Peptides and combination of peptides for use in immunotherapy against non-small cell lung cancer and other cancers

Abstract

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

Claims

1. A method of treating a patient who has hepatocellular cancer, comprising administering to said patient a population of activated T cells that kill cancer cells that present on the surface a peptide consisting of the amino acid sequence of KVLEHVVRV (SEQ ID NO: 1) wherein the activated T cells bind the peptide in a complex with an MHC class I molecule on the surface of the cancer cells.

2. The method of claim 1, wherein the activated T cells are cytotoxic T cells produced by transducing T cells with a T cell receptor (TCR) that binds the peptide in a complex with an MHC class I molecule on the surface of the cancer cells.

3. The method of claim 1, wherein the activated T cells are cytotoxic T cells produced by contacting T cells with an antigen presenting cell that expresses the peptide in a complex with an MHC class I molecule on the surface of the antigen presenting cell, for a period of time sufficient to activate said T cell.

4. The method of claim 1, further comprising administering to said patient at least one adjuvant selected from the group consisting of anti-CD40 antibody, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, interferon-beta, CpG oligonucleotides, poly-(I:C), RNA, sildenafil, particulate formulations with poly(lactide co-glycolide) (PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.

5. The method of claim 4, wherein the at least one adjuvant is IL-2.

6. The method of claim 4, wherein the at least one adjuvant is IL-7.

7. The method of claim 4, wherein the at least one adjuvant is IL-15.

8. The method of claim 4, wherein the at least one adjuvant is IL-21.

9. The method of claim 4, wherein the at least one adjuvant is IL-1.

10. The method of claim 4, wherein the at least one adjuvant is IL-12.

11. A method of eliciting an immune response in a patient who has hepatocellular cancer, comprising administering to said patient a population of activated T cells that kill cancer cells that present on the surface a peptide consisting of the amino acid sequence of KVLEHVVRV (SEQ ID NO: 1) wherein the activated T cells bind the peptide in a complex with an MHC class I molecule on the surface of the cancer cells.

12. The method of claim 11, wherein the activated T cells are cytotoxic T cells produced by transducing T cells with a TCR that binds the peptide in a complex with an MHC class I molecule on the surface of the cancer cells.

13. The method of claim 11, wherein the activated T cells are cytotoxic T cells produced by contacting T cells with an antigen presenting cell that expresses the peptide in a complex with an MHC class I molecule on the surface of the antigen presenting cell, for a period of time sufficient to activate said T cell.

14. The method of claim 11, further comprising administering to said patient at least one adjuvant selected from the group consisting of anti-CD40 antibody, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, interferon-beta, CpG oligonucleotides, poly-(I:C), RNA, sildenafil, particulate formulations with poly(lactide co-glycolide) (PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.

15. The method of claim 14, wherein the at least one adjuvant is IL-2.

16. The method of claim 14, wherein the at least one adjuvant is IL-7.

17. The method of claim 14, wherein the at least one adjuvant is IL-15.

18. The method of claim 14, wherein the at least one adjuvant is IL-21.

19. The method of claim 14, wherein the at least one adjuvant is IL-1.

20. The method of claim 14, wherein the at least one adjuvant is IL-12.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows MAG-003 exon expression in MAGEA4 (tumor versus healthy, RNASeq data). MAGE-003 exon expression in MAGEA4 on healthy tissues was compared to that on various solid tumors (red bars). Healthy tissue subtypes are grouped as high (dark green bars), medium (light green bars) and low risk tissues (grey bars). Each bar represents a single sample. High expression on normal tissues was only found in placenta and testis (low risk). (RPKM=reads per kilobase per million mapped reads)

(2) FIG. 2 shows MAG-003 exon expression on MAGEA8 (tumor versus healthy, RNASeq data). MAGEA8 exon expression on healthy tissues was compared to that on various solid tumors (red bars). Healthy tissue subtypes are grouped as high (dark green bars), medium (light green bars) and low risk tissues (grey bars). Each bar represents a single sample. High expression on normal tissues was only found in placenta (low risk). (RPKM=reads per kilobase per million mapped reads).

(3) FIG. 3 shows IFN release from CD8+ T-cells electroporated with alpha and beta chain RNA of MAG-003 specific TCRs after co-incubation with target cells loaded with MAG-003 peptide (SEQ ID NO:1) or various MAG-003 alanine-substitution variants at positions 1-9 of SEQ ID NO:1 as disclosed herein.

