,


EGP-2, epithelial cell adhesion molecule, Ep-


CAM, KSA, KS1/4 antigen, M4S, tumor antigen


17-1A, EpCAM, CD326)


ERBB2 (epidermal growth factor receptor 2,
Many types of carcinoma, lymphoma, sarcoma


receptor tyrosine-protein kinase erbB-2,
and leukaemia


EGFR2, HER2, HER-2, p185c-erbB2, NEU,


CD340


ERBB3 (receptor tyrosine-protein kinase erbB-
Many types of carcinoma, lymphoma, sarcoma


3, HER3)
and leukaemia


FAP (fibroblast activation protein, alpha)
Many types of carcinoma, lymphoma, sarcoma



and leukaemia.


FCER2 (immunoglobulin E Fc receptor low
Many types of carcinoma, lymphoma, sarcoma


affinity II, Fc epsilon RII, CD23)
and leukaemia in B-cells.


FCGR1 (immunoglobulin G Fc receptor high
Many types of carcinoma, lymphoma, sarcoma


affinity I, Fc gamma RI, CD64, encoded by
and leukaemia.


human FCGR1A, FCGR1B, FCGR1C)


fibrin II beta chain (NH2 terminus)
Many types of carcinoma, lymphoma, sarcoma



and leukaemia.


FLT1 (fms-related tyrosine kinase 1, vascular
Many types of carcinoma, sarcoma, lymphoma,


endothelial growth factor receptor 1, VEGFR-1,
sarcoma and leukaemia. In particular tumour


VEGFR, FLT, FRT, vascular permeability factor
blood vessels.


receptor)


FOLH1 (folate hydrolase, prostate specific
Many types of carcinoma, lymphoma, sarcoma


membrane antigen, PSMA)
and leukaemia in particular prostate carcinoma



and unwanted prostate tissue.


FOLR1 (folate receptor 1, folate receptor FR
Many types of carcinoma, lymphoma, sarcoma


alpha, FR-alpha, adult folate-binding protein,
and leukaemia


FBP, ovarian tumor-associated antigen MOv18)


GD2 ganglioside
Brain tumours and unwanted neuronal tissue.


GD3 ganglioside
Brain tumours and unwanted neuronal tissue.


GLP1R (glucagon-like peptide 1 receptor)
Many types of carcinoma, lymphoma, sarcoma



and leukaemia


GPNMB (glycoprotein transmembrane NMB,
Many types of carcinoma, lymphoma, sarcoma


hematopoeitic growth factor inducible
and leukaemia


neurokinin-1 type, HGFIN)


hapten NP-cap (4-hydroxy-3-nitrophenacetyl
Many types of carcinoma, lymphoma, sarcoma


caproic acid)
and leukaemia


HAVCR1 (hepatitis A virus cellular receptor 1,
Hepatitis A infected cells.


T-cell immunoglobulin and mucin domain-


containing protein 1, TIM1, KIM-1)


HBV (hepatitis B virus)
HBV infected cells


HCMV (human cytomegalovirus) gB
CMV infected cells.


glycoprotein


HCV (hepatitis C virus)
HCV infected cells


heat shock protein 90 homolog
Many types of carcinoma, lymphoma, sarcoma



and leukaemia


HGF (hepatocyte growth factor, scatter factor,
Hepatoma and hepatocellular carcinoma. Also


SF, hepatopoeitin A)
unwanted hepatic tissue.


HIV-1 (human immunodeficiency virus)
HIV infected cells


HLA-DR10 (DRB1*1001)
Autologous or Allogeneic MHC Class-II



expressing cells including tumour cells


HLA-DRB (HLA-DR beta)
Autologous or Allogeneic MHC Class-II



expressing cells including tumour cells


HSV (herpes simplex virus)
HSV infected cells


ICAM1 (intercellular adhesion molecule 1,
Many types of carcinoma, lymphoma, sarcoma


ICAM-1, CD54)
and leukaemia


ICAM3 (intercellular adhesion molecule 3,
Many types of carcinoma, lymphoma, sarcoma


ICAM-3, CD50)
and leukaemia


Membrane Immunoglobulin IgE
IgE secreting B-cells and Plasma cells (cuasing



allergic disease).


IgE Fc
IgE secreting B-cells and Plasma cells (cuasing



allergic disease).


IGF1R (insulin-like growth factor 1 receptor,
Most types of carcinoma, lymphoma, sarcoma


IGF1-R, IGF-1R, CD221)
and leukaemia


IGHE connecting region (CO) M1 prime (in
IgE secreting cells such as B-cells and plasma


alternatively spliced heavy chain of membrane
cells. Particularly unwanted in allergic disease.


IgE on B cells


IL2RA (interleukin-2 receptor, alpha subunit, IL-
B-cells and T-cells in either malignant or


2RA, TAC, CD25)
autoimmune disease.


IL2RB (interleukin-2 receptor beta subunit, IL-
B-cells and T-cells in either malignant or


2RB, p70, CD122)
autoimmune disease.


IL5RA (interteukin 5 receptor alpha subunit,
B-cells and T-cells in either malignant or


CD125)
autoimmune disease.


IL6R (interleukin 6 receptor, IL-6R, CD126)
B-cells and T-cells in either malignant or



autoimmune disease.


ITGA2 {integrin alpha 2, GPIa, subunit of the
Many types of carcinoma, lymphoma,


alpha2beta1 integrin (VLA-2, collagen
leukaemias.


receptor), CD49b)


ITGA2B_ITGB3 (integrin alpha2b_beta3,
Many types of carcinoma, lymphoma,


integrin αIIbβ3, GPIIbIIIa, fibrinogen receptor,
leukaemias.


CD41_CD61)


ITGA4 (integrin alpha 4 subunit, CD49d)
Many types of carcinoma, lymphoma,



leukaemias.


ITGA4_ITGB7 (integrin alpha4_beta7, integrin
Many types of carcinoma, lymphoma,


α4β7, lymphocyte Peyer's patch adhesion
leukaemias.


molecule 1, LPAM-1)


ITGA5 (integrin alpha 5 subunit, CD49e)
Many types of carcinoma, lymphoma,



leukaemias.


ITGAE_ITGB7 (integrin alphaE_beta7, integrin
Many types of carcinoma, lymphoma,


αEβ7, human mucosal lymphocyte antigen 1,
leukaemias.


HML-1)


ITGAL (integrin alpha L subunit, lymphocyte
Many types of carcinoma, lymphoma,


function associated antigen 1, CD11a)
leukaemias.


ITGAV_ITGB3 (integrin alphaV_beta3, integrin
Many types of carcinoma, lymphoma,


αVβ3, CD51_GPIIIa, vitronectin receptor, VNR,
leukaemias.


CD51_CD61


ITGB1 (integrin beta1 subunit, GPIIa, CD29)
Many types of carcinoma, lymphoma,



leukaemias.


ITGB2 (integrin beta2 subunit, LFA-1, MAC-1,
Many types of carcinoma, lymphoma,


CD18)
leukaemias.


KDR (kinase insert domain receptor, vascular
Many types of carcinoma, lymphoma,


endothelial growth factor receptor 2, VEGFR2,
leukaemias.


VEGF-R2, FLK1, CD309)


LTA (lymphotoxin alpha, TNF superfamily
Many types of carcinoma, lymphoma,


member 1, TNFSF1, LT)
leukaemias.


LTB (lymphotoxin beta, TNF superfamily
Many types of carcinoma, lymphoma,


member 3, TNFSF3, p33)
leukaemias.


MET (met proto-oncogene, hepatocyte growth
Many types of carcinoma, lymphoma,


factor HGF receptor, HGFR, scatter factor SF
leukaemias.


receptor, HGF/SF receptor, tyrosine protein


kinase c-met, papillary renal cell carcinoma 2,


RCCP2)


MS4A1 (membrane-spanning 4-domains
Many types of carcinoma, lymphoma,


subfamily A member 1, CD20)
leukaemias.


MSLN (mesothelin, pre-pro-megakaryocyte-
Many types of carcinoma, lymphoma,


potentiating factor, megakaryocyte potentiating
leukaemias.


factor, MPF, CAK1)


MST1R (macrophage stimulating 1 receptor,
Many types of carcinoma, lymphoma,


macrophage stimulating protein receptor, MSP
leukaemias.


receptor, c-met-related tyrosine kinase, protein-


tyrosine kinase 8, PTK8, RON, p185-Ron,


CD136)


MSTN (myostatin, growth differentiation factor
Many types of carcinoma, lymphoma,


8, GDF8)
leukaemias.


MUC1 (mucin 1, polymorphic epithelial mucin,
Many types of carcinoma, lymphoma,


PEM, episialin, CD227)
leukaemias.


MUC1 sialylated carbohydrate, tumour-
Many types of carcinoma, lymphoma,


associated (CA242, cancer antigen 242)
leukaemias.


MUC16 (mucin 16, MUC-16, cancer antigen
Many types of carcinoma, lymphoma,


125, CA125)
leukaemias.


MUC5AC (mucin 5AC, mucin 5 subtypes A and
Many types of carcinoma, lymphoma,


C tracheobronchial/gastric)
leukaemias.


N-glycolyl GM3 ganglioside (N-
Brain tumours and unwanted neural tissue.


glycolylneuraminic acid (NeuGc, NGNA) GM3


ganglioside, NeuGcGM3)


NCA-90 (nonspecific cross-reacting antigens
Many types of carcinoma, lymphoma, sarcoma


90 kDa glycoproteins, granulocyte cell antigen)
and leukaemia


NCAM1 (neural cell adhesion molecule 1,
Brain tumours and unwanted neural tissue also


NCAM-1, NCAM, CD56)
many types of carcinoma and lymphoma.


Nectin-4
Many types of carcinoma, lymphoma, sarcoma



and leukaemia


NGF (nerve growth factor, nerve growth factor
Many types of carcinoma, lymphoma, sarcoma


beta polypeptide, NGFB, beta-NGF)
and leukaemia


NIP-cap (3-iodo-4-hydroxy-5-nitrophenyl-acetyl
Many types of carcinoma, lymphoma, sarcoma


caproic acid)
and leukaemia


NRP1 (neuropilin 1, NRP, vascular endothelial
Many types of carcinoma, lymphoma, sarcoma


cell growth factor 165 receptor, VEGF165
and leukaemia


receptor, VEGF165R, CD304)


PDGFRA (platelet-derived growth factor
Many types of carcinoma, lymphoma, sarcoma


receptor alpha subunit, PDGFR2, CD140a)
and leukaemia


phosphatidylserine
Many types of carcinoma, lymphoma, sarcoma



and leukaemia particularly apoptotic cells.


PSCA (prostate stem cell antigen)
Many types of carcinoma and leukaemia.


RSV (human respiratory syncytial virus,
RSV infected cells


glycoprotein F)


RTN4 (reticulon 4, neurite outgrowth inhibitor,
Many types of carcinoma, lymphoma, sarcoma


NOGO)
and leukaemia


SDC1 (syndecan-1, CD138)
Unwanted plasma cells found in plasma cell



dyscrasias, particularly Myeloma, Amyloidosis



and MGUS.


SELE (E-selectin, CD62E)
Many types of carcinoma and lymphoma.


SELL (L-selectin, CD62)
Many types of carcinoma and lymphoma.


SELP (P-selectin, CD62)
Many types of carcinoma and lymphoma.


SFRP1 (selected frizzled-related protein 1,
Many types of carcinoma and lymphoma.


fusion regulatory protein 1, FRP-1)


SLAMF7 (SLAM family member 7, CD2 subset
Many types of unwanted cells including tumour


1, CS1, CD2-like receptor-activating cytotoxic
cells and those involved in autoimmune


cells, CRACC, 19A24, CD319)
disease.


SLC3A2 (solute carrier family 3 (activators of
Many types of unwanted cells including tumour


dibasic and neutral amino acid transport)
cells and those involved in inflammatory


member 2, 4F2 antigen heavy chain, 4F2HC,
disease.


CD98 heavy chain, CD98hc, CD98)


SOST (sclerostin)
Bone disease including oesteosarcoma and



osteoporosis.


Staphylococcus epidermidis lipoteichoic acid
Staphylococcus infected tissue.


T cell receptor (TR) TR alpha_beta
T-cell lymphoma or autoimmune-causing T-



cells.


TGFB1 (transforming growth factor beta1, TGF
Many types of unwanted cells including tumour


beta)
cells and those involved in fibrotic disease.


TGFB2 (transforming growth factor beta 2)
Many types of unwanted cells including tumour



cells and those involved in fibrotic disease.


TNF (tumor necrosis factor (TNF) superfamily
Many types of unwanted cells including tumour


member 2, TNFSF2, TNF-alpha, TNFA)
cells and those involved in inflammatory



disease.


TNFRSF10A (tumor necrosis factor receptor
Many types of unwanted cells including tumour


(TNFR) superfamily member 10A, death
cells and those involved in inflammatory


receptor 4, DR4, TNF-related apoptosis-
disease.


inducing ligand receptor 1, TRAILR1, TRAIL-


R1, TR-1, CD261)


TNFRSF10B (tumor necrosis factor receptor
Many types of unwanted cells including tumour


(TNFR) superfamily member 10B, death
cells and those involved in inflammatory


receptor 5, DR5, TNF-related apoptosis-
disease.


inducing ligand receptor 2, TRAILR2, TRAIL-


R2, TR-2, CD262)


TNFRSF12A (tumor necrosis factor receptor
Many types of unwanted cells including tumour


(TNFR) superfamily member 12A, fibroblast
cells and those involved in inflammatory


growth factor (FGF)-inducible 14 kDa protein,
disease.


Fn14, TNF-like weak inducer of apoptosis


(Tweak) receptor, Tweak receptor, TweakR,


CD266


TNFRSF8 (tumor necrosis factor receptor
Many types of unwanted cells including tumour


(TNFR) superfamily member 8, CD30)
cells (in particular lymphoma) and those



involved in inflammatory disease.


TNFRSF9 (tumor necrosis factor receptor
Many types of unwanted cells including tumour


(TNFR) superfamily member 9, 4-1BB, T cell
cells and those involved in inflammatory and


antigen ILA, CD137
autoimmune disease.


TNFSF11 (tumor necrosis factor (TNF)
Many types of unwanted cells including tumour


superfamily member 11, osteoclast
cells and those involved in inflammatory and


differentiation factor, ODF, OPGL, RANKL,
autoimmune disease.


TRANCE, CD254)


TNFSF13 (tumor necrosis factor (TNF)
Many types of unwanted cells including tumour


superfamily member 13, a proliferation-
cells and those involved in inflammatory and


including ligand, APRIL, CD256
autoimmune disease.


TNFSF13B (tumor necrosis factor (TNF)
Many types of unwanted cells including tumour


superfamily member 13B, B cell activating
cells and those involved in inflammatory and


factor, BAFF, TALL1, BLyS, B lymphocyte
autoimmune disease.


activator, CD257)


TNFSF14 (tumor necrosis factor (TNF)
Many types of unwanted cells including tumour


superfamily member 14, LIGHT, HVEM-L,
cells and those involved in inflammatory and


CD258)
autoimmune disease.


TNFSF4 (tumor necrosis factor (TNF)
Many types of unwanted cells including tumour


superfamily member 4, OX40 ligand, OX-40L,
cells and those involved in inflammatory and


TAX transcriptionally-activated glycoprotein 1,
autoimmune disease.


TXGP1, gp34, CD252)


TPBG (trophoblast glycoprotein, 5T4)
Multiple carcinomas.


TYRP1 (tyrosinase-related protein 1, 5,6-
Multiple carcinomas.


dihydroxyindole-2-carboxylic acid oxidase,


DHICA oxidase, TRP1, melanoma antigen


gp75)


VAP-1 (vascular adhesion protein)
Multiple carcinomas and hepatomas.


VEGFA (vascular endothelial growth factor A,
Multiple carcinomas and hepatomas.


VEGF-A, VEGF)


VIM (vimentin)
Multiple carcinomas and hepatomas.









Examples of tumour-associated, immune cell-associated and infection reagent-related antigens which may be targeted by the targeting moiety are given in Table 1.







TABLE 1







Cell surface antigens for targeting









Antigen
Antibody
Existing uses










a) Tumour Associated Antigens









Carcino-embryonic
C46 (Amersham)
Imaging and therapy


Antigen
85A12 (Unipath)
of colon/rectum




tumours.


Placental Alkaline
H17E2 (ICRF,
Imaging and therapy


Phosphatase
Travers & Bodmer)
of testicular and




ovarian cancers.


Pan Carcinoma
NR-LU-10 (NeoRx
Imaging and therapy



Corporation)
of various carcino-




mas including small




cell lung cancer.


Polymorphic
HMFG1 (Taylor-
Imaging and therapy


Epithelial Mucin
Papadimitriou,
of ovarian cancer and


(Human milk fat
ICRF)
pleural effusions.


globule)


β-human Chorionic
W14
Targeting of


Gonadotropin

carboxypeptidase to




human xenograft




choriocarcinoma in




nude mice (Searle et




al (1981) Br. J.




Cancer 44, 137-144).


A carbohydrate on
L6 (IgG2a)1
Targeting of alkaline


Human Carcinomas

phosphatase (Senter et




al (1988) PNAS USA




85, 4842-4846.


CD20 Antigen on B
1F5 (IgG2a)2
Targeting of alkaline


Lymphoma (normal

phosphatase (Senter et


and neoplastic)

al (1988) PNAS USA




85, 4842-4846.







b) Immune Cell Antigens









Pan T Lymphocyte
OKT-3 (Ortho)
As anti-rejection


Surface Antigen

therapy for kidney


(CD3)

transplants.


B-lymphocyte
RFB4 (Janossy,
Immunotoxin therapy


Surface Antigen
Royal Free Hospital)
of B cell lymphoma.


(CD22)


Pan T lymphocyte
H65 (Bodmer and
Immunotoxin


Surface Antigen
Knowles, ICRF;
treatment of acute


(CD5)
licensed to Xoma
graft versus host



Corp., USA)
disease, rheumatoid




arthritis.







c) Infectious Agent-Related Antigens









Mumps virus-related
Anti-mumps
Antibody conjugated



polyclonal antibody
to diphtheria toxin




for treatment of




mumps.


Hepatitis B Surface
Anti HBs Ag
Immunotoxin against


Antigen

hepatoma.





1Hellström et al (1986) Cancer Res. 46, 3917-3923


2Clarke et al (1985) Proc. Natl. Acad. Sci. USA 82, 1766-1770


Other antigens include alphafoetoprotein, Ca-125 and prostate specific antigen.






As used herein, the term “antibody” includes but is not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, fragments produced by a Fab expression library and bispecific antibodies. Such fragments include fragments of whole antibodies which retain their binding activity for a target substance, Fv, F(ab′) and F(ab′)2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody. A targeting moiety comprising only part of an antibody may be advantageous by virtue of optimising the rate of clearance from the blood and may be less likely to undergo non-specific binding due to the Fc part. Also included are domain antibodies (dAbs), diabodies, camelid antibodies and engineered camelid antibodies. Furthermore, for administration to humans, the antibodies and fragments thereof may be humanised antibodies, which are now well known in the art (Janeway et at (2001) Immunobiology., 5th ed., Garland Publishing); An et al (2009) Therapeutic Monoclonal Antibodies: From Bench to Clinic, ISBN: 978-0-470-11791-0).
Also included are asymmetric IgG-like antibodies (eg triomab/quadroma, Trion Pharma/Fresenius Biotech; knobs-into-holes, Genentech; Cross MAbs, Roche; electrostatically matched antibodies, AMGEN; LUZ-Y, Genentech; strand exchange engineered domain (SEED) body, EMD Serono; biolonic, Merus; and Fab-exchanged antibodies, Genmab), symmetric IgG-like antibodies (eg dual targeting (DT)-Ig, GSK/Domantis; two-in-one antibody, Genentech; crosslinked MAbs, karmanos cancer center, mAb2, F-star, and Cov X-body, Cov X/Pfizer), IgG fusions (eg dual variable domain (DVD)-Ig, Abbott; IgG-like bispecific antibodies. Eli Lilly; Ts2Ab, Medimmune/AZ; BsAb, ZymoGenetics; HERCULES, Biogen Idec; TvAb, Roche) Fc fusions (eg ScFv/Fc fusions, Academic Institution; SCORPION, Emergent BioSolutions/Trubion, ZymoGenetics/BMS; dual affinity retargeting technology (Fc-DART), MacroGenics; dual (ScFv)2-Fab, National Research Center for Antibody Medicine) Fab fusions (eg F(ab)2, Medarex/AMGEN; dual-action or Bis-Fab, Genentech; Dock-and-Lock (DNL), ImmunoMedics; bivalent bispecific, Biotechnol; and Fab-Fv, UCB-Celltech), ScFv- and diabody-based antibodies (eg bispecific T cell engagers (BiTEs), Micromet; tandem diabodies (Tandab), Affimed; DARTs, MacroGenics; Single-chain diabody, Academic; TCR-like antibodies, AIT, Receptor Logics; human serum albumin ScFv fusion, Merrimack; and COMBODIES, Epigen Biotech), igG/non-lgG fusions (eg immunocytokins, EMDSerono, Philogen, ImmunGene, ImmunoMedics; superantigen fusion protein, Active Biotech; and immune mobilising mTCR Against Cancer, ImmTAC) and oligoclonal antibodies (eg Symphogen and Merus).
The antibody may possess any of the antibody-like scaffolds described by Carter (2006) “Potent antibody therapeutics by design”, Nat Rev Immunol. 6(5):343-57, and Carter (2011) “Introduction to current and future protein therapeutics: a protein engineering perspective”, Exp Cell Res. 317(9): 1261-9. incorporated herein by reference, together with the specificity determining regions described herein. Thus, the term “antibody” also includes affibodies and non-immunoglobulin based frameworks.
The advantages of using antibody fragments, rather than whole antibodies, are several-fold. The smaller size of the fragments may lead to improved pharmacological properties, such as better penetration of solid tissue. Moreover, antigen-binding fragments such as Fab, Fv, ScFv and dAb antibody fragments can be expressed in and secreted from E. coli or yeast, thus allowing convenient production in the laboratory and economical production on a commercial scale.
The antibody may be of any of the IgG, IgE, IgA, IgM and IgD classes and may be derived from any species. If the antibody is an IgG, it may be any of IgG1, IgG2, IgG3 or IgG4. It is preferred, however, that when the agent is for administration to a particular host, that the antibody, or at least the constant regions thereof, are derived from that host. For example, when the agent is to be administered to a human, the antibody is preferably a human antibody or a humanized antibody, and so on.
Suitable antibodies that bind to particular antigens expressed by unwanted cells can be made by the skilled person using technology long-established in the art. Methods of preparation of monoclonal antibodies and antibody fragments are well known in the art and include hybridoma technology (Kohler & Milstein (1975) “Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495-497); antibody phage display (Winter et al (1994) “Making antibodies by phage display technology.” Annu. Rev. Immunol. 12: 433-455); ribosome display (Schaffitzel et al (1999) “Ribosome display: an in vitro method for selection and evolution of antibodies from libraries.” J. Immunol. Methods 231: 119-135); and iterative colony filter screening (Giovannoni et al (2001) “Isolation of anti-angiogenesis antibodies from a large combinatorial repertoire by colony filter screening.” Nucleic Acids Res. 29: E27). Further, antibodies and antibody fragments suitable for use in the present invention are described, for example, in the following publications: “Monoclonal Hybridoma Antibodies: Techniques and Application”, Hurrell (CRC Press, 1982); “Monoclonal Antibodies: A Manual of Techniques”, H. Zola, CRC Press, 1987, ISBN: 0-84936-476-0; “Antibodies: A Laboratory Manual” 1st Edition, Harlow & Lane, Eds, Cold Spring Harbor Laboratory Press, New York, 1988. ISBN 0-87969-314-2; “Using Antibodies: A Laboratory Manual” 2nd Edition, Harlow & Lane, Eds, Cold Spring Harbor Laboratory Press, New York, 1999. ISBN 0-87969-543-9; and “Handbook of Therapeutic Antibodies” Stefan Dübel, Ed., 1st Edition,—Wiley-VCH, Weinheim, 2007. ISBN: 3-527-31453-9.
It is especially preferred if the targeting moiety is a single chain antibody such as a scFv antibody that comprises a heavy chain variable domain and a light chain variable domain, which domains are joined together by a flexible peptide linker. Preferably, the single chain antibody binds to an entity expressed by the unwanted cell, including any of those mentioned above. However, it will also be understood that the targeting moiety may comprise only one antibody variable domain, such as a heavy chain variable domain or a light chain variable domain, and such a domain may bind to an entity expressed by the unwanted cell.
It will be appreciated that the targeting moiety may also be any compound or part thereof that specifically binds, in a non-immune sense, to an entity expressed by unwanted cells or otherwise becomes associated with the unwanted cells. Thus, the specific binding partner may be any of a hormone, a growth factor, a cytokine, or a receptor ligand (e.g. agonist or antagonist).
For example, cytokines have previously been used to target toxins to invading bacterial. Using genetic engineering, recombinant proteins have been produced which contain for example IL-2 and a binding domain-deleted Pseudomonas exotoxin protein (Lorderboum-Galski et al. 1988 (62)). This immunotoxin was effective in experimental animal models (Kozak et al, 1990 (63)). Fusion proteins have also been produced with IL-4, IL-6, alpha-MSH, EGF and TNF-alpha (reviewed in Waldmann 1992 (35)), all of which are appropriate for use as targeting moieties in the present invention.
Particularly useful targeting moieties include cytokines such as IL-2, EGF, VEGF, Flt3L, HGF, IGF, IL-6, or IL-4. IL-2 and IL-4 can target to adult T cell leukaemia/lymphoma cells which express the high affinity IL-2 receptor whereas normal resting T-cells do not, or to T-cells expressing the IL-4 receptor. It has previously been shown that the monoclonal antibody MR6, which binds to the human IL-4 receptor, can inhibit the IL-4 induced proliferation of cloned helper T cells and the production of IgE by polyclonal B cells (Larche et al, 1988 (36)). Such targeting moieties may be used to eliminate a lymphoid cell subpopulation in autoimmune disease or allergy.
Insulin like growth factors (IGF-1 and IGF-11) are preferentially taken up by malignant cells and so may be used to target tumour cells. Similarly EGF can be used to target malignant cells which upregulate the EGF receptor. Also, tumour associated blood vessels overexpress VEGF receptor and so can be targeted by the family of VEGF growth factors.
Flt3 receptor is overexpressed in leukaemias and may be a therapeutic target for acute and chronic leukaemias and myeloproliferative disorders.
Myeloma cells express IL-6 receptor and also secrete IL-6 which acts in an autocrine fashion to stimulate cell proliferation. Thus IL-6 may be used as a targeting moiety for myeloma.
In another example, the targeting moiety is melanoma stimulating hormone (MSH) which binds to the MSH receptor which is expressed in high numbers in melanoma cells.
It is appreciated that a person skilled in the art can readily select suitable binding partners for any given unwanted cell, for example by identifying surface antigens or molecules specific for that unwanted cell and finding a binding partner for that antigen or molecule. Considerable research has been carried out on antibodies and fragments thereof to tumour-associated antigens, immune cell antigens and infectious agents, as described above. Thus, conveniently, selecting an appropriate targeting moiety for a given cell type typically involves searching the literature. Alternatively, an unwanted cell is taken from a patient (e.g. by biopsy), and antibodies directed against the cell prepared. Such ‘tailor-made’ antibodies are already known. It has been demonstrated that antibodies confer binding to tumour cells not only from the patient they have been obtained from but also for a large number of other patients. Thus, a plurality of such antibodies has become commercially available. Other methods of identifying suitable binding partners for a given unwanted cell include genetic approaches (eg microarray), proteomic approaches (eg differential Mass spectrometry), immunological approaches (eg immunising animals with tumour cells and identifying antibody-secreting clones which specifically target malignant cells) and in silico approaches wherein targets are identified using a systems biology approach.
Further selective targets and suitable binding partners are shown in Table 2.







