WO2022093694A1 - Polypeptides ciblant des complexes peptide hpv-cmh et leurs méthodes d'utilisation - Google Patents

Polypeptides ciblant des complexes peptide hpv-cmh et leurs méthodes d'utilisation Download PDF

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WO2022093694A1
WO2022093694A1 PCT/US2021/056462 US2021056462W WO2022093694A1 WO 2022093694 A1 WO2022093694 A1 WO 2022093694A1 US 2021056462 W US2021056462 W US 2021056462W WO 2022093694 A1 WO2022093694 A1 WO 2022093694A1
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hpv
car
seq
cells
antibody
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PCT/US2021/056462
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Aaron D. MARTIN
Xueyin Wang
Mark L. SANDBERG
Han Xu
Carl Alexander Kamb
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A2 Biotherapeutics, Inc.
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    • 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
    • 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
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/464499—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • 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
    • A61K39/464838—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/084—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • 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
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • HPV Human papilloma virus
  • HPV positive cancers include cervical cancers, oropharyngeal cancers, and genital cancers, among others.
  • HPV is a serious human health problem, causing over 600,000 cancers each year worldwide (de Martel et al. Int J Cancer. 2017 141(4):664-670).
  • diagnoses of HPV-positive cancers continue to increase despite the development of prophylactic vaccines (Schiller et al. Vaccine. 2012 30 Suppl 5:F123-38).
  • New therapies directed at HPV- positive tumors are needed (Clark and Trimble. Gynecol Oncol. 2020 156(2):503-510).
  • HPV serotypes 16 and 18 account for about three fourths of HPV-positive cancers and express the E6 protein.
  • E6-derived peptides are produced in HPV-positive cells and cancer, and can be presented on the surface of HPV-positive cells in complex with major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • Cellular therapies targeting E6-derived peptides and E6- derived peptides in complex with MHC (pMHC) provide a novel strategy for treating HPV- positive cells and cancer in humans.
  • New antibodies targeting E6-derived peptides and E6 pMHC complexes are needed to enable cell therapies.
  • the present disclosure relates to novel polypeptides, antibodies, or antigen-binding fragments thereof (antigen binding domains), specific for HPV-E629-3s:peptide-MHC complexes, chimeric antigen receptors comprising the same, and methods of use thereof.
  • the antibody and chimeric antigen receptor (CAR) compositions may be used in the treatment of a variety of cancers.
  • the disclosure provides a polypeptide comprising an antigen binding domain that specifically binds to a peptide:MHC (pMHC) complex displaying a HPV-E629-38 peptide comprising the sequence TUTDIILECV (SEQ ID NO: 1).
  • the polypeptide comprises heavy-chain complementaritydetermining region 1 (CDR-H1) comprising the sequence of SEQ ID NO: 2 or SEQ ID NO: 5; a CDR-H2 comprising the sequence of SEQ ID NO: 3 or SEQ ID NO: 6; and a CDR-H3 comprising the sequence of SEQ ID NO: 4 or SEQ ID NO: 7.
  • the polypeptide disclosed herein comprises: a light-chain complementarity determining region 1 (CDR-L1) comprising the sequence of SEQ ID NO: 8; a CDR-L2 comprising the sequence of SEQ ID NO: 9; and a CDR-L3 comprising the sequence of SEQ ID NO: 10 or SEQ ID NO: 11.
  • the polypeptide comprises a heavy-chain sequence of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 17 or a sequence having at least 95% identity thereto. In some embodiments, the polypeptide comprises a heavy-chain sequence of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 17. In some embodiments, the polypeptide comprises a light-chain sequence of SEQ ID NO: 13 or SEQ ID NO: 15 or a sequence having at least 95% identity thereto. In some embodiments, the polypeptide comprises a light-chain sequence of SEQ ID NO: 13 or SEQ ID NO: 15.
  • the antigen binding domain is an antibody, an antigen binding fragment of an antibody, a single chain variable fragment (scFv) Fab' fragment, single chain antibody fragment, or single domain antibody. In some embodiments, the antigen binding domain is an scFv.
  • the disclosure provides a chimeric antigen receptor (CAR) comprising a polypeptide described herein as an extracellular antigen binding domain.
  • the CAR described herein comprises a transmembrane domain, and an intracellular signaling domain.
  • the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of CD8-alpha, CD4, CD28, CD137, CD80, CD86, CD 152, and PD1.
  • the intracellular signaling domain comprises one or more intracellular signaling domains of an immune effector cell.
  • the intracellular signaling domain comprises one or more intracellular signaling domains from CD3-zeta.
  • the CAR comprises one or more co-stimulatory domains.
  • the one or more co-stimulatory domains are selected from the group consisting of a CD27, CD28, 4-1BB, 0X40, CD30, CD40, CD40L, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, and a DAP10 co-stimulatory domain.
  • the CAR comprises a hinge domain between the extracellular antigen-binding domain and the transmembrane domain.
  • the CAR comprises a signal peptide.
  • the signal peptide comprises a sequence of MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 18).
  • the CAR exhibits sub-nanomolar potency.
  • the disclosure provides an antibody comprising the antigen binding domain described herein.
  • the antibody is a bispecific antibody.
  • the disclosure provides a nucleic acid comprising a polynucleotide sequence encoding the polypeptide described herein, the CAR described herein, or the antibody described herein.
  • the disclosure provides a vector comprising the nucleic acid described herein.
  • the disclosure provides a cell, comprising a nucleic acid or vector described herein.
  • the disclosure provides a cell comprising a CAR described herein.
  • the cell expresses the CAR.
  • the cell is an immune cell.
  • the immune cell is a T cell.
  • the immune cell is an NK cell.
  • the disclosure provides a pharmaceutical composition comprising a polypeptide CAR, antibody nucleic acid or vector described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.
  • the pharmaceutical composition comprises a cell described herein and a pharmaceutically acceptable carrier, diluent, or excipient.
  • the disclosure provides a method for diagnosing a disease involving HPV positive cells, the method comprising detecting or measuring an HPV-derived peptide in cells using a polypeptide or antibody described herein.
  • the disclosure provides a method for treating an HPV positive cancer in a subject in need thereof, the method comprising administering a polypeptide, antibody or pharmaceutical composition comprising same described herein to the subject.
  • the disclosure provides a method for treating an HPV positive cancer in a subject in need thereof, the method comprising administering a composition comprising a plurality of the cells or pharmaceutical composition comprising same described herein to the subject.
  • the cancer is cervical cancer. In some embodiments, the cancer is oropharyngeal cancer. In some embodiments, the cancer is an oral cancer. In some embodiments, the cancer is anal cancer. In some embodiments, the cancer is penile cancer. In some embodiments, the cancer is vaginal cancer. In some embodiments, the cancer is vulvar cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the plurality of cells comprise a therapeutically effective amount. In some embodiments, the HPV positive cancer cells comprise a pMHC complex comprising an HPV-E629-38 peptide.
  • the disclosure provides a kit, comprising the polypeptide, CAR, antibody, nucleic acid or a vector described herein.
  • the kit comprises a plurality of cells described herein or a pharmaceutical composition described herein.
  • FIG. 1 shows a schematic of an integrated V(D)J tripartite recombination substrate.
  • FIGS. 2A-2B show design, generation and analysis of an antibody library generated by V(D)J recombination.
  • FIG. 2A shows a representation of a heavy chain locus having V segments, D segments and J segments linked to an IgGl constant domain, a transmembrane domain (TM) and a puromycin resistance marker. Also shown are a cassette to facilitate Cre- mediated integration (loxP-FLAG-TM-Hygro), a DNTT gene driven by a strong promoter and a common light chain gene.
  • FIG. 2B shows antibody library generation using tetracycline to induce V(D)J recombination and puromycin to select for in-frame heavy chain genes.
  • FIGS. 3A-3D show sequence analysis of heavy chain genes generated by V(D)J recombination.
  • FIG. 3A shows a comparison of heavy chain V gene usage in a library generated by V(D)J recombination (HuTARG) and a previously described library derived from 654 healthy human donors (Human Donors).
  • FIG. 3B shows the frequency of V-J combinations observed in the library generated by V(D)J recombination.
  • FIG. 3C shows the composition of CDR-H3 sequences fifteen amino acids in length present in the library generated by V(D)J recombination and a human CDR-H3 database.
  • FIG. 3D shows the distribution of CDR-H3 amino acid length in the library generated by V(D)J recombination and a human CDR-H3 database.
  • FIGS. 4A-4B show probes and sorting for identifying an HPV-E6 derived monoclonal antibody (mAb) binder.
  • FIG. 4A is a series of diagrams and a table showing strategy and filters to identify pMHC complexes using AlphascreenTM of HPV-derived peptides. 454 unique 9- and 10-mer peptides were tested. For HPV E6, 277 total peptides were screened (139 9-mers and 138 10-mers. For HPV E7, 177 total peptides were screened (89 9-mers and 88 10-mers).
  • FIG. 4B is a plot showing hit selection for AlphaScreenTM.
  • FIG. 4C is a plot showing pMHC probes identified or confirmed in AlphascreenTM and used in binder isolation. *pMHCs reported in literature; all other pMHCs are novel. Red boxes denote peptides that bind multiple HLA alleles. Grey boxes denote sequences of HLA-A*02-restricted HPV E6 pepl (light gray box) and HPV E7 pep2 (dark gray box).
  • Probe sequences from left to right, are: TIHDIILECV (SEQ ID NO: 1), TIHDIILEC (SEQ ID NO: 31), HLDKKQRFH (SEQ ID NO: 32), IVYRDGNPY (SEQ ID NO: 33), IVYRDGNPY (SEQ ID NO: 33), TTLEQQYNK (SEQ ID NO: 35), TTLEQQYNK (SEQ ID NO: 35), YNKPLCDLL (SEQ ID NO: 37), IRGRWTGRC (SEQ ID NO: 38), AFRDLCIVY (SEQ ID NO: 39), VYRDGNPYA (SEQ ID NO: 40), YRDGNPYAV (SEQ ID NO: 41), MLDLQPETT (SEQ ID NO: 42), TLHEYMLDL (SEQ ID NO: 43), TLEDLLMGTL (SEQ ID NO: 44), YMLDLQPET (SEQ ID NO: 45), YMLDLQPET (SEQ ID NO: 45), HVDI
  • FIG. 5 is a table summarizing the incidence of HPV-associated cancers in the United States and contributions of main HPV subtypes. Data are drawn from seer.cancer.gov/statfacts/; seer.cancer.gov/statfacts/ and Doorbar et al. Rev. Med. Virol. 2016; 25: 2-23.
  • FIG. 6 is a table summarizing pMHC targets of CARs screened according to the methods described herein. ’Draper et al. Clin. Cancer Research 2015 21(19):4431-9. 2 Jin et al. JCI Insight 2018 19;3(8). 3 Ramos et at. J Immunotherapy 2013 36(1): 66-76. 4 Kast et. J of Immunology 1994 152:3904-3910. 5 Ressing et al. J Immunology 1995; 154:5934-5943.
  • TIHDIILECV SEQ ID NO: 1
  • YMLDLQPET SEQ ID NO: 45
  • LLMGTLGIV SEQ ID NO: 34
  • TLHEYMLDL SEQ ID NO: 43
  • IVYRDGNPY SEQ ID NO: 33
  • TTLEQQYNK SEQ ID NO: 35
  • FIG. 7 is an illustrative diagram showing an alignment of the HPV 16 and 18 E6 (top) and E7 (bottom) protein sequences. The location of HPV-E629-38 in the sequence of the E6 protein of HPV subtypes 16 and 18 is indicated.
  • E6 top sequence, SEQ ID NO: 36; bottom sequence, SEQ ID NO: 48.
  • E7 top sequence, SEQ ID NO: 49; bottom sequence, SEQ ID NO: 50.
  • FIG. 8 is a series of plots showing an overview of the cell sorting strategy using fluorescence activated cell sorting (FACS) to identify HPV-E629-38 binding antibodies.
  • FACS fluorescence activated cell sorting
  • FIG. 9 is an illustrative diagram showing the workflow for isolating HPV-E629-38 peptide-specific binders with the screening system described herein.
  • FIG. 10 is a pair of plots showing illustrative data characterizing sensitivity and selectivity of HPV-E629-38 primary, light chain optimized, and heavy chain optimized binders. The best binders were identified in the peptide loading Jurkat/T2 assays described herein. CARs are shown in triangles, the HPV-E629-38 TCR is shown as circles.
  • FIG. 11 is series of diagrams showing construction of a library used for optimization ofHPV-E6 2 9 -38 and E7 peptide binders.
  • FIG. 12 is a series of plots showing illustrative data characterizing sensitivity and near and far off-target selectivity of HPV-E629-38 binders with optimized light chain.
  • FIG. 13 is a series of plots and a table showing illustrative data characterizing sensitivity and near and far off-target selectivity of HPV-E629-38 binders with optimized heavy chain.
  • FIG. 14 is a pair of plots showing that light chain optimized HPV-E629-38 binding CARs show minimal near off-target cross-reactivity.
  • FIG. 15 is a table summarizing the characterization of HPV-E629-38 binding CARs, and comparing these CARs with the clinical TCR benchmarks and (un-optimized) parents.
  • Each optimized binder and parent has a unique heavy and light chain.
  • Each fully optimized binder and its parent share their light chain.
  • FIG. 16A is an illustrative diagram of various embodiments of CAR constructs.
  • FIG. 16B is a series of plots showing the sensitivity of different HPV-E629-38 binding CAR formats. CAR variants did not show substantial differences in EC50.
  • FIG. 16C is a series of plots showing the sensitivity of different HPV-E629-38 binding CAR formats.
  • FIG. 17 is a series of plots and diagrams that show characterization of sensitivity and cytotoxicity in acute and repeated antigen-challenge of an HP V-E629-38 targeting CAR.
  • FIG. 17A is a plot that shows acute sensitivity of HPV-E629-38 targeting CAR by peptide loading.
  • FIG. 17B is a diagram of the repeated antigen challenging assay (RACA).
  • FIG. 17C is a plot showing antigen dependent exhaustion in the repeat antigen-challenge assay.
  • FIG. 18 is a table showing near off-target peptides tested with HPV-E629-38 binders.
  • FIG. 19 is a table and a plot showing a standard curve for T2 loading of peptide to estimate pMHCs/cell (original binding data from Purbhoo et al. J Immunol 2006 176(12):7308- 16). Red numbers in the plot are estimated by scaling from the flow cytometry MFI by a factor of 550/20, the values at IE-8 M measurements where the most reliable overlap lies between the single-molecule fluorescence counting and population mean from flow cytometry (MFI).
  • the present disclosure provides novel antibodies and antigen-binding fragments (or domains) thereof that selectively bind to HPV-E629-38 peptides.
  • fusion proteins such as chimeric antigen receptors (CARs) comprising the polypeptides, antibodies, and antigen-binding fragments of the disclosure.
  • CARs chimeric antigen receptors
  • CARs chimeric antigen receptors
  • a cell includes a plurality of cells, including mixtures thereof.
  • the “administration” of an agent, e.g., an anti-HPV E6:pMHC antibody or CAR-expressing cell, to a subject or subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated and target cell or tissue. Non-limiting examples of route of administration include parenteral, enteral, and topical routes of administration. Administration includes self-administration and the administration by another. It is also to be appreciated that the various modes of treatment or prevention of medical conditions as described are intended to mean “substantial”, which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved.
  • the term “animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.
  • the term “mammal” includes both human and non-human mammals.
  • the term “subject” or “patient” includes both human and veterinary subjects, for example, humans, non-human primates, dogs, cats, sheep, mice, horses, and cows.
  • antibody collectively refers to immunoglobulins or immunoglobulin-like molecules including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits and mice, as well as non-mammalian species, such as shark immunoglobulins.
