Peptide pool

Abstract

A pool of peptides comprising at least a first and second peptide derived from keyhole limpet hemocyanin (KLH), wherein both peptides are from 9 to 40 amino acids in length and comprise at least 9 con

Description

Field The invention related to a pool of peptides from KLH, to the use of the pool of peptides in a T cell response assay and to methods for determining the potential of a peptide to stimulate a T cell response. Background Immunogenicity to antibodies and other therapeutic proteins or polypeptides can be problematic. It can cause adverse reactions, loss of efficacy and loss of residual protein or polypeptide function. This immunogenicity is mediated by antibody responses, especially differentiated anti-drug antibody responses (ADA). Mature ADA rely on T cell help to mature and differentiate. While antibodies typically recognize elements of native proteins or peptides with at least secondary structure, T cells recognize relatively short linear peptides derived from such proteins through proteolytic processing, such as those presented on antigen presenting cells (APC). ADA incidence is difficult to predict outside clinical trials and based on animal studies. Animals are often too mismatched to humans, recognising therapeutic proteins or polypeptides and antibodies, that have been designed for human therapy as foreign in any event. Modelling the cascade of events that lead to a differentiated antibody response in silico or in vitro has been challenging. It is however possible to an extent to recapitulate T cell responses that are specific to drug or protein derived peptides in vitro, for example in a cohort of human peripheral blood mononuclear cell (PBMC) donors. This can be achieved by studying sets of overlapping peptides representing a therapeutic antibody or other protein in T cell proliferation assays, or other T cell stimulation assays, to gain an understanding of which peptides may represent functional T cell epitopes that could help in the development of ADA (Jawa et al. (2013) Clinical Immunology 149, 534-555). Such overlapping peptides can be conveniently made using chemical synthesis at high purity. T cell proliferation assays can measure both naive T cell responses and recall T cell responses. Measuring naive responses is important to assess the antigenicity of candidate therapeutic proteins as it is likely that the recipient of a novel therapy will be naive (i.e. not have been pre-exposed) to that therapy. Controls typically used include Keyhole Limpet Hemocyanin (KLH) and Tuberculin Purified Protein Derivative PPD. KLH is a naive antigen protein for humans, as most humans would not have been exposed to that protein, and PPD is a recall antigen in many individuals as a consequence of prior vaccination for tuberculosis. However, neither KLH nor PPD is in the same category of antigen as a synthetic peptide, which is often what is the test article to be tested; KLH is a very large natural product protein and PPD is a protein mixture. With all protein-based controls there is a challenge to control their product quality over time (batch to batch variation, etc.) Being a natural product and a protein mixture, respectively, KLH and PPD present their own challenges with respect to their quality control. It would therefore be desirable to have a positive control for the assay that is comparable in nature to synthetic peptides and nevertheless controls for T cell stimulation over a wide variety of donors. Individual known highly antigenic peptides have been used in the past as controls, such as peptides derived from Influenza Hemagglutinin and Tetanus Toxoid. However, responses to individual peptides are often only sporadically observed in donors and are often rather donor dependent. This is also due to there being only a few known highly antigenic peptides that will bind to a broad spectrum of donor HLA types, whereby they would be able to stimulate a T cell response. Naive T cell proliferation assays are often carried out with PBMCs derived from cohorts of 20-40 independent HLA diverse PBMC donors to reflect the incidence of responses in a diverse population. It is often found that HA and TT single peptide responses often occur in only 5-10% of donors. The low incidence of responses and their distribution, which is thought to follow a Poisson distribution, mean that the actually observed incidence varies widely from donor-cohort study to study, even where both studies are designed in the same way and carried out one after another on different groups of randomly selected donors. Proteins such as KLH and PPD (protein mixture) have very high response rates, usually with close to 100% of donors responding, but these responses are to proteins, not peptides, so it can be argued that they do not represent adequate controls for the category of test article, represented by synthetic peptides. Instead of using individual peptides as controls, protein sequences like those from test antigens can be represented by overlapping synthetic peptides, such as overlapping 15mer peptides, offset by three amino acids to measure the proliferation of helper T cells. For a large protein antigen such as KLH, such overlapping peptide pools are expensive to synthesize. It is also hard to control the quality of the many resulting peptides, and most peptides in the pool would be non-stimulatory in any event. A better strategy would be to find a range of select peptides from a naive antigen such as KLH that are presented by donors with a wide range of HLA Class II types that when routinely used in T cell assays on larger donor cohorts, e.g. of 20-50 donors, that reliably stimulate at least the majority of those donors to a significant extent. The advantage of such a peptide pool is also that the magnitude of the response would be more comparable to that achievable by other naive antigenic peptides, which is a property that protein antigens cannot adequately reflect. One known peptide pool with such properties is the CEFT peptide pool, which is popular as a control for measuring recall T cell responses. CEFT is short for Cytomegalovirus, Epstein Barr Virus, Flu, Tetanus. As the name suggests it consists of HLA Class II -binding peptides derived these pathogens, whereby it is expected that most PBMC donors will have a recall T cell response to one or more of these pathogens, due to likely prior exposure. Thus, while the CEFT pool is a good positive control in peptide form for T cell stimulation assays from peptides to which the donors have been exposed, it is not a good control for naive responses to peptides. Summary The present invention provides a KLH peptide pool which is a robust peptide-based control in T cell stimulation assays, such as proliferation assays because most PBMC donors will respond significantly to said pool without there being a requirement for the donors having to have been exposed to any of the peptides, making it a particularly good control for naive responses. The peptides in the KLH peptide pool typically each comprise at least one CD4+ epitope. A peptide comprising a CD4+ epitope can be presented on Class IIHLA and stimulate a CD4+ response in a T cell assay. The peptides in the KLH peptide pool can typically be presented as antigens on HLA Class II, stimulate responses in T cell assays, and bind to more than one HLA subtype. Each peptide in the pool may bind to one or more HLA subtype. The peptides in the pool as a whole may bind to more than one HLA subtype, such as to two, three, four or more HLA subtypes. The pool is easier to control for manufacture than an overlapping peptide pool because it contains only a few isolated peptides that can be quality controlled individually before mixture at reasonable effort, which have advantageous properties. Accordingly, the present invention provides a pool of peptides comprising a first peptide and a second peptide, wherein: (i) the first peptide is from 9 to 40 amino acids in length and comprises at least 9 contiguous amino acids of the sequence shown in any one of SEQ ID NOs: 1 to 9 or of a sequence having at least 80% identity to the sequence shown in any one of SEQ ID NOs: 1 to 9, wherein the at least 9 contiguous amino acids comprise at least one CD4+ T-cell epitope; and (ii) the second peptide is from 9 to 40 amino acids in length and comprises at least 9 contiguous amino acids of any one of the sequences shown in SEQ ID NOs: 1 to 9 or of a sequence having at least 80% identity to any one of the sequences shown in SEQ ID NOs: 1 to 9, wherein the at least 9 contiguous amino acids comprise at least one CD4+ T-cell epitope, wherein the at least 9 contiguous amino acids comprised in the second peptide are of a different sequence to the at least 9 contiguous amino acids comprised in the first peptide. The CD4+ T-cell epitope comprises at least one anchor residue, such as two, three or four anchor residues, as shown in Table 1. The CD4+ T-cell epitope may comprise one anchor residue labelled as anchor residue 1 in Table 1, typically followed by at least 8 amino acids, which