WO2019086512A1

WO2019086512A1 - Modified receptors

Info

Publication number: WO2019086512A1
Application number: PCT/EP2018/079820
Authority: WO
Inventors: Mark Vickers; Robert Barker; Huan CAO
Original assignee: The University Court Of The University Of Aberdeen; Common Services Agency
Priority date: 2017-10-31
Filing date: 2018-10-31
Publication date: 2019-05-09
Also published as: GB201717974D0

Abstract

The present invention relates to modified receptors. The modified receptors of the invention are chimeras comprising domains derived from an immune molecule and a targeting domain. Cells that express the chimeric receptors of the invention, and medical applications thereof, are also encompassed.

Description

MODIFIED RECEPTORS

TECHNICAL FIELD

BACKGROUND

The innate immune system is an evolutionarily ancient system that allows organisms to detect and destroy invading pathogens. The innate immune system clears pathogens predominantly by phagocytosis, which is performed by phagocytic cells; mainly macrophages, neutrophils and dendritic cells.

Immune receptors of the innate immune system (innate immune receptors) are involved in mediating phagocytosis. Unlike the receptors of the adaptive immune system, such as B and T cell receptors, innate immune receptors do not have antigen binding sequences that are randomly generated at the genetic level to produce receptors that bind very diverse targets. Instead, most innate immune receptors bind common pathogen-associated molecular motifs.

Macrophages are an important part of the innate immune system. They express innate immune receptors that bind pathogen-associated molecular motifs, the recognition of which generally results in uptake via phagocytosis and subsequent digestion within phagosomes. In addition, macrophages play an important role in the maintenance of healthy tissues.

Imaging using immunohistochemistry or transgenic animals such as the MacGreen mouse shows that macrophages are extensively present in almost all tissues. Indeed, they may comprise the most numerous cell type in the body and their cytoplasmic extensions infiltrate all tissues and contact most, perhaps all, cells. Macrophages continually monitor the health of other cells, deciding whether they should be allowed to continue to live, or should be eaten by 'efferocytosis', a term that describes the engulfment and degradation of whole host cells. This decision is made on the basis of the relative strengths of 'eat me' versus 'don't eat me' signals. The scavenging function of macrophages is remarkably efficient: in humans, approximately 2-5xlO^u cells undergo apoptosis per day. These cells are only rarely seen and their disposal is immunologically silent.

Most pathogen-associated 'eat me' signals are glycans that are associated with cell walls. For instance, high mannose structures are found on the surface of procaryotes and fungi, and are mainly recognised by macrophages via C-type lectins (a class of innate immune receptors).

In contrast, the main endogenous (self) 'eat me' ligand is believed to be phosphatidylserine (PS), a phospholipid usually confined to the inner leaflet of plasma membranes by the operation of an active process catalysed by flippases. Cell death by either apoptosis or necrosis results in a redistribution of PS to the outer leaflet, where it can be recognised by receptors expressed on the surface of phagocytic cells, such as macrophages. Several receptors for PS have been described, both receptors that bind directly, such as TIM -4, BAIl and Stabilin 2, or indirectly, recognising bridging molecules, such as GAS6. Other 'eat me' signals have also been identified, such as calreticulin recognised by LRP1 and

thrombospondin recognised by the vitronectin receptor. SUMMARY OF THE INVENTION

In studies related to the present invention, the inventors identified the surprising importance of mannose motifs in the recognition of damaged red blood cells (red cells; erythrocytes) by innate immune receptors expressed by phagocytic cells. The inventors also found that one such immune receptor, CD206 ('the mannose receptor'), is important in erythrocyte uptake. The inventors have gone further and shown that such innate immune receptors can be engineered, by chimerisation with heterologous antigen-binding domains, to recognise alternative target motifs. The present disclosure shows that expression of these chimeric immune receptors (CIR) can direct phagocytic cells to pathogens bearing the alternative motifs. Moreover, this disclosure shows that the redirected phagocytic cells can kill the target cells that express a target surface antigen in an antigen-specific manner. In particular, cancer cells can be effectively and selectively targeted. Accordingly, in a first aspect, this invention provides a chimeric immune receptor (CIR) having the formula I:

B - T - N (I)

wherein;

B is an antigen binding domain,

T is a transmembrane domain, and

N is an intracellular domain,

wherein N is the intracellular domain of CD206. In most embodiments, the transmembrane domain (T) comprises the transmembrane domain of a phagocytic receptor. The CIR of the invention comprises an intracellular domain (N) of the mannose receptor, therefore the CIR of the invention may be termed a chimeric mannose receptor.

The B - T - N structure of the CIR of the invention is not to be construed as precluding further domains or motifs from being present at the N-terminal end, at the C-terminal end and/or between the B and T and/or T and N domains. For instance, an extracellular domain may be present between the B and T domains of the CIR of the invention. More particularly, a 'stalk region' (S) may be present between the B and T domains of the CIR of the invention. In these embodiments, the CIR of the invention has the formula of formula II:

B - S - T - N (II) wherein;

B is an antigen binding domain,

S is a stalk region,

T is a transmembrane domain, and

N is an intracellular domain,

The stalk region the stalk region (S) may correspond with the stalk region of the mannose receptor (CD206), which is a part of the mannose receptor extracellular domain.

