WO2022187963A1 - Agents immunothérapeutiques ciblant brachyury et leurs procédés d'utilisation - Google Patents

Agents immunothérapeutiques ciblant brachyury et leurs procédés d'utilisation Download PDF

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WO2022187963A1
WO2022187963A1 PCT/CA2022/050354 CA2022050354W WO2022187963A1 WO 2022187963 A1 WO2022187963 A1 WO 2022187963A1 CA 2022050354 W CA2022050354 W CA 2022050354W WO 2022187963 A1 WO2022187963 A1 WO 2022187963A1
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seq
targeting agent
hla
antigen targeting
antigen
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Marco A. Marra
Shahrad RASSEKH
Steven Jones
Daniela DI FRANCESCO
Laura Williamson
Robert Holt
Craig M. RIVE
Emma TITMUSS
Janessa LASKIN
Rebecca J. DEYELL
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Provincial Health Services Authority
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464452—Transcription factors, e.g. SOX or c-MYC
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/555—Interferons [IFN]
    • G01N2333/57—IFN-gamma

Definitions

  • Some embodiments relate to peptides, proteins, nucleic acids, cells and/or compositions for use in cancer immunotherapy. Some embodiments relate to methods of using such molecules to assist in selection of a therapy for cancer. Some embodiments relate to methods of using immunotherapy agents to treat, control and/or diagnose cancer.
  • Chordomas are malignant neoplasms derived from notochord cells typically originating in the spine, sacrum or base of the skull, affecting both adults and children. Poorly differentiated chordomas (PDCs) are a recently recognized distinct subgroup of chordoma, which occurs predominantly in children, and is characterized by the expression of brachyury, and by loss of the SWI/SNF chromatin remodelling factor subunit, IN11 , encoded by the SMARCB1 gene 1-5 .
  • Brachyury encoded by the TBXT gene, is an embryonic transcription factor and tumour antigen that is expressed in 90% of chordomas 6 , and in a subset of other tumour types including but not limited to breast, prostate, lung, colorectal and oral squamous cancer 7 . These aggressive tumours are associated with a high risk of rapid local recurrence and distant metastasis and are generally poor candidates for primary resection, highlighting the need for effective systemic therapy.
  • MHC The major histocompatibility complex
  • MHC comprises cell surface proteins essential for the adaptive immune system.
  • MHC class I proteins are expressed in all nucleated cells except red blood cells.
  • MHC class I proteins function to mediate cellular immunity, e.g. to flag tumour cells, infected cells, or damaged cells for destruction.
  • MHC Class I proteins present either self, mutated, or pathogenic peptides to the adaptive immune system, allowing the cell to communicate its condition to the immune system, which can react accordingly .
  • the peptides often result from proteolytic cleavage of mainly endogenous, cytosolic or nuclear proteins, defective ribosomal products, and larger peptides expressed by the cell.
  • T-cell receptor TCR
  • TCR T-cell receptor
  • HLA human leukocyte-antigens
  • subgroups e.g. HLA-A, HLA-B, and HLA-C.
  • HLA alleles are variable in their primary structure. There are thousands of different HLA alleles that are known, and the frequency of alleles within each serotype varies among racial populations. Despite the diversity of HLA alleles across global populations, there is some consistency in the HLA binding groove pockets that hold the antigens. It is believed that the third complementarity determining region (CDR3) of the T-cell receptor is the primary CDR responsible for recognizing processed antigen.
  • CDR3 complementarity determining region
  • One aspect provides antigen targeting agents having an antigen binding site that binds to a brachyury-derived peptide when the brachyury-derived peptide is presented by an MHC protein.
  • the antigen binding site can have first and second chains each having first and second complementarity determining regions (CDRs) that bind to the MHC protein, and the first chain can have a third CDR having the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 13, and the second chain can have a third CDR having the amino acid sequence of SEQ ID NO:16.
  • the first CDR of the first chain can have the sequence of either one of SEQ ID NO:8 or SEQ ID NO:11.
  • the second CDR of the first chain can have the amino acid sequence of either one of SEQ ID NO:9 or SEQ ID NO:12.
  • the first CDR of the second chain can have the amino acid sequence of SEQ ID NO: 14.
  • the second CDR of the second chain can have the amino acid sequence of SEQ ID NO: 15.
  • the first and second chains can be alpha and beta chains of a T-cell receptor.
  • the first and second chains can be gamma and delta chains of a T-cell receptor.
  • the constant regions of the TCR can be murine constant regions and/or can be modified in any manner so as to limit pairing of the engineered TCR chains with endogenous TCR chains.
  • the first and second chains can be provided as part of a chimeric antigen receptor (CAR) construct.
  • the first and second chains can be provided as part of a bi-specific antibody.
  • the MHC protein can be HLA- A*23:01 , HLA-A*31 :01 , HLA-B*40:01 , HLA-B*44:03, HLA-C*03:04, or HLA-C*04:01.
  • the brachyury-derived peptide can have the amino acid sequence RFKELTNEM (SEQ ID NO:20).
  • tumour associated antigen that can be targeted by diagnostic and/or therapeutic agents for cancers involving expression or overexpression of brachyury.
  • the tumour associated antigen can be the brachyury-derived peptide RFKELTNEM (SEQ ID NO:20) presented by HLA-C*04.01.
  • the tumour associated antigen can be used to develop antigen targeting agents.
  • nucleic acids encoding polypeptides having any of the sequences set forth above.
  • One aspect provides recombinant vectors containing such nucleic acids.
  • One aspect provides cells or populations of cells containing such nucleic acids.
  • One aspect provides pharmaceutical compositions comprising antigen targeting agents, nucleic acids, cells or populations of cells as described herein.
  • Antigen targeting agents as described herein can be used in the diagnosis and/or treatment of cancers involving expression or overexpression of brachyury.
  • Antigen targeting agents as described herein can be used in the diagnosis and/or treatment of cancers involving expression or overexpression of brachyury.
  • Figures 1A and 1 B show SMARCBI loss and TBXT expression in pediatric chordoma.
  • Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G and 2H show characterization of clinical patient- derived T cell receptor activation in a patient with TBXT-expressing (i.e. brachyury expressing), poorly differentiated pediatric chordoma.
  • Figures 3A, 3B, 3C, 3D and 3E show representative lentiviral transduction efficacy, CD137 expression and cell killing flow cytometry assays.
  • Figure 4 shows the nucleotide sequence of BTCR 1 (SEQ ID NO:4).
  • Figure 5 shows the amino acid sequence of BTCR 1 (SEQ ID NO:5).
  • Figure 6 shows the nucleotide sequence of BTCR 2 (SEQ ID NO:6).
  • Figure 7 shows the amino acid sequence of BTCR 2 (SEQ ID NO:7).
  • CD8 + T-cells and “TCD8 + ” refer to CD8-positive T-cells.
  • CD8-positive T-cells are able recognize and destroy cells flagged by MHC class I proteins and this ability is known as MHC class l-restriction.
  • CD8-positive T-cells include cytotoxic T- cells (CTLs).
  • CTLs cytotoxic T- cells
  • CD4 + T-cells refers to CD4-positive T-cells.
  • an antigen refers to molecules that can induce an immune response.
  • an antigen may be one that is recognisable by cytotoxic T-cells to stimulate an anti-tumour immune response.
  • epitope refers to the part of an antigen that can stimulate an immune response.
  • an epitope may be a peptide that is bound to a MHC class I protein to thereby form a MHC/peptide complex.
  • the MHC/peptide complex can be selectively recognized by a suitable T-cell receptor of a cytotoxic T-cell to stimulate an anti tumour immune response.
  • DNA refers to deoxyribonucleic acid.
  • the information stored in DNA is coded as a sequence made up generally of four chemical bases: adenine (A), guanine (G), cytosine (C) and thymine (T).
  • A adenine
  • G guanine
  • C cytosine
  • T thymine
  • Other bases and chemically modified bases exist as well and are encompassed within certain embodiments.