(4) FIG. 4 shows IFN release from CD8+ T-cells electroporated with alpha and beta chain RNA of MAG-003 specific TCRs after co-incubation with target cells loaded with MAG-003 peptide (SEQ ID NO:1) or various MAG-003 alanine-substitution variants at positions 1-9 of SEQ ID NO:1 as disclosed herein.

(5) FIG. 5 shows IFN release from CD8+ T-cells electroporated with alpha and beta chain RNA of MAG-003 specific TCRs after co-incubation with target cells loaded with MAG-003 peptide (SEQ ID NO:1) or various MAG-003 alanine-substitution variants at positions 1-9 of SEQ ID NO:1 as disclosed herein.

(6) FIG. 6 shows MAGEA4 expression according to example 7. MAGEA4 mRNA is detectable in the presented cancer specimens. The level of expression covers a range from considerable expression in head and neck cancer and non-small lung cancer specimens (HNSCC062T1, HNSCC064T1 and NSCLC004T1) to rather low expression in non-small lung cancer and ovarian cancer specimens (NSCLC006T1 and OC036T1).

EXAMPLES

(7) Allo-reactive settings can be used to circumvent self-tolerance and yield T-cells with a higher avidity when compared to T-cells derived from autologous settings, i.e., patients. Examples of such settings include in vitro generation of allo-HLA reactive, peptide-specific T-cells (Sadovnikova et al. 1998; Savage et al. 2004; Wilde et al. 2012), and immunization of mice transgenic for human-MHC or human TCR (Stanislawski et al. 2001; Li et al. 2010).

Example 1

(8) In vitro generation of allo-HLA reactive, peptide-specific T-cells (Savage et al. 2004) PBMCs from HLA-A*02-negative healthy donors were used after obtaining informed consent. Recombinant biotinylated HLA-A2 class I monomers and A2 fluorescent tetramers containing MAG-003 were obtained from MBLI (Woburn, MA). PBMCs were incubated with anti-CD20SA diluted in phosphate buffered saline (PBS) for 1 hour at room temperature, washed, and incubated with the biotinylated A2/MAG-003 monomers for 30 minutes at room temperature, washed, and plated at 310.sup.6 cells/well in 24-well plates in RPMI with 10% human AB serum. Interleukin 7 (IL-7; R&D Systems, Minneapolis, MN) was added on day 1 at 10 ng/mL and IL-2 (Chiron, Harefield, United Kingdom) was added at 10 U/mL on day 4. Over a 5-week period cells were restimulated weekly with fresh PBMCs, mixed with responder cells at a 1:1 ratio, and plated at 310.sup.6/well in 24-well plates.

(9) To obtain high avidity T-cells, incubate approximately 10.sup.6 PBMCs with HLA-A2/MAG-003 tetramer-phycoerythrin (PE) (obtained from MBLI) for 30 minutes at 37 C., followed by anti-CD8-fluorescein isothiocyanate (FITC)/allophycocyanin (APC) for 20 minutes at 4 C., followed by fluorescence activated cell sorting (FACS)-Calibur analysis.

(10) Sorting was done with a FACS-Vantage (Becton Dickinson, Cowley, Oxford, United Kingdom). Sorted tetramer-positive cells were expanded in 24-well plates using, per well, 210.sup.5 sorted cells, 210.sup.6 irradiated A2-negative PBMCs as feeders, 210.sup.4 CD3/CD28 beads/mL (Dynal, Oslo Norway), and IL-2 (1000 U/mL). The high avidity T-cells, thus obtained, were then be used to identify and isolate TCRs for amino acid/DNA sequences determination and cloning into expression vectors using methods well known in the art.

Example 2

(11) Immunization of mice transgenic for human-MHC or human TCR

(12) MAG-003 were used to immunize transgenic mice with the entire human TCR gene loci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCR repertoire that compensates for mouse TCR deficiency. (Li et al. 2010). To obtain high avidity T-cells, incubate PBMCs obtained from the transgenic mice with tetramer-phycoerythrin (PE) followed by cell sorting as described above. The high avidity T-cells, thus obtained, were then be used to identify and isolate TCRs for amino acid/DNA sequences determination and cloning into expression vectors using methods well known in the art.