The skilled person will understand that the antigen binding domain (B) and the intracellular domain (N) (and indeed the transmembrane domain (T) and, where present, the stalk region (S)) of the CIR, are not derived from a single molecule. Rather, N is derived from the mannose receptor (CD206) and B is derived from a separate molecule. The mannose receptor is preferably human CD206. The skilled person will understand that a domain is said to be "of, "from" or "derived from" a particular molecule when the amino acid sequence of the domain corresponds with an amino acid sequence of the particular immune molecule.

In most embodiments, the phagocytic receptor is an innate immune receptor. In some embodiments, the transmembrane domain (T) comprises the transmembrane domain of a phagocytic receptor. In some embodiments, a particular innate immune receptor provides both the transmembrane domain (T) and the intracellular domain (N) of the CIR. In other words, T and N might be both derived from the same phagocytic receptor, namely CD206.

The CIR of the invention is able to mediate phagocytosis of an antigen-bearing cell when the antigen binding domain (B) binds the antigen. In preferred embodiments of this invention, the antigen binding domain (B) binds a marker that is present on a pathogenic cell.

Preferably, the marker is a tumour antigen. In some embodiments, the marker is a cell surface protein or glycoprotein, such as a tumour associated protein or glycoprotein. In some embodiments, the marker is CD 19. CD 19 is a marker of B cell non-Hodgkin lymphoma. The skilled person is able to select other markers for targeting other neoplastic conditions, e.g. lymphomas, leukaemias or solid tumours.

The antigen binding domain (B) of the CIR of the invention preferably comprises an antibody variable region heavy chain (VH) and/or an antibody variable region light chain (VL). In some embodiments, B comprises a single-chain variable fragment (scFv). The skilled person will understand that other single chain antibody domains that confer high affinity antigen- specific binding can be used to derive the B domain, such as heavy chain antibodies e.g. heavy chain camelid antibodies.

This disclosure also provides nucleic acids encoding the CIR according to the invention. Preferably, the nucleic acid includes a promotor that is functionally linked to the nucleic acid sequence that encodes the CIR. The promoter may be a tissue specific promoter, e.g. one that is functional in, and may be specific for, myeloid or monocytoid derived cells. For instance use of macrophage specific promoters such as the fms promoter is envisaged.

Cells expressing the CIR of the invention are also provided. The cell that expresses the CIR will comprise a nucleic acid encoding the CIR. The cell may be a bacterial cell, which is useful for amplifying the nucleic acid of the invention using standard molecular biological techniques. The cell may be a eukaryotic cell for instance a yeast or insect cell, which can be used for producing large quantities of the CIR of the invention. Preferably, the cell is a mammalian cell, e.g. a human cell.

In particular, this invention provides phagocytic cells that express the CIR of the invention. The CIR is expressed at the cell surface (plasma membrane) of the phagocytic cell. In some embodiments, the cell is a macrophage. In some embodiments, the cell is a myeloid cell, such as a monocyte or a monocyte-derived cell. In some embodiments, the cell is a macrophage. In some embodiments, the cell is a macrophage that has been derived from a monocyte, e.g. a human monocyte derived macrophage (HMDM). In other embodiments, the cell is a dendritic cell. The skilled person is able to derive macrophages, dendritic cells and other phagocytic cells from progenitors using standard techniques that are known in the art.

This invention also provides methods of producing a cell that expresses the CIR of the invention, the method comprising: (i) providing a cell that has been obtained from a mammalian subject, (ii) introducing the nucleic acid encoding the CIR into the cell, and (iii) propagating the cell. Preferably, the cell is a hematopoietic stem cell or a hematopoietic progenitor cell. The skilled person will understand that the cell is a mammalian cell e.g. as described elsewhere herein. The mammalian cell may have been obtained by standard apheresis. The mammalian cell may have been enriched from peripheral blood using standard techniques in the art, for instance using immunomagnetic separation techniques, or the like. In some embodiments, the mammalian cell is a CD34+ cell and/or a hematopoietic stem or progenitor cell. Preferably, the mammalian cell of step (i), into which the nucleic acid is to be introduced, is capable of differentiating into a phagocytic cell (i.e., the cell into which the nucleic acid is to be introduced is preferably a CD34+ cell and/or a hematopoietic stem or progenitor cell). In some embodiments, the mammalian cell of step (i) is itself a phagocytic cell. In some embodiments, the mammalian cell is a monocyte, a macrophage or a human monocyte derived macrophage (HMDM). In some embodiments the cell is one that can be differentiated into a phagocytic cell. In those embodiments, step (iii) may be performed in culture conditions that are suitable for differentiating the cell into a phagocytic cell, in order to produce phagocytic cells that express the CIR.

The skilled person will appreciate that a gene therapy vector can be used to introduce the nucleic acid of the invention into the recipient mammalian cell. In some embodiments, the gene therapy vector is a viral vector. The viral vector may be an adenoviral vector, an AAV or a lentiviral vector. For some applications, it is advantageous to use a viral vector that is pseudotyped with an envelope protein that facilitates the transduction of hematopoietic stem cells and/or progenitor cells. In some embodiments, the nucleic acid is introduced into the mammalian cell using the CRISPR-CAS9 system.

