WO2011083296A1

WO2011083296A1 - T cell clonotypes

Info

Publication number: WO2011083296A1
Application number: PCT/GB2010/052087
Authority: WO
Inventors: Andrew Sewell
Original assignee: University College Cardiff Consultants Limited
Priority date: 2010-01-09
Filing date: 2010-12-14
Publication date: 2011-07-14
Also published as: GB201000375D0

Abstract

The invention relates to a method for identifying and manufacturing an immunogenic agent or vaccine comprising obtaining a population of T cells; exposing said cells to at least one selected antigen and identifying a good TCR which binds maximally to said antigen and a poor TCR which binds minimally to said antigen; designing or obtaining at least one further improved antigen; exposing said good and poor TCRs to said further antigen and determining the response of said good and said poor TCRs to said antigen; and identifying an antigen as said immunogenic agent or vaccine on the basis that it stimulates a response from said good TCRs but does not stimulate a response from said poor TCRs.

Description

T Cell clonotypes

The invention relates to a method for the identification of an immunogenic agent or vaccine; the manufacture of an immunogenic agent or vaccine; a cell or cell-line involved in the production of or for producing said immunogenic agent or vaccine; a method for treating or vaccinating an individual using said immunogenic agent or vaccine, respectively; an immunogenic agent or vaccine identified or manufactured according to the method of the invention; use of an immunogenic agent or vaccine identified or manufactured according to the method of the invention as a diagnostic indicator; use of an immunogenic agent or vaccine identified or manufactured according to the method of the invention as a therapeutic for treating cancer or an allergic reaction or autoimmune disorder; use of an immunogenic agent or vaccine identified or manufactured according to the method of the invention as an adjuvant for enhancing the performance of an existing vaccine; and a combination therapeutic comprising an immunogenic agent or vaccine identified or manufactured according to the method of the invention in combination with at least one further therapeutic.

Background

There is a constant need to identify and produce immunogenic agents and, in particular, vaccines to assist in the treatment or prevention of a whole range of diseases that are responsive to immunogenic manipulation. Most typically researchers look to identify an agent that can act as an effective antigen for a given disease condition and so stimulate the immune system to respond to said agent and thereby fight the corresponding disease. A traditional vaccine challenges the immune system with an attenuated, non-infectious pathogen.

Advantageously, this process then enables immune memory to mount a secondary response whenever it encounters the real pathogen at a later time. Thus, vaccination takes advantage of immunological memory.

When undertaking investigations researchers spend considerable time and effort modifying or tweaking potential antigens of interest in order to try and increase the efficacy of these known antigens whilst also safeguarding against producing an antigen that is so stimulatory it effectively acts as a disease agent. Implicitly, there is a balance to be achieved and it is best realised when the efficacy of the engineered antigen produces an effective immune response and so treats, prophylactically or curatively, a disease without acting as a disease agent, this is particularly true of vaccines.

In other instances a disease may be so malignant, such as cancer, that it is necessary to make an effective antigen at almost whatever cost. In these circumstances the immunologist's efforts are centred on the production of an antigen so effective that the immune response elicited thereby is sufficient to eradicate the disease and, ideally, provide ongoing protection from any reoccurrence or relapse.

In either case the production of any vaccine involves a consideration of the adaptive immune system. This is a highly evolved system that is able to distinguish between different pathogens and is capable of evolving or 'adapting' during the lifetime of an individual such that immunity becomes better with each successive exposure to pathogen.

Adaptive immunity is controlled by white blood cells called lymphocytes. Lymphocytes have cell surface receptors that recognize antigen. The key to adaptive immunity lies in the somatic gene rearrangement of these antigen receptors which produces an almost infinite array of specific receptors from a finite number of genes.

There are three different sorts of antigen receptor, the B cell receptor (antibody), the αβ T cell receptor (αβ TCR) and the γδ T cell receptor (γδ TCR). These three receptors distinguish the main lymphocyte lineages of the adaptive immune system. Our invention is relevant for αβ T cells.

The antigen receptor on B-cells, undergoes affinity maturation through a process of somatic hypermutation that allows an evolution towards more effective responses over time. In contrast, the T-cell receptor (TCR) does not incorporate this feature and is unchanging. T-cells expressing an αβ-TCR play a pivotal role in immunity to pathogens and cellular malignancies by recognizing short peptide fragments bound to major histocompatibility complex (MHC) molecules at the cell surface.

There are two types of αβ T-cells: CD4+ and CD8+.

CD4+ (T helper) T-cells recognize peptides of 10-20 amino acids long that are derived from exogenous proteins and are presented in the context of MHC class II molecules. Only 'professional' antigen presenting cells (APC) express MHC class II. CD4+ T cells produce cytokines which can help B cells (in antibody production), stimulate macrophages (to promote phagocytosis) and help Cytotoxic T lymphocytes (kill tumour cells and virally infected cells).

CD8+ (Killer) T cells (also known as Cytotoxic T lymphocytes; CTL) recognize peptides of 8-13 amino acids long from endogenous proteins and are presented in the context of MHC class I molecules. Almost all nucleated cells express MHC class I molecules. CTL are able to induce the apoptosis of targets and kill them directly via apoptosis inducing molecules. Thus, CTL can eliminate virally infected cells and tumour cells.

18

As mentioned above, in theory human αβ T cells with approximately 10 possible TCRs could be produced by somatic rearrangement of germline gene segments; a process very similar to antibody gene arrangement. These T cells then undergo a selection process in the Thymus, essentially, involving rejection of either non-interacting TCRs or strongly interacting TCRs. The resulting peripheral T cell repertoire is thus self-MHC -restricted but not self reactive and has the potential to recognize non-self peptide-MHC complexes with high affinity. It is the Complimentarity Determining Regions (CDR) of the TCR that contact the peptide-MHC antigen.

