Patent Application
20250136937
The present disclosure generally relates to the field of oncology, and more particularly to the treatment of cancer such as colorectal cancer.
Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer death worldwide, with over 1.8 million cases and 881,000 deaths estimated in 2018 alone (1). The incidence of CRC is expected to increase as global socioeconomic changes occur, with a predicted 2.2 million cases and 1.1 million deaths occurring annually by 2030 (1,2). This significant disease burden highlights the necessity of developing new and effective treatments against this disease.
The positive correlation between the abundance of tumor-infiltrating lymphocytes (TILs) and increased overall survival in both colon and rectal cancer suggests that T cells can recognize biologically relevant tumor antigens in these tumors (3,4). The potential immunogenicity of these antigens made immune checkpoint inhibition (ICI) a promising treatment for cancer patients; however, early clinical trials evaluating their efficacy in CRC have yielded mixed results. Colorectal tumors characterized by deficiencies in mismatch repair proteins resulting in the accumulation of repetitive DNA sequences (microsatellites), known as microsatellite instability (MSI), have shown relative success in phase II clinical trials with anti-PD1 treatment (5). In contrast, such treatments have had very little efficacy in clinical trials against microsatellite stable (MSS) tumors that do not possess a high mutational burden, which make up approximately 80% of CRC cases (5,6).
Given the significance of the immune response in CRC and the limited success of ICI alone, a promising avenue for research in recent years has been a neoantigen-based vaccine or T cell receptor-based therapy which could be used with or without ICI and would ideally bridge the gap in treatment efficacy across MSI and MSS tumors. For example, tumor-associated antigens (TAAs), antigens overexpressed in cancer cells compared to normal cells, have been previously identified in CRC (7,8). While several TAAs have been tested in vaccine and phase I trials against CRC, most were met with ‘limited success’, likely due to the negative selection of such antigens by the thymus (9). In one study, treatment of metastatic CRC with genetically engineered anti-CEA T cells resulted in tumor regression in one patient but ‘serious inflammatory colitis’ in all patients, demonstrating that an adverse autoimmune response is another possible negative consequence of using TAAs (10).
Due to the mixed responses to TAAs, an effective neoantigen-based therapy would more likely utilize tumor-specific antigens (TSAs), which may be generated through genetic, epigenetic, and post-translational variations, including but not limited to single-nucleotide variants, aberrantly expressed transcripts, or novel splicing events, and are expressed exclusively by the tumor (11). The high prevalence of single nucleotide variants, splice variants, and INDEL mutations in CRC suggests that there is a high probability of unique antigens being presented by the major histocompatibility complex (MHC) of tumors, such that it would be possible to invoke a tumor-specific immune response (12). TSAs have recently been identified in CRC and have demonstrated some success in phase I and II vaccine trials. A 2015 vaccine trial using frameshift antigens originating from MSI-high tumors demonstrated significant and specific immune responses among all patients (13). However, as this study used antigens derived from microsatellite instability frameshifts, these findings are not applicable to the majority of CRC patients. Other studies identifying TSAs in CRC to date have focused exclusively on mutant TSAs (mTSAs) derived from coding regions of the genome (13,14). An investigation of MSS CRC organoids revealed that only 0.5% of non-silent mutations were identified as mTSAs; this was a significantly lower proportion than what was predicted by HLA-binding prediction software (15). In addition, previous studies did not employ mass spectrometry (MS) techniques to quantify the expression of those TSAs on tumor cells, which is information that could influence the therapeutic potential of targeting a given TSA (13,14).
In view of this, there is a pressing need to identify the tumor-specific antigens that can elicit therapeutic immune responses again CRC. Such antigens could be used as vaccines (± immune checkpoint inhibitors) or as targets for T-cell receptor-based approaches (cell therapy, bispecific biologics).
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
In various aspects and embodiments, the present disclosure provides the following items 1 to 62:
or a nucleic acid encoding said TAP.
Other objects, advantages and features of the present disclosure will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
In the appended drawings:
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the technology (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.
The use of any and all examples, or exemplary language (“e.g.”, “such as”) provided herein, is intended merely to better illustrate embodiments of the claimed technology and does not pose a limitation on the scope unless otherwise claimed.
No language in the specification should be construed as indicating any non-claimed element as essential to the practice of embodiments of the claimed technology.
Herein, the term “about” has its ordinary meaning. The term “about” is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value, or encompass values close to the recited values, for example within 10% of the recited values (or range of values).
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into the specification as if they were individually recited herein.
Where features or aspects of the disclosure are described in terms of Markush groups or list of alternatives, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member, or subgroup of members, of the Markush group or list of alternatives.
Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in stem cell biology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).
Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).
In the studies described herein, the present inventors have identified TSA and TAA candidates from CRC cell lines and CRC specimens using a proteogenomic-based approach. A large fraction of the TSAs derived from aberrantly expressed unmutated genomic sequences which are not expressed in normal tissues, such as non-exonic sequences (e.g., intronic and intergenic sequences). The novel CRC TSA and TAA candidates identified herein may be useful, e.g., for CRC T-cell based immunotherapy and vaccines.
Accordingly, in an aspect, the present disclosure relates to a tumor antigen peptide (TAP) (or tumor-specific peptide), and more particularly an isolated TAP, such as a CRC TAP, comprising, or consisting of, one of the following amino acid sequences:
In another aspect, the present disclosure further relates to the use of a TAP (e.g., isolated TAP) comprising, or consisting of, one of the amino acid sequences below, for the treatment of cancer, and more particularly CRC:
In an embodiment, the TAP comprises, or consists of, one of the following amino acid sequences: SEQ ID NOs: 1-23. In an embodiment, the TAP comprises, or consists of, one of the following amino acid sequences: SEQ ID NOs: 1-17, 22, 25, 27, 29, 31, 33, 38, 40, 41 and 46. In a further embodiment, the TAP comprises, or consists of, one of the following amino acid sequences: SEQ ID NOs: 1-17, 22, and 25. In a further embodiment, the TAP comprises, or consists of, one of the following amino acid sequences: SEQ ID NOs: 1-17 and 22.
In general, peptides such as tumor antigen peptides (TAPs) presented in the context of HLA class I vary in length from about 7 or 8 to about 15, or preferably 8 to 14 amino acid residues. In some embodiments of the methods of the disclosure, longer peptides comprising the TAP sequences defined herein are artificially loaded into cells such as antigen presenting cells (APCs), processed by the cells and the TAP is presented by MHC class I molecules at the surface of the APC. In this method, peptides/polypeptides longer than 15 amino acid residues can be loaded into APCs, are processed by proteases in the APC cytosol providing the corresponding TAP as defined herein for presentation. In some embodiments, the precursor peptide/polypeptide that is used to generate the TAP defined herein is for example 1000, 500, 400, 300, 200, 150, 100, 75, 50, 45, 40, 35, 30, 25, 20 or 15 amino acids or less. Thus, all the methods and processes using the TAPs described herein include the use of longer peptides or polypeptides (including the native protein), i.e., tumor antigen precursor peptides/polypeptides, to induce the presentation of the “final” 8-14 TAP following processing by the cell (APCs). In some embodiments, the herein-mentioned TAP is about 8 to 14, 8 to 13, or 8 to 12 amino acids long (e.g., 8, 9, 10, 11, 12 or 13 amino acids long), small enough for a direct fit in an HLA class I molecule. In an embodiment, the TAP comprises 20 amino acids or less, preferably 15 amino acids or less, more preferably 14 amino acids or less. In an embodiment, the TAP comprises at least 7 amino acids, preferably at least 8 amino acids or less, more preferably at least 9 amino acids.
The term “amino acid” as used herein includes both L- and D-isomers of the naturally occurring amino acids as well as other amino acids (e.g., naturally-occurring amino acids, non-naturally-occurring amino acids, amino acids which are not encoded by nucleic acid sequences, etc.) used in peptide chemistry to prepare synthetic analogs of TAPs. Examples of naturally occurring amino acids are glycine, alanine, valine, leucine, isoleucine, serine, threonine, etc. Other amino acids include for example non-genetically encoded forms of amino acids, as well as a conservative substitution of an L-amino acid. Naturally-occurring non-genetically encoded amino acids include, for example, beta-alanine, 3-amino-propionic acid, 2,3-diaminopropionic acid, alpha-aminoisobutyric acid (Aib), 4-amino-butyric acid, N-methylglycine (sarcosine), hydroxyproline, ornithine (e.g., L-ornithine), citrulline, t-butylalanine, t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine (Nle), norvaline, 2-napthylalanine, pyridylalanine, 3-benzothienyl alanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4-tetrahydro-isoquinoline-3-carboxylix acid, beta-2-thienylalanine, methionine sulfoxide, L-homoarginine (Hoarg), N-acetyl lysine, 2-amino butyric acid, 2-amino butyric acid, 2,4,-diaminobutyric acid (D- or L-), p-aminophenylalanine, N-methylvaline, homocysteine, homoserine (HoSer), cysteic acid, epsilon-amino hexanoic acid, delta-amino valeric acid, or 2,3-diaminobutyric acid (D- or L-), etc. These amino acids are well known in the art of biochemistry/peptide chemistry. In an embodiment, the TAP comprises only naturally-occurring amino acids.
In embodiments, the TAPs described herein include peptides with altered sequences containing substitutions of functionally equivalent amino acid residues, relative to the herein-mentioned sequences. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity (having similar physico-chemical properties) which acts as a functional equivalent, resulting in a silent alteration. Substitution for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, positively charged (basic) amino acids include arginine, lysine and histidine (as well as homoarginine and ornithine). Nonpolar (hydrophobic) amino acids include leucine, isoleucine, alanine, phenylalanine, valine, proline, tryptophan and methionine. Uncharged polar amino acids include serine, threonine, cysteine, tyrosine, asparagine and glutamine. Negatively charged (acidic) amino acids include glutamic acid and aspartic acid. The amino acid glycine may be included in either the nonpolar amino acid family or the uncharged (neutral) polar amino acid family. Substitutions made within a family of amino acids are generally understood to be conservative substitutions. The herein-mentioned TAP may comprise all L-amino acids, all D-amino acids or a mixture of L- and D-amino acids. In an embodiment, the herein-mentioned TAP comprises all L-amino acids.
In an embodiment, in the sequences of the TAPs comprising or consisting of one of sequences of SEQ ID NOs: 1-23 and 25-50, the amino acid residues that do not substantially contribute to interactions with the T-cell receptor may be modified by replacement with other amino acid whose incorporation does not substantially affect T-cell reactivity and does not eliminate binding to the relevant MHC.
The TAP may also be N- and/or C-terminally capped or modified to prevent degradation, increase stability, affinity and/or uptake. Thus, in another aspect, the present disclosure provides a modified TAP of the formula Z1—X—Z2, wherein X is a TAP comprising, or consisting of, one of the amino acid sequences of SEQ ID NOs: 1-23 and 25-50.
In an embodiment, the amino terminal residue (i.e., the free amino group at the N-terminal end) of the TAP is modified (e.g., for protection against degradation), for example by covalent attachment of a moiety/chemical group (Z1). Z1 may be a straight chained or branched alkyl group of one to eight carbons, or an acyl group (R—CO—), wherein R is a hydrophobic moiety (e.g., acetyl, propionyl, butanyl, iso-propionyl, or iso-butanyl), or an aroyl group (Ar—CO—), wherein Ar is an aryl group. In an embodiment, the acyl group is a C1-C16 or C3-C16 acyl group (linear or branched, saturated or unsaturated), in a further embodiment, a saturated C1-C6 acyl group (linear or branched) or an unsaturated C3-C6 acyl group (linear or branched), for example an acetyl group (CH3—CO—, Ac). In an embodiment, Z1 is absent. The carboxy terminal residue (i.e., the free carboxy group at the C-terminal end of the TAP) of the TAP may be modified (e.g., for protection against degradation), for example by amidation (replacement of the OH group by a NH2 group), thus in such a case Z2 is a NH2 group. In an embodiment, Z2 may be an hydroxamate group, a nitrile group, an amide (primary, secondary or tertiary) group, an aliphatic amine of one to ten carbons such as methyl amine, iso-butylamine, iso-valerylamine or cyclohexylamine, an aromatic or arylalkyl amine such as aniline, napthylamine, benzylamine, cinnamylamine, or phenylethylamine, an alcohol or CH2OH. In an embodiment, Z2 is absent. In an embodiment, the TAP comprises one of the amino acid sequences of SEQ ID NOs: 1-24 and 26-50. In an embodiment, the TAP consists of one of the amino acid sequences of SEQ ID NOs: 1-24 and 26-50, i.e., wherein Z1 and Z2 are absent.
In another aspect, the present disclosure provides a TAP binding to an HLA-A*02:01 molecule, comprising or consisting of the sequence of SEQ ID NO:6. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the above-identified TAP may further bind to HLA-A*02:05, HLA-A*02:06 and/or HLA-A*02:07 molecules.
