Patent Application
20170327596
The present invention relates to a method for increasing the sensitivity of a T cell expressing a cell-surface antibody. It relates to a method for increasing the ability of such a T cell to recognise low concentrations of target antigen. The antibody may be part of a chimeric antigen receptor (CAR), comprising an extracellular antibody-domain and a cytoplasmic signalling domain.
T-cell-based cancer immunotherapy approaches are commonly based on the capacity of CD8+ cytotoxic T cells (CTLs) to recognize and destroy malignant cells. Cancer cells present peptides derived from tumor-associated antigens (TAAs) at their cell surface in conjunction with major histocompatibility complex (MHC) class I molecules. CD4+ T cells also play an important role in antitumor immunity. For example, CD4+ T cells improve the capacity of dendritic cells (DCs) to induce CTLs by the interaction between CD40 on DCs and CD40 ligand on activated CD4+ T cells. Furthermore, CD4+ T cells provide help for the maintenance and expansion of CTLs by secreting cytokines such as interleukin-(IL-) 2.
In order to extend the recognition specificity of T lymphocytes beyond their classical MHC-peptide complexes, a gene-therapeutic strategy has been developed that allows redirecting T cells to defined tumour cell surface antigens. This strategy uses both the cellular and humoral arm of the immune response by assembling an antigen-binding moiety, most commonly a single chain variable fragment (scFv); together with a lymphocyte-activating signalling chain, such as the CD3 chain. Once this chimeric antigen receptor (CAR) is expressed at the surface of a T cell, the antigen-binding moiety binds to its antigen and an activating signal is transmitted into the T cell, which in turn triggers its effector functions against the target cell.
The use of CARs to redirect T cells specifically against TAA-expressing tumor cells has a number of theoretical advantages over classical T-cell-based immunotherapies. In contrast to the long-lasting procedure of in vitro selection, characterization, and expansion of T-cell clones with native specificity for MHC-tumor peptide complexes, genetic modification of polyclonal T-cell populations allows TAA-specific T cells to be generated in one to two weeks. Engraftment with CARs enables T cells to MHC-independent antigen recognition; thus major immune escape mechanisms of tumors such as downregulation of MHC molecules are efficiently bypassed. Furthermore, proliferation and survival of modified T cells can be improved by the implementation of a multitude of signaling domains from different immune receptors in a single CAR. In addition, T cells can be rendered more resistant against the immunosuppressive milieu in tumor tissue. In addition to cancer immunotherapy, CAR-modified lymphocytes have been successfully applied for the treatment of virus infections, and more recently, first experimental studies have been published using CARs engrafted on regulatory T cells (Tregs) for the treatment of autoimmune diseases.
The first clinical studies using CAR-modified T cells have been reported and a number of clinical trials are currently in progress.
Although CAR technology shows initial promise, improvements are desirable in order to maximise the therapeutic potential of CAR-expressing T cells. In particular, there is a need to improve sensitivity of targeted T cells, so that they can respond to low concentrations of target antigen.
TCR Affinity Ranges and human Jurkat cells and primary T cells transduced with the wild-type and affinity matured TCR constructs
(
T cells transduced with affinity matured TCR down-regulate TCR faster and to a greater level than T cells transduced with Tax-TCR.
(
Up-regulation of phospho-ERK after stimulation of primary T cells transduced with the TCRs constructs and stimulated with pTax/HLA-A2 tetramer
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Kinetics of antigen-specific CD107a up-regulation
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Primary T cells transduced with the high affinity TCR show lower peptide sensitivity compared to Tax-TCR transduced T cells
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The natural affinity range of antibodies is 1,000-100,000 times higher than the natural affinity of T cell receptors (TCR). The present inventors have surprisingly found that increasing the affinity range of TCR by 1000-fold into the affinity range of antibodies did not improve T cell function, and actually increased the concentration of antigen required to trigger T cell activation.
This has major implications in the field of T-cell targetting with antibody constructs, such as chimeric antigen receptors (CARs). In order to achieve optimal T-cell sensitivity, the natural affinity of antibodies needs to be lowered, for example into the natural affinity range of TCRs.
Thus, in a first aspect, the present invention provides a method for increasing the sensitivity of a T cell expressing a cell-surface antibody to a target antigen, which comprises the step of lowering the affinity of the antibody to the target antigen by at least 100-fold.