  • reference to a DNA sequence includes both single and double stranded DNA.
  • a specific sequence refers to (i) a single stranded DNA of such sequence, (ii) a double stranded DNA comprising a single stranded DNA of such sequence and its complement, and (iii) the complement of such sequence.
  • fragment means a portion of a larger whole.
  • a fragment means a portion of the DNA sequence that is less than the complete coding region.
  • the expression product of the fragment may retain substantially the same biological function as the expression product of the complete coding sequence.
  • peptide means a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carbonyl groups of the adjacent amino acid.
  • the peptide can contain naturally occurring, synthetic and/or modified amino acid residues.
  • a peptide may be immunogenic, meaning that the peptide is capable of inducing an immune response, e.g. a T-cell response.
  • isolated means that a material is separated/removed from its original environment.
  • the term “purified” does not mean absolute purity. Instead, it can include preparations that undergo a purification process, e.g. highly purified preparations and partially purified preparations having a purity of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% pure.
  • T-cell response means the proliferation and activation of effector T-cells.
  • T-cell response of MHC class I restricted cytotoxic T-cells may include lysis of target cells, secretion of cytokines, and secretion of effector molecules (e.g. perforins and granzymes).
  • variant means in the context of proteins, one or two or more of the amino acid residues are replaced with other amino acid residues, while the variant retains substantially the same biological function as the unaltered protein.
  • Desired clinical results can include, but are not limited to, reduction or alleviation of at least one symptom of a disease.
  • treatment can be diminishment of at least one symptom of disease, diminishment of extent of disease, stabilization of disease state, prevention of spread of disease, delay or slowing of disease progression, palliation of disease, diminishment of disease reoccurrence, remission of disease, prolonging survival with disease, or complete eradication of disease.
  • cancer cell and “tumor cell” refer to cells, the growth and division of which can be typically characterized as unregulated. Cancer cells can be of any origin, including benign and malignant cancers, metastatic and non-metastatic cancers, and primary and secondary cancers.
  • tumour-infiltrating CD8+ T cells that target cells expressing brachyury and confirmed a brachyury tumour antigen directed immune response in T cells isolated from a patient suffering from poorly differentiated chordoma.
  • the T-cells may similarly be useful in the treatment of patients suffering from other types of cancer involving expression of brachyury.
  • the tumour antigen is the brachyury-derived peptide RFKELTNEM (SEQ ID NO:20).
  • the tumour antigen is presented by an MHC protein.
  • the tumour antigen is the brachyury-derived peptide RFKELTNEM (SEQ ID NO:20) presented by HLA-C*04:01.
  • antigen targeting agents that target the tumour antigen can be used as diagnostic and/or therapeutic agents for cancers that involve expression of brachyury.
  • Some embodiments of the present invention provide a composition and method to treat a patient with cancer with an engineered antigen targeting agent that recognizes brachyury.
  • the brachyury is H LA-restricted brachyury.
  • the brachyury is presented by the HLA.
  • the brachyury is presented by HLA-C*04:01.
  • the brachyury-derived peptide RFKELTNEM (SEQ ID NO:20) is presented by HLA-C*04:01.
  • the invention provides an engineered antigen-binding receptor comprising an antigen binding site configured to specifically bind to brachyury.
  • the invention provides an engineered T cell receptor (TCR) comprising an antigen binding site configured to specifically bind to brachyury.
  • TCR T cell receptor
  • the brachyury is H LA-restricted brachyury.
  • the brachyury is presented by HLA-C*04:01.
  • the brachyury-derived peptide RFKELTNEM (SEQ ID NO:20) is presented by HLA-C*04:01.
  • the engineered antigen-binding receptor comprises an antigen binding site having the amino acid sequences TCR1 (TRA-1 CAVQSNYKLSF (SEQ ID NO:1); and TRB CSASTGRGTEAFF (SEQ ID NO:3)) or TCR2 (TRA-2 CAASNPQAGTALIF (SEQ ID NO:2); and TRB CSASTGRGTEAFF (SEQ ID NO:3)).
  • mouse constant TCR alpha and beta chains are incorporated into the TCRs of the present invention to limit mispairing of the TCR variable alpha and beta chains with endogenous T cell alpha and beta chains.
  • the invention further provides related nucleic acids, recombinant vectors, host cells, populations of cells and pharmaceutical compositions relating to the TCRs, polypeptides and proteins of the invention.
  • HLA typing Methods of identification of patients responsive to treatment by some embodiments of the present invention based on tumour brachyury screening, HLA typing or other methods of patient screening are also provided by some embodiments of the invention.
  • patients are screened for the presence of the following HLA types: HLA-A*23:01 , HLA-B*40:01 , HLA-C*03:04; HLA-A*31 :01 , HLA- B*44:03, or HLA-C*04:01.
  • Methods of detecting the presence of cancer in a mammal and methods of treating or preventing cancer in a mammal are further provided by some embodiments of the invention.
  • Some embodiments of the present invention relate to tumour-associated antigens. Based on the Examples presented herein, the inventors have determined that brachyury- derived peptides presented by MHC proteins provide a tumour-associated antigen that can be targeted in vivo in cancers involving the expression or overexpression of brachyury.
  • cancers include poorly differentiated chordoma, breast cancer, lung cancer, prostate cancer, colorectal cancer and oral squamous cancer, as well as testicular germ cell tumors, lung adenocarcinoma, pediatric Wilms Tumour, pediatric clear cell sarcoma of the kidney, sarcoma, thyroid carcinoma, uterine corpus endometrial carcinoma, colon adenocarcinoma, rectum adenocarcinoma, lymphoid neoplasm diffuse large B-cell lymphoma, stomach adenocarcinoma, lung squamous cell carcinoma, breast invasive carcinoma (including ER-/HER2- and ER+/HER2-), glioblastoma multiforme, esophageal carcinoma, bladder urothelial carcinoma, acute myeloid leukemia, pancreatic adenocarcinoma, ovarian serous cystadenocarcinoma, cervical squamous cell carcinoma, endocervical a
  • the tumour associated-antigen is the brachyury-derived peptide having the amino acid sequence RFKELTNEM (SEQ ID NO:20).
  • the MHC protein that presents the brachyury peptide is HLA- A*23:01 , A*31 :01 , B*40:01 , B*44:03, C*03:04, or C*04:01.
  • the MHC protein that presents the brachyury peptide is HLA-C*04:01.
  • the tumour- associated antigen is the brachyury-derived peptide RFKELTNEM (SEQ ID NO:20) as displayed at the surface of a tumour cell by HLA-C*04:01.
  • a tumour antigen peptide having the amino acid sequence RFKELTNEM (SEQ ID NO:20), which binds to HLA-C*04:01 and is recognized by cytotoxic cells is provided.
  • tumour-associated antigens described herein can be targeted by various antigen targeting agents as described herein, which can be used as diagnostic and/or therapeutic agents for cancers that involve expression of brachyury, and/or to develop new antigen targeting agents that target such tumour-associated antigens.
  • antigen targeting agents including antigen targeting receptors. These antigen targeting agents are configured to target brachyury. In some embodiments, these antigen targeting agents are configured to target a brachyury peptide-MHC complex.
  • the MHC is HLA- A*23:01 , HLA-A*31 :01 , HLA-B*40:01, HLA-B*44:03, HLA-C*03:04, or HLA-C*04:01.
  • the brachyury peptide has the sequence RFKELTNEM (SEQ ID NO:20). In some embodiments, the MHC is HLA-C*04:01.
  • the antigen targeting agents target cytotoxic cells to tumour cells.
  • the antigen targeting agent is a T-cell receptor (TCR) that targets a T-cell incorporating the construct to tumour cells expressing brachyury.
  • the antigen targeting agent is a chimeric antigen receptor (CAR) that targets a cytotoxic cell such as a T-cell to tumour cells expressing brachyury.
  • the antigen targeting agent is an agent such as an antibody that targets the tumour-associated antigens disclosed herein.