In another aspect, the present disclosure provides a TAP binding to an HLA-A*03:01 molecule, comprising or consisting of the sequence of SEQ ID NO:1, 11, 14, 38, 45 or 46, preferably SEQ ID NO:1, 11, or 14. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the above-identified TAP may further bind to HLA-A*03:02, HLA-A*11:01 or HLA-A*30:01 molecules.
In another aspect, the present disclosure provides a TAP binding to an HLA-A*03:02 molecule, comprising or consisting of the sequence of SEQ ID NOs: 3, 5, 7, 16, 23, 31, 32, 33 or 34, preferably SEQ ID NOs: 3, 5, 7, 16 or 23. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the above-identified TAP may further bind to HLA-A*03:01, HLA-A*11:01 or HLA-A*30:01 molecules
In another aspect, the present disclosure provides a TAP binding to an HLA-A*11:01 molecule, comprising or consisting of the sequence of SEQ ID NO:9, 18, 33 or 34, preferably SEQ ID NO:9 or 18. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the above-identified TAP may further bind to HLA-A*03:01, HLA-A*03:02, HLA-A*31:01 and/or HLA-A*68:01 molecules.
In another aspect, the present disclosure provides a TAP binding to an HLA-A*23:01 molecule, comprising or consisting of the sequence of SEQ ID NO:29. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the above-identified TAP may further bind to HLA-A*24:02 molecules.
In another aspect, the present disclosure provides a TAP binding to an HLA-A*24:02 molecule, comprising or consisting of the sequence of SEQ ID NO:26, 27 or 29. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the above-identified TAP may further bind to HLA-A*23:01 molecules.
In another aspect, the present disclosure provides a TAP binding to an HLA-A*30:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 19, 20 or 23. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the above-identified TAP may further bind to HLA-A*30:02 and/or HLA-B*15:02 molecules.
In another aspect, the present disclosure provides a TAP binding to an HLA-A*32:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 8, 37 or 39, preferably SEQ ID NO: 8. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the above-identified TAP may further bind to HLA-B*57:01 and/or HLA-B*58:01 molecules.
In another aspect, the present disclosure provides a TAP binding to an HLA-B*07:02 molecule, comprising or consisting of the sequence of SEQ ID NO:2, 21, 24 or 50, preferably SEQ ID NO:2 or 21. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the above-identified TAP may further bind to HLA-B*35:02, HLA-B*35:03, HLA-B*55:01 and/or HLA-B*56:01 molecules.
In another aspect, the present disclosure provides a TAP binding to an HLA-B*13:02 molecule, comprising or consisting of the sequence of SEQ ID NO: 13 or 48, preferably SEQ ID NO: 13.
In another aspect, the present disclosure provides a TAP binding to an HLA-B*18:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 25. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the above-identified TAP may further bind to HLA-B*40:01, HLA-B*44:02, HLA-B*44:03 and/or HLA-B*45:01 molecules.
In another aspect, the present disclosure provides a TAP binding to an HLA-B*27:05 molecule, comprising or consisting of the sequence of SEQ ID NO: 4. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the above-identified TAP may further bind to HLA-B*27:02
In another aspect, the present disclosure provides a TAP binding to an HLA-B*52:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 10, 12, 15 or 43, preferably SEQ ID NO: 10, 12, or 15. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the above-identified TAP may further bind to HLA-B*51:01.
In another aspect, the present disclosure provides a TAP binding to an HLA-C*06:02 molecule, comprising or consisting of the sequence of SEQ ID NO:17 or 44, preferably SEQ ID NO: 17. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the above-identified TAP may further bind to HLA-B*27:02, HLA-C*07:01 and/or HLA-C*07:02 molecules.
In another aspect, the present disclosure provides a TAP binding to an HLA-C*12:02 molecule, comprising or consisting of the sequence of SEQ ID NO: 47 or 49. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the above-identified TAP may further bind to HLA-B*46:01, HLA-C*03:02, HLA-C*03:03, HLA-C*03:04, HLA-C*08:01, HLA-C*12:03, HLA-C*15:02 and/or HLA-C*16:01 molecules.
In an embodiment, the TAP is encoded by a sequence located a non-protein coding region of the genome. In an embodiment, the TAP is encoded by a sequence located in an untranslated transcribed region (UTR), i.e., a 3′-UTR or 5′-UTR region. In another embodiment, the TAP is encoded by a sequence located in an intron. In another embodiment, the TAP is encoded by a sequence located in an intergenic region. In another embodiment, the TAP is encoded by a sequence located in an exon and originates from a frameshift.
The TAPs of the disclosure may be produced by expression in a host cell comprising a nucleic acid encoding the TAPs (recombinant expression) or by chemical synthesis (e.g., solid-phase peptide synthesis). Peptides can be readily synthesized by manual and/or automated solid phase procedures well known in the art. Suitable syntheses can be performed for example by utilizing “T-boc” or “Fmoc” procedures. Techniques and procedures for solid phase synthesis are described in for example Solid Phase Peptide Synthesis: A Practical Approach, by E. Atherton and R. C. Sheppard, published by IRL, Oxford University Press, 1989. Alternatively, the MiHA peptides may be prepared by way of segment condensation, as described, for example, in Liu et al., Tetrahedron Lett. 37:933-936, 1996; Baca et al., J. Am. Chem. Soc. 117:1881-1887, 1995; Tam et al., Int. J. Peptide Protein Res. 45:209-216, 1995; Schnolzer and Kent, Science 256:221-225, 1992; Liu and Tam, J. Am. Chem. Soc. 116:4149-4153, 1994; Liu and Tam, Proc. Natl. Acad. Sci. USA 91:6584-6588, 1994; and Yamashiro and Li, Int. J. Peptide Protein Res. 31:322-334, 1988). Other methods useful for synthesizing the TAPs are described in Nakagawa et al., J. Am. Chem. Soc. 107:7087-7092, 1985. In an embodiment, the TAP is chemically synthesized (synthetic peptide). Another embodiment of the present disclosure relates to a non-naturally occurring peptide wherein said peptide consists or consists essentially of an amino acid sequences defined herein and has been synthetically produced (e.g., synthesized) as a pharmaceutically acceptable salt. The salts of the TAPs according to the present disclosure differ substantially from the peptides in their state(s) in vivo, as the peptides as generated in vivo are no salts. The non-natural salt form of the peptide may modulate the solubility of the peptide, in particular in the context of pharmaceutical compositions comprising the peptides, e.g., the peptide vaccines as disclosed herein. Preferably, the salts are pharmaceutically acceptable salts of the peptides.
In an embodiment, the herein-mentioned TAP is isolated or substantially pure. A compound such as a peptide or nucleic acid is “isolated” or “substantially pure” when it is separated from the components that are present in the natural environment of the molecule or a naturally occurring source macromolecule (e.g., including other nucleic acids, proteins, lipids, sugars, etc.). Typically, a compound is substantially pure when it is at least 60%, more generally 75%, 80% or 85%, preferably over 90% and more preferably over 95%, by weight, of the total material in a sample. Thus, for example, a polypeptide that is chemically synthesized or produced by recombinant technology will generally be substantially free from its naturally associated components, e.g., components of its source macromolecule. A nucleic acid molecule is substantially pure when it is not immediately contiguous with (i.e., covalently linked to) the coding sequences with which it is normally contiguous in the naturally occurring genome of the organism from which the nucleic acid is derived. A substantially pure compound can be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid molecule encoding a peptide compound; or by chemical synthesis. Purity can be measured using any appropriate method such as column chromatography, gel electrophoresis, HPLC, etc. In an embodiment, the TAP is in solution. In another embodiment, the TAP is in solid form, e.g., lyophilized.
In an embodiment, the TAP is encoded by a sequence located a non-protein coding region of the genome. In an embodiment, the TAP is encoded by a sequence located in an intergenic region. In another embodiment, the TAP is encoded by a non-coding RNA (ncRNA).
In another aspect, the disclosure further provides a synthetic long peptide (SLP) comprising at least one of the TAP described herein. In an embodiment, the SLP comprises at least two TAPs, wherein at least one of the TAP is a TAP as described herein. In an embodiment, the SLP comprises at least two, three, four or five of the TAPs described herein. In an embodiment, the SLP comprises at least one of the TAPs described herein linked to one or more amino acid sequences or domains that confer desired properties to the SLP, such as sequences or domains that stabilize the SLP and/or that improve processing and presentation by MHC molecules, for example a sequence comprising a motif cleavable by cellular proteases such as cathepsins. In another embodiment, the SLP comprises at least one of the TAPs described herein, and a TAP that binds to MHC class II molecules. The TAPs may directly attached to each other, or may be indirectly attached via a linker such as a short amino acid linker. In embodiments, the linker comprises about 4 to about 20 amino acids, or about 4 to about 15 amino acids, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids. In an embodiment, the linker comprises glycine residues, serine residues, proline residues, threonine residues, or a mixture thereof. In an embodiment, the SLP has a length of 200, 150, 100, 90, 80, 70, 60 or 50 amino acids or less. In a further embodiment, the SLP has a length of 20 to 50, 45 or 40 amino acids, for example from 20 or 25 amino acids to 30, 35 or 40 amino acids. “Synthetic”, as used herein, refers to a peptide or nucleic molecule that is not isolated from its natural sources, e.g., which is produced through recombinant technology or using chemical synthesis.
In another aspect, the disclosure further provides a nucleic acid (e.g., isolated) encoding the herein-mentioned TAPs or a tumor antigen precursor-peptide or SLP. In an embodiment, the nucleic acid comprises from about 21 nucleotides to about 45 nucleotides, from about 24 to about 45 nucleotides, for example 24, 27, 30, 33, 36, 39, 42 or 45 nucleotides.
In an embodiment, the nucleic acid (DNA, RNA) encoding the TAP of the disclosure comprises any one of the sequences defined in SEQ ID NOs: 51-73 and 75-100, or SEQ ID NOS: 51-67, 72, 75, 77, 79, 81, 83, 88, 90, 91 and 96, or SEQ ID NOs: 51-67, 72 and 75 (e.g., SEQ ID NOs: 51-67 and 72), or a corresponding RNA sequence (i.e., in which the thymine nucleobases (T) are replaced by uracil nucleobases (U)). In an embodiment, the nucleic acid encoding the TAP is an mRNA molecule. In an embodiment, the nucleic acid is in solution. In another embodiment, the nucleic acid is in solid form, e.g., lyophilized.
A nucleic acid of the disclosure may be used for recombinant expression of the TAP or SLP of the disclosure, and may be included in a vector or plasmid, such as a cloning vector or an expression vector, which may be transfected into a host cell. In an embodiment, the disclosure provides a cloning, expression or viral vector or plasmid comprising a nucleic acid sequence encoding the TAP of the disclosure. Alternatively, a nucleic acid encoding a TAP of the disclosure may be incorporated into the genome of the host cell. In either case, the host cell expresses the TAP or protein encoded by the nucleic acid. The term “host cell” as used herein refers not only to the particular subject cell, but to the progeny or potential progeny of such a cell. A host cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell (e.g., insect cells, yeast cells, plant cells, or mammalian cells) capable of expressing the TAPs described herein. The vector or plasmid contains the necessary elements for the transcription and translation of the inserted coding sequence, and may contain other components such as resistance genes, cloning sites, etc. Methods that are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding peptides or polypeptides and appropriate transcriptional and translational control/regulatory elements operably linked thereto. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y. “Operably linked” refers to a juxtaposition of components, particularly nucleotide sequences, such that the normal function of the components can be performed. Thus, a coding sequence that is operably linked to regulatory sequences refers to a configuration of nucleotide sequences wherein the coding sequences can be expressed under the regulatory control, that is, transcriptional and/or translational control, of the regulatory sequences. “Regulatory/control region” or “regulatory/control sequence”, as used herein, refers to the non-coding nucleotide sequences that are involved in the regulation of the expression of a coding nucleic acid. Thus, the term regulatory region includes promoter sequences, regulatory protein binding sites, upstream activator sequences, and the like. The vector (e.g., expression vector) may have the necessary 5′ upstream and 3′ downstream regulatory elements such as promoter sequences such as CMV, PGK and EF-1a promoters, ribosome recognition and binding TATA box, and 3′ UTR AAUAAA transcription termination sequence for the efficient gene transcription and translation in its respective host cell. Other suitable promoters include the constitutive promoter of simian vims 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), HIV LTR promoter, MoMuLV promoter, avian leukemia virus promoter, EBV immediate early promoter, and Rous sarcoma vims promoter. Human gene promoters may also be used, including, but not limited to the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. In certain embodiments inducible promoters are also contemplated as part of the vectors expressing the TAP. This provides a molecular switch capable of turning on expression of the polynucleotide sequence of interest or turning off expression. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, or a tetracycline promoter. Examples of vectors are plasmid, autonomously replicating sequences, and transposable elements. Additional exemplary vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or PI-derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses. Examples of categories of animal viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40). Examples of expression vectors are Lenti-X™ Bicistronic Expression System (Neo) vectors (Contech), pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DEST™, pLenti6/V5-DEST™, and pLenti6.2N5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. The coding sequences of the TAPs disclosed herein can be ligated into such expression vectors for the expression of the TAP in mammalian cells.
