GB2569692A - T cell antigen receptor chimera - Google Patents

Abstract

A construct for expression in a T cell, the construct providing an antigen receptor chimera (TARC) protein, incorporating the antigen-binding specificity of an alpha beta T cell receptor with a costimulatory-only endodomain is described. The construct may be expressed in gamma delta T cells as shown in the figure or Natural Killer (NK) cells which can allow a tunable response to be provided in such modified cells. Also provided are methods of using such modified cells, and pharmaceutical compositions comprising the same.

Description

(57) A construct for expression in a T cell, the construct providing an antigen receptor chimera (TARC) protein, incorporating the antigen-binding specificity of an alpha beta T cell receptor with a costimulatory-only endodomain is described. The construct may be expressed in gamma delta T cells as shown in the figure or Natural Killer (NK) cells which can allow a tunable response to be provided in such modified cells. Also provided are methods of using such modified cells, and pharmaceutical compositions comprising the same.

Target cell

Figure 2

The claims were filed later than the filing date but within the period prescribed by Rule 22(1) of the Patents Rules 2007.

1/2

Figure 1

Target cell

MHC bound antigen

αβ chains of exogenous TCR

Co-stimulatory signalling domain

2/2

Figure 3 αβ chain portion of TCR

Transmembrane portion

Co-stimulatory domain (signal 2)

B #sc chain

TCR single chain

Linker

Π β chain

ΕΞ

Stalk

CD2S

CDS

Fc

Transmembrane

CD2S

CDS

FceRiy chairs

CD28

41BB

CD2S-418B

DAF18/12

CD27

FceRi γ chain

I

T CELL ANTIGEN RECEPTOR CHIMERA

Field of the invention

The present invention relates to a construct for expression in a T cell, the construct providing an antigen receptor chimera (TARC) protein, incorporating the antigen-binding specificity of an alpha beta T cell receptor with a costimulatory-only endodomain. In particular, the construct may be expressed in gamma delta T cells or Natural Killer (NK) cells which can allow a tunable response to be provided in such modified cells. Also provided are methods of using such modified cells, and pharmaceutical compositions comprising the same.

Background

Several immunotherapy approaches have been used to provide T cells to target cancer or disease cells. Active immunisation or adoptive T-cell transfer are two such approaches. Further therapeutic approaches have utilised modified T cells to provide T cells with exogenous T cell receptors (TCR) or T cells with chimeric antigen receptors (CARs).

T lymphocytes recognise antigen through peptides bound to the major histocompatibility complex (MHC) by means of the T cell receptor (TCR). The TCR expressed on the surface of T cells is a heterodimer with variable domains that is associated with an invariant structure - the CD3 complex. CD3 is considered to be responsible for intracellular signalling following binding to the extracellular portion of the TCR by ligand. Most TCRs include a- and β-chains. The a- and β- TCR chains recognise complexes of MHC and peptide and the CD3 complex results in activation of the T cell. Proposed immunotherapy treatment has considered the provision of exogenous TCRs to T cells wherein the TCRs include a- and β-chains with predefined specificity, generated by several means, including

- modification of the a- and β-chains to promote the preferential association of the chains;

- complexing of the TCR chains by chemical conjugation;

- use of a bispecific antibody to bind exogenous TCR to a T cell;

- transfection of an immune cells with a chimeric TCR wherein the chimeric TCR includes a first segment, either the alpha and beta or gamma and delta chains of a TCR, and a second segment encoding a signal transducing CD3 element of an immune cell (see for example WOOO/312239: Aggen D. H. et al, Single-chain VaVp T-cell receptors function without mispairing with endogenous TCR chains. Gene Therapy (2012) 19, 365-374: Welseng E, et al A TCR-based Chimeric Antigen Receptor Nature Scientific reports 7, 10713: Sharma P et al Recent advances in T-cell engineering for use in Immunotherapy. F1000 Research 2016, 5(F1000 Faculty Rev): 2344).

A number of disadvantages have been determined in providing exogenous TCR to T cells including, TCR mispairing - resulting in approaches using hybrid human TCR, fusion of TCR chains to CD3ζ transmembrane and signalling domains to lead to preferential pairing, substitution of non-native cysteine residues to promote pairing of exogenous TCR chains via formation of an additional disulphide bond. An additional challenge of a TCR gene therapy approach is that levels of exogenous αβ TCRs expressed on the surface of T cells are reduced by concurrent expression of endogenous αβ TCRs, because the total surface levels of TCR are controlled by the availability of the CD3 subunits, in particular the ζ subunits. Previous strategies to overcome this limitation include codon and vector optimisation of exogenous TCR, and providing single-chain TCR chimeras that include the αβ chains and a third domain containing ΰϋ3ζ, for example VαVβCβCD3ζ.

The difficulty of identifying and expanding cancer specific T cell clones or in providing exogenous TCR into T cells led to an alternative immunotherapy approach.

The development of Chimeric Antigen Receptors (CARs) and resulting CAR-T cell therapy is an immunotherapy technique wherein T cells are genetically engineered with a synthetic receptor to recognise and target a particular antigen or protein (cell surface target), independent of HLA restriction. CARs use singlechain variable fragments (scFv) of antibodies to redirect T cell specificity against target antigens independent of TCR-MHC/peptide recognition. In such approaches T cells have been engineered to provide them with chimeric receptors, with a targeting domain, typically using a single-chain fragment variable, a transmembrane domain, and a cytosolic domain that contains signalling elements from a T cell receptor. Such chimeric antigen receptors (CARs) have been developed to include CD3ζ (zeta) or FcsRIy domains (first generation capable of providing signal 1), combinations of the signalling domain of CD3ζ with the cytoplasmic domain of costimulatory receptors, for example from CD28 or TNFR family of receptors (second generation providing signal 1 and signal 2), or multiple costimulatory domains (third generation).