  • the antigen targeting agent is a bi-specific antibody that has a first antigen-binding domain that binds to a brachyury antigen to target the agent to tumour cells and a second antigen-binding domain that targets cytotoxic cells, for example that binds to CD3 to target T-cells to the tumour cells.
  • the tumour cell expressing brachyury is targeted by the antigen targeting agent via the cell-surface display on tumour cells of brachyury peptides by MHC proteins.
  • the MHC is HLA- A*23:01 , HLA-A*31 :01 , HLA- B*40:01 , HLA-B*44:03, HLA-C*03:04, or HLA-C*04:01.
  • the MHC proteins are HLA-C*04:01.
  • Any type of immunotherapy agent that can be used to target cytotoxic cells to tumour cells can be used in various embodiments.
  • bispecific antibodies that bind to both a brachyury antigen and a factor such as CD3 that can be used to target cytotoxic cells such as T-cells to the tumour cells bound by the bispecific antibody can be used.
  • an antigen targeting receptor that can be used to conduct cellular immunotherapy can be used.
  • the antigen targeting receptor is a T-cell receptor (TCR).
  • the antigen targeting receptor is a chimeric antigen receptor (CAR).
  • the antigen targeting receptor is a modified form of TCR-CAR construct with a single chain antigen-binding domain of a TCR fused to the signaling domain of a CAR molecule.
  • the antigen targeting agent is a TCR.
  • the TCR has (i) a first chain having first, second and third complementarity-determining regions (CDR1 , CDR2, and CDR3) and (ii) a second chain having first, second and third complementarity determining regions (CDR1 , CDR2, and CDR3).
  • the first and second chains of the TCR are the alpha chain and beta chain, respectively, of a TCR.
  • the first and second chains of the TCR are the gamma chain and delta chain, respectively, of a TCR.
  • CDR3 third complementarity determining regions
  • CDR1 and CDR2 are believed to play a role in binding to the MHC Class I backbone.
  • each of the first and second chains of the synthetic TCRs has one or more of the following domains: a hinge domain, a transmembrane domain, and an intracellular T-cell signalling domain.
  • the intracellular domains of the TCR do not signal directly, but rather form complexes with other molecules such as CD3 subunits that facilitate signalling.
  • the antigen targeting agent is a T-cell receptor
  • the antigen targeting agent is expressed from a nucleotide construct capable of expressing both chains of the TCR as a single polypeptide.
  • the single polypeptide has a linker peptide linking the first and second chains of the T-cell receptor. The linker peptide may facilitate the expression of a recombinant TCR in a host cell.
  • the single polypeptide incorporating both the first and second chains of the synthetic TCR includes a cleavage sequence interposed between the first and second chains of the TCR, so that the first and second chains will be expressed as a single polypeptide and then cleaved into two separate polypeptides in vivo.
  • the nucleic acid encoding the polypeptide that forms the TCR includes a skipping sequence or a sequence allowing initiation of translation at a site other than the 5’ end of an mRNA molecule, or any other sequence that allows two distinct polypeptides to be translated from a single mRNA, interposed between the nucleic acid encoding the first and second chains of the TCR. Any suitable sequence may be used for this purpose between the first and second chains of the TCR, for example a T2A, P2A, E2A, F2A, or IRES sequence, or the like.
  • first and second chains of the synthetic TCRs in the polynucleotide sequence encoding the TCR and in the resulting polypeptide is interchangeable (i.e in some embodiments, the first chain is provided at the 5’ end of the polynucleotide sequence/the N- terminal direction of the polypeptide, while in other embodiments the second chain is provided at the 5’ end of the polynucleotide sequence/the C-terminal direction of the polypeptide).
  • variable domains of the a chain (V a ) and the b chain (V ) comprise any pairwise combination of the variable regions and/or the CDRs having the amino acid sequences of SEQ ID NOs: 5 and 7 (BTCR-1 and BTCR-2), as shown in Figures 5 and 7.
  • the constant domains of the first and second chains e.g. the alpha chain (C a ) and the beta chain (C ) comprise human constant gene segments.
  • human constant gene segments are replaced with constant gene segments from a different organism, e.g. with murine constant gene segments.
  • An advantage of such replacement is to limit mispairing of the engineered TCR chains, e.g. alpha and beta chains, with the T cell's endogenous T-cell receptor chains, e.g. alpha and beta chains.
  • the constant domains of the first and second chains are further modified in any suitable manner to enhance and/or regulate the interaction therebetween.
  • residues of the transmembrane domains of each of the first and second chains that are positioned adjacent to one another in vivo may be changed to cysteine residues, to encourage the formation of additional disulfide bonds between the engineered first and second chains (while such disulfide bonds would not form with endogenous T-cell receptor chains).
  • the synthetic TCRs are provided with any other suitable protein domain that supports dimerization of the two chains, for example a leucine zipper domain.
  • the first chain and second chains of the TCR have first and second CDRs that bind to an MHC protein, and the first chain has a third CDR having the amino acid sequence of either one of SEQ ID NO:10 or SEQ ID NO:13 (i.e. the CDR3 of BTCR1 or BTCR2).
  • the second chain of the TCR has a third CDR having the amino acid sequence of SEQ ID NO:16 (i.e. CDR3 of BTCR1 and BTCR2).
  • the antigen presenting cell that is bound by the TCR has an HLA genotype including at least one of the following HLA alleles: HLA-A * 23:01 , HLA-B * 40:01 , HLA-C * 03:04 ⁇ HLA-A * 31:01, HLA-B * 44:03, HLA-C * 04:01.
  • the first chain of the TCR contains: a first CDR having the amino acid sequence of either one of SEQ ID NO:8 or SEQ ID NO:11 (i.e. CDR1 of BTCR1 or BTCR2), a second CDR having the amino acid sequence of either one of SEQ ID NO:9 or SEQ ID NO:12 (i.e. CDR2 of BTCR1 or BTCR2), and a third CDR having the amino acid sequence of either one of SEQ ID NO:10 or SEQ ID NO:13 (i.e. CDR3 of BTCR1 or BTCR2).
  • the second chain of the TCR contains: a first CDR having the amino acid sequence of SEQ ID NO:14 (i.e. CDR1 of BTCR1 or BTCR2), a second CDR having the amino acid sequence of SEQ ID NO:15 (i.e. CDR2 of BTCR1 or BTCR2), and a third CDR having the amino acid sequence of SEQ ID NO:16 (i.e. CDR3 of BTCR1 or BTCR2).
  • the first chain of the TCR contains: a first CDR having the amino acid sequence of SEQ ID NO:8 (i.e. CDR1 of BTCR1), a second CDR having the amino acid sequence of SEQ ID NO:9 (i.e. CDR2 of BTCR1), and a third CDR having the amino acid sequence of SEQ ID NO:10 (i.e. CDR3 of BTCR1).
  • the second chain of the TCR contains: a first CDR having the amino acid sequence of SEQ ID NO:14 (i.e. CDR1 of BTCR1), a second CDR having the amino acid sequence of SEQ ID NO: 15 (i.e. CDR2 of BTCR), and a third CDR having the amino acid sequence of SEQ ID NO:16 (i.e. CDR3 of BTCR).
  • the first chain of the TCR contains: a first CDR having the amino acid sequence of SEQ ID NO:11 (i.e. CDR1 of BTCR2), a second CDR having the amino acid sequence of SEQ ID NO:12 (i.e. CDR2 of BTCR2), and a third CDR having the amino acid sequence of SEQ ID NO:13 (i.e. CDR3 of BTCR2).
  • the second chain of the TCR contains: a first CDR having the amino acid sequence of SEQ ID NO:14 (i.e. CDR1 of BTCR2), a second CDR having the amino acid sequence of SEQ ID NO:15 (i.e. CDR2 of BTCR2), and a third CDR having the amino acid sequence of SEQ ID NO: 16 (i.e. CDR3 of BTCR2).