In certain embodiments, the nucleic acids encoding the TAP of the present disclosure are provided in a viral vector. A viral vector can be those derived from adenovirus, vaccinia virus, retrovirus, lentivirus, or foamy virus. As used herein, the term “viral vector” refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the coding sequence for the various proteins described herein in place of nonessential viral genes. In another embodiment, the nucleic acids encoding the TAP of the present disclosure are provided in a self-amplifying or self-replicating RNA (srRNA) vectors. srRNAs are derived from positive-strand RNA viruses where the structural proteins have been removed and replaced with heterologous genes of interest. srRNAs have been successfully derived from flaviviruses, nodamura viruses, nidoviruses, and alphaviruses with therapeutic versions of the technology providing the structural proteins in trans to create single cycle viral replicon particles (VRPs) (see, e.g., Aliahmad et al. Next generation self-replicating RNA vectors for vaccines and immunotherapies. Cancer Gene Ther (2022). https://doi.org/10.1038/s41417-022-00435-8). The vector and/or particle can be utilized for the purpose of transferring DNA, RNA or other nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.
In embodiment, the nucleic acid (DNA, RNA) encoding the TAP of the disclosure is comprised within a vesicle or nanoparticle such as a lipid vesicle (e.g., liposome) or lipid nanoparticle (LNP), or any other suitable vehicle. Thus, in another aspect, the present disclosure provides a vesicle or nanoparticle, such as a lipid vesicle or nanoparticle, comprising a nucleic acid, such as an mRNA, encoding one or more of the TAP described herein.
The term liposome as used herein in accordance with its usual meaning, referring to microscopic lipid vesicles composed of a bilayer of phospholipids or any similar amphipathic lipids (e.g., sphingolipids) encapsulating an internal aqueous medium.
The term “lipid nanoparticle” refers to liposome-like structure that may include one or more lipid bilayer rings surrounding an internal aqueous medium similar to liposomes, or micellar-like structures that encapsulates molecules (e.g., nucleic acids) in a non-aqueous core. Lipid nanoparticles typically contain cationic lipids, such as ionizable cationic lipids. Examples of cationic lipids that may be used for LNPs include DOTMA, DOSPA, DOTAP, ePC, DLin-MC3-DMA, C12-200, ALC-0315, cKK-E12, Lipid H (SM-102), OF-Deg-Lin, A2-Iso5-2DC18, 306Oi10, BAME-O16B, TT3, 9A1P9, FTT5, COATSOME® SS-E, COATSOME® SS-EC, COATSOME® SS-OC and COATSOME® SS-OP (see, e.g., Hou et al., Nature Reviews Materials, volume 6, pages 1078-1094 (2021); Tenchov et al., ACS Nano, 15, 16982-17015 (2021).
Liposomes and lipid nanoparticles typically include other lipid components such as lipids, lipid-like materials, and polymers that can improve liposome or nanoparticle properties, such as stability, delivery efficacy, tolerability and biodistribution. These include phospholipids (e.g., phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, and phosphatidylglycerol) such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and DOPE, sterols (such as cholesterol and cholesterol derivatives), PEGylated lipids (PEG-lipids) such as 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG) and 1,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DSG).
In an embodiment, the lipid nanoparticle according to the present disclosure comprises one or more cationic lipids, such as ionizable cationic lipids. Examples of ionizable cationic lipids include those listed in PCT publications Nos. WO 2017/061150 and WO 2019/188867, which encompassed ionizable cationic lipids commercialized under the tradenames COATSOME® SS-E, COATSOME® SS-EC, COATSOME® SS-OC and COATSOME® SS-OP.
The nucleic acid (e.g., mRNA) encoding one or more of the TAP, may be modified, for example to increase stability and/or reduce immunogenicity. For example, the 5′ end may be capped to stabilize the molecule and decrease immunogenicity (for example, as described in U.S. Pat. Nos. 10,519,189 and 10,494,399). One or more nucleosides of the mRNA may be modified or substituted with 1-methyl pseudo-uridine to either increase stability of the molecule or reduce recognition of the molecule by the innate immune system. A form of modified nucleosides are described in U.S. Pat. No. 9,371,511. Other types of modifications that may be made to the mRNA include incorporation of anti-reverse cap analog (ARCA), 5′-methyl-cytidine triphosphate (m5CTP), N6-methyl-adenosine-5′-triphosphate (m6ATP), 2-thio-uridine triphosphate (s2UTP), pseudouridine triphosphate, N1Methylpseudouridine triphosphate or 5-Methoxyuridine triphosphate (5moUTP). The mRNA may also include additional modifications to the 5′- and/or 3′-untranslated regions (UTRs) and polyadenylation (polyA) tail (see, for example, Kim et al., Molecular & cellular toxicology vol. 18,1 (2022): 1-8). All these modifications and other modifications to the nucleic acid (e.g., mRNA) encoding the TAP are encompassed by the present disclosure.
In another aspect, the present disclosure provides an MHC class I molecule comprising (i.e., presenting or bound to) one or more of the TAP of SEQ ID NOs: SEQ ID NOs: 1-23 and 25-50, preferably SEQ ID NOs: 1-23, such as SEQ ID NOs: 1-17 and 22.
In an embodiment, the MHC class I molecule is an HLA-A2 molecule, in a further embodiment an HLA-A*02:01 molecule. In an embodiment, the MHC class I molecule is an HLA-A3 molecule, in a further embodiment an HLA-A*03:01 or HLA-A*03:02 molecule. In another embodiment, the MHC class I molecule is an HLA-A11 molecule, in a further embodiment an HLA-A*11:01 molecule. In an embodiment, the MHC class I molecule is an HLA-A23 molecule, in a further embodiment an HLA-A*23:01 molecule. In an embodiment, the MHC class I molecule is an HLA-A24 molecule, in a further embodiment an HLA-A*24:02 molecule. In an embodiment, the MHC class I molecule is an HLA-A30 molecule, in a further embodiment an HLA-A*30:01 molecule. In an embodiment, the MHC class I molecule is an HLA-A32 molecule, in a further embodiment an HLA-A*32:01 molecule. In another embodiment, the MHC class I molecule is an HLA-B07 molecule, in a further embodiment an HLA-B*07:02 molecule. In another embodiment, the MHC class I molecule is an HLA-B13 molecule, in a further embodiment an HLA-B*13:02 molecule. In another embodiment, the MHC class I molecule is an HLA-B18 molecule, in a further embodiment an HLA-B*18:01 molecule. In another embodiment, the MHC class I molecule is an HLA-B27 molecule, in a further embodiment an HLA-B*27:05 molecule. In another embodiment, the MHC class I molecule is an HLA-B52 molecule, in a further embodiment an HLA-B*52:01 molecule. In another embodiment, the MHC class I molecule is an HLA-C06 molecule, in a further embodiment an HLA-C*06:02 molecule. In another embodiment, the MHC class I molecule is an HLA-C04 molecule, in a further embodiment an HLA-C*04:01 molecule. In another embodiment, the MHC class I molecule is an HLA-C12 molecule, in a further embodiment an HLA-C*12:02 molecule.
In an embodiment, the TAP (e.g., SEQ ID NOs: SEQ ID NOS: 1-23 and 25-50, preferably SEQ ID NOs: 1-23) is non-covalently bound to the MHC class I molecule (i.e., the TAP is loaded into, or non-covalently bound to the peptide binding groove/pocket of the MHC class I molecule). In another embodiment, the TAP is covalently attached/bound to the MHC class I molecule (alpha chain). In such a construct, the TAP and the MHC class I molecule (alpha chain) are produced as a synthetic fusion protein, typically with a short (e.g., 5 to 20 residues, preferably about 8-12, e.g., 10) flexible linker or spacer (e.g., a polyglycine linker). In another aspect, the disclosure provides a nucleic acid encoding a fusion protein comprising a TAP defined herein fused to an MHC class I molecule (alpha chain). In an embodiment, the MHC class I molecule (alpha chain)-peptide complex is multimerized. Accordingly, in another aspect, the present disclosure provides a multimer of MHC class I molecule loaded (covalently or not) with the herein-mentioned TAP. Such multimers may be attached to a tag, for example a fluorescent tag, which allows the detection of the multimers. A great number of strategies have been developed for the production of MHC multimers, including MHC dimers, tetramers, pentamers, octamers, etc. (reviewed in Bakker and Schumacher, Current Opinion in Immunology 2005, 17:428-433). MHC multimers are useful, for example, for the detection and purification of antigen-specific T cells. Thus, in another aspect, the present disclosure provides a method for detecting or purifying (isolating, enriching) CD8+ T lymphocytes specific for a TAP defined herein, the method comprising contacting a cell population with a multimer of MHC class I molecule loaded (covalently or not) with the TAP; and detecting or isolating the CD8+ T lymphocytes bound by the MHC class I multimers. CD8+ T lymphocytes bound by the MHC class I multimers may be isolated using known methods, for example fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS).
In yet another aspect, the present disclosure provides a cell (e.g., a host cell), in an embodiment an isolated cell, comprising the herein-mentioned nucleic acid, vector or plasmid of the disclosure, i.e., a nucleic acid or vector encoding one or more TAPs. In another aspect, the present disclosure provides a cell expressing at its surface an MHC class I molecule (e.g., an MHC class I molecule of one of the alleles disclosed above) bound to or presenting a TAP according to the disclosure. In one embodiment, the host cell is a eukaryotic cell, such as a mammalian cell, preferably a human cell. a cell line or an immortalized cell. In another embodiment, the cell is an antigen-presenting cell (APC). In one embodiment, the host cell is a primary cell, a cell line or an immortalized cell. In another embodiment, the cell is an antigen-presenting cell (APC). Nucleic acids and vectors can be introduced into cells via conventional transformation or transfection techniques. The terms “transformation” and “transfection” refer to techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection and viral-mediated transfection. Suitable methods for transforming or transfecting host cells can for example be found in Sambrook et al. (supra), and other laboratory manuals. Methods for introducing nucleic acids into mammalian cells in vivo are also known, and may be used to deliver the vector or plasmid of the disclosure to a subject for gene therapy.
Cells such as APCs can be loaded with one or more TAPs using a variety of methods known in the art. As used herein “loading a cell” with a TAP means that RNA or DNA encoding the TAP, or the TAP, is transfected into the cells or alternatively that the APC is transformed with a nucleic acid encoding the TAP. The cell can also be loaded by contacting the cell with exogenous TAPs that can bind directly to MHC class I molecule present at the cell surface (e.g., peptide-pulsed cells). The TAPs may also be fused to a domain or motif that facilitates its presentation by MHC class I molecules, for example to an endoplasmic reticulum (ER) retrieval signal, a C-terminal Lys-Asp-Glu-Leu sequence (see Wang et al., Eur J Immunol. 2004 December; 34 (12): 3582-94).
In another aspect, the present disclosure provides a composition or peptide combination/pool comprising any one of, or any combination of, the TAPs defined herein (or a nucleic acid encoding said peptide(s)). In an embodiment, the composition comprises any combination of the TAPs defined herein (any combination of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more TAPs), or a combination of nucleic acids encoding said TAPs). Compositions comprising any combination/sub-combination of the TAPs defined herein are encompassed by the present disclosure. In another embodiment, the combination or pool may comprise one or more known tumor antigens.
Thus, in another aspect, the present disclosure provides a composition comprising any one of, or any combination of, the TAPs or SLP(s) defined herein (e.g., comprising or consisting of the sequence of SEQ ID NOs: 1-23 and 25-50, preferably SEQ ID NOs: 1-23, such as SEQ ID NOs: 1-17, and 22) and a cell expressing an MHC class I molecule (e.g., an MHC class I molecule of one of the alleles disclosed above). APC for use in the present disclosure are not limited to a particular type of cell and include professional APCs such as dendritic cells (DCs), Langerhans cells, macrophages and B cells, which are known to present proteinaceous antigens on their cell surface so as to be recognized by CD8+ T lymphocytes. For example, an APC can be obtained by inducing DCs from peripheral blood monocytes and then contacting (stimulating) the TAPs, either in vitro, ex vivo or in vivo. APC can also be activated to present a TAP in vivo where one or more of the TAPs of the disclosure are administered to a subject and APCs that present a TAP are induced in the body of the subject. The phrase “inducing an APC” or “stimulating an APC” includes contacting or loading a cell with one or more TAPs, or nucleic acids encoding the TAPs such that the TAPs are presented at its surface by MHC class I molecules. As noted herein, according to the present disclosure, the TAPs may be loaded indirectly for example using longer peptides/polypeptides comprising the sequence of the TAPs (including the native protein), which is then processed (e.g., by proteases) inside the APCs to generate the TAP/MHC class I complexes at the surface of the cells. After loading APCs with TAPs and allowing the APCs to present the TAPs, the APCs can be administered to a subject as a vaccine. For example, the ex vivo administration can include the steps of: (a) collecting APCs from a first subject, (b) contacting/loading the APCs of step (a) with a TAP to form MHC class I/TAP complexes at the surface of the APCs; and (c) administering the peptide-loaded APCs to a second subject in need for treatment.