Typically a “classical” 2^nd or 3^rd generation CAR is designed in a modular fashion, typically comprising an extracellular target binding domain, usually a single chain variable fragment (scFv), a hinge region, a transmembrane domain which anchors the CAR to the cell membrane and one or more intracellular signalling domains. The signalling domain is usually comprised of elements of the CD3 zeta (Οϋ3_ζ) chain activation domain which provides TCR-like stimulation (termed signal 1) and elements of the CD28, CD137 (4-1BB), CD134 (0X40), CD244 or ICOS signalling moieties that provide co-stimulatory signals (termed signal 2). In such typical CAR-expressing T cells, both signal 1 and signal 2 are required to release T cells from a quiescent state. In the absence of additional co-stimulatory signals (signal 2), the presence of signal 1 alone is not sufficient to activate T cells and may render them non-responsive I anergic. Thus, the presence of both signals is necessary to induce T cell activation.

Clinical trials employing CAR expressing T cells (CAR-T) have demonstrated the CAR-T approach and in 2017 anti-CD19 CAR T cell therapy was approved for market by the United States’ FDA. Whilst impressive response rates have been observed in CAR-T trials, currently the technology is somewhat limited by a lack of true disease antigens i.e. antigens expressed only in disease state cells and not in healthy cells. To date, the vast majority of CAR-T therapies have been CD19 targeted. CD19 is expressed in B cell malignancies but it is also expressed on healthy B cells. In this context, CD19-targeted CAR-T treatment can be tolerated, however patients are suffering an increased risk of infections as their immune system is significantly compromised when healthy B cells are targeted by the CAR-expressing T cells. This is not the case for the majority of other tumour types, specifically solid tumours, where the targeting of healthy tissue would be intolerable. Thus, whilst the CAR approach has advantages, it can also result in off target toxicity.

A further limitation of CAR-T therapies trialled clinically to date, is the occurrence of relapse due to the development of resistance to the CAR-T therapy. This has been observed in clinical trials of CD19-targeting CAR-T therapy and is achieved by the emergence of cancer cells which exhibit alternate splicing and/or deleterious mutations of the target (CD19) gene. These ‘escape variants’ result in modified target (CD19) protein which is unrecognisable by the scFv portion of the CAR (whilst retaining sufficient of functionality of the gene). This is a limitation of any single targeting approach; in the context of proliferating cells i.e. cancer, this provides a positive selective pressure to abrogate target antigen expression.

Summary of the invention

Crucially, CAR T-cell approaches have been limited to antigens expressed on the surface of a target cell and are not capable of recognising intracellular-associated antigens. Whilst exogenous TCR are capable of recognising intracellular antigens associated with the MHC, as set out above a limitation of exogenous TCR strategies is the availability of CD3ζ subunits in a cell. Limitations in the number of ΰϋ3ζ subunits can impact on antigen sensitivity.

Whilst multiple preclinical and clinical studies to date have employed transduction of alpha beta (αβ) T cells with modified antigen receptors, few have done so using gamma delta (γδ) T cells.

Gamma delta T lymphocytes represent a minor subset of peripheral blood in humans (less than 10%). For example, Gamma delta T cells expressing νγ9νδ2 (gamma 9 delta 2) T cell receptor recognise the endogenous isopentenyl pyrophosphate (IPP) that is over produced in cancer cells as a result of dysregulated mevalonate pathway. The ability of gamma delta T lymphocytes to produce abundant pro inflammatory cytokines like IFN-gamma, their potent cytotoxic effective function and MHC-independent recognition of antigens makes them an important layer of cancer immunotherapy. Gamma delta T cells have been indicated to be able to kill many different types of tumour cell lines and tumour in vitro, including leukaemia, neuroblastoma, and various carcinomas. Further, it has been demonstrated that gamma delta T cells can recognise and kill many different differentiated tumour cells either spontaneously or after treatment with different bisphosphonates, including zoledronate. Human tumour cells can efficiently present aminobisphosphonate and pyrophosphomonoester compounds to gamma delta T cells inducing their proliferation and IFN-gamma production.

Modified alpha beta (αβ) T cells with exogenous modified antigen receptors have largely been used in studies to date as they have been considered to include the intracellular signalling necessary to allow a response in the TCR when exogenous αβ chains recognise MHC bound peptide. This has been advantageous as it allows specific targeting of the modified T cells.

Unlike alpha beta T cells that recognise peptide provided by MHC, gamma delta cells are MHC independent. Whilst CAR technology has been utilised to allow gamma delta T cells to be specifically targeted to antigen, as indicated above such CARs suffer from the disadvantage that they are not able to bind to intracellular antigen presented by MHC.

The present inventors have designed a chimeric antigen receptor based upon the αβ T cell receptor rather than utilising scFv-mediated CAR recognition of target cells.

Rather than considering the intracellular signalling requirement of the TCR when used in αβ T cells, the present inventors have determined that transduction of gamma delta T cells with a T cell antigen receptor chimera (TARC) which includes a first portion that can bind MHC presented antigen and a second portion that provides for activation of the gamma delta T cell can be advantageous.