preferably include at least one of the anchor residues labelled as anchor residues 3, 4, 6 or 9 in the same sequence in Table 1, more preferably two of the anchor residues labelled as anchor residues 3 and 6, 4 and 6, 4 and 9, 6 and 9 or all three of the anchor residues labelled as anchor residues 4, 6 and 9, most preferably all 8 amino acids following the residue labelled as anchor residue 1 in Table 1. The core CD4+ T-cell epitope may have the amino acid sequence YSLRKAMER (SEQ ID NO: 10), YQAIAGYHG (SEQ ID NO: 11), LINDATYFN (SEQ ID NO: 12), YFNSRSQTF (SEQ ID NO: 13), FFLKYEAFD (SEQ ID NO: 14), VYKYEITQQ (SEQ ID NO: 15), YKYEITQQL (SEQ ID NO: 16), FVKQMEDAL (SEQ ID NO: 17), VKQMEDALA (SEQ ID NO: 18), YDRVFKYDI (SEQ ID NO: 19), FKYDITEKL (SEQ ID NO: 20) or YEHIAGFHG (SEQ ID NO: 21). SEQ ID NO: 10 is comprised in SEQ ID NO: 1 and comprises anchor motifs that bind the HLA allele 15:01. SEQ ID NO: 11 is comprised in SEQ ID NO: 2 and comprises anchor motifs that bind the HLA allele HLA-DRBl*01:01. SEQ ID NOs: 12 and 13 are comprised in SEQ ID NO: 3, and SEQ ID NO: 12 comprises anchor motifs that bind the HLA alleles HLA-DRBl*01:01 and 03:01, whilst SEQ ID NO: 13 comprises anchor motifs that bind the HLA allele HLA-DRB 1*07:01. SEQ ID NO: 14 is comprised in SEQ ID NO: 4 and comprises anchor motifs that bind the HLA allele HLA-DRB1*12:O1. SEQ ID NOs: 15 and 16 are comprised in SEQ ID NO: 5 and comprises anchor motifs that bind the HLA alleles HLA-DRBl*01:01, 07:01 and 15:01. SEQ ID NOs: 17 and 18 are comprised in SEQ ID NO: 6 and comprises anchor motifs that bind the HLA allele HLA-DRBl*07:01. SEQ ID NO: 19 is comprised in SEQ ID NO: 7 and comprises anchor motifs that bind the HLA alleles HLA-DRB1*11:01 and 11:03. SEQ ID NO: 20 is comprised in SEQ ID NO: 8 and comprises anchor motifs that bind the HLA allele HLA-DRBl*04:01. SEQ ID NO: 21 is comprised in SEQ ID NO: 9 and comprises anchor motifs that bind the HLA allele HLA-DRBl*01:01. Hence, the pool of peptides may comprise two or more peptides of up to 40 amino acids in length comprising at least two of the following amino acid sequences: SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12 and / or SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15 and / or SEQ ID NO: 16; SEQ ID NO: 17 and / or SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; and / or SEQ ID NO: 21. The pool of peptides preferably comprises a mixture of peptides that bind to at least two alleles, preferably at least three or four, more preferably five or six, most preferably seven or all of the HLA alleles HLA-DRBl*01:01, 03:01, 04:01, 07:01, 11:01, 11:03, 12:01 and 15:01. The pool of peptides may, for example, comprise a mixture of peptides that bind to at least two different HL A alleles, such as, for example, HLA-DRB1* 15:01 and 01:01, 15:01 and 12:01, 15:01 and 07:01, 15:01 and 04:01, 15:01 and one two or all of 01:01, 03:01 and 07:01; 15:01 and one or both of 11:01 and 11:03; 01:01 and 12:01; 01:01 and 07:01; 01:01 and 04:01; 01:01 and one or both of 11:01 and 11:03; 01:01 and one or both of 03:01 and 07:01; 01:01 and one or both of 15:01 and 07:01; 12:01 and 07:01; 12:01 and 04:01; 12:01 and one or both of 11:01 and 11:03; 12:01 and one two or all of 01:01, 03:01 and 07:01; 12:01 and one two or all of 01:01, 07:01 and 15:01; 07:01 and 04:01; 07:01 and one or both of 11:01 and 11:03; 07:01 and one or both of 01:01 and 03:01; 07:01 and one or both of 01:01 and 15:01; 04:01 and one or both of 11:01 and 11:03; 04:01 and one two or all of 01:01, 03:01 and 07:01; 04:01 and one two or all of 01:01, 07:01 and 15:01; one or both of 11:01 and 11:03 and one two or all of 01:01, 03:01 and 07:01; 11:01 and 11:03 and one two or all of 01:01, 07:01 and 15:01. The invention also provides the use of the pool of peptides according to the invention as a control in an assay for a T cell immune response, such as a naive response. In addition, the invention provides a method for determining the potential of a test peptide to stimulate a T cell response, the method comprising: (a) contacting a first aliquot of a cell suspension comprising T cells derived from at least one donor with a test peptide; (b) contacting a second aliquot of the cell suspension with a pool of peptides according to the invention; (c) measuring the proliferation or stimulation of T cells in the first aliquot of the cell suspension; (d) measuring the proliferation or stimulation of T cells in the second aliquot of the cell suspension; and (e) comparing the measurement obtained in (c) to the measurement obtained in (d), thereby determining the potential of the test peptide to stimulate a T cell response. Preferably, said cell suspension only comprises T cells from one donor. The steps of the method are typically carried out multiple times in parallel using separate cell suspensions each comprising T cells from a different donor, and / or are repeated multiple times using separate cell suspensions each comprising T cells from a different donor. Preferably, the donors are human donors. Also provided by the invention is a method for determining the potential of a protein to stimulate a T cell response, the method comprising: (a) contacting a first aliquot of a cell suspension comprising T cells derived from at least one donor with at least one peptide from the therapeutic protein, and preferably a pool of overlapping peptides from the protein; (b) contacting a second aliquot of the cell suspension with a pool of peptides according to the invention; (c) measuring the proliferation or stimulation of T cells in the first aliquot of the cell suspension; (d) measuring the proliferation or stimulation of T cells in the second aliquot of the cell suspension; and (e) comparing the measurement obtained in (c) to the measurement obtained in (d), thereby determining the potential of the protein to stimulate a T cell response. Preferably, said cell suspension only comprises T cells from one donor. The steps of the method are typically carried out multiple times in parallel using separate cell suspensions each comprising T cells from a different donor, and / or are repeated multiple times using separate cell suspensions each comprising T cells from a different donor. Preferably, the donors are human donors. Figures Figure 1 shows responses from sample test antigens (percentage stimulation above background >0.5%, SEM=2) using the KLH peptide pool as a control. The total Percentage Antigenicity of each test peptide is split into contributing donor segments, where Percentage Stimulation above Background >0.5% has been set. The data are shown with two standard errors above background (SEM=2) applied as a further exclusion criterion when searching for positive events. Each shaded segment on the charts represents a different donor. Figure 2 shows response index (RI) values calculated for each sample test antigen (percentage stimulation above background >0.5%, SEM=2). Figure 3 shows responses from the KLH peptide pool (percentage stimulation above background >0.0.2%) and the responses of each individual KLH peptide KLH-1 through KLH-9, corresponding to SEQ ID NOs: 1 to 9 and in Table 1 below. The total percentage antigenicity of each test peptide is split into contributing donor segments, where percentage stimulation above background >0.02% has been set. Each shaded segment on the charts represents a different donor. Figure 4 shows the response index (RI) values calculated for each sample test antigen (percentage stimulation above background >0.02%). Detailed Description Pool of peptides The invention provides a pool of peptides comprising a first peptide and a second peptide, wherein: (i) the first peptide is from 9 to 40 amino acids in length and comprises at least 9 contiguous amino acids of the sequence shown in any one of SEQ ID NOs: 1 to 9 or of a sequence having at least 80% identity to the sequence shown in any one of SEQ ID NOs: 1 to 9, wherein the at least 9 contiguous amino acids comprise at least one CD4+ T-cell epitope; and (ii) the second peptide is from 9 to 40 amino acids in length and comprises at least 9 contiguous amino acids of any one of the sequences shown in SEQ ID NOs: 1 to 9 or of a sequence having at least 80% identity to any one of the sequences shown in SEQ ID NOs: 1 to 9, wherein the at least 9 contiguous amino acids comprise at least one CD4+ T-cell epitope, wherein the at least 9 contiguous amino acids comprised in the second peptide are of a different sequence to the at least 9 contiguous amino acids comprised in the first peptide. The first peptide and the second peptide are each independently from 9 to 40 amino acids in length, such as from 10 to 35 amino acids in length, 12 to 30 amino acids in length, 14 to 25 amino acids in length, 15 to 23 amino acids in length or 16 to 20 amino acids in length. Preferred length ranges are from 12 to 20, 12 to 23 and 15 to 20 amino acids. The first peptide and the second peptide may each independently comprise: (A) at least 9 contiguous amino acids of the sequence shown in any one of SEQ ID NOs: 1 to 9, such as (i) at least 10, 11, 12, 13, 14, 15 or 16 contiguous amino acids of any one of SEQ ID NOs: 1 to 9, (ii) 17 contiguous amino acids of any one of SEQ ID NOs: 1 to 5 and 7 to 9, (iii) 18 contiguous amino acids of any one of SEQ ID NOs: 1, 3, 5, 7 and 9, (iv) 19 contiguous amino acids of any one of SEQ ID NOs: 3, 7 and 9, or (v) 20, 21, 22 or 23 contiguous amino acids of SEQ ID NO: 3; or (B) at least 9 contiguous amino acids of a sequence having at least 80% identity to any one of the sequences shown in SEQ ID NOs: 1 to 9, such as (i) at least 10, 11, 12, 13, 14, 15 or 16 contiguous amino acids of a sequence having at least 80% identity to any one of the sequences shown in SEQ ID NOs: 1 to 9, (ii) 17 contiguous amino acids of a sequence having at least 80% identity to any one of the sequences shown in SEQ ID NOs: 1 to 5 and 7 to 9, (iii) 18 contiguous amino acids of a sequence having at least 80% identity to any one of the sequences shown in SEQ ID NOs: 1, 3, 5, 7 and 9, (iv) 19 contiguous amino acids of a sequence having at least 80% identity to any one of the sequences shown in SEQ ID NOs: 3, 7 and 9, or (v) 20, 21, 22 or 23 contiguous amino acids of a sequence having at least 80% identity to the sequence shown in SEQ ID NO: 3. The contiguous amino acids comprised in the second peptide are of a different one of SEQ ID NOs: 1 to 9 and sequences having at least 80% identity to SEQ ID NOS: 1 to 9 to the contiguous amino acids comprised in the first peptide. The contiguous amino acids may be from a sequence having at least about 85%, about 90%, about 93% or about 94% identity to any one of SEQ ID NOs: 1 to 9, about 95% identity to SEQ ID NO: 3, 7 or 9, or about 96% identity to SEQ ID NO: 3. The first peptide and the second peptide may each independently comprise at least 9 contiguous amino acids of the sequence shown in any one of SEQ ID NOs: 1 to 9, such as (i) at least 10, 11, 12, 13, 14, 15 or 16 contiguous amino acids of any one of SEQ ID NOs: 1 to 9, (ii) 17 contiguous amino acids of any one of SEQ ID NOs: 1 to 5 and 7 to 9, (iii) 18 contiguous amino acids of any one of SEQ ID NOs: 1, 3, 5, 7 and 9, (iv) 19 contiguous amino acids of any one of SEQ ID NOs: 3, 7 and 9, or (v) 20, 21, 22 or 23 contiguous amino acids of SEQ ID NO: 3. The contiguous amino acids comprised in the second peptide are of a different one of SEQ ID NOs: 1 to 9 to the contiguous amino acids comprised in the first peptide. In preferred embodiments, the first peptide and the second peptide each independently comprise at least 15 contiguous amino acids of the sequence shown in any one of SEQ ID NOs: 1 to 9, such as (i) at least 16 contiguous amino acids of any one of SEQ ID NOs: 1 to 9, (ii) 17 contiguous amino acids of any one of SEQ ID NOs: 1 to 5 and 7 to 9, (iii) 18 contiguous amino acids of any one of SEQ ID NOs: 1, 3, 5, 7 and 9, (iv) 19 contiguous amino acids of any one of SEQ ID NOs: 3, 7 and 9, or (v) 20, 21, 22 or 23 contiguous amino acids of SEQ ID NO: 9. For example the first peptide and the second peptide may each independently comprise (i) at least 15 contiguous amino acids of SEQ ID NO: 6, (ii) at least 16 contiguous amino acids of any one of SEQ ID NOs: 2, 4 and 8, (iii) 17 contiguous amino acids of SEQ ID NO: 1 or 5, (iv) 18 contiguous amino acids of SEQ ID NO: 7 or 9, or (v) 22 contiguous amino acids of SEQ ID NO: 3. In preferred embodiments, the first peptide and the second peptide each independently comprise any one of the sequences shown in SEQ ID NOs: 1 to 9. For example, the first peptide and / or the second peptide may each consist of any one of the sequences shown in SEQ ID NOs: 1 to 9, or one of the first and second peptides may comprise any one of the sequences shown in SEQ ID NOs 1 to 9 and the other of the first and second peptides may consist of any one of the sequences shown in SEQ ID NOs 1 to 9. The first peptide and the second peptide each comprise or consist of a different one of SEQ ID NOs: 1 to 9. The pool of peptides may comprise a third peptide, wherein the third peptide is from 9 to 40 amino acids in length and comprises at least 9 contiguous amino acids of the sequence shown in any one of SEQ ID NOs: 1 to 9 or of a sequence having at least 80% identity to the sequence shown in any one of SEQ ID NOs: 1 to 9, wherein the at least 9 contiguous amino acids comprise at least one CD4+ T-cell epitope, and wherein the at least 9 contiguous amino acids comprised in the third peptide are of a different sequence to the at least 9 contiguous amino acids comprised in the first peptide and in the second peptide. The third peptide may be a peptide as described above in relation to the first and second peptides. The third peptide is a peptide that is different from the first and second peptides. The pool of peptides may comprise a fourth peptide, fourth and fifth peptides, fifth and sixth peptides, fifth, sixth and seventh peptides, fifth, sixth, seventh and eighth peptides or fifth, sixth, seventh, eighth and ninth peptides, wherein the fourth peptide, and the optional fifth, sixth, seventh, eighth and ninth peptides, are each from 9 to 40 amino acids in length and each comprise at least 9 contiguous amino acids of the sequence shown in any one of SEQ ID NOs: 1 to 9 or of a sequence having at least 80% identity to the sequence shown in any one of SEQ ID NOs: 1 to 9, wherein the at least 9 contiguous amino acids comprise at least one CD4+ T-cell epitope, and wherein the at least 9 contiguous amino acids comprised in each of the first, second, third, fourth, fifth, sixth, seventh, eighth and ninth peptides are 9 contiguous amino acids of a different one of the sequences shown in SEQ ID NOs: 1 to 9 or of a sequence having at least 80% identity to a different one the sequences shown in SEQ ID NOs: 1 to 9. Each of the fourth, fifth, sixth, seventh, eighth and ninth peptides may independently be a peptide as described above in relation to the first and second peptides. Each of the first to ninth peptides typically comprises a different one of the sequences shown in SEQ ID NOs: 1 to 9. The first to ninth peptides may each consist of a different one of the sequences shown in SEQ ID NOs: 1 to 9. Any one or more, such as 2, 3, 4, 5, 6, 7 or 8, of the first to ninth peptides may comprise a different one of the sequences shown in SEQ ID NOs 1 to 9 and another one or more, such as 2, 3, 4, 5, 6, 7 or 8, of the first to ninth peptides may consist of a different one of the sequences shown in SEQ ID NOs 1 to 9. The pool of peptides may comprise up to 20, up to 15, up to 12 or up to 10 peptides. Preferably, the number of peptides in the pool is from 2 to 9 peptides, such as 3, 4, 5, 6, 7 or 8 peptides. More preferably, the pool consists of 7, 8 or 9 peptides. Most preferably the pool consists of 9 peptides. It is preferred that all of the peptides in the pool are “naive peptides”, i.e. peptides to which humans are not naturally exposed. For example, all of the peptides in the pool are KLH peptides. A KLH peptide is a peptide consisting of a fragment of the KLH protein. A fragment of the KLH protein may consist of from 9 to 40 contiguous amino acids of the KLH protein. Typically, the first, second, third, fourth, fifth, sixth, seventh, eighth and / or ninth peptides above are fragments of the KLH protein. Thus, where the first, second, third, fourth, fifth, sixth, seventh, eighth and / or ninth peptide has a length of 9 amino acids or more (such as length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21,22, 23, 24, 25, 30, 35 or 40 amino acids in length and comprises at least 9 contiguous amino acids of the sequence shown in any one of SEQ ID NOs: 1 to 9 (such as at least 10, 11, 12, 13, 14, 15 or 16 contiguous amino acids of any one of SEQ ID NOs: 1 to 9, at least 17 contiguous amino acids of any one of SEQ ID NOs: 1 to 5 and 7 to 9, at least 18 contiguous amino acids of any one of SEQ ID NOs: 1, 3, 5, 7 and 9, at least 19 contiguous amino acids of any one of SEQ ID NOs: 3, 7 and 9, or at least 20, 21, 22 or 23 contiguous amino acids of SEQ ID NO: 3), the additional one or more amino acids in the peptide are preferably also contiguous amino acids from the KLH protein sequence. At least one, such as 2, 3, 4, 5, 6, 7, 8 or all, of the first to ninth peptides is a HLA antigen (HLA Class II antigen), is capable of stimulating responses in T cell assays, and / or is capable of binding to more than one HLA type. At least one, such as 2, 3, 4, 5, 6, 7, 8 or all, of the first to ninth peptides is a HLA Class Il-binding antigen. At least one, such as 2, 3, 4, 5, 6, 7, 8 or all, of the first to ninth peptides is preferably aHLA Class Il-binding antigen, capable of stimulating responses in T cell assays, and capable of binding to more than one HLA type. The at least 9 contiguous amino acids preferably comprise at least one HLA anchor residue. Anchor residues in each of SEQ ID Nos: 1 to 9 are shown in Table 1. A CD4+ T-cell epitope binds to the groove in a Class IIMHC molecule, with the anchor residues being the most important for determining binding. The minimum length of a CD4+ T-cell epitope is usually 9 amino acids, and anchor residue 1 is the first of those 9 amino acids. Each 9 amino acid stretch beginning at an anchor residue 1 in Table 1 represents a CD4+ T-cell epitope. Each peptide in the pool comprises a CD4+ T-cell epitope. Therefore, the at least 9 contiguous amino acids of one of SEQ ID NOs: 1-9 comprises a 9 amino acid peptide containing at least one anchor residue typically beginning at an amino acid marked as an anchor residue 1 in Table 1. The ends of the peptide outside the 9 amino acid peptide beginning at an anchor residue 1 can be trimmed or augmented whilst preserving Class II MHC binding. The core 9 amino acids in each of SEQ ID Nos: 1 to 9 correspond to one of SEQ ID Nos: 10 to 21. The peptides may, for example, be trimmed to remove one or more amino acid, for example, 2 or 3 amino acids, from one or both ends of one of SEQ ID NOs: 1-9. Although the 9 amino acid peptide beginning at an anchor residue 1 is preferably preserved, some variation in the sequence can be tolerated without preventing binding to Class II MHC. Where there is only one anchor residue, such as for the peptide of SEQ ID NO: 1 (KLH-1 in Table 1), the skilled person would recognise that the single anchor residue should be preserved, but that one or more of the remaining residues in the 9 amino acid CD4+ T-cell epitope could be substituted. Where there are two, three or four anchor residues, the skilled person would try to retain two or more, such as three or four, of those anchor residues. Thus, inside the 9 amino acid CD4+ T-cell epitope, preferably only non-anchor residues are substituted. Outside the 9 amino acid CD4+ T-cell epitope, amino acid substitutions may be made because Class IIMHC binding will be focused on the 9 amino acid CD4+ T-cell epitope. A peptide that comprises a CD4+ T-cell epitope and that has 80% identity to one of SEQ ID NOs: 1-9 over its entire length therefore retains the Class II MHC binding activity of the starting peptide. The at least 9 contiguous amino acids of SEQ ID NO: 1, or of a sequence having at least about 80%, 85%, 90%, 93% or 94% identity to the sequence shown in SEQ ID NO: 1, preferably comprise residue 5 of SEQ ID NO: 1, more preferably residues 5 to 13 of SEQ ID NO: 1. The at least 9 contiguous amino acids of SEQ ID NO: 2, or of a sequence having at least about 80%, 85%, 90%, 93% or 94% identity to the sequence shown in SEQ ID NO: 2, preferably comprise residues 6 and 9 of SEQ ID NO: 2, and more preferably comprise residues 6 to 9 of SEQ ID NO: 2, most preferably residues 6 to 14 of SEQ ID NO: 2. The at least 9 contiguous amino acids of SEQ ID NO: 3, or of a sequence having at least about 80%, 85%, 90%, 93%, 94%, 95% or 96% identity to the sequence shown in SEQ ID NO: 3, preferably comprise residues 4 and 7, residues 4, 7 and 9, and / or residues 10, 13, 15 and 18 of SEQ ID NO: 3. More preferably, the at least 9 contiguous amino acids of SEQ ID NO: 3 comprise residues 4 to 7, 4 to 9, 4 to 12 and / or 10 to 18 of SEQ ID NO: 3. Most preferably, the at least 9 contiguous amino acids of SEQ ID NO: 3 comprise residues 4 to 18 of SEQ ID NO: 3. The at least 9 contiguous amino acids of SEQ ID NO: 4, or of a sequence having at least about 80%, 85%, 90%, 93% or 94% identity to the sequence shown in SEQ ID NO: 4, preferably comprise residues 5, 7 and 11 of SEQ ID NO: 4, and more preferably comprise residues 5 to 11 of SEQ ID NO: 4, most preferably residues 5 to 13 of SEQ ID NO: 4. The at least 9 contiguous amino acids of SEQ ID NO: 5, or of a sequence having at least about 80%, 85%, 90%, 93% or 94% identity to the sequence shown in SEQ ID NO: 5, preferably comprise residues 4 and 7, and / or residues 5, 10 and 13 of SEQ ID NO: 5. More preferably, the at least 9 contiguous amino acids of SEQ ID NO: 5 comprise residues 4 to 7, 4 to 12, 5 to 13, or 4 to 13 of SEQ ID NO: 5. The at least 9 contiguous amino acids of SEQ ID NO: 6, or of a sequence having at least about 80%, 85%, 90%, 93% or 94% identity to the sequence shown in SEQ ID NO: 6, preferably comprise residues 4, 7 and 12, and / or residues 5, 8, 10 and 13 of SEQ ID NO: 6. More preferably, the at least 9 contiguous amino acids of SEQ ID NO: 6 comprise residues 4 to 12, 5 to 13, or 4 to 13 of SEQ ID NO: 6. The at least 9 contiguous amino acids of SEQ ID NO: 7, or of a sequence having at least about 80%, 85%, 90%, 93%, 94% or 95% identity to the sequence shown in SEQ ID NO: 7, preferably comprise residues 9, 12 and 14 of SEQ ID NO: 7, and more preferably comprise residues 9 to 14 of SEQ ID NO: 7, most preferably residues 9 to 17 of SEQ ID NO: 7. The at least 9 contiguous amino acids of SEQ ID NO: 8, or of a sequence having at least about 80%, 85%, 90%, 93% or 94% identity to the sequence shown in SEQ ID NO: 8, preferably comprise residues 5, 8, 10 and 13 of SEQ ID NO: 8, and more preferably comprise residues 5 to 13 of SEQ ID NO: 8. The at least 9 contiguous amino acids of SEQ ID NO: 9, or of a sequence having at least about 80%, 85%, 90%, 93%, 94% or 95% identity to the sequence shown in SEQ ID NO: 9, preferably comprise residues 8, 11 and 13 of SEQ ID NO: 9, and more preferably comprise residues 8 to 13 of SEQ ID NO: 9, most preferably residues 8 to 16 of SEQ ID NO: 9. The peptides in the pool may be produced by any suitable method. Typically, the peptides are produced by peptide synthesis. Such methods are known in the art. Table 1 of the application shows that each of the peptides was presented in the antigen presentation assay of Example 1 by donors with the tissue types as indicated in the column headed “Allele PP*” (see Table legend). The peptide sequences also matched the known anchor motif for the alleles indicated in Table 1 (see Table legend). At the time the application was filed, positive confirmation of binding of the peptides marked with an “x” in the final column of Table 1 to T cells with the indicated allele had also been obtained via HLA tetramer staining with the same allele and peptide combination HLA tetramer after stimulation of a PBMC sample with the same peptide (see Table legend). Residues in each of SEQ ID NOs: 1-9 that match known HLA-DRB1* allele anchor motifs are indicated in Table 1. The HLA-DRB1* alleles for each anchor motif are also indicated in Table 1. Hence Table 1 shows the anchor motifs (amino acid residues) by which the peptides bind to the indicated HLA-DRB1* alleles. Each of the peptides of SEQ ID NOs: 1-9 are detected in in the antigen presentation assay of Example 1. The matching of the anchor motifs as shown in Table 1 explains the reason why these peptides are detected in the antigen presentation assay. The peptide allele combinations were separately validated by HLA tetramer staining, as described further below. All of the sequences allow antigen presentation by way of binding the indicated anchor motifs, and the fact that KLH is a completely foreign antigen to humans with very low sequence homology to any human protein, means that peptides comprising any one of SEQ ID NOs: 1 to 9, or any of the CD4+ T-cell epitopes defined by the anchor sequences as shown in Table 1, could be individually useful as control antigens in a naive T cell assay of the invention. The nine peptides work across broad genotypes; the nine peptides contain epitopes for the HLA alleles HLA-DRBl*01:01, 03:01, 04:01, 07:01, 11:01, 11:03, 12:01 and 15:01. Different alleles are present in different individuals and allele frequencies vary between different populations. The peptides of SEQ ID NOs: 1 to 9 have been chosen to give broad population coverage. However, it is not essential to include all of the peptides in the pool because a pool lacking one or more of the epitopes would still be effective and cover a reasonable proportion of the wider human population. Therefore, for many donor populations, the high frequency of just a couple of HLA types will mean that two of the peptides (2 CD4+ T-cell epitopes) will generally be enough to give the required population coverage, saving unnecessary expense in manufacturing peptides that will have no HLA matches. Arrieta-Bolanos et al (2023) provides an “HLA map of the world”, with Figure 4 of this document illustrating this diversity for Class IIMHC alleles, and the paragraph spanning pages 7 and 8 of Arrieta-Bolanos et al explaining how the high frequency alleles differ between different populations. Many academic studies and transplant centres have established HLA-DR allele frequencies in their target population. An analysis by the present inventors of a range of population studies aggregated by global regions shows that, for example, in the European population the frequencies of the DRB1 locus alleles mentioned in Table 1 are approximately: HLA-DRBl*01:01-15%; 03:01-28%; 04:01-12%; 07:01-22%; 11:01-12%; 11:03-2%; 12:01-3%; 15:01-22%. AstheDRBl alleles *01:01, *03:01, *04:01, *07:01, *15:01 and *11:01 are all high frequency alleles in most populations, all peptides can usefully be included in a peptide pool to be used as a control in an assay performed as described in this disclosure, but