  • antibody includes intact immunoglobulins and “antibody fragments” or “antigen binding fragments” that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 10 3 M -1 greater, at least 10 4 M -1 greater or at least 10 5 M -1 greater than a binding constant for other molecules in a biological sample).
  • antibody also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies).
  • antibody herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity.
  • Antibody fragments or “antigen binding fragments” include proteolytic antibody fragments (such as F(ab')2 fragments, Fab' fragments, Fab'-SH fragments and Fab fragments as are known in the art), recombinant antibody fragments (such as sFv fragments, dsFv fragments, bispecific sFv fragments, bispecific dsFv fragments, F(ab)'2 fragments, single chain Fv proteins (“scFv”), disulfide stabilized Fv proteins (“dsFv”), diabodies, and triabodies (as are known in the art), and camelid antibodies (see, for example, U.S. Pat. Nos.
  • proteolytic antibody fragments such as F(ab')2 fragments, Fab' fragments, Fab'-SH fragments and Fab fragments as are known in the art
  • recombinant antibody fragments such as sFv fragments, dsFv fragments, bispecific sFv fragment
  • An scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.
  • the term “antigen” refers to a compound, composition, or substance that may be specifically bound by the products of specific humoral or cellular immunity, such as an antibody molecule or T-cell receptor.
  • Antigens can be any type of molecule including, for example, haptens, simple intermediary metabolites, sugars (e.g., oligosaccharides), lipids, and hormones as well as macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, and proteins.
  • antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoa and other parasitic antigens, tumor antigens, antigens involved in autoimmune disease, allergy and graft rejection, toxins, and other miscellaneous antigens.
  • binding affinity refers to the tendency of one molecule to bind (typically non-covalently) with another molecule, such as the tendency of a member of a specific binding pair for another member of a specific binding pair.
  • a binding affinity can be measured as a dissociation constant, which for a specific binding pair (such as an antibody/antigen pair) can be lower than 1 * 10 -5 M, lower than 1 * 10 -6 M, lower than 1 * 10 -7 M, lower than 1 * 10 -8 M, lower than 1 X 10 -9 M, lower than l x lO -lo M, lower than l x l0 -11 M or lower than 1 X 10 -12 M.
  • binding affinity is calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol., 16: 101-106, 1979.
  • binding affinity is measured by a binding constant.
  • binding affinity is measured by an antigen/antibody dissociation rate.
  • a high binding affinity is measured by a competition radioimmunoassay.
  • cancer refers to cells that have undergone a malignant transformation that makes them pathological to the host organism.
  • cancers include hematological malignancies and solid tumors.
  • chimeric antigen receptors may refer to artificial T-cell receptors, chimeric T-cell receptors, or chimeric immunoreceptors, for example, and encompass engineered receptors that graft an artificial specificity onto a particular immune effector cell, such as a helper T cell (CD4+), cytotoxic T cell (CD8+) or NK cell.
  • CARs may be employed to impart the specificity of a monoclonal antibody onto a T cell, thereby allowing a large number of specific T cells to be generated, for example, for use in adoptive cell therapy.
  • CARs direct specificity of the cell to a tumor associated antigen, e.g., HPV-E629 -38.
  • CARs comprise an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising a tumor associated antigen binding region.
  • CARs comprise fusions of single-chain variable fragments (scFvs) or scFabs derived from monoclonal antibodies, fused to a transmembrane domain and intracellular signaling domain. Either heavy-light (H-L) and light-heavy (L-H) scFvs may be used.
  • CAR designs may be derived from ligands of receptors (e.g., peptides) or from Dectins.
  • CARs comprise domains for additional co-stimulatory signaling, such as CD27, CD28, 4-1BB, 0X40, CD30, CD40, CD40L, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, and/or DAP10.
  • molecules can be coexpressed with the CAR, including co-stimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally ablate the T cells upon addition of a pro-drug, homing receptors, cytokines, and cytokine receptors.
  • co-stimulatory molecules including co-stimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally ablate the T cells upon addition of a pro-drug, homing receptors, cytokines, and cytokine receptors.
  • an “epitope” or “antigenic determinant” refers to particular chemical groups or contiguous or non-contiguous peptide sequences on a molecule that are antigenic, /. ⁇ ., that elicit a specific immune response.
  • An antibody binds a particular antigenic epitope.
  • Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • polypeptides described herein may be “isolated” polypeptides, meaning that the removed from living host. Isolated polypeptide may be expressed in a host cell, such as an immune cell, as a receptor and be displayed on the surface of that cell.
  • soluble embodiments include monoclonal antibodies, single-chain fragments (scFv), bispecific or multispecific antibodies, and the like. It will be readily understood that the complementarity determining regions (CDRs) of antibodies can be grafted onto various scaffolds including antibody-based scaffolds, designed protein scaffolds, and non-protein scaffolds.
  • polypeptides of the disclosure may also be expressed in vivo (that is, in a subject organism, e.g., a human subject) rather than in vitro, such as by administration to the subject of a vector comprising a polynucleotide encoding the polypeptide.
  • nucleic acids or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, e.g., at least 60% identity, preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., nucleotide sequence encoding an antibody described herein or amino acid sequence of an antibody described herein).
  • Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
  • the alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1.
  • default parameters are used for alignment.
  • a preferred alignment program is BLAST, using default parameters.
  • preferred programs are BLASTN and BLASTP.Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST.
  • the terms “homology” or “identical”, percent “identity” or “similarity” also refer to, or can be applied to, the complement of a test sequence.
  • the terms also include sequences that have deletions and/or additions, as well as those that have substitutions.
  • the preferred algorithms can account for gaps and the like.
  • identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is at least 50-100 amino acids or nucleotides in length.
  • An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure.
  • human antibody as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis or by somatic mutation in vivo).
  • the term “human antibody” as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a rabbit, have been grafted onto human framework sequences.
  • human antibody refers to an antibody in which substantially every part of the protein (e.g., CDR, framework, CL, CH domains (e.g., CHI, CH2, CHS), hinge, VL, VH) is substantially non-immunogenic in humans, with only minor sequence changes or variations.
  • antibodies designated primate monkey, baboon, chimpanzee, etc.
  • rodent mouse, rat, rabbit, guinea pig, hamster, and the like
  • other mammals designate such species, sub-genus, genus, sub-family, family specific antibodies.
  • chimeric antibodies include any combination of the above.
  • a human antibody is distinct from a chimeric or humanized antibody. It is pointed out that a human antibody can be produced by a non-human animal or prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and/or light chain) genes. Further, when a human antibody is a single chain antibody, it can comprise a linker peptide that is not found in native human antibodies.
  • an Fv can comprise a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain.
  • linker peptides are considered to be of human origin.
  • HPV-E629-38 antibody and “anti-HPV-E629-38 antibody” are used interchangeably, and refer to an antibody that specifically binds to an HPV-E629-38 peptide and/or HPV-E629 -38 peptide-MHC complex as antigen.
  • the HPV-E629-38 peptide is an HPV-E629-38 peptide comprising the sequence TIHDIILECV (SEQ ID NO: 1).
  • the antibody specifically binds a HPV-E629-38 peptide-MHC complex and is referred to as “anti-HPV-E629-38:peptide-MHC complex (pMHC) antibody” or “anti-HPV-E629- 38:pMHC antibody”.
  • anti-HPV-E629-38:peptide-MHC complex (pMHC) antibody or “anti-HPV-E629- 38:pMHC antibody”.
  • HPV-E629-38 refers to both HPV-E629-38 peptide and HPV-E629-38 peptide-MHC complex.
  • the term “monoclonal antibody” refers to an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected.
  • Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells.
  • Monoclonal antibodies include humanized monoclonal antibodies.
  • polynucleotide and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown.
  • polynucleotides a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
  • the term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any aspect of this disclosure that is a polynucleotide encompasses both the double-stranded form and each of two complementary single- stranded forms known or predicted to make up the double-stranded form.
  • a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA.
  • A adenine
  • C cytosine
  • G guanine
  • T thymine
  • U uracil
  • polynucleotide sequence is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
  • protein refers to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics.
  • the subunits may be linked by peptide bonds. In another aspect, the subunit may be linked by other bonds, e.g., ester, ether, etc.
  • a protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein’s or peptide’s sequence.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
  • treating or “treatment” of a disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable.
  • polypeptides comprising anti-HPV-E629-3s:peptide- MHC complex (pMHC) antibodies and antigen-binding fragments thereof, antigen-binding domains, chimeric antigen receptors (CARs), CAR-expressing cells, and compositions comprising.
  • pMHC peptide- MHC complex
  • CARs chimeric antigen receptors
  • pMHCs are a rich source of targets for new medicines directed at peptides derived from proteins whose tissue expression pattern and cellular compartment are well known. Although the therapeutic opportunity is apparent, the limited literature on this modality reflects the technical difficulty of this target class.
  • the historical challenges of generating TCR antibody therapeutics are supported by the fact that only one molecule has advanced to the clinic: Eureka Therapeutics’ ET14O2L1-ARTEMISTM2 T cells for alpha fetoprotein (AFP) expressing hepatocellular carcinoma is currently in a Phase 1/2 clinical trial for liver cancer.
  • AFP alpha fetoprotein
  • V(D)J recombination was employed to isolate and engineer fully human monoclonal antibodies capable of binding pMHCs with high avidity and selectivity.
  • polypeptides comprising anti-HPV-E629-3s:peptide- MHC complex (pMHC) antigen binding domains, antibodies and antigen-binding fragments thereof.
  • pMHC peptide- MHC complex
  • an immunoglobulin monomer comprises two heavy chains and two light chains connected by disulfide bonds. Each heavy chain is paired with one of the light chains to which it is directly bound via a disulfide bond. Each heavy chain comprises a constant region (which varies depending on the isotype of the antibody) and a variable region.
  • the variable region comprises three hypervariable regions (or complementarity determining regions) which are designated CDRH1, CDRH2 and CDRH3 and which are supported within framework regions.
  • Each light chain comprises a constant region and a variable region, with the variable region comprising three hypervariable regions (designated CDRL1, CDRL2 and CDRL3) supported by framework regions in an analogous manner to the variable region of the heavy chain.
  • the hypervariable regions of each pair of heavy and light chains mutually cooperate to provide an antigen binding site that is capable of binding a target antigen.
  • the binding specificity of a pair of heavy and light chains is defined by the sequence of CDR1, CDR2 and CDR3 of the heavy and light chains.
  • an antibody that binds to HPV-E629-38 is provided herein.
  • the HPV early gene product E6 plays a key role in carcinogenesis. E6 promotes cellular transformation by targeting several proteins, including the DNA damage response protein p53, thereby interfering with cell cycle arrest, apoptosis, and promoting genomic instability (Ghittoni et al. Ecancermedicalscience . 9:526 (2015)). Various E6 peptides are known to elicit immune response by display on the cell surface, bound to specific HLA class I molecules. E6 is not expressed in HPV negative normal adult tissues, and does not compromise the tumor specificity of E6 because these HPV negative cells do not bear HLA class I molecules that present E6 antigen. Consequently, E6 antigen is a genuinely selective target for tumorspecific active immunotherapy.
  • EC50 half maximal effective concentration
  • the EC50 is less than 100 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, or less than 1 nM. In some embodiments, the EC50 is less than 5 nM.
  • the antibody binds to the peptide within the context of a peptide-MHC complex.
  • the peptide having SEQ ID NO: 1 was identified as an HLA-A*02:01 restricted peptide.
  • the antibody binds to the peptide within the context of a peptide-HLA-A*02:01 complex.
  • the antibody binds to the peptide within the context of a peptide-HLA-A*02 complex.
  • the antibody has a higher EC50 (decreased binding) for a peptide that differs from that of SEQ ID NO: 1 by one amino acid, two amino acids, three amino acids, four amino acids, five amino acids, or more. In some embodiments, the antibody has a two-fold higher, five-fold higher, 10-fold higher, 15-fold higher, 20-fold higher, 50-fold higher, or 100-fold higher ECso for a peptide that differs from SEQ ID NO: 1.
  • the antibody that binds HPV-E629-38 is a single domain antibody.
  • the antibody that binds, such as specifically binds, HPV-E629- 38 is a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a single chain variable fragment (scFv), or a disulfide stabilized variable fragment (dsFv).
  • the antibody is an immunoglobulin molecule.
  • the antibody is an IgG.
  • the antibody is a fully human antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a chimeric antibody.
  • the antibodies disclosed herein can be of any isotype.
  • the antibody can be, for example, an IgM or an IgG antibody, such as IgGi or an IgG2.
  • the class of an antibody that specifically binds HPV-E629-38 can be switched with another (for example, IgG can be switched to IgM), according to well-known procedures. Class switching can also be used to convert one IgG subclass to another, such as from IgGi to IgG2.
  • Antibody fragments or antigen binding domains are also encompassed by the present disclosure, such as single-domain antibodies (e.g., VH domain antibodies), Fab, F(ab')2, and Fv. These antibody fragments retain the ability to selectively bind with the antigen. These fragments include:
  • Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
  • Fab' the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;
  • (Fab')2 the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction
  • F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds
  • Fv a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
  • Single chain antibody such as scFv
  • scFv Single chain antibody
  • a dimer of a single chain antibody (scFv2), defined as a dimer of a scFv (also known as a “miniantibody”);
  • VH single-domain antibody an antibody fragment consisting of a heavy chain variable domain
  • a single chain Fab fragment (scFab), which can be formed by the introduction of a polypeptide linker between the Fd and the light chain to result in the formation of a single chain Fab fragment.
  • antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in a host cell (such as E. colt) of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly (see U.S. Pat. No. 4,036,945 and U.S. Pat. No. 4,331,647).
  • antibodies of the present disclosure can be purified to homogeneity.
  • the separation and purification of the antibodies can be performed by employing conventional protein separation and purification methods.
  • the antibody can be separated and purified by appropriately selecting and combining use of chromatography columns, filters, ultrafiltration, salt precipitation, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, and the like.
  • Strategies for Protein Purification and Characterization A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual . Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988).
  • Non-limiting examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography.
  • chromatography can be performed by employing liquid chromatography such as HPLC or FPLC.
  • the polypeptides, antigen binding domains, antibodies, or antibody fragments described herein comprise CDRs comprising a heavy chain.
  • the heavy chain comprises at least one of SEQ ID NOs: 2-7, or a sequence comprising at least 80%, 90%, 95%, or 98% identity thereto.
  • the heavy chain comprises SEQ ID NOs: 2-4, or sequences comprising at least 80%, 90%, 95%, or 98% identity thereto.
  • the heavy chain comprises SEQ ID NOs: 5-7, or sequences comprising at least 80%, 90%, 95%, or 98% identity thereto.
  • Illustrative sequences of anti-HPV-E629-38 antibodies are provided herein.
  • the heavy chain comprises a CDR VH-1 selected from the group consisting SEQ ID NOS: 2 and 5, a CDR VH-2 selected from the group consisting of SEQ ID NOS: 3 and 6, and a CDR VH-3 selected from the group consisting of SEQ ID NOS: 4 and 7, or sequences having no more than 1, 2, or 3 substitutions, insertions or deletions relative thereto.
  • Table 1 provide non-limiting illustrative heavy chain CDR sequences for antibodies according to the disclosure.
  • Table 1 Illustrative heavy chain CDR sequences for antibodies that specifically bind HPV- E629-38 pMHC complex
  • the polypeptides, antigen binding domains, antibodies, or antibody fragments described herein comprise CDRs comprising a light chain.
  • the light chain comprises at least one of SEQ ID NOs: 8-11, or a sequence comprising at least 80%, 90%, 95%, or 98% identity thereto.
  • the light chain comprises SEQ ID NOs: 8-10, or sequences comprising at least 80%, 90%, 95%, or 98% identity thereto.