Accordingly a T cell antigen receptor chimera (TARC) construct is provided that incorporates the antigen-binding capacity of the alpha beta T cell receptor with signalling domains associated with co-stimulation of T cells (signal 2), including but not limited to the signalling domains ofCD27, CD28, CD137 (4-1BB), CD134 (0X40), CD244, DAP10, DAP12 or ICOS. Suitably the construct is not provided with a CD3 domain, or the signalling domain to provide signal 1. Suitably the construct may be provided with a non-functional CD3 domain which is not capable of providing a signal 1. Suitably, the introduction of the TARC construct may be to a gamma delta T cell, providing the modified cell with an antigenspecific costimulatory signal from the TARC to complement endogenous gamma delta TCR signalling induced by cancerous, stressed or infected target cells.

Suitably, the construct comprises a transmembrane domain interposed between the first portion that can bind MHC presented antigen, suitably incorporating alpha beta T cell receptor chains with MHC dependent antigen binding specificity and the second portion that provides for signalling domains associated with costimulation of T cells (signal 2), including but not limited to the signalling domains of CD27, CD28, CD137 (4-1 BB), CD134 (0X40), CD244, DAP10, DAP12 or ICOS. Suitably the construct does not comprise a Οϋ3ζ domain or comprises a non-functional CD3 domain and thus cannot provide for signal 1. Optionally the construct may further comprise spacer portions and or hinge portions interposed between the transmembrane domain and the first portion.

Suitably the construct may be formed by providing a soluble TCR in which the TCR αβ chains were truncated at the level of their transmembrane region, cysteines were added on their constant regions and the two chains were linked by a peptide sequence. A transmembrane domain and a domain comprising a co-stimulatory signalling portion (signal 2 but not signal 1) was also provided. Provision of equimolar amounts of the soluble TCR and the separate transmembrane domain and domain comprising a co-stimulatory signalling portion allows formation of a construct allowing specific binding by the TCR αβ chains to a peptide MHC complex and signal transduction through the signalling domains to provide signal 2.

Suitably a second aspect of the present invention provides a construct encoded by a nucleic acid wherein the nucleic acid comprises a signalling domain comprising a sequence selected from but not limited to:

CD27 endodomain CAACGAAGGAAATATAGATCAAACAAAGGAGAAAGTCCTGTGGAGCCTGCA GAGCCTTGTCATTACAGCTGCCCCAGGGAGGAGGAGGGCAGCACCATCCC CATCCAGGAGGATTACCGAAAACCGGAGCCTGCCTGCTCCCCCTGA

CD28 endodomain

AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCC CGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACG CGACTTCGCAGCCTATCGCTCCTGA

CD137 (41BB) endodomain AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGAC CAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAG AAGAAGAAGGAGGATGTGAACTGTGA

DAP10 endodomain

CTGTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTA CATCAACATGCCAGGCAGGGGCTGA

Suitably a second aspect of the present invention provides a construct encoded by a nucleic acid wherein the nucleic acid comprises an alpha beta TCR with binding specificity to a disease antigen, for example

Suitably T-cell receptor (TCR) sequences may be provided by using known published details of the T-cell antigen specificities as provided for example by https://vdjdb.cdr3.net/. Alternatively, suitable TCR sequences may be selected by analysis of TCR sequences using a panel of peptide and major histocompatibility complexes, for example as discussed in Glanville, J. et al Nature 547, 94-98 (06 July 2017) doi:10.1038/nature22976

Suitably mutagenesis of known TCR sequences or de novo TCR design may also be used.

Suitably the T-cell receptor alpha and I or beta chain constant region can be a full length, native T cell alpha or beta chain constant region or any modified portion thereof.

Suitably modified constant portions can be selected for optimised expression.

Suitably a second aspect of the present invention provides a construct encoded by a nucleic acid wherein the nucleic acid comprises a transmembrane domain encoded by, for example

CD8a chain transmembrane domain

ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCA CTGGTTATCACCCTTTAC

CD28 transmembrane domain TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTA GTAACAGTGGCCTTTATTATTTTCTGGGTG

Suitably, the nucleic acid construct may be provided in combination with a promotor region. The promotor region may be responsive to signalling through a T cell receptor for example, but not limited to, CMV, EF1a, MSCV, MND, PGK, CAG, IRES orUBC.

Suitably the nucleic acid sequence may be provided with a suitable leader sequence to ensure that the construct encoded by the nucleic acid is transported to the cell membrane. Suitable leader sequences will be known in the art.

In embodiments, the signalling domain, alpha beta TCR or transmembrane domain may comprise one or more amino acid mutations, such as insertions, deletions or substitutions, suitably up to 5 mutations selected from insertions, deletions or substitutions to insert/remove/replace one or more amino acids. Suitably at least one, two, three, four or five mutations may be provided wherein the endodomain of the TARC has one, two, three, four or five fewer or greater or different amino acids than: the wild- type signal signalling domain sequence from which it is derived.

In a third aspect, the present invention provides a vector comprising a nucleic acid construct according to the second aspect of the invention. The vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon based vector or synthetic mRNA.