  • the first chain of the TCR has the amino acid sequence of TRAV20*01 (SEQ ID NO: 17) containing the first and second CDRs and a third CDR having the amino acid sequence of either one of SEQ ID NO: 10 or SEQ ID NO: 13 (i.e. CDR3 of BTCR1 or BTCR2).
  • the second chain of the TCR has the amino acid sequence of TRBV20*01 (SEQ ID NO:18) containing the first and second CDRs and a third CDR having the amino acid sequence of SEQ ID NO:16 (i.e. CDR3 of BTCR1).
  • the first chain of the TCR has the amino acid sequence of TRAV13-1*01 (SEQ ID NO:19) containing the first and second CDRs and a third CDR having the amino acid sequence of either one of SEQ ID NO: 10 or SEQ ID NO: 13 (i.e. CDR3 of BTCR1 or BTCR2).
  • the second chain of the TCR has the amino acid sequence of TRBV20*01 (SEQ ID NO: 18) containing the first and second CDRs and a third CDR having the amino acid sequence of SEQ ID NO:16 (i.e. CDR3 of BTCR2).
  • the first and second chains of the antigen targeting agent have an amino acid sequence with at least 90% sequence identity to either one of SEQ ID NO:5 or SEQ ID NO:7, while the first and second chains of the antigen targeting agent also have any of the first, second and third CDRs as described herein.
  • a nucleic acid encoding a TCR is provided.
  • the nucleic acid is DNA.
  • the nucleic acid has the nucleotide sequence of either one of SEQ ID NO:4 or SEQ ID NO:6 (i.e. BTCR1 or BTCR2).
  • the nucleic acid encodes a polypeptide having the amino acid sequence of either one of SEQ ID NO:5 or SEQ ID NO:7 (i.e. BTCR1 or BTCR2).
  • the nucleic acid encodes a TCR having a first chain having the amino acid sequence of either one of SEQ ID NO:10 or SEQ ID NO:13 (i.e. CDR3 of BTCR1 or BTCR2). In some such embodiments, the nucleic acid encodes a TCR having a second chain having the amino acid sequence of SEQ ID NO:16 (i.e. CDR3 of BTCR).
  • the nucleic acid encodes a polypeptide that is a first chain of a TCR having a first CDR having the amino acid sequence of either one of SEQ ID NO:8 or SEQ ID NO:11 (i.e. CDR1 of BTCR1 or BTCR2), a second CDR having the amino acid sequence of either one of SEQ ID NO:9 or SEQ ID NO:12 (i.e. CDR2 of BTCR1 or BTCR2), and a third CDR having the amino acid sequence of either one of SEQ ID NO:10 or SEQ ID NO: 13 (i.e. CDR3 of BTCR1 or BTCR2).
  • the nucleic acid encodes a polypeptide that is the second chain and containing a first CDR having the amino acid sequence of SEQ ID NO:14 (i.e. CDR1 of BTCR1 or BTCR2)], a second CDR having the amino acid sequence of SEQ ID NO:15 (i.e. CDR2 of BTCR1 or BTCR2), and a third CDR having the amino acid sequence of SEQ ID NO:16 (i.e. CDR3 of BTCR1 or BTCR2).
  • the nucleic acid encodes a polypeptide that is a first chain of a TCR having: a first CDR having the amino acid sequence of SEQ ID NO:8 (CDR1 of BTCR1), a second CDR having the amino acid sequence of SEQ ID NO:9 (CDR2 of BTCR1), and a third CDR having the amino acid sequence of SEQ ID NO:10 (CDR3 of BTCR1).
  • the nucleic acid encodes a polypeptide that is a second chain of the TCR having: a first CDR having the amino acid sequence of SEQ ID NO:14 (CDR1 of BTCR1), a second CDR having the amino acid sequence of SEQ ID NO:15 (CDR2 of BTCR1), and a third CDR having the amino acid sequence of SEQ ID NO: 16 (CDR3 of BTCR1).
  • the nucleic acid encodes a polypeptide that is a first chain of the TCR having: a first CDR having the amino acid sequence of SEQ ID NO:11 (CDR1 of BTCR2), a second CDR having the amino acid sequence of SEQ ID NO:12 (CDR2 of BTCR2), and a third CDR having the amino acid sequence of SEQ ID NO:13 (CDR3 of BTCR2).
  • the nucleic acid encodes a polypeptide that is a second chain of the TCR having: a first CDR having the amino acid sequence of SEQ ID NO: 14 (CDR1 of BTCR2), a second CDR having the amino acid sequence of SEQ ID NO:15 (CDR2 of BTCR2), and a third CDR having the amino acid sequence of SEQ ID NO: 16 (CDR3 of BTCR2).
  • the nucleic acid encodes a polypeptide that is a first chain of a TOR having the amino acid sequence of TRAV20*01 (SEQ ID NO: 17) containing first and second CDRs and a third CDR having the amino acid sequence of either one of SEQ ID NO: 10 or SEQ ID NO: 13 (CDR3 of BTCR1 or BTCR2).
  • the nucleic acid encodes a polypeptide that is a second chain of the TOR having the amino acid sequence of TRBV20*01 (SEQ ID NO:18) containing the first and second CDRs and a third CDR having the amino acid sequence of SEQ ID NO:16 (CDR3 of BTCR1).
  • the nucleic acid encodes a polypeptide that is a first chain of a TOR having the amino acid sequence of TRAV13-1*01 (SEQ ID NO:19) containing first and second CDRs and a third CDR having the amino acid sequence of either one of SEQ ID NO:10 or SEQ ID NO:13 (CDR3 of BTCR1 or BTCR2).
  • the nucleic acid encodes a polypeptide that is a second chain of the TCR having the amino acid sequence of TRBV20*01 (SEQ ID NO:18) containing the first and second CDRs and a third CDR having the amino acid sequence of SEQ ID NO:16 (CDR3 of BTCR2).
  • Suitable variations on such constructs can be made by those skilled in the art, for example the antigen-binding domains of a T-cell receptor can be inserted into a CAR construct in place of the typical scFv fragment together so that the single-chain antigen binding domain interacts with the signaling domain of the CAR construct to cause the desired cytotoxic activity towards cancer cells.
  • the antigen targeting agent is a chimeric antigen receptor (CAR).
  • the CAR is structured to provide a single-chain antigen binding domain equivalent to the TCR binding domain described above having the first and second chains (e.g. alpha and beta chains) of the TCR (each having three complementarity determining regions, which may be any of the complementarity determining regions described above for the TCR construct) joined together as a single polypeptide and linked together to a single hinge region, transmembrane domain and signalling domain, as well as a suitable co-stimulatory domain, (e.g. CD27, CD28, 4-1 BB, ICOS, 0X40, MYD88, IL1 R1 , CD70, or the like), as well as any other domains intended to enhance the characteristics of the CAR construct.
  • a suitable co-stimulatory domain e.g. CD27, CD28, 4-1 BB, ICOS, 0X40, MYD88, IL1 R1 , CD70,
  • the antigen targeting agent is an antibody, including as an example a bispecific antibody, wherein the bispecific antibody has a first antigen-binding domain that binds to a factor such as CD3 that can be used to recruit T-cells and a second antigen-binding domain that binds to a brachyury-derived peptide displayed by an antigen presenting cell.
  • the second domain of the bispecific antibody has as a single polypeptide the first and second chains (e.g. alpha and beta chains) of a TCR as described herein (each having three complementarity determining regions, which may be any of the complementarity determining regions described herein for the TCR construct) to provide the second antigen-binding domain.
  • the antigen targeting agent is an antibody
  • the antibody has an antigen-biding domain that binds to a brachyury-derived peptide displayed by an antigen presenting cell as described herein.
  • the antibody has an antigen binding site that contains any combination of the first, second and third CDRs for the first and second chains as described herein.
  • Some embodiments of the present invention relate to nucleic acids, recombinant vectors, host cells, populations of cells and pharmaceutical compositions relating to, incorporating or encoding the TCRs, polypeptides and proteins described above.
  • a recombinant vector for expressing an antigen targeting agent as described herein is provided.