TABLE 2







Binding partners for tumour-selective


targets and tumour-associated antigens









Target
Binding Partner
Disease





Truncated EGFR
anti-EGFR mAb
Gliomas


Idiotypes
anti-id mAbs
B-cell lymphomas


EGFR (c-erbB1)
EGF, TGFα anti-
Breast cancer



EGFR mAb


c-erbB2
mAbs
Breast cancer


IL-2 receptor
IL-2
Lymphomas and



anti-Tac mAb
leukaemias


IL-4 receptor
IL-4
Lymphomas and




leukaemias


IL-6 receptor
IL-6
Lymphomas and




leukaemias


MSH (melanocyte-
α-MSH
Melanomas


stimulating hormone)


receptor


Transferrin receptor
Transferrin anti-TR
Gliomas


(TR)
mAb


gp95/gp97
mAbs
Melanomas


p-glycoprotein cells
mAbs
drug-resistant


cluster-1 antigen
mAbs
Small cell lung


(N-CAM)

carcinomas


cluster-w4
mAbs
Small cell lung




carcinomas


cluster-5A
mAbs
Small cell lung




carcinomas


cluster-6 (LeY)
mAbs
Small cell lung




carcinomas


PLAP (placental
mAbs
Some seminomas


alkaline phosphatase)

Some ovarian; some




non small cell lung




cancer


CA-125
mAbs
Lung, ovarian


ESA (epithelial
mAbs
carcinoma


specific antigen)


CD 19, 22, 37
mAbs
B-cell lymphomas


250 kDa
mAbs
Melanoma


proteoglycan p55
mAbs
Breast cancer


TCR-IgH fusion
mAbs
Childhood T-cell




leukaemia


Blood gp A antigen
mAbs
Gastric and colon


(in B or O

tumours


individuals)


Mucin protein core
mAbs
Breast cancer









Further targets useful in preventing or treating various cancers are provided below.














Target
Cancer









EpCam
Bladder



PMSA
Prostate



EGFR
Breast




Lung




Glioblastoma




Colon



CD20
Lymphoma



CD22
Lymphoma



CD52
Lymphoma




Leukaemia










As an alternative to the targeting moiety being a specific binding partner, the targeting moiety may be a non-specific molecule that is capable, following administration to a subject, of accumulating in the vicinity of the unwanted cells. For example, it is known that macromolecules accumulate non-specifically in tumours. Macromolecules known to accumulate in tumours non-specifically include albumin, immunoglobulins, transferrin, liposomes, nanoparticles (eg colloidal nanoparticles) and biodegradable polymers including dextrans, polyethylene glycol, polylysine and hydroxypropylmethylacrylamide. Macromolecules accumulate in human xenografted tumours in nude mice up to about 2.0% of administered dose per gram of tumour. Macromolecules such as polyethylene glycol and dextrans have been found to modify the clearance rate of substances to which they are attached and modify their concentration in tumours (Melton et al. 1987; Eno-Ammoquaye et al, 1996). In exceptional tumours, a non-specific macromolecule may accumulate in greater concentration than an antibody directed at the secreted antigen (Searle et al, 1981).
The discovery that such macromolecules accumulate in tumours has been called the Enhanced Permeability and Retention (EPR) effect, and has been attributed to the leakiness of tumour capillaries and deficient lymphatic drainage (Matsumura & Macda, 1986).
Thus, when the unwanted cells are tumour cells, the targeting moiety may be any of these macromolecules which accumulate in tumours. Preferably, the macromolecule used in the invention is hydrophilic and is characterised by being soluble in body fluids and in conventional fluids for parenteral administration. Suitably, the macromolecule is biodegradable so that systemic accumulation during repeated administration is avoided. Clearly, however, it must not be degraded so fast as to fail to accumulate at the site of the unwanted cells (e.g. tumour). Preferably, the molecular weight and size of the agent comprising such a macromolecule targeting moiety exceeds that of the renal threshold for urinary excretion (MW 60 000), as this helps the blood concentration to be sufficient to provide an effective blood:tumour concentration gradient. A molecular weight of up to at least 800 000 is generally suitable, for example up to 160 000. The macromolecule is preferably one which is not readily captured by the reticuloendothelial system. The molecular weights given exclude any water of hydration.
Macromolecules that are available as sub-units and are not biodegradable may be linked by biodegradable linking units so that the non-biodegradable components are filtered through the kidneys and excreted in the urine.
Alternatively, it is preferred if the polymer used to make the macromolecule is not biodegradable such that the molecular weight of any non-biodegradable portion of the conjugate should be less than the renal threshold (circa 70000) so that after degradation of the biodegradable portion the residual non-biodegradeable portion is excreted through the kidneys.
Conveniently, the macromolecule may be any of a dextran; a polyamino acid; a nanoparticle (eg colloidal nanoparticle), or a non-tumour-specific protein such as an immunoglobulin, an albumin or a transferrin. Suitably, it may be a copolymer of styrene and maleic anhydride, or may be polyaspartic acid, poly-L-lysine, polyethyleneimine or polyethylene glycol.
It is appreciated that such macromolecules are used in melanocyte-directed enzyme prodrug therapy (MDEPT), as described in WO 1998/024478.
In addition to directly targeting an unwanted cell, the targeting moiety may be one that is capable of indirectly targeting to unwanted cells by being capable of binding to a moiety that is capable of targeting to unwanted cells. For example, a moiety that is capable of directly targeting to an unwanted cell (including any of those described above) may comprise a first binding partner. A targeting moiety that comprises a second binding partner, capable of binding to the first binding partner, then has the ability to indirectly target the unwanted cells by virtue of it being able to bind to the first binding partner. In this case, the targeting moiety does not bind to an entity expressed by or associated with the unwanted cell directly, however it does so indirectly by binding to a moiety that does bind to an entity expressed by or associated with the unwanted cell. It will be understood that the molecule of the invention may comprise targeting moieties that indirectly target unwanted cells in this way, as well as those targeting moieties that target unwanted cells directly. By the first and second binding partners, we include the meaning of any two moieties which bind to each other selectively. Most preferably, the first and second binding partners only bind to each other and not to any other moieties. Non-covalent binding such as between biotin/avidin or streptavidin, or immunological bindings are preferred. Thus, the first binding partner may be biotin and the second binding partner may be avidin, and vice versa. Alternatively, the first binding partner may be an antigen and the second binding partner may be an antibody specific for that antigen, and vice versa. However, any pair of first and second binding partners that selectively bind to each other may be used, and suitable pairs will be known to the skilled person.
Unwanted Cell
The unwanted cell may be any cell whose presence in a host is undesired. Thus, the cell may be a tumour cell (benign or malignant), a cell from a tumour microenvironment such as tumour fibroblasts or tumour blood vessels, a virally infected cell, a cell introduced as part of gene therapy, or a normal cell which one wishes to destroy for a particular reason. For instance, it may be desirable to eliminate a subpopulation of immune cells such as T lymphocytes in autoimmune disease or such as B lymphocytes in allergic disease.
Preferably, the unwanted cell is one whose presence characterises a condition in a patient. By a condition characterised by the presence of unwanted cells we include any biological or medical condition or disorder in which at least part of the pathology is mediated by the presence of unwanted cells. The condition may be caused by the presence of the unwanted cells or else the presence of the unwanted cells may be an effect of the condition. Examples of particular conditions include tumours (benign or malignant), autoimmune conditions, cardiovascular diseases, degenerative diseases, diabetes, allergic disease (eg asthma), neurodegenerative diseases such as Alzheimer's, transplantation patients and infectious diseases.
For autoimmune disease, the unwanted cells may represent cells of the adaptive or innate immune response, preferably T cells, but more preferably B cells. For cardiovascular disease, the unwanted cells may represent cells within atheromatous lesions such as macrophages. For degenerative diseases, the unwanted cells may represent cells which induce the neurodegenerative changes, for instance in Alzheimer's disease they may be microglia or astrocytes. For other degenerative diseases any cell which facilitates the process of degeneration or apoptosis may be considered a target. For processes such as aging where unwanted tissue builds up, for example in benign prostatic hyperplasis, non-malignant prostatic tissue would be a preferred target. For allergic disease, cells which participate in the allergic reaction such as tissue mast cells may be considered an ideal target, but also IgE secreting cells such as plasma cells or B cells. In transplantation, alloreactive lymphocytes would represent a preferred target cell. In the context of infectious disease, any cell harbouring a virus, bacteria or fungal pathogen may be considered a preferred target cell for example an HIV infected cell.
Immune Cell Binding Region of Further Moiety
By “immune cell binding region” we include the meaning of a region of the further moiety that is capable of binding to an immune cell. Preferably, the immune cell binding region is capable of selectively binding to an immune cell. For example, it is preferred if the immune cell binding region binds to an immune cell to a greater extent than it does any other type of cell, and most preferably binds to an immune cell only.
Generally, the immune cell binding region binds to an entity expressed by an immune cell, and preferably binds selectively to that entity. Thus, the further moiety may be a binding partner for an entity expressed on an immune cell wherein the immune cell binding region is the region of the further moiety that is responsible for binding. It is preferred if the immune cell binding region has a Kd value for binding to an entity expressed by a given immune cell which is at least five or ten times lower than for at least one other entity expressed by another cell type, and preferably more than 100 or 500 times lower. More preferably, the immune cell binding region has a Kd value for binding to an entity expressed by a given immune cell which is at least 1000 or 5000 times lower than for at least one other entity expressed by another cell type.
By ‘immune cell’, we include the meaning of any immune cell within the natural repertoire of cells in the immune system (e.g. human immune system) which, when activated, is able to bring about a change in the viability of a target cell. By ‘viability of a target cell’ we include the meaning of the target cell's ability to survive, proliferate and/or interact with other cells. Such interaction may either be direct, for example when the target cell contacts another cell, or indirect, for example when the target cell secretes substances which have an influence on the functioning of another distant cell. Generally, the immune cell is one that reduces one or more of the target cell's ability to survive, proliferate and/or interact with other cells, and preferably, the immune cell is one that kills the target cell. It will be appreciated therefore that the immune cell is preferably an immune effector cell.
In an embodiment, the immune cell is a member of the lymphoid cell lineage, and so may be any of a T cell or a natural killer (NK) cell. Advantageously, such cells will have a cytotoxic or an apoptotic effect on the unwanted cell.
In another embodiment, the immune cell is a member of the myeloid lineage, and so may be any of a mononuclear phagocyte (eg monocyte or macrophage), a neutrophilic granulocyte or a dendritic cell. Advantageously, such cells will have a cytotoxic, phagocytic or an apoptotic effect on the unwanted cell.
It is especially preferred if the immune cell is a T cell (eg cytotoxic T cell), and so the immune cell binding region of the further moiety is a T cell binding region. Thus, the invention provides a molecule comprising: (i) a targeting moiety capable of directly or indirectly targeting to unwanted cells, and (ii) a further moiety that has a masked T cell binding region so as to prevent binding of the further moiety to a T cell, wherein the masked immune cell binding region is capable of being selectively unmasked when the molecule is in the vicinity of the unwanted cells so as to allow binding of the further moiety to an immune cell.
By ‘T cell’, we include all types of T cell including CD4+, CD8+, γδ T cells and NK-T cells. Advantageously, the T cell is a cytotoxic T cell such that a cytotoxic T cell response is recruited. In this way, a cytotoxic effect can be exerted on the unwanted cells.
Entities expressed on the surface of T cells include any of CD3, T cell receptor (TCR), CD4, CD8 and CD28, and so when immune cell binding region is one that binds to T cells, the further moiety may be capable of binding to any of these antigens. For example, the further moiety may be an antibody that binds specifically to any of CD3, TCR, CD4, CD8 and CD28.
Entities expressed on the surface of other immune cells are well known in the art and, as with those on the surface of T cells, can be readily identified by interrogating the scientific literature. Examples include CD16, CD56, CD8, NK cell receptor (eg CD94:NKG2 heterodimer, Ly49 homodimer. γδ T cell receptor, pathogen recognition receptor, stress surveillance receptor, Killer-cell Ig-like receptor (KIR) or leukocyte inhibitory receptor), NKG2D, NKp46, CD2, CD28 and CD25.
The entity that is targeted by the immune cell binding region of the further moiety is preferably one which, when bound, leads to activation of the corresponding immune cell (eg T cell). Activation of an immune cell (eg T cell) can be determined by contacting isolated peripheral mononuclear blood cells with the further moiety comprising the immune cell binding region and using standard assays for cell proliferation known in the art. Suitable assays for determining the extent of an immune response include ELISpot, intracellular cytokine staining, HLA-peptide tetramer staining, proliferation assay, activation assays (eg CD69), CD107 mobilisation assays or metabolic assays (eg MTT). Also suitable are assays to detect activation-induced secreted cytokines, for example using ELISA or multiplexed bead technologies.
For instance, in the preferred embodiment when the immune cell binding region of the further moiety is a T cell binding region, it is preferred if the T cell binding region is capable of binding to the CD3 antigen and/or TCR on T cells which are known to activate T cells. CD3 is present on all T cells and consists of subunits designated γ, δ, ε, ζ and η. The cytoplasmic tail of CD3 is sufficient to transduce the signals necessary for T cell activation in the absence of the other components of the TCR receptor complex. Normally, activation of T cell cytotoxicity depends first on binding of the TCR with a major histocompatibility complex (MHC) protein, itself bound to a foreign antigen, located on a separate cell. Only when this initial TCR-MHC binding has taken place can the CD3-dependent signally cascade responsible for T cell clonal expansion and, ultimately, T cell cytotoxicity ensue. However, when the further moiety of the molecule of the present invention binds to CD3 and/or the TCR, activation of cytotoxic T cells in the absence of independent TCR-MHC can take place by virtue of the crosslinking of the CD3 and/or TCR molecules mimicking an immune synapse formation. This means that T cells may be cytotoxically activated in a clonally independent fashion, ie in a manner which is independent of the specific TCR clone carried by the T cell. This allows for activation of the entire T cell compartment rather than only specific T cells of a certain clonal identity.
Conveniently, therefore, the immune cell binding region (eg T cell binding region) is one that binds to the immune cell in a way that mimicks the natural antigen binding to that immune cell which is known to activate the immune cell, eg to exert its effect on the viability of the target cell such as cytotoxicity.
The further moiety may be a polypeptide or a peptide. Preferably, the further moiety that contains the immune cell binding region is an antibody that specifically binds to an entity expressed by an immune cell (eg T cell). In a particularly preferred embodiment, the further moiety comprises one or more antibody variable domains (eg VH and VL domains) that specifically bind to an immune cell antigen. The further moiety may comprise aVH domain and a VL domain that, when paired, specifically bind to an immune cell antigen, or it may comprise a single VH domain or a single VL domain that is capable of binding to an immune cell antigen. Thus, the further moiety may be a single chain antibody construct such as a scFv antibody. It is especially preferred if the further moiety is an antibody (e.g. one that comprises at least one antibody variable domain such as a scFv antibody) that specifically binds to the CD3 antigen on T cells. A specific example is muromonab, but it will be appreciated that any anti-CD3 antibody may also be used. As will become clearer below and from the Figures, the further moiety may comprise two or more separate parts, such as polypeptide domains that are not necessarily encoded by contiguous polynucleotide sequences.
By the term ‘masked’, we include the meaning that the immune cell binding functionality of the immune cell binding region in the further moiety is substantially reduced or preferably inhibited completely. The masking of the immune cell binding region may be reversed by selective unmasking in the vicinity of the unwanted cells. Thus, in the masked state, the immune cell binding region is preferably unable to bind to an immune cell, while in the unmasked state the immune cell binding region is able to bind to an immune cell.
As described further below and exemplified in the figures, in a particularly preferred embodiment, the masking of the immune cell binding region is controlled by selective cleavage of one or more cleavage sites in the molecule. Thus, the molecule may contain one or more cleavage sites that are selectively cleavable when the molecule is in the vicinity of the unwanted cells. Suitable cleavage sites and agents that may cleave them are described in more detail below.
When the immune cell binding region is masked, the further moiety typically binds to an immune cell with an affinity at least 5 times less than when the immune cell binding region is unmasked, and more typically at least 10, 50 or 100 times less than when the immune cell binding region is unmasked. Most preferably, there is no detectable binding between the further moiety and an immune cell when the immune cell binding region is masked. Methods for assessing binding of moieties to cells are common in the art and include, for example, radiolabelled assays, fluorescently labelled techniques and flow cytometry. Conveniently, the further moiety is labelled and added to an immune cell under conditions conducive to binding in both the unmasked and masked states, and the extent of binding between the immune cell and further moiety in the masked and unmasked states compared. Biophysical techniques such as fluorescence correlation spectroscopy, fluorescence resonance energy transfer and analytical ultracentrifugation may also be used.
Preferably, binding of the immune cell binding region to an immune cell activates the immune cell. Thus, when the immune cell binding region is masked, the further moiety typically activates an immune cell at least 5 times less than when the immune cell binding region is unmasked, and more typically at least 10, 50 or 100 times less than when the immune cell binding region is unmasked. Most preferably, there is no detectable activation of immune cells when the immune cell binding region is masked. Methods for assessing activation of immune cells are standard in the art and include cell proliferation and cytokine secretion assays as described above.
In one embodiment, the immune cell binding region is masked by virtue of the further moiety being locked in a particular conformation in which the immune cell binding region is not accessible to an immune cell. The immune cell binding region may then be unmasked, for example, by selective cleavage of one or more cleavage sites in the molecule, when in the vicinity of the unwanted cells, which induces a conformational change in the further moiety that unmasks the immune cell binding region.
An example of this embodiment is shown in FIG. 1, where each of the targeting moiety and further moiety are respective scFv antibodies expressed on a single polypeptide chain. In FIG. 1, the targeting moiety (1) corresponds to one scFv unit that comprises two antibody variable domains (depicted as ovals in the figures): a first heavy chain variable domain (VH) and a first light chain variable domain (VL), which domains can pair together so as to form a first functional epitope binding site specific for an antigen expressed on an unwanted cell. The targeting moiety is attached to a further moiety (3) which corresponds to another scFv unit that comprises two antibody variable domains (depicted as ovals in the figures): a second VH domain and a second VL domain which can pair together so as to form a second functional epitope binding site specific for an antigen expressed on an immune cell (e.g. CD3 on a T cell). Thus, in FIG. 1, the immune cell binding region (4) of the further moiety corresponds to the second functional epitope binding site that is formed when the second VH domain pairs with the second VL domain. The second VH domain and second VL domain of the further moiety (3) in FIG. 1 are covalently joined by a polypeptide linker that is of an insufficient length to allow pairing between the two domains, which has the effect of locking the further moiety (3) in a particular conformation in which the immune cell binding region (4) is masked. However, when the molecule is in the vicinity of the unwanted cells (2), cleavage of a protease cleavage site (5) within the polypeptide linker joining the second VH domain and second VL domain, breaks the covalent linkage between the second VH domain and second VL domain so that the domains can pair and form the second functional epitope binding site. In other words, cleavage of the protease cleavage site has led to a conformation change in the further moiety (3) which unmasks the immune cell binding region (4).
Accordingly and as exemplified in FIG. 1, in one embodiment, the further moiety is a scFv antibody (3) in which the linker that joins the VH and VL domains is of insufficient length to allow pairing of the VH and VL domains such that the scFv antibody (3) cannot bind to the immune cell, and wherein selective cleavage of one or more cleavage sites (5) in said linker, when in the vicinity of the unwanted cells (2), induces pairing of the VH and VL domains such that the scFv antibody (3) can bind to the immune cell. Preferably, the immune cell binding region is a T cell binding region such as one that binds to the CD3 antigen on a T cell.
Preferably, the linker in this and other embodiments described herein is a peptide linker. However other linkers including polymers, nucleotides, nucleic acids, polysaccharide chains, lipid organic species (eg polyethylene glycol) may also be used.
Importantly, the linker that joins the VH and VL domains of the further moiety must be of insufficient length to allow pairing between the VH and VL domains.
By ‘pairing between VH and VL domains’ we include the meaning of correctly juxtaposing the matched VH and VL domains in a conformation that is identical to or approximates to the native paired state in a corresponding whole IgG parent antibody or scFv antibody such that the paired domains bind antigen with similar affinity to the corresponding whole IgG parent antibody or scFv antibody. Pairing of VH and VL domains may be assessed by any binding assay that monitors antibody/antigen binding, known in the art. Suitable techniques include ELISA, SPR, flow cytometry, FRET and structural studies. Competitive assays may also be employed where an excess of one or other of the VH or VL domains is used. If the excess causes binding to increase, this would imply that domains are unpaired. Additionally, functional immune cell assays may be used to assess pairing of VH and VL domains of the further moiety by investigating their ability to bind to, and activate, the corresponding immune cell. For example, if the VH and VL domains of the further moiety were specific for CD3 antigen on T cells, it may be appropriate to monitor correct pairing by performing functional T cell assays.
Generally, peptide linkers that join the VH domain and VL domain of a scFv antibody which are 14 amino acids or less are of insufficient length to allow pairing between the VH and VL domains. Thus, when the linker is a peptide, the peptide linker is typically 14 amino acids or less, such as 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid in length.
Preferably, the targeting moiety of the embodiment in FIG. 1 comprises a VH domain and a VL domain which domains can pair together so as to form a functional epitope binding site specific for an antigen expressed on an unwanted cell. In this case, it is understood that the targeting moiety may be joined to the further moiety such that the molecule can be expressed as a single polypeptide chain.
In an alternative embodiment, the immune cell binding region is masked by virtue of the molecule comprising one or more masking moieties that prevent access of the immune cell binding region to the T cell. Thus, the molecule may comprise at least 1 or 2 or 3 or 4 or more masking moieties.
The one or more masking moieties may be any chemical moiety including a polypeptide or a peptide or an antibody or a small molecule. It is appreciated that any moiety that sterically blocks the immune cell binding region of the further moiety may be used as a masking moiety. Examples of suitable polypeptides include albumin and haemoglobulin. Such moieties can be readily identified by the skilled person. For example, antibodies against an antibody known to activate an immune cell (e.g a T cell activating scFv fragment) may be selected by phage display technology or by immunising a mouse with the appropriate antibody. Similarly, small molecule libraries may be screened to identify inhibitors of a known activator of an immune cell, for example in immune cell activation assays. Other techniques that may be used include molecular computer modelling and ribosomal display methods.
In one embodiment, and as exemplified in FIGS. 1, 2 and 4, the one or more masking moieties are immunoglobulin domains. The immunoglobulin domains may be either heavy chain or light chain domains derived from an antibody, and equally they may be either variable or constant domains derived from an antibody. Thus, the one or more masking moieties may be any one or more of a heavy chain variable domain (VH), a light chain variable domain (VL), a heavy chain constant domain (CH) and a light chain constant domain (CL). For example, the molecule may comprise two masking moieties which are a VL domain and a VH domain, or which are a CL domain and a CH domain. When the one or more masking moieties are a VL domain and a VH domain, the two domains may correspond to a scFv construct. It will be appreciated that when the one or more masking moieties are immunoglobulin domains, they may have an idiotype specific for the immune cell binding region of the targeting moiety. Preferably, however, the immunoglobulin domains used as masking moieties do not specifically bind any other protein in the body, and so are ones that have low toxicity.
In another embodiment, and as exemplified in FIG. 3, the masking moiety is an immune cell surface antigen which the immune cell binding region of the further moiety is capable of binding to. For example, if the immune cell binding region of the further moiety binds to CD3 antigen on a T cell, the masking moiety may be the CD3 antigen (or one of the subunits thereof). In this scenario, the masking moiety competes with the T cell for binding to the immune cell binding region of the further moiety. It will be appreciated that the masking moiety need not be the entire immune cell surface antigen but may comprise only a portion of the immune cell surface antigen provided that the portion is capable of binding to the immune cell binding region. Suitable portions include the external parts of the immune cell surface antigens or portions thereof that can bind to the immune cell binding region. Thus, an external part of one of the subunits of the CD3 antigen (eg CD c) may be used. Various methods may be used to determine binding between an immune cell surface antigen and an immune cell binding region, including, for example, enzyme linked immunosorbent assays (ELISA), surface plasmon resonance assays, chip-based assays, immunocytofluorescence, yeast two-hybrid technology and phage display which are common practice in the art and are described, for example, in Plant et al (1995) Analyt Biochem, 226(2), 342-348 and Sambrook et al (2001) Molecular Cloning A Laboratory Manual. Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Such portions of immune cell surface antigens are included in the definition immune cell surface antigen. Accordingly, the masking moiety may comprise an antigenic portion of the immune cell surface antigen (eg CD3 antigen). Generally, such portions comprise a stretch of amino acid residues of at least 8 amino acids, generally at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, and more generally at least 25, 50, 75 or 100 amino acids.
Typically, the one or more masking moieties are covalently attached to the molecule by one or more linkers. Such linkers may contain the one or more cleavage sites which are selectively cleavable when the molecule is in the vicinity of the unwanted cells, as described further below. Alternatively, the one or more masking moieties may be non-covalently attached to the molecule. In this case, the one or more cleavage sites described below may be located at one or more positions in the further moiety, such that upon cleavage, the part of the further moiety that is non-covalently bound to a masking moiety is released from the molecule, and the part of the further moiety that contains the immune cell binding region is retained in the molecule. As such, cleavage of the one or more cleavage sites has caused the immune cell binding region to be unmasked.
The one or more masking moieties may operate by simply blocking the immune cell binding region, so that the region is not accessible to an immune cell. In this instance there is no conformational change in the further moiety upon unmasking, and removal of the one or more masking moieties simply exposes the underlying T cell binding region so that it may bind an immune cell. It will be appreciated that the masking moiety may be any suitable chemical moiety that sterically blocks the immune cell region, as discussed above. Examples of this embodiment are shown in FIGS. 2 and 3, where the masking moiety is either an immunoglobulin domain or an immune cell surface antigen respectively.
In FIG. 2, as in FIG. 1, each of the targeting moiety and further moiety comprise respective pairs of VH and VL domains. Although FIG. 2 depicts a molecule containing two polypeptide chains, it will be appreciated that each of the targeting moiety and further moiety may also be respective scFv antibodies that are expressed on a single polypeptide chain. In the figure, the targeting moiety (1) comprises a first VH domain and a first VL domain which domains can pair together so as to form a first functional epitope binding site specific for an antigen expressed on an unwanted cell. The targeting moiety (1) is attached to a further moiety (3) that comprises a second VH domain and a second VL domain which can pair together so as to form a second functional epitope binding site specific for an antigen expressed on an immune cell (e.g. CD3), i.e. the immune cell binding region (4). However, the immune cell binding region (4) is masked by virtue of the second VH domain and second VL domain of the further moiety being joined to, respectively, a first masking moiety (6) comprising a CH domain and a second masking moiety (6) comprising a CL domain, which act to block the immune cell binding region. When the molecule is in the vicinity of the unwanted cells, cleavage of the protease cleavage sites (5) within the respective polypeptide linkers joining the second VH domain to the CH domain of the first masking moiety, and the second VL domain to the CL domain of the second masking moiety, acts to release the CH domain and CL domain masking moieties (6), such that the second functional epitope binding site becomes accessible to an immune cell. In other words, cleavage of the protease cleavage sites has unmasked the immune cell binding region.
Accordingly, and as exemplified in FIG. 2, in one embodiment, the molecule of the invention comprises:

    
    
        a further moiety (3) which comprises a VH domain and a VL domain that are capable of specifically binding to an immune cell,
        a first linker which joins the VH domain of the further moiety (3) to a CH domain of a first masking moiety (6), and
        a second linker which joins the VL domain of the further moiety (3) to a CL domain of a second masking moiety (6),
        such that the CH and CL domains of the first and second masking moieties (6) mask the binding regions of the VH and VL domains (4) so as to prevent binding of the further moiety to an immune cell, and
        wherein selective cleavage of one or more cleavage sites in said linkers (5), when in the vicinity of the unwanted cells (2), releases the first and second masking moieties (6) from the further moiety (3) so as to allow binding of the further moiety (3) to an immune cell. Preferably, the immune cell binding region is a T cell binding region such as one that binds to the CD3 antigen on a T cell. Preferably, the targeting moiety comprises a VH domain and a VL domain which domains can pair together so as to form a functional epitope binding site specific for an antigen expressed on an unwanted cell. The targeting moiety may or may not be expressed as a single polypeptide with the further moiety. It is appreciated in this embodiment that the first and second masking moieties may be any chemical moieties capable of preventing binding of the immune cell binding region to the immune cell, for example by steric blocking. For the avoidance of doubt therefore, although FIG. 2 depicts the masking moieties as CH and CL domains, it is understood that any one or more suitable masking moieties may be used, including any combination of immunoglobulin domains.
    
    


In FIG. 3, each of the targeting moiety and further moiety are respective scFv antibodies expressed as a single polypeptide chain. However, it will be appreciated that the respective VH and VL domains of the targeting moiety and further moiety of the molecule shown in FIG. 3 may equally be present on two polypeptide chains that pair together (eg scFv-like molecules or diabodies). In the figure, the targeting moiety (1) corresponds to one scFv unit that comprises a first VH domain and a first VL domain which domains can pair together so as to form a first functional epitope binding site specific for an antigen expressed on an unwanted cell. The targeting moiety is attached to a further moiety (3) that corresponds to another scFv unit which comprises a second VH domain and a second VL domain which can pair together so as to form a second functional epitope binding site specific for an antigen expressed on an immune cell (e.g. CD3), i.e. the immune cell binding region (4). However, the immune cell binding region is masked by virtue of a linker joining one of the second VH domain or second VL domain of the further moiety (3) to an immune cell surface antigen, i.e. the masking moiety (6), such that the immune cell surface antigen binds to and blocks the immune cell binding region (4). When the molecule is in the vicinity of the unwanted cells (2), cleavage of the cleavage site (5) within the linker allows the immune cell surface antigen (6) to leave the immune cell binding region, such that the second functional epitope binding site (4) becomes accessible to an immune cell. In other words, cleavage of the cleavage site (5) has unmasked the immune cell binding region (4). This embodiment involves competition between an immune cell and the immune cell surface antigen (6) within the molecule for binding to the immune cell binding region (4) of the further moiety (3). When not in the vicinity of unwanted cells (2), the immune cell surface antigen (6) is joined to the molecule by means of a linker which increases the local concentration of the immune cell surface antigen (6) and so favours binding of the immune cell surface antigen (6), rather than an immune cell, to the immune cell binding region (4). When the molecule is in the vicinity of unwanted cells (2), the linker joining the immune cell surface antigen (6) to the molecule is cleaved and so the immune cell surface antigen (6) is free to leave the immune cell binding region (4) such that an immune cell can now bind to it.
Accordingly, the molecule of the invention may comprise:

    
    
        a further moiety (3) which comprises a VH domain and a VL domain that are capable of specifically binding to an immune cell, and
        a linker which joins the further moiety (3) to an immune cell surface antigen (6),
        such that the immune cell surface antigen (6) binds to and masks the immune cell binding region (4) of the VH and VL domains so as to prevent binding of the further moiety (3) to an immune cell,
        wherein selective cleavage of one or more cleavage sites (5) in the linker, when in the vicinity of the unwanted cells (2), releases the immune cell surface antigen (6) from the immune cell binding region (4) of the VH and VL domains so as to allow binding of the further moiety (3) to an immune cell. Preferably, the immune cell binding region (4) is a T cell binding region such as one which specifically binds to the CD3 antigen of a T cell, and the immune cell surface antigen is a T cell surface antigen such as CD3 antigen or part thereof. Preferably, and as exemplified in FIG. 3, the targeting moiety comprises a VH domain and a VL domain which domains can pair together so as to form a functional epitope binding site specific for an antigen expressed on an unwanted cell. In this way, the targeting moiety and further moiety may correspond to two separate scFv units that can be expressed as a single polypeptide chain.
    
    


In an alternative embodiment, the one or more masking moieties operate by promoting a conformation of the further moiety such that the immune cell binding region is not accessible to an immune cell. In this case, unmasking does involve a conformational change in the further moiety such that the immune cell binding region now becomes accessible for binding to an immune cell. For example, when the further moiety comprises a pair of VH and VL domains that specifically bind to an antigen on an immune cell (e.g. CD3 antigen), it is appreciated that the one or more masking moieties may act to prevent pairing of the component VH and VL domains of the further moiety such that the VH and VL domains are not in the desired three dimensional conformation to bind to the immune cell antigen. Various methods may be used to prevent pairing of the VH and VL domains of a further moiety. One such method is exemplified in FIG. 4 which makes use of dummy variable domains that pair with the VH and VL domains of the further moiety such that the VH and VL domains of the further moiety cannot pair with each other. However, any suitable masking moiety that prevents pairing of the VH and VL domains of the further moiety, for example by binding to VH-VL interface protein surface, may be used. In all cases, the prevention of pairing of the VH and VL domains is lifted by cleavage of one or more cleavage sites within the molecule when the molecule is in the vicinity of the wanted cells.
In FIG. 4, the targeting moiety (1) corresponds to a scFv unit that comprises a first VH domain and a first VL domain which domains can pair together so as to form a first functional epitope binding site specific for an antigen expressed on an unwanted cell. A first linker joins the first VL domain of the targeting moiety (1) to a second VH domain of a further moiety (3), a second linker joins the first VH domain of the targeting moiety (1) to a first masking moiety comprising a VH domain (6), a third linker joins the second VH domain of the further moiety (3) to a second masking moiety comprising a VL domain (6), and a fourth linker joins the VH domain of the first masking moiety to a second VL domain of the further moiety (3); wherein the second VH and second VL domains of the further moiety, when paired, form a second functional epitope binding site specific for an antigen expressed on an immune cell (e.g. CD3 antigen on a T cell), i.e. the immune cell binding region (4); and wherein the third and fourth linkers are of a sufficient length so that the second VH domain of the further moiety (3) is paired with the VL domain of the second masking moiety (6), and the second VL domain of the further moiety (3) is paired with the VH domain of the first masking moiety (6). Thus, the first and second masking moieties in FIG. 4 prevent the second VH and second VL domain of the further moiety from pairing and so mask the immune cell binding region (4). In this way, the variable domains of the masking moieties act as ‘dummy’ VH and VL domains which pair with the second VH and VL domains of the further moiety, and the third and fourth linkers stabilise this configuration by preventing domain exchange. However, when the molecule is in the vicinity of the unwanted cells (2), cleavage of one or more cleavage sites (5) present in the third and fourth linkers release the second VH domain and second VL domain from pairing with the VL domain of the second masking moiety (6) and the VH domain of the first masking moiety (6), respectively, so that the second VH domain and second VL domain of the further moiety can pair to form the immune cell binding region (4). In other words, cleavage of the cleavage sites (5) present in the third and fourth linkers has allowed conformational rearrangement of the molecule wherein the second VH domain and second VL domain are correctly paired so that the immune cell binding ability is recovered. The immune cell binding region (4) has been unmasked.
Accordingly, in one embodiment, the molecule of the invention comprises:

    
    
        a further moiety (3) which comprises separately a first VH and a first VL domain which when paired are capable of specifically binding to an immune cell,
        a linker which joins the first VH domain to a second VL domain of a first masking moiety (6), and
        a linker which joins the first VL domain to a second VH domain of a second masking moiety (6),
        such that the first VH and first VL domains of the further moiety are not paired and the further moiety cannot bind to the immune cell, and
        wherein selective cleavage of one or more cleavage sites (5) in said linkers, when in the vicinity of the unwanted cells (2), allows pairing of the first VH and first VL domains such that the further moiety can bind to the immune cell. Preferably, the immune cell binding region (4) is a T cell binding region such as one that specifically binds to the CD3 antigen on a T cell. Given that the masking moieties mask the immune cell binding region, it will of course be appreciated in this embodiment, that the second VH domain and second VL domain of the masking moieties are not ones which are capable of respectively pairing with the first VL domain and first VH domain of the further moiety so as to form a functional epitope binding site specific for an antigen expressed on an immune cell (e.g. an antigen expressed on a T cell such as CD3 antigen). In any event, it will further be appreciated that any suitable masking moiety that prevents pairing of the VH and VL domains of the further moiety may be substituted for the VH and VL masking moieties shown in FIG. 4. Preferably, and as exemplified in FIG. 4, the targeting moiety comprises a VH domain and a VL domain which domains can pair together so as to form a functional epitope binding site specific for an antigen expressed on an unwanted cell. In this case, it is understood that the targeting moiety may be joined to the further moiety and the one or more masking moieties such that the molecule can be expressed as a single polypeptide chain. The targeting moiety may be in the middle of the polypeptide chain flanked by the further moiety and/or one or more masking moieties. For example, the targeting moiety may be a scFv unit, one domain of which is joined to a first VL domain of the further moiety and the other domain of which is joined to a second VL domain of a first masking moiety, or the targeting moiety may be a scFv unit, one domain of which is joined to the first VH domain of the further moiety and the other domain of which is joined to second VH domain of the second masking moiety. Alternatively, the targeting moiety may be at either end of the polypeptide chain.
    
    