not all of the nine peptides are necessary. From the nine peptides of SEQ ID NOs: 1 to 9, peptides could be selected to form pools with fewer than nine peptides without sacrificing much of the response rate of the pool as a whole. For example, a pool of two peptides comprising the CD4+ T-cell epitope in KLH-1 (SEQ ID NO: 1), covering *15:01, and the CD4+ T-cell epitopes in KLH-3, covering HLA-DRBl*01:01, 03:01 and 07:01 (SEQ ID NO: 3), would still cover 96.3% of the European population. The skilled person can select which two or more of the peptides of SEQ ID NOs: 1 to 9 (or CD4+ T-cell epitopes from SEQ ID NOs: 1 to 9) are needed to provide adequate coverage (e.g. at least 20%, 40%, 60%, 80%, 90%, 95% coverage) in a peptide pool control for use in any given population. Reasons for omitting peptides from the pool of nine could include cost, when the pool can thus be limited to only peptides that bind to the most frequent HLA types in the target population. Assay The invention provides the use of the pool of peptides according to the invention as a control in an assay for a T cell immune response. The T cell response may be a naive response. Any suitable assay for a T cell response may be used. The assay may, for example, be a T cell stimulation assay, such as a naive T cell proliferation assay, a DC T cell proliferation assay, an ELISpot assay, such as IL-2 ELISpot assay. Other assay types may be used, such as those measuring signal transduction, transcription factor translocation or RNA synthesis (e.g. of cytokine products). Such assays are described in the art. T cell stimulation assays, such as T cell and DC T cell proliferation assays and the other assays mentioned above can help identify the presence or absence of potential T cell epitopes in a protein or peptide sequence. Such assays can help develop a picture of “relative antigenicity” between structurally similar molecules, such as therapeutic proteins, for example, therapeutic antibodies, that are comparable in their application, formulation, mode of action, and route of exposure. The assays can serve to distinguish between different but very similar peptides because the assay assists in the identification of epitope sequences that can elicit helper T cell proliferation and therefore potentially result in the development of a helper T cell immune response. Helper T cell responses can play a pivotal role in the development of antibody responses against a peptide, protein or antibody. Such antibody responses are often unwanted in the context of therapeutic applications. However, the development of the extent of anti-therapeutic peptides or protein antibody responses is difficult to test ex vivo, i.e. prior to undertaking human clinical trials. Measuring potential T cell responses to peptides and proteins can be carried out ex vivo on T cells that are collected from human donors as PBMC. Since significant T cell responses are required for antibody responses to mature, assessing T cell responses in isolation ex vivo has become a widely used means to gain a general picture for a peptide or protein to be able to elicit a naive antibody response when administered in vivo. In one preferred method, T cell proliferation is measured using the flow cytometric method of analysing cell proliferation by tracking cells that have been labelled with the cell dye carboxyfluorescein succinimidyl ester (CFSE). The merits of such a CFSE assay, as compared to the prior art radioactive 3H-thymidine incorporation assay to measure T cell proliferation, is further described in the literature, such as in Mannering S. I., et al., Journal of Immunological Methods 283 (2003) 173- 183. The applicant was the first provider to adapt this assay for a commercial service to assess protein antigenicity. The CFSE-based T cell assay allows for reliable, reproducible and accurate determination of potential epitopes from CD4+ T cells. CD4 is a transmembrane glycoprotein expressed on T helper cells with an ability to bind to HLA Class II molecules on antigen presenting cells. Unlike the prior art radioactive 3H-thymidine incorporation assay to measure T cell proliferation, the CFSE method ensures that only the desired sub-set of T cells are analysed, and that the total percentage of proliferating CD4+ T cells is measured, with proliferation events being captured irrespective of when they happen during the assay interval. This method also provides the potential for combining CFSE labelling with different antibody detection prior to fixation of the cells. This could allow T cell proliferation to be sub-classified into further cell phenotypes or groups, which otherwise cannot be detected using a conventional 3H-thymidine incorporation assay. CFSE T cell proliferation assays are particularly suitable for assessing the potential of naive T cells to respond to a new peptide T cell antigen challenge. This is important for evaluating the T cell antigenicity of novel therapeutic proteins, such as therapeutic antibodies, since the potential recipient of these therapies will usually be naive to this new protein and peptide content. In such assays, cell proliferation is determined using CFSE, which forms an intracellular fluorescent conjugate following cellular uptake. Fluorescence intensity of CFSE is halved through each consecutive cell division, thus allowing measurement of proliferation as a function of altered fluorescence of CFSE containing cells. The peptide pool of the invention comprised of peptides derived from the KLH protein is included in the assay is used as a control in these assays. Other positive controls for cell proliferation include whole proteins Tuberculin Purified Protein Derivative (PPD) and Keyhole Limpet Hemocyanin (KLH); two synthetic peptide pool controls, ProMix™ CEFT Peptide Pool, and these can also be included in the assay. T cell proliferation assays can measure both naive T cell responses and recall T cell responses. Measuring naive responses is important to assess the antigenicity of candidate therapeutic proteins and antibody as it is likely that the recipient of a novel therapy will be naive (i.e. not have been pre-exposed) to that therapy. The peptide pool of the invention is advantageous as a positive control in such assays. The peptide pool of the invention is comparable in nature to synthetic peptides and nevertheless controls for T cell stimulation over a wide variety of donors. The peptide pool typically does not contain a non-naive CD4+ T-cell epitope. Naive T cell proliferation assays may be carried out with PBMCs derived from cohorts of 2-300, such as 10-100, 20-50, or 30-40, independent HLA diverse PBMC donors to reflect the incidence of responses in a diverse population. It is often found that HA and TT single peptide responses often occur in only 5-10% of donors. The low incidence of responses and their distribution, which is thought to follow a Poisson distribution, mean that the actually observed incidence varies widely from donor-cohort study to study, even where both studies are designed in the same way and carried out one after another on different groups of randomly selected donors. The peptide pool of the invention comprises select peptides from the naive antigen KLH that are presented by donors with a wide range of Class-II HLA types. When used in T cell assays on larger donor cohorts, e.g. of 20-50 donors, the peptide pool typically stimulates the majority of those donors to a significant extent. The peptide pool also has the advantage of producing a response having a magnitude comparable to that achievable by other naive antigenic peptides, which is a property that protein antigens cannot adequately reflect. A therapeutic protein or peptide is a protein or peptide having an effect useful in treating a disease in a human or animal, particularly in a human. Many types of therapeutic proteins and peptides have been approved for use in the treatment of human disease. Therapeutic proteins include purified blood products, recombinant cytokines, growth factors, enzyme replacement factors, monoclonal antibodies, fusion proteins, hormones, and chimeric fusion proteins. Method The invention provides a method for determining the potential of a test peptide to stimulate a T cell response, the method comprising: (a) contacting a first aliquot of a cell suspension comprising T cells derived from at least one donor with a test peptide; (b) contacting a second aliquot of the cell suspension with a pool of peptides according to the invention; (c) measuring the proliferation or stimulation of T cells in the first aliquot of the cell suspension; (d) measuring the proliferation or stimulation of T cells in the second aliquot of the cell suspension; and (e) comparing the measurement obtained in (c) to the measurement obtained in (d); thereby determining the potential of the test peptide to stimulate a T cell response. The test peptide may be a fragment of a therapeutic protein or polypeptide, such as an antibody molecule. Examples of therapeutic proteins or polypeptides from which the test peptide may be