The first subject and the second subject may be the same subject (e.g., autologous vaccine), or may be different subjects (e.g., allogeneic vaccine). Alternatively, according to the present disclosure, use of a TAP described herein (or a combination thereof) for manufacturing a composition (e.g., a pharmaceutical composition) for inducing antigen-presenting cells is provided. In addition, the present disclosure provides a method or process for manufacturing a pharmaceutical composition for inducing antigen-presenting cells, wherein the method or the process includes the step of admixing or formulating the TAP, or a combination thereof, with a pharmaceutically acceptable carrier. Cells such as APCs expressing an MHC class I molecule (e.g., any of the above-noted HLA molecules) loaded with any one of, or any combination of, the TAPs defined herein, may be used for stimulating/amplifying CD8+ T lymphocytes, for example autologous CD8+ T lymphocytes. Accordingly, in another aspect, the present disclosure provides a composition comprising any one of, or any combination of, the TAPs defined herein (or a nucleic acid or vector encoding same); a cell expressing an MHC class I molecule and a T lymphocyte, more specifically a CD8+ T lymphocyte (e.g., a population of cells comprising CD8+ T lymphocytes).
In an embodiment, the composition further comprises a buffer, an excipient, a carrier, a diluent and/or a medium (e.g., a culture medium). In a further embodiment, the buffer, excipient, carrier, diluent and/or medium is/are pharmaceutically acceptable buffer(s), excipient(s), carrier(s), diluent(s) and/or medium (media). As used herein “pharmaceutically acceptable buffer, excipient, carrier, diluent and/or medium” includes any and all solvents, buffers, binders, lubricants, fillers, thickening agents, disintegrants, plasticizers, coatings, barrier layer formulations, lubricants, stabilizing agent, release-delaying agents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and the like that are physiologically compatible, do not interfere with effectiveness of the biological activity of the active ingredient(s) and that are not toxic to the subject. The use of such media and agents for pharmaceutically active substances is well known in the art (Rowe et al., Handbook of pharmaceutical excipients, 2003, 4th edition, Pharmaceutical Press, London UK). Except insofar as any conventional media or agent is incompatible with the active compound (peptides, cells), use thereof in the compositions of the disclosure is contemplated. In an embodiment, the buffer, excipient, carrier and/or medium is a non-naturally occurring buffer, excipient, carrier and/or medium. In an embodiment, one or more of the TAPs defined herein, or the nucleic acids (e.g., mRNAs) encoding said one or more TAPs, are comprised within or complexed to a vesicle such as a lipid vesicle or liposome, e.g., a cationic lipid vesicle or liposome (see, e.g., Vitor M T et al., Recent Pat Drug Deliv Formul. 2013 August; 7 (2): 99-110) or suitable other carriers.
In another aspect, the present disclosure provides a composition comprising one of more of the any one of, or any combination of, the TAPs or SLP(s) defined herein (e.g., comprising or consisting of the sequence of SEQ ID NOs: 1-23 and 25-50, preferably SEQ ID NOs: 1-23 such as SEQ ID NOs: 1-17 and 22) (or nucleic acid(s) encoding said peptide(s) or SLP(s)), and a buffer, an excipient, a carrier, a diluent and/or a medium. For compositions comprising cells (e.g., APCs, T lymphocytes), the composition comprises a suitable medium that allows the maintenance of viable cells. Representative examples of such media include saline solution, Earl's Balanced Salt Solution (Life Technologies®) or PlasmaLyte® (Baxter International®). In an embodiment, the composition (e.g., pharmaceutical composition) is an “immunogenic composition”, “vaccine composition” or “vaccine”. The term “Immunogenic composition”, “vaccine composition” or “vaccine” as used herein refers to a composition or formulation comprising one or more TAPs or vaccine vector and which is capable of inducing an immune response against the one or more TAPs present therein when administered to a subject. Vaccination methods for inducing an immune response in a mammal comprise use of a vaccine or vaccine vector to be administered by any conventional route known in the vaccine field, e.g., via a mucosal (e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract) surface, via a parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route, or topical administration (e.g., via a transdermal delivery system such as a patch). In an embodiment, the TAP (or a combination thereof) is conjugated to a carrier protein (conjugate vaccine) to increase the immunogenicity of the TAP(s). The present disclosure thus provides a composition (conjugate) comprising a TAP (or a combination thereof), or a nucleic acid encoding the TAP or combination thereof, and a carrier protein. For example, the TAP(s) or nucleic acid(s) may be conjugated or complexed to a Toll-like receptor (TLR) ligand (see, e.g., Zom et al., Adv Immunol. 2012, 114:177-201) or polymers/dendrimers (see, e.g., Liu et al., Biomacromolecules. 2013 Aug. 12; 14 (8): 2798-806). In an embodiment, the immunogenic composition or vaccine further comprises an adjuvant. “Adjuvant” refers to a substance which, when added to an immunogenic agent such as an antigen (TAPs, nucleic acids and/or cells according to the present disclosure), nonspecifically enhances or potentiates an immune response to the agent in the host upon exposure to the mixture. Examples of adjuvants currently used in the field of vaccines include (1) mineral salts (aluminum salts such as aluminum phosphate and aluminum hydroxide, calcium phosphate gels), squalene, (2) oil-based adjuvants such as oil emulsions and surfactant based formulations, e.g., MF59 (microfluidised detergent stabilised oil-in-water emulsion), QS21 (purified saponin), AS02 [SBAS2] (oil-in-water emulsion+MPL+QS-21), (3) particulate adjuvants, e.g., virosomes (unilamellar liposomal vehicles incorporating influenza haemagglutinin), AS04 ([SBAS4] aluminum salt with MPL), ISCOMS (structured complex of saponins and lipids), polylactide co-glycolide (PLG), (4) microbial derivatives (natural and synthetic), e.g., monophosphoryl lipid A (MPL), Detox (MPL+M. Phlei cell wall skeleton), AGP [RC-529] (synthetic acylated monosaccharide), DC_Chol (lipoidal immunostimulators able to self-organize into liposomes), OM-174 (lipid A derivative), CpG motifs (synthetic oligonucleotides containing immunostimulatory CpG motifs), modified LT and CT (genetically modified bacterial toxins to provide non-toxic adjuvant effects), (5) endogenous human immunomodulators, e.g., hGM-CSF or hIL-12 (cytokines that can be administered either as protein or plasmid encoded), Immudaptin (C3d tandem array) and/or (6) inert vehicles, such as gold particles, and the like.
In an embodiment, the TAP(s) or SLP(s) (e.g., comprising or consisting of the sequence of SEQ ID NOs: 1-23 and 25-50, preferably SEQ ID NOs: 1-23) (or a nucleic acid such as a mRNA encoding said peptide(s)) or composition comprising same is/are in lyophilized form. In another embodiment, the TAP(s), SLP(s), nucleic acid(s) or composition comprising same is/are in a liquid composition. In a further embodiment, the TAP(s), SLP(s), or nucleic acid(s) is/are at a concentration of about 0.01 μg/mL to about 100 μg/mL in the composition. In further embodiments, the TAP(s), SLP(s), or nucleic acid(s) is/are at a concentration of about 0.2 μg/mL to about 50 μg/mL, about 0.5 μg/mL to about 10, 20, 30, 40 or 50 μg/mL, about 1 μg/mL to about 10 μg/mL, or about 2 μg/mL, in the composition.
As noted herein, cells such as APCs that express an MHC class I molecule loaded with or bound to any one of, or any combination of, the TAPs defined herein, may be used for stimulating/amplifying CD8+ T lymphocytes in vivo or ex vivo. Accordingly, in another aspect, the present disclosure provides T cell receptor (TCR) molecules capable of interacting with or binding the herein-mentioned MHC class I molecule/TAP complex, and nucleic acid molecules encoding such TCR molecules, and vectors comprising such nucleic acid molecules. A TCR according to the present disclosure is capable of specifically interacting with or binding a TAP loaded on, or presented by, an MHC class I molecule, preferably at the surface of a living cell in vitro or in vivo.
The term TCR as used herein refers to an immunoglobulin superfamily member having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail; see, e.g., Janeway et al, Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 4:33, 1997) capable of specifically binding to an antigen peptide bound to a MHC receptor. A TCR can be found on the surface of a cell and generally is comprised of a heterodimer having α and β chains (also known as TCRα and TCRβ, respectively). Like immunoglobulins, the extracellular portion of TCR chains (e.g., α-chain, β-chain) contain two immunoglobulin regions, a variable region (e.g., TCR variable a region or Vα and TCR variable β region or Vβ; typically amino acids 1 to 116 based on Rabat numbering at the N-terminus), and one constant region (e.g., TCR constant domain a or Cα and typically amino acids 117 to 259 based on Rabat, TCR constant domain β or Cβ, typically amino acids 117 to 295 based on Rabat) adjacent to the cell membrane. Also, like immunoglobulins, the variable domains contain complementary determining regions (CDRs. 3 in each chain) separated by framework regions (FRs). In certain embodiments, a TCR is found on the surface of T cells (or T lymphocytes) and associates with the CD3 complex.
A TCR and in particular nucleic acids encoding a TCR of the disclosure may for instance be applied to genetically transform/modify T lymphocytes (e.g., CD8+ T lymphocytes) or other types of lymphocytes generating new T lymphocyte clones that specifically recognize an MHC class I/TAP complex. In a particular embodiment, T lymphocytes (e.g., CD8+ T lymphocytes) obtained from a patient are transformed to express one or more TCRs that recognize a TAP and the transformed cells are administered to the patient (autologous cell transfusion). In a particular embodiment, T lymphocytes (e.g., CD8+ T lymphocytes) obtained from a donor are transformed to express one or more TCRs that recognize a TAP and the transformed cells are administered to a recipient (allogenic cell transfusion). In another embodiment, the disclosure provides a T lymphocyte e.g., a CD8+ T lymphocyte transformed/transfected by a vector or plasmid encoding a TAP-specific TCR. In a further embodiment the disclosure provides a method of treating a patient with autologous or allogenic cells transformed with a TAP-specific TCR. In certain embodiments, TCRs are expressed in primary T cells (e.g., cytotoxic T cells) by replacing an endogenous locus, e.g., an endogenous TRAC and/or TRBC locus, using, e.g., CRISPR, TALEN, zinc finger, or other targeted disruption systems.
In another embodiment, the present disclosure provides a nucleic acid encoding the above-noted TCR. In a further embodiment, the nucleic acid is present in a vector, such as the vectors described above. In a further embodiment, the nucleic acid is an mRNA molecule.
In yet a further embodiment the use of a tumor antigen-specific TCR in the manufacture of autologous or allogenic cells for the treating of cancer (e.g., colorectal cancer) is provided.