(13) In an aspect, MAG-003 and its variants, i.e., p286-1Y2L (having 2 amino acid substitutions, SEQ ID NO:2) and p286-1Y2L9L (having 3 amino acid substitutions, SEQ ID NO:3) exhibit potent binding affinity and stability towards HLA-A*0201 molecule. In particular, p2861Y2L9L showed the capability to induce specific CTLs which, in an aspect, lyse the target cancer cells from both PBMCs of healthy donors and HLA-A2.1/Kb transgenic mice. See, for example, (Wu et al. 2011), the content of which is hereby incorporated by reference in its entirety.

(14) To obtain high avidity TCRs for MHC I or II/p286-1Y2L or p286-1Y2L9L complexes, these peptides can be used in methods described in Examples 1 and 2. The high avidity T-cells, thus obtained, were then be used to identify and isolate TCRs for amino acid/DNA sequences determination and cloning into expression vectors using methods well known in the art.

(15) High avidity TCR variants can also be selected from a library of CDR mutants by yeast, phage, or T-cell display (holler et al. 2003; Li et al. 2005; Chervin et al. 2008). Candidate TCR variants, thus, provide guidance to design mutations of the TCR's CDRs to obtain high avidity TCR variants (Robbins et al. 2008; Zoete et al. 2007).

Example 3: Cloning of TCRs

(16) Methods of cloning TCRs are known in the art, for example, as described in U.S. Pat. No. 8,519,100, which is hereby incorporated by reference in its entirety for said methods. The alpha chain variable region sequence specific oligonucleotide A1 which encodes the restriction site NdeI, an introduced methionine for efficient initiation of expression in bacteria, and an alpha chain constant region sequence specific oligonucleotide A2 which encodes the restriction site SalI are used to amplify the alpha chain variable region. In the case of the beta chain, a beta chain variable region sequence specific oligonucleotide which encodes the restriction site e.g. NdeI, an introduced methionine for efficient initiation of expression in bacteria, and a beta chain constant region sequence specific oligonucleotide B2 which encodes the restriction site e.g. AgeI are used to amplify the beta chain variable region.

(17) The alpha and beta variable regions were cloned into pGMT7-based expression plasmids containing either C or C by standard methods described in (Molecular Cloning a Laboratory Manual Third edition by Sambrook and Russell). Plasmids were sequenced using an Applied Biosystems 37301 DNA Analyzer.

(18) The DNA sequences encoding the TCR alpha chain cut with NdeI and SalI were ligated into pGMT7+C vector, which was cut with NdeI and XhoI. The DNA sequences encoding the TCR beta chain cut with NdeI and AgeI was ligated into separate pGMT7+C vector, which was also cut with NdeI and AgeI. Ligated plasmids are transformed into competent Escherichia coli strain XL1-blue cells and plated out on LB/agar plates containing 100 g/ml ampicillin. Following incubation overnight at 37 C., single colonies are picked and grown in 10 ml LB containing 100 g/ml ampicillin overnight at 37 C. with shaking. Cloned plasmids are purified using a Miniprep kit (Qiagen) and the insert is sequenced using an automated DNA sequencer (Lark Technologies).

Example 4: Autologous T-Cell Engineering

(19) T-cells can be engineered to express high avidity TCRs (so-called TCR therapies) or protein-fusion derived chimeric antigen receptors (CARs) that have enhanced antigen specificity to MHC I/MAG-003 complex or MHC II/MAG-003 complex. In an aspect, this approach overcomes some of the limitations associated with central and peripheral tolerance, and generates T-cells that will be more efficient at targeting tumors without the requirement for de novo T-cell activation in the patient.

(20) To obtain T-cells expressing TCRs of the present description, nucleic acids encoding the tumor specific TCR-alpha and/or TCR-beta chains identified and isolated, as described in Examples 1-3, were cloned into expression vectors, such as gamma-retrovirus or lentivirus. The recombinant viruses were generated and then tested for functionality, such as antigen specificity and functional avidity. An aliquot of the final product is then used to transduce the target T-cell population (generally purified from patient PBMCs), which is expanded before infusion into the patient.

(21) TCR chains introduced into a peripheral T-cell may compete with endogenous TCR chains for association with the CD3 complex, which is necessary for TCR surface expression. Because a high level of TCR surface expression is essential to confer appropriate sensitivity for triggering by cells expressing the target tumor antigen (Cooper et al., 2000; Labrecque et al., 2001), strategies that enhance TCR-alpha and TCR-beta gene expression levels are an important consideration in TCR gene therapy.