derived include without limitation purified blood products, recombinant cytokines, growth factors, enzyme replacement factors, monoclonal antibodies, hormones, and fusion proteins, such as chimeric fusion proteins. The test peptide may be of any suitable length, such as, for example from 9 to 40 amino acids, 10 to 30 amino acids, 12 to 25 amino acids or 12 to 20 amino acids. Class II binding sequences are most commonly found to be in the region of 12-20 amino acids in length, but an extended sequence of more than 20 amino acids is also capable of binding, so long as the linear MHC-binding stretch is still exposed. Peptides of up to 40 amino acids typically do not have significant ternary structure, which could otherwise cause problems with direct Class II MHC binding, or with binding of the MHC-peptide complex to a TCR. Extensive analytical tools are available to those skilled in the art to assess sequences as suitable in this regard. Therefore, the character of the CD4+ T cell epitopes in the peptides in the pool is preserved when a peptide comprising the amino acid sequence of one of SEQ ID NOs: 1 to 9 is extended up to 40 amino acids. The amino acid sequence outside the core CD4+ epitope(s) in the peptides and extending beyond the peptides of SEQ ID NOs: 1 to 9 is preferably an amino acid sequence from KLH, but may be any amino acid sequence that does not contain a non-naive CD4+ T-cell epitope. The invention also provides a method for determining the potential of a protein or polypeptide to stimulate a T cell response, the method comprising: (a) contacting a first aliquot of a cell suspension comprising T cells derived from at least one donor with at least one peptide from the protein or polypeptide; (b) contacting a second aliquot of the cell suspension with a pool of peptides according to the invention; (c) measuring the proliferation or stimulation of T cells in the first aliquot of the cell suspension; (d) measuring the proliferation or stimulation of T cells in the second aliquot of the cell suspension; and (e) comparing the measurement obtained in (c) to the measurement obtained in (d); thereby determining the potential of the protein or polypeptide to stimulate a T cell response. In step (a) of this method, the first aliquot of the cell suspension comprising T cells derived from at least one donor is contacted with one or more peptides, typically with one peptide only at a time in a separate sub-aliquot of the T cells derived from said aliquot, such as from 1 to 100, 5 to 70, 10 to 60, 20 to 50 or 30 to 40 peptides, which are fragments of the protein or polypeptide. The multiple peptides may be a pool of overlapping peptides from the protein or polypeptide. The pool of overlapping peptides typically spans the entire protein or polypeptide. The peptides in the pool may overlap by any number of amino acids, such as for example by 9 to 12 amino acids. The length of the peptides in the overlapping pool are not limited, and the lengths of the peptides may be the same or different. The lengths of the overlapping peptides may be, for example, from 9 to 40 amino acids, such as 15 to 20 amino acids. The multiple peptides may be fragments of the protein produced by proteolytic cleavage in human dendritic cells in culture, isolated from peptide-HLA complexes and identified by mass spectrometry analysis, for example by comparing to a human proteome peptide fragment pattern. Preferably, the multiple peptides are produced for the assay by chemical synthesis, which may be crude synthesis for high throughput objectives, or purified peptide synthesis with peptide purities produced of at least about 70%, 75%, 80%, 85%, 90%, 95%, or 98%. The test protein or polypeptide may be an antibody or other therapeutic protein or polypeptide. For example, the protein may be a purified blood product, a recombinant cytokine, a growth factor, an enzyme replacement factor, a monoclonal antibody, a hormone, or a fusion protein, such as a chimeric fusion protein. In the methods of the invention, the steps may be carried out in accordance with the steps of a known T cell response assay, such as a T cell stimulation assay (for example a naive T cell proliferation assay), a DC T cell proliferation assay, an ELISpot assay (for example IL-2 ELISpot). The cell suspension comprising T cells used in the methods is derived from at least one donor. The cell suspension may comprise T cells from only one donor. The cell suspension may comprise T cells from two or more donors, such as from 2, 3, 4, 5 or 10 donors. The T cells comprised in the cell suspension may be from HLA Class-II matched donors, or from a range of HLA Class II types. The donors providing the T cells typically have a wide range of HLA Class II types. The cell suspension may comprise T cells from a donor cohort. The donor cohort may, for example, comprise 20-50 donors. The method may be carried out multiple (such as from 2 to about 60 times, from about 10 to about 50 times, about 20 to about 40 times), for example in parallel, using separate cell suspensions from different donors in the donor cohort. In this situation, each cell suspension preferably comprises T cells from only one donor. However, where the method is carried out multiple times using the same test peptide the cell suspension may comprise T cells from two or more donors, such as from 2, 3, 4, 5 or 10 donors. Typically, steps (a) and (b) of the methods are carried out as part of the same experiment. Typically, steps (c) and (d) are carried out as part of the same experiment. After carrying out steps (a) and (b), the cell suspension that has been contacted with the peptide and the cell suspension that has been contacted with the peptide pool of the invention are incubated for a suitable period of time. The skilled person will be able to determine a suitable incubation period. A typical incubation period may, for example, be from minutes (for example 5-30 minutes) or hours (for example 1-12 hours) to 21 days, such as from 1 day to 18 days, 3 days to 14 days, or from 5 to 10 days, for example for 6, 7 or 8 days. The incubation time between steps (a) and (c) and the incubation time between steps (b) and (d) are typically the same. After carrying out steps (a) and (b), the cell suspension that has been contacted with the peptide and the cell suspension that has been contacted with the peptide pool of the invention are incubated under suitable conditions. The skilled person will be able to determine suitable incubation conditions. These will typically be 37°C, 5% CO2. In the methods, proliferation or stimulation of T cells may be measured by any suitable means, including without limitation for example by flow cytometry, or by measuring signal transduction, transcription factor translocation or RNA synthesis (e.g. of cytokine products). The flow cytometric method of analysing cell proliferation may comprise tracking cells that have been labelled with the cell dye CFSE. ELISpot assays are carried out according to standard protocols known well in the art and those published by the companies CTL Immunospot and Mabtech AB and analysed with ELISpot readers such as those provided by those companies. After measuring T cell proliferation or stimulation, the measurement obtained following incubation of the T cells with the test peptide and the measurement obtained following incubation of the T cells with the peptide pool of the invention are compared. The potential of test peptide, or peptides, and hence, if applicable, the protein from which the test peptide is, or peptides are, derived, to stimulate a T cell response, for example a naive response, may be determined from the results. Where proliferation or stimulation of T cells in the presence of the peptide pool of the invention is observed at the expected incidence rate within the donor cohort tested, the assay can be considered to have passed at least this control criteria, which among other additional controls used in the assay, depending on the objective of the assay, may be the basis of determining that the results of the assay may provide a valid assessment as to whether the test peptide has, or peptides have, the potential to stimulate a T cell response in humans. Hence the potential antigenicity of the protein or polypeptide from which the test peptide is, or peptides are, derived can be determined. Where proliferation or stimulation of T cells in the presence of the peptide pool of the invention is not observed, the assay is unsuccessful and cannot be used to conclude that a test peptide, or peptides, do not have the potential to stimulate a T cell response in humans. Examples Example 1: Identification of immunogenic KLH peptides Potentially immunogenic regions of KLH1 and KLH2 were determined. Whole KLH protein comprising the KLH1 and KLH2 peptide chains were each added to cultures of monocyte-derived dendritic cells isolated from normal healthy blood donors. Peptide fragments of KLH1 and KLH2 naturally produced by proteolytic cleavage in the dendritic cells, and consequently presented by HLA Class II molecules, such as HLA-DR molecules on the dendritic cells, were released from harvested dendritic cells by lysis. The solubilises HLA-peptide complexes were isolated by an immunoaffinity step which specifically recovers HLA complexes. Peptides were eluted from the complexes and identified using mass spectrometry (LC-MS / MS)-based analysis in which MS data were analysed against human proteome peptide fragment patterns and a fragment pattern from KLH1 or KLH2. 