There is a lot of evidence to suggest that we can mount an immune response to the vast majority of foreign peptide-MHC complexes. Indeed, it is possible to make a large number of peptides from the 20 proteogenic amino acids, (e.g. 1.6xl0 1ⁱ8^O 14 -mers, 4x1015

12-mers, lxl0 1¹3^J 10 -mers, 5x1011 9-mers). Many of these peptides are capable of binding to individual self-MHC. Thus, the number of potential antigenic peptides we could encounter and present is vast.

Theoretically we can make T-cells with 10 18 different TCRs so it should be possible to have a different TCR for each antigenic peptide. However, something as simple as the sheer weight of this number of cells precludes this option, (A very high level of crossreactivity is an essential feature of the T-cell receptor". D. Mason 1998 Immunol Today 19, 395-404). This means a T cell must be capable of recognising many thousands of peptides. In addition, there must be many thousands of T cells expressing each TCR because the number of naive T-cells with the capacity to react to a foreign peptide must be high enough to ensure the rapid mobilization of an antigen- specific T-cell response, (A very high level of crossreactivity is an essential feature of the T-cell receptor". D. Mason 1998 Immunol Today 19, 395-404). In simple terms, there is a requirement for many T-cells to express each potentially useful TCR so that a pathogen encounters a T- cell that can recognize it within the first few hours of infection.

T-cells that express that same TCR are said to be a 'clone' (referred to hereinafter as clonotype). Direct estimates of human αβ TCR diversity suggest that only ~2.5xl 0⁷ different antigen receptors are present in the naive T-cell pool ("A direct estimate of the human αβ T cell receptor diversity". Arstila et al. (1999). Science 286, 958-61).

These facts suggest TCRs must be extremely cross-reactive. Indeed, we have evidence that individual CD8+ T-cells can recognise > 1,00,000 different peptides of 10 amino acids in length. If each TCR is capable of responding to hundreds of thousands of peptides then the chances of the actual disease-derived peptide it recognizes being the best peptide are almost vanishingly small. Therefore, it should be possible to alter almost any peptide antigen so as to improve its binding to host TCRs.

It is notable that, although αβ T cells are essential for effective vaccination, current vaccine technologies do not consider the individual clonotypes (or αβ TCRs) that vaccines induce and, further, current strategies almost invariably aim for the biggest measured immune response. However, we have speculated that the 'quality' of a T cell response is more important than its quantity. Thus, the best vaccine should induce the best T-cell receptor(s). 'Best' in the context of tumour immunity, where TCRs are known to bind weakly (Cole et al. 2007. Human T-Cell Receptor Binding Affinity is Governed by Major Histocompatibility Complex Class Restriction. Journal of Immunology, 178, 5727-34), might be the highest affinity TCR that can ideally recognize target cells bearing a low density of cognate pMHC on their surface. 'Best' in another context will be the most difficult TCR to escape from in the case of pathogens of variable antigenicity like HIV.

Our technology therefore goes against conventional wisdom by involving the specific selection of the best T-cells that induce the most effective immunity and/or the deselection of T-cells that do not induce effective immunity.

In our invention we have exploited the inherent plasticity and substantial degeneracy of the TCR to identify a (or those) clonotype(s) that produce(s) the best immune response and to then design the best vaccine with this/these clonotype(s) in mind.

Our methodology involves, essentially, three considerations (in no particular order of importance).

(1) To have widespread application the best clonotype must occur in most individuals within the population that express the relevant HLA molecule (as above, best in this context is disease specific and so, for example, for cancer it may be the most sensitive, whereas for HIV it may be the most relentless or difficult to escape from). These T-cell receptors are said to be 'public' .

(2) The best clonotype must be selected; and/or

(3) Ideally, poor clonotypes should be de-selected as they do not contribute to immue quality

An immunogenic agent or vaccine is engineered, designed or selected to bind specifically or selectively to, and activate, the best T-cell clonotype(s) so as to positively influence the propagation of that clonotype(s) during the vaccination process or the disease response. In short, our method gives us activation and expansion of the best clonotype(s).

This 'quality over quantity' approach is in substantial contrast to the prior art where an immunogenic agent or vaccine is chosen without any regard to its clonotypic influence but merely on the magnitude of its immune response, such as the number of T cells activated, thus any number of clonotypes are produced in response to a conventional vaccine (or disease) and, further, not all these clonotypes are effective at producing an immune response.

The invention described herein represents a departure from conventional wisdom in that it concerns addressing the problem of how to identify and manufacture a new antigen by considering, and exploiting, its clonotypic influence so as to induce T cells with the highest quality.

The invention thus concerns identifying both the best and the least sensitive T cells (having regard to the nature of the disease to be treated), ideally, but not exclusively, naturally occurring T cells, to a given antigen and then studying, ideally isolating, the T cell receptor (TCR) of said best sensitive T cell and using this TCR as a template or scaffold for the design, identification or selection of an optimal antigen, ideally, but not exclusively, a peptide or peptide-like compound that binds to this TCR well while binding to other (non-best/least sensitive) TCRs poorly. Once this optimal antigen has been designed, testing it to ensure that it activates only the best sensitive T cells and not the least sensitive T cells. If least sensitive T cells are also activated by the antigen, the antigen is further altered until this criterion has been satisfied i.e. it does not activate/propagate least sensitive (or non best) T cells. Once this has been shown, the antigen can be used as an immunogenic agent that, after administration to an individual to be treated, will skew a repertoire of naturally occurring T-cells towards that clonotype best (having regard to the nature of the disease to be treated) at recognizing and/or removing the antigen in question. The invention therefore concerns manipulating an existing immune system by identifying which type of T cells (clonotype(s)) in that system ought to be selected for further production and arranging matters so that these selected types of T cells are over produced with respect to other cells in said system. The invention therefore concerns the selection and use of an optimal antigen that provides an overall immune response with a substantially better clonotypic architecture than that of current vaccines or adjuvants.

