CA2993746C - Therapeutic agents - Google Patents

Abstract

An immunoresponsive cell, such as a T-cell expressing (i)a second generation chimeric antigen receptor comprising: (a) a signalling region; (b) a co-stimulatory signalling region; (c) a transmembrane domain; and (d) a binding element that specifically interacts with a first epitope on a target antigen; and (ii)a chimeric costimulatory receptor comprising (e) a co-stimulatory signalling region which is different to that of (b); (f) a transmembrane domain; and (g) a binding element that specifically interacts with a second epitope on a target antigen. This arrangement is referred to as parallel chimeric activating receptors (pCAR). Cells of this type are useful in therapy, and kits and methods for using them as well as methods for preparing them are described and claimed.

Description

Therapeutic Agents The present invention relates to nucleic acids encoding novel chimeric antigen receptors (CARs), as well as to the CARs 5 themselves, in therapy, cells incorporating the nucleic acids and their use in particular to methods in which they are used to facilitate a T-cell response to a selected target.

Background of the Invention 10 Chimeric antigen receptors (CARs), which may also be referred to as artificial T cell receptors, chimeric T cell receptors (TCR) or chimeric immunoreceptors are engineered receptors, are well known in the art.

They are used primarily to transform immune effector cells, in particular T-cells, so as to provide those 15 cells with a particular specificity.

They are particularly under investigation in the field of cancer immunotherapy where they may be used in techniques such as adoptive cell transfer.

In these therapies, T-cells are removed from a patient and modified so that they express receptors specific to the antigens 20 found in a particular form of cancer.

The T cells, which can then recognize and kill the cancer cells, are reintroduced into the patient.

First generation CARs provide a TCR-like signal, most commonly 25 using CD3 zeta (z) and thereby elicit tumouricidal functions.

However, the engagement of CD3z-chain fusion receptors may not suffice to elicit substantial IL-2 secretion and/ or proliferation in the absence of a concomitant co-stimulatory signal.

In physiological T-cell responses, optimal lymphocyte 30 activation requires the engagement of one or more co-stimulatory receptors (signal 2) such as CD28 or 4-lBB.

Consequently, T cells have also been engineered so that they receive a costimulatory signal in a tumour antigen-dependent manner. 35 An important development in this regard has been the successful design of 'second generation CARs' that transduce a functional antigen-dependent co-stimulatory signal in human primary T 2 cells, permitting T-cell proliferation in addition to turnouricidal activity.

Second generation CARs most commonly provide co-stimulation using modules derived from CD28 or 4 lBB.

The combined delivery of co-stimulation plus a CD3 zeta signal 5 renders second generation CARs clearly superior in terms of function, when compared to their first generation counterparts (CD3z signal alone).

An example of a second generation CAR is found in us Patent No 7,446,190. 10 More recently, so-called 'third generation CARs' have been prepared.

These combine multiple signalling domains, such as CD28+4-1BB+CD3z or CD28+OX40+CD3z, to further augment potency.

In the 3rd generation CARs, the signalling domains are aligned in series in the CAR endodomain and placed upstream of CD3z.

In general however, the results achieved with these third generation CARs have disappointingly represented only a marginal improvement over 2nd generation configurations. 20 The use of cells transformed with multiple constructs has also been suggested.

For example, Kloss et al.

Nature Biotechnology 2012, doi:10.1038/nbt.2459 describes the transduction of T-cells with a CAR comprising a signal activation region (CD3 zeta chain) that targets a first antigen and a chimeric co- 25 stimulatory receptor (CCR) comprising both CD28 and 4 lBB costimulatory regions which targets a second antigen.

The two constructs bind to their respective antigens with different binding affinities and this leads to a 'tumour sensing' effect that may enhance the specificity of the therapy with a view to 30 reducing side effects.

It is desirable to develop systems whereby T-cells can be maintained in a state that they can grow, produce cytokines and deliver a kill signal through several repeated rounds of 35 stimulation by antigen-expressing tumour target cells.

Provision of sub-optimal co-stimulation causes T-cells to lose these effector functions rapidly upon re-stimulation, entering a state 3 known as "anergy".

When CART-cells are sequentially restimulated in vitro, they progressively lose effector properties (eg IL-2 production, ability to proliferate) and differentiate to become more effector-like - in other words, less likely to 5 manifest the effects of co-stimulation.

This is undesirable for a cancer immunotherapy since more differentiated cells tend to have less longevity and reduced ability to undergo further growth/ activation when they are stimulated repeatedly in the tumour microenvironment.

Summary of the Invention The applicants have found that effective T-cell responses may be generated using a combination of constructs in which multiple co-stimulatory regions are arranged in distinct constructs.

According to a first aspect of the present invention, there is provided an immuno-responsive cell expressing (i) a second generation chimeric antigen receptor comprising: (a) a signalling region; (b) a co-stimulatory signalling region; (c) a transmembrane domain; and (d) a binding element that specifically interacts with a first epitope on a target antigen; and (ii) a chimeric costimulatory receptor comprising (e) a co-stimulatory signalling region which is different to that of (b); (f) a transmembrane domain; and (g) a binding element that specifically interacts with a second epitope on a target antigen.

The applicants have found that the efficacy of this system is good and in particular may be better than that achieved using conventional third generation CARs having similar elements. 35 Constructs of the type of the invention may be called 'parallel chimeric activating receptors' or 'pCAR'. 4 In addition, the proliferation of the cells, their ability to maintain their cytotoxic potency and to release IL-2 is maintained over many repeated rounds of stimulation with antigen-expressing tumour cells.

Without being bound by theory, the arrangement of the elements in the pCARs may be facilitating activity.

For example, by definition, one of the co-stimulatory modules in a 3rd generation CAR must be placed away from its natural location 10 close to the inner leaflet of the plasma membrane.

This may cause it not to signal normally owing to impaired access to obligate membrane-associated partner molecules.

Alternatively, close proximity of 2 co-stimulatory signalling modules in a 3rd generation CAR might lead to steric issues, preventing full 15 engagement of one or more downstream signalling pathways.

Both of these issues are avoided in the arrangement of the invention.

Both the signalling moieties (b) and (e) may be fused directly to a transmembrane domain, ensuring that they are both adjacent to the plasma membrane within the cell.

Furthermore, they may be 20 spaced at distinct sites within the cell so that will not interact sterically with each other.

Suitable immuno-responsive cells for use in the first aspect of the invention include T-cells such as cytotoxic T-cells, helper 25 T-cells or regulatory T-cells and Natural Killer (NK) cells.

In particular, the immuno-responsive cell is a T-cell.

Suitable elements (a) above may include any suitable signalling region, including any region comprising an Immune-receptor- 30 Tyrosine-based-Activation-Motif (ITAM), as reviewed for example by Love et al.

Cold Spring Harbor Perspect.

Biol 2010 2(6)1 a002485.