(22) To increase the expression of TCR of the present description, strong promoters, such as retroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murine stem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), -actin, ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter (Cooper et al., 2004; Jones et al., 2009), elongation factor (EF)-1a (Tsuji et al., 2005) and the spleen focus-forming virus (SFFV) promoter (Joseph et al., 2008), can be used in the present description.

(23) In addition to strong promoters, many TCR expression cassettes contain additional elements that can enhance transgene expression, including a central polypurine tract (cPPT), which promotes the nuclear translocation of lentiviral constructs (Follenzi et al., 2000), and the woodchuck hepatitis virus posttranscriptional regulatory element (wPRE), which increases the level of transgene expression by increasing RNA stability (Zufferey et al., 1999).

(24) Achieving high-level TCR surface expression requires that both the TCR-alpha and TCR-beta chains of the introduced TCR be transcribed at high levels. To do so, the TCR-alpha and TCR-beta chains of the present description may be cloned into bicistronic constructs in a single vector, which has been shown to be capable of overcoming this obstacle. The use of a viral intraribosomal entry site (IRES) between the TCR-alpha and TCR-beta chains results in the coordinated expression of both chains, because the TCR-alpha and TCR-beta chains are generated from a single transcript that is broken into two proteins during translation, ensuring that an equal molar ratio of TCR-alpha and TCR-beta chains are produced. (Schmitt et al. 2009).

(25) Another modification that has proven to be beneficial for increasing TCR transgene expression is codon optimization. Redundancy in the genetic code allows some amino acids to be encoded by more than one codon, but certain codons are less optimal than others because of the relative availability of matching tRNAs as well as other factors (Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta gene sequences such that each amino acid is encoded by the optimal codon for mammalian gene expression, as well as eliminating mRNA instability motifs or cryptic splice sites, has been shown to significantly enhance TCR-alpha and TCR-beta gene expression (Scholten et al., 2006).

(26) Furthermore, mispairing between the introduced and endogenous TCR chains may result in the acquisition of specificities that pose a significant risk for autoimmunity. For example, the formation of mixed TCR dimers may reduce the number of CD3 molecules available to form properly paired TCR complexes, and therefore can significantly decrease the functional avidity of the cells expressing the introduced TCR (Kuball et al., 2007).

(27) To reduce mispairing, the C-terminus domain of the introduced TCR chains of the present description may be modified in order to promote interchain affinity, while decreasing the ability of the introduced chains to pair with the endogenous TCR. These strategies may include replacing the human TCR-alpha and TCR-beta C-terminus domains with their murine counterparts (murinized C-terminus domain); generating a second interchain disulfide bond in the C-terminus domain by introducing a second cysteine residue into both the TCR-alpha and TCR-beta chains of the introduced TCR (cysteine modification); swapping interacting residues in the TCR-alpha and TCR-beta chain C-terminus domains (knob-in-hole); and fusing the variable domains of the TCR-alpha and TCR-beta chains directly to CD3 (CD3 fusion). (Schmitt et al. 2009).

(28) The present description provides TCR proteins that are useful in treating cancers/tumors, preferably non-small cell lung cancer that over- or exclusively present MAG-003.

Example 5: Allogeneic T-Cell Engineering

(29) Gamma delta () T cells, which are non-conventional T lymphocyte effectors implicated in the first line of defense against pathogens, can interact with and eradicate tumor cells in a MHC-independent manner through activating receptors, among others, TCR-gamma and TCR-delta chains. These T cells display a preactivated phenotype that allows rapid cytokine production (IFN-, TNF-) and strong cytotoxic response upon activation. These T-cells have anti-tumor activity against many cancers and suggest that T cell-mediated immunotherapy is feasible and can induce objective tumor responses. (Braza et al. 2013).

(30) Recent advances using immobilized antigens, agonistic monoclonal antibodies (mAbs), tumor-derived artificial antigen presenting cells (aAPC), or combinations of activating mAbs and aAPC have been successful in expanding gamma delta T-cells with oligoclonal or polyclonal TCR repertoires. For example, immobilized major histocompatibility complex Class-I chain-related A was a stimulus for T-cells expressing TCR1 isotypes, and plate-bound activating antibodies have expanded V1 and V2 cells ex vivo. Clinically sufficient quantities of TCR1, TCR2, and TCR1.sup.neg TCR2.sup.neg have been produced following co-culture on aAPC, and these subsets displayed differences in memory phenotype and reactivity to tumors in vitro and in vivo. (Deniger et al. 2014).