When working the invention to see which T cells are activated by a selected antigen activation means examining said T cell so see if they exhibit effector function such as, without limitation, release of soluble lymphokines or effector cell proliferation or upregulation of surface activation markers or target cell lysis.

Notably, our invention can be employed in isolation of other, or established, vaccines or as an adjuvant or enhancer thereof. In the latter instance, for example, an immunogenic agent or vaccine identified using our invention could be administered to an individual prior to, after or at the same time as the administration of a conventional vaccine. When used in this way our technology would act as an adjuvant to select for the production of desirable T cell clonotypes within an individual when using a conventional vaccine thus ensuring the greater success of the conventional vaccine which, ideally, would be an attenuated pathogen. This skewing of the repertoire of T cells could, therefore, be used to generate key memory responses with the best clonotype for immunity prior to, after or at the same time as the administration of a conventional vaccine. When relevant, the interval between the administration of our technology and a conventional vaccine would be well know to those in the art and would be dependent upon the nature of the disease to be treated.

Also notably, whilst in some instances, the invention has been described having regard to the use of one T cell, one TCR and so one optimised antigen, it will be apparent to those skilled in the art that multiple T cells, TCR's and antigens may be identified and used together so that a given disease may be defeated using more than one optimised antigen administered simultaneously or successively, or both. Notably further still, although the invention has been explained having regard to peptide antigens it will also be apparent to those skilled in the art that the invention applies to any antigen that is recognised by αβ T cells and so may include non-peptide antigens.

Significantly, therefore, the technology can be applied to any T cell and revolutionizes vaccination by allowing the specific tailoring or skewing of the induced T cell response towards the most effective T cells for targeting disease.

Statements of Invention

According to the invention there is therefore provided a method for manufacturing, an immunogenic agent or vaccine comprising:

a) obtaining a population of T cells;

b) exposing said cells to at least one selected antigen and identifying a good TCR, or TCR's, expressed by said T cells which binds to said selected antigen with maximum affinity or avidity or sensitivity or efficacy and a poor TCR, or TCR's, expressed by said T cells which binds to said selected antigen with minimum affinity or avidity or sensitivity or efficacy ;

c) designing or obtaining at least one further improved antigen that binds to said good TCR, or TCR's, of part b) with maximum affinity or avidity or sensitivity or efficacy;

d) exposing said good and poor TCRs of part b) to said further antigen and

determining the response of said good and said poor TCRs to said antigen;

e) identifying an antigen as said immunogenic agent or vaccine on the basis that it stimulates under part d) a response from said good TCRs but does not stimulate a response from said poor TCRs; and

f) using the antigen identified under part e) in the manufacture of said

immunogenic agent or vaccine.

Reference herein to affinity is reference to the tendency of a molecule to associate with another.

Reference herein to avidity is reference to the combined strength of bond affinities in a complex. Reference herein to sensitivity is reference to the dose-response of a TCR to its antigen.

Reference herein to efficacy is reference to the capacity of a TCR to produce a desired effect.

According to a further aspect of the invention there is therefore provided a method for identifying an immunogenic agent or vaccine comprising:

a) obtaining a population of T cells;

d) exposing said good and poor TCRs of part b) to said further antigen and

determining the response of said good and said poor TCRs to said antigen; and e) identifying an antigen as said immunogenic agent or vaccine on the basis that it stimulates under part d) a response from said good TCRs but does not stimulate a response from said poor TCRs.

In either of the above aspects of the invention said response in parts d) and/or e) above involves or includes a measurement of T cell activation such as, without limitation, lymphokine release or upregulation of surface activation markers or effector cell proliferation or target cell lysis.

In an alternative embodiment of the invention said good TCR can be defined as a TCR characteristic of a T cell that has some other desirable property, in addition to or instead of its binding properties recited above, such as its ability to sustain a response, prevent immune escape or induce a desirable effector function. In a preferred method of either aspect of the invention part b) may be undertaken in silico i.e. as an analysis performed using computers in conjunction with informatics capabilities. Ideally, part b) involves the use of a particularly efficacious or immunogenic antigen and, more ideally still, the naturally occurring antigen for the disease to be treated by the said immunogenic agent or the vaccine. Further still, said good TCR in part b) is one present in the population to be treated and ideally in the majority of the population to be treated that express the relevant HLA molecule, accordingly, the above method may further include the determination of this fact wherein the good TCR is investigated in order to ensure it is present in the population, ideally the majority of said population, to be treated. Moreover, reference to the antigen in part b) above may also include reference to a co-presenting molecule such as a MHC molecule of either class I or II. Ideally, when designing or obtaining the further antigen under part c) above, regard is had to the Complimentarity Determining Regions (CDR) of the TCR that contact the peptide or peptide-MHC or antigen. Accordingly, said further antigen is designed or obtained, ideally, on the basis of its binding to the CDR of said good TCR, or TCR's, of part b).

In a preferred method of either aspect of the invention, where the antigen is a peptide or peptide-like molecule, part c) involves scanning combinatorial chemistry (peptide) library analysis to determine the optimal antigen for T-cells with maximum affinity or avidity or sensitivity or efficacy. However, other conventional methods may be employed for this purpose, such as, without limitation, structural and/or thermodynamic studies, provided they allow one to design or select an antigen that will bind to said TCR in an effective way and so produce the desired immunogenic response.

In the above methods said antigen under part b), and/or said antigen under part c) and d) may be a peptide and it may also be a peptide that is a MHC co-presented antigen and may be either a MHC class I or class II antigen. Further it may be an antigen that is co- presented by, for example, common HLA types such as, without limitation, HLA- A*0201, the most highly expressed Caucasian HLA type. However, the invention is not intended to be limited to any particular HLA type, but rather, is for use with those, typically, widespread among the population. An example of an immunogenic agent or vaccine manufactured or identified using the method of the invention is FATGIGIITV which is a peptide antigen for treating malignant melanoma.