472 uniquely presented peptide sequences were identified in KLH1 and 687 in KLH2, all of which are potential immunogenic regions. Example 2: T cell proliferation assay (TCA) PBMC were used to provide both T cells and antigen presenting cells (APC) in the assay. Cultures were set up in multi-well plates. Each peptide was tested at a final assay concentration of 5pM per well. Cells were labelled with CFSE prior to incubation with test peptides. Each test peptide was cultured with each of the donor PBMC samples in six replicate wells. A panel of donors was selected so that HLA class II alleles known to be highly expressed in the global population were well represented. Donors were predominantly selected by DRB1 allele expression. Each plate included six unstimulated control wells. The following reference antigens comprising known HLA Class II epitopes were used in this study: Tuberculin Purified Protein Derivative (PPD) is a derivative of Mycobacterium tuberculosis, and was used at a final assay concentration of 5mg / ml. 80-100% of donors are expected to react to this protein as a result of previous vaccination, i.e. through a memory immune response. - Keyhole Limpet Hemocyanin (KLH) is a recognized and potent naive protein immunogen, used at a final concentration of 0.25mg / ml in the assay. Typically, between 70-100% of donors might be expected to react to this protein, presumably driven by a naive immune response. The ProMix™ CEFT Peptide Pool consists of 24 peptides, each corresponding to a defined HLA class Il-restricted T cell epitope from cytomegalovirus, Epstein-Barr virus, influenza and tetanus toxoid. This pool is commonly used as a positive control in cellular assays such as ELISpot. It is used at a final assay concentration of 2.5 pM. Between 60-90% of donors might be expected to respond significantly to CEFT peptide pool. A KLH Peptide Pool of the invention comprising 9 peptides comprising putative CD4+ T cell epitopes identified from HLA-DR presented peptides from the KLH protein, identified as described in Example 1, wherein the peptides have the sequences shown in SEQ ID NOs: 1 to 9 and in the Table below. The KLH peptide pool is used at a final assay concentration of 2.5 pM. Table 1: Peptides comprised in the KLH pool used in the TCA Name Peptide sequence MW mg purity Allele PP* Tet** KLH-1 AEETYSLRKAMERFQNDK 1 2216.48 0.5 96.47 15:01 X KLH-2 DSNQGYQAIAGYHGVPT 1 4 1777.88 0.5 96.57 01: 01 X KLH-3 LPSiLNDATYFNSRSQTFDPNPF 1 4 14 6 14 6 9 2644.9 0.5 95.49 01: 01 03: 01 07 : 01 KLH-4 VGDNFFLKYEAFDLNGG 13 6 1906.1 0.5 95.39 12 : 01 KLH-5 YDRVYKYEITQQLHDLDL 1 4 1 6 9 2312.58 0.5 95.76 01: 01 07 : 01 15 : 01 X KLH-6 HRLFVKQMEDALAAHG 14 9 14 6 9 1823.12 0.5 96.27 07 : 01 X KLH-7 DENEMPWAYDRVFKYDITE 14 6 2421.64 0.5 95.42 11: 01 11: 03 KLH-8 YDRVFKYDITEKLHDLK 14 6 9 2183.51 0.5 95.50 04:01 X KLH-9 NDESHGGYEHIAGFHGYPN 14 6 2101.15 0.5 96.14 01:01 X Average 2154.15 0.5 95.89 Table legend: Allele PP*: Indicates the donor HLA-DRB1 locus alleles in the antigen presentation 5 assay of Example 1 for donors presenting the peptide with high significance, whereby the peptide sequence also matches the known anchor motif for the same HLA-DRB1 allele (see the HLA FactsBook, AP Factsbook Series ISBN 0-12-545025-7). Anchor motif matches are marked-up underneath each peptide sequence in bold or cursive or underlined, with the anchor position number indicated in each case and same style marking indicated in the Allele PP* column on the 10 matching allele(s). Tet**: ‘x’ indicates positive confirmation of peptide with T cell Tetramer staining using Tetramers with peptides as indicated and alleles matching at least one of the alleles indicated for such peptide of T cells following primary stimulation with the relevant peptides in one or more donors. 15 After 7 days incubation with peptides or control proteins, cells were stained with anti-CD4 antibody, then washed and fixed for flow cytometric analysis. Proliferation was determined by measuring a decrease in CFSE intensity. A positive response in 2 or more independent donor samples is considered indicative of a potential antigen. In this study, where data from 41 donors were utilized, this sets a threshold of 4.9% antigenicity. The results are shown in Table 2 below: Table 2: Frequency of Response - Percentage Stimulation above Background >0.5%, SEM=2 Reference Antigen % Antigenicity Donors Responding Ctrl 1 PPD 100.00 41 Ctrl 2 KLH 100.00 41 Ctrl 3 HA 48.78 20 Ctrl 4 TT 17.07 7 CEFT Peptide pool 97.56 40 KLH Peptide pool 73.17 30 The KLH peptide pool is a pool of a few well-characterised and well-controlled peptides making it a very reliable control for measuring peptide-based T cell stimulation responses in a range of assays that measure T cell stimulation. Between 40-80% of donors might typically be expected to respond to KLH peptide pool. The T cell proliferation assay as described has been carried out using the KLH peptide pool as a control, as well as the other controls, to identify immunogenic peptides, which are fragments of a test protein. Immunogenic peptides (potential antigens) have been identified where a positive response has been observed in 2 or more independent donor samples. Figure 1 shows a sample response from test antigens (Percentage Stimulation above Background >0.5%, SEM=2) using the KLH peptide pool as a control. A positive response in 2 or more independent donor samples was considered indicative of a potential antigen. In this study, 41 donors were utilized, setting a threshold of 4.9% antigenicity. Figure 1 shows the total Percentage Antigenicity of each test peptide, split into contributing donor segments, where Percentage Stimulation above Background >0.5% has been set. The data are shown with either two standard errors above background (SEM=2) applied as a further exclusion criterion when searching for positive events. Each shaded segment on the charts represents a different donor. One-way analysis of variance (ANOVA) was performed to determine whether the response to test antigens was significantly different from the unstimulated control. ANOVA (p) values <0.05 are considered to be significant. The strength of positive donor cell responses was determined by taking an average of the percentage stimulation value obtained across accepted donors for each protein. This allowed the strength as well as the frequency of the response to be considered when calculating antigenicity. As some binding responses may be pan-allele, but not be very strong, determining a Response Index (RI) through the multiplication of strength and frequency values is more representative of the level of antigenicity. Response Index values (RI) are calculated from Percentage Antigenicity values multiplied by the strength of response (i.e. the mean Percentage Stimulation above Background) values divided by 100. Figure 2 shows sample response index (RI) values calculated for the test antigens shown in Figure 1 (percentage stimulation above background >0.5%, SEM=2). Example 3: Follow-up T cell proliferation assay (TCA) on individual KLH peptides PBMC were used to provide both T cells and antigen presenting cells (APC) in a follow-up assay. The same conditions as described in Example 2 were used for the reference antigens: Tuberculin PPD, KLH (whole protein), ProMix™ CEFT Peptide Pool, and the KLH Peptide Pool comprising peptides of the sequences shown in SEQ ID NOs: 1 to 9 and as shown as KLH-1 to KLH-9 in Table 1. In addition, each individual peptide KLH-1 through KLH-9, corresponding to SEQ ID NOs: 1 to 9 and in Table 1 were also tested at a final assay concentration of 5pM per well. Figure 3 shows a sample response from test antigens (Percentage Stimulation above Background >0.02%) using the KLH peptide pool as a control and each individual peptide KLH-1 through KLH-9. In this study, 20 donors were utilized. Figure 3 shows the total Percentage Antigenicity of each test peptide, split into contributing donor segments, where Percentage Stimulation above Background >0.02% has been set to resolve the responses for individual KLH peptides. Each shaded segment on the charts represents a different donor. The results demonstrate that all of the KLH-1 5 trough KLH-9 peptides comprise T cell epitopes capable of stimulating a T cell response in the assay in two or more donors. Response Index values (RI) were calculated for each of the test antigens as described in Example 2. Figure 4 shows sample response index (RI) values calculated for test antigens 10 shown in Figure 3 (percentage stimulation above background >0.02%), including each individual peptide KLH-1 through KLH-9.