(31) In addition, T-cells are amenable to genetic modification as evidenced by introduction of TCR-alpha and TCR-beta chains. (Hiasa et al. 2009). Another aspect of the present description relates to production of T-cells expressing TCR-alpha and TCR-beta that bind to MAG-003. To do so, T-cells are expanded by methods described by Deniger et al. 2014, followed by transducing the recombinant viruses expressing the TCRs that bind to MAG-003 (as described in Example 3) into the expanded T-cells. The virus-transduced T-cells are then infused into the patient.

Example 6: Immunogenicity and Functional T-Cell Data

(32) The immunogenicity of MAG-003 was tested using protocols that mimic the manufacturing procedure for a pharmaceutical product. Priming of MAG-003-specific T-cells was observed for healthy donors. Generated T cells were able to kill peptide loaded target cells demonstrating their functionality. The data demonstrated that 1) MAG-003 is an immunogenic target and 2) that generated T cells against MAG-003 are functional.

(33) Additional data as generated provided evidence that MAG-003 is a peptide with very good binding to HLA-A*02:01.

Example 7: MAGEA4 mRNa Expression in Tissues

(34) In situ hybridization (ISH) is used to detect mRNA expression directly in formalin-fixed or frozen tissue sections. Due to its high sensitivity and its spatial resolution, it is a suitable method to determine cell type specific target expression and the distribution or frequency of target expression within cancer tissue sections.

(35) ISH has been performed to detect MAGEA4 mRNA using the BaseScope technology developed by Advanced Cell Diagnostics (ACD). The BaseScope technology is based on the hybridization of on to four pairs of Z-shaped oligonucleotide probes to the target sequence. Signal amplification is achieved by branched DNA amplification, which is based on multiple hybridization steps of oligonucleotides, ultimately building up a branched DNA (bDNA) tree. Finally, a great number of label probes hybridize to the branches of the bDNA tree and the enhanced signal can be detected. The chromogenic BaseScope Detection Kit (RED) includes label probes which are linked to an enzyme (alkaline phosphatase). Signal detection depends on the enzymatic conversion of the chromogenic substrate FastRed, which additionally amplifies the original signal. BaseScope is a very sensitive technology, which is due to the efficient process of signal amplification, paired with the high sensitivity and the robust binding of the Z probe pairs to the target mRNA, even if it is partially crosslinked or degraded. According to ACD, binding of one single probe pair to each single mRNA molecule is enough to generate a detectable ISH signal.

(36) Each ISH experiment is subdivided into two methodological processes: 1) Tissue pretreatment for target retrieval, and 2) Target hybridization, signal amplification and detection. Optimal pretreatment conditions are critical for successful target detection in FFPE tissue sections. The fixation process induces crosslinking of proteins, DNA and RNA in cells and tissues and thereby masks hybridization sites. Thus, to assure accessibility of the target mRNA and proper binding of the probe set, these crosslinks have to be removed prior to target hybridization. Tissue pretreatment includes three discrete steps: 1) Blocking of endogenous alkaline phosphatase by hydrogen peroxide treatment, 2) target retrieval by boiling in target retrieval reagent, and 3) target retrieval by protease digestion. As the extent of fixation and crosslinking may vary between different FFPE blocks, the optimal target retrieval conditions have to be determined experimentally for each individual FFPE block. Therefore, tissue sections were exposed to different boiling and protease digestion times followed by hybridization with a positive and a negative control probe set. The optimal conditions were determined by microscopic evaluation of specific signal intensity in the positive control, unspecific background in the negative control and tissue morphology. Tissue pre-treatment was performed according to the manufacturer's protocols. Pretreatment reagents are included in the BaseScope reagent kits. After completion of the different pretreatment steps, target expression was assessed by hybridization of specific probe sets to the mRNA of interest with subsequent branched DNA signal amplification and chromogenic or fluorescent signal detection. All assays were performed according to the manufacturer's protocols.

(37) TABLE-US-00011 TABLE 10 Expression analysis Sample Tissue MAGEA4 expression HNSCC062T1 Head and neck cancer ++ HNSCC064T1 Head and neck cancer ++ NSCLC004T1 Non-small cell lung cancer ++ NSCLC006T1 Non-small cell lung cancer + OC036T1 Ovarian cancer + Overall expression level of MAGEA4 in the respective section: very low, + low to moderate, ++ strong, +++ very strong

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