In some embodiments, patients treated with the compositions (e.g., pharmaceutical compositions) of the disclosure are treated prior to or following treatment with an anti-tumor agent and/or immunotherapy (e.g., CAR therapy). Compositions of the disclosure include: allogenic T lymphocytes (e.g., CD8+ T lymphocyte) activated ex vivo against a TAP; allogenic or autologous APC vaccines loaded with a TAP; TAP vaccines and allogenic or autologous T lymphocytes (e.g., CD8+ T lymphocyte) or lymphocytes transformed with a tumor antigen-specific TCR. The method to provide T lymphocyte clones capable of recognizing a TAP according to the disclosure may be generated for and can be specifically targeted to tumor cells expressing the TAP in a subject (e.g., graft recipient), for example an ASCT and/or donor lymphocyte infusion (DLI) recipient. Hence the disclosure provides a CD8+ T lymphocyte encoding and expressing a T cell receptor capable of specifically recognizing or binding a TAP/MHC class I molecule complex. Said T lymphocyte (e.g., CD8+ T lymphocyte) may be a recombinant (engineered) or a naturally selected T lymphocyte. This specification thus provides at least two methods for producing CD8+ T lymphocytes of the disclosure, comprising the step of bringing undifferentiated lymphocytes into contact with a TAP/MHC class I molecule complex (typically expressed at the surface of cells, such as APCs) under conditions conducive of triggering T cell activation and expansion, which may be done in vitro or in vivo (i.e. in a patient administered with a APC vaccine wherein the APC is loaded with a TAP or in a patient treated with a TAP vaccine). Using a combination or pool of TAPs bound to MHC class I molecules, it is possible to generate a population CD8+ T lymphocytes capable of recognizing a plurality of TAPs. Alternatively, tumor antigen-specific or targeted T lymphocytes may be produced/generated in vitro or ex vivo by cloning one or more nucleic acids (genes) encoding a TCR (more specifically the alpha and beta chains) that specifically binds to a MHC class I molecule/TAP complex (i.e. engineered or recombinant CD8+ T lymphocytes). Nucleic acids encoding a TAP-specific TCR of the disclosure, may be obtained using methods known in the art from a T lymphocyte activated against a TAP ex vivo (e.g., with an APC loaded with a TAP); or from an individual exhibiting an immune response against peptide/MHC molecule complex. TAP-specific TCRs of the disclosure may be recombinantly expressed in a host cell and/or a host lymphocyte obtained from a graft recipient or graft donor, and optionally differentiated in vitro to provide cytotoxic T lymphocytes (CTLs). The nucleic acid(s) (transgene(s)) encoding the TCR alpha and beta chains may be introduced into a T cells (e.g., from a subject to be treated or another individual) using any suitable methods such as transfection (e.g., electroporation) or transduction (e.g., using viral vector). The engineered CD8+ T lymphocytes expressing a TCR specific for a TAP may be expanded in vitro using well known culturing methods.
The present disclosure provides methods for making the immune effector cells which express the TCRs as described herein. In one embodiment, the method comprises transfecting or transducing immune effector cells, e.g., immune effector cells isolated from a subject, such as a subject having a colorectal cancer (e.g., colon cancer, rectal cancer), such that the immune effector cells express one or more TCR as described herein. In certain embodiments, the immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells can then be directly re-administered into the individual. In further embodiments, the immune effector cells are first activated and stimulated to proliferate in vitro prior to being genetically modified to express a TCR. In this regard, the immune effector cells may be cultured before or after being genetically modified (i.e., transduced or transfected to express a TCR as described herein).
Prior to in vitro manipulation or genetic modification of the immune effector cells described herein, the source of cells may be obtained from a subject. In particular, the immune effector cells for use with the TCRs as described herein comprise T cells. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cell can be obtained from a unit of blood collected from the subject using any number of techniques known to the skilled person, such as FICOLL™ separation. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocyte, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing. In one embodiment of the invention, the cells are washed with PBS. In an alternative embodiment, the washed solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. As would be appreciated by those of ordinary skill in the art, a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated flow-through centrifuge. After washing, the cells may be resuspended in a variety of biocompatible buffers or other saline solution with or without buffer. In certain embodiments, the undesirable components of the apheresis sample may be removed in the cell directly resuspended culture media. In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. A specific subpopulation of T cells, such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method for use herein is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD8+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD4. Flow cytometry and cell sorting may also be used to isolate cell populations of interest for use in the present disclosure. PBMC may be used directly for genetic modification with the TCRs using methods as described herein. In certain embodiments, after isolation of PBMC, T lymphocytes are further isolated and in certain embodiments, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and/or expansion.
The present disclosure provides isolated immune cells such as CD8+ T lymphocytes that are specifically induced, activated and/or amplified (expanded) by a TAP (i.e., a TAP bound to MHC class I molecules expressed at the surface of cell), or a combination of TAPs. The present disclosure also provides a composition comprising CD8+ T lymphocytes capable of recognizing a TAP, or a combination thereof, according to the disclosure (i.e., one or more TAPs bound to MHC class I molecules) and said TAP(s). In another aspect, the present disclosure provides a cell population or cell culture (e.g., a CD8+ T lymphocyte population) enriched in CD8+ T lymphocytes that specifically recognize one or more MHC class I molecule/TAP complex(es) as described herein. Such enriched population may be obtained by performing an ex vivo expansion of specific T lymphocytes using cells such as APCs that express MHC class I molecules loaded with (e.g. presenting) one or more of the TAPs disclosed herein. “Enriched” as used herein means that the proportion of tumor antigen-specific CD8+ T lymphocytes in the population is significantly higher relative to a native population of cells, i.e., which has not been subjected to a step of ex vivo-expansion of specific T lymphocytes. In a further embodiment, the proportion of TAP-specific CD8+ T lymphocytes in the cell population is at least about 0.5%, for example at least about 1%, 1.5%, 2% or 3%. In some embodiments, the proportion of TAP-specific CD8+ T lymphocytes in the cell population is about 0.5 to about 10%, about 0.5 to about 8%, about 0.5 to about 5%, about 0.5 to about 4%, about 0.5 to about 3%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 5% or about 3% to about 4%. Such cell population or culture (e.g., a CD8+ T lymphocyte population) enriched in CD8+ T lymphocytes that specifically recognizes one or more MHC class I molecule/peptide (TAP) complex(es) of interest may be used in tumor antigen-based cancer immunotherapy, as detailed below. In some embodiments, the population of TAP-specific CD8+ T lymphocytes is further enriched, for example using affinity-based systems such as multimers of MHC class I molecule loaded (covalently or not) with the TAP(s) defined herein. Thus, the present disclosure provides a purified or isolated population of TAP-specific CD8+ T lymphocytes, e.g., in which the proportion of TAP-specific CD8+ T lymphocytes is at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.
In another aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof, or a soluble TCR, that specifically binds to a complex comprising a TAP as described herein bound to an HLA molecule, such as the HLA molecules defined herein. Such antibodies are commonly referred to as TCR-like antibodies. The term “antibody or antigen-binding fragment thereof” as used herein refers to any type of antibody/antibody fragment including monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, humanized antibodies, CDR-grafted antibodies, chimeric antibodies and antibody fragments so long as they exhibit the desired antigenic specificity/binding activity. Antibody fragments comprise a portion of a full-length antibody, generally an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules (e.g., single-chain Fv, scFv), single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments, single-chain diabodies (scDbs), bispecific T cell engagers (BiTEs), dual affinity retargeting molecules (DARTs), bivalent scFv-Fcs, and trivalent scFv-Fcs. Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, VH regions (VH, VH-VH), anticalins, PepBodies, antibody-T-cell epitope fusions (Troybodies) or Peptibodies. In an embodiment, the antibody or antigen-binding fragment thereof is a single-chain antibody, preferably a single-chain Fv (scFv). In an embodiment, the antibody or antigen-binding fragment thereof comprises at least one constant domain, e.g., a constant domain of a light and/or heavy chain, or a fragment thereof. In a further embodiment, the antibody or antigen-binding fragment thereof comprises a Fragment crystallizable (Fc) fragment of the constant heavy chain of an antibody. In an embodiment, the antibody or antigen-binding fragment is a scFv comprising a Fc fragment (scFV-Fc). In an embodiment, the scFv component is connected to the Fc fragment by a linker, for example a hinge. The presence of an Fc region is useful to induce a complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC) response against a tumor cell.
In an embodiment, the antibody or antigen-binding fragment thereof is a multispecific antibody or an antigen-binding fragment thereof, such as a bispecific antibody or an antigen-binding fragment thereof, wherein at least one of the antigen-binding domains of the multispecific antibody or antibody fragment recognize(s) a complex comprising a TAP as described herein bound to an HLA molecule. In an embodiment, at least one of the antigen-binding domains of the multispecific antibody or antibody fragment recognize(s) an immune cell effector molecule. The term “immune cell effector molecule” refers to a molecule (e.g., protein) expressed by an immune cell and whose engagement by the multispecific antibody or antibody fragment leads to activation of the immune cells. Examples of immune cell effector molecules include the CD3 signaling complex in T cells such as CD8 T cells and the various activating receptors on NK cells (NKG2D, KIR2DS, NKp44, etc.). In a further embodiment, at least one of the antigen-binding domains of the multispecific antibody or antibody fragment recognize(s) and engage(s) the CD3 signaling complex in T cells (e.g., anti-CD3). In a further embodiment, the multispecific antibody or antibody fragment is a single-chain diabody (scDb). In a further embodiment, the scDb comprises a first antibody fragment (e.g., scFv) that binds to a complex comprising a TAP as described herein bound to an HLA molecule and a second antibody fragment (e.g., scFv) that binds to and engages an immune cell effector molecule, such as the CD3 signaling complex in T cells (e.g., anti-CD3 scFv). Such constructs may be used for example to induce the cytotoxic T cell-mediated killing of tumor cells expressing the tumor antigen/MHC complex recognized by the multispecific antibody or antibody fragment. Antibodies or antigen-binding fragments thereof may also be used as a chimeric antigen receptor (CAR) to produce CAR T cells, CAR NK cells, etc. CAR combines a ligand-binding domain (e.g., antibody or antibody fragment) that provides specificity for a desired antigen (e.g., MHC/TAP complex) with an activating intracellular domain (or signal transducing domain) portion, such as a T cell or NK cell activating domain, providing a primary activation signal. Antigen-binding fragments of antibodies, and more particularly scFv, capable of binding to molecules expressed by tumor cells are commonly used as ligand-binding domains in CAR. Thus, in another aspect, the present disclosure provides a host cell, preferably an immune cell such as a T cell or NK cell, expressing the antibody or antibody fragment (e.g., scFv) described herein.
In an embodiment, the soluble TCR is a soluble therapeutic bispecific TCR (see, e.g., Robinson et al., FEBS J. 2021 November; 288 (21): 6159-6173; Dilchert et al., Antibodies (Basel). 2022 May 10; 11 (2): 34).
The present disclosure further relates to a pharmaceutical composition or vaccine comprising the above-noted immune cell (CD8+ T lymphocytes, CAR T cell) or population of TAP-specific CD8+ T lymphocytes. Such pharmaceutical composition or vaccine may comprise one or more pharmaceutically acceptable excipients and/or adjuvants, as described above.
The present disclosure further relates to the use of any TAP or SLP (e.g., comprising or consisting of any of the sequences of SEQ ID NOs: 1-23 and 25-50, preferably SEQ ID NOs: 1-23, such as SEQ ID NOs: 1-17 and 22), nucleic acid, expression vector, T cell receptor, antibody/antibody fragment, cell (e.g., T lymphocyte, APC, CAR T cell), and/or composition according to the present disclosure, or any combination thereof, as a medicament or in the manufacture of a medicament. In an embodiment, the medicament is for the treatment of cancer, e.g., cancer vaccine. The present disclosure relates to any TAP, SLP, nucleic acid, expression vector, T cell receptor, antibody/antibody fragment, cell (e.g., T lymphocyte, APC), and/or composition (e.g., vaccine composition) according to the present disclosure, or any combination thereof, for use in the treatment of cancer e.g., as a cancer vaccine. The TAP sequences identified herein may be used for the production of synthetic peptides to be used i) for in vitro priming and expansion of tumor antigen-specific T cells to be injected into tumor patients and/or ii) as vaccines to induce or boost the anti-tumor T cell response in cancer patients, such as CRC patients. In an embodiment, the cancer (e.g., CRC) expresses one or more of the TAPs described herein.
In another aspect, the present disclosure provides the use of a TAP described herein (e.g., SEQ ID NOs: 1-23 and 25-50, preferably SEQ ID NOs: 1-23, such as SEQ ID NOs: 1-17 and 22), or a combination thereof (e.g., a peptide pool), or of one or more nucleic acid(s) encoding the TAP(s), as a vaccine for treating cancer, such as a CRC, in a subject. The present disclosure also provides the TAP described herein, or a combination thereof (e.g., a peptide pool), or of one or more nucleic acid(s) encoding the TAP(s), for use as a vaccine for treating cancer, such as a CRC, in a subject. In an embodiment, the subject is a recipient of TAP-specific T lymphocytes (e.g., CD8+ T lymphocytes). Accordingly, in another aspect, the present disclosure provides a method of treating cancer, such as a CRC (e.g., of reducing the number of tumor cells, killing tumor cells), said method comprising administering (infusing) to a subject in need thereof an effective amount of T lymphocytes (e.g., CD8+ T lymphocytes) recognizing (i.e., expressing a TCR that binds) one or more MHC class I molecule/TAP complexes (expressed at the surface of a cell such as an APC). In an embodiment, the method further comprises administering an effective amount of the TAP, or a combination thereof, or of one or more nucleic acid(s) encoding the TAP(s), and/or a cell (e.g., an APC such as a dendritic cell) expressing MHC class I molecule(s) loaded with the TAP(s), to said subject after administration/infusion of said CD8+ T lymphocytes. In yet a further embodiment, the method comprises administering to a subject in need thereof a therapeutically effective amount of a dendritic cell loaded with one or more TAPs. In yet a further embodiment the method comprises administering to a patient in need thereof a therapeutically effective amount of an allogenic or autologous cell that expresses a recombinant TCR that binds to a TAP presented by an MHC class I molecule.