The affinity of the antibody to the target antigen may be lowered by at least 1000-fold.
The affinity of the antibody may be lowered by mutagenesis of the starting antibody, followed by in vitro selection for low affinity variants.
The affinity of the antibody for the target antigen, following lowering, (i.e. the affinity for the variant antibody) may be in the range of 100-1 μM.
The variant antibody, following affinity lowering, may have a dissociation half time from the target antigen in the range of 5-60 seconds.
The antibody may be part of a chimaeric antigen receptor (CAR) comprising:
In a second aspect, the present invention provides a T cell made by a method according to the first aspect of the invention.
The T cell may express a variant cell surface antibody derived from a starting antibody, wherein the variant cell surface antibody has an affinity for its target antigen which is at least 100-fold less than that of the starting antibody.
The T cell may express a cell-surface antibody having an affinity for the target antigen in the range of 100-1 μM.
The T cell may express a cell-surface antibody having a dissociation half-time from the target antigen which is in the range of 5-60 seconds.
In a third aspect, the present invention provides a chimeric antigen receptor (CAR) for expression in a T cell, comprising an antibody domain and a signalling domain, in which the antibody domain has an affinity for a target antigen in the range of 100-1 μM.
In a second embodiment of this aspect of the invention, there is provided a chimeric antigen receptor (CAR) for expression in a T cell, comprising an antibody domain and a signalling domain, in which the antibody domain binds to a target antigen with a dissociation half time in the range of 5-60 seconds.
In a fourth aspect, the present invention provides a nucleic acid sequence encoding a cell-surface antibody as described above. The nucleic acid sequence may, for example, encode a CAR according to the third aspect of the invention.
In a fifth aspect, the present invention provides a vector comprising a nucleic acid sequence according to the fourth aspect of the invention, which is capable of expressing a CAR in a T cell.
In a sixth aspect, the present invention provides a T cell which expresses a CAR according to the third aspect of the invention.
In a seventh aspect, there is provided a vector according to the fifth aspect of the invention, or a T cell according to the second or sixth aspects of the invention for use in the treatment of a disease.
In an eighth aspect, the present invention provides a method for treating a disease in a subject, which comprises the step of administering a vector according to the fifth aspect of the invention, or a T cell according to the second or sixth aspects of the invention to the subject.
There is also provided the use of a vector according to the fifth aspect of the invention, or a T cell according to the second or sixth aspects of the invention in the manufacture of a medicament for use in the treatment of a disease.
The disease may be cancer, viral infection or autoimmunity.
In a first aspect, the present invention relates to a method for increasing the sensitivity of a T cell.
In particular, it relates to a method for increasing the ability of a T cell expressing a cell-surface antibody to recognise low concentrations of target antigen.
Antigen recognition may be monitored by looking at T cell activation. So a T cell with increased sensitivity will be activated by a lower concentration of antigen.
T cell activation may be monitored by looking at one of the signalling events in T cell activation, such as ERK phosphorylation. Alternatively one may look at an antigen-specific effector function, such as CD107 upregulation, cytokine production or cytotoxicity.
The method of the present invention increases the sensitivity of a T cell expressing a cell-surface antibody, by lowering the affinity of the antibody to the target antigen. An increase in sensitivity may be determined by comparing the concentration of antigen necessary for T cell activation with the starting high-affinity antibody, with the concentration of antigen necessary for T cell activation with the affinity-lowered antibody. Alternatively, an increase in sensitivity may be determined by comparing the level of T cell activation achieved at a given (low) antigen concentration a) with the starting high-affinity antibody, and b) with the affinity-lowered antibody.
The sensitivity of a T cell expressing an affinity-lowered antibody may, for example, be 10, 50 or 100-fold higher than the sensitivity of a T cell expressing the equivalent natural affinity antibody.
Without wishing to be bound by theory, the present inventors predict that T cells expressing lower-affinity antibodies have higher sensitivity because they are better suited to T cell activation by serial triggering than T cells expressing high affinity antibodies.
In the serial triggering model of T cell activation, one cell-surface antibody sequentially interacts with target antigen a plurality of times, leading to a plurality of triggering events. In this model, dissociation of the antibody from the antigen is an essential step. Antibodies with low affinity and short dissociation half lives (t1/2) will therefore be better suited to serial triggering than antibodies with high affinity and long t1/2.