Preferably, and as exemplified in FIG. 4, the linker which joins the first VH domain to a second VL domain of a first masking moiety, is of sufficient length to allow the first VH domain to be paired with the second VL domain, and the linker which joins the first VL domain to a second VH domain of a second masking moiety is of sufficient length to allow the first VL domain to be paired with the second VH domain. Typically, the linkers are peptides and so must have a sufficient number of amino acids to allow pairing between the VH and VL domains. Generally, peptide linkers that join a VH domain and a VL domain of a scFv antibody which are 15 amino acids or more are of sufficient length to allow pairing between the VH and VL domains. Thus, when said linkers are peptides, they are typically 15 amino acids or more in length, such as at least 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acids in length. Pairing between VH and VL domains can be assessed by routine methods known in the art, including those described above.
Selective Unmasking of Immune Cell Binding Region
By ‘selective unmasking’ we include the meaning that the immune cell binding region is unmasked by the presence of an agent that selectively resides in the in the vicinity of the unwanted cells, which agent acts to unmask the immune cell binding region. Preferably, the agent that unmasks the immune cell binding region resides in the vicinity of the unwanted cells at least five or ten times higher than the concentration of the agent outside the vicinity of the unwanted cells, and more preferably at least 100 or 500 or 1000 times higher. Most preferably, the agent that unmasks the immune cell binding region binding region is found only in the vicinity of the unwanted cells.
In a preferred embodiment, the immune cell binding region is unmasked by selective cleavage of one or more cleavage sites in the molecule when in the vicinity of the unwanted cells. In this case, the agent is one that cleaves the one or more cleavage sites. In other words, the immune cell binding region may be unmasked by means of one or more cleavage sites within the molecule being cleaved selectively in the vicinity of the unwanted cells.
Thus, the invention includes a molecule comprising (i) a targeting moiety capable of directly or indirectly targeting to unwanted cells, and (ii) a further moiety that has a masked immune cell binding region so as to prevent binding of the further moiety to an immune cell, wherein selective cleavage of one or more cleavage sites in the molecule when in the vicinity of the unwanted cells unmasks the immune cell binding region so as to allow binding of the further moiety to an immune cell. Preferably, the immune cell binding region is a T cell binding region including one that binds to the CD3 antigen and/or TCR on a T cell. Accordingly, it is appreciated that the invention provides a molecule comprising (i) a targeting moiety capable of directly or indirectly targeting to unwanted cells, and (ii) a further moiety that has a masked T cell binding region so as to prevent binding of the further moiety to a T cell, wherein selective cleavage of one or more cleavage sites in the molecule when in the vicinity of the unwanted cells unmasks the T cell binding region so as to allow binding of the further moiety to a T cell.
By “cleavage site that is cleavable selectively in the vicinity of the unwanted cells” we include the meaning of a site that can only be cleaved by an agent which resides selectively in the vicinity of the unwanted cells, so as to unmask the immune cell binding region. Preferably, the agent that cleaves the one or more cleavage sites resides in the vicinity of the unwanted cells at a concentration at least five times or ten times higher than the concentration of the agent outside the vicinity of the unwanted cells, and more preferably at a concentration at least 100 or 500 or 1000 times higher. Most preferably, the agent that cleaves the one or more cleavage sites is found only in the vicinity of the unwanted cells. For example, when the unwanted cells are particular tumour cells (e.g. breast tumour cells), the one or more cleavage sites may be ones that are cleaved by an agent which resides selectively in the particular tumour (e.g. breast tumour) but which agent does not reside outside the vicinity of the particular tumour (e.g. breast tumour).
By ‘in the vicinity of cells’, we include the meaning of either at or near to the surface of the cells, or both, or in the environment that immediately surrounds the cells e.g. blood, lymph, and other body fluids.
The one or more cleavage sites are selectively cleaved in the vicinity of the unwanted cells so that the immune cell binding region of the further moiety is preferentially unmasked in the vicinity of the unwanted cells so as to recruit immune cells preferentially to unwanted cells rather than wanted cells. Thus, it is preferred that the one or more cleavage sites are ones that are selectively cleaved such that the immune cell binding region is unmasked in the vicinity of the unwanted cells at least five times or ten times more than the extent to which it is unmasked in the vicinity of wanted cells, and more preferably at least 100 or 500 or 1000 times more. Most preferably, the immune cell binding region is not unmasked in the vicinity of wanted cells, and therefore immune cells are not recruited to wanted cells.
For a given unwanted cell, the skilled person will be able to identify appropriate one or more cleavage sites that are selectively cleavable in the vicinity of the unwanted cell, using established methods in the art. For example, which proteases cleave which peptides can be assessed by consulting peptide libraries and studying an MS analysis of the fragmentation profile following cleavage. Also, published literature of protease cleavage motifs and peptide cleavage data can be searched as described further below. Gene expression and proteomic data may also be analysed to identify which proteases are expressed by particular unwanted cells.
By virtue of the one or more cleavage sites being selectively cleavable in the vicinity of the unwanted cells, the immune cell binding region is selectively unmasked in the vicinity of the unwanted cells.
The one or more cleavage sites may be located between separate parts of the further moiety (e.g. separate immunoglobulin domains), such that upon cleavage of the cleavage sites a conformational change is induced whereby the separate parts of the further moiety are rearranged so as to unmask the immune cell binding region (see FIG. 1). The one or more cleavage sites may be located between the one or more masking moieties and either the further moiety and/or targeting moiety. Thus, the one or more cleavage sites may be located between the one or more masking moieties and the further moiety (see FIGS. 2 and 3), or they may be located between the one or more masking moieties and the targeting moiety (see FIG. 4), or they may be located between the one or more masking moieties and each of the targeting moiety and further moiety (see FIG. 4). When any of the further moiety, targeting moiety and masking moiety comprise one or more immunoglobulin domains, it is appreciated that the one or more cleavage sites should be located between the immunoglobulin domains to allow movement of whole immunoglobulin domains rather than movement of only parts of the domains, upon unmasking of the immune cell binding region.
The cleavage site may be one that is cleavable by an enzyme such as any of a protease, a nuclease, a lipase, a lyase, a phosphatase or a carbohydrase, which may or may not be membrane bound. Accordingly, it will be appreciated that the cleavage site may be one that is cleavable by an enzyme that is bound to the membrane of, or secreted by, the unwanted cell.
Generally, the cleavage site is a protease cleavage site. Thus, when the unwanted cells are tumour cells, the one or more cleavage sites may be cleavable selectively by proteases that reside in the vicinity of the tumour cells. In other words, the protease cleavage site may be one that is cleavable by a tumour associated protease. It is well known that during tumour development, tumours aberrantly express proteases which allow them to invade local tissues and eventually metastasise.
The protease may include any of a cysteine protease (including the Cathepsin family B, L, S etc), an aspartyl protease (including Cathepsin D and E) and a serine protease (including Cathepsin A and G, Thrombin, Plasmin, Urokinase, Tissue Plasminogen Activator). The protease may be a metalloproteinase (MMP1-28) including both membrane bound (MMP14-17 and MMP24-25) and secreted forms (MMP1-13 and MMP18-23 and MMP26-28). The protease may belong to the A Disintegrin and Metalloproteinase (ADAM) and A Disintegrin, or Metalloproteinase with Thrombospondin Motifs (ADAMTS) families of proteases. Other examples include CD10 (CALLA) and prostate specific antigen (PSA). It is appreciated that the proteases may or may not be membrane bound.
Protease cleavage sites are well known in the scientific literature, and linker sequences comprising such cleavage sites can be readily constructed using established genetic engineering techniques, or by synthetic synthesis techniques known in the art.
Protease cleavage sites may be ones that are cleavable by any of the proteases listed in Table 4 below, which indicates the expression of selected proteases in various tumour types. Candidate substrates for the proteases are provided. Thus, in order to treat a particular tumour type, the skilled person will typically select one or more protease cleavage sites that are selectively cleaved by a protease known to be highly expressed in that tumour type, as seen from the table. For example, to treat breast cancer, it is preferred to use a protease cleavage site cleavable by any of uPA, tPA, matriptase, matriptase 2, Cathepsin K, Cathepsin O, MMP1, MMP2, MMP3, MMP11, MMP12, MMP17, ADAM9, ADAM12, ADAM15, ADAM17, ADAM28 or ADAMTS15, and so on. It will be appreciated that the one or more protease cleavage sites selected by the skilled person include ones that are cleaved by different proteases, which may help to improve the specificity of the molecule of the invention further.
Similarly, Table 5 lists tumour sites in which ADAM protease overexpression has been reported, and so in an embodiment, the one or more cleavage sites are selectively cleavable by one of the ADAM proteases listed in Table 5. Accordingly, the molecule may be used to prevent or treat the corresponding tumour type.
The one or more cleavage sites may be selectively cleavable by any of the following human proteases (MEROPS peptidase database number provided in parentheses; Rawlings N. D., Morton F. R., Kok, C. Y., Kong, J. & Barrett A. J. (2008) MEROPS: the peptidase database. Nucleic Acids Res 36 Database issue, D320-325): pepsin A (MER000885), aastricsin (MER000894), memapsin-2 (MER005870), renin (MER000917), cathepsin D (MER000911), cathepsin E (MER000944), memapsin-1 (MER005534), napsin A (MER004981), Memame-AA034 peptidase (MER014038), pepsin A4 (MER037290), pepsin A5 (Homo sapiens) (MER037291), hCG1733572 (Homo sapiens)-type putative peptidase (MER107386), napsin B pseudogene (MER004982), CYMP g.p. (Homo sapiens) (MER002929), subfamily A1A unassigned peptidases (MER181559), mouse mammary tumor virus retropepsin (MER048030), rabbit endogenous retrovirus endopeptidase (MER043650), S71-related human endogenous retropepsin (MER001812), RTVL-H-type putative peptidase (MER047117), RTVL-H-type putative peptidase (MER047133), RTVL-H-type putative peptidase (MER047160), RTVL-H-type putative peptidase (MER047206), RTVL-H-type putative peptidase (MER47253), RTVL-H-type putative peptidase (MER047260), RTVL-H-type putative peptidase (MER047291), RTVL-H-type putative peptidase (MER047418), RTVL-H-type putative peptidase (MER047440), RTVL-H-type putative peptidase (MER047479), RTVL-H-type putative peptidase (MER047559), RTVL-H-type putative peptidase (MER047583), RTVL-H-type putative peptidase (MER015446), human endogenous retrovirus retropepsin homologue 1 (MER015479), human endogenous retrovirus retropepsin homologue 2 (MER015481), endogenous retrovirus retropepsin pseudogene 1 (Homo sapiens chromosome 14) (MER029977), endogenous retrovirus retropepsin pseudogene 2 (Homo sapiens chromosome 8) (MER029665), endogenous retrovirus retropepsin pseudogene 3 (Homo sapiens chromosome 17) (MER002660), endogenous retrovirus retropepsin pseudogene 3 (Homo sapiens chromosome 17) (MER030286), endogenous retrovirus retropepsin pseudogene 3 (Homo sapiens chromosome 17) (MER047144), endogenous retrovirus retropepsin pseudogene 5 (Homo sapiens chromosome 12) (MER029664), endogenous retrovirus retropepsin pseudogene 6 (Homo sapiens chromosome 7) (MER002094), endogenous retrovirus retropepsin pseudogene 7 (Homo sapiens chromosome 6) (MER029776), endogenous retrovirus retropepsin pseudogene 8 (Homo sapiens chromosome Y) (MER030291), endogenous retrovirus retropepsin pseudogene 9 (Homo sapiens chromosome 19) (MER029680), endogenous retrovirus retropepsin pseudogene 10 (Homo sapiens chromosome 12) (MER002848), endogenous retrovirus retropepsin pseudogene 11 (Homo sapiens chromosome 17) (MER004378), endogenous retrovirus retropepsin pseudogene 12 (Homo sapiens chromosome 11) (MER003344), endogenous retrovirus retropepsin pseudogene 13 (Homo sapiens chromosome 2 and similar) (MER029779), endogenous retrovirus retropepsin pseudogene 14 (Homo sapiens chromosome 2) (MER029778), endogenous retrovirus retropepsin pseudogene 15 (Homo sapiens chromosome 4) (MER047158), endogenous retrovirus retropepsin pseudogene 15 (Homo sapiens chromosome 4) (MER047332), endogenous retrovirus retropepsin pseudogene (Homo sapiens chromosome 4) (MER003182), endogenous retrovirus retropepsin pseudogene 16 (MER047165), endogenous retrovirus retropepsin pseudogene 16 (MER047178), endogenous retrovirus retropepsin pseudogene 16 (MER047200), endogenous retrovirus retropepsin pseudogene 16 (MER047315), endogenous retrovirus retropepsin pseudogene 16 (MER047405), endogenous retrovirus retropepsin pseudogene 16 (MER030292), endogenous retrovirus retropepsin pseudogene 17 (Homo sapiens chromosome 8) (MER005305), endogenous retrovirus retropepsin pseudogene 18 (Homo sapiens chromosome 4) (MER030288), endogenous retrovirus retropepsin pseudogene 19 (Homo sapiens chromosome 16) (MER01740), endogenous retrovirus retropepsin pseudogene 21 (Homo sapiens) (MER047222), endogenous retrovirus retropepsin pseudogene 21 (Homo sapiens) (MER047454), endogenous retrovirus retropepsin pseudogene 21 (Homo sapiens) (MER047477), endogenous retrovirus retropepsin pseudogene 21 (Homo sapiens) (MER004403), endogenous retrovirus retropepsin pseudogene 22 (Homo sapiens chromosome X) (MER030287), subfamily A2A non-peptidase homologues (MER047046), subfamily A2A non-peptidase homologues (MER047052), subfamily A2A non-peptidase homologues (MER047076), subfamily A2A non-peptidase homologues (MER047080), subfamily A2A non-peptidase homologues (MER047088), subfamily A2A non-peptidase homologues (MER047089), subfamily A2A non-peptidase homologues (MER047091), subfamily A2A non-peptidase homologues (MER47092) subfamily A2A non-peptidase homologues (MER047093), subfamily A2A non-peptidase homologues (MER047094), subfamily A2A non-peptidase homologues (MER047097), subfamily A2A non-peptidase homologues (MER047099), subfamily A2A non-peptidase homologues (MER047101), subfamily A2A non-peptidase homologues (MER047102), subfamily A2A non-peptidase homologues (MER047107), subfamily A2A non-peptidase homologues (MER047108), subfamily A2A non-peptidase homologues (MER047109), subfamily A2A non-peptidase homologues (MER047110), subfamily A2A non-peptidase homologues (MER047111), subfamily A2A non-peptidase homologues (MER047114), subfamily A2A non-peptidase homologues (MER047118), subfamily A2A non-peptidase homologues (MER047121), subfamily A2A non-peptidase homologues (MER047122), subfamily A2A non-peptidase homologues (MER047126), subfamily A2A non-peptidase homologues (MER047129) subfamily A2A non-peptidase homologues (MER047130), subfamily A2A non-peptidase homologues (MER047134), subfamily A2A non-peptidase homologues (MER047135), subfamily A2A non-peptidase homologues (MER047137), subfamily A2A non-peptidase homologues (MER047140), subfamily A2A non-peptidase homologues (MER047141), subfamily A2A non-peptidase homologues (MER047142), subfamily A2A non-peptidase homologues (MER047148), subfamily A2A non-peptidase homologues (MER047149), subfamily A2A non-peptidase homologues (MER047151), subfamily A2A non-peptidase homologues (MER047154), subfamily A2A non-peptidase homologues (MER047155), subfamily A2A non-peptidase homologues (MER047156), subfamily A2A non-peptidase homologues (MER047157), subfamily A2A non-peptidase homologues (MER047159), subfamily A2A non-peptidase homologues (MER047161), subfamily A2A non-peptidase homologues (MER047163), subfamily A2A non-peptidase homologues (MER047166), subfamily A2A non-peptidase homologues (MER047171), subfamily A2A non-peptidase homologues (MER047173), subfamily A2A non-peptidase homologues (MER047174), subfamily A2A non-peptidase homologues (MER047179), subfamily A2A non-peptidase homologues (MER04183), subfamily A2A non-peptidase homologues (MER047186), subfamily A2A non-peptidase homologues (MER047190), subfamily A2A non-peptidase homologues (MER047191), subfamily A2A non-peptidase homologues (MER047196), subfamily A2A non-peptidase homologues (MER147198), subfamily A2A non-peptidase homologues (MER047199), subfamily A2A non-peptidase homologues (MER47201), subfamily A2A non-peptidase homologues (MER047202), subfamily A2A non-peptidase homologues (MER047203), subfamily A2A non-peptidase homologues (MER047204), subfamily A2A non-peptidase homologues (MER047205), subfamily A2A non-peptidase homologues (MER047207), subfamily A2A non-peptidase homologues (MER047208), subfamily A2A non-peptidase homologues (MER047210), subfamily A2A non-peptidase homologues (MER047211), subfamily A2A non-peptidase homologues (MER047212), subfamily A2A non-peptidase homologues (MER047213), subfamily A2A non-peptidase homologues (MER047215), subfamily A2A non-peptidase homologues (MER047216), subfamily A2A non-peptidase homologues (MER047218), subfamily A2A non-peptidase homologues (MER047219), subfamily A2A non-peptidase homologues (MER047221), subfamily A2A non-peptidase homologues (MER047224), subfamily A2A non-peptidase homologues (MER047225), subfamily A2A non-peptidase homologues (MER047226), subfamily A2A non-peptidase homologues (MER047227), subfamily A2A non-peptidase homologues (MER047230), subfamily A2A non-peptidase homologues (MER47232), subfamily A2A non-peptidase homologues (MER047233), subfamily A2A non-peptidase homologues (MER047234), subfamily A2A non-peptidase homologues (MER047236), subfamily A2A non-peptidase homologues (MER047238), subfamily A2A non-peptidase homologues (MER047239), subfamily A2A non-peptidase homologues (MER047240), subfamily A2A non-peptidase homologues (MER047242), subfamily A2A non-peptidase homologues (MER047243), subfamily A2A non-peptidase homologues (MER047249), subfamily A2A non-peptidase homologues (MER047251), subfamily A2A non-peptidase homologues (MER047252), subfamily A2A non-peptidase homologues (MER047254), subfamily A2A non-peptidase homologues (MER047255), subfamily A2A non-peptidase homologues (MER047263), subfamily A2A non-peptidase homologues (MER047265), subfamily A2A non-peptidase homologues (MER047266), subfamily A2A non-peptidase homologues (MER047267), subfamily A2A non-peptidase homologues (MER047268), subfamily A2A non-peptidase homologues (MER047269), subfamily A2A non-peptidase homologues (MER047272), subfamily A2A non-peptidase homologues (MER047273), subfamily A2A non-peptidase homologues (MER47274), subfamily A2A non-peptidase homologues (MER047275), subfamily A2A non-peptidase homologues (MER047276), subfamily A2A non-peptidase homologues (MER047279), subfamily A2A non-peptidase homologues (MER047280), subfamily A2A non-peptidase homologues (MER047281), subfamily A2A non-peptidase homologues (MER047282) subfamily A2A non-peptidase homologues (MER047284), subfamily A2A non-peptidase homologues (MER047285), subfamily A2A non-peptidase homologues (MER047289), subfamily A2A non-peptidase homologues (MER47290), subfamily A2A non-peptidase homologues (MER47294), subfamily A2A non-peptidase homologues (MER047295), subfamily A2A non-peptidase homologues (MER047298), subfamily A2A non-peptidase homologues (MER047300), subfamily A2A non-peptidase homologues (MER047302), subfamily A2A non-peptidase homologues (MER47304) subfamily A2A non-peptidase homologues (MER047305), subfamily A2A non-peptidase homologues (MER047306), subfamily A2A non-peptidase homologues (MER047307), subfamily A2A non-peptidase homologues (MER047310), subfamily A2A non-peptidase homologues (MER047311), subfamily A2A non-peptidase homologues (MER047314), subfamily A2A non-peptidase homologues (MER047318), subfamily A2A non-peptidase homologues (MER047320), subfamily A2A non-peptidase homologues (MER047321), subfamily A2A non-peptidase homologues (MER047322), subfamily A2A non-peptidase homologues (MER047326), subfamily A2A non-peptidase homologues (MER047327), subfamily A2A non-peptidase homologues (MER047330), subfamily A2A non-peptidase homologues (MER047333), subfamily A2A non-peptidase homologues (MER047362), subfamily A2A non-peptidase homologues (MER047366), subfamily A2A non-peptidase homologues (MER047369), subfamily A2A non-peptidase homologues (MER047370), subfamily A2A non-peptidase homologues (MER047371), subfamily A2A non-peptidase homologues (MER047375), subfamily A2A non-peptidase homologues (MER047376), subfamily A2A non-peptidase homologues (MER047381), subfamily A2A non-peptidase homologues (MER047383), subfamily A2A non-peptidase homologues (MER047384), subfamily A2A non-peptidase homologues (MER047385), subfamily A2A non-peptidase homologues (MER047388), subfamily A2A non-peptidase homologues (MER047389), subfamily A2A non-peptidase homologues (MER047391), subfamily A2A non-peptidase homologues (MER047394), subfamily A2A non-peptidase homologues (MER47396), subfamily A2A non-peptidase homologues (MER047400), subfamily A2A non-peptidase homologues (MER047401), subfamily A2A non-peptidase homologues (MER047403), subfamily A2A non-peptidase homologues (MER047406), subfamily A2A non-peptidase homologues (MER047407), subfamily A2A non-peptidase homologues (MER047410), subfamily A2A non-peptidase homologues (MER047411), subfamily A2A non-peptidase homologues (MER047413), subfamily A2A non-peptidase homologues (MER047414), subfamily A2A non-peptidase homologues (MER047416), subfamily A2A non-peptidase homologues (MER047417), subfamily A2A non-peptidase homologues (MER047420), subfamily A2A non-peptidase homologues (MER047423), subfamily A2A non-peptidase homologues (MER047424), subfamily A2A non-peptidase homologues (MER047428), subfamily A2A non-peptidase homologues (MER047429), subfamily A2A non-peptidase homologues (MER047431), subfamily A2A non-peptidase homologues (MER047434), subfamily A2A non-peptidase homologues (MER047439), subfamily A2A non-peptidase homologues (MER047442), subfamily A2A non-peptidase homologues (MER047445), subfamily A2A non-peptidase homologues (MER047449), subfamily A2A non-peptidase homologues (MER047450), subfamily A2A non-peptidase homologues (MER047452), subfamily A2A non-peptidase homologues (MER047455), subfamily A2A non-peptidase homologues (MER047457), subfamily A2A non-peptidase homologues (MER047458), subfamily A2A non-peptidase homologues (MER07459), subfamily A2A non-peptidase homologues (MER047463), subfamily A2A non-peptidase homologues (MER047468), subfamily A2A non-peptidase homologues (MER047469), subfamily A2A non-peptidase homologues (MER047470), subfamily A2A non-peptidase homologues (MER047476), subfamily A2A non-peptidase homologues (MER047478), subfamily A2A non-peptidase homologues (MER047483), subfamily A2A non-peptidase homologues (MER047488), subfamily A2A non-peptidase homologues (MER047489), subfamily A2A non-peptidase homologues (MER047490), subfamily A2A non-peptidase homologues (MER047493), subfamily A2A non-peptidase homologues (MER047494), subfamily A2A non-peptidase homologues (MER047495), subfamily A2A non-peptidase homologues (MER047496), subfamily A2A non-peptidase homologues (MER047497), subfamily A2A non-peptidase homologues (MER047499), subfamily A2A non-peptidase homologues (MER047502), subfamily A2A non-peptidase homologues (MER047504), subfamily A2A non-peptidase homologues (MER047511), subfamily A2A non-peptidase homologues (MER047513), subfamily A2A non-peptidase homologues (MER047514), subfamily A2A non-peptidase homologues (MER047515), subfamily A2A non-peptidase homologues (MER047516), subfamily A2A non-peptidase homologues (MER47520), subfamily A2A non-peptidase homologues (MER047533), subfamily A2A non-peptidase homologues (MER047537), subfamily A2A non-peptidase homologues (MER047569), subfamily A2A non-peptidase homologues (MER047570), subfamily A2A non-peptidase homologues (MER047584), subfamily A2A non-peptidase homologues (MER047603), subfamily A2A non-peptidase homologues (MER047604), subfamily A2A non-peptidase homologues (MER047606), subfamily A2A non-peptidase homologues (MER047609), subfamily A2A non-peptidase homologues (MER047616), subfamily A2A non-peptidase homologues (MER047619), subfamily A2A non-peptidase homologues (MER047648), subfamily A2A non-peptidase homologues (MER047649), subfamily A2A non-peptidase homologues (MER047662), subfamily A2A non-peptidase homologues (MER048004), subfamily A2A non-peptidase homologues (MER048018), subfamily A2A non-peptidase homologues (MER048019), subfamily A2A non-peptidase homologues (MER048023), subfamily A2A non-peptidase homologues (MER048037), subfamily A2A unassigned peptidases (MER047164), subfamily A2A unassigned peptidases (MER047231), subfamily A2A unassigned peptidases (MER047386), skin aspartic protease (MER057097), oresenilin 1 (MER005221), presenilin 2 (MER005223), impas 1 peptidase (MER019701), impas 1 peptidase (MIER147.2), impas 4 peptidase (MER019715), impas 2 peptidase (MER019708), impas 5 peptidase (MER019712), impas 3 peptidase (MER019711), possible family A22 pseudogene (Homo sapiens chromosome 18) (MER029974), possible family A22 pseudogene (Homo sapiens chromosome 11) (MER023159), cathepsin V (MER004437), cathepsin X (MER004508), cathepsin F (MER004980), cathepsin L (MER000622), cathepsin S (MER000633), cathepsin O (MER001690), cathepsin K (MER000044), cathepsin W (MER003756), cathepsin H (MER000629), cathepsin B (MER000686), dipeptidyl-peptidase I (MER001937), bleomycin hydrolase (animal) (MER002481), tubulointerstitial nephritis antigen (MER016137), tubulointerstitial nephritis antigen-related protein (MER021799), cathepsin L-like pseudogene 1 (Homo sapiens) (MER002789), cathepsin B-like pseudogene (chromosome 4, Homo sapiens) (MER029469), cathepsin B-like pseudogene (chromosome 1, Homo sapiens) (MER029457), CTSLL2 g.p. (Homo sapiens) (MER005210), CTSLL3 g.p. (Homo sapiens) (MER005209), calpain-1 (MER000770), calpain-2 (MER000964), calpain-3 (MER001446), calpain-9 (MER004042), calpain-8 (MER021474), calpain-15 (MER004745), calpain-5 (MER002939), calpain-11 (MER005844), calpain-12 (MER029889), calpain-10 (MER013510), calpain-13 (MER020139), calpain-14 (MER029744), Mername-AA253 peptidase (MER005537), calpamodulin (MER000718), hypothetical protein flj40251 (MER003201), ubiquitinyl hydrolase-L1 (MER000832), ubiquitinyl hydrolase-L3 (MER000836), ubiquitinyl hydrolase-BAPi (MER003989), ubiquitinyl hydrolase-UCH37 (MER005539), ubiquitin-specific peptidase 5 (MER002066), ubiquitin-specific peptidase 6 (MER000863), ubiquitin-specific peptidase 4 (MER001795), ubiquitin-specific peptidase 8 (MER001884), ubiquitin-specific peptidase 13 (MER002627), ubiquitin-specific peptidase 2 (MER004834), ubiquitin-specific peptidase 11 (MER002693), ubiquitin-specific peptidase 14 (MER002667), ubiquitin-specific peptidase 7 (MER002896), ubiquitin-specific peptidase 9X (MER005877), ubiquitin-specific peptidase 10 (MER004439), ubiquitin-specific peptidase 1 (MER004978), ubiquitin-specific peptidase 12 (MER005454), ubiquitin-specific peptidase 16 (MER005493), ubiquitin-specific peptidase 15 (MER005427), ubiquitin-specific peptidase 17 (MER002900), ubiquitin-specific peptidase 19 (MER005428), ubiquitin-specific peptidase 20 (MER005494), ubiquitin-specific peptidase 3 (MER005513), ubiquitin-specific peptidase 9Y (MER004314), ubiquitin-specific peptidase 18 (MER005641), ubiquitin-specific peptidase 21 (MER006258), ubiquitin-specific peptidase 22 (MER012130), ubiquitin-specific peptidase 33 (MER014335), ubiquitin-specific peptidase 29 (MER012093), ubiquitin-specific peptidase 25 (MER011115), ubiquitin-specific peptidase 36 (MER014033), ubiquitin-specific peptidase 32 (MER014290), ubiquitin-specific peptidase 26 (Homo sapiens-type) (MER014292), ubiquitin-specific peptidase 24 (MER005706), ubiquitin-specific peptidase 42 (MER011852), ubiquitin-specific peptidase 46 (MER014629), ubiquitin-specific peptidase 37 (MER014633), ubiquitin-specific peptidase 28 (MER014634), ubiquitin-specific peptidase 47 (MER014636), ubiquitin-specific peptidase 38 (MER014637), ubiquitin-specific peptidase 44 (MER014638), ubiquitin-specific peptidase 50 (MER030315), ubiquitin-specific peptidase 35 (MER014646), ubiquitin-specific peptidase 30 (MER014649), Mername-AA091 peptidase (MER014743), ubiquitin-specific peptidase 45 (MER030314), ubiquitin-specific peptidase 51 (MER014769), ubiquitin-specific peptidase 34 (MER014780), ubiquitin-specific peptidase 48 (MER064620), ubiquitin-specific peptidase 40 (MER015483), ubiquitin-specific peptidase 41 (MER045268), ubiquitin-specific peptidase 31 (MER015493), Memame-AA129 peptidase (MER016485), ubiquitin-specific peptidase 49 (MER016486), Mername-AA187 peptidase (MER052579), USP17-like peptidase (MER030192), ubiquitin-specific peptidase 54 (MER028714), ubiquitin-specific peptidase 53 (MER27329), ubiquitin-specific endopeptidase 39 [misleading](MER064621), Memame-AA090 non-peptidase homologue (MER014739), ubiquitin-specific peptidase 43 [misleading](MER030140), ubiquitin-specific peptidase 52 [misleading](MER030317), NEK2 pseudogene (MER014736), C19 pseudogene (Homo sapiens: chromosome 5) (MER029972) Mername-AA088 peptidase (MER014750), autophagin-2 (MER013564), autophagin-1 (MER013561), autophagin-3 (MER014316), autophagin-4 (MER064622), Cezanne deubiquitinylating peptidase (MER029042), Cezanne-2 peptidase (MER029044), tumor necrosis factor alpha-induced protein 3 (MER029050), trabid peptidase (MER029052), VCIP135 deubiquitinating peptidase (MER152304), otubain-1 (MER02905), otubain-2 (MER029061), CylD protein (MER030104), UfSP1 peptidase (MER042724), UfSP2 peptidase (MER060306), DUBA deubiquitinylating enzyme (MER086098), KIAA0459 (Homo sapiens)-like protein (MER122467), Otud1 protein (MER125457), alycosyltransferase 28 domain containing 1, isoform CRA_c (Homo sapiens)-like (MER123606), hin1L g.p. (Homo sapiens) (MER139816), ataxin-3 (MER099998), ATXN3L putative peptidase (MER115261) Josephin domain containing 1 (Homo sapiens) (MER125334), Josephin domain containing 2 (Homo sapiens) (MER124068), YOD1 peptidase (MER116559), legumain (plant alpha form) (MER044591), legumain (MER001800), glycosylphosphatidylinositol:protein transamidase (MER002479), legumain pseudogene (Homo sapiens) (MER029741), family C13 unassigned peptidases (MER175813), caspase-1 (MER000850), caspase-3 (MER000853), caspase-7 (MER002705), caspase-6 (MER002708), caspase-2 (MER001644), caspase-4 (MER001938), caspase-5 (MER002240), caspase-8 (MER002849), caspase-9 (MER002707), caspase-10 (MER002579), caspase-14 (MER012083), paracaspase (MER019325), Memame-AA143 peptidase (MER21304) Mername-AA186 peptidase (MER020516), putative caspase (Homo sapiens) (MER021463), FLIP protein (MER003026), Memame-AA142 protein (MER021316), caspase-12 pseudogene (Homo sapiens) (MER019698), Memame-AA093 caspase pseudogene (MER014766), subfamily C14A non-peptidase homologues (MER185329), subfamily C14A non-peptidase homologues (MER179956), separase (Homo sapiens-type) (MER011775), separase-like pseudogene (MER014797), SENP1 peptidase (MER011012), SENP3 peptidase (MER011019), SENP6 peptidase (MER011109), SENP2 peptidase (MER012183), SENP5 peptidase (MER014032), SENP7 peptidase (MER014095), SENP8 peptidase (MER016161), SENP4 peptidase (MER005557), pyroglutamyl-peptidase I (chordate) (MER011032), Memame-AA073 peptidase (MER029978), Sonic hedgehog protein (MER002539), Indian hedgehog protein (MER002538), Desert hedgehog protein (MER012170), dipeptidyl-peptidase III (MER004252), Memame-AA164 protein (MER020410), LOC138971 g.p. (Homo sapiens) (MER020074), Atp23 peptidase (MER060642), prenyl peptidase 1 (MER004246), aminopeptidase N (MER000997), aminopeptidase A (MER001012), leukotriene A4 hydrolase (MER001013), pyroglutamyl-peptidase II (MER012221), cytosol alanyl aminopeptidase (MER002746), cystinyl aminopeptidase (MER002060), aminopeptidase B (MER001494), aminopeptidase PILS (MER005331), arginyl aminopeptidase-like 1 (MER012271), leukocyte-derived arginine aminopeptidase (MER002968), aminopeptidase Q (MER052595), aminopeptidase O (MER019730), Tata binding protein associated factor (MER026493), angiotensin-converting enzyme peptidase unit 1 (MER004967), angiotensin-converting enzyme peptidase unit 2 (MER001019), angiotensin-converting enzyme-2 (MER011061), Memame-AA153 protein (MER020514), thimet oligopeptidase (MER001737), neurolysin (MER010991), mitochondrial intermediate peptidase (MER003665), Memame-AA154 protein (MER021317), leishmanolysin-2 (MER014492), leishmanolysin-3 (MER180031), matrix metallopeptidase-1 (MER001063), matrix metallopeptidase-8 (MER001084), matrix metallopeptidase-2 (MER001080), matrix metallopeptidase-9 (MER001085), matrix metallopeptidase-3 (MER001068), matrix metallopeptidase-10 (Homo sapiens-type) (MER001072), matrix metallopeptidase-11 (MER001075), matrix metallopeptidase-7 (MER001092), matrix metallopeptidase-12 (MER001089), matrix metallopeptidase-13 (MER001411), membrane-type matrix metallopeptidase-1 (MER001077), membrane-type matrix metallopeptidase-2 (MER002383), membrane-type matrix metallopeptidase-3 (MER002384), membrane-type matrix metallopeptidase-4 (MER002595), matrix metallopeptidase-20 (MER003021), matrix metallopeptidase-19 (MER002076), matrix metallopeptidase-23B (MER004766), membrane-type matrix metallopeptidase-5 (MER005638), membrane-type matrix metallopeptidase-6 (MER012071), matrix metallopeptidase-21 (MER006101), matrix metallopeptidase-22 (MER014098), matrix metallopeptidase-26 (MER012072), matrix metallopeptidase-28 (MER013587), matrix metallopeptidase-23A (MER037217), macrophage elastase homologue (chromosome 8, Homo sapiens) (MER030035), Memame-AA156 protein (MER021309), matrix metallopeptidase-like 1 (MER045280), subfamily M10A non-peptidase homologues (MER175912), subfamily M10A non-peptidase homologues (MER187997), subfamily M10A non-peptidase homologues (MER187998), subfamily M10A non-peptidase homologues (MER180000), meprin alpha subunit (MER001111), meprin beta subunit (MER005213), procollagen C-peptidase (MER001113), mammalian tolloid-like 1 protein (MER005124), mammalian-type tolloid-like 2 protein (MER005866), ADAMTS9 peptidase (MER012092), ADAMTS14 peptidase (MER016700), ADAMTS15 peptidase (MER017029), ADAMTS16 peptidase (MER015689), ADAMTS17 peptidase (MER016302), ADAMTS18 peptidase (MER016090), ADAMTS19 peptidase (MER015663), ADAM8 peptidase (MER003902), ADAM9 peptidase (MER001140), ADAM10 peptidase (MER002382), ADAM12 peptidase (MER005107), ADAM19 peptidase (MER012241), ADAM15 peptidase (MER002386), ADAM17 peptidase (MER003094), ADAM20 peptidase (MER004725), ADAMDEC1 peptidase (MER000743), ADAMTS3 peptidase (MER005100), ADAMTS4 peptidase (MER005101), ADAMTS1 peptidase (MER005546), ADAM28 peptidase (Homo sapiens-type) (MER005495), ADAMTS5 peptidase (MER005548), ADAMTS8 peptidase (MER005545), ADAMTS6 peptidase (MER005893).