In another aspect, the present disclosure provides the use of T lymphocytes (e.g., CD8+ T lymphocytes) that recognize one or more MHC class I molecules loaded with (presenting) a TAP, or a combination thereof, for treating cancer (e.g., of reducing the number of tumor cells, killing tumor cells), such as CRC, in a subject. In another aspect, the present disclosure provides the use of T lymphocytes (e.g., CD8+ T lymphocytes) that recognize one or more MHC class I molecules loaded with (presenting)) a TAP, or a combination thereof, for the preparation/manufacture of a medicament for treating cancer (e.g., for reducing the number of tumor cells, killing tumor cells), such as CRC, in a subject. In another aspect, the present disclosure provides T lymphocytes (e.g., CD8+ T lymphocytes) that recognize one or more MHC class I molecule(s) loaded with (presenting) a TAP, or a combination thereof, for use in the treatment of cancer (e.g., for reducing the number of tumor cells, killing tumor cells), such as CRC, in a subject. In a further embodiment, the use further comprises the use of an effective amount of a TAP (or a combination thereof), and/or of a cell (e.g., an APC) that expresses one or more MHC class I molecule(s) loaded with (presenting) a TAP, after the use of said TAP-specific CD8+ T lymphocytes.
The present disclosure also provides a method of generating an immune response against tumor cells (e.g., colorectal cancer cells) expressing human class I MHC molecules loaded with any of the TAP disclosed herein (e.g., SEQ ID NOs: 1-23 and 25-50, preferably SEQ ID NOs: 1-23, such as SEQ ID NOs: 1-17 and 22) or combination thereof in a subject, the method comprising administering cytotoxic T lymphocytes that specifically recognizes the class I MHC molecules loaded with the TAP or combination of TAPs. The present disclosure also provides the use of cytotoxic T lymphocytes that specifically recognizes class I MHC molecules loaded with any of the TAP or combination of TAPs disclosed herein for generating an immune response against tumor cells expressing the human class I MHC molecules loaded with the TAP or combination thereof.
In an embodiment, the cancer is colorectal cancer. In a further embodiment, the cancer is colon cancer. In another embodiment, the cancer is rectal cancer. In an embodiment, the colorectal cancer is characterized by microsatellite instability (MSI). In an embodiment, the colorectal cancer is characterized by microsatellite stability (MSS). In an embodiment, the colorectal cancer is characterized by RAS (e.g., KRAS) and/or RAF mutations. In another embodiment, the colorectal cancer is resistant/refractory to chemotherapy. In another embodiment, the colorectal cancer is resistant/refractory to EGFR inhibitors.
In an embodiment, the methods or uses described herein further comprise determining the HLA class I alleles expressed by the patient prior to the treatment/use, and administering or using TAPs that bind to one or more of the HLA class I alleles expressed by the patient. For example, if it is determined that the patient expresses HLA-A2*01 and HLA-B07*02, any combinations of the TAPs of SEQ ID NO:6 (that bind to HLA-A2*01) and/or SEQ ID NO:2, 21, 24, and/or 50 (that bind to HLA-B07*02) may be administered or used in the patient.
In an embodiment, the TAP, SLP, nucleic acid, expression vector, T cell receptor, antibody/antibody fragment, cell (e.g., T lymphocyte, CAR T or NK cell, APC), and/or composition according to the present disclosure, or any combination thereof, may be used in combination with one or more additional active agents or therapies to treat cancer (e.g., CRC), such as chemotherapy (e.g., vinca alkaloids, agents that disrupt microtubule formation (such as colchicines and its derivatives), anti-angiogenic agents, therapeutic antibodies, EGFR targeting agents, tyrosine kinase targeting agent (such as tyrosine kinase inhibitors), transitional metal complexes, proteasome inhibitors, antimetabolites (such as nucleoside analogs), alkylating agents, platinum-based agents, anthracycline antibiotics, topoisomerase inhibitors, macrolides, retinoids (such as all-trans retinoic acids or a derivatives thereof), geldanamycin or a derivative thereof (such as 17-AAG), surgery, immune checkpoint inhibitors or immunotherapeutic agents (e.g., PD-1/PD-L1 inhibitors such as anti-PD-1/PD-L1 antibodies, CTLA-4 inhibitors such as anti-CTLA-4 antibodies, B7-1/B7-2 inhibitors such as anti-B7-1/B7-2 antibodies, TIM3 inhibitors such as anti-TIM3 antibodies, BTLA inhibitors such as anti-BTLA antibodies, CD47 inhibitors such as anti-CD47 antibodies, GITR inhibitors such as anti-GITR antibodies), antibodies against tumor antigens (e.g., anti-CD19, anti-CD22 antibodies), cell-based therapies (e.g., CAR T cells, CAR NK cells), and cytokines such as IL-2, IL-7, IL-21, and IL-15. In an embodiment, the TAP, nucleic acid, expression vector, T cell receptor, cell (e.g., T lymphocyte, APC), and/or composition according to the present disclosure is administered/used in combination with an immune checkpoint inhibitor. In an embodiment, the TAP, nucleic acid, expression vector, T cell receptor, cell (e.g., T lymphocyte, APC), and/or composition according to the present disclosure is administered/used in combination one or more therapies used for the treatment of CRC (e.g., surgery, chemotherapy (e.g., using 5-fluorouracil, capecitabine, oxaliplatin, irinotecan, raltitrexed, trifluridine, tipiracil), radiation therapy, bevacizumab, cetuximab, panitumumab, regorafenib.
The additional therapy may be administered prior to, concurrent with, or after the administration of the TAP, SLP, nucleic acid, expression vector, T cell receptor, antibody/antibody fragment, cell (e.g., T lymphocyte, CAR T or NK cell, APC), and/or composition according to the present disclosure.
The present disclosure is illustrated in further details by the following non-limiting examples.
Cell lines. Four colorectal cancer cell lines [COLO 205 (ATCC® CCL-222™), HCT 116 (ATCC® CCL-247™), RKO (ATCC® CRL-2577™), SW620 [SW-620] (ATCC® CCL-227™)] and 1 normal fetal small intestine cell line [HIEC6 (ATCC® CRL3266™)] were obtained from the American Type Culture Collection (ATCC). COLO205, HCT116, and SW620 were grown in RPMI-1640 (Gibco) supplemented with 10% Fetal bovine serum (FBS), RKO was grown in Eagle's Minimum Essential Medium (EMEM) (ATCC®) supplemented with 10% FBS, and HIEC-6 was grown in OptiMEM® 1 Reduced Serum Medium (Gibco) supplemented with 20 mM HEPES (Gibco), 10 mM GlutaMAX® (Gibco), 10 ng/mL epidermal growth factor (EGF) (Gibco), and FBS to a final concentration of 4%. All cells were maintained at 37° C. with 5% CO2. For collection, cells were rinsed with warm PBS before being trypsinized with TrypLET Express Enzyme (1×) (Gibco) for 5-15 minutes at 37° C. with 5% CO2. Harvested material was then spun at 1000 rpm for 5 minutes, rinsed once with warm PBS, then resuspended in ice-cold PBS. After cell count, replicates of 2×108 CRC cells were pelleted and frozen at −80° C. until further use. MHC class I surface density of the CRC cell lines was determined by Qifikit™ (Agilent) using the W6/32 anti-HLA class I antibody (BioXCell), according to the manufacturer's instructions.
Primary tissues. Six pairs of primary human samples consisting of matched colon adenocarcinoma tumor and normal adjacent tissue (NAT) were purchased from Tissue Solutions. Tissue samples were taken from patients receiving surgery as first line of treatment and were flash-frozen in liquid nitrogen. More information about primary tissue samples can be found in Table 2.
RNA extraction. For RNA extraction of cell lines, 1-2 million cells were collected and washed once with ice-cold PBS. The cells were then resuspended in Trizol™ (Invitrogen). For cell lines and primary tissue samples, total RNA was isolated using the AllPrep™ DNA/RNA/miRNA Universal kit (Qiagen) or the RNeasy™ Mini kit (Qiagen) as recommended by the manufacturer.
RNA sequencing. 500 ng of total RNA was used for library preparation. RNA quality control was assessed with the Bioanalyzer™ RNA 6000 Nano assay on the 2100 Bioanalyzer™ system (Agilent Technologies) and all samples had a RIN above 8. Libraries were prepared with the KAPA mRNAseq Hyperprep™ kit (Roche). Ligation was made with Illumina dual-index UMI (IDT). After being validated on BioAnalyzer™ DNA1000 chip and quantified by QuBit and qPCR, libraries were pooled to equimolar concentration and sequenced with the Illumina Nextseq500 using the Nextseq™ High Output 150 (2×75 bp) cycles kit. A mean of 129 and 95 million paired-end PF reads were generated for the cell lines and tissue samples, respectively. Library preparation and sequencing was performed at the Genomic Platform of the Institute for Research in Immunology and Cancer (IRIC).
Bioinformatic analyses. Sequences were trimmed using Trimmomatic version 0.35 (17) and aligned to the reference human genome version GRCh38 (gene annotation from Gencode version 33, based on Ensembl 99) using STAR version 2.7.1a (18). Gene expressions were obtained both as readcount directly from STAR as well as computed using RSEM (19) in order to obtain normalized gene and transcript level expression, in TPM values, for these stranded RNA libraries.
HLA genotyping. HLA genotyping of cell lines and tissues was performed using OptiType (https://github.com/FRED-2/OptiType) (20).
Microsatellite instability prediction. MSI status of the primary tumor samples was predicted using the MSIsensor program using paired tumor and NAT (https://github.com/ding-lab/msisensor) (21).
Differential expression analysis. DESeq2 version 1.22.2 (22) was used to normalize gene readcounts and compute differential expression between tumor and normal samples. Principal component analyses (PCA) were generated using normalized log readcounts for the first two most significant components. For differential expression analysis of the cell lines, fold changes were computed between the mean expression of the four CRC cell lines compared to the normal cell line (HIEC-6). Significant differentially expressed genes (DEGs) are (those with padj lower than 0.05) and with |log 2 fold change|>1 were considered for GO terms using the Metascape tool (23). For paired differential expression analysis of the tissues, TPM normalized values were used to compare tumor/NAT pairs. As only a single replicate of the tissues was sequenced, rather than filtering by adjusted p-value, only genes that were significantly differentially expressed in all six subjects for GO term analysis with |log 2 fold change|>1 were selected. When examining differentially expressed genes between MSS and MSI tissues, the same fold change thresholds were applied. For GO term analysis of MSI DEGs, genes were selected that were exclusively differentially expressed in both MSI tissues (i.e., not considered DEGs in any MSS tissues). For GO term analysis of MSS DEGs, genes were considered if they were differentially expressed in 3 or more MSS tissues.
Transcriptome analysis of tissue samples. The proportion of various biotypes in the transcriptome of tissue samples was determined as previously described (24). Briefly, following quantification and alignment of Ensembl annotated transcripts by Kallisto (19), transcripts and repetitive elements were annotated using a Kallisto index containing Ensembl annotated transcripts supplemented with genetic repeat identifications from the USCS Table Browser GRCh38 repeat masker database (25).
Mutation profiles/‘genetic variant annotation’. Genetic variant calling was performed for both cell line and primary biopsies using SNPEff (https://pcingola.github.io/SnpEff/#snpeff) (26).
Database generation. Global cancer databases were constructed as previously described (16). In brief, RNA-seq reads were trimmed using Trimmomatic version 0.35 (17) and aligned to the reference human genome version GRCh38 (gene annotation from Gencode version 33, based on Ensembl 99) using version STAR 2.7.1a (18). Kallisto (https://pachterlab.github.io/kallisto) was used to quantify transcript expression in TPM (19). Sample-specific exomes were constructed by integrating single nucleotide variants (quality >20) identified with Freebayes (https://github.com/ekg/freebayes) into PyGeno (27). Annotated open reading frames with TPM>0 were then translated in silico and added to the canonical proteome in fasta format. To generate the cancer-specific proteome, RNA-seq reads were cut into 33-nucleotide sequences known as kmers and only kmers present <2 in mTECs or matched NAT for cell lines and tissues, respectively, were kept. Overlapping kmers were assembled into contigs, which were then 3-frame translated in silico. Of note, short peptide sequences generated through the kmer approach were then concatenated into longer sequences of approximately ten thousand amino acids. These peptides were concatenated using the ‘JJ’ sequence as a separator, which is recognized internally by the PeaksX+ software to split sequences upon occurrence of this sequence. Then, the canonical proteome and the cancer-specific proteome were concatenated to create the global cancer databases. Cell line databases consisted of 3.38×106 sequences on average.