The second aspect of the invention relates to a T cell.
The T cell is engineered to express a cell-surface antibody.
The T cell may, for example, be a cytotoxic T cell (CD8+), a helper T cell (CD4+), a regulatory T cell, or a Th17 T cell.
The T cell may be from a T cell line or a primary T cell. Primary T cells are thought to have increased peptide sensitivity compared to hybridoma cells expressing the same TCR.
As used herein, “antibody” includes a whole immunoglobulin molecule or a part thereof, or a bioisostere, mimetic or derivative thereof. Examples of antibody fragments include: Fab, F(ab)′2, and Fv. Examples of a bioisostere include single chain Fv (ScFv) fragments, chimeric antibodies, bifunctional antibodies.
The term “antibody” also includes monobodies, diabodies and triabodies, minibodies and single domain antibodies.
The starting antibody may be an antibody which has previously been expressed at the cell-surface of a T cell, for example as part of a chimeric antigen receptor (CAR—see below).
The antibody is expressed at the surface of a T cell, so may be associated with a domain that enables this expression, such as a transmembrane domain. The antibody may be expressed as a fusion protein with a transmembrane domain, such that it is expressed at the T cell surface.
Chimeric antigen receptors comprise an extracellular binding domain, transmembrane domain and a cytoplasmic signalling domain. They may also comprise an extracellular hinge and spacer element.
The binding domain may comprise or consist of an antibody, such as an scFv.
An scFv commonly comprises the light (VL) and heavy (VH) variable regions of an antibody joined by a flexible linker.
ScFvs against tumour associated antigens (TAAs) have been used to produce CARs to redirect T cells against TAAs expressed at the surface of tumour cells from various malignancies including leukaemia, lymphomas and solid tumours. A major advantage of endowing T cells with non-MHC-restricted, antibody-derived specificity is that the potential target structures are no longer restricted to protein-derived peptides, but rather comprise every surface molecule on tumour cells including proteins with varying glycosylation patterns and non-protein structures such as gangliosides or carbohydrate antigens. Thus, the panel of potential tumor-specific targets is enlarged.
Other binding moieties than scFvs have also been introduced into CARs and successfully used for predefined targeting of lymphocytes. For example, chimeric receptors incorporating receptor ligands like a vascular endothelial growth factor polypeptide, an integrin-binding peptide, heregulin, or an IL-13 mutein have been explored and tumor regression was demonstrated in mouse models. Another group has successfully retargeted murine and human lymphocytes against hematopoetic tumors using a chimeric NKG2D receptor fused to the CD3 chain.
Most CARs comprise a hinge region between the binding and the transmembrane domain. The rationale for including a spacer region is to provide more flexibility and accessibility for the binding moiety, which might be otherwise buried by the dense glycocalyx shell covering T cells. Examples of extracellular spacer regions include immunoglobulin domains like the Fc regions of antibodies or immunoglobulin-like domains derived from the extracellular portions of CD8α, CD28, TCRβ chain, or NKG2D.
The transmembrane region of CARs may be derived from homo- or heterodimeric type I membrane proteins like CD4, CD8, CD28, CD3, or Fc gamma.
Much attention has been paid to the intracellular signaling domains of chimeric receptors. The first generation of CARs only contains a single signaling unit, in most cases derived from the CD3 chain. Second generation CARs contain an additional costimulatory domain (CM I), predominantly the CD28 domain. Signaling through this costimulatory domain leads to enhanced proliferation, cytokine secretion, and renders engrafted T cells resistant to immunosuppression and induction of AICD. Recent developments fused the intracellular part of a second costimulatory molecule (CM II) in addition to CD28 and ITAM-bearing signaling chains, thus generating tripartite signaling CARs. T cells engrafted with third generation CARs have been reported to have superior qualities regarding effector functions and in vivo persistence.
The method of the first aspect of the invention involves lowering the affinity of an antibody for its target antigen.
Techniques for altering the affinity of antibodies are known in the art. For example, mutations may be introduced into the antibody-encoding gene, and the resulting variant antibodies screened for low-affinity binders, by a technique such as yeast display or phage display.
The mutation step may be random, or targeted to specific residues in the antigen binding pocket.
The process may involve successive rounds of mutagenesis and screening, for example as part of an in vitro evolution process.
The term “antigen” in terms of target antigen means an entity which is recognised (i.e. binds specifically) to the antibody expressed at the T cell surface.