In some aspects, the invention provides a therapeutic agent for use in a method of treating a disease in a mammalian subject. The method comprises administering the therapeutic agent to the subject, and the therapeutic agent is either a phagocytic cell that expresses the CIR of the invention or a nucleic acid that is capable of expressing the CIR of the invention in a phagocytic cell. For instance, the therapeutic agent may be a gene therapy vector comprising the nucleic acid that expresses the CIR. In preferred embodiments, the invention provides a therapeutic agent for use in a method of treating a disease in a mammalian subject, wherein the method comprises administering a non-phagocytic cell containing a transgene that encodes the CIR of the invention. The non- phagocytic cell is capable of differentiating into a phagocytic cell that expresses the CIR of the invention. In related aspects, the invention provides a method of treating a disease in a mammalian subject, the method comprising administering the therapeutic agent to the subject, wherein the therapeutic agent is either a phagocytic cell that expresses the CIR of the invention or a nucleic acid that is capable of expressing the CIR of the invention in a phagocytic cell. For instance, the therapeutic agent may be a gene therapy vector comprising the nucleic acid that expresses the CIR.

In embodiments in which the therapeutic agent is a nucleic acid, it may comprise a tissue specific promoter that is adapted to selectively express the CIR in a cell of hematopoietic lineage, for instance in a monocyte or a monocyte-derived cell. This can be advantageous in directing expression of the CIR to the appropriate cell types. Suitable tissue specific promoters include the fms promoter.

In embodiments in which the therapeutic agent is a nucleic acid that is provided as part of a gene therapy vector, this may be a viral vector. Appropriate types of viral vectors include adenoviral vectors, AAV and lentiviral vectors. It may be advantageous to use a viral vector that is pseudotyped with an envelope protein that facilitates transduction of hematopoietic stem cells and/or progenitor cells, to direct transduction to the appropriate cell type. In some embodiments of these aspects, the disease that is treated is cancer. The cancer may be a lymphoma, a leukaemia or a solid tumour.

In some aspects, this invention provides methods of neutralising a pathogenic cell by contacting it, under conditions that allow phagocytosis, with a phagocytic cell that expresses the CIR of the invention. In some embodiments, the pathogenic cell is a cancer cell. The skilled person will appreciate that, in these aspects, the antigen binding domain (B) binds a marker that is present on the pathogenic cell, e.g. a tumour associated antigen that is present on a cancer cell.

Further embodiments according to the present invention will be apparent to the person skilled in the art, e.g. in light of the figures, detailed description and examples that follow.

FIGURES

Figure 1: Panel A shows a schematic showing two closely related exemplary chimeric mannose receptors of the invention, denoted "ChiMRl" (top) and "ChiMRl Martin" (below). Each chimeric mannose receptor is formed of the intracellular (I) and transmembrane domains (T) of the mannose receptor, fused to an anti-CD 19 scFv. Panel B shows a chimeric mannose receptor of the invention present in the plasma membrane of a cell.

Figure 2: Amino acid and cDNA sequences of two examples of anti-CD 19-mannose receptor chimeras of the invention. Restriction endonuclease sites are highlighted (yellow and magenta boxes, respectively) and are shown on both DNA and amino acid sequences for reference. Triple stop codon sequence TGATGATGA is also shown on both DNA and amino acid sequences for reference. The CD 19 scFv amino acid sequence is shown in blue lettering (DIQMT... VTVSS). The stalk 9 amino acid sequence of the mannose receptor is shown in purple lettering (PKIID...MDPSK). The amino acid sequence of the mannose receptor transmembrane is shown in black lettering (PSSNV... AYFFY). The amino acid sequence of the mannose receptor intracellular domain is shown in green lettering

(KKRRV...EHSVI).

Figure 3: Expression plasmid structure and cloning strategy of a chimeric mannose receptor of the invention. Figure 4: Flow cytometry plots show expression of CIR of the invention in THP-1 cells. Panel A shows the gating strategy. Panel B shows overlaid histograms of water-transfected THP-1 (left), ChiMRl transfected THP-1 (centre; tallest peak) and ChiMRl -Martin transfected THP-1 (right). Panel C shows the fluorescence intensity of Flag-PE measured for individual groups under the same conditions shown as dot plots of the non-transfected control, Martin transfected THP-1 and ChiMRl transfected THP-1.

Figure 5: Panel A shows the grid selection for Raji on day 2 with Annexin FITC and A780. The same principle has been used for every dot plot of Panel B, which shows the percentage of apoptosis in Raji, Daudi and Jurkat cell lines. Figure 6: Fluorescence microscopy image of phagocytosis by THP-1 in green and Raji in red. In the yellow circle a phagocytotic event is clearly visible.