  • the light chain comprises SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 or SEQ ID NO: 11, or sequences comprising at least 80%, 90%, 95%, or 98% identity thereto.
  • the light chain comprises SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 or SEQ ID NO: 11, or sequences having no more than 1, 2, or 3 substitutions, insertions or deletions relative thereto.
  • Table 2 provides non-limiting illustrative light chain CDR sequences for antibodies according to the disclosure.
  • the polypeptides, antigen binding domains, antibodies, or antibody fragments described herein comprise CDRs comprising a heavy chain and a light chain.
  • the heavy chain comprises one of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 17, or a sequence comprising at least 80%, 90%, 95%, or 98% identity thereto.
  • the light chain comprises one of SEQ ID NO: 13 or SEQ ID NO: 15, or a sequence comprising at least 80%, 90%, 95%, or 98% identity thereto.
  • the heavy chain comprises SEQ ID NOs: 12 and the light chain comprises SEQ ID NO: 13, or sequences comprising at least 80%, 90%, 95%, or 98% identity thereto.
  • the heavy chain comprises SEQ ID NOs: 14 and the light chain comprises SEQ ID NO: 15, or sequences comprising at least 80%, 90%, 95%, or 98% identity thereto.
  • the heavy chain comprises SEQ ID NOs: 16 and the light chain comprises SEQ ID NO: 13, or sequences comprising at least 80%, 90%, 95%, or 98% identity thereto.
  • the heavy chain comprises SEQ ID NOs: 17 and the light chain comprises SEQ ID NO: 15, or sequences comprising at least 80%, 90%, 95%, or 98% identity thereto.
  • Table 3 provides illustrative heavy and light chain sequences for antibodies according to the disclosure.
  • one or more amino acid residues in a CDR of the antibodies provided herein are substituted with another amino acid.
  • the substitution may be “conservative” in the sense of being a substitution within the same family of amino acids.
  • the naturally occurring amino acids may be divided into the following four families and conservative substitutions will take place within those families.
  • Amino acids with basic side chains lysine, arginine, histidine.
  • Amino acids with acidic side chains aspartic acid, glutamic acid
  • Amino acids with uncharged polar side chains asparagine, glutamine, serine, threonine, tyrosine.
  • Amino acids with nonpolar side chains glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, cysteine.
  • one or more amino acid residues are added to or deleted from one or more CDRs of an antibody. Such additions or deletions may occur at the N or C termini of the CDR or at a position within the CDR.
  • antibodies of the disclosure comprising such varied CDR sequences may still bind HPV-E629-38 with similar specificity and sensitivity profiles. This may be tested by way of the binding assays disclosed in Examples described herein.
  • the constant regions of antibodies may also be varied.
  • antibodies may be provided with Fe regions of any isotype: IgA (IgAl, IgA2), IgD, IgE, IgG (IgGl, IgG2, IgG3, IgG4) or IgM.
  • IgA IgAl, IgA2
  • IgD IgD
  • IgE IgG
  • IgM IgM
  • Such conservative variants employed in antibody fragments will retain critical amino acid residues necessary for correct folding and stabilizing between the VH and the VL regions.
  • the variants will retain the charge characteristics of the residues, for example.
  • Amino acid substitutions (such as at most one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, at most ten, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, or at most 20 amino acid substitutions) can be made in the VH and/or the VL regions to increase yield.
  • Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:
  • Amino acid substitutions, deletions, and additions, and other such sequence variations may be performed based on sequence alignment techniques using existing sequence alignment tools.
  • the term “detectable label” refers to a molecule or material that can produce a detectable (such as visually, electronically or otherwise) signal that indicates the presence and/or concentration of the label in a sample.
  • the detectable label can be used to locate and/or quantify the target to which the specific binding molecule is directed. Thereby, the presence and/or concentration of the target in a sample can be detected by detecting the signal produced by the detectable label.
  • a detectable label can be detected directly or indirectly, and several different detectable labels conjugated to different specific-binding molecules can be used in combination to detect one or more targets.
  • a first detectable label conjugated to an antibody specific to a target can be detected indirectly through the use of a second detectable label that is conjugated to a molecule that specifically binds the first detectable label.
  • Multiple detectable labels that can be separately detected can be conjugated to different specific binding molecules that specifically bind different targets to provide a multiplexed assay that can provide simultaneous detection of the multiple targets in a sample.
  • a detectable signal can be generated by any mechanism including absorption, emission and/or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons).
  • Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected through antibody-hapten binding interactions using additional detectably labeled antibody conjugates, and paramagnetic and magnetic molecules or materials.
  • catalysts such as enzymes
  • haptens that can be detected through antibody-hapten binding interactions using additional detectably labeled antibody conjugates, and paramagnetic and magnetic molecules or materials.
  • Non-limiting examples of detectable labels include enzymes such as horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, P-galactosidase or p -glucuronidase; fluorphores such as fluoresceins, luminophores, coumarins, BODIPY dyes, resorufins, and rhodamines (many additional examples of fluorescent molecules can be found in The Handbook — A Guide to Fluorescent Probes and Labeling Technologies, Molecular Probes, Eugene, Oreg.); nanoparticles such as quantum dots (obtained, for example, from QuantumDot Corp, Invitrogen Nanocrystal Technologies, Hayward, Calif.; see also, U.S.
  • enzymes such as horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, P-galactosidase or p -glucuroni
  • detectable label includes an enzyme
  • a detectable substrate such as a chromogen, a fluorogenic compound, or a luminogenic compound can be used in combination with the enzyme to generate a detectable signal (A wide variety of such compounds are commercially available, for example, from Invitrogen Corporation, Eugene Oreg.).
  • Non-limiting examples of chromogenic compounds include diaminobenzidine (DAB), 4-nitrophenylphospate (pNPP), fast red, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), BCIP/NBT, fast red, AP Orange, AP blue, tetramethylbenzidine (TMB), 2,2'-azino-di-[3-ethylbenzothiazoline sulphonate] (ABTS), o- dianisidine, 4-chloronaphthol (4-CN), nitrophenyl-P-D-galactopyranoside (ONPG), o- phenylenediamine (OPD), 5-bromo-4-chloro-3-indolyl-P-galactopyranoside (X-Gal), methylumbelliferyl-P-D-galactopyranoside (MU-Gal), p-nitrophenyl-a-D-gal
  • an enzyme can be used in a metallographic detection scheme.
  • Metallographic detection methods include using an enzyme such as alkaline phosphatase in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redox-active agent reduces the metal ion, causing it to form a detectable precipitate.
  • Metallographic detection methods include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate.
  • an oxido-reductase enzyme such as horseradish peroxidase
  • Haptens are small molecules that are specifically bound by antibodies, although by themselves they will not elicit an immune response in an animal and must first be attached to a larger carrier molecule such as a protein to generate an immune response. Examples of haptens include di -nitrophenyl, biotin, digoxigenin, and fluorescein.
  • the detectable label comprises a non-endogenous hapten (e.g. not biotin), such as, for example, the haptens disclosed in U.S. Pat. Nos.
  • 7,695,929, 8,618,265 and 8,846,320 (incorporated herein by reference), including for example pyrazoles, nitrophenyl compounds, benzofurazans, triterpenes, ureas and thioureas, rotenone and rotenone derivatives, oxazoles and thiazoles, coumarin and coumarin derivatives, and cyclolignans.
  • the antibodies of the present disclosure may be multimerized to increase the affinity for an antigen.
  • the antibody to be multimerized may be one type of antibody or a plurality of antibodies which recognize a plurality of epitopes of the same antigen.
  • binding of the IgG CH3 domain to two scF v molecules, binding to streptavidin, introduction of a helix-turn-helix motif and the like can be exemplified.
  • the antibody compositions of the present disclosure may be in the form of a conjugate formed between any of these antibodies and another agent (immunoconjugate).
  • the antibodies of the present disclosure are conjugated to radioactive material.
  • the antibodies of the present disclosure can be bound to various types of molecules such as polyethylene glycol (PEG).
  • the disclosed monoclonal antibodies specific for HPV-E629-38 can be conjugated to a therapeutic agent or effector molecule including, but are not limited to, molecules in which there is a covalent linkage of a therapeutic agent to an antibody.
  • a therapeutic agent is an agent with a particular biological activity directed against a particular target molecule or a cell bearing a target molecule.
  • therapeutic agents can include various drugs such as vinblastine, daunomycin and the like, cytotoxins such as native or modified Pseudomonas exotoxin or Diphtheria toxin, encapsulating agents (such as liposomes) which themselves contain pharmacological compositions, radioactive agents such as 125 1, 32 P, 14 C, 3 H and 35 S and other labels, target moieties and ligands.
  • the choice of a particular therapeutic agent depends on the particular target molecule or cell, and the desired biological effect.
  • the therapeutic agent can be a cytotoxin that is used to bring about the death of a particular target cell (such as a tumor cell).
  • the therapeutic agent can be conjugated to a non-lethal pharmacological agent or a liposome containing a non-lethal pharmacological agent.
  • nucleic acids encoding antibodies and conjugates and fusion proteins thereof.
  • Effector molecules can be linked to an antibody of interest using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means may be used.
  • the procedure for attaching an effector molecule to an antibody varies according to the chemical structure of the effector.
  • Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine ( — NH2) or sulfhydryl ( — SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule.
  • the antibody is derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of known linker molecules.
  • the linker can be any molecule used to join the antibody to the effector molecule.
  • the linker is capable of forming covalent bonds to both the antibody and to the effector molecule.
  • Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers.
  • the linkers may be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.
  • immunoconjugates will comprise linkages that are cleavable in the vicinity of the target site. Cleavage of the linker to release the effector molecule from the antibody may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site.
  • the antibodies or antibody fragments disclosed herein can be derivatized or linked to another molecule (such as another peptide or protein).
  • the antibody or antibody fragment (such as a VH domain) is fused to a heterologous protein, for example an Fc protein.
  • the antibody or antibody fragment is fused to a part of a chimeric antigen receptor (CAR) protein.
  • the antibody or antibody fragment is fused to a CD8 hinge, a CD28 transmembrane domain and a CD28-4-lBB-CD3( ⁇ signaling domain.
  • the antibodies or portion thereof is derivatized such that the binding to the target antigen is not affected adversely by the derivatization or labeling.
  • the antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (for example, a bispecific antibody or a diabody), a detection agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
  • One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types, such as to create bispecific antibodies).
  • Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl suberate).
  • Such linkers are commercially available.
  • An antibody that binds (for example specifically binds) HPV-E629-38 or a fragment thereof can be labeled with a detectable moiety.
  • Non-limiting examples of detection agents include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5- dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like.
  • Bioluminescent markers are also of use, such as luciferase, Green fluorescent protein (GFP), Yellow fluorescent protein (YFP).
  • GFP Green fluorescent protein
  • YFP Yellow fluorescent protein
  • An antibody can also be labeled with enzymes that are useful for detection, such as horseradish peroxidase, P-galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like.
  • an antibody When an antibody is labeled with a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is visually detectable.
  • An antibody may also be labeled with biotin, and detected through indirect measurement of avidin or streptavidin binding. It should be noted that the avidin itself can be labeled with an enzyme or a fluorescent label.
  • An antibody may be labeled with a magnetic agent, such as gadolinium.
  • Antibodies can also be labeled with lanthanides (such as europium and dysprosium), and manganese.
  • Paramagnetic particles such as superparamagnetic iron oxide are also of use as labels.
  • An antibody may also be labeled with a predetermined polypeptide epitopes recognized by a secondary reporter (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
  • labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
  • An antibody can also be labeled with a radiolabeled amino acid.
  • the radiolabel may be used for both diagnostic and therapeutic purposes.
  • the radiolabel may be used to detect HPV-E629-38 by x-ray, emission spectra, or other diagnostic techniques.
  • Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3 H, 14 C, 15 N, 35 S, 90 Y, "Tc, in In, 125 I, 131 I.
  • An antibody can also be derivatized with a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups may be useful to improve the biological characteristics of the antibody, such as to increase serum half-life or to increase tissue binding.
  • Toxins can be employed with the monoclonal antibodies described herein to produce immunotoxins.
  • Exemplary toxins include ricin, abrin, diphtheria toxin and subunits thereof, as well as botulinum toxins A through F. These toxins are readily available from commercial sources (for example, Sigma Chemical Company, St. Louis, Mo.). Contemplated toxins also include variants of the toxins described herein (see, for example, see, U.S. Pat. Nos. 5,079,163 and 4,689,401).
  • the toxin is Pseudomonas exotoxin (PE) (U.S. Pat. No. 5,602,095).
  • Pseudomonas exotoxin refers to a full-length native (naturally occurring) PE or a PE that has been modified. Such modifications can include, but are not limited to, elimination of domain la, various amino acid deletions in domains lb, II and III, single amino acid substitutions and the addition of one or more sequences at the carboxyl terminus (for example, see Siegall et al., J. Biol. Chem. 264: 14256-14261, 1989).
  • PE employed with the monoclonal antibodies described herein can include the native sequence, cytotoxic fragments of the native sequence, and conservatively modified variants of native PE and its cytotoxic fragments.
  • Cytotoxic fragments of PE include those which are cytotoxic with or without subsequent proteolytic or other processing in the target cell. Cytotoxic fragments of PE include PE40, PE38, and PE35.
  • Cytotoxic fragments of PE include PE40, PE38, and PE35.
  • the antibodies described herein can also be used to target any number of different diagnostic or therapeutic compounds to cells expressing HPV-E629-38 or HPV-E629-38 peptide- MHC complex.
  • an antibody of the present disclosure can be attached directly or via a linker to a drug that is to be delivered directly to cells expressing cell-surface HPV-E629-38 peptide-MHC complex. This can be done for therapeutic, diagnostic or research purposes.
  • Therapeutic agents include such compounds as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, lipids, carbohydrates, or recombinant viruses.
  • Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking with single or duplex DNA, and triplex forming oligonucleotides.
  • the molecule linked to an anti-HPV-E629-38 antibody can be an encapsulation system, such as a liposome or micelle that contains a therapeutic composition such as a drug, a nucleic acid (for example, an antisense nucleic acid), or another therapeutic moiety that is preferably shielded from direct exposure to the circulatory system.
  • a therapeutic composition such as a drug, a nucleic acid (for example, an antisense nucleic acid), or another therapeutic moiety that is preferably shielded from direct exposure to the circulatory system.
  • Means of preparing liposomes attached to antibodies are well known to those of skill in the art (see, for example, U.S. Pat. No. 4,957,735; Connor et al., Pharm. Ther. 28:341-365, 1985).
  • Antibodies described herein can also be covalently or non-covalently linked to a detectable label.
  • Detectable labels suitable for such use include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels include magnetic beads, fluorescent dyes (for example, fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (for example, 3 H, 125 1, 35 S, 14 C, or 32 P), enzymes (such as horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (such as polystyrene, polypropylene, latex, and the like) beads.
  • fluorescent dyes for example, fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, and the like
  • radiolabels for example, 3 H, 125 1, 35 S, 14 C, or 32 P
  • enzymes such as horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA
  • colorimetric labels such as colloidal gold or colored glass or plastic (such as polystyren
  • radiolabels may be detected using photographic film or scintillation counters
  • fluorescent markers may be detected using a photodetector to detect emitted illumination
  • Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
  • the present disclosure provides chimeric antigen receptors (CARs) that bind to HPV- E629-38.
  • the CAR binds to a HPV-E629-38 peptide, the HPV-E629-38 peptide comprising the sequence of TIHDIILECV (SEQ ID NO: 1).
  • the CAR binds to a peptide of SEQ ID NO: 1 within the context of a peptide-MHC complex.
  • the CAR may comprise different domains, including an extracellular, antigen-binding domain; a hinge domain; a transmembrane domain; an intracellular signaling domain; and a co-stimulatory domain. It may comprise one or more of each of these domains. It may comprise a subset of these domains.