An “epitope” is the portion of a molecule which is recognised by antibody. In the sense of the present invention, an antigen is or comprises at least one epitope.
An antigen may be a complete molecule, or a fragment thereof. The antigen may be or be derivable from a naturally occurring molecule.
The antigen may be or be derivable from, for example, a protein, glycoprotein, glycolipid, or carbohydrate.
Where the gene modified T cell is for use in the treatment of cancer, the antibody may recognise an antigen that is or is part of a tumour associated antigen (TAA).
Some known TAAs which are recognised by antibodies are listed in Table 1.
The T cell of the present invention may express a cell-surface antibody which recognises an epitope from an antigen listed in Table 1.
Where the gene modified T cell is for use in the treatment of chronic infection, the antibody may recognise an antigen that is or is part of a virus associated antigen.
Where the gene modified Treg cell is for use in the treatment of autoimmunity, the antibody may recognise a tissue-specific antigen that is expressed in the tissue affected by autoimmunity.
Affinity may be expressed in terms of the dissociation constant (KD).
The natural affinity of antibodies is usually in the range 10 nM-10 pM (10−8-10−11 M).
TCR: MCH/peptide interactions are commonly in the range 100-1 μM (10−4-10−6 M).
In the method of the present invention, the affinity of the antibody may be lowered at least 100-fold, at least 1000-fold or at least 10,000-fold.
The affinity of the antibody may be lowered such that it has an affinity for antigen which is similar to the affinity a TCR has for a MHC/peptide complex.
The T cell of the present invention may comprise an antibody or antibody domain having an affinity for target antigen in the range 100 nm-1 μM.
Affinity may alterternatively be expressed in terms of the dissociation half time (t1/2).
The present inventors have shown that increasing the half time from 9.3 seconds to 348, 1320 and 3120 seconds resulted in a loss of sensitivity.
The dissociation half time of the antibody described herein may be less than 100 seconds, or less than a minute. It may be in the range 5 to 60 seconds. It may be in the range 10-30 seconds.
In a fourth aspect, the present invention provides a nucleic acid sequence encoding a cell-surface antibody as described above.
The nucleic acid sequence may be an RNA or DNA sequence or a variant thereof.
The nucleic acid sequence may encode a CAR according to the third aspect of the invention. In this respect, the nucleic acid sequence may comprise a sequence encoding an antibody domain operably linked to a sequence encoding a signalling domain.
The nucleic acid sequence may also comprise a nucleic acid sequence encoding a hinge region; a nucleic acid sequence encoding a spacer; and/or a nucleic acid sequence encoding a transmembrane region.
Where the nucleic acid sequence encodes a plurality of distinct sequences, such as VL and VH antibody domains, or cytoplasmic signalling domains; the nucleic acid sequence may comprise a plurality of separate sequences; a single sequence capable of producing more than one product (e.g. joined by an IRES); or a single sequence capable of producing a fused product (e.g. an scFv).
In a fifth aspect, the present invention provides a vector. The vector may comprise the nucleic acid sequence of the third aspect of the invention. The vector may be capable of expressing an antibody as defined herein in a T cell.
The vector may express the antibody at the cell-surface of the T cell.
The vector may comprise a nucleic acid sequence encoding the antibody and a transmembrane region.
The vector may be capable of expressing a chimeric antigen receptor, which comprises an antibody domain which is expressed at the T cell surface and a cytoplasmic signalling domain.
The vector may be any agent capable of delivering or maintaining nucleic acid in a host cell, and includes viral vectors, plasmids, naked nucleic acids, nucleic acids complexed with polypeptide or other molecules and nucleic acids immobilised onto solid phase particles.
The antibody is “expressed” in the host T cell by being produced as a result of translation, and optionally transcription, of the nucleic acid. Thus, the antibody is produced in situ in the cell prior to display at the cell surface.
Non-viral vectors may be introduced into T cells using a variety of non-viral techniques known in the art, such as transfection, transformation, electroporation and biolistic transformation.
Alternatively, the vector may be introduced into a T cell using a viral method, for example infection with a recombinant viral vector such as one derived from a retrovirus, lentivirus, herpes simplex virus or adenovirus.
Retroviral vectors and plasmids have both previously been used to express CARs in T cells.
T cells may be transfected or transformed with vector in vitro, ex vivo or in vivo.