Figure 7: Graphs of the incident rate (expressed as a percentage) of phagocytosis or binding of Raji, Daudi and Jurkat cell lines incubated with THP-1 derived macrophages for 4 hours (left) or 24 hours (right). Figure 8: Graphs showing phagocytosis and binding of primary chronic lymphatic leukaemia B cells:

Left hand graph shows, in the first two columns, percentage phagocytosis of CLL B cells by THP-1 derived macrophages transfected with a CIR of the invention and, in the third column, by THP-1 derived macrophages mock transfected with water. Left hand graph shows, in the fourth and fifth columns, percentage binding of CLL B cells by THP-1 derived macrophages transfected with a CIR of the invention and, in the sixth column, by THP-1 derived macrophages mock transfected with water.

Right hand graph shows, in the first two columns, the increase in phagocytosis in THP-1 derived macrophage cells expressing the CIR of the invention as a multiple of the extent of phagocytosis in THP-1 derived macrophage cells subjected to the water (mock) transfection. The first column shows a 2- to 4-fold increase in phagocytosis over that by the mock transfected controls. Right hand graph shows, in the third and fourth columns, the extent of binding of CLL B cells by THP-1 derived macrophage cells expressing the CIR of the invention as a multiple of the extent of binding in THP-1 derived macrophage cells subjected to the water (mock) transfection. No substantial change in binding is observed. DETAILED DESCRIPTION

This invention provides chimeric receptors that combine the intracellular domains from the phagocytosis-initiating mannose receptor (CD206) with an extracellular domain that replaces (at least part of) the original binding domain with ones that recognise ligands expressed on the surface of pathogenic cells, e.g. malignant cells. As part of a project to identify the ligands involved in the uptake of damaged red blood cells by macrophages, the inventors identified an important role for high mannose motifs and identified certain lectins, including CD206, which was not previously considered to be a phagocytic receptor, as being suitable for chimerisation according to this invention. The inventors have shown that CD206 ('the mannose receptor') is important in erythrocyte uptake via efferocytosis.

The clustering of homologous plasma membrane receptors causes co-operative enzymatic activity from their intracytoplasmic (and transmembrane) domains and subsequently triggers phagocytosis. The process is not generally dependent on any functional property of the external ligand binding domains, besides recognition of the ligand. Chimerisation of these receptors to redirect them towards other antigens (particularly pathogenic markers), to thus 'reprogram' phagocytic cells that express the chimeric immune receptor (CIR), represents an important new therapeutic approach. The inventors have shown that such engineering of phagocytic receptors is possible and that reprogrammed phagocytic cells can clear target cells expressing the corresponding target ligand. The antigen binding domain (B) of the chimeric immune receptor (CIR) can be an scFv

(single chain variable fragment) sequence. Many scFv sequences have previously been used in the generation of chimeric T cell receptors (often termed chimeric antigen receptors;

CAR), which are considered to represent an approach to harness effector cells of the adaptive immune system to attack cancers. In contrast, the present invention harnesses the innate immune system. This approach has several advantages over CAR T cells:

Firstly, many tissues do not normally contain T cells and it has proven difficult to induce CAR T cells to enter malignancies arising from these tissues. In contrast, all tissues contain macrophages. Secondly, activated CAR T cells often cause substantial side effects, resulting in stays in intensive care and sometimes causing deaths, which have resulted in the suspension of several current and previous clinical trials. In contrast, the endogenous homeostatic mechanism of phagocytosis is immunologically and metabolically silent. The present invention can be used in therapeutic approaches against any disease mediated by pathogenic cells on which an externally expressed ligand (markers; antigens) is known. Cancers are an important example. A large number of cancer antigens are known, and many of these represent targets for the CIRs of the invention. In particular, the CIR of the invention can direct phagocytic cells to target pathogenic cells displaying a surface antigen. Particular applications include the selective deletion of pathogenic B or T cells in immune mediated disease (e.g. autoimmune disorders) or lymphoproliferative diseases.

Abbreviations

ACD Acid Citrate Dextrose

CIR Chimeric Immune Receptor (may also be referred to as CIIR; chimeric innate immune receptor)

CR Cysteine Rich region

CRD Carbohydrate Recognition Domain

CLL Chronic Lymphocytic Leukaemia

CTFR Cell Trace Far Red

DAMP Damage Associated Molecular Pattern

FITC Fluorescein Isothiocyanate

GNA Galanthus nivalis Lectin

GPA Glycophorin A

HMDM Human Monocyte Derived Macrophages (may also be referred to as MDMO)

NPL Narcissus pseudonarcissus Lectin

O-GlcNAc O-linked N-acetylglucosamine

PBS Phosphate Buffered Saline

PFA Paraformaldehyde

RBC Red Blood Cells

SIM Structured Illumination Microscopy scFv Single Chain Variable Fragment antibody SCD Sickle Cell Disease

TEM Transmission Electron Microscopy

Degree of correspondence

In the context of this disclosure, two sequences are said to correspond with each other when they share at least 50% sequence identity. Sequence identity is determined across the entire length of either sequence. For instance, a given domain might correspond with the intracellular domain of a phagocytic receptor across the full length of the intracellular domain of the phagocytic receptor. In some instances, the degree of sequence identity may be exactly 100%). In some instances, the degree of sequence identity may be at least 50%>, 60%>, 70%>, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% (wherein sequence identity is determined across the full length of either sequence). The skilled person will understand that a given domain that corresponds to a reference domain will typically retain the function of the reference domain. In the context of this disclosure, "at least 50%, 60%, 70%, 75%, etc..." means "at least 50%, at least 60%, at least 70%, at least 75%, etc. Pharmaceutical compositions