In a particular embodiment, the signalling region comprises the intracellular domain of human CD3 [zeta] chain as described for example in US Patent No 7,446,190, or a variant 35 thereof.

In particular, this comprises the domain, which spans amino acid residues 52 163 of the full-length human CD3 zeta chain.

It has a number of polymorphic forms (e.g.

Sequence ID: gblAAF34793.1 and gblAAA60394.1), which are shown respectively as SEQ ID NO 1 5 and 2: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO 1) 10 RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO 2) 15 As used herein, the term 'variant' refers to a polypeptide sequence which is a naturally polymorphic form of the basic sequence as well as synthetic variants, in which one or more amino acids within the chain are inserted, removed or replaced.

However, the variant produces a biological effect which is similar to that of the basic sequence.

For example, the variant mentioned above will act in a manner similar to that of the intracellular domain of human CD3 [zeta] chain.

Amino acid substitutions may be regarded as "conservative" where an amino acid is replaced with a different amino acid in the same class with broadly similar properties.

Non-conservative substitutions are where amino acids are replaced with amino acids of a different type or class.

Amino acid classes are defined as follows: Class Amino acid examples Nonpolar: A, v, L, I, P, M, F, w Uncharged polar: G, s, T, C, Y, N, Q Acidic: D, E Basic: K, R, H.

As is well known to those skilled in the art, altering the primary structure of a peptide by a conservative substitution 6 may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. 5 This is so even when the substitution is in a region which is critical in determining the peptide's conformation.

Non-conservative substitutions may also be possible provided that these do not interrupt the function of the polypeptide as 10 described above.

Broadly speaking, fewer non-conservative substitutions will be possible without altering the biological activity of the polypeptides.

In general, variants will have amino acid sequences that will be 15 at least 70%, for instance at least 71%, 75%, 79%, 81%, 84%, 87%, 90%, 93%, 95%, 96% or 98% identical to the basic sequence, for example SEQ ID NO 1 or SEQ ID NO 2.

Identity in this context may be determined using the BLASTP computer program with SEQ ID NO 2 or a fragment, in particular a fragment as described 20 below, as the base sequence.

The co-stimulatory signal sequence (b) is suitably located between the transmembrane domain (c) and the signaling region (a) and remote from the binding element (d).

Similarly the co- 25 stimulatory signal sequence (e) is suitably located adjacent the transmembrane domain (f) and remote from the binding element (g) . 30 Suitable co-stimulatory signalling regions for use as elements (b) and (e) above are also well known in the art, and include members of the B7/CD28 family such as B7-1, B7-2, B7-Hl, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA, CD28, CTLA-4, Gi24, ICOS, PD- 1, PD-L2 or PDCD6; or ILT/CD85 family proteins such as LILRA3, 35 LILRA4, LILRBl, LILRB2, LILRB3 or LILRB4; or tumour necrosis Date Re9ue/Date Received 2023-01-03 7 ly members such as 4-lBB, BAFF, BAFF R, CD27, CD30, CD40, DR3, GITR, HVE:M, LIGHT, Lymphotoxi OX4D, R.ELT, TACI, TLlA, TNF-·alpha or TNF RII; or members of the SLAM fan,j_ly such as 2B4, BL;01,1E, CD2, CD2F"-10, CD48, CD58, CD84, 5 C'.D229, CRACC, NTB-_A_ or SLAM:; er members of the Tilv1 family su_ch as TJM-l, TIM-3 or TJM:-4; or other co-stimu1atory mo1ecu les such a_;:; c;u7 r CD96, CD160, C".D200, C':D300a.! CRTI-uVI, DliPl2, Dec-:tir1~1, DPPIV, n 4 i;eta J. r n 4 L;eta The selection of the co-stirr~latory signalling r c,n.s rna :l be selected depending upon the particular use intended for the transfo:t:rned cells.

In_ parti_culax, the co--·stimulato1::{ ~:iqnaJ_1inq regions selected fer (b) and (e) above are these which may work or synerqistically For the co-stirnula ling s .·frJ:r: ( .1--.J) ar1d. ( e) rnay~ .be se1ected from CD28, CD27, ICOS, 4·-:LBB, OX40, CD30, G:I:TR, HVEl'.-'1, DR.3 <..>.r CD40., 20 Ir1 a. pa~ct.icLtlar- ern}Jociirner1t, or1e of· (b) c_r (e) is C[)2B arid tf1e other is 4-lBB or OX40.

In a particular embodiment, (b) is CD28. 25 .Ln another parti.cu1a.r ernbod:i.ment (e) 1.s tJ--lBB or OX40 and :c.n p,:n:ticular, is 4··-:LBB.

In another ,:-:rabodiment, (e) j_s CD27.

Ttte t.:r:ansmembrane domains of (c) and (f) abo,.,e rnay be the saine or different but in particular are different to ensure 30 sepa:r:ation of the corwtructs on the :su:i::face of the cel1.

Selection of different transmembrane domains may also enhance stability of the vector since inclusion of a direct repeat nucleic acid sequence in the viral vector renders it prone to rearrangement, with deletion of sequences between the direct repeats.

Where the transmembrane domains of (c) and (f) are the same however, this risk can be reduced by modifying or 8 "wobbling" the codons selected to encode the same protein sequence.

Suitable transmembrane domains are known in the art and include 5 for example, CD8a, CD28, CD4 or CD3z transmembrane domains.

Where the co-stimulatory signalling region comprises CD28 as described above, the CD28 transmembrane domain represents a suitable option.

The full length CD28 protein is a 220 amino 10 acid protein of SEQ ID NO 3 MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVE VCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKS NGTIIHVKGKHLCPSPLFPGPSKPE'WVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYM 15 NMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO 3) where the transmembrane domain is shown in bold type.

In particular, one of the co-stimulatory signalling regions is 20 based upon the hinge region and suitably also the transmembrane domain and endodomain of CD28.

In particular, which comprises amino acids 114-220 of SEQ ID NO 3, shown below as SEQ ID NO 4.

IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIF 25 WVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO 4) In a particular embodiment, one of the co-stimulatory signalling regions (b) or (e) above is a modified form of SEQ ID NO 4 which includes a c-myc tag of SEQ ID No 5.

The c-myc tag is well known and is of SEQ ID NO 5 EQKLISEEDL (SEQ ID NO 5) 35 The c-myc tag may be added to the co-stimulatory signalling region (b) or (e) by insertion into the ectodomain or by 9 replacement of a region in the ectodomain, which is therefore within the region of amino acids 1-152 of SEQ ID NO 3.

In a particularly preferred embodiment, the c-myc tag replaces 5 MYPPPY motif in the CD28 sequence.

This motif represents a potentially hazardous sequence.

It is responsible for interactions between CD28 and its natural ligands, CD80 and CD86, so that it provides potential for off-target toxicity when CAR T-cells encounter a target cell that expresses either of 10 these ligands.