The vectors and/or antibody-expressing T cells of the present invention may be used to treat and/or prevent a disease.
To “treat” means to administer the vector/cell to a subject having an existing disease in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.
To “prevent” means to administer the vector/cell to a subject who has not yet contracted the disease and/or who is not showing any symptoms of the disease to prevent or impair the cause of the disease (e.g. infection) or to reduce or prevent development of at least one symptom associated with the disease.
The vector/T cell of the present invention may be used to treat and/or prevent cancer, an infectious disease or an autoimmune disease in a subject.
The vector may be administered directly to the subject, or may be administered in vitro to T cells. In an ex vivo approach, T cells may be isolated from the subject, treated with vector such that they express low-affinity antibody, and then returned to the subject.
T cells expressing antibodies against TAAs can recognize tumor cells by binding of the CAR to a TAA. As a result, T cells are activated and can eliminate tumor cells by the secretion of perforin and granzymes as well as the expression of FasL and tumor necrosis factor-related apoptosis inducing ligand (TRAIL). In addition, other tumor-infiltrating immune cells can be activated by the secretion of various cytokines.
Several clinical trials have been conducted or are in progress using CAR-modified T cells to treat cancer patients, including the following:
(i) the use of T cells expressing a CAR recognizing carbonic anhydrase IX, to treat metastatic renal cell carcinoma (RCC);
(ii) the use of T cells expressing an α-folate receptor-specific CAR to treat metastatic ovarian cancer
(iii) the use of T cells expressing a L1-cell adhesion molecule to treat neuroblastoma patients
(iv) the use of T cells expressing a CD20-specific CAR to treat B-cell lymphoma or mantle cell lymphoma; and
(v) the use of EBV-specific T cells engineered with a CAR recognizing the diasialoganglioside GD2 to treat neuroblastoma.
The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.
Antigen recognition by T cells is via the T cell receptor (TCR) that binds peptides displayed by MHC molecules. Compared to antibodies the affinity of TCR for MHC/peptide is relatively low, with dissociation constants KD ranging from 100-1 μM. Unlike antibodies, TCR do not naturally undergo somatic hypermutation to enhance the affinity for the TCR recognised antigen.
The present inventors explored in detail how TCR/antigen affinities spanning the natural range and extending 700-fold into the supra-physiologic range affected the kinetic and the sensitivity of primary human CD8+ T cell responses. They used variants of an HLA-A0201-restricted TCR specific for a peptide derived from the Tax antigen of Human T Lymphotopic virus I (HTLV1). The affinity matured variants were generated by phage display and they retained peptide-specificity when introduced into primary CD8+ T cells. The functional analysis of transduced T cells revealed that although affinity matured TCR mediated faster responses than wild type TCR, this, paradoxically, led to an inability to recognise low concentration of antigen. The data strongly suggest that affinity maturation beyond the physiologic range accelerates human CD8+ T cell responses but disrupts the serial TCR triggering that is required to maximise recognition of low density peptide ligands.
The HLA-A0201-restricted TCR specific for a peptide (pTax) derived from the Tax protein of HTLV-1 was used in this study. Recently, phage display technology was employed to isolate affinity-matured versions of the Tax-TCR. Soluble TCR molecules and HLA/peptide complexes were analysed in the surface plasmon resonance system to measure the dissociation constant KD of the TCR-HLA/peptide binding and the half-life t1/2 of the interaction. The binding of the wild type Tax-TCR to HLA/pTax had a KD of 1.8 μM and a t1/2 of 9.3 seconds. The t1/2 of the three affinity matured versions M1-TCR, M2-TCR and M3-TCR increased to 348 sec, 1320 sec and 3120 sec, respectively, with a KD for the M3-TCR of 2.5 nM (
Human Jurkat cells and primary peripheral blood T cells were transduced with retroviral constructs encoding the Tax-TCR or the affinity matured M1, M2 and M3-TCR. Jurkat cells were stained with anti-CD3 and Vβ13 (the beta variable region segment which is used by the Tax-TCR) Abs, whilst T cells were stained with anti-CD3 and anti-CD8 and either anti-Vβ13 or HLA-A2/pTax tetramer. TCR constructs were readily expressed in human Jurkat cells (
The present inventors performed experiments to test whether TCR affinity affected the kinetic of peptide-specific immune responses. In these experiments saturating concentrations of peptide were used to provide optimal TCR stimulation over defined periods of time.