  • the CAR, or a host cell expressing such a CAR binds to a HPV-E629 -3s:pMHC with an EC50 of less than 10 nM. In some embodiments, the CAR, or a host cell expressing such a CAR, binds to a HPV-E629-38:pMHC with an EC50 of less than 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM.
  • the CAR or a host cell comprising, binds to a HPV-E629-38:pMHC with an EC50 of less than 5 nM. In some embodiments, the CAR shows a 1000-5000 fold improvement in EC50 compared to the parent antibody from which the antigen-binding domain is derived. In some embodiments, the CAR shows a 1000 fold improvement in EC50 compared to the parent antibody from which the antigen-binding domain is derived.
  • the antigen-binding domain of the CAR of the present disclosure may be derived from an anti- HPV-E629-38 antibody or antigen-binding fragment thereof as disclosed herein.
  • the antibody binds to a HPV-E629-38 peptide having the sequence of SEQ ID NO: 1.
  • the antibody binds to a HPV-E629-38 peptide having the sequence of SEQ ID NO : 1 within the context of a peptide-MHC complex.
  • the antigen-binding domain comprises an scFv fragment of an antibody disclosed herein.
  • the antigen-binding domain comprises an scFab fragment of an antibody disclosed herein.
  • the antigen-binding domain comprises a single chain fragment antigen-binding fragment of an antibody according to the present disclosure.
  • antibody fragments that may be comprised by the antigen-binding domain include, but are not limited to, fragment antigen-binding (Fab) fragments, single chain Fab (scFab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, single chain variable fragments (scFv), single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments, diabodies, and multi-specific antibodies formed from antibody fragments.
  • the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs.
  • the antigen-binding domain may comprise an scFv having a VH-linker-VL orientation. In some embodiments, the antigen-binding domain may comprise an scFv having a VL-linker-VH orientation.
  • the antigen-binding domain further comprises a leader sequence or signal peptide.
  • the signal peptide may be positioned at the amino terminus of the scFv.
  • the signal peptide when the heavy chain variable region is N-terminal, the signal peptide may be positioned at the amino terminus of the heavy chain variable region.
  • the signal peptide when the light chain variable region is N-terminal, the signal peptide may be positioned at the amino terminus of the light chain variable region.
  • the signal peptide may comprise any suitable signal peptide.
  • the signal peptide comprises the sequence of MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 18).
  • the CAR comprises a linker, spacer, or hinge sequence between the antigen-binding domain and the transmembrane domain.
  • a hinge sequence is a short sequence of amino acids that, in at least some instances, facilitates flexibility (see, e.g., Woof et al., Nat. Rev. Immunol., 4(2): 89-99 (2004)).
  • the hinge sequence can be any suitable sequence derived or obtained from any suitable molecule.
  • the length of the hinge sequence may be optimized based on the distance between the CAR and the HPV-E629-38 or HPV-E629-38 peptide-MHC complex (pMHC) binding epitope, e.g., longer hinges may be optimal for membrane proximal HPV-E629- 38 or HPV-E629-38 peptide-MHC complex (pMHC) epitopes.
  • pMHC peptide-MHC complex
  • the CAR such as the antigen-binding portion thereof, further includes a hinge, linker or spacer.
  • the hinge may be derived from or include at least a portion of an immunoglobulin Fc region, for example, an IgGl Fc region, an IgG2 Fc region, an IgG3 Fc region, an IgG4 Fc region, an IgE Fc region, an IgM Fc region, or an IgA Fc region.
  • the spacer domain includes at least a portion of an IgGl, an IgG2, an IgG3, an IgG4, an IgE, an IgM, or an IgA immunoglobulin Fc region that falls within its CH2 and CH3 domains.
  • the spacer domain may also include at least a portion of a corresponding immunoglobulin hinge region.
  • the hinge is derived from or includes at least a portion of a modified immunoglobulin Fc region, for example, a modified IgGl Fc region, a modified IgG2 Fc region, a modified IgG3 Fc region, a modified IgG4 Fc region, a modified IgE Fc region, a modified IgM Fc region, or a modified IgA Fc region.
  • the modified immunoglobulin Fc region may have one or more mutations (e.g., point mutations, insertions, deletions, duplications) resulting in one or more amino acid substitutions, modifications, or deletions that cause impaired binding of the spacer domain to an Fc receptor (FcR).
  • the modified immunoglobulin Fc region may be designed with one or more mutations which result in one or more amino acid substitutions, modifications, or deletions that cause impaired binding of the spacer domain to one or more FcR including, but not limited to, FcyRI, FcyR2A, FcyR2Bl, FcyR2B2, FcyR3A, FcyR3B, FcsRI, FcsR2, FcaRI, Fca/pR, or FcRn.
  • a portion of the immunoglobulin constant region serves as a spacer region between the antigen-binding domain, e.g., scFv, and transmembrane domain.
  • the spacer can be of a length that provides for increased responsiveness of the cell following antigenbinding, as compared to in the absence of the spacer.
  • Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between the endpoints of any of the listed ranges.
  • a spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less.
  • the spacer is at or about 12 amino acids in length.
  • Exemplary spacers include a CD28 hinge, IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain.
  • Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, international patent application publication number W02014031687, U.S. Pat. No. 8,822,647 or published app. No.
  • the hinge sequence is derived from the human CD8-alpha molecule or a CD28 molecule. In some embodiments, the hinge sequence is derived from CD8- alpha. In some embodiments, the CD8a hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 21). In some embodiments, the CD8a hinge comprises SEQ ID NO: 21. In some embodiments, the CD8a hinge consists essentially of SEQ ID NO: 21. In some embodiments, the CD8a hinge is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of
  • the CD8a hinge is encoded by SEQ ID NO: 22.
  • the CD28 hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 23). In some embodiments, the CD28 hinge comprises or consists essentially of SEQ ID NO: 23.
  • the CD28 hinge is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of TGTACCATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGA ACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCT TCTAAGCCC (SEQ ID NO: 24).
  • the CD28 hinge is encoded by SEQ ID NO: 24.
  • a short oligo- or polypeptide linker may form the linkage between the transmembrane domain and the intracellular signaling domain(s) of the CAR.
  • a glycine-serine doublet may provide a suitable linker.
  • poly-glycine and poly-serine sequences may provide suitable linkers.
  • the polypeptide linker is between 2 and 10 amino acids in length.
  • the CAR can be designed to comprise a transmembrane domain that is fused to the antigen-binding domain of the CAR.
  • the transmembrane domain that naturally is associated with one of the domains in the CAR is used.
  • the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
  • the transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Typically, the transmembrane domain denotes a single transmembrane alpha helix of a transmembrane protein, also known as an integral protein. Transmembrane regions of particular use in this invention may be derived from (i.e.
  • the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and valine can be used at one or both ends of a synthetic transmembrane domain.
  • a transmembrane domain of the invention can be thermodynamically stable in a membrane. It may be a single alpha helix, a transmembrane beta barrel, a beta-helix of gramicidin A, or any other structure. In some embodiments, transmembrane helices are about 20 amino acids in length.
  • the transmembrane domain in the CAR of the invention is the CD28 transmembrane domain.
  • the CARs comprise a CD28 transmembrane domain.
  • the CD28 transmembrane domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 29).
  • the CD28 transmembrane domain comprises or consists essentially of SEQ ID NO: 29.
  • the CD28 transmembrane domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of
  • the CD28 transmembrane domain is encoded by SEQ ID NO: 30.
  • the intracellular signaling domain or otherwise the cytoplasmic domain of the CAR of the invention triggers or elicits activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed.
  • effector function refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
  • intracellular signaling domain refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While the entire intracellular signaling domain may be employed, in many cases it is not necessary to use the entire chain.
  • intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.
  • intracellular signaling domains for use in the CAR of the invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.
  • TCR T cell receptor
  • T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigenindependent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).
  • Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way.
  • Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs.
  • ITAM-containing primary cytoplasmic signaling sequences that are useful as intracellular signaling domains according to the present disclosure include those derived from an intracellular signaling domain of a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor subunit, an IL-2 receptor subunit, CD3( ⁇ , FcRy, FcRP, CD3y, CD36, CD3s, CD5, CD22, CD79a, CD79b, CD66d, CD278(ICOS), FcsRI, DAP10, and DAP12.
  • the intracellular signaling domain in the CAR of the invention comprises a cytoplasmic signaling sequence derived from CD3.
  • the CD3( ⁇ activation domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 25).
  • the CD3( ⁇ activation domain comprises or consists essentially of SEQ ID NO: 25.
  • the CD3( ⁇ activation domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACC AGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAG CGTAGAGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGG AAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATT GGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGAC TCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTC GC (SEQ ID NO: 26).
  • the CD3( ⁇ activation domain is encoded by SEQ ID NO: 26).
  • the CAR comprises a costimulatory domain.
  • the costimulatory domain refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule.
  • a costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen.
  • Non-limiting examples of co-stimulatory molecules include an MHC class I molecule, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, a Toll ligand receptor, B7-H3, BAFFR, BTLA, BLAME (SLAMF8), CD2, CD4, CD5, CD7, CD8alpha, CD8beta, CD 11 a, LFA-1 (CDl la/CD18), CD 11b, CD 11c, CD l id, CD 18, CD 19, CD 19a, CD27, CD28, CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CD100 (SEMA4D), CD103, CRTAM, 0X40 (CD134), 4-1BB (CD137), SLAM (SLAMF1, CD150, IPO-3), CD160 (BY55),
  • the intracellular domains of CARs of the instant disclosure comprise at least one co-stimulatory domain.
  • the co-stimulatory domain is isolated or derived from CD28.
  • the CD28 co-stimulatory domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 27).
  • the CD28 co-stimulatory domain comprises or consists essentially of SEQ ID NO: 27.
  • the CD28 co-stimulatory domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of AGGAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCCGGAG GCCTGGCCCCACCCGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGATTTCGCCGC CTACCGGAGC (SEQ ID NO: 28).
  • the CD28 co-stimulatory domain is encoded by SEQ ID NO: 28.
  • the cytoplasmic signaling sequences within the intracellular signaling domain of the CAR of the invention may be linked to each other in a random or specified order.
  • a short oligo- or polypeptide linker may form the linkage.
  • the linker comprises glycine-serine doublet.
  • the linker is between 2 and 10 amino acids in length.
  • the intracellular domain comprises the intracellular signaling domain of CD3-( ⁇ and a costimulatory domain derived from CD28.
  • the intracellular domain comprises the intracellular signaling domain of CD3-( ⁇ and a costimulatory domain derived from 4-1BB.
  • the intracellular domain comprises the intracellular signaling domain of CD3-( ⁇ and costimulatory domains derived from both CD28 and 4-1BB.
  • the CARs of the present invention may be further modified, engineered, optimized, or appended in order to provide or select for various features. These features may include, but are not limited to, efficacy, persistence, target specificity, reduced immunogenicity, multi-targeting, enhanced immune response, expansion, growth, reduced off-tumor effect, reduced subject toxicity, improved target cytotoxicity, improved tumor infiltration, detection, selection, targeting, and the like.
  • the cells may be engineered to express another CAR, a suicide mechanism, and may be modified to remove or modify expression of an endogenous receptor or molecule such as a TCR and/or MHC molecule.
  • the vector or nucleic acid sequence encoding the CAR further encodes other genes.
  • the vector or nucleic acid sequence may be constructed to allow for the coexpression of multiple genes using a multitude of techniques including co-transfection of two or more plasmids, the use of multiple or bidirectional promoters, or the creation of bicistronic or multi ci stronic vectors.
  • the construction of multi ci stronic vectors may include the encoding of IRES elements.
  • the CAR expressing cell may further comprise a disruption to one or more endogenous genes.
  • the endogenous gene encodes TCRa, TCRP, CD52, glucocorticoid receptor (GR), deoxycytidine kinase (dCK), or an immune checkpoint protein such as, for example, programmed death-1 (PD-1).
  • the CARs of the present invention and cells expressing these CARs may be further modified to improve efficacy against solid tumors.
  • This increased efficacy may be measured by an increase in tumor cytotoxicity, tumor infiltration, and evasion of or resistance to tumor immunosuppressive mediators.
  • enhanced anti-tumor efficacy may be characterized by increased TCR signaling, increased cytokine release, enhanced killing of tumor cells, increased T cell infiltration of established tumors, improved tumor trafficking, attenuated tumor-induced hypofunction, and improved migration and chemotaxis.
  • the CAR expressing cells are further modified to evade or neutralize the activity of immunosuppressive mediators, including, but not limited to prostaglandin E2 (PGE2) and adenosine.
  • this evasion or neutralization is direct.
  • this evasion or neutralization is mediated via the inhibition of protein kinase A (PKA) with one or more binding partners, for example ezrin.
  • PGE2 prostaglandin E2
  • the CAR-expressing cells further express the peptide “regulatory subunit I anchoring disruptor” (RIAD).
  • RIAD is thought to inhibit the association of protein kinase A (PKA) with ezrin, which thus prevents PKA-mediated inhibition of TCR activation (Newick et al. Cancer Res 2016 August; 76(15 Suppl): Abstract nr B27).
  • PKA protein kinase A
  • the CAR expressing cells of the invention may induce a broad antitumor immune response consistent with epitope spreading.
  • the CAR expressing cells of the invention further comprise a homing mechanism.
  • the cell may transgenically express one or more stimulatory chemokines or cytokines or receptors thereof.
  • the cells are genetically modified to express one or more stimulatory cytokines.
  • one or more homing mechanisms are used to render the inventive cells resistant to an inhibitory tumor microenvironment.
  • the CAR expressing cells are further modified to release inducible cytokines upon CAR activation, e.g., to attract or activate innate immune cells to a targeted tumor (so-called fourth generation CARs or TRUCKS).
  • CARs may co-express homing molecules, e.g., CCR4 or CCR2b, to increase tumor trafficking. Controlling CAR Expression
  • apoptosis using, e.g., a caspase fused to a dimerization domain (see, e.g, Di et al., N Engl. J. Med. 2011 Nov. 3; 365(18): 1673-1683), can be used as a safety switch in the CAR therapy of the instant invention.
  • CAR- expressing cells can also express an inducible Caspase-9 (iCaspase-9) molecule that, upon administration of a dimerizer drug (e.g., rimiducid (also called API 903 (Bellicum Pharmaceuticals) or AP20187 (Ariad)) leads to activation of the Caspase-9 and apoptosis of the cells.
  • a dimerizer drug e.g., rimiducid (also called API 903 (Bellicum Pharmaceuticals) or AP20187 (Ariad)
  • the iCaspase-9 molecule contains a chemical inducer of dimerization (CID) binding domain that mediates dimerization in the presence of a CID. This results in inducible and selective depletion of CAR-expressing cells.
  • CID chemical inducer of dimerization
  • the iCaspase-9 molecule is encoded by a nucleic acid molecule separate from the CAR-encoding vector(s). In some cases, the iCaspase-9 molecule is encoded by the same nucleic acid molecule as the CAR-encoding vector.
  • the iCaspase-9 can provide a safety switch to avoid any toxicity of CAR-expressing cells. See, e.g., Song et al. Cancer Gene Ther. 2008; 15(10):667-75; Clinical Trial Id. No. NCT02107963; and Di Stasi et al. N. Engl. J. Med. 2011; 365: 1673-83.
  • Alternative strategies for regulating the CAR therapy of the instant invention include utilizing small molecules or antibodies that deactivate or turn off CAR activity, e.g., by deleting CAR-expressing cells, e.g., by inducing antibody dependent cell-mediated cytotoxicity (ADCC).
  • CAR-expressing cells described herein may also express an antigen that is recognized by molecules capable of inducing cell death, e.g., ADCC or compliment-induced cell death.