By replacement of this motif with a tag sequence as described above, the potential for unwanted side-effects is reduced.

Thus in a particular embodiment, the co-stimulatory signalling 15 region (b) of the construct is of SEQ ID NO 6 IEVEQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVA FIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO 6) 20 Furthermore, the inclusion of a c-myc epitope means that detection of the CART-cells using a monoclonal antibody is facilitated.

This is very useful since flow cytometric detection had proven unreliable when using some available antibodies.

In addition, the provision of a c-myc epitope tag could facilitate the antigen independent expansion of targeted CARTcells, for example by cross-linking of the CAR using the appropriate monoclonal antibody, either in solution or 30 immobilised onto a solid phase (e.g. a bag).

Moreover, expression of the epitope for the anti-human c-myc antibody, 9e10, within the variable region of a TCR has previously been shown to be sufficient to enable antibody- 35 mediated and complement mediated cytotoxicity both in vitro and in vivo (Kieback et al. (2008) Proc.

Natl.

Acad.

Sci. USA, 105(2) 623-8).

Thus, the provision of such epitope tags could also be used as a "suicide system", whereby an antibody could be used to deplete CART-cells in vivor in the event of toxicity.

The binding elements (d) and (g) will be different and will bind 5 the same, overlapping or different epitopes.

In a particular embodiment the first and second epitopes are associated with the same receptor or antigen.

Thus the first and second epitopes as described above may, in some cases, be the same, or overlapping so that the binding elements (d) and (g) will 10 compete in their binding.

Alternatively, the first and second epitopes may be different and associated with the same or different antigens depending upon the particular therapy being envisaged.

In one embodiment, the antigens are different but may be associated with the same disease such as the same 15 specific cancer.

As used herein, the term 'antigen' refers to any member of a specific binding pair that will bind to the binding elements.

Thus the term includes receptors on target cells.

Thus suitable binding elements (d) and (g) may be any element which provides the pCAR with the ability to recognize a target of interest.

The target to which the pCARs of the invention are directed can be any target of clinical interest to which it 25 would be desirable to induce a T cell response.

This would include markers associated with cancers of various types, including for example, one or more ErbB receptors or the av~6 integrin, markers associated with prostate cancer (for example using a binding element that binds to prostate-specific membrane 30 antigen (PSMA) ), breast cancer (for example using a binding element that targets Her-2 (also known as ErbB2)) and neuroblastomas (for example using a binding element that targets GD2), melanomas, small cell or non-small cell lung carcinoma, sarcomas and brain tumours.

In a particular embodiment the 35 target is one or more ErbB dimers as described above or the receptor for colony stimulating factor-1 (CSF-lR) or the Uv~6 11 integrin, all of which have been implicated in several solid tumours.

The binding elements used in the pCARs of the invention may 5 comprise antibodies that recognize a selected target.

For convenience, the antibody used as the binding element is preferably a single chain antibody (scFv) or single domain antibody, from a camelid, human or other species.

Single chain antibodies may be cloned from the V region genes of a hybridoma 10 specific for a desired target.

The production of such hybridomas has become routine, and the procedure will not be repeated here.

A technique which can be used for cloning the variable region heavy chain (VH) and variable region light chain (VL) has been described in Orlandi et al., Proc.

Natl Acad.

Sci. (USA) 86: 15 3833-3837 (1989).

Briefly, mRNA is isolated from the hybridoma cell line, and reverse transcribed into complementary DNA (cDNA), for example using a reverse transcriptase polymerase chain reaction (RT-PCR) kit.

Sequence-specific primers corresponding to the sequence of the VH and VL genes are used. 20 Sequence analysis of the cloned products and comparison to the known sequence for the VH and VL genes can be used to show that the cloned VH gene matched expectations.

The VH and VL genes are then attached together, for example using an oligonucleotide encoding a (gly4-ser)3 linker.

Alternatively, a binding element of a pCAR may comprise ligands such as the TlE peptide (binds ErbB homo- and heterodimers), colony-stimulating factor-1 (CSF-1) or IL-34 (both bind to the CSF-1 receptor).

The TlE peptide is a chimeric fusion protein 30 composed of the entire mature human EGF protein, excluding the five most N-terminal amino acids (amino acids 971-975 of proepidermal growth factor precursor (NP 001954.2)), which have been replaced by the seven most N-terminal amino acids of the mature human TGF-~ protein (amino acids 40-46 of pro- 35 transforming growth factor alpha isoform 1 (NP_003227.1)) 12 In another embodiment, a binding element of a pCAR comprises an av~6 integrin-specific binding agent.

The integrin av~6 is now regarded as a target in cancer as it has been found to be strongly upregulated in many types of cancer. av~6 has been 5 identified as a receptor for foot-and-mouth disease virus (FMDV) in vitro by binding through an RGD motif in the viral capsid protein, VPl.

As a result, as described for example in US Patent No. 8,383,593, a range of peptides derived from FMDV and in particular, peptides originating from the VPl protein of FMDV 10 and comprising an RGD motif showed increased binding potency and binding specificity. sequence motif In particular, these peptides comprise the (SEQ ID NO 7) or RGDLX5X6I (SEQ ID NO 8), wherein LX5X6L or LX5X6I is contained within an alpha helical structure, wherein X5 and X6 are helix promoting residues, 20 which have a conformational preference greater than 1.0 for being found in the middle of an [alpha]-helix (from Creighton, 1993 and Pace C. N. and Scholtz J.M. (1998), Biophysical Journal, Vol. 75, pages 422-427).

In particular such residues are independently selected from the group consisting of Glu, 25 Ala, Leu, Met, Gln, Lys, Arg, Val, Ile, Trp, Phe and Asp.

Specific examples of such sequences include SEQ ID Nos 9-11 or variants thereof: YTASARGDLAHLTTTHARHL GFTTGRRGDLATIHGMNRPF or NAVPNLRGDLQVLAQKVART (SEQ ID NO 9) (SEQ ID NO 10) (SEQ ID NO 11) 35 These peptides may form a particular group of binding elements for the CARs of the present application. 13 For selected malignancies such as Hodgkin's lymphoma and some breast cancers, two natural ligands are CSF-1 and IL-34 and these form particularly suitable binding elements for (d) and 5 (g).

They do however bind with different affinities.

The affinity of binding can impact on the activity observed.

It may be beneficial in this case to ensure that the binding element with the lower binding is used as binding element (b) and that with the higher binding affinity is used as binding 10 element (g).

In particular, in an embodiment, the relative affinity of the second generation CAR (i) for its cognate target is lower than that of the partnering TNFR-based chimeric costimulatory receptor (ii).

This does not preclude the use of high or low affinity targeting moieties in each position provided that 15 this relative affinity relationship is maintained.