ADAMTS7 peptidase (MER005894), ADAM30 peptidase (MER006268), ADAM21 peptidase (Homo sapiens-type) (MER004726), ADAMTS10 peptidase (MER014331), ADAMTS12 peptidase (MER014337), ADAMTS13 peptidase (MER015450), ADAM33 peptidase (MER015143), ovastacin (MER029996), ADAMTS20 peptidase (Homo sapiens-type) (MER026906), procollagen I N-peptidase (MER004985), ADAM2 protein (MER003090), ADAM6 protein (MER047044), ADAM7 protein (MER005109), ADAM18 protein (MER012230), ADAM32 protein (MER026938), non-peptidase homologue (Homo sapiens chromosome 4) (MER029973), family M12 non-peptidase homologue (Homo sapiens chromosome 16) (MER047654), family M12 non-peptidase homologue (Homo sapiens chromosome 15) (MER047250), ADAM3B protein (Homo sapiens-type) (MER005199), ADAM11 protein (MER001146), ADAM22 protein (MER005102), ADAM23 protein (MER005103), ADAM29 protein (MER006267), protein similar to ADAM21 peptidase preproprotein (Homo sapiens) (MER026944), Memame-AA225 peptidase homologue (Homo sapiens) (MER047474), putative ADAM pseudogene (chromosome 4, Homo sapiens) (MER029975), ADAM3A g.p. (Homo sapiens) (MER005200), ADAM11 g.p. (Homo sapiens) (MER003912), subfamily M12B non-peptidase homologues (MER188210), subfamily M12B non-peptidase homologues (MER188211), subfamily M12B non-peptidase homologues (MER188212), subfamily M12B non-peptidase homologues (MER188220), neprilysin (MER001050), endothelin-converting enzyme 1 (MER001057), endothelin-converting enzyme 2 (MER004776), DINE peptidase (MER005197), neprilysin-2 (MER013406), Kell blood-group protein (MER154), PHEX peptidase (MER002062), i-AAA peptidase (MER001246), i-AAA peptidase (MER005755), araplegin (MER0004454), Afg3-like protein 2 (MER005496), Afg3-like protein 1A (MER014306), pappalysin-1 (MER002217), pappalysin-2 (MER014521), famesylated-protein converting enzyme 1 (MER002646), metalloprotease-related protein-1 (MER030873), aminopeptidase AMZ2 (MER011907), aminopeptidase AMZ1 (MER058242), carboxypeptidase A1 (MER001190), carboxypeptidase A2 (MER001608), carboxypeptidase B (MER001194), carboxypeptidase N (MER001198), carboxypeptidase E (MER001199), carboxypeptidase M (MER001205), carboxypeptidase U (MER001193), carboxypeptidase A3 (MER001187), metallocarboxypeptidase D peptidase unit 1 (MER003781), metallocarboxypeptidase Z (MER003428), metallocarboxypeptidase D peptidase unit 2 (MER004963), carboxypeptidase A4 (MER013421), carboxypeptidase A6 (MER013456), carboxypeptidase A5 (MER017121), metallocarboxypeptidase O (MER016044), cytosolic carboxypeptidase-like protein 5 (MER033174), cytosolic carboxypeptidase 3 (MER033176), cytosolic carboxypeptidase 6 (MER033178), cytosolic carboxypeptidase 1 (MER033179), cytosolic carboxypeptidase 2 (MER037713), metallocarboxypeptidase D non-peptidase unit (MER004964), adipocyte-enhancer binding protein 1 (MER003889), carboxypeptidase-like protein X1 (MER013404), carboxypeptidase-like protein X2 (MER078764), cytosolic carboxypeptidase (MER026952), family M14 non-peptidase homologues (MER199530), insulysin (MER001214), mitochondrial processing peptidase beta-subunit (MER004497), nardilysin (MER003883), eupitrilysin (MER004877), mitochondrial processing peptidase non-peptidase alpha subunit (MER001413), ubiquinol-cytochrome c reductase core protein I (MER003543), ubiquinol-cytochrome c reductase core protein II (MER003544), ubiquinol-cytochrome c reductase core protein domain 2 (MER043998), insulysin unit 2 (MER046821), nardilysin unit 2 (MER046874), insulysin unit 3 (MER078753), mitochondrial processing peptidase subunit alpha unit 2 (MER124489), nardilysin unit 3 (MER142856), LOC133083 g.p. (Homo sapiens) (MER021876), subfamily M16B non-peptidase homologues (MER188757), leucyl aminopeptidase (animal) (MER003100), Memame-AA040 peptidase (MER003919), leucyl aminopeptidase-1 (Caenorhabditis-type) (MER013416), methionyl aminopeptidase 1 (MER001342), methionyl aminopeptidase 2 (MER001728), aminopeptidase P2 (MER004498), Xaa-Pro dipeptidase (eukaryote) (MER001248), aminopeptidase P1 (MER004321), mitochondrial intermediate cleaving peptidase 55 kDa (MER013463), mitochondrial methionyl aminopeptidase (MER014055), Memame-AA020 peptidase homologue (MER010972), proliferation-association protein 1 (MER005497), chromatin-specific transcription elongation factor 140 kDa subunit (MER026495), proliferation-associated protein 1-like (Homo sapiens chromosome X) (MER029983), Mername-AA226 peptidase homologue (Homo sapiens) (MER056262), Memame-AA227 peptidase homologue (Homo sapiens) (MER047299), subfamily M24A non-peptidase homologues (MER179893), aspartyl aminopeptidase (MER003373), Gly-Xaa carboxypeptidase (MER033182), camosine dipeptidase II (MER014551), camosine dipeptidase I (MER015142), Memame-AA161 protein (MER021873), aminoacylase (MER001271), olutamate carboxypeptidase II (MER002104), NAALADASE L peptidase (MER005239), alutamate carboxypeptidase III (MER005238), plasma glutamate carboxypeptidase (MER005244), Memame-AA103 peptidase (MER015091), Fxna peptidase (MER029965), transferrin receptor protein (MER002105), transferrin receptor 2 protein (MER005152), alutaminyl cyclise (MER015095), glutamate carboxypeptidase II (Homo sapiens)-type non-peptidase homologue (MER026971), nicalin (MER044627), membrane dipeptidase (MER001260), membrane-bound dipeptidase-2 (MER013499), membrane-bound dipeptidase-3 (MER013496), dihydro-orotase (MER005767), dihydropyrimidinase (MER033266), dihydropyrimidinase related protein-1 (MER030143), dihydropyrimidinase related protein-2 (MER030155), dihydropyrimidinase related protein-3 (MER030151), dihydropyrimidinase related protein-4 (MER030149), dihydropyrimidinase related protein-(MER030136), hypothetical protein like 5730457F11RIK (MER033184), 1300019j08rik protein (MER033186)), guanine aminohydrolase (MER037714), Kae1 putative peptidase (MER001577), OSGEPL1-like protein (MER013498), S2P peptidase (MER004458), subfamily M23B non-peptidase homologues (MER199845), subfamily M23B non-peptidase homologues (MER199846), subfamily M23B non-peptidase homologues (MER199847), subfamily M23B non-peptidase homologues (MER137320), subfamily M23B non-peptidase homologues (MER201557), subfamily M23B non-peptidase homologues (MER199417), subfamily M23B non-peptidase homologues (MER199418), subfamily M23B non-peptidase homologues (MER199419), subfamily M23B non-peptidase homologues (MER199420), subfamily M23B non-peptidase homologues (MER175932), subfamily M23B non-peptidase homologues (MER199665), Poh1 peptidase (MER020382), Jab1/MPN domain metalloenzyme (MER022057), Memame-AA165 peptidase (MER021865), Brcc36 isopeptidase (MER021890), histone H2A deubiquitinase MYSM1 (MER021887), AMSH deubiquitinating peptidase (MER030146), putative peptidase (Homo sapiens chromosome 2) (MER029970), Memame-AA168 protein (MER021886), COP9 signalosome subunit 6 (MER030137), 26S proteasome non-ATPase regulatory subunit 7 (MER030134), eukaryotic translation initiation factor 3 subunit 5 (MER030133. IFP38 peptidase homologue (MER301032), subfamily M67A non-peptidase homologues (MER191181), subfamily M67A unassigned peptidases (MER191144), aranzyme B (Homo sapiens-type) (MER000168), testisin (MER005212), tryptase beta (MER000136), kallikrein-related peptidase 5 (MER005544), corin (MER005881), kallikrein-related peptidase 12 (MER006038), DESC1 peptidase (MER006298), tryptase gamma 1 (MER011036), kallikrein-related peptidase 14 (MER011038), hyaluronan-binding peptidase (MER003612), transmembrane peptidase, serine 4 (MER011104), intestinal serine peptidase (rodent) (MER016130), adrenal secretory serine peptidase (MER003734), tryptase delta 1 (Homo sapiens) (MER005948), matriptase-3 (MER029902), marapsin (MER006119), tryptase-6 (MER006118), ovochymase-1 domain 1 (MER099182), transmembrane peptidase, serine 3 (MER005926), kallikrein-related peptidase 15 (MBR000064), Memame-AA031 peptidase (MER014054), TMPRSS13 peptidase (MER014226), Memame-AA038 peptidase (MER062848), Mername-AA204 peptidase (MER029980), cationic trypsin (Homo sapiens-type) (MER000020), elastase-2 (MER000118), mannan-binding lectin-associated serine peptidase-3 (MER031968), cathepsin G (MER000082), myeloblastin (MER000170), granzyme A (MER001379), granzyme M (MER001541), chymase (Homo sapiens-type) (MER000123), tryptase alpha (MER000135), granzyme K (MER001936), granzyme H (MER000166), chymotrypsin B (MER000001), elastase-1 (MER003733), pancreatic endopeptidase E (MER000149), pancreatic elastase II (MER000146), enteropeptidase (MER002068), chymotrypsin C (MER000761), prostasin (MER002460), kallikrein 1 (MER000093), kallikrein-related peptidase 2 (MER000094), kallikrein-related peptidase 3 (MER000115), mesotrypsin (MER000022), complement component C1r-like peptidase (MER016352), complement factor D (MER000130), complement component activated C1r (MER000238), complement component activated Cis (MER000239), complement component C2a (MER000231), complement factor B (MER000229), mannan-binding lectin-associated serine peptidase 1 (MER000244), complement factor I (MER000228), pancreatic endopeptidase E form B (MER000150), pancreatic elastase IIB (MER000147), coagulation factor XIIa (MER000187), plasma kallikrein (MER000203) coagulation factor Xia (MER000210), coagulation factor IXa (MER000216), coagulation factor Vila (MER000215), coagulation factor Xa (MER000212), thrombin (MER000118), protein C (activated) (MER000222), acrosin (MER000078), hepsin (MER000156), hepatocyte growth factor activator (MER000186), mannan-binding lectin-associated serine peptidase 2 (MER002758), u-plasminogen activator (MER000195), t-plasminogen activator (MER000192), plasmin (MER000175), kallikrein-related peptidase 6 (MER002580), neurotrypsin (MER004171), kallikrein-related peptidase 8 (MER005400), kallikrein-related peptidase 10 (MER003645), epitheliasin (MER003736), kallikrein-related peptidase 4 (MER005266), prosemin (MER004214), chymopasin (MER001503), kallikrein-related peptidase 11 (MER004861), kallikrein-related peptidase 11 (MER216142), trypsin-2 type A (MER000021), HtrA1 peptidase (Homo sapiens-type) (MER002577), HtrA2 peptidase (MER208413), HtrA2 peptidase (MER004093), HtrA3 peptidase (MER014795), HtrA4 peptidase (MER016351), Tysnd1 peptidase (MER050461), TMPRSS12 peptidase (MER017085), HAT-like putative peptidase 2 (MER021884), trypsin C (MER021898), kallikrein-related peptidase 7 (MER002001), matriptase (MER003735), kallikrein-related peptidase 13 (MER005269), kallikrein-related peptidase 9 (MER005270), matriptase-2 (MER005278), umbelical vein peptidase (MER005421), LCLP peptidase (MER001900), spinesin (MER014385), marapsin-2 (MER021929), complement factor D-like putative peptidase (MER056164), ovochymase-2 (MER022410), HAT-like 4 peptidase (MER044589), ovochymase 1 domain 1 (MER022412), epidermis-specific SP-like putative peptidase (MER029900), testis serine peptidase 5 (MER029901), Memame-AA258 peptidase (MER000285), polyserase-IA unit 1 (MER030879), Dolyserase-IA unit 2 (MER030880), testis serine peptidase 2 (human-type) (MER033187), hypothetical acrosin-like peptidase (Homo sapiens) (MER033253), HAT-like 5 peptidase (MER028215), polyserase-3 unit 1 (MER061763), polyserase-3 unit 2 (MER061748), peptidase similar to tryptophan/serine protease (MER056263), polyserase-2 unit 1 (MER061777), Memame-AA123 peptidase (MER021930), HAT-like 2 peptidase (MER099184), hCG2041452-like protein (MER099172), hCG22067 (Homo sapiens) (MER099169), brain-rescue-factor-1 (Homo sapiens) (MER098873), hCG2041108 (Homo sapiens) (MER099173), polyserase-2 unit 2 (MER061760), polyserase-2 unit 3 (MER065694), Memame-AA201 (peptidase homologue) MER09175, secreted trypsin-like serine peptidase homologue (MER030000), polyserase-1A unit 3 (MER029880), azurocidin (MER000119), haptoglobin-1 (MER000233), haptoglobin-related protein (MER000235), macrophage-stimulating protein (MER001546), hepatocyte growth factor (MER000185), protein Z (MER000227), TESP1 protein (MER047214), LOC136242 protein (MER016132), plasma kallikrein-like protein 4 (MER016346), PRSS35 protein (MER016350), DKFZp586H2123-like protein (MER066474), apolipoprotein (MER000183), osi-KLK1 pseudogene (Homo sapiens) (MER033287), tryptase pseudogene I (MER015077), tryptase pseudogene II (MER015078), tryptase pseudogene III (MER015079), subfamily S1A unassigned peptidases (MER216982), subfamily S1A unassigned peptidases (MER216148), amidophosphoribosyltransferase precursor (MER003314), alutamine-fructose-6-phosphate transaminase 1 (MER003322), glutamine:fructose-6-phosphate amidotransferase (MER012158), Memame-AA144 protein (MER021319), asparagine synthetase (MER033254), family C44 non-peptidase homologues (MER159286), family C44 unassigned peptidases (MER185625) family C44 unassigned peptidases (MER185626), secemin 1 (MER045376), secemin 2 (MER064573), secemin 3 (MER064582), acid ceramidase precursor (MER100794), N-acylethanolamine acid amidase precursor (MER141667), oroteasome catalytic subunit 1 (MER000556), proteasome catalytic subunit 2 (MER002625), proteasome catalytic subunit 3 (MER002149), proteasome catalytic subunit ii (MER000552), proteasome catalytic subunit 2i (MER001515), proteasome catalytic subunit 3i (MER000555), proteasome catalytic subunit 5t (MER026203), protein serine kinase c17 (MER026497), proteasome subunit alpha 6 (MER000557), oroteasome subunit alpha 2 (MER000550), oroteasome subunit alpha 4 (MER000554), proteasome subunit alpha 7 (MER033250), proteasome subunit alpha 5 (MER000558), proteasome subunit alpha 1 (MER000549), proteasome subunit alpha 3 (MER000553), proteasome subunit XAPC7 (MER004372), proteasome subunit beta 3 (MER001710), proteasome subunit beta 2 (MER002676), proteasome subunit beta 1 (MER000551), proteasome subunit beta 4 (MER001711), Mername-AA230 peptidase homologue (Homo sapiens) (MER047329), Memame-AA231 pseudogene (Homo sapiens) (MER047172), Memame-AA232 pseudogene (Homo sapiens) (MER047316), glycosylasparaginase precursor (MER003299), isoaspartyl dipeptidase (threonine type) (MER031622), taspase-1 (MER016969), gamma-glutamyltransferase 5 (mammalian-type) (MER001977), gamma-glutamyltransferase 1 (mammalian-type) (MER001629), aamma-glutamyltransferase 2 (Homo sapiens) (MER001976), aamma-glutamyltransferase-like protein 4 (MER002721), aamma-glutamyltransferase-like protein 3 (MER016970), similar to gamma-glutamyltransferase 1 precursor (Homo sapiens) (MER026204), similar to gamma-glutamyltransferase 1 precursor (Homo sapiens) (MER026205), Mername-AA211 putative peptidase (MER026207), gamma-glutamyltransferase 6 (MER159283), gamma-glutamyl transpeptidase homologue (chromosome 2, Homo sapiens) (MER037241), polycystin-1 (MER126824), KIAA1879 protein (MER159329), polycystic kidney disease 1-like 3 (MER172554), aamma-glutamyl hydrolase (MER002963), guanine 5′′-monophosphate synthetase (MER043387), carbamoyl-phosphate synthase (Homo sapiens-type) (MER078640), dihydro-orotase (N-terminal unit) (Homo sapiens-type) (MER060647), DJ-1 putative peptidase (MER003390), Mername-AA100 putative peptidase (MER014802), Mername-AA101 non-peptidase homologue (MER014803), KIAA0361 protein (Homo sapiens-type) (MER042827), FLJ34283 protein (Homo sapiens) (MER044553), non-peptidase homologue chromosome 21 open reading frame 33 (Homo sapiens) (MER160094), family C56 non-peptidase homologues (MER177016), family C56 non-peptidase homologues (MER176613), family C56 non-peptidase homologues (MER176918), EGF-like module containing mucin-like hormone receptor-like 2 (MER037230), CD97 antigen (human type) (MER037286), EGF-like module containing mucin-like hormone receptor-like 3 (MER037288), EGF-like module containing mucin-like hormone receptor-like 1 (MER037278), EGF-like module containing mucin-like hormone receptor-like 4 (MER037294), cadherin EGF LAG seven-pass G-type receptor 2 precursor (Homo sapiens) (MER045397), Gpr64 (Mus musculus)-type protein (MER123205), GPR56 (Homo sapiens)-type protein (MER122057), latrophilin 2 (MER122199), latrophilin-1 (MER126380), latrophilin 3 (MER124612), protocadherin Flamingo 2 (MER1242391. ETL protein (MER126267), G protein-coupled receptor 112 (MER126114), seven transmembrane helix receptor (MER125448), Gpr114 protein (MER159320), GPR126 vascular inducible G protein-coupled receptor (MER140015), GPR125 (Homo sapiens)-type protein (MER159279), GPR116 (Homo sapiens)-type G-protein coupled receptor (MER159280), GPR128 (Homo sapiens)-type G-protein coupled receptor (MER162015), GPR133 (Homo sapiens)-type protein (MER159334), GPR110 G-protein coupled receptor (MER159277), GPR97 protein (MER159322), KPG_006 protein (MER161773), KPG_008 protein (MER161835), KPG_009 protein (MER159335), unassigned homologue (MER166269), GPR113 protein (MER159352), brain-specific angiogenesis inhibitor 2 (MER159746), PIDD auto-processing protein unit 1 (MER020001), PIDD auto-processing protein unit 2 (MER063690), MUC1 self-cleaving mucin (MER074260), dystroglycan (MER054741), proprotein convertase 9 (MER022416), site-1 peptidase (MER001948), furin (MER000375), proprotein convertase 1 (MER000376), proprotein convertase 2 (MER000377), proprotein convertase 4 (MER028255), PACE4 proprotein convertase (MER000383), proprotein convertase 5 (MER002578), proprotein convertase 7 (MER002984), tripeptidyl-peptidase II (MER000355), subfamily S8A non-peptidase homologues (MER201339), subfamily S8A non-peptidase homologues (MER191613), subfamily S8A unassigned peptidases (MER191611), subfamily S8A unassigned peptidases (MER191612), subfamily SBA unassigned peptidases (MER191614), tripeptidyl-peptidase I (MER003575), prolyl oligopeptidase (MER000393), dipeptidyl-peptidase IV (eukaryote) (MER000401), acylaminoacyl-peptidase (MER000408), fibroblast activation protein alpha subunit (MER000399), PREPL A protein (MER004227), dipeptidyl-peptidase 8 (MER013484), dipeptidyl-peptidase 9 (MER004923), FLJ1 putative peptidase (MER017240), Memame-AA194 putative peptidase (MER017353), Memame-AA195 putative peptidase (MER017367), Memame-AA196 putative peptidase (MER017368), Mername-AA197 putative peptidase (MER017371), C14orf29 protein (MER033244), hypothetical protein (MER033245), hypothetical esterase/lipase/thioesterase (MER047309), protein bat5 (MER037840), hypothetical protein flj40219 (MER033212), hypothetical protein flj37464 (MER033240), hypothetical protein flj33678 (MER033241), dipeptidylpeptidase homologue DPP6 (MER000403), dipeptidylpeptidase homologue DPP10 (MER005988), protein similar to Mus musculus chromosome 20 open reading frame 135 (MER037845), kynurenine formamidase (MER046020), thyroglobulin precursor (MER011604), acetylcholinesterase (MER033188), cholinesterase (MER033198), carboxylesterase D1 (MER033213), liver carboxylesterase (MER033220) carboxylesterase 3 (MER033224), carboxylesterase 2 (MER033226), bile salt-dependent lipase (MER033227), carboxylesterase-related protein (MER033231), neuroligin 3 (MER033232), neuroligin 4, X-linked (MER033235), neuroligin 4, Y-linked (MER033236), esterase D (MER043126), arylacetamide deacetylase (MER033237), KIAA1363-Iike protein (MER033242), hormone-sensitive lipase (MER033274), neuroligin 1 (MER033280), neuroligin 2 (MER033283), family S9 non-peptidase homologues (MER212939), family S9 non-peptidase homologues (MER211490), subfamily S9C unassigned peptidases (MER192341), family S9 unassigned peptidases (MER20911) family S9 unassigned peptidases (MER200434), family S9 unassigned peptidases (MER209507), family S9 unassigned peptidases (MER209142), serine carboxypeptidase A (MER000430), vitellogenic carboxypeptidase-like protein (MER005492), RISC peptidase (MER010960), family S15 unassigned peptidases (MER199442), family S15 unassigned peptidases (MER200437), family S15 unassigned peptidases (MER212825), lysosomal Pro-Xaa carboxypeptidase (MER000446), dipeptidyl-peptidase II (MER004952), thymus-specific serine peptidase (MER005538), epoxide hydrolase-like putative peptidase (MER031614), Loc328574-like protein (MER033246), abhydrolase domain-containing protein 4 (MER031616), epoxide hydrolase (MER000432), mesoderm specific transcript protein (MER199890), mesoderm specific transcript protein (MER017123), cytosolic epoxide hydrolase (MER029997), cytosolic epoxide hydrolase (MER213866), similar to hypothetical protein FLJ22408 (MER031608), CGI-58 putative peptidase (MER030163), Williams-Beuren syndrome critical region protein 21 epoxide hydrolase (MER031610), epoxide hydrolase (MER031612), hypothetical protein flj22408 (epoxide hydrolase) (MER031617), monoglyceride lipase (MER033247), hypothetical protein (MER033249), valacyclovir hydrolase (MER033259), Ccg1-interacting factor b (MER210738), glycosylasparaginase precursor (MER003299), isoaspartyl dipeptidase (threonine type) (MER031622), taspase-1 (MER016969), aamma-glutamyltransferase 5 (mammalian-type) (MER001977), gamma-glutamyltransferase 1 (mammalian-type) (MER001629), aamma-glutamyltransferase 2 (Homo sapiens) (MER001976), gamma-glutamyltransferase-like protein 4 (MER002721), aamma-glutamyltransferase-like protein 3 (MER016970), similar to gamma-glutamytransferase 1 precursor (Homo sapiens) (MER026204), similar to gamma-glutamyltransferase 1 precursor (Homo sapiens) (MER026205), Memame-AA211 putative peptidase (MER026207), gamma-glutamyltransferase 6 (MER159283), aamma-glutamyl transpeptidase homologue (chromosome 2. Homo sapiens) (MER037241), polycystin-1 (MER126824), KIAA1879 protein (MER159329), polycystic kidney disease 1-like 3 (MER172554), gamma-glutamyl hydrolase (MER002963), guanine 5′′-monophosphate synthetase (MER043387), carbamoyl-phosphate synthase (Homo sapiens-type) (MER078640), dihydro-orotase (N-terminal unit) (Homo sapiens-type) (MER060647), DJ-1 putative peptidase (MER003390), Memame-AA100 putative peptidase (MER014802), Memame-AA101 non-peptidase homologue (MER014803), KIAA0361 protein (Homo sapiens-type) (MER042827), FLJ34283 protein (Homo sapiens) (MER044553), non-peptidase homologue chromosome 21 open reading frame 33 (Homo sapiens) (MER160094), family C56 non-peptidase homologues (MER177016), family C56 non-peptidase homologues (MER176613), family C56 non-peptidase homologues (MER176918), EGF-like module containing mucin-like hormone receptor-like 2 (MER037230), CD97 antigen (human type) (MER037286), EGF-like module containing mucin-like hormone receptor-like 3 (MER037288), EGF-like module containing mucin-like hormone receptor-like 1 (MER037278), EGF-like module containing mucin-like hormone receptor-like 4 (MER037294), cadherin EGF LAG seven-pass G-type receptor 2 precursor (Homo sapiens) (MER045397), Gpr64 (Mus musculus)-type protein (MER123205), GPR56 (Homo sapiens)-type protein (MER122057), latrophilin 2 (MER122199), latrophilin-1 (MER126380), latrophilin 3 (MER124612), protocadherin Flamingo 2 (MER124239), ETL protein (MER126267), G protein-coupled receptor 112 (MER126114), seven transmembrane helix receptor (MER125448), Gpr114 protein (MER159320), GPR126 vascular inducible G protein-coupled receptor (MER140015), GPR125 (Homo sapiens)-type protein (MER159279), GPR116 (Homo sapiens)-type G-protein coupled receptor (MER159280), GPR128 (Homo sapiens)-type G-protein coupled receptor (MER162015), GPR133 (Homo sapiens)-type protein (MER159334) GPR110 G-protein coupled receptor (MER159277), GPR97 protein (MER159322), KPG_006 protein (MER161773) KPG_008 protein (MER161835), KPG_009 protein (MER159335), unassigned homologue (MER166269), GPR113 protein (MER159352), brain-specific angiogenesis inhibitor 2 (MER159746), PIDD auto-processing protein unit 1 (MER020001). PIDD auto-processing protein unit 2 (MER063690), MUC1 self-cleaving mucin (MER74260), dystroglycan (MER054741), proprotein convertase 9 (MER022416), site-1 peptidase (MER001948), furin (MER000375), proprotein convertase 1 (MER000376), proprotein convertase 2 (MER000377), proprotein convertase 4 (MER028255), PACE4 proprotein convertase (MER000383), proprotein convertase 5 (MER002578), proprotein convertase 7 (MER002984), tripeptidyl-peptidase II (MER000355), subfamily S8A non-peptidase homologues (MER201339), subfamily S8A non-peptidase homologues (MER191613), subfamily S8A unassigned peptidases (MER191611), subfamily S8A unassigned peptidases (MER191612), subfamily S8A unassigned peptidases (MER191614), tripeptidyl-peptidase I (MER003575), prolyl oligopeptidase (MER000393), dipeptidyl-peptidase IV (eukaryote) (MER000401), acylaminoacyl-peptidase (MER000408), fibroblast activation protein alpha subunit (MER000399), PREPL A protein (MER004227), dipeptidyl-peptidase 8 (MER013484), dipeptidyl-peptidase 9 (MER004923), FLJ1 putative peptidase (MER017240), Mername-AA194 putative peptidase (MER017353), Memame-AA195 putative peptidase (MER0173671. Memame-AA196 putative peptidase (MER017368), Memame-AA197 putative peptidase (MER017371), C14orf29 protein (MER033244), hypothetical protein (MER033245), hypothetical esterase/lipase/thioesterase (MER047309), protein bat5 (MER037840), hypothetical protein flj40219 (MER033212), hypothetical protein flj37464 (MER033240), hypothetical protein flj33678 (MER033241), dipeptidylpeptidase homologue DPP6 (MER000403), dipeptidylpeptidase homologue DPP10 (MER005988), protein similar to Mus musculus chromosome 20 open reading frame 135 (MER037845), kynurenine formamidase (MER046020), thyroglobulin precursor (MER011604), acetylcholinesterase (MER033188), cholinesterase (MER033198), carboxylesterase D1 (MER033213), liver carboxylesterase (MER033220), carboxylesterase 3 (MER033224), carboxylesterase 2 (MER033226), bile salt-dependent lipase (MER033227), carboxylesterase-related protein (MER033231), neuroligin 3 (MER033232), neuroligin 4, X-linked (MER033235), neuroligin 4, Y-linked (MER033236), esterase D (MER043126), arylacetamide deacetylase (MER033237), KIAA1363-like protein (MER033242), hormone-sensitive lipase (MER033274), neuroligin 1 (MER033280), neuroligin 2 (MER033283), family S9 non-peptidase homologues (MER212939), family S9 non-peptidase homologues (MER211490), subfamily S9C unassigned peptidases (MER192341), family S9 unassigned peptidases (MER209181), family S9 unassigned peptidases (MER200434), family S9 unassigned peptidases (MER209507), family S9 unassigned peptidases (MER209142), serine carboxypeptidase A (MER000430), vitellogenic carboxypeptidase-like protein (MER005492), RISC peptidase (MER010960), family S15 unassigned peptidases (MER199442), family S15 unassigned peptidases (MER200437), family S15 unassigned peptidases (MER212825), lysosomal Pro-Xaa carboxypeptidase (MER000446), dipeptidyl-peptidase II (MER004952), thymus-specific serine peptidase (MER005538), epoxide hydrolase-like putative peptidase (MER031614), Loc328574-like protein (MER033246), abhydrolase domain-containing protein 4 (MER031616), epoxide hydrolase (MER000432), mesoderm specific transcript protein (MER199890), mesoderm specific transcript protein (MER017123), cytosolic epoxide hydrolase (MER029997), cytosolic epoxide hydrolase (MER213866), similar to hypothetical protein FLJ22408 (MER031608), CGI-58 putative peptidase (MER030163), Williams-Beuren syndrome critical region protein 21 epoxide hydrolase (MER031610), epoxide hydrolase (MER031612), hypothetical protein flj22408 (epoxide hydrolase) (MER031617), monoglyceride lipase (MER033247), hypothetical protein (MER033249), valacyclovir hydrolase (MER033259), Ccg1-interacting factor b (MER210738).
It will be appreciated that for a given unwanted cell type, the skilled person can readily determine an appropriate one or more protease cleavage sites to use, for example by consulting scientific literature to determine which proteases are overexpressed by that cell type. Oncomine (https://www.oncomine.org) is an online cancer gene expression database, and so when the molecule of the invention is for treating cancer, the skilled person may search the Oncomine database to identify a particular one or more protease cleavage sites that will be appropriate for treating a given cancer type. Alternative databases include European Bioinformatic Institute (http://www.ebi.ac.uk) in particular (http://www.ebi.ac.uk/gxa). Protease databases include PMAP (http://www.proteolysis.org), ExPASy Peptide Cutter (http://ca.expasy.org/tools/peptide cutter) and PMAP.Cut DB (http://cutdb.bumham.org).
It is noted that it may be desirable to screen a library of peptides incorporating multiple potential cleavage sites and evaluating the optimal cleavage site for a given unwanted cell (eg tumour). Such peptides may be useful as linkers, for example, to join the further moiety to one or more masking moieties, or to promote a particular conformation of the further moiety so as to mask T cell binding region of the further moiety (see embodiment exemplified by FIG. 1).