Therapeutic agents according to the present disclosure are preferably provided as

pharmaceutical compositions. Pharmaceutical compositions according to the present disclosure, and for use in accordance with the present disclosure, may comprise, in addition to the active ingredient, i.e. a therapeutic agent according to the claims a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous, or intravenous. Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may comprise a solid carrier such as gelatin. For intravenous, cutaneous or subcutaneous injection, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Formulations suitable for parenteral administration (e.g., by injection), include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the therapeutic agent is dissolved, suspended, or otherwise provided (e.g. in a liposome or other microparticulate). Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes that render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required. Dosage

It will be appreciated by one of skill in the art that appropriate dosages of the therapeutic agent, and compositions comprising these active elements, can vary from subject to subject. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health, and prior medical history of the subject. The amount of compound and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.

In general, a suitable dose of the therapeutic agent is in the range of about 100 ng to about 25 mg (more typically about 1 μg to about 10 mg) per kilogram body weight of the subject per day. Flat dosing may also be considered (i.e. not dependent on body weight or body surface area). Where the active compound is a salt, an ester, an amide, a prodrug, or the like, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.

Duration of treatment

A treatment regimen based may preferably extend over a sustained period of time. The particular duration would be at the discretion of the physician. For example, the duration of treatment may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or longer, at least 2, 3, 4, 5 years, or longer. In some embodiments, the duration of treatment will be between 6 and 12 months. In some embodiments, the duration of treatment will be between 1 and 5 years.

Antibodies The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies {e.g., bispecific antibodies), intact antibodies (also described as "full-length" antibodies) and antibody fragments, so long as they exhibit the desired biological activity, for example, the ability to bind a first target protein (Miller et al (2003) Jour, of Immunology 170:4854-4861).

Antibodies may be murine, human, humanized, chimeric, or derived from other species such as rabbit, goat, sheep, horse or camel.

An antibody is a protein generated by the immune system that is capable of recognising and binding to a specific antigen. (Janeway, C, Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen generally has numerous binding sites, also called epitopes, recognized by Complementarity Determining Regions (CDRs) on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. An antibody may comprise a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. The CIR of the present invention is particularly suited to utilising the immunologically active portion of an immunoglobulin as its antigen binding domain (B). The immunoglobulin can be of any type (e.g. IgG, IgE, IgM, IgD, and IgA), class (e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass, or allotype (e.g. human Glml, Glm2, Glm3, non-Glml [that, is any allotype other than G lml], Glml7, G2m23, G3m21, G3m28, G3ml l, G3m5, G3ml3, G3ml4, G3ml0, G3ml5, G3ml6, G3m6, G3m24, G3m26, G3m27, A2ml, A2m2, Kml, Km2 and Km3) of immunoglobulin molecule. The immunoglobulins can be derived from any species, including human, murine, or rabbit origin. "Antibody fragments" comprise a portion of a full-length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')₂, and scFv fragments; diabodies; linear antibodies; fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementary determining region), and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler et al (1975) Nature 256:495, or may be made by recombinant DNA methods (see, US 4816567). The monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al (1991) Nature, 352:624-628; Marks et al (1991) J. Mol. Biol., 222:581-597 or from transgenic mice carrying a fully human immunoglobulin system (Lonberg (2008) Curr. Opinion 20(4):450-459). The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (US 4816567; and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81 :6851-6855).

Chimeric antibodies include "primatized" antibodies comprising variable domain antigen- binding sequences derived from a non-human primate (e.g. Old World Monkey or Ape) and human constant region sequences.

An "intact antibody" herein is one comprising VL and VH domains, as well as a light chain constant domain (CL) and heavy chain constant domains, CHI, CH2 and CH3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variant thereof. The intact antibody may have one or more "effector functions", which refer to those biological activities attributable to its Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody.

Examples of antibody effector functions include Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down regulation of cell surface receptors such as B cell receptor and BCR.

Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different "classes." There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into "subclasses" (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

Pharmaceutical compositions and their use in medicine The pharmaceutical compositions and formulations described herein are useful, for example, in methods of treatment of a disorder as described herein.

Use in Methods of Therapy

Another aspect of the present invention pertains to a pharmaceutical composition or formulation, as described herein, for use in a method of treatment of the human or animal body by therapy, for example, for use a method of treatment of a disorder as described herein. Use in the Manufacture of Medicaments

Another aspect of the present invention pertains to use of a pharmaceutical composition, as described herein, in the manufacture of a pharmaceutical formulation, as described herein, for the treatment of a disorder {e.g., cancer), as described herein. In one embodiment, the medicament comprises the therapeutic agent as described herein.

Methods of Treatment

Another aspect of the present invention pertains to a method of treatment, for example, of a disorder {e.g., cancer) as described herein, comprising administering to a patient in need of treatment a therapeutically effective amount of a pharmaceutical composition or formulation, as described herein.