Thus in the case of the present invention, in a particular embodiment, binding element (b) is CSF-1 which has a relatively low binding affinity, whilst binding element (g) comprises IL-34 which has a higher binding affinity.

Suitably the binding element is associated with a leader sequence which facilitates expression on the cell surface. leader sequences are known in the art, and these include the macrophage colony stimulating factor receptor (FMS) leader 25 sequence or CD124 leader sequence.

In a further embodiment, the cells expressing the pCAR are engineered to co-express a chimeric cytokine receptor, in particular the 4a~ chimeric cytokine receptor.

In 4a~, the Many 30 ectodomain of the IL-4 receptor-a chain is joined to the transmembrane and endodomains of IL-2/15 receptor-~.

This allows the selective expansion and enrichment of the genetically engineered T-cells ex vivo by the culture of these cells in a suitable support medium, which, in the case of 4a~, would 35 comprise IL-4 as the sole cytokine support.

Similarly, the system can be used with a chimeric cytokine receptor in which 14 the ectodomain of the IL-4 receptor-a chain is joined to the transmembrane and endodomains of another receptor that is naturally bound by a cytokine that also binds to the common y chain.

As discussed, these cells are useful in therapy to stimulate a T-cell mediated immune response to a target cell population.

Thus a second aspect of the invention provides a method for stimulating a T cell mediated immune response to a target cell 10 population in a patient in need thereof, said method comprising administering to the patient a population of immuno-responsive cells as described above, wherein the binding elements (d) and (g) are specific for the target cell. 15 In a third aspect of the invention, there is provided a method for preparing an immuno-responsive cell according to any one of the preceding claims, said method comprising transducing a cell with a first nucleic acid encoding a CAR of structure (i) as defined above; and also a second nucleic acid encoding a CAR of 20 structure (ii) as defined above.

In particular, in this method, lymphocytes from a patient are transduced with the nucleic acids encoding the CARs of (i) and (ii).

In particular, T-cells are subjected to genetic 25 modification, for example by retroviral mediated transduction, to introduce CAR coding nucleic acids into the host T-cell genome, thereby permitting stable CAR expression.

They may then be reintroduced into the patient, optionally after expansion, to provide a beneficial therapeutic effect.

Where the 30 cells such as the T-cells are engineered to co-express a chimeric cytokine receptor such as 4ap, the expansion step may include an ex vivo culture step in a medium which comprises the cytokine, such as a medium comprising IL-4 as the sole cytokine support in the case of 4ap.

Alternatively, the chimeric cytokine 35 receptor may comprise the ectodomain of the IL-4 receptor-a chain joined to the endodomain used by a common y cytokine with distinct properties, such as IL-7.

In this setting, expansion of the cells in IL-4 may result in less cell differentiation, capitalizing on the natural ability of IL-7 to achieve this effect.

In this way, selective expansion and enrichment of 5 genetically engineered T-cells with the desired state of differentiation can be ensured.

In a fourth aspect of the invention, there is provided a combination of a first nucleic acid encoding a CAR of (i) above 10 and a second nucleic acid encoding a CCR of (ii) above.

As indicated previously, this combination is referred to as a pCAR.

Suitable sequences for the nucleic acids will be apparent to a skilled person.

The sequences may be optimized for use in the required immuno-responsive cell.

However, in some cases, as 15 discussed above, codons may be varied from the optimum or 'wobbled' in order to avoid repeat sequences.

Particular examples of such nucleic acids will encode the preferred embodiments described above. 20 In order to achieve transduction, the nucleic acids of the fourth aspect of the invention are suitably introduced into a vector, such as a plasmid or a retroviral vector.

Such vectors including plasmid vectors, or cell lines containing them form a further aspect of the invention.

The first and second nucleic acids or vectors containing them may be combined in a kit, which is supplied with a view to generating immuno-responsive cells of the first aspect of the invention in situ.

Parallel chimeric activating receptors (pCAR) encoded by the nucleic acids described above form a further aspect of the invention. 35 Detailed Description of the Invention The invention will now be particularly described by way of example and with reference to the following Figures in which: 16 Figure 1 is a schematic diagram showing a panel of CARs and pCARs (named C34B and 34CB) embodying the invention.

All CARs and pCARs were co-expressed in the SFG retroviral vector with 5 4ap, a chimeric cytokine receptor in which the IL-4 receptor-a ectodomain has been fused to the transmembrane and endodomain of IL-2 receptor-p.

Use of 4ap allows selective enrichment and expansion of gene-modified T-cells by culture in IL-4, since it recruits the gamma c (ye) chain.

Figure 2 shows the results of an experiment using CARs shown in Figure 1. T-cells (1 x 106 cells) expressing these CARs and pCARs (or untransduced (UT) as control) were co-cultivated in vitro for 24 hours with T47D tumour cells that express (T47D-FMS) or 15 lack (T47D) the cognate target antigen (Colony-stimulating factor-1 receptor (CSF-lR), encoded by c-fms).

Residual viable tumour cells were then quantified by MTT assay.

Figure 3 shows a representative experiment in which T-cells that 20 express CARs and pCARs of Figure 1 (or untrans(duced) T-cells as control) were subjected to successive rounds of Ag stimulation in the absence of exogenous cytokine.

Stimulation was provided by weekly culture on T47D FMS monolayers and T-cell numbers were enumerated at the indicated intervals.

Figure 4 shows pooled data from 7 similar replicate experiments to that shown in Figure 3, indicating the fold expansion of CAR T-cells that occurred in the week after each cycle of stimulation.

Figure 5 shows illustrative cytotoxicity assays performed at the time of stimulation cycles 2, 6, 9 and 12 in the experiment shown in Figure 3.

This follows from the testing of T-cells for their ability to to kill T47D FMS and unmodified T47D monolayers 35 (MTT assay), twenty four hours after the time of each restimulation cycle. 17 Figure 6 shows the results of testing of supernatant, removed from cultures one day after each cycle of stimulation, for IL-2 and IFN-y content by ELISA.

Figure 7 demonstrates the establishment of an in vivo xenograft model of CSF-lR-expressing anaplastic large cell lymphoma, which allowed subsequent testing of anti-tumour activity of CAR and pCAR-engineered T-cells.

The model was established using K299 10 cells, engineered to express firefly luciferase (luc) and red fluorescent protein (RFP).

Figure 7A shows tumour formation following the intravenous injection of the indicated doses of K299 luc cells, quantified by bioluminescence imaging (BLI).

Representative BLI images are shown in Figure 7B in mice that 15 received 2 million tumour cells.

Expression of RFP+ tumour cells (Figure 7C) in the indicated tissues are shown, demonstrating that tumours only formed in lymph nodes in this model.

Expression of the CSF-lR on five representative lymph node tumours is shown in Figure 7D.

Figure S shows the results of therapeutic studies in which K299 luc cells were injected intravenously in SCID Beige mice (n=9 per group, divided over 2 separate experiments).