  • the recombinant vector includes any one of the nucleic acid sequences described above.
  • such nucleic acids or recombinant vectors are incorporated into a host cell.
  • the host cells are amplified to produce a population of host cells that can be used as, or can be used to express, an antigen targeting agent as described herein.
  • pharmaceutical compositions comprising such cells are provided.
  • pharmaceutical compositions comprising recombinantly expressed antigen targeting agents as described herein are provided.
  • pharmaceutical compositions comprising populations of cells that act as antigen targeting agents are provided.
  • the antigen targeting agents described above such as TCRs or CARs, are introduced into cytotoxic cells in any suitable manner, to provide a cytotoxic cell that specifically targets and kills cells expressing brachyury.
  • cytotoxic cells examples include tumour infiltrating lymphocytes (TILs), including CD8 + T-cells, CD4 + T-cells, natural killer (NK) cells, and the like. Any cell that can be engineered to carry out cellular immunotherapy via an antigen targeting agent can be used in alternative embodiments.
  • TILs tumour infiltrating lymphocytes
  • NK natural killer cells
  • the antigen targeting construct can be introduced into the cytotoxic cell using any suitable technigue now known or later developed.
  • the antigen targeting construct is introduced into the cytotoxic cell using plasmid or RNA transfection, transduction by viral vectors, direct editing via programmable nucleases (e.g. CRISPR systems (clustered regularly interspaced short palindromic repeats), TALENs (transcription activator-like effector nucleases), zinc finger nucleases), and so on as known to those skilled in the art, whether such systems are currently known or developed in future.
  • the antigen targeting construct is introduced into the cytotoxic cell by transduction with a suitable a vector, e.g.
  • the antigen targeting construct is introduced into the cytotoxic cell using a transposon system or electroporation.
  • the desired antigen targeting receptor is used to generate engineered cytotoxic cells using autologous adoptive cell therapy. That is, the cytotoxic cells are harvested from a mammalian subject, genetically engineered to express the antigen targeting receptor, expanded ex vivo, and then the expanded cells are introduced back into the subject to treat the cancer associated with cells expressing brachyury.
  • the mammalian subject is a human.
  • the desired antigen targeting receptor is used to generate engineered cytotoxic cells using universal adoptive cell therapy using allogenic cells.
  • a bank of cells from an allogenic donor are genetically modified to express the desired antigen targeting receptor, such as a TCR or CAR as described herein.
  • the modified allogenic cells are then introduced into a patient to treat a cancer associated with cells expressing brachyury.
  • the patient can be a mammalian subject, e.g. a human.
  • the desired antigen targeting receptor is introduced into a mammalian subject, e.g. a human, using systemic gene therapy.
  • a replication incompetent viral vector containing a nucleotide sequence for expressing the antigen targeting receptor is directly infused into a patient to directly transduce T-cells in situ to treat a cancer associated with cells expressing brachyury.
  • the desired antigen targeting agent is provided as a suitable soluble immunotherapy agent, for example a bi specific antibody such as a bi-specific T-cell engager (BiTE®), that can be directly administered to a mammalian subject.
  • a bi specific antibody such as a bi-specific T-cell engager (BiTE®)
  • the portions of the first and second chains that form the antigen-binding region are combined together as a single polypeptide that targets tumour cells expressing brachyury (including via presentation of brachyury-derived peptides by HLA proteins of such tumour cells), and are expressed as a fusion protein together with a second antigen binding domain, e.g. an scFv that binds to T-cells e.g. via the CD3 receptor.
  • the resulting fusion protein is purified and administered to the subject in any suitable manner to direct cytotoxic T-cells to the tumour cells.
  • the cytotoxic cells are administered together with one or more additional suitable chemotherapeutic and/or immunotherapeutic agents.
  • Methods of administration of the cellular immunotherapy agents and immunotherapy agents described herein are known in the art, and may include, for example, intravenous or subcutaneous injection.
  • the likelihood that a mammalian subject will benefit from therapy using an antigen targeting agent described herein is assessed prior to commencing such therapy. For example, a sample from the subject is evaluated to determine if the subject may have potentially cancerous cells that express brachyury. For example, a sample of a tumour from the patient may be subjected to RNA sequencing, immunohistochemistry or other appropriate analytical techniques to determine the presence of brachyury expression.
  • the mammalian subject is also subjected to HLA typing, to determine if the subject has an HLA genotype including at least one of the following HLA alleles: HLA-A * 23:01, HLA-B * 40:01, HLA-C * 03:04 ⁇ HLA-A * 31:01, HLA- B * 44:03, or HLA-C * 04:01 and/or which HLA alleles the subject has.
  • the subject has both potentially cancerous cells that are expressing brachyury and at least one of an HLA- A * 23:01, HLA-B * 40:01 , HLA-C * 03:04 ⁇ HLA-A * 31:01, HLA-B * 44:03, or HLA-C * 04: 01 allele, then the subject is a potential candidate for immunotherapy using the antigen targeting agents described herein.
  • the subject has at least one of an HLA-A * 23:01 , HLA- B * 40:01, HLA-C * 03:04 ⁇ HLA-A * 31:01, HLA-B * 44:03, or HLA-C * 04: 01 allele.
  • the subject has at least one of an HLA-A * 31 :01 or HLA-C * 04:01 allele.
  • the subject has an HLA-C * 04:01 allele.
  • the HLA-C * 04:01 allele is a relatively common HLA allele across many different ethnic groups 8 10 .
  • engineered TCRs as described herein are incorporated into CD8+ T cells.
  • the T-cell receptor recognizes and binds to brachyury antigens presented by tumour cells expressing brachyury
  • the CD8+ T cells are activated and can bind to the tumour cells and initiate a cytotoxic response to kill the tumour cells, e.g. through lysis of the tumour cells, secretion of cytokines, and/or secretion of effector molecules (e.g. perforins and granzymes).
  • the T-cell receptors are synthesized and reconstituted in CD8+ T cells using lentiviral transduction.
  • the lentiviral transduction uses a nucleotide vector encoding a receptor comprising an antigen binding domain capable of binding to brachyury antigens presented by a MHC protein.
  • the nucleotide vector includes nucleotides having a DNA sequence of any one of SEQ ID NOs:4 or 6 (BTCR1 or BTCR2) or any of the nucleotide sequences set forth above.
  • immune cells capable of binding to brachyury antigens and initiating a cytotoxic response are made.
  • Such cells are made by first isolating the immune cells from a source of cells and genetically engineering the immune cells to express a receptor comprising an antigen binding domain capable of binding to brachyury antigens as displayed at the cell surface by MHC proteins.
  • the genetic engineering can be carried out using a lentiviral vector.
  • the engineered immune cells can be introduced into the body of a patient suffering from cancer or another disorder involving expression of brachyury to treat the cancer or the disorder.
  • the patient has an HLA genotype including at least one of the following HLA alleles: HLA-A * 23:01 , HLA- B * 40:01, HLA-C * 03:04 ⁇ HLA-A * 31:01, HLA-B * 44:03, or HLA-C * 04:01.
  • the engineered CD8+ T cells may be used to treat a patient with cancer and/or to screen for cancer. Focusing on an example illustrating the treatment aspect, because brachyury expression is a feature of poorly differentiated chordoma (PDC) and other types of cancer including breast cancer, lung cancer, prostate cancer, colorectal cancer, oral squamous cancer, testicular germ cell tumors, lung adenocarcinoma, pediatric Wilms Tumour, pediatric clear cell sarcoma of the kidney, sarcoma, thyroid carcinoma, uterine corpus endometrial carcinoma, colon adenocarcinoma, rectum adenocarcinoma, lymphoid neoplasm diffuse large B-cell lymphoma, stomach adenocarcinoma, lung squamous cell carcinoma, breast invasive carcinoma (including ER-/HER2- and ER+/HER2-), glioblastoma multiforme, esophageal carcinoma, bladder urotheli
  • the antigen targeting agents targeting brachyury antigens are used in a patient having an HLA genotype including at least one of an HLA-A * 23:01 , HLA- B * 40:01, HLA-C * 03:04 ⁇ HLA-A * 31:01, HLA-B * 44:03, or HLA-C * 04:01 subtype in a method for treating or preventing cancer.