The vector backbone may contain a bacterial origin of replication such as, for example, pBR322 and a selectable marker conferring resistance to an antibiotic, such as, but not limited to, the beta-lactamase gene conferring resistance to the antibiotic ampicillin to allow for sufficient propagation of the plasmid DNA in a bacterial host. Optionally, the vector may include the bacterial and phage attachment sites (attB and attP) of an integrase such as phiC31 in combination with the recognition sites of an endonuclease such as l-Scel to allow the production of minicircles devoid of the bacterial backbone. The vector will also include a sequence which encodes for expression of the TARC linked to a suitable promoter sequence for expression in the target cell of interest, most preferably a gamma delta T cell or NK cell. Optionally, the vector may include an antibiotic resistance gene, for positive selection in mammalian cells and may also include a reporter gene for identification of expression such as, but not limited to, green fluorescence protein (GFP). Additional reporter and/or selection gene expression may be driven from individual promoters, a bi-directional promoter or achieved by use of an IRES or self-cleaving T2A sequence.

The vector may be capable of transfecting or transducing a gamma delta T cell or NKcell.

In a fourth aspect, the present invention provides a cell comprising a construct according to the first aspect of the invention or a vector according to the third aspect of the invention.

Suitably the present invention may provide a cell which expresses at least a first T cell antigen receptor chimera (TARC), the TARC comprising:

(i) an antigen-binding domain based on the alpha beta T cell receptor;

(ii) optionally a spacer or hinge portion;

(iii) a trans-membrane domain; and (iv) an intracellular T activation cell signaling domain (endodomain) wherein the intracellular T cell signalling domain comprises the signalling domain of the CD27, CD28, CD137 (4-1BB), CD134 (0X40), CD244, DAP10, DAP12 or ICOS or combinations of them, but which does not provide a signal 1 signalling domain (for example as would be provided for by Οϋ3ζ).

In embodiments, the process provides a modified cell that expresses a T cell antigen receptor chimera, wherein the TARC has binding specificity to a diseaseassociated antigen. Suitably a disease associated antigen may be selected from a cancer cell associated antigen, a viral antigen, a bacterial antigen, a fungal antigen or a protozoan antigen.

Suitably, a modified T cell may be provided which comprises a T cell antigen receptor chimera wherein the TARC comprises an extracellular antigen binding domain derived from an αβ T cell receptor with binding specificity to a disease antigen, a hinge, a transmembrane domain, and (i) one or more co-stimulatory signalling regions but absent of a CD3 zeta domain (tuneable TARC), or (ii) a CD3 zeta activation domain, or (iii) one or more co-stimulatory signalling regions and a functional CD3 zeta activation domain.

Suitably the cell may be an immune cell such as a gamma delta (γδ) T cell or a natural killer (NK) cell. Suitably, in embodiments the γδ T cell expresses a TCR of any gamma delta TCR pairing from νγ1 to 9 and νδ1 to 8. In embodiments the γδ T cell is of the νγ9νδ2 subtype.

Advantageously presentation of TCRs with binding specificity to peptide MHC complexes on the surface of the modified cell will allow a higher degree of sensitivity to the peptide antigen than similar CAR technologies. Advantageously the TCRs will be able to recognise intracellular foreign peptides that can be bound to an MHC molecule and transported to the surface. This is particularly advantageous over CAR technologies which use MHC independent targeting, but thus typically cannot bind to intracellular targets.

Cells may suitably be obtained from PBMC isolations. The cells may be provided with TARC immediately after isolation, or at a later stage during in vitro culture, for example during in vitro expansion

In a fifth aspect, the present invention provides a method for making a cell according to the fourth aspect of the invention, which comprises the step of introducing: a nucleic acid construct according to the second aspect of the invention or a vector according to the third aspect of the invention, into a cell.

The cell may be part of or derived from a sample isolated from a subject.

The cell used in the method of the fifth aspect of the invention may be from a sample isolated from a patient, a related or unrelated haematopoietic transplant donor, a completely unconnected donor, from peripheral blood, cord blood, differentiated from an embryonic cell line, differentiated from an inducible progenitor cell line, or derived from a transformed cell line.

In a sixth aspect, the present invention there is provided a pharmaceutical composition comprising a plurality of cells according to the fifth aspect of the invention. The composition may be a combination of gamma delta T cell and/or NK cell composition.

The method of genetically modifying a T and/or NK cell to incorporate the nucleic acid encoding the T cell antigen receptor chimera (TARC) may include any technique known to those skilled in the art.

Suitable methodologies include, but are not restricted to, viral transduction with viruses e.g. lentiviruses/retroviruses/adenoviruses, cellular transfection of nucleic acids by electroporation, nucleofection, lipid-based transfection reagents, nanoparticles, calcium chloride based transfection methods or bacterially-derived transposons, DNA transposons or retrotransposons, TALENS or CRISPR/Cas9 technologies.

Suitably, the genetic information may take the form of DNA (cDNA, plasmid, linear, episomal, minicircle), RNA or in vitro transcribed (IVT) RNA. In addition to the genetic information encoding the T cell antigen receptor chimera (TARC), the genetic information may also encode for proteins/enzymes/sequences required to aid integration of the genetic information into the host genome.

When lentiviruses/retroviruses/adenoviruses are employed for transduction, inclusion of chemical reagents as would be understood by those skilled in the art to enhance this process can be used. These include for example, but are not limited to, hexadimethrine bromide (polybrene), fibronectin, recombinant humanfibronectin (such as RetroNectin-Takara Clontech), DEAE dextran and TransPlus Virus Transduction Enhancer (ALSTEM Cell Advancements).

Suitably, incorporation of nucleic acids encoding a T cell antigen receptor chimera (TARC) may be introduced to T cells, peripheral blood mononuclear cells (PBMCs), cord blood mononuclear cells (CBMCs) or tissue derived expanded T cells at any time-point over the culturing period.

In a seventh aspect, the present invention provides a method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition according to the sixth aspect of the invention to a subject.