Claims

1. A pool of peptides comprising a first peptide and a second peptide, wherein:(i) the first peptide is from 9 to 40 amino acids in length and comprises at least 9 contiguous amino acids of the sequence shown in any one of SEQ ID NOs: 1 to 9 or of a sequence having at least 80% identity to the sequence shown in any one of SEQ ID NOs: 1 to 9, wherein the at least 9 contiguous amino acids comprise at least one CD4+ T-cell epitope which comprises the amino acid labelled as anchor residue 1 in Table 1 followed by at least 8 amino acids; and(ii) the second peptide is from 9 to 40 amino acids in length and comprises at least 9 contiguous amino acids of any one of the sequences shown in SEQ ID NOs: 1 to 9 or of a sequence having at least 80% identity to any one of the sequences shown in SEQ ID NOs: 1 to 9, wherein the at least 9 contiguous amino acids comprise at least one CD4+ T-cell epitope which comprises the amino acid labelled as anchor residue 1 in Table 1 followed by at least 8 amino acids,wherein the at least 9 contiguous amino acids comprised in the second peptide are of a different sequence to the at least 9 contiguous amino acids comprised in the first peptide.

2. A pool of peptides according to claim 1, wherein the at least 8 amino acids include at least one of the amino acids labelled as anchor residues 3, 4, 6 or 9 in the same sequence as anchor residue 1 in Table 1.

3. A pool of peptides according to claim 2, wherein the at least 8 amino acids include two of the amino acids labelled as anchor residues 3 and 6, 4 and 6, 4 and 9, 6 and 9, all three of the anchor residues labelled as anchor residues 4, 6 and 9.

4. A pool of peptides according to any one of claims 1 to 3, wherein the first peptide and / or the second peptide comprises at least 9 contiguous amino acids of any one of the sequences shown in SEQ ID NOs: 1 to 9.

5. A pool of peptides according to claim 4, wherein the at least 9 contiguous amino acids comprise a CD4+ T-cell epitope having the amino acid sequence shown in any one of SEQ ID NOs: 10 to 21.

6. A pool of peptides according to any one of the preceding claims, wherein the first peptide and / or the second peptide comprises any one of the sequences shown in SEQ ID NOs: 1 to 9.

7. A pool of peptides according to any one of the preceding claims, wherein the first peptide and / or the second peptide consists of any one of the sequences shown in SEQ ID NOs: 1 to 9.

8. A pool of peptides according to any one of the preceding claims, which comprises a third peptide, wherein the third peptide is from 9 to 40 amino acids in length and comprises at least 9 contiguous amino acids of the sequence shown in any one of SEQ ID NOs: 1 to 9 or of a sequence having at least 80% identity to the sequence shown in any one of SEQ ID NOs: 1 to 9, wherein the at least 9 contiguous amino acids comprise at least one CD4+ T-cell epitope which comprises the amino acid labelled as anchor residue 1 in Table 1 followed by at least 8 amino acids, and wherein the at least 9 contiguous amino acids comprised in the third peptide are of a different sequence to the at least 9 contiguous amino acids comprised in the first peptide and in the second peptide.

9. A pool of peptides according to claim 8, which comprises a fourth peptide, wherein the fourth peptide is from 9 to 40 amino acids in length and comprises at least 9 contiguous amino acids of the sequence shown in any one of SEQ ID NOs: 1 to 9 or of a sequence having at least 80% identity to the sequence shown in any one of SEQ ID NOs: 1 to 9, wherein the at least 9 contiguous amino acids comprise at least one CD4+ T-cell epitope which comprises the amino acid labelled as anchor residue 1 in Table 1 followed by at least 8 amino acids, and wherein the at least 9 contiguous amino acids comprised in each of the first, second, third and fourth peptides are 9 contiguous amino acids of a different one of the sequences shown in SEQ ID NOs: 1 to 9 or of a sequence having at least 80% identity to a different one the sequences shown in SEQ ID NOs: 1 to 9.

10. A pool of peptides according to claim 9, which comprises a fifth peptide, fifth and sixth peptides, fifth, sixth and seventh peptides, fifth, sixth, seventh and eighth peptides or fifth, sixth, seventh, eighth and ninth peptides, wherein the fifth, sixth, seventh, eighth and ninth peptides, are each from 9 to 40 amino acids in length and each comprise at least 9 contiguous amino acids of the sequence shown in any one of SEQ ID NOs: 1 to 9 or of a sequence having at least 80% identity to the sequence shown in any one of SEQ ID NOs: 1 to 9, wherein the at least 9 contiguous amino acids comprise at least one CD4+ T-cell epitope which comprises the amino acid labelled as anchor residue 1 in Table 1 followed by at least 8 amino acids, and wherein the at least 9 contiguous amino acids comprised in each of the first, second, third, fourth, fifth, sixth, seventh, eighth and ninth peptides are 9 contiguous aminoacids of a different one of the sequences shown in SEQ ID NOs: 1 to 9 or of a sequence having at least 80% identity to a different one the sequences shown in SEQ ID NOs: 1 to 9.

11. A pool of peptides according to claim 10, wherein the at least 8 amino acids include at least one of the amino acids labelled as anchor residues 3, 4, 6 or 9 in the same sequence as anchor residue 1 in Table 1.

12. A pool of peptides according to claim 11, wherein the at least 8 amino acids include two of the amino acids labelled as anchor residues 3 and 6, 4 and 6, 4 and 9, 6 and 9, all three of the anchor residues labelled as anchor residues 4, 6 and 9.

13. A pool of peptides according to claim 12, wherein the at least 9 contiguous amino acids comprise a CD4+ T-cell epitope having the amino acid sequence shown in any one of SEQ ID NOs: 10 to 21.

14. The pool of peptides according to claim 10, wherein each of the first to ninth peptides comprises a different one of the sequences shown in SEQ ID NOs: 1 to 9.

15. The pool of peptides according to any one of claims 10 to 14, wherein each of the first to ninth peptides consists of a different one of the sequences shown in SEQ ID NOs: 1 to 9.

16. Use of the pool of peptides according to any one of claims 1 to 15 as a control in an assay for a T cell immune response.

17. Use according to claim 16, wherein the T cell immune response is a naive response.

18. A method for determining the potential of a test peptide to stimulate a T cell response,the method comprising:(a) contacting a first aliquot of a cell suspension comprising T cells derived from at least one donor with a test peptide;(b) contacting a second aliquot of the cell suspension with a pool of peptides according to any one of claims 1 to 15;(c) measuring the proliferation or stimulation of T cells in the first aliquot of the cell suspension;(d) measuring the proliferation or stimulation of T cells in the second aliquot of the cell suspension; and(e) comparing the measurement obtained in (c) to the measurement obtained in (d);thereby determining the potential of the test peptide to stimulate a T cell response.

19. A method for determining the potential of a polypeptide or protein to stimulate a T cell response, the method comprising:(a) contacting a first aliquot of a cell suspension comprising T cells derived from at least one donor with at least one peptide from the polypeptide or protein;(b) contacting a second aliquot of the cell suspension with a pool of peptides according to any one of claims 1 to 15;(c) measuring the proliferation or stimulation of T cells in the first aliquot of the cell suspension;(d) measuring the proliferation or stimulation of T cells in the second aliquot of the cell suspension;(e) comparing the measurement obtained in (c) to the measurement obtained in (d);thereby determining the potential of the polypeptide or protein to stimulate a T cell response.

20. The method of claim 19, wherein in step (a) the first aliquot of the cell suspension comprising T cells derived from at least one donor is contacted with a pool of overlapping peptides from the polypeptide or protein.

21. The method of any one of claims 18 to 20, wherein the test peptide is a fragment of a therapeutic polypeptide or therapeutic protein or the polypeptide or protein is a therapeutic polypeptide or therapeutic protein.

22. The method of claim 21, wherein the therapeutic polypeptide or protein is an antibody molecule.

23. The method of any one of claims 18 to 22, wherein the T cell immune response is a naive response.

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