  • CAR expressing cells described herein may also express a receptor capable of being targeted by an antibody or antibody fragment.
  • receptors examples include EpCAM, VEGFR, integrins (e.g., integrins avP3, a4, aI3/4p3, a4p7, a5pi, avP3, av), members of the TNF receptor superfamily (e.g., TRAIL-R1, TRAIL-R2), PDGF Receptor, interferon receptor, folate receptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG-72, IL-6 receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CD5, CD11, CDl la/LFA-1, CD15, CD18/ITGB2, CD19, CD20, CD22, CD23/lgE Receptor, CD25, CD28, CD30, CD33, CD38, CD40, CD41, CD44, CD51, CD52, CD62L, CD74, CD80, CD125, CD147/basigin, CD152/CTLA-4, CD154/
  • CAR-expressing cells described herein may also express a truncated epidermal growth factor receptor (EGFR) which lacks signaling capacity but retains the epitope that is recognized by molecules capable of inducing ADCC, e.g., cetuximab (ERBITUX.RTM.), such that administration of cetuximab induces ADCC and subsequent depletion of the CAR-expressing cells (see, e.g., WO2011/056894, and Jonnalagadda et al., Gene Then 2013; 20(8)853-860).
  • EGFR epidermal growth factor receptor
  • the CAR cell comprises a polynucleotide encoding a suicide polypeptide, such as for example RQR8. See, e.g., WO2013153391 A, which is hereby incorporated by reference in its entirety.
  • the suicide polypeptide may be expressed at the surface of a CAR cell.
  • the suicide polypeptide may also comprise a signal peptide at the amino terminus.
  • Another strategy includes expressing a highly compact marker/ suicide gene that combines target epitopes from both CD32 and CD20 antigens in the CAR-expressing cells described herein, which binds rituximab, resulting in selective depletion of the CAR-expressing cells, e.g., by ADCC (see, e.g., Philip et al., Blood. 2014; 124(8)1277-1287).
  • Other methods for depleting CAR-expressing cells described herein include administration of CAMPATH, a monoclonal anti-CD52 antibody that selectively binds and targets mature lymphocytes, e.g., CAR-expressing cells, for destruction, e.g., by inducing ADCC.
  • the CAR-expressing cell can be selectively targeted using a CAR ligand, e.g., an anti -idiotypic antibody.
  • the anti -idiotypic antibody can cause effector cell activity, e.g, ADCC or ADC activities, thereby reducing the number of CAR- expressing cells.
  • the CAR ligand, e.g., the anti -idiotypic antibody can be coupled to an agent that induces cell killing, e.g., a toxin, thereby reducing the number of CAR- expressing cells.
  • the CAR molecules themselves can be configured such that the activity can be regulated, e.g., turned on and off as described below.
  • a regulatable CAR where the CAR activity can be controlled is desirable to optimize the safety and efficacy of a CAR therapy.
  • a RCAR comprises a set of polypeptides, typically two in the simplest embodiments, in which the components of a standard CAR described herein, e.g., an antigenbinding domain and an intracellular signaling domain, are partitioned on separate polypeptides or members.
  • the set of polypeptides include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigen-binding domain to an intracellular signaling domain. Additional description and exemplary configurations of such regulatable CARs are provided herein and in International Publication No. WO 2015/090229, hereby incorporated by reference in its entirety.
  • an RCAR comprises two polypeptides or members: I) an intracellular signaling member comprising an intracellular signaling domain, e.g., a primary intracellular signaling domain described herein, and a first switch domain; 2) an antigen-binding member comprising an antigen-binding domain, e.g., that specifically binds a tumor antigen described herein, as described herein and a second switch domain.
  • the RCAR comprises a transmembrane domain described herein.
  • a transmembrane domain can be disposed on the intracellular signaling member, on the antigen-binding member, or on both.
  • the order can be as provided, but other orders are included as well.
  • the order is as set out in the text, but in other embodiments, the order can be different.
  • the order of elements on one side of a transmembrane region can be different from the example, e.g, the placement of a switch domain relative to an intracellular signaling domain can be different, e.g, reversed.
  • the CAR expressing immune cell may only transiently express a CAR.
  • the cells of the invention may be transduced with mRNA comprising a nucleic acid sequence encoding an inventive CAR.
  • the present invention also includes an RNA construct that can be directly transfected into a cell.
  • a method for generating mRNA for use in transfection involves in vitro transcription (IVT) of a template with specially designed primers, followed by poly A addition, to produce a construct containing 3’ and 5’ untranslated sequences (“UTRs”), a 5’ cap and/or Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length.
  • RNA so produced can efficiently transfect different kinds of cells.
  • the template includes sequences for the CAR.
  • an RNA CAR vector is transduced into a cell by electroporation.
  • the CAR expressing cells of the present invention may further comprise one or more additional CARs. These additional CARs may or may not be specific for HPV-E629-38 or HPV- E629-38 peptide-MHC complex. In some embodiments, the one or more additional CARs may act as inhibitory or activating CARs. In some aspects, the HPV-E629-38-targeting or HPV-E629-38 peptide-MHC complex-targeting CAR is the stimulatory or activating CAR; in other aspects, it is the costimulatory CAR. In some embodiments, the cells further include inhibitory receptors such as inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl.
  • an inhibitory CAR recognizing an antigen other than HPV-E629-38 or HPV-E629 -38 peptide-MHC complex, whereby an activating signal delivered through the HPV- E629-38-targeting or HPV-E629-38 peptide-MHC complex-targeting CAR is diminished or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.
  • the CAR expressing cells of the present invention may further comprise one or more additional CARs that may target one or more antigens selected from the group of: BCMA; BCR-Abl; BST2; CAIX; CD19; CD20; CD22; CD123; CD171; CD30; CD33; CD38; CD44v6; CD44v7/8; CEA; CLL-1; EGFRvIII; EGP-2; EGP-40; ERBB2 (Her2/neu); EPC AM; fetal acetylcholine receptor, FBP; FLT3; Folate receptor alpha; GD2; GD3; Her3 (ErbB3); Her4 (ErbB4); k-light chain; KDR; MAD-CT-1; MAD-CT-2; MAGE-A1; MARTI; ML-IAP; MYCN; Oncofetal antigen (h5T4); NKG2D ligands PDK1; PDL1; PSCA; PSMA
  • the antigen-binding domain of the inventive CAR is affinity tuned.
  • the affinity of the anti- HPV-E629-38 or anti- HPV-E629-3s:peptide-MHC complex CAR antigen-binding domain is adjusted to discriminate cells overexpressing HPV- E629-38, e.g. tumor cells, from normal tissues which express low or minimal physiological levels ofHPV-E6 2 9 -38. This may be accomplished, e.g., through the use of a CAR-expressing T cell with target antigen affinities varying over three orders of magnitude (Liu et al. Cancer Res 2015 September; 75( 17): 3596-607).
  • the antigen-binding domain of the CAR is or is part of an immunoconjugate, in which the antigen-binding domain is conjugated to one or more heterologous molecule(s), such as, but not limited to, a cytotoxic agent, an imaging agent, a detectable moiety a multimerization domain or other heterologous molecule.
  • Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211, 1131, 1125, Y90, Rel86, Rel88, Sml53, Bi212, P32, Pb212 and radioactive isotopes of Lu); chemotherapeutic agents (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins.
  • radioactive isotopes e.g., At211, 1131, 1125, Y90, Rel86, Rel88, Sml53, Bi212, P32, Pb212 and radioactive isotopes
  • the antigen-binding domain is conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g. , protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
  • cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g. , protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
  • the CAR expressing cells of the invention may be further genetically modified to express the dominant negative form of the transforming growth factor (TGF) beta receptor (DNR).
  • TGF transforming growth factor beta receptor
  • the CAR expressing cell may be specific for another antigen, including a tumor antigen in some cases.
  • the transformed host cells may be selected for specificity for one or more strong viral antigens or may be transformed to exhibit specificity for these antigens.
  • the cells are pp65CMV-specific T cells, CMV-specific T cells, EBV-specific T cells, Varicella Virus-specific T cells, Influenza Virusspecific T cells and/or Adenovirus-specific T cells.
  • the cells of the invention may be further modified to overexpress pro-survival signals, reverse anti-survival signals, overexpress Bcl-xL, overexpress hTERT, lack Fas, or express a TGFp dominant negative receptor. Persistence may also be facilitated by the administration of cytokines, e.g., IL-2, IL-7, and IL-15.
  • cytokines e.g., IL-2, IL-7, and IL-15.
  • the term “functional portion” when used in reference to a CAR refers to any part or fragment of the CAR of the invention, which part or fragment retains the biological activity of the CAR of which it is a part (the parent CAR).
  • Functional portions encompass, for example, those parts of a CAR that retain the ability to recognize target cells, or detect, treat, or prevent a disease, to a similar extent, the same extent, or to a higher extent, as the parent CAR.
  • the functional portion can comprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of the parent CAR.
  • the functional portion can comprise additional amino acids at the amino or carboxyl terminus of the portion, or at both termini, which additional amino acids are not found in the amino acid sequence of the parent CAR.
  • the additional amino acids do not interfere with the biological function of the functional portion, e.g., recognize target cells, detect cancer, treat or prevent cancer, etc.
  • the additional amino acids enhance the biological activity, as compared to the biological activity of the parent CAR.
  • the term “functional variant” as used herein refers to a CAR, polypeptide, or protein having substantial or significant sequence identity or similarity to a parent CAR, which functional variant retains the biological activity of the CAR of which it is a variant.
  • Functional variants encompass, for example, those variants of the CAR described herein (the parent CAR) that retain the ability to recognize target cells to a similar extent, the same extent, or to a higher extent, as the parent CAR.
  • the functional variant can, for instance, be at least about 30%, 50%, 75%, 80%, 90%, 98% or more identical in amino acid sequence to the parent CAR.
  • a functional variant can, for example, comprise the amino acid sequence of the parent CAR with at least one conservative amino acid substitution.
  • the functional variants can comprise the amino acid sequence of the parent CAR with at least one non-conservative amino acid substitution.
  • the non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent CAR.
  • Amino acid substitutions of the inventive CARs are preferably conservative amino acid substitutions.
  • Conservative amino acid substitutions are known in the art, and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid that has the same or similar chemical or physical properties.
  • the conservative amino acid substitution can be an acidic/negatively charged polar amino acid substituted for another acidic/negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Vai, He, Leu, Met, Phe, Pro, Trp, Cys, Vai, etc.), a basic/positively charged polar amino acid substituted for another basic/positively charged polar amino acid (e.g.
  • Lys, His, Arg, etc. an uncharged amino acid with a polar side chain substituted for another uncharged amino acid with a polar side chain (e.g, Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid with a betabranched side-chain substituted for another amino acid with a beta-branched side-chain (e.g, He, Thr, and Vai), an amino acid with an aromatic side-chain substituted for another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.
  • amino acids may be added or removed from the sequence based on vector design.
  • the CAR can consist essentially of the specified amino acid sequence or sequences described herein, such that other components, e.g., other amino acids, do not materially change the biological activity of the functional variant.
  • the CARs of embodiments of the invention can be of any length, /. ⁇ ., can comprise any number of amino acids, provided that the CARs (or functional portions or functional variants thereof) retain their biological activity, e.g., the ability to specifically bind to antigen, detect diseased cells in a mammal, or treat or prevent disease in a mammal, etc.
  • the CAR can be about 50 to about 5000 amino acids long, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length.
  • the CARs of embodiments of the invention can comprise synthetic amino acids in place of one or more naturally-occurring amino acids.
  • Such synthetic amino acids are known in the art, and include, for example, aminocyclohexane carboxylic acid, norleucine, a-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4- aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, P- phenylserine P-hydroxyphenylalanine, phenylglycine, a-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid
  • the CARs of embodiments of the invention can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N- acylated, cyclized via, e.g., a disulfide bridge, or converted into an acid addition salt and/or optionally dimerized or polymerized, or conjugated, or undergo additional post-translational modifications.
  • the CARs of embodiments of the invention can be obtained by methods known in the art.
  • the CARs may be made by any suitable method of making polypeptides or proteins. Suitable methods of de novo synthesizing polypeptides and proteins are described in references, such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Pat. No. 5,449,752.
  • polypeptides and proteins can be recombinantly produced using the nucleic acids described herein using standard recombinant methods. See, for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, N Y, 1994. Further, some of the CARs of the invention (including functional portions and functional variants thereof) can be isolated and/or purified from a source, such as a plant, a bacterium, an insect, a mammal, e.g., a rat, a human, etc.
  • a source such as a plant, a bacterium, an insect, a mammal, e.g., a rat, a human, etc.
  • the CARs described herein can be commercially synthesized by companies.
  • the inventive CARs can be synthetic, recombinant, isolated, and/or purified.
  • the CARs described herein comprise a sequence of SEQ ID NO: 19, or a sequence comprising at least 80%, 90%, 95%, or 98% identity thereto. In some embodiments, the CARs described herein comprise a sequence of SEQ ID NO: 20, or a sequence comprising at least 80%, 90%, 95%, or 98% identity thereto. Table 4 provides non-limiting illustrative CAR sequences according to the disclosure.
  • the present disclosure also provides nucleic acids encoding the polypeptides, antibodies, antigen-binding antibody fragments, or chimeric antigen receptors as disclosed herein.
  • the present invention also provides vectors in which a nucleic acid of the present invention is inserted.
  • Vectors derived from retroviruses are suitable tools to achieve long-term gene transfer since they allow for genetic stability and high expression, in addition to having a flexible genome. Furthermore, clinical experience with retroviral vectors provides guidance for optimizing efficacy and safety in their use.
  • the expression of natural or synthetic nucleic acids encoding CARs is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide or portions thereof to a promoter, and incorporating the construct into an expression vector.
  • the vectors can be suitable for replication and integration in eukaryotes.
  • Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.
  • the expression constructs of the present invention may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties.
  • the invention provides a gene therapy vector.
  • the nucleic acid can be cloned into a number of types of vectors.
  • the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • the expression vector may be provided to a cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals.
  • Viruses, which are useful as vectors include, but are not limited to, retroviruses, gammaretroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retroviral systems are known in the art.
  • adenovirus vectors are used.
  • a number of adenovirus vectors are known in the art.
  • adeno-associated virus vectors are used.
  • retrovirus vectors are used.
  • Additional promoter elements e.g., enhancers, regulate the frequency of transcriptional initiation.
  • these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.
  • Various promoter sequences may be used, including, but not limited to the immediate early cytomegalovirus (CMV) promoter, Elongation Growth Factor- la (EF-la), simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.
  • CMV immediate early cytomegalovirus
  • EF-la Elongation Growth Factor- la
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HSV human immunodeficiency virus
  • LTR human immunodeficiency virus
  • inducible promoters are also contemplated as part of the invention.
  • the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired.
  • inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.
  • Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, betagalactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).
  • Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
  • the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter.
  • Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
  • the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art.
  • the expression vector can be transferred into a host cell by physical, chemical, or biological means.
  • Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
  • Viral vectors, and especially retroviral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.
  • Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.
  • Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in produce is a liposome (e.g., an artificial membrane vesicle).
  • an exemplary delivery vehicle is a liposome.
  • lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo).
  • the nucleic acid may be associated with a lipid.
  • the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • Lipids suitable for use can be obtained from commercial sources.
  • DMPC dimyristyl phosphatidylcholine
  • DCP dicetyl phosphate
  • Choi cholesterol
  • DMPG dimyristyl phosphatidylglycerol
  • Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20 degrees Celsius.
  • Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10).
  • compositions that have different structures in solution than the normal vesicular structure are also encompassed.
  • the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.
  • lipofectamine-nucleic acid complexes are also contemplated.
  • assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.
  • molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR
  • biochemical assays such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.