Throughout this specification non-natural amino acid residues in antigenic peptides are indicated by bold text.

In a preferred method of either aspect of the invention said T cell population is a naturally occurring population or a cell line such as, without limitation, MEL5.

The invention also relates to the use of a good TCR or immunogenic agent, identified according to the method of the invention under parts b) and e) above, respectively, as a diagnostic indicator, particularly, but not exclusively, in autoimmune conditions such as where T-cells, such as killer T-cells, have a particular T-cell receptor for a native antigen (and so being equipped to attack native tissue). As will be appreciated by those skilled in the art, particularly potent TCRs of these deleterious T-cells can be identified and these TCRs and/or their antigen binding agent can be used as diagnostic indicators for a selected condition.

The invention further relates to an immunogenic agent, identified according to the method of the invention under part e) above, as a therapeutic to treat an allergic reaction or autoimmune disorder such as where T-cells, such as killer T-cells, have a particular T- cell receptor for a native antigen (and so are equipped to attack native tissue). As will be appreciated by those skilled in the art, a sufficiently large dose of the immunogenic agent can be used to, effectively, overwhelm the immune system and so shut down the clonotypes that respond to the immunogenic agent, thereby overcoming the condition.

An allergic reaction occurs when the immune system attacks a normally harmless substance, for example, without limitation, gluten in the diet leading to the development of Celiac disease. An autoimmune disorder is a condition that occurs when the immune system mistakenly attacks and destroys healthy body tissue. Such disorders include, without limitation, Hashimoto's thyroiditis, Pernicious anemia, Addison's disease, Type I diabetes, Rheumatoid arthritis, Systemic lupus erythematosus, Dermatomyositis, Sjogren syndrome, Lupus erythematosus, Multiple sclerosis, Myasthenia gravis, Reactive arthritis, and Grave's disease.

According to a further aspect of the invention there is provided a method for treating an individual comprising administering to said individual at least one immunogenic agent or vaccine identified or manufactured according to the method of the invention.

According to a further aspect of the invention there is provided an immunogenic agent or vaccine identified or manufactured according to the method of the invention.

In a preferred embodiment said agent or vaccine is a peptide antigen for treating malignant melanoma which is 66.66%, ideally, 70%, more ideally 77.77%, 80%, 88.88% or, more preferably, 90% identical with a naturally occurring peptide antigen, such as AAGIGILTV or EAAGIGILTV. In a further preferred embodiment of the invention said peptide antigen is FATGIGIITV.

According to a further aspect of the invention there is provided an immunogenic agent or vaccine comprising an antigen that elicits an immune response from a selected T cell clonotype wherein said clonotype comprises only those T cells that produce an effective immune response. In this instance effective immune response is taken to mean the eradication of invading pathogen or neoplastic cells, or the prevention of immune escape or immune tolerance of a specific T-cell clonotype.

According to a further aspect of the invention there is provided an immunogenic agent or vaccine identified or manufactured according to the method of the invention for use as an adjuvant.

In a preferred embodiment said agent or vaccine is FATGIGIITV. According to a further aspect of the invention there is provided an adjuvant comprising an immunogenic agent or vaccine identified or manufactured according to the method of the invention.

In a preferred embodiment said agent or vaccine is FATGIGIITV.

According to a further aspect of the invention there is provided a combination therapeutic comprising an immunogenic agent or vaccine produced according to the method of the invention in combination with at least one further therapeutic, wherein said further therapeutic may be any known medicament and/or an alternative or additional vaccine.

Typically, in this latter aspect of the invention the combination therapeutic may comprise any conventional therapeutic for treating or alleviating the symptoms of the disease to be treated in combination with the immunogenic agent or vaccine of the invention.

According to a yet further aspect of the invention there is provided a cell or cell line that expresses a high affinity TCR for either a naturally occurring disease associated pathogen or an immunogenic agent of the invention. Ideally, said TCR exists in the majority of the population. In the instance where the invention is used to identify or manufacture an immunogenic agent or vaccine to treat malignant melanoma said cell line or clonotype is MEL5 clone.

According to a further aspect of the invention there is provided a TCR isolated from said cell or cell line.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprises", or variations such as "comprises" or "comprising" is used in an inclusive sense i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. All references, including any patent or patent application, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art.

Preferred features of each aspect of the invention may be as described in connection with any of the other aspects.

Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus, features, integers, characteristics, compounds or chemical moieties described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.

Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.

An embodiment of the invention will now be described by way of example only with reference to the following figures wherein: -

Figure 1 shows the majority of Melan A reactive T-cells exhibit poor recognition of the natural ligand(s) (A) and only one T-cell clone that gives the profile in (B), ELAGIGILTV (black squares) EAAGIGILTV (hashed squares) and AAGIGILTV (empty squares), the latter 2 represent natural epitopes;

Figure 2 shows a diagrammatic representation of the structure of the Mel TCR ELAGIGILTV (Structural recognition of the most studied human cancer antigen J. Biol. Chem. (2009), 284, 27281-9);

Figure 3 shows an enhanced view of a part of Figure 2 wherein the binding site of the peptide is shown in greater detail (Cole et al. Germline-governed recognition of a cancer epitope by an immunodominant human T-cell receptor. Journal of Biological Chemistry, 284, 27181-9);

Figure 4 shows the selection of an antigen for producing the best clonotypes for fighting melanoma, the ILAGIGILTV peptide is the best agonist for the 'good' CTL clone but it is an even better agonist for 'poor' CTL, in contrast peptide FATGIGIITV is a super- agonist for the good CTL clone but does not activate poor CTL clones;

Figure 5 shows how designed antigens compare with naturally occurring ligands when eliciting an immune response and importantly is can be seen that the peptide made in accordance with our invention is superior to the other competeing peptides made using conventional techniques;

Figure 6 shows that peptide made in accordance with our invention FATGIGIITV (empty graph/red histograms) primes CTL lines that see the natural peptide AAGIGILTV better that those primed with current technology the ELAGIGILTV heteroclitic peptide (hashed graph/black histograms), these cells also exhibit better recognition of HLA A2+ melanoma cells (526/624) (right panel);

Figure 7 shows Melan A peptide analogues that preferentially activate the MEL5 CTL clone. A-C. 3x104 MEL187.c5 or MEL5 CTL were incubated overnight with 6x104 C1R A2 B cells pre-pulsed with various concentrations of indicated peptide. Supernatant was subsequently harvested and assayed for ΜΙΡΙβ by ELISA.