The subject/patient

The subject/patient may be a chordate, a vertebrate, a mammal, a placental mammal, a marsupial {e.g., kangaroo, wombat), a rodent {e.g., a guinea pig, a hamster, a rat, a mouse), murine {e.g., a mouse), a lagomorph {e.g., a rabbit), avian {e.g., a bird), canine {e.g., a dog), feline {e.g., a cat), equine {e.g., a horse), porcine {e.g., a pig), ovine {e.g., a sheep), bovine {e.g., a cow), a primate, simian {e.g., a monkey or ape), a monkey {e.g., marmoset, baboon), an ape {e.g., gorilla, chimpanzee, orangutan, gibbon), or a human. Furthermore, the subject/patient may be any of its forms of development, for example, a foetus.

In one preferred embodiment, the subject/patient is a human. Examples

The following examples serve to illustrate and support the claimed invention and are not to be construed as limiting in any way. A person who is skilled in the art will appreciate that modifications of the embodiments described herein can be made without departing from the scope of the claimed invention. CD206 / anti-CD19 chimeras

As noted above, the present inventors have shown that the mannose receptor (CD206) can mediate phagocytosis of human erythrocytes. The inventors have constructed a chimeric gene that encodes the intracellular (N) and transmembrane (T) domains of CD206 fused to a single chain antibody domain (B) that binds CD 19 (the extracellular ligand binding domains of CD206 were replaced with the anti-CD 19 scFv sequence). CD 19 is an antigen expressed on several cancers derived from B lymphocytes. The anti-CD 19 scFv (single chain variable fragment antibody) sequence has been previously used in the generation of chimeric T cell receptors (often termed chimeric antigen receptors; CAR), which are considered to represent an approach to harness effector cells of the adaptive immune system to attack cancers. As noted elsewhere herein, the present invention differs from the CAR approach by harnessing the innate immune system, rather than the adaptive immune system.

Expression of the chimeric gene in a macrophage-like cell line enabled these cells to phagocytose CD19 expressing cells, but not control lymphocytes that did not express CD19.

Materials and Methods

Chimera design Two variants of the chimera immune receptor (CIR) were prepared. The design of both constructs includes changes of the extracellular domain of the original mannose receptor (Figure 2). Part of the extracellular domain was replaced by a CD 19 targeting ScFv. The transmembrane region and intracellular domain correspond with these domains of human CD206. A leader peptide was also included in the CIR to promote the correct orientation of the protein in the membrane. Codon optimised cDNA sequences encoding the chimeras were provided by GENEWIZ (NJ, USA) and these were cloned into the shuttle vector px3FLAG- CMV-9 (Sigma- Aldrich). The Flag tag that is present on this vector is detectable with an antibody, which allows confirmation of successful trans fections.

Cells THP-1, Raji, Daudi and Jurkat cell lines were obtained from Sigma- Aldrich. Raji and Daudi were chosen because they are both B-cell lines coming from humans with B-cell lymphomas. The Jurkat cell line was as a negative control.

Transfected cells were assayed for FLAG expression using mouse anti-FLAG PE antibody. Primary cells were collected from 50 ml of blood from chronic lymphatic leukaemia (CLL) patients by venepuncture into Lithium heparin and silicone coated serum (Greiner Bio-One) vacutainers. B cells were extracted from this sample. All donors gave informed consent and the study was approved by the North-East of Scotland Research Ethics Committee

(Application number: REC Ref 14/NS/0009).

Fluorescence microscopy

Fluorescence microscopy was used to visualise the phagocytosis of target cells by cultured THP-1 cells. All images were taken with a Zeiss Axio Observer Zl fluorescent microscope. Samples were visualised at x20 magnification using the DAPI, FITC, and Cy5 channel.

Images were captured using AxioVision 4.8.2 SP3 software.

Flow cytometry

Flow cytometry was used in conjunction with immunostaining of the aimed molecular targets to quantify their expression in the sample. Its major advantage is the fast production of quantitative data and justifies the use of it in the investigations during this project. BD FACScalibur and BD LSRFortessa were used for sample analysis. Unstained and isotype antibodies were used to measure the background signal obtained by each sample.

Macrophage phagocytosis assay

The vectors ChiMRl and ChiMRl -Martin described herein were transfected into THP-1 cells using SG Cell Line 4D-Nucleofector® X Kit L (Lonza) and following the by Lonza recommended protocol. 48 hours after transfection, the THP-1 cells were activated with ^g/ml of Phorbol myristate acetate (PMA) in RPMI containing FCS and PS. Adherent transfected THP-1 cells were cells were fluorescently stained with ΙμΙ_^ CellTracker™ Green CMFDA Dye (Thermofisher) diluted in 1 mL RPMI/PS, which is detectable using

fluorescence microscopy in the 'FITC channel'. Daudi, Raji and Jurkat cells were fluorescently stained using 2μί Cell Trace Far Red Cell Proliferation Kit (Thermofisher) diluted in 2 mL RPMI/PS, which is detectable using fluorescence microscopy, in the 'Cy5 channel'.

The following detection criteria were used to identify binding, phagocytosis and the uptake of degraded cells. ASSIGNED CELL DEFINITION

NUMBER TYPE/EVENT

1 THP-1 All green fluorescent cells

2 Phagocytosis One or more target cells inside a THP-1 (pink, FarRed stained cell in a green, cTG stained cell. Without FITC filter only a black hole is visible)

3 Binding Pink, FarRed stained cells at the outline of green, cTG stained THP-1.

4 Borderline Pink, FarRed stained cells interacting with green, cTG stained cells. Due to dye fading not possible to define as phagocytosis.