TABLE 4





Matrix showing preferred protease cleavage sites for treating particular tumours











































Mel-

















Endo-
Cer-
Blad-

ano-
Lung- 
Lung- 
Pros-



Oesoph-
Gas-
Pancre-



Protease

Substrate
Breast
Ovarian
metrial
vical
der
Renal
ma
NSLC
SLC
tate
Testicular
Thyroid
Brain
ageal
tric
atic
Colorectal





Serine





















urokinase-type
uPA
CPGR-VVGG
•
•
•
•
•

•
•

•



•
•
•
•


plasminogen

(SEQ ID No: 1)



















activator






















tPA
CPGR-VVGG
•




















(SEQ ID No: 1)




















Cathepsin A







•













Cathepsin G





















Plasmin
GGR-X 





















(SEQ ID No: 2)




















C1s
YLGR-SYKV





















(SEQ ID No: 3) 





















or MQLGR-X





















(SEQ ID No: 4)




















MASP2
SLGR-KIQI





















(SEQ ID No: 5)




















Thrombin
LVPRGS 





















(SEQ ID No: 6)




















Trypsin
XXXR-X 





















(SEQ ID No: 7)




















Chymotrypsin





















Elastase 1




















Leucocyte/Neut 
Elastase 2
AAPV-X 



















Elastase

(SEQ ID No: 8)



















Leucocyte/Neut 
Elastase 2
AAPV-X 



















Elastase

(SEQ ID No: 8)



















MT-SP1/ST14
Matriptase
KQLR-VVNG
•
•







•






•




(SEQ ID No: 9)





















or KQSR-





















(SEQ ID No: 10 )





















KFVP





















(SEQ ID No: 11)



















MT-SP2
Matriptase 2

•


















TMPRSS1
Hepsin


•



•



•










TMPRSS2
GGR-X 









•











(SEQ ID No: 2)




















TMPRSS3





















TMPRSS4















•
•
•


PSA
Prostate 
SSKYQ 









•










Specific
(SEQ ID No: 12) 




















Antigen
or HSSKLQL





















(SEQ ID No: 13)



















Leucocyte/Neut 
Elastase 2
AAPV-X 



















Elastase

(SEQ ID No: 8)



















MT-SP1/ST14
Matriptase
KQLR-VVNG
•
•







•






•




(SEQ ID No: 9)



















MT-SP2
Matriptase 2

•


















TMPRSS1
Hespin


•



•



•










TMPRSS2
GGR-X 









•











(SEQ ID No: 2)




















TMPRSS3





















TMPRSS4















•
•
•


PSA
Prostate 
SSKYQ 









•










Specific
(SEQ ID No: 12) 




















Antigen
or HSSKLQL





















(SEQ ID No: 13)



















Cysteine






















Cathepsin B
GGGG-F 

•





•
•



•








(SEQ ID No: 14)




















Cathepsin L





















Cathepsin F





















Cathepsin H










•










Cathepsin K

•





•


•










Cathepsin L1





















Cathepsin L2





















Cathepsin O

•



















Cathepsin W





















Cathepsin S













•







Cathepsin Z 





















(or X)




















Aspartic






















Cathepsin D







•




•
•







Cathepsin E




















Metallo





















Collagenase 1
MMP1
PLG-LLG
•






•
•
•



•
•
•
•




(SEQ ID No: 15)



















Gelatinase A
MMP2
PQG-IAGQ
•
•

•
•




•




•






(SEQ ID No :16) 





















or PVGLIG





















(SEQ ID No: 17)



















Stromelysin
MMP3

•



















MMP4





















MMP5





















MMP6




















Matrilysin
MMP7















•

•


Collagenase 2
MMP8




















Gelatinase B
MMP9
(PQG-IAGQ




•

•


•











(SEQ ID No: 16) 





















or PRA-LY





















(SEQ ID No: 18)




















MMP10





















MMP11

•



















MMP12

•



















MMP13





















MMP14
PRH-LR

•

•
•


•
•
•

•
•

•
•
•




(SEQ ID No: 19)




















MMP15

•






•

•










MMP16





















MMP17

•



















MMP18





















MMP19





















MMP20





















MMP21





















MMP23A





















MMP23B

















•



MMP24













•







MMP25





















MMP26



•






•










MMP27





















MMP28















•
•
•














•










ADAM2





















ADAM7





















ADAM8


•



•

•
•



•


•




ADAM9








•
•
•




•
•




ADAM10


•
•






•



•


•


Metallo






















ADAM11





















ADAM12

•



•







•

•

•



ADAM15

•






•
•
•




•





ADAM17

•
•



•

•
•
•




•
•
•



ADAM18/27





















ADAM19






•






•







ADAM20





















ADAM21/31





















ADAM22





















ADAM23





















ADAM28

•




•














ADAM29





















ADAM30





















ADAM33





















ADAMTS1





















ADAMTS2





















ADAMTS3





















ADAMTS4





















ADAMTS5/11





















ADAMTS6





















ADAMTS7





















ADAMTS8

















•



ADAMTS9





















ADAMTS10





















ADAMTS12





















ADAMTS13





















ADAMTS14





















ADAMTS15

•



















ADAMTS16





















ADAMTS17





















ADAMTS18





















ADAMTS19





















ADAMTS20



























Leu-














kae-
My-





Model 



Protease

Substrate
Liver
mia
eloma
NHL
Hodgkin's
AML
ALL
CLL
Cell line






Serine














urokinase-type
uPA
CPGR-VVGG


•
•
•
•
•
•




plasminogen

(SEQ ID No: 1)












activator















tPA
CPGR-VVGG














(SEQ ID No: 1)













Cathepsin A














Cathepsin G














Plasmin
GGR-X 














(SEQ ID No: 2)













C1s
YLGR-SYKV














(SEQ ID No: 3) 














or MQLGR-X














(SEQ ID No: 4)













MASP2
SLGR-KIQI














(SEQ ID No: 5)













Thrombin
LVPRGS 














(SEQ ID No: 6)













Trypsin
XXXR-X 














(SEQ ID No: 7)













Chymotrypsin














Elastase 1













Leucocyte/Neut 
Elastase 2
AAPV-X 












Elastase

(SEQ ID No: 8)












Leucocyte/Neut 
Elastase 2
AAPV-X 












Elastase

(SEQ ID No: 8)












MT-SP1/ST14
Matriptase
KQLR-VVNG














(SEQ ID No: 9)














or KQSR-














(SEQ ID No: 10 )














KFVP














(SEQ ID No: 11)












MT-SP2
Matriptase 2













TMPRSS1
Hepsin














TMPRSS2
GGR-X 














(SEQ ID No: 2)













TMPRSS3














TMPRSS4













PSA
Prostate 
SSKYQ 













Specific
(SEQ ID No: 12) 













Antigen
or HSSKLQL














(SEQ ID No: 13)












Leucocyte/Neut 
Elastase 2
AAPV-X 












Elastase

(SEQ ID No: 8)












MT-SP1/ST14
Matriptase
KQLR-VVNG














(SEQ ID No: 9)












MT-SP2
Matriptase 2













TMPRSS1
Hespin














TMPRSS2
GGR-X 














(SEQ ID No: 2)













TMPRSS3














TMPRSS4













PSA
Prostate 
SSKYQ 













Specific
(SEQ ID No: 12) 













Antigen
or HSSKLQL














(SEQ ID No: 13)












Cysteine















Cathepsin B
GGGG-F 














(SEQ ID No: 14)













Cathepsin L














Cathepsin F














Cathepsin H














Cathepsin K














Cathepsin L1














Cathepsin L2














Cathepsin O














Cathepsin W














Cathepsin S














Cathepsin Z 














(or X)













Aspartic















Cathepsin D

•












Cathepsin E













Metallo














Collagenase 1
MMP1
PLG-LLG


•





PMA Activated





(SEQ ID No: 15)








U937 and MCF7 cells, 














MDA-MB231



Gelatinase A
MMP2
PQG-IAGQ

•



•
•

Colo205, HT29





(SEQ ID No :16) 














or PVGLIG














(SEQ ID No: 17)












Stromelysin
MMP3














MMP4














MMP5














MMP6













Matrilysin
MMP7


•










Collagenase 2
MMP8













Gelatinase B
MMP9
(PQG-IAGQ


•





MCF7, PC3





(SEQ ID No: 16) 














or PRA-LY














(SEQ ID No: 18)













MMP10














MMP11














MMP12














MMP13














MMP14
PRH-LR
•













(SEQ ID No: 19)













MMP15














MMP16














MMP17














MMP18














MMP19









THP-1, HL-60




MMP20














MMP21














MMP23A














MMP23B














MMP24














MMP25














MMP26














MMP27














MMP28














ADAM2














ADAM7














ADAM8














ADAM9

•












ADAM10


•










Metallo















ADAM11














ADAM12

•












ADAM15














ADAM17

•







LNCap, 














MDA-MB231,














MCF7 express














ADAM17.














Colo205 express














barely any




ADAM18/27














ADAM19














ADAM20














ADAM21/31














ADAM22














ADAM23














ADAM28














ADAM29














ADAM30














ADAM33














ADAMTS1














ADAMTS2














ADAMTS3














ADAMTS4














ADAMTS5/11














ADAMTS6














ADAMTS7














ADAMTS8














ADAMTS9














ADAMTS10














ADAMTS12














ADAMTS13














ADAMTS14














ADAMTS15














ADAMTS16














ADAMTS17














ADAMTS18














ADAMTS19














ADAMTS20
















TABLE 5







Tumour sites in which ADAM overexpression has been reported










Protein
Tumour expression







ADAM8
Brain, kidney, lung, pancreas



ADAM9
Breast gastric, liver, lung, pancreas,




prostate



ADAM10
Colon, gastric, leukaemia, prostate, uterus,




ovary



ADAM12
Bladder, brain, breast, colon, gastric, liver



ADAM15
Breast, gastric, lung, prostate



ADAM17
Brain, breast, colon, gastric, kidney, liver,




lung, ovary, pancreas, prostate



ADAM19
Brain, kidney



ADAM28
Breast, kidney, lung







A number of the proteolytic ADAMS (a disintegrin and metalloproteinase) have been detected in cancers and mRNA or protein levels have been found to be upregulated relative to normal tissue (adapted from Nature Reviews Cancer 8, 932-941 (December 2008 | doi:10.1038/nrc2459).



In one emobodiement, the protease may be an esterase.