The method may comprise the following steps:

(i) isolation of a gamma delta T cells and/or NK cell-containing sample from a subject;

(ii) transduction or transfection of the gamma delta T cells and/or NK cells with: a construct according to the first aspect of the invention or a nucleic acid according to a first aspect or a vector according to the third aspect of the invention; and (iii) administering the gamma delta T cells and/or NK cells from (ii) to a subject.

There is also provided a pharmaceutical composition according to the sixth aspect of the invention for use in treating and/or preventing a disease.

There is also provided a cell according to the fourth aspect of the invention for use in treating and/or preventing a disease, for example a cancer, a viral disease, a bacterial disease, a fungal disease or a protozoan disease. Suitably a cancer that may be treated may be, but are not limited to, breast cancer, lung cancer, leukaemia, for example mixed lineage leukaemia, chronic lymphocytic leukaemia or acute lymphoblastic leukaemia. A cancer for treatment may include blastomas, melanomas, sarcomas, haematological cancers, lymphoid malignancies, benign and malignant tumours and malignancies.

Procedures for isolating, genetically modifying and administering T cells to a subject are known in the art. Suitably isolated T cells may be expanded and or activated. Suitably T cells may be transduced or transfected with nucleic acids as discussed herein. T cells can be autologous with respect to the subject or allogeneic, syngeneic or xenogeneic with respect to the subject.

Additionally, there is provided the use of a cell according to the fourth aspect of the invention in the manufacture of a medicament for treating and/or preventing a disease.

Embodiments of the present invention will now be described by way of example only with reference to the accompanying figures in which

Figure 1 illustrates a construct of the present invention in modified cell;

Figure 2 illustrates a construct of the present invention in a modified gamma delta T cell and the resulting signalling when the exogenous αβ chains specifically bind to a peptide MHC complex and the gamma delta TCR binds to its target, for example when a Vy9VS2 (gamma 9 delta 2) T cell receptor recognise the endogenous isopentenyl pyrophosphate (IPP); and

Figure 3 illustrates a nucleic acid construct encoding a construct of the present invention.

Detailed description

CARs are chimeric type I transmembrane proteins which connect an extracellular antigen-recognizing domain to an intracellular signalling domain (endodomain). The antigen recognising domain is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other formats which comprise an antibody-like antigen binding site. A spacer domain is usually necessary to isolate the binder from the membrane and to allow it a suitable orientation. A common spacer domain used is the Fc of lgG1. More compact spacers can suffice e.g. the stalk from CD8a and even just the lgG1 hinge alone, depending on the antigen. A transmembrane domain anchors the protein in the cell membrane and connects the spacer to the endodomain.

First generation CAR designing comprised endodomains derived from the intracellular parts of either the γ chain of the Fc sR1 or CD3ζ. These designs were mostly applied to alpha beta T cells. The activation signal initiated by these domains was sufficient to trigger T-cell killing of cognate target cells in in vitro assays but failed to fully activate the T-cell to proliferate and survive. It was soon discovered that the use of these domains resulted in alpha beta T cell anergy. Second generation CAR designs comprised endodomains which included fusion of the intracellular part of a T-cell co-stimulatory molecule to that of CD3ζ. These can transmit an activating and co-stimulatory signal simultaneously after antigen recognition. The co-stimulatory domain most commonly used is that of CD28. This supplies the most potent co-stimulatory signal - namely immunological ‘signal 2’ - in addition to ‘signal T activation through the CD3ζ endodomain, which triggers T-cell proliferation. Alternative receptor endodomains have also been described, which include TNF receptor family endodomains, such as the closely related 0X40 and 4-1BB which transmit survival signals. The most commonly used costimulatory domains used in clinical trials are CD28 or 4-1 BB (CD137).

Further CAR designs have been made in which the CAR construct utilizes only the co-stimulatory signal domains and lacks a Οϋ3ζ domain or has a Οϋ3ζ domain that is functionally inactive, as described for example by WO 2016/166544.

Crucially, current CAR designs are based on the use of a scFv antigen-binding domain capable of binding a membrane-associated target antigen on tumour or infected cells. Whilst therapeutic application of these CARs have shown excellent clinical efficacy in certain leukemias and lymphomas, their successes in solid tumours have been limited and such CAR constructs show limited use in targeting infection or tumour-associated antigens from an intracellular source. CARs represent a high-affinity receptor, but have a high threshold of target antigens, requiring expression of at least 1,000 molecules on the surface of a target cell.

An alternative approach has been to use technologies based on the T cell Receptor (TCR). Whilst the affinity of the TCR/pMHC interaction is low relative to a CAR, the sensitivity of the T cell receptor is significantly higher and is able to detect only 10 peptide/MHC targets per cell. Unlike CAR technologies, TCRbased approaches are not limited to the detection of surface antigens. As most expressed proteins expressed by a cell can be degraded and presented in the context of the Major Histocompatibility Complex (MHC), the TCR can potentially recognize antigens across the whole proteome, including mutated variants of self-proteins or antigens generated by pathogens.