A. Compares recognition of the ELAGIGILTV sequence to IF, 3T and 81 single substitutions. B. Compares recognition ELAGIGILTV and FLTGIGIITV by each clone. C. Compares recognition ELAGIGILTV and FATGIGIITV.

Figure 8 shows binding of Melan A analogue peptides to Melan A-specific TCRs. SPR equilibrium binding of soluble MEL5 TCR to HLA A*0201 -FLTGIGIITV (A) and HLA A*0201 -FATGIGIITV (B) and binding of soluble MEL187.c5 to (C) HLA A*0201- FLTGIGIITV and (B) HLA A*0201 -FATGIGIITV. The mean response for each concentration is plotted (n = 2). Table I: Binding affinities of MEL187.c5 and MEL5 TCRs to Melan A-peptide variants. Summarized data from this study.

Examples Melanoma

Malignant melanoma is by far the leading cause of skin cancer-related deaths worldwide. Melanona immunotherapy efforts have been largely focused on an 18 kDA melanocyte- specific transmembrane protein called Melanoma Antigen (Melan A) or Melanoma Antigen Recognised by Tcells (MART-1); a good vaccine candidate for the treatment of melanoma. There is a large HLA A*0201 -restricted naive Melan A specificT-cell pool available for manipulation in both melanoma patients and normal donors.

The MART-1, also known as Melan A, protein is expressed in more than 60% of melanoma cells. The natural sequence of the HLA A2-restricted Melan A27-35 9 amino acid peptide is AAGIGILTV. One study also claimed to find the longer Melan A26-35 10 amino acid peptide EAAGIGILTV on the surface of tumour cells.

The 9 mer AAGIGILTV peptide is a very poor immugen and it is very difficult to raise T-cell responses with this peptide. It is possible to raise immune responses using the EAAGIGILTV peptide.

The EAAGIGILTV peptide does not incorporate a good position 2 anchor for HLA A2 binding. Immunologists have therefore substituted p2 for a leucine. The ELAGIGILTV 'heteroclitic' peptide is now in widespread use in melanoma vaccination trials as it is capable of inducing a very large immune response.

Unfortunately, the response generated by the commonly used ELAGIGILTV peptide is not good at recognizing real melanoma cells (which express the shorter AAGIGILTV peptide). Our clonotypic analysis indicates that the ELAGIGILTV peptide induces (or 'primes') T-cells that express hundreds of different TCRs. Most of these TCRs are very poor at recognizing the natural AAGIGILTV peptide (KD >80 μΜ).

Current melanoma vaccine strategies therefore generate large numbers of CD8+ T-cells that recognize melanoma poorly. Our aim was to use our invention in order to induce T- cells with the best clonotype (TCR) for recognizing the natural peptide antigen known to be present on the surface of Melanoma cells. In order to design an analogue peptide that stimulates T-cell clonotypes superior in the detection and destruction of melanoma cells, we grew a number of Melan A specific T-cell clones and selected a candidate T-cell clone, MEL5, that efficiently recognizes the dominant natural epitope, AAGIGILTV.

We have expressed a soluble TCR derived from an A2 Mel specific T cell clone and studied the TCR/ELAGIGILTV interaction structurally and thermodynamically. Furthermore, we have used a randomized peptide library screen in order to determine the optimal peptide sequence for the ELAGIGILTV specific TCR.

The crystal structure and the peptide screen show that, although the TCR makes contact with seven of the ten Mel peptide residues, it is exquisitely specific for the positions four-six, which form the central peptide bulge. The other TCR-peptide interactions show a great degree of flexibility and so represent a basis to develop a peptide with greater immunogenic potential for use in anti-melanoma therapies.

METHODS

Generation and maintenance of HLA-A*0201 -restricted Melan A26-35 specific CTL With reference to Figure 1, a population of T cells was isolated from a blood sample, using conventional techniques, and clones were established using conventional techniques. (See Sewell et. al (1997). European Journal of Immunology, 27, 2323-29 for methods). Briefly, the generation of HLA A2-restricted MARTI -specific CTL lines were generated essentially as follows. CD8+ T-cell lines specific for HLA A*0201- restricted Melan A epitopes were generated by pulsing 6x106 PBMCs from healthy HLA A*0201+ individuals with 10 μΜ peptide (ELAGIGILTV or AAGIGILTV or EAAGIGILTV or FATGIGIITV) for 1 hour at 37°C; cells were subsequently washed and resuspended in RIO media (RPMI 1640 supplemented with 100 U/ml penicillin (LifeTechnologies, Paisley, UK), 100 μg/ml streptomycin (Life Technologies) and 10% heat inactivated FCS (Life Technologies). After day 3, increasing amounts of IL-2 were added to the media reaching a maximum concentration of 20 IU/ml by day 14, lines were then tested by pMHC class I (pMHCI) tetramer staining. CD8+ T-cells were subsequently maintained in R10 with 2.5% cellkines (Helvetica Healthcare, Geneva, Switzerland), 20 IU/ml IL-2 and 25 ng/ml IL-15 (Peprotech, London,UK).

Clones MEL5 and MEL187.c5 were used in this study.