After 5 days, mice were treated with CART-cells.

Pooled bioluminescence 25 emission from tumours is shown in Figure SA.

Bioluminescence emission from individual mice is shown in Figure 8B and survival of mice shown in Figure SC.

Figure 9 shows the weights of animals used in the therapeutic 30 study over time.

Figures 10-13 show the results of analysis of the expression of 'exhaustion markers' from dual CAR (C34B) expressing T-cells of the invention where Figure 10 shows the results for PDl 35 analysis, Figure 11 shows the results for TIM3 analysis, Figure 18 12 shows the results of LAG3 analysis and Figure 13 shows the results for 2B4 analysis.

Figure 14 is a schematic diagram of a panel of CARs and 5 constructs targeted to the integrin av~6 which have been prepared including a pCAR (named SFG TIE-41BB/A20-28z) embodying the invention. A20-28z is a second generation CAR that is targeted using the A20 peptide derived from foot and mouth disease virus. A20 binds with high affinity to av~6 and with 85- 10 1000 fold lower affinity to other RGD-binding integrins. C20-28z is a matched control in which key elements of A20 have been mutated to abrogate integrin binding activity.

All CARs have been co-expressed with 4ap, as described in Figure 1. 15 Figure 15 is illustrating Cells were a series of histograms obtained by flow cytometry integrin expression in A37 5 puro and Panel cells. stained with anti-~6 (Biogen Idec) followed by secondary conjugated, anti-mouse PE, Bio-Techne) . anti-av~3 Gates were or set anti-av~5 based on (both APC secondary 20 antibody alone or isotype controls.

Figure 16 is a series of graphs illustrating the cytotoxicity of CARs including the pCARs of the invention targeted to av~6.

Tcells expressing the indicated CARs and pCARs were co-cultivated 25 with av~6-negative (Panel and A375 puro) or av~6-positive (Bxpc3 and A375 puro ~6) tumour cells.

Data show the mean± SEM of 2-7 independent experiments, each performed in **p<0.0l; ***p<0.001. *p<0.05; 30 Figure 1 7 is a series of graphs showing production of IFN-y by CARs including pCARs of the invention, targeted to av~6. T-cells expressing the indicated CARs and pCARs were co-cultivated with av~6-negative (Panel and A375 puro) or av~6-positive (Bxpc3 and A375 puro ~6) tumour cells.

Data show the mean ± SEM of 5-6 35 independent experiments, each performed in duplicate. *p<0. 05; **p<0.0l; ***p<0.001; ns - nots 19 Figure 18 shows the results of re-stimulation experiments using the CAR and pCAR-engineered T-cells described above and indicating the ability of A20-28z/T1E-41BB pCAR T-cells to 5 undergo repeated antigen stimulation, accompanied by expansion of T-cells and destruction of target cells that do (Bxpc3) or do not (Panel) express the av~6 integrin.

Figure 19 shows the results of re-stimulation experiments using 10 pCAR-engineered T-cells in which A20-28z was co-expressed with T1E-41BB, T1E-CD27 or T1E-CD40, allowing the comparative evaluation of co-stimulation by additional members of the TNF receptor family.

Control T-cells were non-transduced (NT) while CARs contained truncated (tr) endodomains. T-cells were re- 15 stimulated on target cells that do ( Bxpc3) or do not (Panel) express the av~6 integrin, making comparison with unstimulated T-cells.

In the case of Bxpc3 cells, superior expansion (Figure 19A) accompanied by sustained cytotoxic activity (Figure 19B) was observed with A2 0-2 8 z/T1E-CD27 T-cells.

By contrast, with Panel cells, superior expansion (Figure 19A) accompanied by sustained cytotoxic activity (Figure 19B) was observed with A20- 28z/T1E-CD27 T-cells.

These data demonstrate that additional members of the TNF receptor family can also deliver costimulation using the pCAR format.

Example 1 A panel of CARs targeted against the CSF-1 receptor (encoded by c-FMS), which is over-expressed in Hodgkin's lymphoma, 30 anaplastic large cell lymphoma and some solid tumours such as triple negative breast cancer were prepared and are illustrated schematically in Figure 1.

The panel of CARs included both second and third generation CARs with either of the two natural ligands, CSF-1 or IL-34, as the targeting moieties.

Although 35 both CSF-1 and IL-34 bind to CSF-1 receptor, IL-34 binds with much higher affinity (34-fold higher than CSF-1).

The constructs SFG C28 ~ and SFG CTr were cloned in the SFG retroviral vector as NcoI/XhoI fragments, ensuring that their start codons are at the site of the naturally occurring NcoI site, previously occupied by the deleted env gene.

Gene 5 expression is achieved from the Moloney murine leukaemia virus (MoMLV) long terminal repeat (LTR), which has promoter activity and virus packaging of the RNA is ensured by the MoMLV * packaging signal, which is flanked by splice donor and acceptor sites.

All other constructs Polymerase Incomplete PIPE cloning method is were designed and cloned using the Primer Extension (PIPE) cloning method. a PCR-based alternative to conventional restriction enzyme- and ligation-dependent cloning methods.

It 15 eliminates the need to incorporate restriction sites, which could encode additional unwanted residues into expressed proteins.

The PIPE method relies on the inefficiency of the amplification process in the final cycles of a PCR reaction, possibly due to the decreasing availability of dNTPs, which 20 results in the generation of partially single-stranded (PIPE) PCR products with overhanging 5'ends. A set of vector-specific primers was used for PCR vector linearization and another set of primers with 5' -vector-end overlapping sequences then used for insert amplification, generating incomplete extension products 25 by PIPE.

In a following step, the PIPE products were mixed and the single-stranded overlapping sequences annealed and assembled as a complete SFG CAR construct.

Successful cloning was confirmed by diagnostic restriction digestion. DNA sequencing was performed on all constructs to confirm that the predicted 30 coding sequence was present, without any PCR-induced mutations (Source Bioscience, UK).

The panel included two ''dual targeted" Chimeric Activating Receptors (pCARS) in which CSF-1 or IL-34 are coupled to 28z and 35 4-lBB, or vice versa.

The dual targeted pCAR combinations were then stoichiometrically co-expressed in the same T-cell population using a Thosea Asigna (T)2A-containing retroviral 21 vector.

One of these CARs was designated 'C34B' (CSF1-28z plus IL34-41BB) and the other was named '34CB' (IL34-28z plus CSFl 41BB). 5 In these dual targeted CAR T-cells, both co-stimulatory motifs (CD28/ 4-lBB) are placed in their natural location, close to the membrane, physically separated from each other and co-expressed in the same T-cell. 10 All CARs were co-expressed with an IL-4 responsive 4aJ3 receptor using an additional T2A element in the vector.

This enables enrichment/ expansion of T-cells using IL-4, making it easier to compare the function of these diverse cell populations after selection.