  • cells, cell populations, and compositions containing the cells e.g., cells comprising a nucleic acid sequence encoding an anti-HPV-E629-38 or anti-HPV-E629- 3s:peptide-MHC complex chimeric antigen receptor.
  • the cells can be immune cells, such as T cells or NK cells.
  • pharmaceutical compositions and formulations for administration such as for adoptive cell therapy.
  • therapeutic methods for administering the cells and compositions to subjects e.g., patients.
  • the cells generally are eukaryotic cells, such as mammalian cells, and typically are human cells, more typically primary human cells, e.g., allogeneic or autologous donor cells.
  • the cells for introduction of the CAR may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject.
  • the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered.
  • the subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
  • the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immune systems, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells.
  • Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs).
  • the cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen.
  • the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4 + cells, CD8 + cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation.
  • T cells or other cell types such as whole T cell populations, CD4 + cells, CD8 + cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation.
  • the cells may be allogeneic and/or autologous.
  • the methods included are off-the-shelf methods.
  • the cells are pluripotent and/or multipotent, such as stem cells, induced pluripotent stem cells (iPSCs), or T cells that either lack or are engineered to be deficient in T cell receptor function.
  • the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them, as described herein, and re-introducing them into the same patient, before or after cry opreservation.
  • T cells and/or of CD4 + and/or of CD8 + T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.
  • TN naive T
  • TSCM stem cell memory T
  • TCM central memory T
  • TEM effector memory T
  • TIL tumor-infiltrating lymphocyte
  • the cells are natural killer (NK) cells, Natural Killer T (NKT) cells, cytokine-induced killer (CIK) cells, tumor-infiltrating lymphocytes (TIL), lymphokine- activated killer (LAK) cells, or the like.
  • the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.
  • the cells are derived from cell lines, e.g., T cell lines.
  • the cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, nonhuman primate, and pig.
  • the methods of the present disclosure relate, in part, to the discovery by the present applicant of antibodies that binds to HPV-E629-38 peptide-MHC complexes and their incorporation into CARs.
  • an advantage of some embodiments of the methods of the present disclosure is that they specifically bind to HPV-E629-38 antigen or pMHC complex thereof over other peptide antigens.
  • There are no known antibodies and receptors targeting HPV- E629-38 suggesting the need to identify novel binding agents for use in therapeutics and diagnostics. This need is satisfied by the present disclosure.
  • the present disclosure provides methods of using the disclosed anti- HPV-E629 -38:pMHC antibodies and antigen binding fragments thereof for the treatment of cancer.
  • the present disclosure provides methods of using the disclosed anti-HPV- E629-38:pMHC chimeric antigen receptors (CARs) and cells comprising same for the treatment of cancer.
  • the compositions of the present disclosure may be used in diagnostic methods to detect and/or quantify the presence of HPV positive cells.
  • the method comprises administering a composition.
  • the composition comprises a plurality of cells, polypeptides, antibodies, antibody fragments, CARs, nucleic acids, and/or vectors described herein.
  • the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers, diluents, or excipients.
  • the disclosure provides a method for diagnosing a disease involving HPV positive cells, the method comprising detecting or measuring an HPV-derived peptide in cells using a polypeptide described herein, an antibody described herein or a bispecific antibody described herein.
  • the disclosure provides a method for treating an HPV positive cancer in a subject in need thereof, the method comprising administering a polypeptide described herein, an antibody described herein, or a pharmaceutical composition described herein to the subject.
  • the disclosure provides a method for treating an HPV positive cancer in a subject in need thereof, the method comprising administering a composition comprising a plurality of the cells described herein or the pharmaceutical composition described herein to the subject.
  • HPV positive cancers can be used to treat HPV positive cancers.
  • the methods may be used to treat HPV positive cervical cancer, HPV positive oropharyngeal cancer, HPV positive oral cancer, HPV positive anal cancer, HPV positive penile cancer, HPV positive vaginal cancer, HPV positive vulvar cancer, or other HPV positive solid tumors.
  • HPV positive tumor cells may be identified via known methods.
  • HPV positive tumor cells may be identified via PCR, fluorescence in situ hybridization, and real-time PCR, DNA sequencing, or other methods known in the art to detect HPV associated polynucleotides or polypeptides.
  • HPV positive cancer cells may be detected using the antibodies described herein.
  • Cancers that may be treated include tumors containing cells that are HPV positive.
  • the cancers may be vascularized, or not yet substantially vascularized, as well as vascularized tumors.
  • the cancers may comprise non-solid tumors (such as hematological tumors, for example, leukemias and lymphomas) or may comprise solid tumors.
  • Types of cancers to be treated with the CARs of the invention include, but are not limited to, solid tumors, epithelial cancers, and hematological malignancies.
  • the cancer is a hematologic malignancy, multiple myeloma, an epithelial cancer, a solid tumor, melanoma, head and neck cancer, breast cancer, lung cancer, or synovial sarcoma.
  • the cancer may be selected from a carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas.
  • sarcomas e.g., sarcomas, carcinomas, and melanomas.
  • adult tumors/cancers and pediatric tumor s/cancers are also included.
  • Hematologic cancers are cancers of the blood or bone marrow.
  • hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin’s disease, nonHodgkin’s lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom’s macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.
  • Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas).
  • solid tumors such as sarcomas and carcinomas
  • solid tumors include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing’s tumor, leciomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wil
  • cytokines e.g., interferon-y, granulocyte/monocyte colony stimulating factor (GM-CSF), tumor necrosis factor a (TNF-a) or interleukin 2 (IL-2)
  • cytokines e.g., interferon-y, granulocyte/monocyte colony stimulating factor (GM-CSF), tumor necrosis factor a (TNF-a) or interleukin 2 (IL-2)
  • GM-CSF granulocyte/monocyte colony stimulating factor
  • TNF-a tumor necrosis factor a
  • IL-2 interleukin 2
  • a biopsy is the removal of tissue and/or cells from an individual. Such removal may be to collect tissue and/or cells from the individual in order to perform experimentation on the removed tissue and/or cells. This experimentation may include experiments to determine if the individual has and/or is suffering from a certain condition or disease-state.
  • the condition or disease may be, e.g., cancer.
  • the sample comprising cells of the host can be a sample comprising whole cells, lysates thereof, or a fraction of the whole cell lysates, e.g., a nuclear or cytoplasmic fraction, a whole protein fraction, or a nucleic acid fraction. If the sample comprises whole cells, the cells can be any cells of the host, e.g., the cells of any organ or tissue, including blood cells or endothelial cells.
  • E6 A mechanism by which HPV causes cancer involves a gene carried by the virus, known as E6.
  • E6 is a classic viral oncogene that inactivates host cancer defenses.
  • E6 binds the tumor suppressor p53 (Scheffner et al. PNAS. 1991 88(13):5523-7).
  • Expression of E6 is required for continued survival and growth of tumor cells (DeFilippis et al. J Virol. 2003 77(2): 1551-63).
  • E6 has little homology to human proteins so it is an especially attractive targets for drug discovery, as it affords absolute discrimination from normal cells. However, it reside in the cytoplasm, inaccessible to antibodies and other large molecules.
  • E6 is not considered conventionally druggable; /. ⁇ ., inhibitable by small molecules.
  • various E6 peptides are known to elicit immune response by display on the cell surface, bound to specific HLA class I molecules (Altmann et al. Eur J Cancer. 1992 28(2-3):326-33; Blatnik et al. Proteomics. 2018 18(1 l):el700390).
  • HLA class I molecules Altmann et al. Eur J Cancer. 1992 28(2-3):326-33; Blatnik et al. Proteomics. 2018 18(1 l):el700390.
  • serotypes 16 and 18 have attracted special attention.
  • E6 is pursued in the tumor-infiltrating lymphocyte (TIL) and TCR-T fields (Stefanovic et al. J Clin Oncol. 2015 33(14): 1543-50; Doran et al. J Clin Oncol. 2019 37(30):2759-2768
  • the subject referred to herein may be any living subject.
  • the subject is a mammal.
  • the mammal referred to herein can be any mammal.
  • the term “mammal” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits.
  • the mammals may be from the order Carnivora, including Felines (cats) and Canines (dogs).
  • the mammals may be from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses).
  • the mammals may be of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes).
  • the subject, to whom the cells, cell populations, or compositions are administered is a primate, such as a human.
  • the primate is a monkey or an ape.
  • the subject can be male or female and can be any suitable age, including infantjuvenile, adolescent, adult, and geriatric subjects.
  • the patient or subject is a validated animal model for disease, adoptive cell therapy, and/or for assessing toxic outcomes such as cytokine release syndrome (CRS).
  • CRS cytokine release syndrome
  • the subject has persistent or relapsed disease, e.g., following treatment with another immunotherapy and/or other therapy, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT.
  • HSCT hematopoietic stem cell transplantation
  • the administration effectively treats the subject despite the subject having become resistant to another therapy.
  • the subject has not relapsed but is determined to be at risk for relapse, such as at a high risk of relapse, and thus the compound or composition is administered prophylactically, e.g., to reduce the likelihood of or prevent relapse.
  • the methods include administration of CAR expressing cells or a composition containing the cells to a subject, tissue, or cell, such as one having, at risk for, or suspected of having a disease, condition or disorder associated with HPV infection or HPV positive cells.
  • the cells, populations, and compositions are administered to a subject having the particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy.
  • the cells or compositions are administered to the subject, such as a subject having or at risk for the disease or condition.
  • the methods thereby treat, e.g., ameliorate one or more symptom of the disease or condition, such as by lessening tumor burden in an HPV positive cancer.
  • compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired.
  • administration may be topical, parenteral, or enteral.
  • adoptive cell therapy methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions.
  • adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.
  • compositions of the invention are suitable for parenteral administration.
  • parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ.
  • Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like.
  • parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intratumoral, intrasynovial injection or infusions; and kidney dialytic infusion techniques.
  • parenteral administration of the compositions of the present invention comprises intravenous or intraarterial administration.
  • compositions comprising polypeptides comprising the disclosed anti-HPV-E629-3s:pMHC antigen binding domains, anti-HPV-E629- 3s:pMHC chimeric antigen receptors (CARs) and cells comprising same, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the disclosure provides a pharmaceutical composition comprising a polypeptide described herein, a CAR described herein, an antibody described herein, a nucleic acid described herein or a vector described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.
  • the pharmaceutical composition comprises a cell described herein and a pharmaceutically acceptable carrier, diluent, or excipient.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide described herein, a CAR described herein, an antibody described herein, a nucleic acid described herein or a vector described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.
  • the pharmaceutical composition comprises a cell described herein and a pharmaceutically acceptable carrier, diluent, or excipient.
  • the disclosure provides a method for diagnosing a disease involving HPV positive cells, the method comprising detecting or measuring an HPV-derived peptide in cells using a polypeptide described herein, an antibody described herein or a bispecific antibody described herein.
  • the disclosure provides a method for treating an HPV positive cancer in a subject in need thereof, the method comprising administering a polypeptide described herein, an antibody described herein, or a pharmaceutical composition described herein to the subject.
  • Formulations of a pharmaceutical composition suitable for parenteral administration typically generally comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
  • a suitable vehicle e.g. sterile pyrogen-free water
  • Parenteral formulations also include aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents, but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.
  • parenteral administration forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired.
  • Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, or in a liposomal preparation.
  • Formulations for parenteral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
  • oral refers to administration of a compound or composition to an individual by a route or mode along the alimentary canal.
  • oral routes of administration of a composition include, without limitation, swallowing liquid or solid forms of a composition from the mouth, administration of a composition through a nasojejunal or gastrostomy tube, intraduodenal administration of a composition, and rectal administration, e.g., using suppositories for the lower intestinal tract of the alimentary canal.
  • compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, semisolids, monophasic compositions, multiphasic compositions (e.g., oil-in-water, water-in-oil), foams, microsponges, liposomes, nanoemulsions, aerosol foams, polymers, fullerenes, and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • compositions and formulations for parenteral, intrathecal, or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carder compounds and other pharmaceutically acceptable carriers or excipients.
  • compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.
  • compositions of the present invention may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, aerosols, and enemas.
  • the compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the pharmaceutical compositions may be formulated and used as foams.
  • Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product.
  • Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention.
  • cationic lipids such as lipofectin (U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and poly cationic molecules, such as polylysine (WO 97/30731), also enhance the cellular uptake of oligonucleotides.
  • compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions.
  • the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • additional materials useful in physically formulating various dosage forms of the compositions of the present invention such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • such materials when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention.
  • the formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • Formulations comprising anti-HPV-E629-38 antibodies or anti-HPV-E629-3s:pMHC CAR expressing cells may include pharmaceutically acceptable excipient(s).
  • Excipients included in the formulations will have different purposes depending, for example, on the CAR construct, the subpopulation of cells used, and the mode of administration. Examples of generally used excipients include, without limitation: saline, buffered saline, dextrose, water-for-infection, glycerol, ethanol, and combinations thereof, stabilizing agents, solubilizing agents and surfactants, buffers and preservatives, tonicity agents, bulking agents, and lubricating agents.
  • formulations comprising anti-HPV-E629-38 antibodies or anti-HPV-E629-3s:pMHC CAR expressing cells will typically have been prepared and cultured in the absence of any non-human components, such as animal serum (e.g., bovine serum albumin).
  • animal serum e.g., bovine serum albumin
  • the formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the binding molecules or cells, preferably those with activities complementary to the binding molecule or cell, where the respective activities do not adversely affect one another.
  • active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
  • the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.
  • chemotherapeutic agents e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.
  • the pharmaceutically active agents or drugs may comprise immune checkpoint inhibitors, e.g., drugs that target PD-1, MAGE-A3, PD-L2, LAG3, CTLA4, KIR, CD244, B7-H3, B7-H4, BTLA, HVEM, GAL9, TIM3, and/or A2aR.
  • immune checkpoint inhibitors e.g., drugs that target PD-1, MAGE-A3, PD-L2, LAG3, CTLA4, KIR, CD244, B7-H3, B7-H4, BTLA, HVEM, GAL9, TIM3, and/or A2aR.
  • inhibitors include, but are not limited to, pidilizumab, nivolumab, pembrolizumab, atezolizumab, MDX-1105, BMS-936559, MED14736, MPDL3280A, MSB0010718C, tremelimumab, and ipilimumab, which may be administered alone or in combination with other agents, e.g., GM-CSF.
  • the pharmaceutical composition in some aspects can employ time-released, delayed release, and sustained release delivery systems such that the delivery of the composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated.
  • Many types of release delivery systems are available and known. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician.
  • Administration can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.
  • the pharmaceutical composition in some embodiments contains the anti-HPV-E629-38 antibodies or anti-HPV-E629-38 CAR cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount.
  • Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined.
  • the desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.
  • the dose or the frequency of administration varies depending on the objective therapeutic effect, administration method, treating period, age, body weight and the like, in some embodiments, the dose may be between 10 pg/kg to 10 mg/kg per day.
  • a subject in the context of genetically engineered cells expressing the CARs, is administered the range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells
  • the cells or population of cells can be administrated in one or more doses.
  • said effective amount of cells can be administrated as a single dose.
  • said effective amount of cells can be administrated as more than one dose over a period time. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the patient.
  • the cells or population of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of optimal ranges of effective amounts of a given cell type for a particular disease or conditions within the skill of the art.
  • An effective amount means an amount which provides a therapeutic or prophylactic benefit.
  • the dosage administrated will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired.
  • an effective amount of cells or composition comprising those cells are administrated parenterally.
  • administration can be an intravenous administration.
  • administration can be directly done by injection within a tumor.
  • the amount or dose of the inventive CAR material administered should be sufficient to effect a therapeutic or prophylactic response in the subject or animal over a reasonable time frame.