SPR Analysis

Inducing effective CTL responses against cancer

Soluble MEL5 or MEL187.c5 derived TCRs were manufactured as previously described (16, 42). Binding analysis was performed using a BIAcore T3000TM equipped with a CM5 sensor chip. Between 200 and 400 response units (RUs) of biotinylated pMHCI was immobilized to streptavidin, which was chemically linked to the chip surface. The pMHCI was injected at a slow flow rate (ΙΟμΙ/min) to ensure uniform distribution on the chip surface. Combined with the small amount of pMHCI bound to the chip surface, this reduced the likelihood of off-rate limiting mass transfer effects. The MEL5 TCR and MEL187.c5 TCR were purified and concentrated to -100 μΜ on the same day of SPR analysis to reduce the likelihood of TCR aggregation affecting the results. For equilibrium analysis, eight serial dilutions were carefully prepared in triplicate for each sample and injected over the relevant sensor chips at 25°C. The TCRs were injected over the chip surface at a flow rate of 45 μΐ/min. Results were analyzed using BIA evaluation 3.1TM, Microsoft ExcelTM and Origin 6.1TM. The equilibrium binding constant (KD) values were calculated using a nonlinear curve fit (y = (P1JC)/(P2 + x).

The MEL5 CTL clonotype exhibits the best recognition of the dominant natural AAGIGILTV peptide that we have seen (unpublished observations). We are unaware of any other clonotype that exhibits better recognition of the AAGIGILTV peptide. The MEL5 clone appears to compensate for the reduced HLA A*0201 binding of EAAGIGILTV and AAGIGILTV natural peptides (Figure IB) suggesting that its TCR must bind better to these variants than to HLA A*0201-ELAGIGILTV. Indeed, the MEL5 TCR binds to the natural epitope, HLA A*0201 -EAAGIGILTV, with an affinity within the range normally seen for TCRs that recognise pathogen-derived epitopes (KD ~ 6.4 μΜ) (Table I). To the best of our knowledge, this is the highest affinity ever described for TCR binding to a non-MHC anchor modified self-peptide. MEL5 TCR binds to HLA A*0201 -AAGIGILTV with an affinity of KD 14 μΜ compared to KD 17 μΜ for HLA A*0201-ELAGIGILTV (Table I). Overall, we conclude that the MEL5 TCR is an outlier within the population of TCRs able to engage HLA A*0201 Melan- A27-35 and Melan-A26-35 as it exhibits a strong preference for the natural antigen over the heteroclitic variant. We reasoned that T-cells bearing TCRs with specificities and binding affinities similar to MEL5, that can recognize the dominant natural antigen on the surface of melanoma cells efficiently, would represent ideal clonotypes to induce during therapeutic vaccination against melanoma. We next devised a strategy to skew the repertoire of HLA A*0201 -restricted Melan A-specific T-cells towards the MEL5 clonotype and those with similar properties.

Inducing effective CTL responses against cancer

CTL effector function assays: MlPlfi, CBA analysis and degranulation assay

MEL5 or MEL187.c5 CTL clones were incubated overnight at 37°C with C1R A2 cells (36) previously pulsed for 1 hour with indicated peptide at various concentrations. Subsequent to incubation, supernatant was harvested and assayed for ΜΙΡΙβ by ELISA according to the manufacturer's instructions (R&D systems, Abingdon, UK). Remaining supernatant was assayed with the human TH1/TH2 cytokine kit (BD Biosciences, San Jose, CA) according to the manufacturer's instructions; data were acquired using a FACSCalibur flow cyto meter and analyzed with CBA software (BD Biosciences). Surface CD 107a mobilization was used to assess degranulation as described previously (37). Briefly MEL5 or MEL187.c5 CTLs were incubated for 4 hours at 37°C with C1R A2*0201 cells previously pulsed with various concentrations of indicated peptide. Both FITC-conjugated anti-CD107a (clone H4A3; BD Biosciences) and 0.7 μΐ/ml monensin (GolgiStop™; BD Biosciences) were added prior to incubation. Subsequent to incubation, the cells were washed twice and resuspended in PBS. Data were acquired using a FACSCalibur flow cytometer and analyzed with FlowJo software (Tree Star Inc. Ashland, OR).

The T cell responses were measured in terms of MIPip(pg/ml) and as a result a clone showing poor recognition of the natural ligands EAAGIGILTV and AAGIGILTV was identified MEL187.c5 and a clone showing good recognition of these ligands was also identified (MEL5) (Figure 1). The public TCR (i.e TCR present in the majority of individuals to be treated) from this T-cell clone (MEL5) bound to the AAGIGILTV peptide well (KD = 18 μΜ). T-cells with this TCR are able to kill Melanoma cells well.

Crystallization, diffraction data collection and model refinement

HLA A*0201 -FATGIGIITV crystals were grown at 18°C by vapor diffusion via hanging drop technique. Data from HLA-A*0201- FATGIGIITV crystals were collected at 100K on beamline 103 at the Diamond Light Source (DLS), Oxfordshire, UK. The HLA-A*0201 -FATGIGIITV crystals dataset was collected at a wavelength of 0.976A using a ADSC Q315 CCD detector. Reflection intensities were estimated with the MOSFLMpackage (44) and the data were scaled, reduced and analyzed with SCALA and the CCP4 package(45). The structure was solved with Molecular Replacement using PHASER (46). The model sequence was adjusted with COOT (47) and the model refined with REFMAC5 (48). Graphical representations were prepared with PYMOL (49). Data reduction and refinement statistics are shown in Supplementary Table I. The reflection data and final model coordinates were deposited with the PDB database, assigned accession code PDB: 3GH1 . The PDB details are 1 0.221 0/ pdb3hq l / pdb located at

http ://www.pdb.org/pdb/ explore/ explore.do?structurel d= 3 HG1 We also solved the structure of this MEL5 TCR which is shown in Figures 2 and 3. With reference to Figure 3 it can be seen that the amino acids at positions 1, 3 and 8 of the peptide do not make optimal TCR contact and therefore these represent positions where optimisation can be achieved.