As expected, stimulation of TCR-transduced bulk Jurkat T cells with the pTax peptide resulted in downmodulation of the Tax-TCR and the affinity matured versions. However, there was a dramatic difference in the speed of TCR downmodulation. Within 5 minutes of peptide stimulation the affinity matured M3-TCR showed a substantial reduction of TCR-positive T cells from 55% to 20% (
Next, the present inventors explored whether the kinetic of early signalling is different for the Tax-TCR and the affinity matured variants. For these experiments transduced primary human T cells were stimulated with saturating concentrations of HLA-A2/pTax tetramers for short time periods followed by intracellular staining for ERK phosphorylation, an early signalling event in T cell activation. Tetramers were used to achieve uniform TCR stimulation within a short time period, which was difficult to achieve with peptide loaded T2 cells. The transduced T cell populations used in these experiments contained similar numbers of TCR-positive T cells, as determined by the percentage of tetramer-positive cells (Tax-TCR=83%, M1-TCR=88%, M2-TCR=92% and M3-TCR=94%). Un-stimulated T cells showed little background phospo-ERK staining (see time point 0 in
In order to determine if the faster signalling and TCR down-regulation of the affinity matured TCR correlated with accelerated antigen-specific effector function, the present inventors investigated the upregulation of CD107, a molecule that is exposed on the surface of T cells releasing effector molecules such as perforin and cytokines from intracellular granules. Primary T cells expressing similar levels of the Tax-TCR or the affinity matured variants were stimulated with antigen-presenting T2 cells loaded with saturating concentrations of pTax peptide. Over a two-hour stimulation period, all TCR transduced populations showed an increase in the percentage of CD107 positive cells (
In order to analyse the kinetics of CD107 upregulation within the natural affinity range, primary T cells expressing the Tax-TCR or the M3-TCR were stimulated with T2 cells coated with saturating concentrations of the pHuD peptide. As before, the fastest response was seen when the M3-TCR was stimulated with the pTax peptide, followed by Tax-TCR stimulation with pTax peptide. The response of M3-TCR stimulated with pHuD was approximately 2 hours slower than the same TCR stimulated with pTax. The response of the Tax-TCR stimulated by pHuD was approximately 4 hours slower compared to stimulation with pTax (
The present inventors next explored whether the accelerated response displayed by affinity matured TCR correlated with improved performance at low concentrations of peptide antigen. To explore this, transduced T cells were stimulated with decreasing peptide concentrations for 18 hours, a time period sufficient to initiate responses irrespective of the kinetic of the individual TCR/antigen combinations.
The peptide sensitivity of the CD107 response was assessed using transduced T cells expressing similar levels of the Tax-TCR or the affinity matured versions. The T cells were stimulated with T2 cells loaded with decreasing concentrations of the pTax or pHuD peptide. The low affinity Tax-TCR/pHuD combination required approximately 10-fold more peptide to trigger a CD107 response than the medium affinity M3-TCR/pHuD combination (
The present inventors used IFNγ production to explore if the unexpected loss in T cell sensitivity was peculiar to CD107 upregulation or also affected other T cells effector functions. Again, T cells expressing the Tax-TCR produced IFNγ at lower concentrations of pTax peptide than T cells expressing the affinity matured TCR (
Finally, the present inventors determined the peptide concentrations required to mediate TCR down-regulation. Although the speed of down-regulation was much faster for the affinity matured TCR (see
In the final experiments the present inventors explored the ability of the Tax-TCR and the affinity matured TCR to recognise naturally processed Tax antigen. HLA-A2 positive HeLa cells were transfected with a plasmid vector encoding full length Tax protein and GFP as marker, or with a control vector encoding GFP only. The transfected HeLa cells were used to stimulate T cells expressing the Tax-TCR or affinity matured TCR followed by measurement of Tax-specific CD107 induction. A robust CD107 up-regulation was seen when T cells expressing the Tax-TCR and the M1-TCR were stimulated with the Tax-expressing HeLa cells (
The data presented here show that within the ‘natural’ TCR affinity range there is a good correlation between dissociation constant KD of the TCR/ligand interaction and the speed and peptide sensitivity of T cell responses. However, beyond the ‘natural’ affinity range there is a separation of the speed and the sensitivity of CD8+ T cell responses. Whilst affinity matured TCR trigger faster responses, this was linked to a reduction in peptide sensitivity. Optimal peptide sensitivity was achieved with the Tax-TCR (t1/2 9.3 sec) and increasing the dissociation half time t1/2 to 348, 1320 and 3120 seconds resulted in a progressive loss of peptide sensitivity. A recent study with the human NY-ESO-TCR showed that an increase in t1/2 from 2.2 sec for the wild type TCR to 19 sec for an affinity matured TCR improved the ability of TCR transduced CD8+ T cells to recognise NY-ESO expressing tumour cells, while this improvement was not seen with an affinity matured TCR with a t1/2 of 41 sec (Robbins et al. (2008) J Immunol. 180:6116-6131). Together, these observations suggest that affinity maturation may be beneficial when the half-life of the TCR-HLA/peptide interaction is increased to >10 sec, but is likely to impair peptide sensitivity when the t1/2 is enhanced into the range of minutes.