Other cleavage sites include linkages which are labile under certain conditions in the vicinity of unwanted cells (eg tumour microenvironment). For example, the one or more cleavage sites may comprise disulphide bonds, which can be reduced in the hypoxic tumour microenvironment, or may comprise pH sensitive moieties that break in acidic conditions. It will be understood, however, that the one or more cleavage sites must be selectively cleavable in the vicinity of the unwanted cells and so such linkages must be more labile and preferably only labile in the vicinity of unwanted cells compared to in the vicinity of wanted cells.
Alternatively, the one or more cleavage sites may comprise nucleic acid (eg DNA or RNA) that is selectively cleavable in the vicinity of unwanted cells (eg by nucleases). Other cleavage sites include phosphate, lipid or disulphide containing moieties that may be cleavable by appropriate enzymes.
Synthesis of Molecule of Invention
Conveniently, the targeting moiety is joined to the further moiety by one or more linkers. Thus, by ‘linker’ we include the meaning of a chemical moiety that attaches the targeting moiety to the further moiety. Preferably, the linker is a peptide linker. It will be appreciated that such linkers may contain one or more cleavage sites that are selectively cleaved in the vicinity of the unwanted cells so as to unmask the immune cell binding region of the further moiety.
As described above, it is possible for the further moiety to have two or more separate parts. For example, the further moiety may comprise two or more polypeptide domains including immunoglobulin domains of an antibody (e.g. scFv antibody). Such parts may be linked together by one or more linkers. Thus, by ‘linker’, we also include the meaning of any chemical moiety that attaches the separate parts of a further moiety to each other. Preferably, the linker is a peptide linker. For example, the further moiety of the molecule illustrated in FIG. 1 comprises a VH domain and a VL domain which domains are joined by a linker. These linkers may contain one or more cleavage sites that are selectively cleaved in the vicinity of the unwanted cells so as to unmask the T cell binding region of the further moiety
Also conveniently, when the molecule comprises one or more masking moieties, these are attached to the targeting moiety and/or further moiety by means of one or more linkers. Thus, by ‘linker’ we also include the meaning of a chemical moiety that attaches the targeting moiety to a masking moiety and a chemical moiety that attaches the further moiety to a masking moiety. Preferably, the linker is a peptide linker. For example, FIGS. 2 and 3 depict examples of linkers between a further moiety and a masking moiety, and FIG. 4 depicts examples of linkers between a further moiety and a masking moiety, and between a targeting moiety and a masking moiety. Such linkers may also contain one or more cleavage sites that are selectively cleaved in the vicinity of the unwanted cells so as to unmask the T cell binding region of the further moiety
Examples of suitable linkers include peptides, polymers, nucleotides, nucleic acids, polysaccharides and lipid organic species (eg polyethylene glycol). Most preferably, however, the linkers described herein are peptide linkers. Generally, the peptide linkers have between 2 and 100 amino acid residues, such as more than or less than 10, 20, 30, 40, 50, 60, 70, 80 and 90 amino acids. Further detail of the preferred length of particular linkers is given below.
It is appreciated that the targeting moiety may either be bound covalently or non-covalently to the further moiety. Likewise, it is appreciated that the targeting moiety and/or further moiety may either be bound covalently or non-covalently to the one or more masking moieties.
Typically, the targeting moiety is covalently bound to the further moiety, and when the molecule comprises one or more masking moieties, the one or more masking moieties are covalently bound to the further moiety and/or targeting moiety.
In one embodiment, the targeting moiety and further moiety are covalently attached by a linker (eg peptide linker). When the molecule comprises one or more masking moieties, the one or more masking moieties may be covalently attached to the further moiety and/or targeting moiety by one or more linkers (eg peptide linkers).
It is appreciated that the targeting moiety, further moiety (including separate parts thereof) and one or more masking moieties do not need to be linked directly to each other, but may be attached via one or more spacer moieties (eg peptides). For example, the targeting moiety may be linked to a chemical moiety (eg peptide) which in turn is linked to the further moiety. Similarly, the further moiety may be linked to one or more chemical moieties (eg peptides) which in turn are linked to one or more respective masking moieties. In one embodiment, such spacer moieties may comprise a cleavage site that is cleavable selectively in the vicinity of the unwanted cells, as discussed above. It will be appreciated that the spacer moiety may serve to prevent steric hindrance and facilitate protease cleavage.
In view of the above, it is appreciated that the invention provides a molecule comprising: (i) a targeting moiety capable of directly or indirectly targeting to unwanted cells, (ii) a further moiety that has a masked immune cell binding region so as to prevent binding of the further moiety to an immune cell, (iii) one or more masking moieties attached to the further moiety and/or targeting moiety which one or more masking moieties act to mask the immune cell binding region of the further moiety, and (iv) one or more cleavable sites between the one or more masking moieties, and the further moiety and/or the targeting moiety, wherein the one or more cleavage sites can be selectively cleaved in the vicinity of the unwanted cells so as to unmask the immune cell binding region.
In a preferred embodiment where the targeting moiety, further moiety and, if present, the one or more masking moieties are covalently attached and where all moieties are peptides or polypeptides, it is appreciated that the component moieties of the molecule may be part of one or more fusion polypeptides that may be encoded by a respective one or more nucleic acid molecules. The invention includes such a nucleic acid molecules and host immune cells containing them. For example, a targeting moiety (eg antibody) may be genetically engineered to contain the further moiety and, if present, the one or more masking moieties, using genetic engineering techniques well established in the art. Thus, it will be appreciated that the further moiety (including separate parts thereof) and, if present, the one or more masking moieties, may be embedded within the polypeptide sequence of the targeting moiety, provided that the immune cell binding region of the further moiety can be unmasked when the molecule is in the vicinity of unwanted cells, such that the further moiety can bind to an immune cell. It will be appreciated that the each of the targeting moiety, further moiety and, if present, one or more masking moieties need not be encoded by contiguous sections of polynucleotide. For example, although the further moiety may be fused to the terminus of the targeting moiety, and, if present, the one or more masking moieties fused to the terminus of the further moiety, it is also contemplated that the various moieties of the molecule may be encoded by non-contiguous polynucleotide sequences, and between these non-contiguous polynucleotide sequences, polynucleotides sequences may exist that encode other moieties of the molecule. For example, it is clear from FIG. 4 that separate parts of the further moiety are encoded by non-contiguous sections of polynucleotide sequence and between these sections there is a polynucleotide sequence that encodes the targeting moiety.
It is further appreciated that the molecule of the invention may comprise two or more polypeptide chains that fold into the correct conformation where the two or more polypeptide chains are mixed. Each of the two or more polypeptides may include one or more of the targeting moiety, further moiety (including separate parts thereof) and, if present, the one or more masking moieties. This is illustrated by the molecule depicted in FIG. 2 and is particularly relevant when any of the targeting moiety, further moiety and, if present, the one or more masking moieties comprise one or more immunoglobulin domains, since the domains on one polypeptide chain (eg VH domain) may pair with domains on another polypeptide chain (eg VL domain). Pairing of immunoglobulin domains derived from different polypeptide chains is well known in the art, and forms the basis of the preparation of diabodies and triabodies.
It follows that when in the specific embodiment where the targeting moiety, further moiety and, if present, the one or more masking moieties are covalently attached and where all moieties are peptides or polypeptides, the molecule of the invention may be considered to be a bispecific antibody (eg a BiTE bispecific antibody). For example, the bispecific antibody may comprise (i) a first portion that is capable of specifically binding to an unwanted cell and (ii) a second portion that comprises a masked immune cell binding region so as to prevent binding of the second portion to an immune cell, wherein the masked immune cell binding region is capable of being selectively unmasked when the bispecific antibody is in the vicinity of the unwanted cells so as to allow binding to an immune cell. In this instance, the first portion of the bispecific antibody may correspond to the targeting moiety described above and the second portion of the bispecific antibody may correspond to the further moiety described above. Preferably, the antibody is a single chain antibody construct; however, it is appreciated that the antibody may comprise two or more polypeptide chains, as discussed above.
Preferably, the immune cell binding region of the bispecific antibody is a T cell binding to region. Thus, the bispecific antibody may comprise (i) a first portion that is capable of specifically binding to an unwanted cell and (ii) a second portion that comprises a masked T cell binding region so as to prevent binding of the second portion to a T cell, wherein the masked T cell binding region is capable of being selectively unmasked when the bispecific antibody is in the vicinity of the unwanted cells so as to allow binding to a T cell. Typically, the T cell binding region is one that can bind to the CD3 antigen and/or TCR present on a T cell.
It is also preferred that the immune cell binding region is unmasked by selective cleavage of one or more cleavage sites in the molecule when in the vicinity of the unwanted cells. Thus, the bispecific antibody may comprise (i) a first portion that is capable of specifically binding to an unwanted cell and (ii) a second portion that comprises a masked immune cell binding region so as to prevent binding of the second portion to an immune cell, wherein the masked immune cell binding region is unmasked by selective cleavage of one or more cleavage sites in the molecule when in the vicinity of the unwanted cells so as to allow binding to an immune cell.
Accordingly, the invention provides a bispecific antibody comprising (i) a first portion that is capable of specifically binding to an unwanted cell and (ii) a second portion that comprises a masked T cell binding region so as to prevent binding of the second portion to a T cell, wherein the masked T cell binding region is unmasked by selective cleavage of one or more cleavage sites in the molecule when in the vicinity of the unwanted cells so as to allow binding to a T cell. Typically, the T cell binding region is one that can bind to the CD3 antigen and/or TCR present on a T cell.
Suitably, the targeting moiety, further moiety and, if present, the one or more masking moieties are joined so that all moieties retain their respective activities such that the molecule may be targeted to an unwanted cell and the immune cell binding region may be unmasked within the vicinity of the unwanted cells so as to bind to an immune cell.
The targeting moiety and further moiety are typically joined by a linker peptide. Preferably, the linker joining the targeting moiety and further moiety contains between 2 and 100 amino acid residues, such as more than or less than 10, 20, 30, 40, 50, 60, 70, 80 and 90 amino acids. More preferably, the linker joining the targeting moiety and further moiety contains between 2 and 50 amino acids and still more preferably between 4 and 20 amino acids. Thus, the linker peptide may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 amino acids.
The one or more masking moieties are typically joined to the further moiety (including separate parts thereof) by a respective one or more linker peptides. The lengths of such linker peptides may depend on how the one or more masking moieties are operating to mask the T cell binding region. For example, if the one or more masking moieties are immunoglobulin domains that are intended to pair with variable domains of a further moiety (e.g. FIG. 4), the linkers that join the one or more immunoglobulin domains of the masking moieties with the respective variable domains of the further moiety must be of a sufficient length to allow pairing between the respective immunoglobulin domains of the one or more masking moieties and the respective variable domains of the further moiety. Generally, therefore, the linkers are at least 15 amino acids in length, such as at least 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acids in length. If the one or more masking moieties are simply blocking access to the underlying T cell binding region of the further moiety (e.g. FIGS. 2 and 3), it will be appreciated that the one or more linkers joining the respective one or more masking moieties to the further moiety may be shorter, or even zero length. Typical amino acid lengths include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 amino acids. However, longer linkers may also be used especially when the masking moiety is an immune cell surface antigen (e.g. FIG. 3). Thus, the linker may be at least 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acids in length. If the one or more masking moieties are immunoglobulin domains that are intended to prevent pairing of variable domains of a further moiety but without the immunoglobulin domains of the one or more masking moieties pairing with the variable domains of the further moiety, the linkers that join the one or more immunoglobulin domains of the masking moieties with the respective variable domains of the further moiety must be of an insufficient length to allow pairing between the respective immunoglobulin domains of the one or more masking moieties and the respective variable domains of the further moiety. Thus, the linkers are generally 14 amino acids or less, such as 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid in length.
In some embodiments, the one or more masking moieties may be joined to the targeting moiety (e.g. FIG. 4). Typically, the one or more masking moieties are joined to the targeting moiety by a respective one or more linker peptides. Preferably, the linker joining the targeting moiety and further moiety contains between 2 and 100 amino acid residues, more preferably between 2 and 50 and still more preferably between 4 and 20 amino acids (eg at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 amino acids).
Suitable linker peptides are those that typically adopt a random coil conformation, for example the polypeptide may contain alanine or proline or a mixture of alanine plus proline residues.
Polynucleotides which encode suitable targeting moieties and further moieties are known in the art or can be readily designed from known sequences such as from sequences of proteins known to interact with surface markers expressed on unwanted cells or immune cells or contained in nucleotide sequence databases such as the GenBank, EMBL, ImMunoGeneTics (IMGT), and dbEST databases. Polynucleotides which encode suitable one or more masking moieties are also known in the art or can readily be designed from known sequences or sequences in the above databases and made.
Polynucleotides which encode suitable linker peptides can readily be designed from linker peptide sequences and made.
Thus, polynucleotides which encode the molecules used in the invention can readily be constructed using well known genetic engineering techniques.
The nucleic acid is then expressed in a suitable host to produce a molecule of the invention. Thus, the nucleic acid encoding the molecule of the invention may be used in accordance with known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the molecule of the invention of the invention.
It is appreciated that the nucleic acid encoding the molecule of the invention may be joined to a wide variety of other nucleic acid sequences for introduction into an appropriate host. The companion nucleic acid will depend upon the nature of the host, the manner of the introduction of the nucleic acid into the host, and whether episomal maintenance or integration is desired, as is well known in the art.
In an alternative embodiment, the targeting moiety, further moiety (including separate parts thereof) and one or more masking moieties may be non-covalently attached. For non-covalent bindings, immunological bindings or such binding as via biotin/avidin or streptavidin, respectively, are preferred. For example, the targeting moiety may be a bispecific antibody, one specificity of which is directed to an entity expressed by the unwanted cell and one specificity of which is directed to the further moiety or part thereof. Also, it is possible to couple the further moiety to another substance against which, in turn, the specificity of the bispecific antibody will be directed to. For instance, the further moiety may contain further peptidic sequences which are recognised by the bispecific antibody. Another possibility involves coupling the targeting moiety, for example to streptavidin whilst the further moiety is coupled to biotin, and vice versa. Other means by which non-covalent interactions can be formed include leucine zipper sequences or affinity bonds. In any event, it is preferred that there are one or more cleavage sites within the molecule that are cleavable selectively when the molecule is in the vicinity of the unwanted cells, such that the immune cell binding region can be unmasked.
Although less preferred, the targeting moiety, further moiety (including separate parts thereof) and one or more masking moieties may be conveniently linked to each other by any of the conventional ways of cross-linking molecules, such as those generally described in O'Sullivan et al Anal. Biochem. (1979) 100, 100-108. For example, one of the targeting moiety or further moiety (including separate parts thereof) or masking moiety or spacer moiety may be enriched with thiol groups and the moiety it is to be linked to reacted with a bifunctional agent capable of reacting with those thiol groups, for example the N-hydroxysuccinimide ester of iodoacetic acid (NHIA) or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), a heterobifunctional cross-linking agent which incorporates a disulphide bridge between the conjugated species. Amide and thioether bonds, for example achieved with m-maleimidobenzoyl-N-hydroxysuccinimide ester, are generally more stable in vivo than disulphide bonds.
It is known that bis-maleimide reagents allow the attachment of a thiol group (e.g. thiol group of a cysteine residue of an antibody) to another thiol-containing moiety (e.g. thiol group of an immune cell antigen or a linker intermediate), in a sequential or concurrent fashion. Other functional groups besides maleimide, which are reactive with a thiol group include iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate.
Further useful cross-linking agents include S-acetylthioglycolic acid N-hydroxysuccinimide ester (SATA) which is a thiolating reagent for primary amines which allows deprotection of the sulphydryl group under mild conditions (Julian et al (1983) Anal. Biochem. 132, 68), dimethylsuberimidate dihydrochloride and N,N′-o-phenylenedimaleimide.
Particularly preferred crosslinking agents include sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Sulfo-SMCC), sulfosuccinimidyl 6-(3′-[2-pyridyldithio]-propionamido) hexanoate (Sulfo-LC-SPDP) and N-[β-Maleimidopropionic acid]hydrazide, trifluoroacetic acid salt (BMPH).
It will be understood that a large number of homobifunctional and heterobifunctional crosslinking chemistries would be appropriate to join the various moieties of the molecule together, and any such chemistry may be used. For example, Click Chemistry using Staudinger Ligation Chemistry (phosphine-azido chemistry) may be used.
Amino acid residues described herein are generally in the natural “L” isomeric form, which is preferred. However, residues in the “D” isomeric form can be substituted for L-amino acid residues in certain situations, provided that the agent of the invention still retains its function, namely to prevent or treat a condition characterised by the presence of unwanted cells. The definition also includes, unless otherwise specifically indicated, chemically-modified amino acids, including amino acid analogues (such as penicillamine, 3-mercapto-D-valine), naturally-occurring non-proteogenic amino acids (such as norleucine), beta-amino acids, azapeptides, N-methylated amino acids and chemically-synthesised compounds that have properties known in the art to be characteristic of an amino acid. The term “proteogenic” indicates that the amino acid can be incorporated into a protein in a cell through well-known metabolic pathways. The definition also includes amino acids in which the functional side group has been chemically derivatised. Such derivatised molecules include, for example, those molecules in which free amino groups have been derivatised to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatised to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatised to form O-acyl or O-alkyl derivatives. Also included as derivatives are those peptide portions that contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids.
Accordingly, it is appreciated that the peptide portions of the molecule of the invention can be peptide “mimetics”, i.e. peptidomimetics which mimic the structural features of peptides comprising or consisting of the amino acid sequence as described herein. Peptidomimetics can be even more advantageous in therapeutic use, in the resistance to degradation, in permeability or in possible oral administration.
A primary goal in the design of peptide mimetics has been to reduce the susceptibility of mimetics to cleavage and inactivation by peptidases. In one approach, such as disclosed by Sherman et al (1990), one or more amide bonds have been replaced in an essentially isosteric manner by a variety of chemical functional groups. This stepwise approach has met with some success in that active analogues have been obtained. In some instances, these analogues have been shown to possess longer biological half-lives than their naturally-occurring counterparts. In another approach, a variety of uncoded or modified amino acids such as D-amino acids and N-methyl amino acids have been used to modify mammalian peptides. Alternatively, a presumed bioactive conformation has been stabilised by a covalent modification, such as cyclization or by incorporation of γ-lactam or other types of bridges (Veber et al, 1978) and Thorsett et al, 1983). Another approach, disclosed by Rich (1986) has been to design peptide mimics through the application of the transition state analogue concept in enzyme inhibitor design. For example, it is known that the secondary alcohol of statine mimics the tetrahedral transition state of the sessile amide bond of the pepsin substrate. Other approaches include the use of azapeptides and beta-amino acids.
Also included in the definition of ‘peptidomimetics’, are retro-inverso peptides. By retro-inverso peptides (also known as all-D-retro or retro-enantio peptides) we include the meaning of a peptide in which all of the L-amino acids are replaced with D-amino acids and the peptide bonds are reversed. Thus, the peptides are composed of D-amino acids assembled in the reverse order from that of the parent L-sequence. Retro-inverso peptides can be synthesised by methods known in the art, for example such as those described in Meziere et al (1997) J. Immunol. 159 3230-3237. This approach involves making pseudopeptides containing changes involving the backbone, and not the orientation of side chains which remain very similar to the parent peptide. Retro-inverse peptides are much more resistant to proteolysis.
Therefore, it will be appreciated that when any of the targeting moiety, further moiety, one or more masking moieties, cleavage site, and spacer moieties as described herein are peptides or polypeptides, any one or more of those peptides or polypeptides may be substituted for a corresponding peptidomimetic that retains the respective activity of the parent peptide or polypeptide. This may help to confer protease resistance on the agent of the invention and thereby improve its stability. Thus, for example, when a targeting moiety is attached to a further moiety via one or more peptide spacer moieties, it may be desirable for one or more of those spacer moieties to be peptidomimetics, e.g. wherein one or more of the naturally occurring amino acids of the spacer moieties are replaced or modified, for example, to improve stability.
Another approach to increase stability of peptide portions of the agent of the invention is to have stabilising groups at one or both termini. Typical stabilising groups include amido, acetyl, benzyl, phenyl, tosyl, alkoxycarbonyl, alkyl carbonyl, benzyloxycarbonyl and the like end group modifications. Additional modifications include using a “D” amino acid in place of a “L” amino acid at the termini, and amide rather than amino or carboxy termini or acetyl rather than amino termini, to inhibit exopeptidase activity. Thus, it is appreciated that whenever the agent of the invention has an exposed peptide terminus, that terminus may have a capping moiety, preferably a moiety that is less than 200 Da in molecular weight. Further capping moieties include a naftyl group or a polyethylene glycol group. It is appreciated that retro-inverso peptides are already relatively stable and so may not require additional capping moieties.
Preferably, the molecule of the invention has a half-life in plasma of at least 24 hours at 37° C.
It may be desirable to modify the molecule of the invention so that it can be more easily detected, for example by biotinylating it or by incorporating any detectable label known in the art such as radiolabels, fluorescent labels or enzymatic labels.
As discussed above, the molecules of the invention have utility in redirecting the body's immune machinery to eradicate or neutralise particular unwanted cells in a specific manner. Since any unwanted cell may be targeted in this way, the molecules of the invention offer significant therapeutic potential.
Accordingly, a second aspect of the invention provides a method of preventing or treating a condition characterised by the presence of unwanted cells, the method comprising administering a molecule according to the first aspect of the invention to a subject.
Thus, the method may involve identifying a subject who has a condition or who is at risk of developing a condition characterised by unwanted cells (eg cancer), administering the molecule according to the first aspect of the invention to the subject, and monitoring the levels of the unwanted cells in the subject either by conducting tests to determine the number of unwanted cells or by monitoring the clinical symptoms of the subject. Depending on the results of the monitoring step, it may be necessary to administer more of the agent.
Similarly, the invention includes a molecule according to the first aspect of the invention for use in preventing or treating a condition characterised by the presence of unwanted cells.
The invention also includes the use of a molecule according to the first aspect of the invention in the manufacture of a medicament for preventing or treating a condition characterised by the presence of unwanted cells.
Preferences for the condition and unwanted cells, are as described above with respect to the first aspect of the invention. Examples of particular conditions include tumours (benign or malignant), autoimmune conditions, cardiovascular diseases, degenerative diseases, diabetes, allergic disease (eg asthma), neurodegenerative diseases such as Alzheimer's, transplantation patients and infectious diseases. Preferably, the unwanted cells are cancer cells and the condition is a cancer. The cancer may be any cancer such as breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, renal cancer, melanoma, lung cancer, prostate cancer, testicular cancer, thyroid cancer, brain cancer, oesophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, liver cancer, leukaemia, myeloma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute myeloid leukaemia, acute lymphoblastic leukaemia, chronic lymphoblastic leukaemia, lymphoproliferative disorder, myelodysplastic disorder, myeloproliferative disease and premalignant disease. It will be appreciated that the molecule of the invention also has utility in regenerative medicine (eg laboratory grown organs or tissues).
By preventing or treating a condition we include the meaning of reducing or alleviating symptoms in a patient (i.e. palliative use), preventing symptoms from worsening or progressing, treating the disorder (e.g. by inhibition or elimination of the causative agent), or prevention of the condition or disorder in a subject who is free therefrom.
It will be appreciated that the molecules of the invention lend themselves to personalised medicine in the clinic whereby the most appropriate molecule to be administered to the patient is determined, and either selected or prepared in the clinic. For example, it may be desirable to acquire an expression profile of the unwanted cell in a patient so that the optimum targeting moiety for that patient can be selected. Assessing the expression profile of the unwanted cell may be carried out on a biopsy sample using routine assays for measuring nucleic acid (e.g. DNA or RNA transcripts) or protein levels. For example, transcriptomic or proteomic techniques may be used. In this way, it will be possible to identify tailored targeting moieties that bind specifically to, for example, surface markers expressed by the unwanted cell. It may also be possible to identify appropriate protease cleavage sites that may be selectively cleaved in the vicinity of the unwanted cells. For example, it may be that a molecule containing one or more MMP2 cleavage sites would not be activated by a given patient's cancer proteases, but a molecule containing one or more MMP9 cleavage sites would be activated.
Thus the method of the second aspect of the invention may include the steps of (i) identifying a subject who has a condition, or who is at risk of developing a condition characterised by the presence of unwanted cells (eg cancer), (ii) taking a sample from the subject, (iii) analysing the sample to identify the optimum targeting moiety and/or cleavage site for preventing or treating the condition in that subject, (iii) preparing the molecule of the invention, (iv) administering the molecule to the subject, and (v) monitoring the levels of unwanted cells in the subject either by conducting tests to determine the number of unwanted cells or by monitoring the clinical symptoms of the subject.
It is appreciated that an apparatus may be used to select and optionally prepare the most appropriate molecule to be used for a particular patient. For example, the apparatus may perform an automated analysis of one or more samples from the subject, and based on this analysis select and optionally prepare a tailor-made molecule for that subject. Thus the apparatus may carry out an expression profile of unwanted cells from the subject (eg from a biopsy sample) so as to determine a suitable targeting moiety that will bind to the unwanted cell and/or determine a suitable cleavage site that will be selectively cleaved in the vicinity of the unwanted cell.
By performing any one or more of these steps in the clinic a molecule tailored for a particular subject can be prepared. For example, the molecule can contain a targeting moiety that is known to bind selectively to surface markers expressed by the unwanted cell, and/or a cleavage site (e.g. protease cleavage site) that allows unmasking of the immune cell binding region (eg T cell binding region) when the molecule is in the vicinity of the unwanted cells.
In one embodiment, the subject is administered a further therapeutic agent in addition to the molecule according to the first aspect of the invention. For example, when administering the molecule to prevent or treat a particular condition, a further therapeutic agent known to be useful for combating that condition may be administered. As an example, when the molecule is for treating cancer, a further anti-cancer agent (eg anti-neoplastic chemotherapy) may be administered to the subject alongside the molecule of the invention. Similarly, the further therapeutic agent may be one that is known to have therapeutic application in allergic disease, inflammatory disease, regenerative medicine and neuroregenerative disease.
It is appreciated that the further therapeutic agent may be administered at the same time as the molecule of the invention (i.e. simultaneous administration optionally in a co-formulation) or at a different time to the molecule of the invention (i.e. sequential administration).
The further therapeutic agent may be any one or more of a vaccine; an immuno stimulatory drug; an anti-cancer agent; an agent inhibiting an antibody response against the agent of the invention; and/or a protease inhibitor.
For example, it may be desirable to administer immunostimulating agents such as IL-2, IL-7, IFNα, GM-CSF, metformin, lenalidomide; and/or administer anti-immunoregulatory agents such as Ipilimumab; all of which may be considered as further therapeutic agents.
It is also appreciated that if the subject is one to whom is administered immunosuppressive agents, that these immunosuppressive agents may be withdrawn from the subject (e.g. by suspending treatment) when or before being administered the agent of the invention. This is particularly true where the subject is one to whom is administered immunosuppressive agents to ablate T cells.
Similarly, it may be desirable to employ methods aimed at circumventing any immunogenicity issues relating to the molecule of the invention whereby an adverse antibody response is elicited in vivo. For example, the subject may also be administered one or more agents that are known to inhibit the activity of B cells, such as any of Rituximab, cyclophosphamide, Syk inhibitors, an anti-BAFF antibody (eg Belimumab), an anti-CD22 antibody, an anti-CD20 antibody and an anti-CD19 antibody, all of which may be considered as further therapeutic agents. In this case, it is particularly preferred if the inhibitor of B cells is administered to the subject prior to the molecule of the invention, eg as a pre-treatment to ablate B cells.
In another embodiment, where the molecule comprises one or more protease cleavage sites, it may be appropriate to administer a particular protease inhibitor so as to improve the target selectivity of the molecule of the invention. For example, if a targeting moiety is known to bind cells in both the heart and breast tissue, but only those in the breast are to be targeted, it may be desirable to administer an agent that selectively inhibits the protease responsible for unmasking the immune cell binding region, in the heart but not the breast. In other words, an agent is administered to inhibit a protease that is capable of unmasking the immune cell binding region but which protease resides in the vicinity (eg at or near the surface of) of wanted cells but not in the vicinity (eg at or near the surface of) of unwanted cells. This is particularly useful in the event that a protease cleavage site within the molecule of the invention is cleavable by multiple proteases, some of which reside in the vicinity of unwanted cells and some of which reside in the vicinity of wanted cells. In this case, targeting specificity may be improved by administering a protease inhibitor that inhibits a protease that resides in the vicinity of wanted cells but nevertheless is capable of cleaving the cleavage site and therefore unmasking the immune cell binding region. The effect of administering the inhibitor would be to ensure that the immune cell binding region is preferentially unmasked in the vicinity of the unwanted cells. For instance, if a subject with cancer also has active rheumatoid arthritis where MMP2 and other proteases are active, and the one or more cleavage sites in the molecule of the invention are cleavable by multiple proteases including MMP2, it may be beneficial to inhibit MMP2 to prevent cleavage of the molecule at the arthritic joint but retain cleavage at the cancer site by another protease.
The invention thus includes a composition comprising (i) a molecule according to the first aspect of the invention and (ii) a further therapeutic agent, for use in preventing or treating a condition characterised by the presence of unwanted cells. Given that the molecule of the invention and the further therapeutic agent may be administered simultaneously or sequentially, it will be appreciated that the invention includes a molecule according to the first aspect of the invention for use in preventing or treating a condition characterised by the presence of unwanted cells in a subject who is administered a further therapeutic agent. It also follows that the invention includes a therapeutic agent for use in preventing or treating a condition characterised by the presence of unwanted cells in a subject who is administered a molecule according to the first aspect of the invention.
Similarly, the invention includes a use of a composition comprising (i) a molecule according to the first aspect of the invention and (ii) a further therapeutic agent, in the manufacture of a medicament for preventing or treating a condition characterised by the presence of unwanted cells. Again, given that the molecule of the invention and the further therapeutic agent may be administered simultaneously or sequentially, it will be appreciated that the invention includes the use of a composition comprising a molecule according to the first aspect of the invention in the manufacture of a medicament for preventing or treating a condition characterised by the presence of unwanted cells in a subject who is administered a further therapeutic agent. It also follows that the invention includes the use of a therapeutic agent in the manufacture of a medicament for preventing or treating a condition characterised by the presence of unwanted cells in a subject who is administered a molecule according to the first aspect of the invention.
The invention also provides a composition comprising (i) a molecule according to the first aspect of the invention and (ii) a further therapeutic agent suitable for preventing or treating the same condition characterised by the presence of unwanted cells. It is appreciated that the therapeutic agent mentioned in the immediately preceding two paragraphs may be agents suitable for treating the same condition characterised by the presence of unwanted cells, as treatable by the molecules of the invention.
A third aspect of the invention provides a molecule according to the first aspect of the invention for use in medicine.
A fourth aspect of the invention a pharmaceutical composition comprising a molecule according to the first aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.
Whilst it is possible for the molecule of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be “acceptable” in the sense of being compatible with the therapeutic agent and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free.
Where appropriate, the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient (agent for treating or preventing a condition characterised by unwanted cells) with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.
A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.
Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
The molecule of the invention can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder. The molecule may also be transdermally administered, for example, by the use of a skin patch.
Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of an active ingredient.
It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.
The amount of the molecule which is administered to the individual is an amount effective to combat the particular individual's condition. The amount may be determined by the physician.
Preferably, in the context of any aspect of the invention described herein, the subject to be treated is a human. Alternatively, the subject may be an animal, for example a domesticated animal (for example a dog or cat), laboratory animal (for example laboratory rodent, for example mouse, rat or rabbit) or an animal important in agriculture (i.e. livestock), for example horses, cattle, sheep or goats.
The invention provides a kit of parts for preventing or treating a condition characterised by the presence of unwanted cells, the kit comprising:

    
    
        (i) a targeting moiety capable of directly targeting to unwanted cells and which is attached to a first binding partner, and
        (ii) a further moiety that has a masked immune cell binding region so as to prevent binding of the further moiety to an immune cell, and which further moiety is attached to a second binding partner which is capable of binding to the first binding partner,
        wherein the masked immune cell binding region is capable of being selectively unmasked when the further moiety is in the vicinity of the unwanted cells so as to allow binding of the further moiety to an immune cell. Preferably, the immune cell binding region is a T cell binding region (eg one that binds to CD3 antigen and/or TCR on a T cell). Preferably, the immune cell binding region is unmasked by selective cleavage of one or more cleavage sites in the further moiety when in the vicinity of the unwanted cells.
    