Decamer combinatorial peptide library (CPL) scan

The decamer combinatorial peptide library contains a total of 9.36x10 12 (10+19) xl99) different decamer peptides (38, 39) and was divided into 200 different peptide mixtures (40). For CPL screening, MEL5 CTL were washed and rested overnight in R10 in 96- well U-bottom plates, 6xl0⁴ CIR cells were pulsed with various library mixtures (at 100μ§/ιη1) in duplicate for 2 hours at 37°C. Following peptide pulsing, 3xl0⁴ MEL5 CTL were added and the assay was incubated overnight at 37°C. Subsequently, the supernatant was harvested and assayed for ΜΙΡΙβ by ELISA according to the manufacturer's instructions (R&D systems).

Combinatorial peptide library screens of the MEL5 TCR suggested that this clone prefers a 10 amino acid long peptide to other lengths. This may partly explain why the AAGIGILTV natural 9 amino acid antigen does not readily prime this TCR. These screens indicated that this TCR likes an isoleucine at position 1 to the natural 10 mer pi glutamic acid.

Natural 9-mer AAGIGILTV

Natural 10-mer EAAGIGILTV

Heteroclitic ELAGIGILTV

TCR optimized ILAGIGILTV

The best agonist we have seen for the Mel5 T-cell clone is the ILAGIGILTV peptide. Testing of the TCR optimized peptide ILAGIGILTV showed that it was 100 x more potent an agonist for the Mel 5 CD8+ T-cell clone than the ELAGIGILTV peptide currently in vaccine trials. Unfortunately, several T-cells with 'poor' TCRs also preferred the sequence ILAGIGILTV. This data is shown in Figure 4, left hand graph. Mutation of the TCR optimized peptide ILAGIGILTV indicated substitutions at position 1, 3 and 8 of the natural 10-mer (FATGIGIITV) produced a peptide only 10-fold less potent than the ILAGIGILTV peptide at activating MEL 5 T-cells but that was not favoured by the TCRs of clontypes that were poor at recognizing the AAGIGILTV natural antigen. This data is shown in Figure 4, right hand graph. Additionally, these substitutions of Phenylalanine (F), Threonine (T) and Isoleucine (I) at position 1, 3 and 8 (FATGIGIITV) produced a potent stimulation of the MEL5 CTL clone that has a superior ability to recognize natural tumor epitopes but a poor stimulation of MEL187.c5, a CTL clone that is only efficient at recognizing the heteroclitic peptide (Figure 7C).

Single amino acid substitutions made to the ELAGIGILTV peptide showed a range of effects on the ability of the peptide to activate MEL187.c5 and MEL5 CTL clones (Figure 7A). A triple substitution (IF, 3T and 81) in the ELAGIGILTV peptide produced a peptide (FLTGIGIITV) that showed enhanced ability to activate both MEL187.c5 and MEL5 clonotypes (Figure 7B). In contrast, the same triple substitution (IF, 3T and 81) in the natural EAAGIGILTV epitope produced a peptide analogue (FATGIGIITV) with different properties.

We thus proceeded with the FATGIGIITV peptide in the Melan A system as this peptide is a super agonist (>100x better than the natural ligand) for a 'good' Melan A TCR while being a poor agonist for all 'poor' Melan A TCRs tested. This peptide thus fulfils all of the requirements for optimizing or producing the best T cell response.

In Figure 6 Melan A-specific T-cell lines were primed from HLA A2+ve peripheral blood mononuclear cells. Primed CTL lines were assayed for degranulation by flow cytometric assessment of cell surface expression of CD 107. In figure 6 we show tests on the optimized peptide and it can be seen that our optimized peptide induced a good immune response when compared with either the natural ligand AAGIGILTV or the peptide currently being used to produce vaccines ELAGIGILTV. Moreover, the peptide FATGIGIITV primes CTL lines that see the natural peptide AAGIGILTV better that those primed with current technology (i.e. the ELAGIGILTV heteroclitic peptide). Further, these cells also exhibit better recognition of HLA A2+ melanoma cells.

Moreover, introducing the triple substitution (IF, 3T and 81) into the hetreoclitic peptide to produce the FLTGIGIITV peptide results in stronger binding to both MEL5 and MEL187.C5 TCRs (KD of 16μΜ and 5.1μΜ, respectively) (Figure 8A&C) consistent with the observation that this peptide has an enhanced ability to activate both MEL187.c5 and MEL5 when compared to activation with the ELAGIGILTV peptide (Figures 7B). In contrast, introducing the triple substitution (IF, 3T and 81) into the EAAGIGILTV natural sequence to generate the FATGIGIITV sequence increases the strength of the interaction with MEL5 TCR by ~6 fold (to a KD of 3 μΜ) but weakens the strength of the interaction with MEL187.c5 TCR by ~2 fold (to a KD of 35 μΜ) (Figure 8B&D).

Summary

We have previously shown that TCRs can differentiate between natural and anchor- modified heteroclitic peptides enabling T-cells to exhibit a strong preference for either type of antigen (29). Thus, MHC anchor- modified heteroclitic peptides can induce T-cell populations that are clonotypically distinct from those induced by natural tumor eptiopes (29, 51). It is therefore important that the T-cell clonotypes induced by any altered peptide ligand (APL)-based immune intervention are carefully evaluated after ex vivo priming to ensure efficacy prior to studies in vivo. The successful approach adopted here incorporates some important fundamental differences to the attempts that preceded it. The first difference is that we started by selecting a CTL clonotype with superior ability to recognize the natural target. Our rationale was that we might then be able skew the expanded CTL population towards the type of clonotype known to be superior in recognition and destruction of tumor cells. We describe this approach as 'TCR Optimized Peptide Skewing Of the Repertoire of T-cells' (TOPSORT). In the examples described above, we selected a Melan A- specific CTL clone (MEL5) with superior ability to recognize the natural tumor epitopes EAAGIGILTV and AAGIGILTV (AAGIGILTV being the most abundantly cell surface expressed of these two antigens); then utilised CPL scan technology to identify analogue peptides that exhibited improved recognition by this clonotype. The same technique can be applied to the identification of any other new, improved antigen.