The presented data support the serial triggering model of T cell activation, which predicts that one MHC/peptide complex can sequentially engage several TCR molecules to achieve a critical threshold of approximately 200 triggered TCR that is required to achieve T cell activation. In this model, the dissociation of the TCR is an essential step, which is progressively impaired with increasing the KD and half-live of the TCR-MHC/peptide complex. The experimental data presented here are fully compatible with the serial triggering model and provide an explanation for the relative low affinity and short t1/2 of the natural TCR repertoire. Our data show that affinity matured TCR do not improve the ability of T cells to recognise small numbers of MHC presented peptide epitopes, an essential function of high avidity T cells. The ability of T cells to recognise low concentration of peptide was found to correlate with an improved ability to control the growth of tumor cells and the spread of virus infection in vivo, supporting the biological importance of high T cell avidity (Alexander-Miller et al (1996) Proc Natl Acad Sci USA. 93:4102-4107; Zeh et al (1999) J Immunol. 162:989-994). Similarly, the present inventors recently demonstrated a correlation between the functional avidity of Tax-specific CTL, assayed directly ex vivo, and the efficiency of control of HTLV-1 replication in vivo (Kattan et al (2009) J Immunol. 182:5723-5729).
Unless otherwise stated all culture medium was RPMI (Cambrex, UK) supplemented with 10% heat inactivated FCS (Sigma, UK), 1% penicillin/streptomycin (Invitrogen, UK) and 1% L-glutamine (Invitrogen). Cell lines used were the HLA-A2 positive T2 cell line that is deficient in TAP (transporter associated with antigen processing) and can be efficiently loaded with exogenous peptides and HeLa cells previously stably transduced to express HLA-A2. HLA-A2 positive peripheral blood mononuclear cells (PBMCs) were obtained from volunteer donors from the National Blood Service, Collindale, London, UK. Flow cytometry antibodies were anti-human PE CD107a, PerCP CD3 and APC CD8 (BD Biosciences, UK), PE Vβ13.1 (Immunotech, UK), phosphor-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (197G2) rabbit mAb (Cell Signaling Technology, UK) and Alexa Fluor 488 F(ab)2 fragment of goat anti-rabbit IgG (Invitrogen, UK). PE-labelled HLA-A2/Tax tetramers were obtained from Beckman Coulter. The peptides used in this study were the HLA-A2 binding peptides pTax (LLFGYPVYV), pHuD (LGYGFVNYI) and pWT235 (CMTWNQMNL) and were synthesised by Prolmmune (Oxford, UK).
Only the beta TCR chain underwent affinity maturation and was accomplished as detailed previously by Li et al (2005). The TCR alpha □and beta □chain constructs were cloned together into retroviral pMP71 vectors and were linked via a viral p2A sequence. The alpha and beta TCRs contained an additional inter-chain disulphide bound in the constant domains. This was achieved by replacing residue 48 of the alpha chain from a threonine to a cysteine and residue 57 of the beta chain from a serine to a cysteine as previously described (REF). The TCR construct was codon optimised.