A major obstacle to generating TCR-based cell therapies is the complicated signalling associated with TCR ligation, with a highly-structured complex of membrane-bound and cytosolic proteins recruited into an immunological synapse that is essential for full T cell activation. Whilst approaches in the art have shown it is feasible to introduce a specific TCR a and/or β chain into T cells in order to redirect T cell specificity towards a defined antigen these approaches have made use of endogenous signalling molecules, allowing the redirected T cell to signal effectively upon antigen recognition. However, an important issue with introduction of a novel TCR-based therapy into αβ T cells is the potential of the new TCR chains to pair with endogenous a or β TCR chains, creating new, unexpected antigen-specificities within targeted T cells with the potential to be pathogenic. Some studies have overcome this hurdle by introducing a singlechain construct incorporating the variable domains of both a and β TCR chains, linked by a short linker sequence. These single-chain TCR variable (scTv) fragments have also been expressed as a membrane tethered protein in αβ T cells, incorporating intracellular CD28 and CD3-zeta domains to provide a maximal stimulatory signal to modified T cells upon engagement of the relevant peptide/MHC complex by the scTv. Crucially, to avoid the risk of on-target, offtumour side-effects, antigens targeted by such an approach would be restricted to those specifically associated with tumour cells. The inclusion in such a construct of the CD3-zeta domain (required to fully activate the αβ T cell) means that any cell presenting the relevant antigen would be targeted by a modified T cell and destroyed. The successful development of such a technology therefore requires an alternative approach.

The present invention exploits the natural ability of γδ T cells to specifically recognise stressed I diseased cells (i.e. cancer cells or infected cells) and combines this with the potent, antigen-directed cytotoxic effector function of T cell antigen receptor chimera technology. A γδ T cell transduced with a classical CAR including a CD3 zeta domain exhibits enhanced effector functions against target cells expressing the CAR-triggering antigen and increased persistence in vivo. The inventors consider gamma delta T cells modified to express a T cell antigen receptor chimera would render cancerous or infected cells, which may be resistant to normal γδ T cells, susceptible to γδ T cell-mediated killing. Suitably, without wishing to be bound by theory, the inventors consider that a γδ T cell coexpressing a TARC will be capable of recognising, and therefore targeting for cytolysis, cells expressing either phosphoantigens or a TARC-binding antigen presented in the context of MHC, thus increasing the range of cells which may be targeted by such modified gamma delta T cells. It is further considered that a limitation of existing CAR-T therapies, the development of resistance due to positive selection of cancer cells that do not express the CAR antigen, will be mitigated by TARC-expressing γδ T cells that will have dual antigen specificity. Suitably such TARC-modified gamma delta T cells could be provided to an allogenic subject, i.e. a different subject to that from which the gamma delta T cells were first obtained.

In embodiments a T cell antigen receptor chimera (TARC) that incorporates a non-functional or inactive CD3zeta domain or a TARC lacking a CD3zeta domain is unable to provide a signal 1 as discussed herein. Suitably, there may be provided a modified γδ T cell comprising a T cell antigen receptor chimera wherein the TARC comprises an extracellular antigen binding domain with binding specificity to an intracellular disease-associated antigen, optionally a hinge, a transmembrane domain, one or more co-stimulatory signalling regions and absence of a functional signal 1 providing domain. As would be appreciated by a person of skill in the art, signal 1 may be provided by a CD3 zeta domain or the like.

In such embodiments the T cell antigen receptor chimera is incapable of providing signal 1 whilst retaining signal 2 function from the presence of one or more co-stimulatory domains. Advantageously this leads to inability for the TARC to elicit cytotoxic effector function in the absence of a signal via the endogenous γδ T cell receptor/ signal 1. Without wishing to be bound by theory, the inventors consider that whilst this costimulatory-only TARC design would be ineffective in a polyclonal αβ T cell population due to lack of signal 1, the expression of such a TARC in an in vitro expanded γδ T cell population provides for an effective costimulatory signal that synergises with signal 1 when the γδ TCR is activated. Suitably a γδ T cell of the νγ9νδ2 isotype expressing a TARC in which there is an absence of CD3ζ domain, or in which the CD3ζ domain is unable to interact with the TARC or has rendered inactive or non-functional, can be provided. In such embodiments, signal 1 can be provided by phosphoantigen stimulation of the endogenous νγ9νδ2 TCR. In such embodiments, suitably only in the presence of phosphoantigens may the TARC elicit cell-mediated cytolysis and cytokine production. Suitably, such a TARC design may exploit the defined specificity of νγ9νδ2 T cells towards phosphoantigens (present only on stressed cells) and permit activation which can be tuned by T cell receptor signalling.

In embodiments it is proposed a gamma delta T cell of the present invention, in particular a νγ9\/δ2 cell or cell populations of gamma delta T cells can be modified to comprise a TARC to direct the gamma delta T cell against a particular antigen presented in the context of an MHC molecule. Suitably the target antigen peptide/MHC complex will be joined to the target cell. This allows the TARC-expressing gamma delta T cell to be brought into proximity with the target cell, in particular a target cell including presenting both peptide/MHC complexes and phosphoantigens and to trigger the activation of the gamma delta T cell. Such T cell antigen receptor chimera (TARC) gamma delta T cells form an aspect of the present invention.

Suitably the antigen recognition domain of the TARC construct can be derived from the alpha beta (αβ) T cell receptor and may be expressed as a full receptor comprised of each chain of the receptor or expressed as only the variable domains of the a- and β-chains expressed as a single construct through a linker sequence to form a single chain TCR (scTv) and selected from libraries of T celL receptors that specifically recognise and are capable of binding to a selected MHC molecules loaded with a specific disease associated antigen.