  • the method may be chimeric antigen receptor (CAR) T-cell therapy, T-cell receptor (TCR) T-cell therapy, or immunotherapy using an antibody such as a BITE® bi-specific antibody, using an antigen targeting agent as described herein.
  • methods of identification of patients likely to be responsive to treatment by the present invention based on tumour screening for expression of brachyury, HLA typing or other methods of patient screening are also provided.
  • the antigen targeting agents targeting brachyury antigens displayed at the cell surface by MHC proteins are used to detect the presence of tumour cells in a sample such as a patient biopsy.
  • detection is made by conducting an assay to evaluate the ability of cytotoxic cells expressing the antigen targeting receptor to kill tumour cells in a tumour cell culture derived from the sample, or by evaluating the expression of molecules that indicate activation of cytotoxic cells, such as interferon-gamma, when such cells are co-cultured with tumour cells (e.g. using ELISpot).
  • the antigen targeting agents targeting brachyury antigens are used to detect the presence of tumour cells in a sample such as blood, for example by detecting such antigens displayed on episomes, i.e. membrane fragments that have been shown to be present in blood.
  • an in vitro assay using the synthetic TCRs for example using the TCR as a labelled soluble reagent or expressed in a cell with a reporter system as described below can detect the presence of such antigens displayed on episomes.
  • the engineered antigen targeting receptors are used for detecting the presence of cancer in a mammal.
  • the engineered antigen targeting receptors may be brought into contact with a sample having one or more cells or episomes. If the cells express brachyury antigens that are displayed by MHC proteins, the engineered antigen targeting receptors will bind to the brachyury antigens and thereby form a complex. The detection of the complex is indicative of the presence of potentially cancerous or pre-cancerous cells.
  • the detection of the complex may take place through any number of ways known in the art.
  • the engineered antigen targeting agents and/or their related polypeptides, proteins, nucleic acids, recombinant expression vectors, or engineered cells
  • a detectable and/or visual label e.g. a radioisotope or a fluorophore.
  • the engineered antigen targeting receptors are reconstituted in immortalized T-cell lines (e.g. Jurkat cells) to support in vitro high throughput screening assays, for example for use in research and development and/or drug discovery.
  • the antigen targeting receptors are reconstituted in a soluble tetrameric form of an ab TCR, i.e. a TCR multimer, and used diagnostically, e.g. to visualize cells exposed to infectious agents or cellular transformation and/or therapeutically, e.g. for the delivery of drugs to compromised cells, for example as described by Low et al. 11 .
  • the engineered antigen targeting receptors are reconstituted in reporter cells derived from the T cell lymphoma line Jurkat as reported by Rydzek et al. 12 .
  • MRI and subsequent PET imaging of patient 1 revealed a fluorodeoxyglucose (FDG)-avid soft tissue mass of the neck, with bone erosion of C1 and C2.
  • FDG fluorodeoxyglucose
  • a differential diagnosis of I NI1 -negative spindle cell neoplasm and epithelioid sarcoma was proposed, however a diagnosis of poorly differentiated chordoma was favoured following positive brachyury staining.
  • Imaging of patient 2 demonstrated an FDG-avid, unresectable, multi-lobulated expansile soft tissue mass centered at the left anterior arch and lateral mass of the C1 vertebral body, involving the clivus, occipital condyle and dens and two lung nodules, later confirmed to be metastatic disease.
  • a transoral needle biopsy of the C1 mass demonstrated an epithelioid malignancy, which was positive for AE1/AE3 pan-cytokeratin and EMA.
  • Tumour cells showed loss of nuclear SMARCB1/INI1 protein expression and strong nuclear staining for brachyury. Based on these histopathological findings, a diagnosis of poorly differentiated chordoma was established.
  • WGTA detected low somatic tumour mutation burdens (TMB) for both cases (1.83 and 1.75 mutations per megabase for patients 1 and 2, respectively).
  • Notable alterations in both cases included single copy losses affecting SMARCB1 (Figure 1A), which were associated with low expression of SMARCB1 compared to both TCGA sarcoma 13 data and a pan-cancer cohort of adult cancers, referred to as POG570, sequenced as part of British Columbia’s POG program 14 ( Figure 1 B).
  • Figures 1A and 1 B show SMARCB1 loss and TBXT expression in pediatric chordoma.
  • Figure 1A shows tumour/normal ratios of binned read depths and copy states for chromosome 22 (hg19 chr22: 18000000-30000000) using CNAseq 15 are plotted for patient 1 (top) and 2 (bottom). Regions defined as single copy loss are denoted in darker circles (within square regions in Figure 1A).
  • the SMARCB1 gene location is indicated by the black bar on the lower track.
  • Log2- transformed TPM is plotted on the x-axis and sample densities on the y-axis.
  • the median expression of SMARCB1 and TBXT in pediatric chordomas is represented by the vertical line in each panel.
  • SMARCB1 expression is 30.7 TPM for patient 1 and 19.7 TPM for patient 2 compared to median values of 56.5 and 70.4 TPM for the POG570 and TCGA sarcoma cohorts, respectively;
  • TBXT expression is 148.2 TPM for patient 1 and 69.3 TPM for patient 2 compared to median values of 0 TPM for both POG570 and TCGA sarcoma cohorts.
  • tumour biopsy samples were analysed using multiplex IHC staining. Staining with a T cell panel confirmed the presence of CD3+CD8-, CD3+CD8+ and granzyme B positive cytotoxic CD8+ T cells in both samples.
  • PD-L1 was expressed by both tumour and non-tumour cells as evidenced by the presence of PD-L1+/TBXT+ tumour cells in patient 2, and PD-L1+/CD68+ macrophages in patients 1 and 2. Both samples also harboured CD79a+ B and plasma cells, which have been associated with ICI response in other cancers 24 ’ 25 .
  • TRA T Cell receptor alpha chain
  • TRB 110 beta chain
  • Brachyury is a protein that is normally restricted to the developing embryo but can be mis-expressed in tumours and elicit an immune response 2931 .
  • Peptide-MHC (pMHC) binding predictions of brachyury (TBXT-203) with patient-derived HLA alleles identified 169 and 172 potential unique pMHCs for patient 1 and 2, respectively (Table 1).
  • the inventors therefore investigated the ability of patient MHC class l-matched antigen presenting cells (APCs) expressing brachyury to activate T cells harbouring the identified T cell receptor sequences (TCR1 comprised of TRA-1 (SEQ ID NO:1) and TRB (SEQ ID NO:3); TCR2 comprised of TRA-2 (SEQ ID NO:2) and TRB (SEQ ID NO:3))
  • APCs patient MHC class l-matched antigen presenting cells
  • cytotoxic T cells and evidence for brachyury tumour antigen- mediated immune activity, along with high expression of PD-L1 , suggests that the observed responses to nivolumab may have been driven by disinhibition of this brachyury-specific T cell clone and potentially other tumor-reactive T cells.
  • Figure 2A shows the frequency of unique TCR alpha (TRA) and TCR beta (TRB) chain sequences which were inferred using bulk tumour RNA sequencing data from patient 2 and MiXCR 28 . Estimated fractions of respective T cell clones are shown. The relative fraction of each sequence compared to all TRA and TRB aligned sequences is plotted on the y-axis.
  • TRA TCR alpha
  • TRB TCR beta
  • TRA-1 CAVQSNYKLSF SEQ ID NO:1
  • TRA-2 CAASNPQAGTALIF SEQ ID NO:2
  • CSASTGRGTEAFF SEQ ID NO:3
  • FIG. 2B shows ELISpot analysis of two reconstituted T cells (TCR1 and TCR2) expressing a patient-derived TRB chain and either of the two patient-derived TRA chains, and synthetic K562 antigen presenting cells (APCs), which were transduced with one set of the patient’s classical HLA Class I alleles (Set1 and Set2, see Methods) and TBXT (TBXT-203 ENST00000366876).