  • the dose of the inventive CAR material should be sufficient to bind to antigen, or detect, treat or prevent disease in a period of from about 2 hours or longer, e.g., about 12 to about 24 or more hours, from the time of administration. In certain embodiments, the time period could be even longer.
  • the dose will be determined by the efficacy of the particular inventive CAR material and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated.
  • an assay which comprises, for example, comparing the extent to which target cells are lysed or IFN-y is secreted by T cells expressing the inventive CAR, polypeptide, or protein upon administration of a given dose of such T cells to a mammal, among a set of mammals of which is each given a different dose of the T cells, could be used to determine a starting dose to be administered to a mammal.
  • the extent to which target cells are lysed or IFN-y is secreted upon administration of a certain dose can be assayed by methods known in the art.
  • the cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent.
  • another therapeutic intervention such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent.
  • the cells or antibodies in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order.
  • the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa.
  • the cells or antibodies are administered prior to the one or more additional therapeutic agents.
  • the cells or antibodies are administered after to the one or more additional therapeutic agents.
  • a lymphodepleting chemotherapy is administered to the subject prior to, concurrently with, or after administration (e.g., infusion) of CAR cells.
  • the lymphodepleting chemotherapy is administered to the subject prior to administration of the cells.
  • the lymphodepleting chemotherapy ends 1-4 days (e.g., 1, 2, 3, or 4 days) prior to CAR cell infusion.
  • multiple doses of CAR cells are administered, e.g., as described herein.
  • a lymphodepleting chemotherapy is administered to the subject prior to, concurrently with, or after administration (e.g., infusion) of a CAR-expressing cell described herein.
  • lymphodepletion examples include, but may not be limited to, nonmyeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, etc.
  • lymphodepleting agents include, but are not limited to, antithymocyte globulin, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD8 antibodies, anti-CD52 antibodies, anti-CD2 antibodies, TCRaP blockers, anti-CD20 antibodies, anti-CD19 antibodies, Bortezomib, rituximab, anti-CD 154 antibodies, rapamycin, CD3 immunotoxin, fludarabine, cyclophosphamide, busulfan, melphalan, Mabthera, Tacrolimus, alefacept, alemtuzumab, OKT3, OKT4, OKT8, OKT11, fmgolimod, anti-CD40 antibodies, anti- BR3 antibodies, Campath-IH, anti-CD25 antibodies,
  • the antibodies of the disclosure are useful in methods known in the art relating to the localization and/or quantitation of a HPV-E629-38 polypeptide or HPV-E629-38 peptide-MHC complex (e.g., for use in measuring levels of the HPV-E629-38 polypeptide within appropriate physiological samples, for use in diagnostic methods, for use in imaging the polypeptide, and the like).
  • the antibodies of the disclosure are useful in isolating a HPV-E629-38 polypeptide or HPV- E629-38 peptide-MHC complex by standard techniques, such as affinity chromatography, immunoprecipitation or immunohistochemistry.
  • HPV-E629-38 antibody of the disclosure can facilitate the purification of natural HPV-E629-38 peptide-MHC complex from biological samples, e.g., mammalian sera or cells as well as recombinantly-produced HPV-E629-38 peptide-MHC complex expressed in a host system.
  • HPV-E629-38 antibody can be used to detect a HPV-E629 -38 polypeptide or HPV-E629-38 peptide-MHC complex (e.g., in plasma, a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the polypeptide.
  • HPV-E629-38 antibodies of the disclosure can be used diagnostically to monitor HPV-E629 -38 levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen.
  • the detection can be facilitated by coupling (z.e., physically linking) the HPV-E629-38 antibody of this disclosure to a detectable substance.
  • the antibodies of the disclosure are useful for detection of HPV-E629-38 polypeptide or HPV-E629-38 peptide-MHC complex.
  • An exemplary method for detecting the level of HPV- E629-38 polypeptides or HPV-E629-38 peptide-MHC complex in a biological sample involves obtaining a biological sample from a subject and contacting the biological sample with a HPV- E629-38 antibody of the present disclosure which is capable of detecting the HPV-E629-38 polypeptides or HPV-E629-38 peptide-MHC complex.
  • the HPV-E629-38 antibodies or fragments thereof are detectably labeled.
  • the term “labeled”, with regard to the antibody is intended to encompass direct labeling of the antibody by coupling (z.e., physically linking) a detectable substance to the antibody, as well as indirect labeling of the antibody by reactivity with another compound that is directly labeled.
  • Non-limiting examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin.
  • the detection method of this disclosure can be used to detect expression levels of HPV-E629 -38 polypeptides or HPV-E629-38 peptide-MHC complex in a biological sample in vitro as well as in vivo.
  • In vitro techniques for detection of HPV-E629-38 polypeptides or HPV-E629-38 peptide-MHC complex include enzyme linked immunosorbent assays (ELISAs), Western blots, flow cytometry, immunoprecipitations, radioimmunoassay, and immunofluorescence (e.g., IHC).
  • HPV-E629-38 polypeptides or HPV-E629-38 peptide-MHC complex include introducing into a subject labeled anti-HPV-E629-38 antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the biological sample contains polypeptide molecules from the test subject.
  • a HPV-E629-38 antibody of the present disclosure can be used to assay HPV-E629-38 polypeptide or HPV-E629-38 peptide-MHC complex levels in a biological sample (e.g., a cell or tissue sample) using antibody -based techniques.
  • a biological sample e.g., a cell or tissue sample
  • protein expression in tissues can be studied with classical immunohistochemical (IHC) staining methods. Jalkanen, M. et al., J. Cell. Biol. 101 : 976-985 (1985); Jalkanen, M. et al., J. Cell. Biol. 105: 3087-3096 (1987).
  • antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes or other radioactive agents, such as iodine ( 125 I, 121 I, 133 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium (“mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • HPV-E629-38 polypeptide or HPV-E629-38 peptide-MHC complex levels can also be detected in vivo by imaging.
  • Labels that can be incorporated with anti-HPV-E629-38 antibodies for in vivo imaging of HPV-E629-38 polypeptide or HPV-E629-38 peptide-MHC complex levels include those detectable by X-radiography, NMR or ESR.
  • suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject.
  • suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which can be incorporated into the HPV-E629-38 antibody by labeling of nutrients for the relevant scF v clone.
  • a HPV-E629-38 antibody which has been labeled with an appropriate detectable imaging moiety such as a radioisotope (e.g., 131 1, 112 In, "mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (e.g., parenterally, subcutaneously, or intraperitoneally) into the subject.
  • a radioisotope e.g., 131 1, 112 In, "mTc
  • mTc 131 1, 112 In, "mTc
  • a radio-opaque substance e.g., a radio-opaque substance, or a material detectable by nuclear magnetic resonance
  • the quantity of imaging moiety needed to produce diagnostic images.
  • the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of "mTc.
  • HPV-E629-38 antibody will then preferentially accumulate at the location of cells which contain the specific target polypeptide or HPV-E629-38 peptide-MHC complex.
  • specific target polypeptide or HPV-E629-38 peptide-MHC complex For example, in vivo tumor imaging is described in S. W. Burchiel et al., Tumor Imaging: The Radiochemical Detection of Cancer 13 (1982).
  • HPV-E629-38 antibodies containing structural modifications that facilitate rapid binding and cell uptake and/or slow release are useful in in vivo imaging detection methods.
  • the HPV-E629-38 antibody contains a deletion in the CH2 constant heavy chain region of the antibody to facilitate rapid binding and cell uptake and/or slow release.
  • a Fab fragment is used to facilitate rapid binding and cell uptake and/or slow release.
  • a F(ab)'2 fragment is used to facilitate rapid binding and cell uptake and/or slow release.
  • HPV-E629 -38 antibody compositions of the disclosure are useful in diagnostic and prognostic methods.
  • the present disclosure provides methods for using the antibodies of the disclosure useful in the diagnosis of HPV-E629-38-related medical conditions in a subject.
  • Antibodies of the disclosure may be selected such that they have a high level of epitope binding specificity and high binding affinity to the HPV-E629-38 polypeptide or HPV-E629-38 peptide- MHC complex.
  • the higher the binding affinity of an antibody the more stringent wash conditions can be performed in an immunoassay to remove nonspecifically bound material without removing the target polypeptide.
  • HPV-E629-38 antibodies of the disclosure useful in diagnostic assays usually have binding affinities of at least IO -6 , KT 7 , KT 8 , KT 9 , KT 10 , KT 11 , or 10 -12 M.
  • HPV-E629-38 antibodies used as diagnostic reagents have a sufficient kinetic on-rate to reach equilibrium under standard conditions in at least 12 hours, at least 5 hours, at least 1 hour, or at least 30 minutes.
  • Some methods of the disclosure employ polyclonal preparations of anti-HPV-E629-38 antibodies and anti- HPV-E629-38 antibody compositions of the disclosure as diagnostic reagents, and other methods employ monoclonal isolates.
  • the preparation typically contains an assortment of HPV-E629-38 antibodies, e.g., antibodies, with different epitope specificities to the target polypeptide.
  • the monoclonal anti-HPV-E629-38 antibodies of the present disclosure are useful for detecting a single antigen in the presence or potential presence of closely related antigens.
  • HPV-E629 -38 antibodies of the present disclosure can be used as diagnostic reagents for any kind of biological sample.
  • the HPV-E629-38 antibodies disclosed herein are useful as diagnostic reagents for human biological samples.
  • HPV-E629-38 antibodies can be used to detect HPV-E629-38 polypeptides or HPV-E629-38 peptide-MHC complex in a variety of standard assay formats. Such formats include immunoprecipitation, Western blotting, ELISA, radioimmunoassay, flow cytometry, IHC and immunometric assays. See Harlow & Lane, Antibodies, A Laboratory Manual (Cold Spring Harbor Publications, New York, 1988); U.S. Pat.
  • Bio samples can be obtained from any tissue (including biopsies), cell or body fluid of a subject.
  • the disclosure also provides for prognostic (or predictive) assays for determining whether a subject is at risk of developing a medical disease or condition associated with increased HPV-E629-38 polypeptide or HPV-E629-38 peptide-MHC complex expression or activity (e.g., detection of a precancerous cell).
  • prognostic or predictive assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a medical disease or condition characterized by or associated with HPV- E629-38 polypeptide or HPV-E629-38 peptide-MHC complex expression.
  • Another aspect of the disclosure provides methods for determining HPV-E629-38 expression in a subject to thereby select appropriate therapeutic or prophylactic compounds for that subject.
  • the prognostic assays can be utilized to identify a subject having or at risk for developing HPV positive cervical cancer, HPV positive oropharyngeal cancer, HPV positive oral cancer, HPV positive anal cancer, HPV positive penile cancer, HPV positive vaginal cancer, HPV positive vulvar cancer, or other HPV positive solid tumor.
  • the disclosure provides a method for identifying a disease or condition associated with increased HPV-E629 -38 polypeptide or HPV-E629-38 peptide-MHC complex expression levels in which a test sample is obtained from a subject and the HPV-E629-38 polypeptide or HPV-E629-38 peptide-MHC complex detected, wherein the presence of increased levels of HPV-E629-38 polypeptides or HPV-E629 -38 peptide-MHC complex compared to a control sample is predictive for a subject having or at risk of developing a disease or condition associated with increased HPV-E629-38 polypeptide or HPV-E629-38 peptide-MHC complex expression levels.
  • the disease or condition associated with increased HPV-E629-38 polypeptide expression or HPV-E629- 38 peptide-MHC complex levels is selected from the group consisting of HPV positive cervical cancer, HPV positive oropharyngeal cancer, HPV positive oral cancer, HPV positive anal cancer, HPV positive penile cancer, HPV positive vaginal cancer, HPV positive vulvar cancer, and other HPV positive solid tumors.
  • the disclosure provides methods for determining whether a subject can be effectively treated with a compound for a disorder or condition associated with increased HPV-E629 -38 polypeptide or HPV-E629-38 peptide-MHC complex expression wherein a biological sample is obtained from the subject and the HPV-E629-38 polypeptide or HPV-E629-38 peptide- MHC complex is detected using the HPV-E629-38 antibody.
  • the expression level of the HPV- E629-38 polypeptide or HPV-E629-38 peptide-MHC complex in the biological sample obtained from the subject is determined and compared with the HPV-E629-38 or HPV-E629-38 peptide-MHC complex expression levels found in a biological sample obtained from a subject who is free of the disease. Elevated levels of the HPV-E629-38 polypeptide or HPV-E629-38 peptide-MHC complex in the sample obtained from the subject suspected of having the disease or condition compared with the sample obtained from the healthy subject is indicative of the HPV-associated disease or condition in the subject being tested.
  • HPV- E629-38 polypeptides or HPV-E629-38 peptide-MHC complex there are a number of disease states in which the elevated expression level of HPV- E629-38 polypeptides or HPV-E629-38 peptide-MHC complex is known to be indicative of whether a subject with the disease is likely to respond to a particular type of therapy or treatment.
  • the method of detecting a HPV-E629-38 polypeptide or HPV-E629-38 peptide-MHC complex in a biological sample can be used as a method of prognosis, e.g., to evaluate the likelihood that the subject will respond to the therapy or treatment.
  • the level of the HPV-E629-38 polypeptide or HPV-E629 -38 peptide-MHC complex in a suitable tissue or body fluid sample from the subject is determined and compared with a suitable control, e.g., the level in subjects with the same disease but who have responded favorably to the treatment.
  • the present disclosure provides for methods of monitoring the influence of agents (e.g., drugs, compounds, or small molecules) on the expression of HPV-E629-38 polypeptides or HPV-E629-38 peptide-MHC complex.
  • agents e.g., drugs, compounds, or small molecules
  • Such assays can be applied in basic drug screening and in clinical trials.
  • the effectiveness of an agent to decrease HPV-E629- 38 polypeptide or HPV-E629-38 peptide-MHC complex levels can be monitored in clinical trials of subjects exhibiting HPV infection, e.g., patients diagnosed with cancer.
  • An agent that affects the expression of HPV-E629-38 polypeptides or HPV-E629-38 peptide-MHC complex can be identified by administering the agent and observing a response.
  • the expression pattern of the HPV-E629 -38 polypeptide or HPV-E629-38 peptide-MHC complex can serve as a marker, indicative of the physiological response of the subject to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the subject with the agent.
  • the present disclosure provides for methods of monitoring or predicting the efficacy of therapeutic agents that target HPV-E629-38 polypeptides or HPV-E629-38 peptide- MHC complex.
  • Automated Embodiments A person of ordinary skill in the art will appreciate that aspects of the methods for using the HPV-E629-38 antibodies disclosed herein can be automated. Ventana Medical Systems, Inc. is the assignee of a number of United States patents disclosing systems and methods for performing automated analyses, including U.S. Pat. Nos. 5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901 and 6,943,029, and U.S. published application Nos. 20030211630 and 20040052685, each of which is incorporated herein by reference. Particular aspects of HPV-E629-38 staining procedures can be conducted using various automated processes.
  • kits comprising polypeptides comprising the disclosed anti- HPV-E629 -38:pMHC antigen binding domains or CDRs, or polynucleotides or vectors encoding same.
  • kits comprising the disclosed anti-HPV-E629-38:pMHC antibodies or polynucleotides or vectors encoding same.
  • kits comprising the disclosed anti-HPV-E629-38:pMHC chimeric antigen receptors (CARs) or polynucleotides or vectors comprising same, or cells comprising same.
  • CARs chimeric antigen receptors
  • kits further comprise positive controls (e.g. HPV-E629-38 antigen), negative controls, appropriate buffers and instructions for use.
  • positive controls e.g. HPV-E629-38 antigen
  • negative controls e.g. HPV-E629-38 antigen
  • the human IGH locus is large and complex, spanning approximately 1.3 Mb and including V, D, J and constant domain sequences.