Suitably a disease antigen bound by a T cell antigen receptor chimera as discussed herein can be an intracellular target antigen or an extracellular antigen acquired by the targeted cell. Suitably, the disease associated antigen can be, for example, an antigen associated with a disease state, for example in cancer or in an infection wherein the disease associated antigen can be on or in the vicinity of a cell to be targeted by the γδ T cell such that the γδ T cell can target said cell to be targeted. Suitably a target antigen can be an antigen found in an intracellular infection, bacterial infection, fungal infection or protozoan infection or can be an active or inactivated viral fragment, a peptide, a protein, an antigenic segment or the like from such a virus. Alternatively the target antigen of the TARC may include a tumour-specific antigen and/or tumour associated antigen.

Suitably the TARC, for example αβ T cell receptor domains provided on the extracellular surface of the gamma delta T cell can be connected via a spacer or fused directly to a transmembrane domain which traverses the cell membrane and connects to an intracellular signalling domain.

Suitably the TARC, for example the αβ T cell receptor domains can be fused via a transmembrane domain to a CD3zeta (providing signal 1) and co-stimulatory immunoreceptor tyrosine-based activation motif (ITAM) (signal 2). Suitably such a TARC can be provided to a gamma delta T cell or an NK cell.

A TARC is generally considered to redirect the T cell specificity to a target antigen and overcomes issues relating to T cell tolerance. As will be appreciated, the target antigen typically can be selected to ensure targeting of the gamma delta T cell towards cells of interest, e.g. tumour cells or virally infected cells, in preference to healthy cells.

As discussed above, in other CAR targeting systems where the chimeric antigen receptor fuses signal 1 and signal 2 components into a single construct, they can provide highly potent and sensitive target-dependent effector responses. However, such CAR targeting systems are not tuneable to the level of cell surface target expressed on a target cell.

It is considered that endogenous Vy9VS2 TCR-mediated recognition provides a TARC-independent activation pathway that allows the TARC response to be “tuned” such that a stimulating signal from the TARC will translate to a functional response only in the context of Vy9VS2 TCR stimulation. This allows, for example a broad range of markers of stressed target cells (for example phosphoantigens) to be targeted specifically with an additional antigendependent mechanism through recognition via a TARC -modified gamma delta T cell. These TARC modified gamma delta T cells of the invention provide a tunable response, wherein for a given tumour-associated antigen, pyrophosphate I phosphonate (drug) dose there is generated a suboptimal signal 1 strength response, with an ability to synergise with signal 2 from a specific “co-stimulatory TARC”.

In embodiments in vitro activation assays may be used to determine relevant drug doses for optimal “tuning response”, to allow maximum discrimination between target tumour lines expressing high levels of the tumour associated antigen (higher signal 2 strength) and relevant non-transformed cells expressing lower levels (generating lower signal strength). In embodiments, multiple TARCs could be utilised to target different tumour I cell types. Such multiple TARCs could be provided on one gamma delta T cell, in particular Vy9VS2 cell, or multiple gamma delta T cells, in particular Vy9VS2 cells, with different TARCs on each cell (a treatment bank) could be generated and the respective TARC gamma delta cell used for a particular tumour and I or cell type.

In embodiments a gamma delta T cell can further comprise an inhibitory T cell antigen receptor chimera (iTARC), wherein the iTARC minimises activation in offtarget cells e.g. non tumour cells wherein the cell surface target is a tumourassociated, but not tumour-specific antigen. To minimise for example, such “on target, off tumour toxicity” the presence of a second antigen on an off target cell which can be bound by the inhibitory TARC will cause the signal provided by any binding of the TARC to the cell surface target to be inhibited.

In embodiments, the gamma delta cell can comprise a further CAR or TARC capable of binding to a different antigen present on a target cell or to soluble signalling proteins present in, for example, a tumour or virally infected cell environment, e.g. IL-12 that can stimulate gamma delta T cell activation and recruitment.

In embodiments, the nucleic acid can encode an extracellular antigen-binding domain that is a single chain protein comprised of the variable domains of the alpha beta T cell receptor that specifically recognises and is capable of binding to an antigen presented in the context of the major histocompatibility complex.

In embodiments, the antigen binding domain of the TARC binds to a peptide antigen derived from a tumour antigen and/or tumour associated antigen. In embodiments a target peptide can be an antigen found in a cancerous cell infection, bacterial infection, fungal infection, protozoan infection or virus infection or can be an active or inactivated viral fragment, a peptide, a protein, an antigenic segment or the like from such a virus.

Suitably, in embodiments of the present invention the extracellular antigen binding domain of the TARC can recognise and bind to a tumour-specific antigen which is present only on tumour cells and not on any other cells and/or a tumour associated antigen which is present on some tumour cells and also some normal cells. Such tumour specific antigens may include, but are not limited to, CD19, EGFRvRIII, ErbB2, GM3, GD2, GD3, CD20, CD22, gp100, NY-ESO-1, carbonic anhydrase IX, WT1, carcinoembryonic antigen, CA-125, MUC-1, MUC-3, epithelial tumour antigen and a MAGE-type antigen including MAGEA1, MAGEA3, MAGEA4, MAGEA12, MAGEC2, BAGE, GAGE, XAGE1B, CTAG2, CTAG1, SSX2, or LAGE1 or viral antigens or combinations thereof or posttranslationally modified proteins that may include, but are not limited to, carbamylated and citrullinated proteins.

Provision of T cell antigen receptor chimera to a gamma delta T cell can be by means known in the art to provide chimeric antigen receptors or other genetic modifications to T cells, for example through use of lentivirus, retrovirus, nonviral systems, transposons or the like.