    


Preferences for the unwanted cells, targeting moiety, further moiety and cleavage sites are as defined above. It is particularly preferred if the kit of parts is for preventing or treating cancer. It is also appreciated that the kit of parts may comprise one or more masking moieties that mask the immune cell binding region of the further moiety. Preferences for the masking moiety are as defined above, and include both masking moieties that promote a conformation of the further moiety in which conformation the immune cell binding region is not accessible to an immune cell, as well as masking moieties that simply block an underlying immune cell binding region of the further moiety.
By the first and second binding partners, we include the meaning of any two moieties which bind to each other selectively. Most preferably, the first and second binding partners only bind to each other and not to any other moieties. Non-covalent binding such as between biotin/avidin or streptavidin, or immunological bindings are preferred. Thus, the first binding partner may be biotin and the second binding partner may be avidin, and vice versa. Alternatively, the first binding partner may be an antigen and the second binding partner may be an antibody specific for that antigen, and vice versa. However, any pair of first and second binding partners that selectively bind to each other may be used, and suitable pairs will be known to the skilled person.
It will be appreciated that the kit allows one to first administer the targeting moiety that directly targets the unwanted cells to the subject, and establish the correct localisation of the targeting moiety in the subject (for example by the targeting moiety being detectably labelled (eg radiolabelled)) before administering the further moiety. The further moiety is then targeted to the unwanted cells by virtue of the second binding partner binding to the first binding partner. Once in the vicinity of the unwanted cells, the immune cell binding region of the further moiety is unmasked, for example by selective cleavage of one or more cleavage sites within the further moiety, allowing an immune cell (eg T cell) to be recruited to the unwanted cells. It will be understood that the further moiety that is attached to a second binding partner may be considered a targeting moiety that is capable of indirectly targeting to an unwanted cell as mentioned above in relation to the first aspect of the invention.
Accordingly, the invention further provides a method of preventing or treating a condition characterised by the presence of unwanted cells, the method comprising administering

    
    
        (i) a targeting moiety capable of directly targeting to unwanted cells and which is attached to a first binding partner, and
        (ii) a further moiety that has a masked immune cell binding region so as to prevent binding of the further moiety to an immune cell, and which further moiety is attached to a second binding partner which is capable of binding to the first binding partner,
        wherein the masked immune cell binding region is capable of being selectively unmasked when the further moiety is in the vicinity of the unwanted cells so as to allow binding of the further moiety to an immune cell. Preferably, the immune cell binding region is a T cell binding region (eg one that binds to CD3 antigen and/or TCR on a T cell). It is also preferred that the immune cell binding region is unmasked by selective cleavage of one or more cleavage sites in the further moiety when in the vicinity of the unwanted cells. Preferably, the targeting moiety is administered before the further moiety, for example to allow to correct localisation of the targeting moiety at the unwanted cells to be established. However, the targeting moiety may be administered at the same time as the further moiety. It is appreciated that the targeting moiety and further moiety may be attached to each other by binding of the first and second binding partners.
    
    


Similarly, the invention provides a composition comprising:

    
    
        (i) a targeting moiety capable of directly targeting to unwanted cells and which is attached to a first binding partner, and
        (ii) a further moiety that has a masked immune cell binding region so as to prevent binding of the further moiety to an immune cell, and which further moiety is attached to a second binding partner which is capable of binding to the first binding partner,
        wherein the masked immune cell binding region is capable of being selectively unmasked when the further moiety is in the vicinity of the unwanted cells so as to allow binding of the further moiety to an immune cell,
        for use in preventing or treating a condition characterised by unwanted cells in a subject. Preferably, the immune cell binding region is a T cell binding region (eg one that binds to CD3 antigen and/or TCR on a T cell). It is also preferred that the immune cell binding region is unmasked by selective cleavage of one or more cleavage sites in the further moiety when in the vicinity of the unwanted cells
    
    


Similarly, the invention provides a use of a composition comprising:

    
    
        (i) a targeting moiety capable of directly targeting to unwanted cells and which is attached to a first binding partner, and
        (ii) a further moiety that has a masked immune cell binding region so as to prevent binding of the further moiety to an immune cell, and which further moiety is attached to a second binding partner which is capable of binding to the first binding partner,

wherein the masked immune cell binding region is capable of being selectively unmasked when the further moiety is in the vicinity of the unwanted cells so as to allow binding of the further moiety to an immune cell,

        in the manufacture of a medicament for preventing or treating a condition characterised by the presence of unwanted cells. Preferably, the immune cell binding region is a T cell binding region (eg one that binds to CD3 antigen and/or TCR on a T cell). It is also preferred that the immune cell binding region is unmasked by selective cleavage of one or more cleavage sites in the further moiety when in the vicinity of the unwanted cells.
    
    


In an embodiment of the invention, the targeting moiety is an anti-MUC1 antibody, and the further moiety is an anti-CD3 antibody.
In an embodiment of the invention, the targeting moiety is an anti-CD19 antibody, and the further moiety is an anti-CD3 antibody.



The invention will be described in further detail with the aid of the following Figures and Examples.
FIG. 1: Schematic diagram illustrating molecule of the invention. The targeting moiety (1) comprises VH and VL domains specific for an unwanted cell (2), and the further moiety (3) comprises VH and VL domains specific for CD3 antigen. The VH and VL domains of the further moiety are attached by a linker which is of insufficient length to allow pairing, and so the T cell binding region of the further moiety is masked. Cleavage of a cleavage site (5) in the linker when the molecule is in the vicinity of the unwanted cell (2) allows unmasking of the T cell binding region (4).
FIG. 2: Schematic diagram illustrating molecule of the invention. The targeting moiety (1) comprises paired VH and VL domains specific for an antigen on the unwanted cell (2). The further moiety (3) comprises VH and VL domains specific for CD3 antigen. However, the T cell binding region of the further moiety is blocked by the presence of two masking moieties, a CH domain and a CL domain (6) that are joined to the further moiety by respective linkers. Cleavage of cleavage sites (5) in the linkers when the molecule is in the vicinity of the unwanted cell (2) allows unmasking of the T cell binding region (4).
FIG. 3: Schematic diagram illustrating molecule of the invention. The targeting moiety (1) comprises paired VH and VL domains specific for an antigen on the unwanted cell (2). The further moiety (3) comprises VH and VL domains specific for CD3 antigen. However, the T cell binding region of the further moiety is blocked by the presence of a masking moiety (6) which is an immune cell antigen that binds to the T cell binding region, and which is securely tethered to the further moiety by a covalent linker. Cleavage of a cleavage site (5) in the linker when the molecule is in the vicinity of the unwanted cell (2) allows the masking moiety to leave the T cell binding region and so the T cell binding region (4) is unmasked.
FIG. 4: Schematic diagram illustrating molecule of the invention. The targeting moiety (1) comprises paired VH and VL domains specific for an antigen on the unwanted cell (2). The further moiety (3) comprises VH and VL domains specific for CD3 antigen. However, the VH and VL domains of the further moiety (3) are unable to pair due to the presence of two masking moieties (6) respectively comprising a VL and a VH domain. The VL and VH domains of the masking moieties act as dummy domains that pair with the VH and VL domains of the further moiety and so prevent the VH and VL domains of the further moiety from pairing. The T cell binding region (4) is therefore masked. Cleavage of cleavage sites (5) in linkers that join the further moiety to the one or more masking moieties when the molecule is in the vicinity of the unwanted cell (2) releases the VH and VL domains from pairing with the dummy VH and VL domains of the masking moiety and so the VH domain and VL domain of the further moiety can pair. The T cell binding region (4) is thereby unmasked.
FIG. 5: (A) Schematic diagram illustrating a molecule of the invention. The VH and VL regions encompassing the scFv of the targeting moiety are linked via a single peptide to linker to the VH region of the further moiety which in turn is linked via a single peptide linker containing a protease cleavage site to the CH region of the masking moiety. The VL region of the further moiety is linked via a single peptide linker containing a protease cleavage site to the CL region of the masking moiety. The VH and VL regions of the further moiety and the CH and CL regions of the masking moiety will pair when the proteins are incubated together. Thus, the CH and CL regions of the masking moiety prevent the VH and VL regions of the further moiety from interacting with its natural ligand (eg CD3) and from activating T cells. However, after binding to unwanted cells via the target moiety, specific proteolytic cleavage will occur in the vicinity of unwanted cells and the masking moiety will be thereby released allowing interaction of T cells with the unwanted cell. (B) Schematic diagram denoting the genetic make-up of the protein chains depicted in (A). Rickymab (Heavy) comprises the VH of the further moiety and the CH region of the masking moiety and this will be molecularly cloned together with the scFv of the target moiety. Rickymab (Light) comprises the VL region of the further moiety and the CL region of the masking moiety which will pair with the VH and CH regions of Rickymab (Heavy) after introducing the DNA into cells capable of generating the embodiment.
FIG. 6: (A) Schematic diagram that denotes the regions of DNA combined together to form the polypeptide chain seen in FIG. 4. Further indications by arrow denote protease cleavage sites and sites of DNA enzyme cleavage (BamH, Sac & Xho) allowing for movement of regions within the molecule to generate the molecules seen in FIGS. 1, 2, 4 and 5. (B) Schematic diagram that denotes the regions of DNA that make up the VH and CH regions of the target moiety in (A) coupled to a fluorescent dye. The VH and CH regions of the other molecules (eg FIGS. 1-5) could also be linked to the fluorescent dye by enzymatic digestion of the DNA and subsequent ligation to the fluorescent dye. Once the labelled molecule is used to label cells, those cells can be viewed using flow cytometry. This allows demonstration that the target moiety binds to the unwanted cells of interest and not any wanted cell types.
FIG. 7: Peripheral blood mononuclear cells (PBMCs) from 2 donors (with different HLA types) were incubated with the molecule exemplified in FIG. 3 (comprising an anti-CD19 scFv (targeting moiety) linked to an anti-CD3 scFv (the further moiety) further linked to the CD3 epsilon domain (the masking moiety)—B.mab) and incubated overnight at 37° C. with the addition of a protease to release the active site of the further moiety. The following day, cells were labelled with antibodies against anti-CD3 to determine the presence of T cells after incubation with the molecule and also anti-CD19 to determine the presence/absence of B cells (see Example 2). Incubation with the molecule removes all B cells from the PBMCs as would be expected.
FIG. 8: Target cells, B lymphoblastoid cell line (B-LCL)) are labelled with a molecule comprising an anti-CD19 scFv (targeting moiety) linked to an anti-CD3 scFv (the further moiety) further linked to the CD3 epsilon domain (the masking moiety) (FIG. 3). After removing unbound embodiment, the B-LCL cells were incubated overnight with or without T cells with the B-LCLs producing the protease which cleaves the masking moiety releasing the further moiety. As a control measure, the T cells were incubated with the molecule without target cells to determine background activation of unbound molecule. After overnight incubation, the amount of interferon gamma (IFN-γ) in each culture was assayed to determine the extent of T cell activation. There was only activation of T cells when target cells were labelled with the molecule (see Example 1).
FIG. 9: Conditionally activated bispecific construct. Peripheral blood mononuclear cells from a healthy donor were assessed using flow cytometry following overnight culture. Cells were stained using anti-CD3 (Y-axis) and anti-CD19 (x-axis) to reveal numbers of T-cells and B-cells. The scFv1-scFv2-CD3e construct contained a protease cleavable section between the CD3e cap and the scFv1-scFv2 activating region which could be cleaved using the Factor Xa protease. scFv1 is the anti-CD19 entity while scFV2 has affinity toward CD3e. (A) PBMCs cultured overnight in media only; (B) PBMCs+scFv1-scFv2-CD3e construct; (C) PBMCs+scFv1-scFv2-CD3e construct+Factor Xa protease (0.01 ug/ml); (D) PBMCs+scFv1-scFv2-CD3e construct+Factor Xa protease (0.1 ug/ml); (E) PBMCs+scFv1-scFv2-CD3e construct+Factor Xa protease (0.5 ug/ml); (F) PBMCs+scFv1-scFv2-CD3e construct+Factor Xa protease (1 ug/ml). As can be seen from the figure, only when the protease is added is the cap cleaved off, thereby allowing depletion of B cells. The construct comprised an anti-CD19 scFv (targeting moiety) linked to an anti-CD3 scFv (the further moiety) further linked to the CD3 epsilon domain (the masking moiety).
FIG. 10: Conditionally activated bispecific construct through removal of masking domain. (A) Design of 2 constructs Ricky2A (SEQ ID No: 30) and Ricky2B (SEQ ID No: 31) which when co-expressed in mammalian expression systems (293T) produce a scFv1-scFv2-Cap where the anti-CD3 binding region (scFv2) is masked by an immunoglobulin constant region (CH,CL). The constant region is cleaved by tumour-associated proteases (MMP2) through inclusion of an MMP2 recognition site IPVSLR (SEQ ID No: 32) between the masking and binding domains. In this case an anti-MUC1 (scFv1) and anti-CD3 (scFv2) domains are used for the construct; (B) T-cells used for experiment stained using CD3-PE (x-axis) and anti-CD138-APC (y-axis) as assessed using flow cytometry; (C) Muc1-expressing U266 cells used for experiment stained using CD3-PE (x-axis) and anti-CD138-APC (y-axis) as assessed using flow cytometry; (D) Co-culture of U266 cells with T-cells; (E) Co-culture of U266 cells with T-cells+Ricky2A/2B activatable masked bispecific showing 12% reduction of U266 population.



EXAMPLE 1
Use of Molecule of Invention
Target cells modelling B cell malignancy (B lymphoblastoid cell line (B-LCL)) are targeted with a molecule comprising an anti-CD19 scFv (targeting moiety) linked to an anti-CD3 scFv (the further moiety) further linked to the CD3 epsilon domain (the masking moiety) (eg molecule in FIG. 3). Once targeted, excess molecule is washed from the target cells and the target cells are cultured with T cells (CD4+ and CD8+) overnight at 37° C. After incubation, supernatant from the culture is tested for IFN-gamma release which would demonstrate the activation of the T cells in recognising the molecule on the surface of the target B-LCLs. Activation of T cells would only be seen if proteolytic cleavage of the linker between the further moiety and CD3 epsilon masking moiety has occurred, thus unmasking the binding region of the anti-CD3 binding region. After incubation of the molecule with either target cells alone or with T cells alone, supernatant from the cultures demonstrated no IFN-gamma production. However, after incubation of targeted B-LCLs with T cells, there was a large production of IFN-gamma demonstrating the release of the masking moiety in the presence of unwanted target cells (B-LCL) and the recognition of the unwanted cells by T cells.
EXAMPLE 2
Use of Molecule of Invention
Targeting B cells in a mixed population of human peripheral blood mononuclear cells (containing B cells, T cells, monocytes, macrophages and NK cells), with a molecule comprising an anti-CD19 scFv (targeting moiety) linked to an anti-CD3 scFv (the further moiety) further linked to the CD3 epsilon domain (the masking moiety). Once the molecule has been added to the mixed population of cells, thereby labelling B cells, an exogenous protease (e.g. trypsin) is added to the cells to release the masking agent before culturing at 37° C. overnight. After culture, the cells are labelled with fluorescently labelled antibodies anti-CD3 and anti-CD19 and analysed using flow cytometry. The results demonstrate the presence of B cells ( ̃10%) in the cells after overnight culture without the molecule but the complete ablation of B cells in the cells cultured overnight in the presence of the molecule. This result demonstrates the targeted cytotoxicity of unwanted B cells in a mixed population of peripheral blood mononuclear cells.
EXAMPLE 3
Synthesis of Molecule of Invention
The production of the molecules shown in the figure comprises generating an protein sequence of the target moiety (VH and VL domains) and the protein sequence of the further moiety (VH and VL domains) and linking these domains using standard proteomic linkages (Gly-Gly-Gly-Ser repeats: SEQ ID No: 33). The scFv domains may then be linked to one or more masking moieties using similar techniques. Where required, one or more protease cleavage sequences can be inserted into the linker polypeptide sequences. Once the protein sequence has been determined for one or more polypeptide chains, the DNA is synthesised and inserted into a vector suitable for either prokaryotic or eukaryotic expression systems. In a prokaryotic system, E. coli are transformed with the synthesised gene encoding one or more polypeptide chains and after culture overnight, the supernatant is harvested and the polypeptide chains can be purified by standard techniques (e.g. His tag purification). After purification, the one or more polypeptide chains are refolded and can then be used to label target cells. In a eukaryotic expression system, the synthesised gene encoding one or more polypeptide chains is transfected into a suitable cell line in a lentiviral expression system. After 48 hours incubation, supernatant containing the correctly refolded and conformationally correct molecules can be purified using standard techniques (e.g. His tag purification).
An example of a molecule depicted in FIG. 3 contains the following amino acid sequences on a single polypeptide chain:







Anti-CD19 VL


(SEQ ID No: 20)


DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKL


 


LIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPW


 


TFGGGTKLEIK


 


Linker


(SEQ ID No: 21)


GGGGSGGGGSGGGGS


 


Anti-CD19 VH


(SEQ ID No: 22)


QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQ


 


IWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRE


 


TTTVGRYYYAMDYWGQGTTVTVSS


 


Linker


(SEQ ID No: 23)


GGGGSSD


 


Anti-CD3 VH


(SEQ ID No: 24)


IKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYI


 


NPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYD


 


DHYCLDYWGQGTTLTVSS


 


Linker


(SEQ ID No: 25)


VEGGSGGSGGSGGSGGVDD


 


Anti-CD3 VL


(SEQ ID No: 26)


IQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTS


 


KVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGT


 


KLELK


 


Long Linker


(SEQ ID No: 21)


GGGGSGGGGSGGGGS


 


Cleavage site


(SEQ ID No: 27)


TIPVSLR


 


Long Linker


(SEQ ID No: 28)


SGGGGSGGGGSGGGGSDI


 


CD3 epsilon


(SEQ ID No: 29)


QTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDH


 


LSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCEGS






This molecule is illustrative of an embodiment of the invention where the molecule comprises one or more masking moieties that are immune cell surface antigens, in this case CD3 epsilon.
An example of a molecule depicted in FIG. 5 is shown in FIG. 10. This molecule is illustrative of an embodiment of the invention where the molecule comprises one or more masking moieties which are immunoglobulin domains, and more specifically, constant domains. The embodiment is also illustrated in FIG. 2, but whereas in FIG. 2, the VH and VL portions of the targeting moiety are on separate polypeptide chains, in FIG. 5, the VH and VL portions of the targeting moiety are on the same polypeptide chain.

## Claims

1. A composition for redirecting T cells to unwanted cells comprising:
(i) a targeting moiety capable targeting to unwanted cells wherein the targeting moiety is an anti-HER2 antibody or antigen binding fragment thereof and wherein the targeting moiety is not masked, and
(ii) at least one further moiety that has a masked immune cell binding region so as to prevent binding of the further moiety to an immune cell, wherein the immune cell binding region is an anti-CD3 antibody or antigen binding fragment thereof, and wherein the masked immune cell binding region is capable of being selectively unmasked by cleavage of at least one protease cleavage site when the molecule is in the vicinity of the unwanted cells so as to allow binding of the further moiety to an immune cell and,

wherein the further moiety is a scFv antibody in which the linker that joins the heavy chain variable domain (VH) and light chain variable domain (VL) is of insufficient length, optionally a peptide linker of 14 or less amino acids, to allow pairing of the VH and VL domains such that the scFv antibody cannot bind to the immune cell, and wherein, when in the vicinity of the unwanted cells, pairing of the VH and VL domains occurs by selectively cleaving one or more cleavage sites in said linker such that the VH and VL domains from the scFv antibody can bind to the immune cell.

2. A composition according to claim 1, wherein the anti-CD3 antibody or antigen binding fragment thereof is a single chain antibody construct.

3. A composition according to claim 1, wherein each of the further moiety and targeting moiety are parts of a single polypeptide chain.

4. A pharmaceutical composition, comprising a composition according to claim 1, and a pharmaceutically acceptable carrier, diluent or excipient.

5. A method of treating carcinoma expressing HER2 on the cell membrane, the method comprising:
(i) administering a composition according to claim 1 to a subject or (ii) administering a composition according to claim 1 and a further therapeutic agent, which may include one or more of an immunostimulatory drug, an anti-cancer agent, and an inhibitor of an antibody response against the agent of the invention.

6. A method according to claim 5 comprising
(i) a composition according to claim 1 and
(ii) a further therapeutic agent suitable for treating carcinoma, optionally wherein the further therapeutic agent is one or more of an immunostimulatory drug, an anti-cancer agent, and an inhibitor of an antibody response against the agent of the invention.

7. The composition according to claim 1, wherein the anti-HER2 antibody is a full length antibody.

8. The composition according to claim 1, wherein the anti-HER2 antibody is an antigen binding fragment of an antibody.

9. The composition according to claim 1, wherein the anti-HER2 antibody is an scFv, a camelid antibody, or an engineered camelid antibody.

10. The composition according to claim 1, wherein the anti-HER2 antibody or antigen binding fragment thereof is a human antibody or a humanized antibody.

11. The composition according to claim 1, wherein the anti-CD3 scFv is a human scFv or a humanized scFv.

12. The composition according to claim 1, wherein the linker in the further moiety that joins the heavy chain variable domain (VH) and light chain variable domain (VL) of insufficient length is a peptide linker of 14 or less amino acids.

13. The composition according to claim 12, wherein the peptide linker is of 10 amino acids.

14. The composition according to claim 12, wherein the peptide linker is of 9 amino acids.

15. The composition according to claim 12, wherein the peptide linker is of 8 amino acids.

16. The composition according to claim 12, wherein the peptide linker is of 7 amino acids.