The main focus of the experiments was to test the behaviour of the T-cells on repeated re-stimulation with tumour target cells that either express or lack the FMS/ CSF-1 receptor target.

In each cycle, 1 million of the indicated IL-4 expanded CART-cells 20 were suspended in RPMI + human AB serum and cultured with a confluent monolayer (24 well dish) of the antigen-expressing target (T47D FMS) or antigen null target (T47D).

Thereafter, if the CART-cells had persisted and destroyed the 25 monolayer, 1 million T-cells were removed and re-stimulated in an identical manner each week.

Total cell number was extrapolated at each time-point depending on the expansion of Tcells that occurred in each weekly cycle. 30 Throughout all of these experiments, T-cells were cultured in the absence of any exogenous cytokine such as IL-2 or IL-4 - so they had to make their own cytokines in order to persist and expand.

Cytokine ( IFN-y and IL-2) production was measured by ELISA in supernatants harvested from T-cell/ tumour cell co- 35 cultures, providing a second marker of effective co-stimulation. 22 It was found (Figure 2) that on their first exposure to a tumour monolayer that expresses target (FMS encoded CSF-1 receptor), all CARs that are predicted to kill do so (pooled data from 12 expts).

The controls are UT (untransduced), P4 (targets an 5 irrelevant antigen, PSMA) and CT4 in which the endodomain is truncated.

As expected, none of the CAR T-cells kill tumour cells that lack CSF-1 receptor (T47D).

A representative re-stimulation experiment is shown in Figure 3. 10 Pooled re-stimulation data from 7 experiments is shown in Figure 4.

In this case, proliferation on the first cycle was similar for most of the constructs, although the IL-34 targeted second and third generation constructs were poorer.

This may be because the affinity of the IL-34 targeting moiety is too high.

In the later cycles however, the C34B dual pCAR combination (a CSF-1 targeted 28z second generation CAR co-expressed with an IL-34 targeted 4-lBB co-stimulatory motif) consistently emerged as clearly superior.

In the experiment shown 2 4 hours after the time in Figure of each 3, supernatant re-stimulation analysed for cytokine content (IFN-y and IL-2) was collected cycle and was by ELISA.

The percentage of residual tumour cell viability was measured by MTT 25 assay (representative examples shown in Figure 5).

The cytokine production results are shown in Figure 6.

It was found that only the C34B CAR T-cells retained the ability to make IL-2 throughout each cycle of stimulation.

This was lost by all of the other CAR combinations after the first cycle.

Sustained 30 retention of the ability to make IL-2 through recursive restimulation is not usually seen with CAR T-cells and this suggests that this delivery of dual co-stimulation is fundamentally altering the differentiation of these cells in vitro, delaying the onset of anergy. 23 Number of viable T-cells post mono layer destruction on consecutive cycles of Ag-stimulation was also monitored and the results are shown in Figure 5.

After the second cycle of restimulation, all CARs except C34B begin to lose the ability to 5 achieve CSF-lR-dependent tumour cell killing.

By contrast, Tcells that express C34B retain antigen-dependent potency in this cytotoxic assay for up to 13 iterative cycles of re-stimulation, but never elicit cytotoxicity against unmodified T47D cells. 10 Also, so-called "exhaustion markers" on these T-cells (PDl, TIM3, 2B4 and LAG3) were also measured by flow cytometry.

The results are shown in Figures 10-13.

As expected, the percentage of T cells that expressed various exhaustion markers progressively increased on the re-stimulated T-cells, but this 15 did not retard the proliferation, tumour cell destruction or cytokine release by the C34B cells, upon antigen stimulation.

This suggests that the superior function of C34B is not the result of delayed upregulation of exhaustion markers. 20 In summary, the pCAR approach of the invention seems to maintain the cells in a state whereby they retain responsiveness to antigen through more cycles of re-stimulation.

There are indications that it may retard differentiation beyond controlled memory state and it appears to delay the onset of anergy while 25 retaining the ability of the cells to make IL-2 upon activation.

Example 2 Analysis of effects in vivo 30 A panel of CARs used in Example 1 above were tested for antitumour activity using a highly aggressive in vivo xenograft model in which the CSF-1 receptor target is expressed at low levels and in which disease is disseminated throughout lymph nodes (Figure 7).

Tumour cells were tagged with firefly 35 luciferase, allowing the non-invasive monitoring of disease burden. 24 SCID/Beige mice were randomised into 6 groups ( 9 animals per group combined over two independent experiments) and were inoculated intravenously (IV) with 2x10 6 K299 tumour cells, resuspended in 200μL PBS.

On day 5, the groups were treated with 5 one of the therapeutic regimens indicated below: • C4B group: 20x10 6 C4B T-cells IV • C34B group: 20xl06 C34B T-cells IV • 43428Bz: 20xl06 43428Bz T-cells IV • 34CB group: 20x106 34CB T-cells IV 10 • UT (Untransduced) group: 20x10 6 untransduced T-cells IV • NT (Non-treated) group: 200μL PBS IV Tumour growth was monitored using bioluminescence imaging (BLI) at appropriate time-points for the duration of the study.

The results are shown in Figure 8. system was that of the pCAR, C34B, Again, the best performing indicated by lower average BLI emission (Figure 8A-B), delayed tumour progression or tumour regression, leading to prolonged survival of mice (Figure SC).

Animals were weighed throughout the experiment and no significant toxicity was noted (Figure 9).

Example 3 Selection of targeting moieties to engineer pCARs that elicit Tcell activation in an av~6-dependent manner.

A panel of CARs that target av~6 integrin alone or together with the extended ErbB family were prepared and are shown schematically in Figure 14.

The binding element used in this 30 case was A20 peptide (SEQ ID NO 11) derived from the GH-loop of the capsid protein VPl from Foot and Mouth Disease Virus ( serotype 01 BFS) (US8, 927, 5 CJ. J , Th.is was placed downstream of a CD124 signal peptide and fused to CD28 and CD3( endodomains to form A20-28(, a 2nd generation CAR. A control (C20-28() was 35 prepared comprising a similar construct but with a scrambled targeting peptide (named C20) in which the key RGDL motif was replaced with AAA.A. A second control comprised A20 fused to a CD28 truncated endodomain (A20-Tr).

To create the pCAR of the invention (named TIE-41BB/A20-28z), 5 A20-28z was co-expressed with a chimeric co-stimulatory receptor comprising a pan-ErbB targeted peptide (TlE) fused to a CD8cx transmembrane and a 41BB endodomain.

Where indicated, CARs were co-expressed with the 4cxR, chimeric 10 cytokine receptor to allow for IL-4-mediated enrichment in vitro.

Equimolar co-expression of the IL-4-responsive 4cxR, chimeric cytokine receptor, in which the IL-4 receptor ex ectodomain is fused to the transmembrane and endodomain of the shared IL-2/15 receptor R,, was achieved using a Thosea Asigna 15 (T)2A ribosomal skip peptide.