Example 1

An example of a suitable nucleic acid sequence encoding a first portion relating to alpha beta chains of a TCR which binding specificity to a MHC presented antigen, and a second portion comprising a costimulatory signalling domain providing signal 2, but not signal 1 is provided by figure 3.

Suitably such a nucleic acid is provided for expression in a gamma delta T cell for example by means known in the art, for example using lentiviral delivery to provide the nucleic acid to a cell, for example a gamma delta T cell.

Whilst various embodiments and aspects of the present invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only.

04 19

Claims

1. AT cell antigen receptor chimera (TARC) construct comprising an antigen-binding portion of an alpha beta T cell receptor and a signalling domain

Claims (12)

1. AT cell antigen receptor chimera (TARC) construct comprising an antigen-binding portion of an alpha beta T cell receptor and a signalling domain

5 associated with co-stimulation of T cells (signal 2), optionally wherein the signalling domain is selected from CD27, CD28, CD137 (4-1BB), CD134 (0X40), CD244, DAP10, DAP12 or ICOS.

2. The construct of claim 1 wherein the construct is not provided with a CD3 10 signalling domain to provide signal 1.

3. The construct of claim 1 comprising a non-functional CD3 domain which is not capable of providing a signal 1.

15

4. A construct of any one of claims 1 to 3 comprising a transmembrane domain interposed between a first portion that can bind MHC presented antigen, and a second portion that provides for a signalling domain associated with costimulation of T cells (signal 2), wherein the signalling domain is selected from at least one of CD27, CD28, CD137 (4-1 BB), CD134 (0X40), CD244, DAP10, 20 DAP12orlCOS.

5. A cell comprising a T cell antigen receptor chimera construct of any one of claims 1 to 4 comprising an antigen binding portion of an alpha beta T cell receptor and signalling domains associated with co-stimulation of T cells (signal

25 2) or a vector comprising a nucleic acid encoding the T cell antigen receptor chimera construct of any one of claims 1 to 4.

6. A cell of claim 5 which expresses at least a first T cell antigen receptor chimera (TARC), the TARC comprising:

30 (i) an antigen-binding domain based on the alpha beta T cell receptor;

(ii) optionally a spacer or hinge portion;

(iii) a trans-membrane domain; and (iv) an intracellular T activation cell signalling domain (endodomain)

23 04 19 wherein the intracellular T cell signalling domain comprises the signalling domain of the CD27, CD28, CD137 (4-1BB), CD134 (0X40), CD244, DAP10, DAP12 or ICOS or combinations of them, but which does not provide a signal 1 signalling domain (for example as would be provided for by Οϋ3ζ).

7. A modified cell comprising a T cell antigen receptor chimera wherein the TARC comprises an extracellular antigen binding domain derived from an αβ T cell receptor with binding specificity to a disease antigen, a hinge, a transmembrane domain, and

10 (i) one or more co-stimulatory signalling regions but absent of a CD3 zeta domain (tuneable TARC), or (ii) a CD3 zeta activation domain, or (iii) one or more co-stimulatory signalling regions and a functional CD3 zeta activation domain.

8. A modified cell of claim 7 wherein the cell is an immune cell such as a gamma delta (γδ) T cell or a natural killer (NK) cell.

9. A method for making a modified cell comprising a T cell antigen receptor

20 chimera construct comprising an antigen binding portion of an alpha beta T cell receptor and signalling domains associated with co-stimulation of T cells (signal 2) or a vector comprising a nucleic acid encoding the T cell antigen receptor chimera construct wherein the method comprises the step of introducing a nucleic acid construct encoding the construct of any one of claims 1 to 4 into a 25 cell.

10. A method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition comprising a cell of any of claims 5 to 8 to a subject.

11. A construct of any one of claims 1 to 4 or a cell of any one of claims 5 to 8 for use in medicine.

12. A construct of any one of claims 1 to 4 or a cell of any one of claims 5 to 8 for use in the treatment of cancer, a viral disease, a bacterial disease, a fungal disease, or a protozoan disease.

23 04 19

Intellectual Property Office

Application No: GB1817676.8

Claims searched: 1-12

Examiner:

J.P. Bellia

Date of search: 10 May 2019

Patents Act 1977: Search Report under Section 17

Documents considered to be relevant:

Category Relevant to claims Identity of document and passage or figure of particular relevance X,Y 1, 4-7, 9- 12 Gene Therapy, vol. 19, 2012, Aggen et al, Single-chain ValphaVbeta Tcell receptors function without mispairing with endogenous TCR chains, page 365-374 available online at URL www.nature.com/articles/gt2011104 See Figure 1 X,Y 1, 4-7, 9- 12 Scientific reports, vol. 7, 2017, Walseng et al, A TCR based chimeric antigen receptor, available online at URL www. ncbi. nlm. nih. gov/pmc/articles/PMC5 5 87706/ See Figure 1 X,Y 1, 4, 5, 7- 12 WO 2017/197347 Al (ADICET BIO) See page 29 line 10-30 and Examples 19-23 Y 2,3,6 WO 2018/170475 Al (FRED HUTCHINSON CANCER RESEARCH) See page 58 line 30 page 61 line 11

Categories:

X Document indicating lack of novelty or inventive step A Document indicating technological background and/or state of the art. Y Document indicating lack of inventive step if P Document published on or after the declared priority date but combined with one or more other documents of before the filing date of this invention. same category. & Member of the same patent family E Patent document published on or after, but with priority date earlier than, the filing date of this application.

Field of Search:

Search of GB, EP, WO & US patent documents classified in the following areas of the UKCX :

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