  • the functional gene segments can be joined in more than 5000 different V-D-J combinations by recombination; however, the calculated theoretical complexity of the human antibody repertoire is greater than 10 13 .
  • This diversity is largely due to the action of terminal deoxynucleotidyl transferase (TdT), which introduces sequence diversity at V-D and D-J junctions.
  • TdT terminal deoxynucleotidyl transferase
  • FIG. 1 shows the organization of a representative engineered heavy chain locus.
  • the synthetic locus include the use of a strong promoter, a leader sequence derived from a commonly used V gene, a consensus Kozak sequence, codon-optimized V and constant region sequences and high efficiency RSSs. Together, these features contribute to robust rates of recombination and high levels of antibody expression (FIG. 2B).
  • This synthetic locus is integrated into an engineered HEK293 cell line at a pre-selected chromosomal site using Cre-tox recombination.
  • each cell When a plasmid pool is introduced into a cell population by Cre-mediated integration, individual cells receive a single plasmid from the pool encoding a randomly selected V, D and J segment; thus, each cell has the potential to express only one heavy chain gene.
  • the cell population represents the entire segmental complexity present in the chosen V-D-J complement, whether it be that corresponding to a natural heavy chain locus or some other preferred combination or ratio of gene segments.
  • the size of the heavy chain locus is reduced from 1.3 Mb to approximately 5 kb per cell making it amenable to standard molecular cloning techniques.
  • the repertoires described in this report were generated using a single human light chain derived from IGKV1-39/J4.
  • EXAMPLE 2 Engineering a cell line capable of V(D) J recombination
  • RAG1 is expressed by a cytomegalovirus (CMV) promoter under the control of a tetracycline-inducible regulatory system (Yao, F. et al. Human Gene Therapy 9, 1939-1950 (1998)) and RAG2 is expressed by a constitutive CMV promoter. Tetracycline upregulates RAG1 expression allowing for assembly of the RAG1/RAG2 complex. Regulation of RAG levels is important for ensuring that recombination occurs only when a culture has been scaled to the desired cell number, typically greater than ten billion cells, since premature recombination would otherwise limit the complexity of a nascent library and introduce clonal bias.
  • CMV cytomegalovirus
  • junctional diversity is a major contributor to the sequence variation observed in natural antibody repertoires. This is due in part to the activity of TdT, which introduces non- templated nucleotides (N-nucleotides) at gene segment junctions during recombination.
  • N-nucleotides non- templated nucleotides
  • levels of TdT activity were achieved by randomly integrating a CMV promoter- driven DNTT gene into the host cell’s chromosomal DNA. Additional TdT activity was provided by adding the DNTT expression cassette to the in vitro antibody locus.
  • EXAMPLE 3 Design, construction and analysis of a fully human antibody repertoire generated using V(D)J recombination in the engineered cell line
  • FIG. 2A shows a representation of human germline V segments, D segments and human germline J segments. All segments were designed to be present at an even ratio.
  • the in vitro antibody loci were introduced into a modified host HEK293 cell line using Cre-lox recombination. The resulting cells stably maintained the V, D and J segments in an unrecombined state as evidenced by the lack of antibody present on the cell surface (FIG. 2B).
  • tetracycline-mediated induction of V(D)J recombination for six days resulted in approximately 10% of cells displaying cell-surface antibody.
  • HuTARGTM allows generation of an entire heavy-chain antibody repertoire paired with a single light chain, with a diversity in the range of one billion idiotypes (Oh et al. Sci Rep. 2019 9(1): 17291).
  • the top E6 binders were examined in detail for selectivity (FIG. 14). 47 peptides predicted from the human proteome as most similar to HPV-E629-38 were identified by NCBI BLAST and a simple matching algorithm (see A2 Scan, Methods). All of these peptides derive from human proteins with transcript levels between 1-40 TPM in multiple tissues (GTEx website, //www.gtexportal.org/home).
  • EXAMPLE 5 Characterization of fully optimized HPV-E629-38 CARs in Jurkat cell assays [0273] The most enriched fully optimized (light- and heavy-chain) HPV-E629-38 binders were studied to determine sensitivity and selectivity. In Jurkat-cell assays, the two most sensitive binders, CT503 (SEQ ID NO: 19) and CT512 (SEQ ID NO: 20) had EC50s ⁇ 1 nM (FIG. 13, FIG. 15) The improvement over their parental (light-chain optimized only) sequences was ⁇ 10X (FIG. 12-13). CT512 matched the benchmark E6 TCR.
  • HPV-E629 -38 CARs killed target-positive cells throughout multiple cycles of co-culture (4 or 8 cycles of serial co-culture, +/- IL-2, respectively) to the same extent as the TCR (FIG. 17C).
  • A375 cells (CRL-1619), T2 (CTL-1992TM), HeLa (CCL-2TM), and A375 Fireflyluciferase (CRL-1619-LUC2) cells were purchased from ATCC®.
  • Jurkat NF AT -Firefly- Luciferase cells were purchased from BPS Bioscience (#60621).
  • HLA-A2 alpha chain was expressed by transducing Hela cells with HLA-A2 lentivirus and HLA-A2 positive population was enriched by sorting on cells stained with BB7.2 Ab. All cell lines were cultured in media as recommended by the vendors. 100 U/mL Penicillin-streptomycin (Gibco 15140163) (lx P/S) was used in all media. Suspension cells were maintained below a density of lE6/mL. Adherent cells lines were passaged at -80% confluency.
  • the assays utilized streptavidin-conjugated donor beads (6760002B, PerkinElmer) and acceptor beads (6762002, PerkinElmer) conjugated in-house with 1 mg/mL W6/32 (BE0079, Bioxell).
  • the reaction was performed in 15 uL refolding buffer (50 mM Tris-maleate pH 6.6 and 0.03 % Pluronic acid) containing purified alpha subunit and beta subunit (0877095-CF, MP Biomedicals; “B2m” below) and varying concentration of peptides (4 points tested per peptide: 50 uM, 5 uM, 0.05 uM or 0.005 uM; purchased from Genscript).
  • Optimized concentrations of each purified alpha subunit in combination with B2m were used on a per allele basis as follows: 0.3 uM A*01:01 + 0.6 uM B2m, 1 uM A*02:01 + 2 uM B2m, 0.1 uM A*03:01 + 0.2 uM B2m, 0.1 uM A* 11 :01 + 0.2 uM B2m, 0.1 uM C*07:01 + 0.6 uM B2m, or 0.1 uM C*07:02 + 0.6 uM B2m.
  • the 15 uL refolding reaction was sealed and incubated overnight at 37 degrees Celsius.
  • the 15 uL reaction was transferred to a 384-well Proxiplate (6008289, PerkinElmer) and a 5 uL mixture containing streptavidin donor beads and in-house conjugated W6/32 acceptor beads in a 1 : 1 ratio was added at a final total bead concentration of 20 ug/mL.
  • the plates were incubated at room temperature for 1 hour and the “Alpha Signal” was then read via EnSpire 2300 (PerkinElmer).
  • HLA-A*02:01 and beta-2 microglobulin (B2M) were expressed in bacterial cells and purified as previously described (Altman et al., 1996; Garboczi et al., 1992).
  • the extracellular domains of HLA-A*02:01 or HLA-A* 11 :01 were expressed in E. coli BL21 (DE3) pLysS cells (Invitrogen C606003). Protein expression was induced by 1 mM IPTG for 4 hours at 42°C.
  • This centrifugation/wash step was repeated 6-8 times with the final wash step using inclusion-body wash buffer without 0.5% (v/v) Triton-X 100.
  • the inclusion bodies were then solubilized using 25 mM MES, pH 6.0, 8 M urea, and 10 mM EDTA, followed by centrifugation to deplete debris.
  • HLA-A*02:01 and B2M complex To generate peptide, HLA-A*02:01 and B2M complex, peptides freshly dissolved in DMSO, B2M (MP Biomedicals, 08770953), and extracellular domains of HLA class I molecules were added dropwise to fresh refolding buffer (100 mM Tris-HCl, pH 8, 0.5 M L-arginine, 2 mM EDTA, 5 mM reduced glutathione, 0.5 mM oxidized glutathione and 1 : 100 diluted protease inhibitor) in sequence to reach final concentration 1 pM, 2 pM, 3 pM, respectively. The mixture was stirred at 10°C for at least 6-10 hours between each addition.
  • fresh refolding buffer 100 mM Tris-HCl, pH 8, 0.5 M L-arginine, 2 mM EDTA, 5 mM reduced glutathione, 0.5 mM oxidized glutathione and 1 : 100
  • the supernatant was collected and concentrated before purification using an ion-exchange Hi-Trap Q column (GE 3 17115401), followed by a sizeexclusion HiLoad® 16/600 Superdex® 200 column. Post purification, protein concentration and biotinylation were determined by absorbance at 280 nm and HABA assays (Invitrogen 28005), respectively.
  • HuTARGTM primary libraries were from innovative Targeting Solutions, Inc. An in vitro V(D)J repertoire with > 1 billion diversity were generated by expression of RAG- 1 and TdT in the host cells as described previously. pMHC probes were generated as described previously. The library was enriched for cells displaying antibodies that bind specifically to target pMHC probes, but not to off-target pMHC probes using a flow sorter device. Multiple enrichment rounds were performed to increase on-target and decrease off-target binding. In the final round, on-target and off-target binding cells were collected. RNA was extracted from these pools and reverse transcribed into cDNA.
  • PCR fragments containing the CDR regions were generated using the cDNAs as template, followed by targeted NGS to determine the frequency of each binder with a unique CDR region. The degree of enrichment/depletion was determined by comparing the output and input NGS counts.
  • Target-specific binders from the primary libraries were used in some cases as parents to generate optimization libraries to further improve on-target sensitivity and/or reduce off-target cross-reactivity.
  • Optimization libraries were constructed by diversification of CDR-1, CDR-2 or CDR-3 light chains of parent binders by in vitro RAG-mediated V(D)J recombination.
  • the optimization library was enriched for on-target activity and depleted for off-target activity as for enrichment of the primary library.
  • NGS was also used to identify binders enriched as described above.
  • All CAR constructs were created by fusing an scFv LBD to a hinge, a TM and an intracellular signaling domain.
  • the hinge was derived from CD8, the transmembrane domain from CD28, and the signaling domain from CD28, 4- IBB and CD3.
  • Gene segments were combined using Golden Gate cloning and inserted downstream of a human EFla promoter contained in a lentivirus expression plasmid.
  • Preferred amino acids at anchor positions get a score increment of +1
  • tolerated amino acids get a score increment of +0.5
  • unfavorable amino acids get a score decrement of -0.5
  • Incompatibility score penalizes differences in the physicochemical properties of amino acids at each position of the peptides when compared to the target peptide. Cutoffs used for the selection of potential cross-reactive peptides were based on the Titin (TTN) peptide which was proposed to be the most likely cause of the off-target toxicity for a MAGE- A3 pMHC target (Raman et al., 2016).
  • Jurkat NFAT-Firefly-Luciferase cells were transfected on day 1 with TCR and CAR constructs using standard protocols for the Lonza 4D NucleofectorTM (AAF-1002B) or NeonTM transfection system (ThermoFisher, MPK5000).
  • T2 cells were loaded with peptides listed in FIG. 6.
  • Peptides were resuspended in DMSO, and diluted 16 or 20 times serially 3x per step. Serially diluted peptide solutions were added to T2 cells resuspended in peptide-loading media (RPMH640 + 1% BSA + IX P/S).
  • peptide-loaded T2 cells at approximately lE6/mL, with peptide concentrations ranging from ⁇ 10 fM to 100 pM, including a control at 0 pM.
  • Peptide-loaded T2 cells were incubated overnight at 37°C in 384-well plates (Thermo Scientific AB0781). On day 2, the cells were cocultured in a 384-well plate (Corning 3570). Peptide-loaded T2 cells (10,000 cells/well) were added to CAR/TCR-transfected Jurkat-NFAT- Firefly-Luciferase cells (12,000 cells/well) to a final volume of 20 pL.
  • LymphoONETM supplemented with IL-2 (300 lU/ml) was added to transduced cells and cultured for 3 days before transfer to a 24-well G-Rex plate (Wilson Wolf 80192M).
  • Fresh IL-2 300 lU/ml was added every 48 hours with a media change every 7 days during expansion in G-Rex plates. Expression and antigen binding of transduced CARs or TCRs in primary T cells was confirmed by flow cytometry as described above.
  • CAR- or TCR-expressing cells were labeled with protein L-biotin/streptavidin-PE or mTCR-PE, followed by anti-PE microbeads (Miltenyi 130-048-801) according to the manufacturer’s protocol, and subsequently enriched using AutoMACS® Pro Separator (Miltenyi). Enriched cells were grown in G-Rex plates until harvest.
  • CARs and TCRs were determined by flow cytometry using biotinylated protein L (ThermoFisher #29997) followed by fluorescently labeled streptavidin, fluorescently labeled anti-kappa light chain, or fluorescently labeled anti-murine TRBV antibody (Biolegend Cl:H57-597).
  • Antigen binding was determined by staining CAR- or TCR-expressing Jurkat cells with biotinylated-pMHCs probes that were tetramerized and prelabeled with streptavidin conjugated to an appropriate fluorochrome (Biolegend). All staining was carried out at 4 ° C, and median fluorescent intensity (MFI) was determined using a FACS Canto II flow cytometer (BD Biosciences). Relative MFI was used to compare results from different sets of experiments.
  • Target cells MCF-7 renilla-luciferase cells or A375 firefly-luciferase cells, were loaded with target peptides as described above in the Jurkat/T2 section, except that LymphoONETM supplemented with 1% human serum and IX P/S was used. 24 hours after peptide loading, a calibration curve was generated using CellTiter-Glo® (Promega G7570) readout to determine the number of target cells seeded per well. T cells were mixed with target cells at 3: 1 E:T ratio according to the target cell number determined by the calibration curve.
  • cytotoxicity of primary T cells was quantified by the bioluminescent signal using One-StepTM Luciferase assay system firefly (BPS Bioscience, 60690) or Renilla Luciferase Assay System (Promega, E2810) on Tecan Infinite® M1000.
  • Target cells A375 GFP+ cells, were loaded with target peptide (purchased from Genescript) in LymphoONE supplemented with 1% human serum and IX P/S. Briefly, peptides were resuspended in DMSO, and diluted to indicated concentrations. Diluted peptide solutions were added to A375 target cells. This yielded peptide-loaded A375 target cells, which were seeded in either 48-well (for ‘round’ 1) or 96-well (for subsequent rounds) plates at 0.027e6/well or 0.009e6/well, respectively.
  • T cells were mixed with target cells at 3: 1 E:T ratio of “effector” T cells (E) to “target” (T) A375 cells. Plates were placed in the Incucyte Imaging S3 Platform (Sartorius) and imaged every 2 hours for 48- or 72-hours for round 1 and subsequent rounds, respectively. At the end of each co-culture period (referred to as “rounds” of co-culture), cell solutions were aspirated, counted and resuspended at a uniform density. These re-suspended cells were then added to subsequent ‘rounds’ of co-culture with equivalently peptide-loaded target A375 cells and incubated for 72hrs.

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Abstract

La divulgation concerne des domaines de liaison à l'antigène, des anticorps et des récepteurs d'antigènes chimériques qui se lient spécifiquement à un complexe peptide:CHM (pMHC) affichant un peptide HPV-E629-38 comprenant la séquence TIHDIILECV (SEQ ID NO : 1), et des méthodes d'utilisation de ceux-ci dans le diagnostic et le traitement de cancers positifs au HPV.
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WO2024050399A1 (fr) * 2022-09-01 2024-03-07 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anticorps à domaine unique ciblant des complexes peptide/cmh oncogènes hpv e6/e7

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