These chimeric molecules were expressed in human T-cells by retroviral gene transfer.

The integrin expression pattern of cancer cell lines A375 was assessed using flow cytometry (Figure 15), and these were 20 separated into cxvR,6-negative (Panel and A375 puro) or cxvR,6- positive (Bxpc3 and A375 puro R,6) tumour cells.

These cells were co-cultured with CART-cells at an effector:target ratio of 1:1 for either 24, 28 or 72 hours, after which time, cytotoxicity was assessed by MTT assay and expressed relative to 25 untreated tumour cells.

The results are shown in Figure 16.

These data show that A20-28z CART-cells kill all target cells that express cxvR,6 integrin (Bxpc3 and A375 R,6 puro), but spare targets that lack this integrin (Panel and A375 puro).

Secondly, 30 the control CARs C20-28z and A20-Tr are inactive in these assays.

Thirdly, T-cells that express the T1E-41BB/ A20-28z pCAR cause efficient killing of target cells that express cxvR,6 integrin (Bxpc3 and A375 R,6 puro).

All of these results are as expected.

Notably however, T-cells that express the T1E-41BB/ 35 A20-28z pCAR also cause the killing of target cells that lack cxvR,6 (Panel and A375 puro) This indicates that, within a pCAR configuration, the ability of the A20 peptide to bind non-cxvR,6 26 integrins with low affinity is sufficient to trigger the activation of these engineered T-cells.

Production of IFN-y by the pCAR and control engineered T-cells 5 was then assessed.

Tumour cells that lacked av~6 (Panel and A375 puro) or expressed av~6 (Bxpc3 and A375 puro ~6) were cocultured with genetically engineered T-cells at an effector:target ratio of 1:1 and supernatant was collected after 24, 48 or 72 hours.

Levels of IFN-y were quantified by ELISA 10 (eBioscience).

The results are shown in Figure 17.

As expected, the controls did not generate significant quantities of IFN-y while A20-28z CAR T-cells released IFN-y when cultured with av~6-positive (Bxpc3 and A375 puro ~6) tumour cells.

Notably, Tcells that express the pCAR of the invention, TIE-41BB/A2 Oz, 15 produce more IFN-y than A20-28z T-cells when cultured with av~6- positive (Bxpc3) tumour cells.

In addition, TIE-41BB/A2Qz+ Tcells produced IFN-y when cultured with av~6-negative (Panel and A37 5 puro) tumour cells.

Once again, this demonstrates that, within a pCAR configuration, low affinity binding of the A20 20 peptide to non-av~6 integrins is sufficient to trigger the activation of these engineered T-cells.

Next, the CART-cell populations were re-stimulated bi-weekly in the absence of IL-2 support on Panel (av~6 negative) or Bxpc3 25 tumour cells (av~6 positive) Tumour cells were co-cultured with CAR T-cells derived from a patient with pancreatic ductal adenocarcinoma (PDAC) at an effector:target ratio of 1:1 (Figure 18). T-cells were initially added at 2x10 5 cells/well and were counted 72hrs after co-culture to assess expansion (top panels). 30 Cytotoxicity was assessed at 72hrs post-addition of T-cells by MTT assay (bottom panels) If there were a sufficient number of T-cells (2x10 5 ), T-cells were re-stimulated on a fresh tumour monolayer and the process repeated a further 72hrs later. 35 Results are shown in Figure 28z/T1E-41BB+ T-cells undergo 18.

These illustrate that A20- a number of rounds of expansion 27 accompanied by IL-2 release (data not shown) and destruction of cxvl3 6+ Bxpc3 cells.

Once again, they also underwent a number of rounds of expansion accompanied by IL-2 release and destruction of Panel tumour cells.

Overall, the results clearly showed that the pCAR comprising A20-28z/TlE-41BB compared to Furthermore, a the exhibits enhanced generation CAR A20-28z/TlE-41BB+ in vitro targeted T-cells functionality against also cxvl36. undergo 10 activation by Panel or A375 puro cells, which express minimal to undetectable levels of this integrin.

Taken with the findings obtained using the C34B pCAR (examples 1 and 2) , this indicates that the pCAR configuration allows T-cell activation to occur upon serial re-stimulation when a high affinity binding interaction occurs with the 41BB CCR while a lower affinity interaction occurs with the 28z 2nd generation CAR.

Example 4 Use of an alternative TNF receptor family member, CD27 to engineer a functional pCAR.

Using the A20-28z/TlE-41BB pCAR as starting material, additional pCARs were engineered in which the 41BB module was replaced by alternative members of the TNF receptor family, namely CD27 or CD4 0.

Control pCARs were engineered in which endodomains were truncated (tr).

Target cells that express (Bxpc3) or lack (Panel) cxvl36 were plated at a density of 5xl04 cells per well of a 24 well plate.

After 24 hours, 5xl04 pCAR T-cells were added to target cells or empty wells ("unstimulated"), without exogenous cytokine support.

After a further 72 hours, T-cells were 30 harvested from the wells and were counted ( Figure 19A) .

An MTT assay was performed to determine the percentage viability of the residual target cells, making comparison with control target cells that had been plated without addition of T-cells (Figure 19B).

If T-cells proliferated after each cycle of stimulation, 35 they were re-stimulated on fresh target cells, exactly as described above.

Proliferation of pCAR T-cells (Figure 19A) and MTT assay (Figure 19B) were performed after 72 hours as before. s 28 Iterative re-stimulation of pCAR T-cells and assessment of target cell killing was continued in this manner until T-cells no longer proliferated over the course of each 72 hour cycle.

These data once again confirm the superior functionality of the A20-28z/T1E-41BB pCAR when T-cells are stimulated on Panel target cells, indicated by sustained T-cell proliferation and tumour cell killing.

This provides further confirmation that low affinity binding of the A20 peptide to non-cxvl36 integrins is sufficient to trigger the activation of these engineered Tcells.

Notably however, the A20-28z/T1E-CD27 pCAR achieved the greatest level of proliferation (Figure 19A) and sustained tumour cell killing ( Figure 19B) when re-stimulated on cxvl36- expressing Bxpc3 cells.

By contrast, CD40-based pCARs exhibited modest function demonstrate that employed to in these assays.

Together, these data a number of TNF receptor family members can be engineer pCARs that demonstrate superior functionality, exemplified by CD27 or 41BB.