Thus our invention, which uses peptides optimized for good TCRs, but deselected against poor TCRS, induces T-cell responses of better overall quality. As a result of this targeting towards good TCRs, this new technology will result in better immunogenic agents and vaccines.

Table 1 LIGAND TCR binding ΚΟ (μΜ)

MEL5 M EL.187.c5

HLA A*0201-ELAGIGILTV 17 18

HLA A*0201-EAAGIGILTV 6.4 42

HLA A*0201- AAGIGILTV 14 94

HLA A*0201-FLTGIGIITV 5.1 16

HLA A*0201-FATGIGIITV 3 35

References

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Claims

1. A method for identifying, or manufacturing, an immunogenic agent or vaccine comprising:

a) obtaining a population of T cells;

b) exposing said cells to at least one selected antigen and identifying a good TCR, or TCR's, expressed by said T cells which binds to said selected antigen with maximum affinity or avidity or sensitivity or efficacy and a poor TCR, or TCRs expressed by said T cells which binds to said selected antigen with minimum affinity or avidity or sensitivity or efficacy ;

c) designing or obtaining at least one further improved antigen that binds to said identified good TCR, or TCR' s, of part b) with maximum affinity or avidity or sensitivity or efficacy;

d) exposing said good and poor TCRs of part b) to said further antigen and

determining the response of said good and said poor TCRs to said antigen; and e) identifying an antigen as said immunogenic agent or vaccine on the basis that it stimulates under part d) a response from said good TCRs but does not stimulate a response from said poor TCRs; and, where said immunogenic agent or vaccine is to be manufactured,

f) using the antigen identified under part e) in the manufacture of said

immunogenic agent or vaccine.

2. A method according to claim 1 wherein said good TCR is defined as a TCR characteristic of a T cell that has a desirable property, in addition to or instead of the binding properties recited in claim 1, such as its ability to sustain an immune response.

3. A method according to claim 1 or 2 wherein said good TCR of part b) is one present in the majority of the population to be treated.

4. A method according to claims 1-3 wherein part (b) is undertaken in silico.

5. A method according to claims 1 - 4 wherein part (b) involves the use of a particularly efficacious or immunogenic antigen.

6. A method according to any preceding claim wherein the antigen of part b) is naturally occurring.

7. A method according to any preceding claim wherein the antigen of part b) or c) includes a co-presenting molecule.

8. A method according to any preceding claim wherein the antigen of part b) or c) is a peptide.

9. A method according to claim 8 wherein part (c) involves scanning a combinatorial peptide library to determine the optimal or improved peptide for said TCR.

10. A method according to any preceding claim wherein part (c) involves undertaking a structural or thermodynamic analysis of said improved antigen or said TCR.

11. A method according to any preceding claim wherein part (c) involves designing or obtaining said further antigen on the basis of its binding to the CDR of said good TCR.

12. A method according to any preceding claim wherein said antigen of part b) or said further improved antigen of part c) or said peptide of claims 8-11 is a MHC co- presented antigen.

13. A method according to claim 12 wherein said MHC is either class I or class II.

14. Use of a good TCR or immunogenic agent, identified according to claim 1 under parts b) and e), respectively, as a diagnostic indicator.

15. Use according to claim 14 wherein said indicator is for autoimmune conditions.

16. A method for treating an individual comprising administering to said individual at least one immunogenic agent or vaccine identified, or manufactured, in accordance with claims 1-13.

17. An immunogenic agent or vaccine identified or manufactured in accordance with claims 1-13.

18. An immunogenic agent or vaccine wherein said agent or vaccine is a peptide antigen for treating malignant melanoma which is 66.66%, 70%, 77.77%, 80%, 88.88% or 90% identical with a naturally occurring peptide antigen but not including ELAGIGILTV.

19. An immunogenic agent or vaccine wherein said agent or vaccine is selected from the list comprising FATGIGIITV.

20. An immunogenic agent or vaccine comprising an antigen that elicits an immune response from at least one selected T cell clonotype wherein said clonotype comprises only those T cells that produce an effective immune response.

21. A combination therapeutic comprising an immunogenic agent or vaccine produced according to claims 1 - 13 or according to 17-20 in combination with at least one further therapeutic.

22. An adjuvant comprising an immunogenic agent or vaccine produced according to claims 1 - 13 or according to 17-20.

23. An immunogenic agent, identified according to part e) of claim 1, as a therapeutic to treat an allergic reaction or autoimmune disorder.

24. An immunogenic agent according to claim 23 wherein said reaction or disorder is any one or more of the following:Hashimoto's thyroiditis, Pernicious anemia, Addison's disease, Type I diabetes, Rheumatoid arthritis, Systemic lupus erythematosus, Dermatomyositis, Sjogren syndrome, Lupus erythematosus, Multiple sclerosis, Myasthenia gravis, Reactive arthritis, Grave's disease and Celiac disease.

25. A cell line for producing an immunogenic agent or vaccine according to claims 1-13 or 17-20.

26. A cell line according to claim 25 wherein said cell line is MEL5.

27. A method according to claims 1-13; use according to claims 14 or 15; a method according to claim 16; an immunogenic agent or vaccine according to claims 17-20, 23 & 24; a combination therapeutic according to claim 21; an adjuvant according to claim 22; and a cell line according to claims 25 & 26 as substantially herein described and with reference to the accompanying figures.