Isolation of MAPS. CRC cell line pellet samples (2×108 cells per replicate, 4 replicates per cell line) were resuspended with PBS up to 2 mL and then solubilized by adding 2 mL of ice-cold 2× lysis buffer (1% w/v CHAPS). Tumor and normal adjacent tissue samples (between 455 mg and 693 mg) were cut into small pieces (cubes, ˜3 mm in size) and 5 ml of ice-cold PBS containing protein inhibitor cocktail (Sigma, cat #P8340-5 ml) was added. Tissues were first homogenized twice for 20 seconds using an Ultra Turrax T25 homogenizer (IKA-Labortechnik) set at speed 20000 rpm and then 20 seconds using an Ultra Turrax T8 homogenizer (IKA-Labortechnik) set at speed 25000 rpm. Then, 550 μl of ice-cold 10× lysis buffer (5% w/v CHAPS) was added to each sample. After 60-minute incubation with tumbling at 4° C., tissue samples and CRC cell line samples were spun at 10000 g for 30 minutes at 4° C. Supernatants were transferred into new tubes containing 1 mg of W6/32 antibody covalently-cross-linked protein A magnetic beads and MAPs were immunoprecipitated as previously described (28). MAP extracts were then dried using a Speed-Vac and kept frozen before MS analyses.
TMT labeling. MAP extracts were resuspended in 200 mM Hepes buffer pH 8.1. 50 μg of TMT reagent (Thermo Fisher Scientific) in anhydrous acetonitrile was added to samples as follows: CRC cell line replicates were labeled with TMT6plex (lot #UG287166) channels TMT6-126-129; Tissue samples were labeled with TMT10plex (lot #UH285228) −126 (NAT) and −127N (tumor). Samples were gently vortexed and reacted at room temperature for 1.5 hours. Samples were then quenched with 50% hydroxylamine for 30 minutes at room temperature, then were diluted with 4% FA/H2O. CRC cell line replicates and individual NAT-tumor pairs were combined. Samples were then desalted on homemade C18 membrane (Empore) columns and stored at −20° C. until injection. To quantify MAPs of interest in primary tissue samples, synthetic peptides at concentrations of 0.75 to 192 fmoles were labeled with TMT 10plex channels −128N, −128C, −129N, −129C, −130N, −130C, −131, while the NAT and CRC tissues were labeled with −126 and 127N, respectively. Note that the channel −127C was left empty to assess contamination between channels.
Liquid Chromatography-tandem MS analyses. Dried peptide extracts were resuspended in 4% FA and loaded on a homemade C18 analytical column (20 cm×150 μm i.d. packed with C18 Jupiter Phenomenex) with a 106-minute gradient from 0% to 30% ACN (0.2% FA) and a 600 nL/min flow rate on an EASY-nLC II system. Samples were analyzed with an Orbitrap Exploris 480 spectrometer (Thermo Fisher Scientific) in positive ion mode with Nanoflex source at 2.8 kV. Each full MS spectrum, acquired with a 240,000 resolution was followed by 20 MS/MS spectra, where the most abundant multiply charged ions were selected for MS/MS sequencing with a resolution of 30,000, an automatic gain control target of 100%, an injection time of 700 ms, and collisional energy of 40%.
MAP Identification. Database searches were conducted using the PeaksX+ software (Bioinformatics Solutions Inc.) (29). Error tolerances for precursor mass and fragment ions were set to 10.0 ppm and 0.01 Da, respectively. A non-specific digest mode was used. TMT6plex or 10plex was set as a fixed PTM, and variable modifications included phosphorylation (STY), Oxidation (M), Deamidation (NQ), and TMT6plex or 10plex STY. Peaks searches were then loaded into MAPDP (30), which was used to apply the following filters: selecting peptides of 8-11 amino acids in length, with rank eluted ligand threshold≤2% based on NetMHCpan-4.1b predictions, using a 5% FDR.
Quantification of MAP coding sequences in RNA-Seq data. MAP coding sequences (MCSs) were quantified in RNA-seq data as previously described (31). Briefly, MCSs were reverse translated into all possible nucleotide sequences with an in-house python script (deposited on Zenodo at DOI: 3739257). The nucleotide sequences were then mapped onto the genome with GSNAP (32) to determine all possible genomic locations able to code for a given MAP. MCSs were also mapped onto the transcriptome to account for MAPs overlapping splice sites, and portions of the transcriptome corresponding to these MAPs were then also mapped onto the reference genome with GSNAP. For MAPs of interest, genomic alignment of all reads containing the MCS was performed. GSNAP output was filtered to keep only perfect matches between the sequence and reference, resulting in a file containing all possible genomic regions able to code for a given MAP. The number of reads containing the MCSs at their respective genomic locations in each desired RNA-Seq sample (such as CRC and NAT, GTEx, or TCGA samples), aligned on the reference genome with STAR, was summed. Lastly, all read counts for a given MAP were summed and normalized on the total number of reads sequenced in each sample of interest to obtain a reads-per-hundred-million (RPHM) count.
Determination of MAP source transcripts. To investigate what proportion of tissue sample MAPs were derived from certain transcript biotypes, the most abundant putative source transcript based on kmer-per-hundred-million (KPHM) quantification was determined. For peptides from the cancer-specific (kmer) database, the MCSs were reverse translated into all possible nucleotide sequences and all possible genomic regions able to code for a given MAP were identified (see ‘Quantification of MAP coding sequences in RNA-Seq data’ above). Finally, Kallisto was used to determine the most expressed transcript at that location, which was then assigned as the most probable transcript for the given peptide. Peptides that had more than one putative source transcript were excluded from the analysis.
Identification of TSA candidates. TSA candidates were identified through a stringent TSA identification pipeline. First, MAPs underwent peptide classification in which the peptide sequence accessions were retrieved from the protein database and used to extract the nucleotide sequences of each peptide. RNA-Seq data from each cancer and normal samples were transformed into 24-nucleotide-long k-mer databases with Jellyfish 2.2.3 (using the -C option) and used to query each TSA candidate coding sequence's 24-nucleotide-long k-mer set. The number of reads fully overlapping a given peptide-coding sequence was estimated using the k-mer set's minimum occurrence (kmin), as in general, one k-mer always originates from a single RNA-Seq read. This kmin value was then transformed into several k-mers detected per 108 reads sequenced (kphm) using the following formula: kphm=(kmin×108) rtot, with rtot representing the total number of reads sequenced in a given RNA-Seq experiment. Peptides were kept only if their RNA coding sequences were expressed at least 10-fold higher in cancer than in normal (pooled mTEC samples for cell lines, matched NAT for tissues), and expressed <2 KPHM in normal. Subsequent filtering removed any peptides with indistinguishable isoleucine/leucine variants; a peptide with an IL variant was kept only if the most expressed variant met the above-mentioned criteria. The MCSs of the remaining peptides were quantified in RNA-seq data as described above and were kept only if their expression was <8.55 RPHM in mTECs and other normal tissues (GTEx). Genomic localization for each peptide was assigned by mapping reads containing each MCS to the reference genome (GRCh38.99) using BLAT (https://genome.ucsc.edu/cgi-bin/hgBlat, Kent W J. BLAT—the BLAST-like alignment tool. Genome Res. 2002 April; 12 (4): 656-64. PMID: 11932250). Peptides were excluded if the genomic localization was unclear or if they mapped to a hypervariable region (HLA, Ig, or T cell receptor (TCR) genes). Finally, the MS/MS spectra of the remaining candidates were manually validated. Peptides were classified as mTSAs if their amino acid sequence was different from the reference, and if the mutation was not a known germline polymorphism. Peptides were classified as aeTSAs if they were overexpressed ≥10-fold in tumor compared to normal and ≤0.2 KPHM in mTECs (and NAT in the case of tissues) and as TAAs if they were overexpressed ≥10-fold in cancer but the expression in mTECs and/or NAT was >0.2 KPHM. Ultimately, the transcript of origin of each TSA/TAA was attributed by selecting the most highly expressed peptide-overlapping transcript from the kallisto quantification file (see Database Generation section).
Intertumoral sharing. To examine the intertumoral distribution of TSA and TAA sequences in other CRC tumors, the log (RPHM+1) expression of the peptide coding sequences in 151 colon adenocarcinoma (COAD) samples from TCGA was determined (see ‘Quantification of MAP coding sequences in RNA-Seq data’).
Immunogenicity prediction. The predicted immunogenicity of MAPs of interest was determined with the R package Repitope v3.0.1 (https://github.com/masato-ogishi/Repitope) (34).
Validation of TSA peptide candidates. Synthetic peptides of TSA and select TAA sequences were obtained from Genscript. Synthetic peptides were solubilized in DMSO to a concentration of 1 nmol/μL and all synthetic peptides were combined in a stock solution at a concentration of 10 pmol/μL. The stock solution was desalted in aliquots of 150 pmol on homemade C18 membrane (Empore) columns and dried using a Speed-Vac. Dried peptide extracts were labeled with a TMT10plex channel as described (see ‘TMT labeling’ section), desalted, and dried down in Speed-Vac. Labeled synthetic peptides were resuspended in 4% FA and 1 pmol of each synthetic peptide was loaded on a homemade C18 analytical column (20 cm×150 μm i.d. packed with C18 Jupiter Phenomenex) with a 76-minute gradient from 0% to 30% ACN (0.2% FA) and a 600 nL/min flow rate on an EASY-nLC II system. Samples were analyzed with an Orbitrap Exploris 480 spectrometer (Thermo Fisher Scientific) in positive ion mode with Nanoflex source at 2.8 kV. Each full MS spectrum was acquired with a 120 000 resolution, and an inclusion list was used to select ions for fragmentation with 40% collision energy and an isolation window of 1 m/z. MS/MS were acquired with a resolution of 30 000. MS/MS correlations were computed as previously described (17). Briefly, expected peptide fragments were computed with pyteomics v4.0.1 (https://bitbucket.org/levitsky/pyteomics) and reproducibly detected peptide fragments were identified. Root scaled intensities of these fragments were correlated between endogenous and synthetic peptide scan pairs and SciPy v1.2.1 (https://www.scipy.org/) was used to compute Pearson correlation coefficient, p-value, and confidence intervals. Mirror plots of the scan pair with the lowest p-value were generated for each peptide using spectrum_utils v0.2.1 (https://github.com/bittremieux/spectrum_utils).
Relative quantification. To relatively quantify MAPs of interest in primary tissue samples, synthetic peptides at concentrations of 10, 100, or 1000 fmol labeled with TMT 10plex-129N, 130N, and 131N, respectively, were spiked into remaining purified MAPs from NAT and CRC tissue samples labeled with TMT10plex-126 and 127N, respectively. Note that the channel TMT 10plex-127C was left empty to assess contamination. Samples were analyzed with an Orbitrap Fusion Tribrid spectrometer (Thermo Fisher Scientific) in positive ion mode with Nanoflex source at 2.4 kV. For synchronous precursor selection MS3 (SPS-MS3), full MS scans were acquired with a range of 300-1000 m/z, Orbitrap resolution of 120 000, automatic gain control (AGC) of 5.0e5, and a maximum injection time of 50 ms, using an inclusion list for the peptides of interest. A 3s top speed approach for MS2 was used in the ion trap, with an isolation window of 0.4m/z, collision induced dissociation of 35%, a ‘normal’ ion trap scan rate mode, 2.0e4 AGC target, and 50 ms maximum injection time. This was followed by the selection of eight synchronous precursor ions for MS3 acquisition, which was done with a scan range of 110-500 m/z, Orbitrap resolution of 50 000, AGC of 1.0e5, maximum injection time of 300 ms, an isolation window of 2.0m/z, and 65% HCD collision energy. LC-MS instrument was controlled using Xcalibur version 4.4 (Thermo Fisher Scientific, Inc). Error tolerances for precursor mass and fragment ions were set to 10.0 ppm and 0.5 Da, respectively. A non-specific digest mode was used. TMT10plex was set as a fixed PTM, and variable modifications included phosphorylation (STY), Oxidation (M), Deamidation (NQ), and TMT10plex STY. For quantification, PSMs were filtered to exclude those with contamination in the TMT10plex-127C channel, and to select those within the 70th intensity percentile. MS2 precursor profiles and intensity profiles of all relevant channels were manually inspected to select peptides for quantification. Intensity ratios for each peptide were calculated using the average TMT10plex-127N and TMT10plex-126 intensities of good quality PSMs.
Data analysis and visualization.