  • TCR1 and TCR2 synthetic K562 antigen presenting cells
  • IFNy production measured by the number of spot forming units (SFU) per million cells, correlates with the level of T cell activation upon co-culture of engineered T cells and APCs.
  • Figures 2C and 2D show activation of T cells expressing patient-derived TCR1 and TCR2 sequences following co-culture with APCs expressing TBXT and patient-derived HLA Set1 and Set2.
  • a significant increase in T cell activation in response to TBXT-ex pressing APCs compared to control APCs that did not express TBXT was observed for both TCR1 and TCR2 (unpaired T-test, p ⁇ 0.05 for all comparisons with control TSXT-negative APCs).
  • Figure 2F shows varying concentration of brachyury peptide RFKELTNEM (SEQ ID NO:20) with HLA-C*04:01 APCs with TCR1 T cells.
  • Box plots Figures 2C, 2D, 2E, 2G and 2H represent median, upper and lower quartiles, and whiskers represent limits of the distributions (1.5-times interquartile range).
  • TCR1 and TCR2 T cells are activated by antigen presenting cells expressing HLA-A*23:01 , HLA-A*31 :01 , HLA-B*40:01 , HLA-B*44:03, HLA-C*03:04, or HLA- C*04:01 , and that presentation of the brachyury derived peptide RFKELTNEM (SEQ ID NQ:20) by HLA-C*04:01 activates TCR1 T cells.
  • the inventors reviewed expression data for the TCGA and Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative (phs000218, managed by the National Cancer Institute) disease cohorts and identified those in which TBXT (brachyury) was expressed in at least one sample with an expression value greater than one transcript per million (TPM).
  • TPM transcript per million
  • One transcript per million was confirmed to be a reasonable threshold for determining that brachyury was expressed based on the distribution of the data.
  • Results for the frequency (%) of samples meeting the 1 TPM threshold are shown in Table 2.
  • cancers in addition to poorly differentiated chordoma in which brachyury is expressed include: testicular germ cell tumors, lung adenocarcinoma, pediatric Wilms T umour, pediatric clear cell sarcoma of the kidney, sarcoma, thyroid carcinoma, uterine corpus endometrial carcinoma, colon adenocarcinoma, rectum adenocarcinoma, lymphoid neoplasm diffuse large B-cell lymphoma, stomach adenocarcinoma, lung squamous cell carcinoma, breast invasive carcinoma (including ER- /HER2- and ER+/HER2-), glioblastoma multiforme, esophageal carcinoma, bladder urothelial carcinoma, acute myeloid leukemia, pancreatic adenocarcinoma, ovarian serous cystadenocarcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma,
  • Nucleic acid was extracted from fresh frozen using RLT buffer (Qiagen). Constitutional DNA representing normal cells was extracted from peripheral blood. To minimize library bias and coverage gaps associated with PCR amplification of high GC or AT-rich regions, a version of the TruSeq DNA PCR-free kit (E6875-6877B-GSC, New England Biolabs), automated on a Microlab NIMBUS liquid handling robot (Hamilton) was employed. Briefly, 500ng of genomic DNA was arrayed into wells in a 96-well microtitre plate and subjected to shearing by sonication (Covaris LE220).
  • RNA samples were determined using an Agilent Bioanalyzer RNA Nanochip or Caliper RNA assay and arrayed into a 96-well plate (Thermo Fisher Scientific). Polyadenylated (poly(A)) RNA was purified using the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490L, NEB) from 350 ng (case 1) and 192 ng (case 2) ng total RNA normalized in 35 pL for DNase l-treatment (1 Unit, Invitrogen).
  • NEBNext Poly(A) mRNA Magnetic Isolation Module E7490L, NEB
  • RNA MagClean DX beads Aline Biosciences, C-1005-250
  • Microlab NIMBUS liquid handler Hamilton Robotics, USA.
  • Messenger RNA (mRNA) selection was performed using NEBNext Oligod(T)25 beads (NEB).
  • NEB NEBNext Oligod(T)25 beads
  • rRNA cytoplasmic and mitochondrial ribosomal RNA species from total RNA NEBNext rRNA Depletion Kit for Human/Mouse/Rat was used (NEB, E6310X).
  • the rRNA in DNA hybrids were digested using RNase H in a 20 pL reaction incubated in a thermocycler at 37C for 30 minutes.
  • First-strand cDNA was synthesized from the purified polyadenylated mRNA or rRNA depleted total RNA using the Maxima H Minus First Strand cDNA Synthesis kit (Thermo-Fisher, FSSP9760210) and random hexamer primers at a concentration of 5mM along with a final concentration of 1 mg/mL Actinomycin D, followed by PCRClean DX bead purification on a Microlab NIMBUS robot (Hamilton Robotics, USA).
  • Second strand cDNA was synthesized following the NEBNext Ultra Directional Second Strand cDNA Synthesis protocol (NEB) which incorporates dUTP in the dNTP mix, allowing the second strand to be digested using USERTM enzyme (NEB) in the post-adapter ligation reaction, thus achieving strand specificity.
  • cDNA was fragmented using Covaris LE220 sonication to achieve 200-250 bp average fragment lengths.
  • the paired-end sequencing library was prepared using a strand- specific, plate-based library construction protocol on a Microlab NIMBUS robot (Hamilton Robotics, USA).
  • the sheared cDNA was subject to end-repair and phosphorylation in a single reaction using an enzyme premix (NEB) containing T4 DNA polymerase, Klenow DNA Polymerase and T4 polynucleotide kinase.
  • End-repaired cDNA was purified in 96- well format using PCRClean DX beads, and 3’ A-tailed (adenylation) using Klenow fragment (3’ to 5’ exo minus).
  • Illumina PE adapters were ligated and adapter-ligated products were purified using PCR Clean DX beads, then digested with USERTM enzyme (1 U/pL, NEB) followed by 13 cycles of indexed PCR using Phusion DNA Polymerase (Thermo Fisher Scientific Inc.
  • Tumor genomes were on HiSeqX using v2.5 chemistry and paired-end 150 base reads.
  • Transcriptomes were sequenced on NextSeq500 using v2 chemistry to a length of 75 bp. Sequence coverage is summarized in Table 3. Table 3. Average sequence coverage for each patient.
  • RNA-seq reads were aligned using STAR (v.2.5.2b) and expression was quantified using RSEM (v.1.3.0) as TPM.
  • RSEM v.1.3.0
  • STAR and RSEM were generated from the hg38 reference genome (available from the University of California Santa Cruz) and gene annotations were based on EnsEMBL v.85 32 .
  • SVs Structural variants in RNA-seq data were identified using the assembly-based tools ABySS v1.3.4 38 and TransABySS (v1.4.10) 39 and alignment-based tools Chimerascan 40 (vO.4.5) and DeFuse 41 (vO.6.2); SVs in the DNA sequence data were identified using assembly-based tools ABySS and Trans ABySS and alignment-based tools Manta 42 vl .0.0 and Delly 43 vO.7.3. Putative SV calls identified from the DNA and RNA sequences were merged into a consensus caller MAVIS 44 (v2.1.1), where they were clustered, computationally validated and annotated against constitutional DNA to provide somatic and germline structural variant calls.
  • MAVIS 44 v2.1.1
  • Both DNA- and RNA-derived structural variant calls were additionally filtered to identify those called by more than one tool, and for which a contig could be assembled that aligned across a candidate genomic breakpoint.
  • DNA SV calls were further filtered to exclude events with identical genomic breakpoints in multiple samples, removing potentially confounding germline variants and technical artifacts.
  • Variants were annotated to genes using SNPEff (v3.2) 45 with the EnsEMBL database (v69). Mutation burden is reported as the sum of all SNVs and INDELS passed by Strelka divided by the size of the whole genome (2864785220 bases). TCR alpha and beta clones were detected from RNA sequencing data using MiXCR (v3.0.5) 28 .