Transduction of Retroviral TCR Constructs into Jurkat Cells and Primary T Cells:
For retroviral transduction, 2×106 phoenix amphotropic packaging cells were cultured in 10 cm culture plates for 24 h at 37° C., 5% CO2 in DMEM media supplemented with 10% heat-inactivated FCS, 1% penicillin/streptomycin and 1% L-glutamine. The phoenix amphotropic packaging cells were cultured in fresh DMEM media and transfected with 16 μg of the vector constructs using calcium phosphate precipitation (Invitrogen). 24 h after transfection the media was replaced with RPMI culture media and incubated for a further 24 h. The viral supernate was then harvested. Jurkat cells were split 24 h prior to retroviral transduction and PBMNs were activated for 48 h using the anti-CD3 antibody OKT3 at 30 ng/ml and interleukin (IL)-2 at 600 U/ml (Chiron, CA). Retroviral transductions were conducted on retronectin (Takara, Japan) coated 24 well plates. 1 ml of virus supernate was added per well and the plates was spun at 2,000 g, 32° C., 2 h. Wells were washed twice with PBS and cells were seeded at 5×105 per well in 2 ml of culture media. PBMC were transduced in the presence of IL-2 at 600 U/ml. After 24 h of incubation, the media was replaced with fresh culture media. Transduction efficiency was determined by flow cytometry analysis, conducted on a LSR II flow cytometer after a further 48 h culture period. FACS data was analysed using FACSdiva.
TCR transduced primary T cells were stimulated and expanded every 7-10 days. The stimulations were conducted in 24 well plates in 2 ml culture media containing 10% non-heat inactivated FCS and 10 U/ml IL-2 (Roche, UK), at 37° C., 5% CO2. Each well contained, 5×105 transduced cells, 2×105 irradiated T2 cells loaded for 2 h with 100 μM of the peptide pTax and 2×106 irradiated autologous PBMCs as feeder cells.
2×105 TCR transduced T cells or Jurkat cells were co-cultured with 2×105 T2 cells pre-loaded with peptide in round-bottom 96 well plates in 200 μl culture media. After each co-culturing time point the reaction was immediately stopped by washing in ice-cold PBS. Jurkat cells were stained with anti-Vβ13.1 Abs and T cells were stained with anti-CD8 Abs and tetramer and FACs analysed.
2×105 TCR transduced T cells were cultured with 1 in 500 dilution of tetramer in 100 μl of culture media for the stated times. After each time point the reaction was immediately stopped by adding 2% paraformaldehyde for 10 min at 37° C. Cells were washed, permeabilised with 90% methanol (ice, 30 min) and then stained with anti-phosphor-p44/42 MAPK, followed by Alexa Fluor 488 F(ab)2 fragment of goat anti-rabbit IgG for FACs analysis.
For assays using T2 cells as stimulator cells, 2×105 T2 cells were pre-loaded with peptide. For assays using HLA-A2 positive HeLa cells as stimulator cells, the HeLa cells were transfected with either a pGFPC1 plasmid (Clontech) or a full length Tax gene encoded within the pGFPC1 plasmid30. Cells were transfected using 8 μg plasmid DNA and 24 μl Fugene (Roche, UK). 24 h after transfection the GFP expression was determined and was typically 20-25%. For stimulations, the ratio of TCR-positive T cells to GFP positive HeLa cells was 1:1. As positive control TCR-positive T cells were stimulated with transfected HeLa cells loaded with saturating concentration of pTax peptide (100 μM) at a ratio of 1:1. T2 cells or HeLa cells were co-cultured with 2×105 TCR transduced T cells in round-bottom 96 well plates in 250 μl culture media containing 1 μg/ml brefeldin A (Sigma, UK), 0.7 μg/ml BD GolgiStop (BD Bioscience Pharmingen, UK) and 2 μl of CD107a PE. After each co-culturing time point the reaction was immediately stopped by washing in ice-cold PBS. T cells were stained with anti-CD8 and FACS analysed.
For cytokine secretion assays, 1×105 TCR transduced T cells were stimulated with 1×105 irradiated T2 cells loaded for 2 h with peptide. Assays were conducted in triplicates in round-bottom 96 well plates in 250 μl culture media. After 18 h incubation at 37° C., 5% CO2, supernatant was harvested and tested for secreted IFNγ using a human enzyme linked immunosorbent assay (ELISA) kit (BD Biosciences) as per the manufacturer's instructions. The data was analysed using excel software.
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in cancer therapy, immunology, molecular biology or related fields are intended to be within the scope of the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1108236.9 | May 2011 | GB | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14117947 | Feb 2014 | US |
| Child | 15606789 | US |