Experimental Design and Statistical Rationale. To effectively elucidate the MHC I immunopeptidome of colorectal cancer, 4 CRC cell lines were selected and 6 samples from human subjects consisting of both matched tumor and normal adjacent tissue (NAT) were acquired. NAT was used as an approximation of healthy tissue, as it is the most effective control for each respective tumor. Since no matched samples were available for cell lines, a pool of 6 mTEC samples was used in the creation of global cancer databases, to obtain a wide berth of approximate normal RNA expression. All instances of p-values are determined using two-sample t-test, except in the determination of significance for immunogenicity scores, in which case the Mann-Whitney test was used as the data did not have a normal distribution, as determined by the Shapiro test. For t-tests, f-tests were performed to determine whether the dataset had significant variation; if yes, then the t-test assuming variation was used, and otherwise the t-test assuming no variation was used. For CRC-derived cell lines, four technical replicates of 2×108 cells were prepared, which were TMT labeled and multiplexed prior to injection. Due to limited tissue material, half of the purified MAPs from primary samples were injected to obtain global immunopeptidomic data, and the remaining sample was used for targeted analysis with synthetic peptides to confirm the sequences and abundance of putative TSAs and select TAAs. To select high quality PSMs for quantification, those of low intensity or with contamination in an empty TMT channel were excluded. Further, only peptides with favorable MS2 precursor and intensity profiles were quantified.
To determine the composition of the immunopeptidome of colorectal cancer, a collection of samples comprised of 4 colorectal cancer-derived cell lines and 6 sets of primary adenocarcinoma samples, which consist of matched tumour and normal adjacent tissue (Tables 1 and 2), were analyzed. Paired-end RNA sequencing (RNA-seq) allowed the creation of a global cancer database, consisting of a canonical cancer proteome as well as a cancer-specific proteome for each sample, created through the generation of cancer-specific kmers which are translated into 3 reading frames to encompass non-canonical sequences from any genomic origin (
MHC I: peptide complexes were isolated through immunoprecipitation, and the eluted MHC I-associated peptides (MAPs) were labeled with tandem mass tag (TMT) isobaric labeling reagent, as TMT labeling was recently shown to enhance the detection of MAPs by increasing their charge state and hydrophobicity (37). MAPs were then sequenced and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) using the personalized cancer databases generated through RNA-seq. Identified MAPs then underwent a rigorous series of classifications and validations to identify putative TSAs and TAAs. Tumor antigens identified in CRC tissues were then validated and quantified with synthetic peptides to determine to what extent they were overexpressed at the cell surface of tumors, and their predicted immunogenicity and intertumoral distribution were also investigated to obtain a sense of their clinical potential (
In the present study, 4 CRC-derived cell lines with different alleles and characteristics were used as summarized in Table 1. HCT116 and RKO are derived from primary tumors and are characterized by microsatellite instability (MSI), whereas Colo205 and SW620 are derived from metastases of ascites and lymph node, respectively, and are both microsatellite stable (MSS). These cell lines have a wide range of MHC I surface expression ranging from 1.44×105 to 5.07×105 MHC I molecules/cell and a diversity of HLA alleles. Among the 4 cell lines are mutations in several key genes, such as BRAF, RAS, SMAD4, TP53, and PI3CA. These cell lines have a varying MHC I surface expression ranging from 1.44×105 to 5.07×105 MHC I molecules/cell, as determined by Qifikit, and a diversity of HLA alleles which were identified using OptiType, an HLA genotyping tool that uses RNA-Seq data to predict a sample's HLA alleles, in combination with the HLA alleles for these cell lines documented in the literature (Table 1) (22).
All of the primary tumor samples are derived from stage II adenocarcinomas, which vary only slightly in tumor grade and TNM (tumor-node-metastases) classification (Table 2). The primary samples had a tumor content of 95-100% and an average mass of 0.6625 grams. The tumors are all derived from the sigmoid colon, with the exception of S1 (cecum) and S5 (ascending colon). All patients are female, with the exception of S2, and the patients range in age from 43-85, with a mean age of 62. Similar to the cell lines, the tissue samples also possess a variety of HLA alleles. A visualization of the number of HLA alleles unique to or shared by cell line and tissue samples is available in
Because the outcome for CRC patients within a given disease stage differs greatly based on the molecular characteristics of the tumor (40, 41), RNA sequencing data were used to characterize the molecular heterogenicity of the samples. After first examining the mutational status of key biomarkers (such as KRAS, NRAS, or BRAF) which are commonly used to guide therapeutic decisions and prognosis in the clinics (40, 41) (Table 1), the microsatellite statuses of cell lines and primary samples were respectively determined from the literature (42, 43) and using the MSIsensor package (46) to expand the knowledge of the molecular characteristics of these samples. While MSI is found in a limited subset of CRC tumors (i.e., 15% of sporadic CRC and 90% of nonpolyposis colorectal cancer) (47), in this study, 50% of the tumorigenic cell lines and 33% of the primary biopsies present this phenotype (Table 3). Although several elements in the literature suggest that MSI and MSS tumors are immunologically different (5, 11, 48, 49), this study provides a comparison of MSI and MSS colorectal tumors at the immunopeptidomic level.
Principal component analysis of the top 500 varying genes between normal and tumor biopsy samples (
Next, the degree of immune infiltration of each sample was estimated via two independent approaches using the immune infiltration score from the ESTIMATE package (37) (
Because TSAs can arise from a wide range of cancer-specific events/dysregulations (11, 12) and that the immunopeptidome contribution of each antigenic source varies significantly across malignancies (11), RNA sequencing data were also used to inform which TSA classes might be enriched in the samples. By looking at the genomic origin of the transcripts, it was observed that both the proportion and the absolute abundance of non-coding polyadenylated RNAs are significantly increased in tumors compared to NATs (
To elucidate the MHC I immunopeptidomes of CRC-derived cell lines, MAPs from 4 replicates of 2×108 cells were immunoprecipitated for each line, and each replicate was derivatized with a separate TMT6plex channel (126, 127, 128, 129) for cell lines or with TMT10plex-126 and -127N for primary NAT and tissue samples, respectively. The four replicates of each cell line, and half of the respective NAT and tumor MAPs from each subject, were multiplexed and analyzed by LC-MS/MS. The median labeling efficiencies were 72.4% or 87.8% for cell lines and tissue samples, respectively. The lower efficiency of labeling in cell lines was ascribed to meager MAP yields. 5281 and 27 583 unique MAPs were identified in the cell line and tissue datasets, respectively, with a mean of 1433 unique MAPs per cell line and 5855 per tissue (
When taking the cell line and tissue samples together, a total of 30485 unique MAPs were identified. Within the MAP repertoire of each sample, 32-68% of the peptides are sample-specific, and even in comparing only cell line or primary samples, there are very few shared MAPs (
To contextualize these comparisons, the HLA alleles of the samples can again be considered. Out of the 2079 MAPs shared by S1 and S6, 1542 MAPs are predicted to bind the same allele in both samples, and that allele is HLA-B*07:02 in over 93% of these cases. Similarly, 136 out of 152 MAPs shared by COLO205 and SW620 are bound by the same allele, and that allele is HLA-A*02:01 in 113 of these cases. Thus, the MHC I immunopeptidomes of the samples is mainly influenced by the HLA repertoire.
At the gene level, peptides derived from over 8000 unique source genes were identified, with an average of 1014 and 3168 source genes per cell line and tissue sample, respectively (
To obtain an overview of the genomic origin of the MHC I immunopeptidome and investigate the shared nature of source genes, GO term analysis was performed on all the source genes identified in the cell lines as well as those identified in 4 or more tissues. Several common features between cell lines and tissues are detectable at the immunopeptidome level, including a significant enrichment of genes involved in RNA metabolism, ribonucleoprotein complex biogenesis, translation, and cellular responses to stress (
To investigate what proportion of MAPs from the tissue samples were from non-coding transcripts, it was first determined, for each peptide, the most abundant putative source transcript (Ensembl Annotation 99). For peptides from the cancer-specific database, the MCS were mapped onto the genome, and determined the most expressed transcript at that location (see ‘Quantification of MAP coding sequences in RNA-Seq data’ in Methods section). It was thus determined that on average, 95.3% of the MAPs from tissue samples were from protein coding transcripts (i.e. UTR or CDR) (
Following the identification of over 30,000 unique MAPs, peptide coding sequences were filtered to select those overexpressed at least 10-fold in cancer and expressed <2rphm in pooled mTEC samples or matched NAT, for cell lines and primary samples, respectively. A recent immunopeptidomic study in acute myeloid leukemia (AML) demonstrated that MCSs with RPHM <8.55 have less than 5% probability to generate MAPs (18). The MAP coding sequences in RNA-data were then quantified, and only those that were expressed less than 8.55rphm in mTECs and other normal tissues (GTEx) were kept. Following manual validation of the remaining peptides, peptides were classified as aberrantly expressed TSAs (aeTSAs) if they were overexpressed at least 10-fold in tumor and were expressed <0.2kphm in mTECs (and NAT in the case of tissues). MAPs were classified as TAAs if they were also overexpressed at least 10-fold in cancer but their expression in mTEC and/or NAT was greater than 0.2kphm.
While the TSA identification in CRC-derived cell lines was relatively meager, possibly due in part to the low MAP identification, an average of 3 TSAs was identified per primary tissue sample (
While the primary objective was to identify putative TSAs in CRC, an average of 6.33 TAAs were also identified in the CRC tissue samples, though none were identified in the CRC-derived cell lines (
It was initially expected to identify an above average number of both TSAs and TAAs in MSI tissues. This was the case in S5, however the same was not true for the other MSI tissue (
To determine if any of the putative TSAs or TAAs have been previously identified, it was verified if the peptide sequences were reported in the Immune Epitope Database (IEDB), the HLA Ligand Atlas (64), and 2 previous publications that sought to identify tumor antigens in CRC from Löffler et al. 2018 (8) and Newey et al. 2019 (15). Of note, none of the putative aeTSAs, mTSAs, or non-canonical TAAs have been previously reported in any of these resources. Of the 26 putative canonical TAAs identified, 24 of them were reported either in IEDB, Löffler et al 2018 (PXD009602), Newey et al 2019 (PXD014017), or some combination of the three (
1peptides identified in more than one sample may have multiple predicted alleles
2not identified as tumor antigens
Following the identification of putative TSAs and TAAs, the validation of all the TSAs and a subset of 11 TAAs, which were selected based on favorable initial TMT intensity ratios and precursor ion fractions in cancer vs. matched NAT was performed, prior to validation with synthetic peptides. First, the expression, in TPM, of the source transcripts in their respective tumor samples compared to the matched NAT, as well as the mean average of that transcript in the CRC/NAT sample, were studied (
To evaluate the specificity of the putative tumor antigens, the mean expression of the peptide-coding sequences in the large dataset of healthy tissues provided by the Genotype-Tissue Expression project (GTEx) was determined (
The putative TSAs and TAAs were validated by MS with their corresponding synthetic peptides. These TAAs were selected based on favorable initial TMT intensity ratios and precursor ion fractions in cancer vs matched NAT. These candidates all had MS/MS that correlated well with those of the synthetic peptides, with Pearson correlation score ≥0.6. Synthetic peptides were labelled with TMT10plex-129N, 130N, and 131 at concentrations of 10, 100, and 1000 fmol, respectively, and spiked into remaining purified MAPs from tissue samples that were labeled with TMT126 (NAT) and 127N (CRC). SPS-MS3 was then used to quantify peptides of interest in these samples. Despite the decreased sensitivity of SPS-MS3, it was possible to quantify seven TSAs and seven TAAs. Good quality PSMs were selected for quantification, and all were more abundant in their respective CRC compared to NAT (Table 8). Determining the ratio of intensity of TMT127N peptides compared to TMT126 peptides revealed that TSAs had a median intensity fold change of 16.96 in CRC compared to NAT, while TAAs had a fold change of 6.93. In addition, the TSA with sequence RYLEKFYGL was also overexpressed in the S1 tumor, despite only passing the transcriptomic thresholds for S6. Thus, it was possible to demonstrate that the TSA identification methodology used in this study successfully identified TSA and TAA sequences that are more highly abundant at the surface of cancer cells than that of NAT. TSAs and TAAs candidates that were validated with synthetic peptides are listed in Tables 9A-9B.
Tables 9A and 9B depict the TSAs and TAAs candidates that were validated with synthetic peptides.
To examine the intertumoral distribution of these TSAs and TAAs in other CRC tumors, the log (RPHM+1) expression of the peptide coding sequences in 151 colon adenocarcinoma samples from The Cancer Genome Atlas (TCGA) (
Another important consideration in the identification of tumor antigens is whether these peptides are able to invoke an effective anti-tumor immune response. Repitope predictions of immunogenicity revealed that the aeTSAs are predicted to be significantly more immunogenic than a set of thymic peptides which are presumed non-immunogenic (67) (
Finally, an approximation of the proportion of individuals who possess the alleles that are predicted to bind and present the tumor antigens was sought (
Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the word “comprising” is used as an open-ended term, substantially equivalent to the phrase “including, but not limited to”. The singular forms “a”, “an” and “the” include corresponding plural references unless the context clearly dictates otherwise.
The present application claims the benefits of U.S. provisional patent application No. 63/261,315 filed on Sep. 17, 2021, which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2022/051377 | 9/16/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63261315 | Sep 2021 | US |