  • RNA sequencing reads were aligned and processed using JAGuaR (v2.0.3) 46 as described in Jones and colleagues (2017) 47 .
  • Gene level RPKM reads per kilobase per million mapped reads
  • Immune cell type deconvolution was performed with the CIBERSORT R package (v1.04) 19 , using the LM22 cell subtype signature in absolute mode, with 1 ,000 permutations and no quantile normalization as described in Pender and colleagues (2020) 20 .
  • TBXT protein sequence was downloaded from Uniprot (J3KP65-1). Class I HLA genotypes for each patient were derived using OptiType 48 run separately on the tumour WGS, tumour RNA-seq, and normal RNA-seq datasets. NetMHCpan 4.0 49 was used to predict pMHC binding for 8-11mer peptides from TBXT. NetMHCpan was run in Eluted Ligand mode, which predicts the likelihood that a peptide-MHC would be observed on the surface of a cell. The inventors selected peptide-MHC combinations with Rank ⁇ 2% as predicted binders.
  • Recombinant TCRs were designed based on the RNA sequencing data from patient 2 using Geneious (v.8.1.4) and the IMGT/GENE Database 50 . To minimize mismatch pairing with the endogenous TCRs, mouse constant regions were used for both the alpha and beta chains as previously described 51 .
  • Recombinant HLAs were designed based on the patient 2 derived HLA-class I alleles and the sequences were retrieved from the EMBL-EBI Immune Polymorphism Database 52 .
  • HLA-class I A, B and C alleles were split into two sets to facilitate testing (HLA Set-1 : A*23:01 , B*40:01 , C*03:04; HLA Set-2: A*31 :01 , B*44:03, C*04:01).
  • the nucleotide sequence for TBXT was retrieved from NCBI (Gene ID:
  • NM_001366285.1 (SEQ ID NO:21).
  • TCR, HLA, and the TBXT sequences were separately cloned into pMND lentivirus vectors.
  • the patient derived TCR alpha and beta chains were synthesized de novo and cloned into lentiviral transfer plasmid as a biscistronic alpha- beta gene cassette containing a downstream mStrawberry reporter gene, giving two recombinant TCRs: TCR1 (TRA-1 (SEQ ID NO:1) and TRB (SEQ ID NO:3)) and TCR2 (TRA-2 (SEQ ID NO:2) and TRB (SEQ ID NO:3)).
  • the TBXT sequence was synthesized de novo and cloned into lentiviral transfer plasmids containing a downstream mStrawberry reporter gene.
  • the HLA sequences were synthesized de novo and cloned into lentiviral transfer plasmids.
  • each lentivirus 80 pg of the specific transfer plasmid was incubated for 30 minutes at room temperature, with 72 pg of the pCMV-AR8.91 plasmid, 8 pg of the pCMV-VSV-G plasmid, and 430 pl_ of TranslT-LT1 reagent (Mirus, MIR2305) in a mix made up to 8 ml. with OptiMEM (Gibco, 31985062). HEK 293T/17 cells (ATCC® CRL-11268TM) were transfected. Viral supernatants were collected and virus resuspended in OptiMEM.
  • HLA-ABC Set1 and Set2 HLA-ABC Set1 and Set2 and transduced with the TBXT lentivirus.
  • Donor CD8 + T cells were isolated from a source of PBMCs using the Miltenyi MACS human CD8 + isolation kit, as per the manufacturer’s protocol (Miltenyi, 130-096-495) and using an in-house MACS separation buffer.
  • CD8 + T cells were resuspended in RPMI-1640 supplemented media with 300U/mL rhlL-2 (Peprotech, 200-02 and StemCell, 78036.3), 100 ng/mL of anti-CD3 (eBiosciences, 16-0037-85) and 100 ng/mL anti-CD28 (BioLegend, 302943) soluble antibodies.
  • CD8+ T cells were transduced with lentivirus carrying TCR1 and TCR2 sequences and sorted for mStrawberry expression via flow cytometry (BD Aria and BD FACSDiva software).
  • TCR1 and TCR2 transduced CD8 + T cells were expanded in RPMI-1640 supplemented media with 300U/mL rhlL-2 (Peprotech, 200- 02 and StemCell, 78036.3) and allogenic irradiated feeder cells at a ratio of 1 TCR T cell to 200 irradiated feeder cells.
  • ELISpot plates were coated with IFNy capture antibody (2 pg/ml per well, Mabtech mAb 1-D1 K, 3420-7) in D-PBS and then blocked with RPMI-1640 supplemented media.
  • Co culture of effector Set1 or Set2 APCs co-expressing TBXT or negative control with TCR1 or TCR2 T cells at a ratio of 1 :1 was followed by detection of IFNy with anti-IFNy biotinylated antibody (1 pg/mL per well; Mabtech, mAb 7-B6-1 , 3420-9H), streptavidin-HRP (diluted 1 :100 with D-PBS, Mabtech, 3310-9-1000), and 3'3'5'5'-Tetramethylbenzidine (TMB substrate) (Mabtech, 3652-F10).
  • the ELISpot plate was imaged on AID automated microplate ELISpot reader and associated software (AID ELISpot Version 7.0).
  • McPherson, A. et al. deFuse an algorithm for gene fusion discovery in tumor RNA- Seq data. PLoS Comput. Biol. 7, e1001138 (2011).
  • Cingolani, P. et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin) 6, 80-92 (2012).

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Abstract

L'invention concerne des agents ciblant des antigènes, qui ciblent brachyury, restreints et présentés par des protéines MHC. L'agent ciblant les antigènes peut être une cellule T, une construction CAR, un anticorps, un anticorps bispécifique ou analogue. L'invention porte sur des procédés de traitement de cancers, impliquant l'expression ou la surexpression de brachyury et sur des procédés de criblage des patients susceptibles de bénéficier d'un tel traitement. L'invention porte également sur un nouvel antigène associé à une tumeur, comprenant un peptide brachyury présenté par HLA-C*04:01.
PCT/CA2022/050354 2021-03-10 2022-03-10 Agents immunothérapeutiques ciblant brachyury et leurs procédés d'utilisation WO2022187963A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2125868B1 (fr) * 2007-02-28 2015-06-10 The Govt. Of U.S.A. As Represented By The Secretary Of The Department Of Health And Human Services Polypeptides brachyury et procédés d'utilisation
US20180214525A1 (en) * 2015-08-03 2018-08-02 Globeimmune, Inc. Modified yeast-brachyury immunotherapeutic compositions

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2125868B1 (fr) * 2007-02-28 2015-06-10 The Govt. Of U.S.A. As Represented By The Secretary Of The Department Of Health And Human Services Polypeptides brachyury et procédés d'utilisation
US20180214525A1 (en) * 2015-08-03 2018-08-02 Globeimmune, Inc. Modified yeast-brachyury immunotherapeutic compositions

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
TUCKER JO A ET AL: "Identification and characterization of a cytotoxic T-lymphocyte agonist epitope of brachyury, a transcription factor involved in epithelial to mesenchymal transition and metastasis.", CANCER IMMUNOLOGY, IMMUNOTHERAPY, SPRINGER, BERLIN, DE, vol. 63, no. 12, 1 December 2014 (2014-12-01), Berlin, DE , pages 1307 - 1317, XP002763346, ISSN: 1432-0851 *
WILLIAMSON LAURA M., RIVE CRAIG M., DI FRANCESCO DANIELA, TITMUSS EMMA, CHUN HYE-JUNG E., BROWN SCOTT D., MILNE KATY, PLEASANCE ER: "Clinical response to nivolumab in an INI1-deficient pediatric chordoma correlates with immunogenic recognition of brachyury", NPJ PRECISION ONCOLOGY, vol. 5, no. 1, 20 December 2021 (2021-12-20), pages 103, XP055969717, DOI: 10.1038/s41698-021-00238-4 *

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