5 Degraded cell Small pink dots present in green, cTG stained THP-1.

uptake Without FITC filter, there are small black holes

present.

Table 1. Surface bound Raji, Daudi or Jurkat cells were classified as those associated with a THP-1 cells but not creating green fluorescence obstruction.

The nature of a macrophage is to phagocytose apoptotic cells. Therefore, viable target cells were used in the phagocytosis assays to avoid phagocytosis via the wild type mechanism. Healthy cells were selected based on Annexin and A780 stains (Figure 5).

Results

Cell transfection and expression of chimeras

Flow cytometry analysis revealed that transfection of THP-1 cells was successful, as shown in Table 2. However, the level of expression of the construct did vary within experiments due transient transfection being used for these experiments. CONTROL MARTIN CHIMR1

% HIGH LEVEL EXPRESSION 0.99 11.1 5.37

GMFI 1252 2346 2018

NORMALISED GMFI 1124 2157 1791

Table 2. Percentage of high level expression (first row). Geometrical Median Fluorescence Intensity (second row) and normalised GMFI (third row).

Target cell viability

The analysis of Raji, Daudi and Jurkat cells in showed no rise in apoptosis between day 1 and day 2 but a dramatic increase of apoptosis in all three cell lines on day 3 (Figure 5B).

Phagocytosis assays used target cells on day 2 after passage at the latest to avoid substantial numbers of apoptotic target cells.

Detection of phagocytosis

The success of targeted phagocytosis of CD19-positive cells, such as Raji or Daudi was investigated through the addition of Raji or Daudi cells and compared with the non-CD 19 expressing T cell line Jurkat, when incubated with ChiMRl or Martin transfected THP-1 (also called CIIR THP-1). All phagocytosis data is presented as the percentage of THP-1 in which phagocytosis has occurred. The criteria for detecting phagocytosis are stated in Table 1, above. Figure 6 illustrates that phagocytosis of Raji (shown in the red channel) by THP-1 (shown in the green channel) is clearly visible (indicated by the circle). Figure 7 shows that

phagocytosis of target cells by THP-1 derived macrophages expressing the CIR of the invention is increased. This effect is most pronounced in the ChiMRl -Martin expressing phagocytes incubated with CD 19+ Raji target cells for 24 hours (top-right panel). Increased phagocytosis of CD 19+ Daudi cells and CD 19- Jurkat cells is also observed (top-right panel).

The inventors now consider Jurkat cells to be of limited use as a control because, in general, macrophages tend to phagocytose Jurkat resulting in a higher phagocytosis rate that is difficult to compare with the other groups (Figure 7; left-hand panels). Knocking-out CD 19 expression in an otherwise CD 19+ target cell would yield a more appropriate negative control cell line. The efficacy of the invention was more pronounced in experiments on primary CLL B cells. Figure 8 shows highly significant CIR mediated phagocytosis and indicate that CIR expressing macrophages are able to phagocytose human primary target cells: The left hand graph shows in the first three columns that the percentage phagocytosis of CLL B cells by THP-1 derived macrophages transfected with a CIR of the invention is elevated with respect to the percentage phagocytosis of CLL B cells by THP-1 derived macrophages mock transfected with water. The first two columns of the right hand graph of Figure 3 shows the increase in phagocytosis by THP-1 derived macrophage cells expressing the CIR of the invention, in proportional terms, as a multiple of the extent of phagocytosis in THP-1 derived macrophage cells subjected to the water (mock) trans fection. These experiments demonstrate the surprising effectiveness of the CIR of the invention.

References

1. Mogensen TH. Pathogen recognition and inflammatory signaling in innate immune defenses. Clin Microbiol Rev. 2009;22(2):240-73, Table of Contents. 2. Abbas AK, Lichtman AH, Shiv P. Basic Immunology - Functions and Disorders of the Immune System. 4th Edition ed: Elsevier Saunders; 2014.

3. Kerrigan AM, Brown GD. C-type lectins and phagocytosis. Immunobiology.

2009;214(7):562-75.

4. Cambi A, Figdor CG. Dual function of C-type lectin-like receptors in the immune system. Curr Opin Cell Biol. 2003;15(5):539-46.

5. Barclay AN, Van den Berg TK. The interaction between signal regulatory protein alpha (SIRP alpha) and CD47: structure, function, and therapeutic target. Annu Rev Immunol. 2014;32:25-50.

6. Chmielewski M, Hombach AA, Abken H. Of CARs and TRUCKs: chimeric antigen receptor (CAR) T cells engineered with an inducible cytokine to modulate the tumor stroma.

Immunol Rev. 2014;257(l):83-90.

7. Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nat Rev Immunol. 2008;8(12):958-69. 8. Sharpe M, Mount N. Genetically modified T cells in cancer therapy: opportunities and challenges. Dis Model Mech. 2015;8(4):337-50.