Claims (33)

1. CLAIMS: 1. An immuno-responsive T-cell or Natural Killer (NK)-cell expressing: (i) a second generation chimeric antigen receptor (CAR) comprising: (a) a signalling region; (b) a co-stimulatory signalling region; (c) a transmembrane domain; and (d) a binding element that specifically interacts with a first epitope on a target antigen; and (ii) a chimeric costimulatory receptor (CCR) comprising (e) a co-stimulatory signalling region which is different to that of (b); (f) a transmembrane domain; and (g) a binding element that specifically interacts with a second epitope on a target antigen.
2. 2. The immuno-responsive cell according to claim 1, which is a T-cell.
3. 3. The immuno-responsive cell according to claim 1 or claim 2, wherein the signalling region (a) comprises the intracellular domain of human CD3 [zeta] chain.
4. 4. The immuno-responsive cell according to claim 3, wherein the signalling region (a) is selected from SEQ ID NO:1 or SEQ ID NO:2.
5. 5. The immuno-responsive cell according to any one of claims 1 to 4, wherein the costimulatory signalling regions for (b) and (e) are selected from CD28, CD27, inducible Tcell costimulator (ICOS), 4-lBB, OX40, CD30, glucocorticoid-induced TNFR-related protein (GITR), herpes virus entry mediator (HVEM), death domain receptor 3 (DR3) or CD40.
6. 6. The immuno-responsive cell according to claim 5, wherein one of co-stimulatory signalling regions (b) or (e) is CD28 and the other co-stimulatory signalling region is 4- lBB, CD27 or OX40.
7. 7. The immuno-responsive cell according to claim 5 or claim 6, wherein one of costimulatory signalling regions (b) or (e) is CD28 and the other is 4-lBB or OX40.
8. 8. The immuno-responsive cell according to claim 5, wherein (b) is CD28. Date Re9ue/Date Received 2023-09-25
9. 9. The immuno-responsive cell according to claim 5, wherein (e) is 4-lBB or CD27.
10. 10. The immuno-responsive cell according to any one of claims 1 to 9, wherein the transmembrane domains of (c) and (f) are selected from CD8a and CD28 transmembrane domains.
11. 11. The immuno-responsive cell according to any one of claims 1 to 10, wherein the first and second epitopes are associated with the same receptor or antigen.
12. 12. The immuno-responsive cell according to any one of claims 1 to 11, which co-expresses a chimeric cytokine receptor.
13. 13. The immuno-responsive cell according to claim 12, wherein the chimeric cytokine receptor is 4a~.
14. 14. The immuno-responsive cell according to any one of claims 1 to 13, wherein at least one of binding element (d) or binding element (g) is a ligand for an ErbB dimer, a receptor for colony stimulating factor-1 (CSF-1R) or is an av~6 integrin-specific binding agent.
15. 15. The immuno-responsive cell according to any one of claims 1 to 14, wherein binding element (d) comprises colony stimulating factor-1 (CSF-1) and binding element (g) comprises IL-34.
16. 16. The immuno-responsive cell according to any one of claims 1 to 14, wherein one of the binding elements (d) and (g) is a TlE peptide, wherein the TlE peptide is a chimeric fusion protein composed of the entire mature human epidermal growth factor (EGF) protein, but wherein the five most N-terminal amino acids of said EGF protein are replaced by the seven most N-terminal amino acids of the mature human TGF-a isoform 1 protein.
17. 17. The immuno-responsive cell according to claim 16, wherein binding element (d) is an av~6 integrin-specific binding agent which is a peptide comprising the sequence motif RGDLX5X6L (SEQ ID NO 7) or RGDLX5X6I (SEQ ID NO 8), wherein LX5X6L or LX5X6I is contained within an alpha helical structure, wherein X5 and X6 are helix promoting residues; and binding element (g) is a TlE peptide. Date Re9ue/Date Received 2023-09-25 31
18. 18. The immuno-responsive cell according to any one of claims 1 to 17, wherein binding affinity of binding element (d) for the first epitope is lower than that of binding element (g) for the second epitope.
19. 19. A method for preparing the immuno-responsive cell as defined in any one of claims 1 to 18, said method comprising transducing a cell with a first nucleic acid encoding the second generation CAR; and also a second nucleic acid encoding the CCR; and culturing the cell in conditions suitable for expression of the second generation CAR and the CCR.
20. 20. The method according to claim 19 wherein the immuno-responsive cell further comprises a chimeric cytokine receptor, and wherein the culturing step is in a medium comprising said cytokine.
21. 21. A combination of (i) a first nucleic acid encoding a second generation chimeric antigen receptor (CAR) comprising (a) a signalling region; (b) a co-stimulatory signalling region; (c) a transmembrane domain; and (d) a binding element that specifically interacts with a first epitope on a target antigen and (ii) a second nucleic acid encoding a chimeric costimulatory receptor (CCR) comprising: (e) a co-stimulatory signalling region which is different to that of (b); (f) a transmembrane domain; and (g) a binding element that specifically interacts with a second epitope on a target antigen.
22. 22. A vector comprising the combination as defined in claim 21.
23. 23. A vector set comprising the combination as defined in claim 21, wherein the vector set comprises: (a) a first vector comprising the first nucleic acid; and (b) a second vector comprising the second nucleic acid. Date Re9ue/Date Received 2023-09-25 32
24. 24. A kit comprising: (a) the combination as defined in claim 21, the vector as defined in claim 22 or the vector set as defined in claim 23; and (b) instructions for use.
25. 25. The immuno-responsive cell according to any one of claims 1 to 18, for use in immunotherapy of a target cancer cell population in a patient, wherein the binding elements (d) and (g) specifically bind to the same or different antigen(s) of the target cell.
26. 26. The immuno-responsive cell as defined in any one of claims 1 to 18, for use in the treatment of cancer in a patient.
27. 27. The immuno-responsive cell for use of claim 26, wherein the cancer is selected from the group consisting of prostate cancer, breast cancer, neuroblastoma, melanoma, small cell or non-small cell lung carcinoma, sarcoma and brain tumours.
28. 28. A use of the immuno-responsive cell as defined in any one of claims 1 to 18, for effecting immunotherapy of a target cancer cell population in a patient, wherein the binding elements (d) and (g) specifically bind to the same or different antigen(s) of the target cell.
29. 29. A use of the immuno-responsive cell as defined in any one of claims 1 to 18 for treatment of cancer in a patient.
30. 30. The use of claim 29, wherein the cancer is selected from the group consisting of prostate cancer, breast cancer, neuroblastoma, melanoma, small cell or non-small cell lung carcinoma, sarcoma and brain tumours.
31. 31. A use of the immuno-responsive cell as defined in any one of claims 1 to 18 for preparation of a medicament for effecting immunotherapy of a target cancer cell population in a patient, wherein the binding elements (d) and (g) specifically bind to the same or different antigen(s) of the target cell.
32. 32. A use of the immuno-responsive cell as defined in any one of claims 1 to 18 for preparation of a medicament for treatment of cancer in a patient. Date Re9ue/Date Received 2023-09-25 33
33. 33. The use of claim 32, wherein the cancer is selected from the group consisting of prostate cancer, breast cancer, neuroblastoma, melanoma, small cell or non-small cell lung carcinoma, sarcoma and brain tumours. Date Re9ue/Date Received 2023-09-25