9. p3XFLAG-CMV^™-9 Expression Vector: Sigma-Aldrich; [Available from:

10. Kaur G, Dufour JM. Cell lines: Valuable tools or useless artifacts. Spermatogenesis. 2012;2(l): l-5.

Claims

1. A chimeric immune receptor (CIR) having the formula I:

B - T - N (I)

wherein;

B is an antigen binding domain, T is a transmembrane domain, and N is an intracellular domain; wherein the intracellular domain (N) is an intracellular domain of CD206.

2. The CIR according to claim 1 , wherein the antigen binding domain (B) binds a marker that is present on a pathogen or pathogenic cell.

3. The CIR according to claim 2, wherein the marker is a tumour antigen.

4. The CIR according to claim 2 or claim 3, wherein the marker is a cell surface protein or glycoprotein.

5. The CIR according to claim 2, wherein the marker is CD 19.

6. The CIR according to any one of the preceding claims, wherein antigen binding domain (B) comprises an antibody variable region heavy chain (VH) and/or light chain (VL).

7. The CIR according to claim 6, wherein antigen binding domain (B) comprises a single-chain variable fragment (scFv).

8. A nucleic acid encoding the CIR according to any one of the preceding claims.

9. A cell comprising the nucleic acid according to claim 8.

10. The cell according to claim 9, wherein the cell is a phagocytic cell and wherein a CIR according to any one claims 1-6 is expressed at the cell surface.

11. The phagocytic cell according to claim 10, wherein the cell is a macrophage.

12. The phagocytic cell according to claim 11, wherein the macrophage is a human monocyte derived macrophage (HMDM).

13. The phagocytic cell according to claim 10, wherein the cell is a dendritic cell.

14. The cell according to claim 9, wherein the cell is a CD34+ cell, and/or a hematopoietic stem or progenitor cell.

15. A method of producing the cell according to any one of claims 9-14, the method comprising

(i) providing a precursor cell that has been obtained from a mammalian subject, (ii) introducing the nucleic acid encoding the CIR into the cell, and

(iii) propagating the cell.

16. The method according to claim 15, wherein the precursor cell is the CD34+ cell, the hematopoietic stem cell or hematopoietic progenitor cell according to claim 14.

17. The method according to claim 15 or claim 16, wherein the cell is a CD34+ cell.

18. The method according to claim 15, wherein the cell is a monocyte, a macrophage or a human monocyte derived macrophage (HMDM).

19. The method according to any one of claims 15-18, wherein the nucleic acid is introduced into the cell using a gene therapy vector.

20. The method according to claim 19, wherein the gene therapy vector is a viral vector.

21. The method according to claim 20, wherein the viral vector is an adenoviral vector, an AAV or a lentiviral vector.

22. The method according to any one of claims 15-21, wherein the nucleic acid is introduced into the cell using a CRISPR-CAS9 system.

23. The method according to any one of claims 15-22, wherein the nucleic acid of (ii) comprises a tissue specific promotor that is functionally linked to the nucleic acid sequence that encodes the CIR.

24. The method according to any one of claims 15-23, wherein the cell is a phagocytic cell, or wherein step (iii) is performed in culture conditions that differentiate the cell into a phagocytic cell.

25. A therapeutic agent for use in a method of treating a disease in a mammalian subject, the method comprising administering the therapeutic agent to the subject, wherein the therapeutic agent is;

(a) the phagocytic cell according to any one of claims 10-14,

(b) the phagocytic cell produced by the method of claim 24,

(c) the nucleic acid according to claim 8, or

(d) a gene therapy vector comprising the nucleic acid according to claim 8.

26. A method of treating a disease in a mammalian subject, the method comprising administering the therapeutic agent to the subject, wherein the therapeutic agent is;

(a) the phagocytic cell according to any one of claims 10-14,

(b) the phagocytic cell produced by the method of claim 24,

(c) the nucleic acid according to claim 8, or

(d) a gene therapy vector comprising the nucleic acid according to claim 8.

27. The therapeutic agent for the use according to claim 25 or the method of treatment according to claim 26, wherein the disease is cancer.

28. The therapeutic agent for the use, or the method of treatment, according to any one of the preceding claims, wherein the nucleic acid comprises a tissue specific promoter that is adapted to selectively express the CIR in a myeloid cell such as a neutrophil, monocyte or a monocyte-derived cell.

29. The therapeutic agent for the use, or the method of treatment, according to any one of the preceding claims, wherein the therapeutic agent is a gene therapy vector according to claim 25(d) or claim 26(d) which is a viral vector.

30. The therapeutic agent for the use, or the method of treatment, according to claim 29, wherein the viral vector is an adenoviral vector, an AAV or a lentiviral vector.

31. The therapeutic agent for the use, or the method of treatment, according to claim 29 or claim 30, or the method according to claim 20 or claim 21, wherein the viral vector is pseudotyped with an envelope protein that facilitates transduction of hematopoietic stem cells and/or progenitor cells.

32. A method of neutralising a pathogenic cell by contacting it with the phagocytic cell according to any one of claims 10-14, or with the phagocytic cell produced by the method of claim 24, under conditions that allow phagocytosis.

33. The method of neutralising the pathogenic cell according to claim 32, wherein the pathogenic cell is a cancer cell.