WO2019140278A1 - Immunothérapie ciblant des antigènes du facteur de liaison du noyau - Google Patents
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Classifications
-
- 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
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
- A61K2239/48—Blood cells, e.g. leukemia or lymphoma
-
- 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
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464401—Neoantigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464452—Transcription factors, e.g. SOX or c-MYC
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
- A61K39/001196—Fusion proteins originating from gene translocation in cancer cells
Definitions
- AML Acute myeloid leukemia
- CBF core binding factor
- CBF AMLs carry fusion gene mutations involving the alpha (RUNX1) or beta (CBFP) CBF components and respective fusion partners; i.e. RUNXl/RUNXTlin t(8:2l) and CBFp/MYHl lin inv(l6) and t(l6: 16) (pl3;q22) cases.
- CBF AML can be successfully treated with chemotherapy alone, but approximately 15-50% of patients with CBF AML do not achieve a complete molecular remission (CMR) with induction
- CCT allogeneic hematopoietic stem cell transplantation
- Figure 1A shows a schematic illustration of a normal Core Binding
- CBF Transcription Factor
- RUNX1 CBFa subunit
- CBFP Transcription Factor
- Figure IB shows a schematic illustration of mutant forms of CBF that are associated with Acute Myeloid Leukemia.
- Left A mutant CBF complex produced by an inversion on chromosome 16 (invl6).
- the rearranged chromosome encodes a chimeric transcript consisting of the N-terminus of CBFP in a fusion with the C- terminal portion of the smooth muscle myosin heavy chain 11 (MYH11).
- MYH11 smooth muscle myosin heavy chain 11
- the fusion protein complexes with CBFa subunit (RUNX1).
- Figure 1C shows the frequency of occurrence (y-axis) of certain mutations (key at right) in AML patients within the indicated age ranges (x-axis, years).
- the CBF mutations illustrated in Figure 1B are indicated with arrows.
- Figure 2 shows the prevalence (second column from left), in CBF AML patients, of inv(l6) and t(8;2l) cytogenetic events, and of co-occuring KIT, NRAS, and KRAS mutations. Variant subtypes of the indicated mutations and their respective frequencies within the mutation category are indicated in the four right-most columns. Data is based on an unpublished merged TARGET, ECOG, TCGA dataset (-50% pediatric and adolescent patients, -50% adult patients [age > 18]).
- Figures 3A-3C provide schematic illustrations of an inv(l6) chromosomal translocation event producing a CBFpXIYH l 1 (Type A) fusion amino acid sequence from which candidate neoantigenic peptides can arise, and Human Leukocyte Antigen (HLA) subtypes to which the candidate neoantigens are computationally predicted to bind.
- A (Left) wild-type human chromosome 16 prior to an inv(l6) event. The MYH11 and ('BI'b loci are indicated (not to scale).
- B (Top) genomic DNA encoding a CBFpXIYH l 1 fusion sequence.
- Figures 4A-4E show cytotoxicity of high-avidity T cell clones specific for CBFp YHl 1 neoantigen peptides.
- A Percent lysis of peptide-pulsed target cells (autologous lymphoblastoid cell line pulsed with a pool of candidate neoantigen peptides) by individual CD8 + T cell lines from two HLA-B*40:0l + donors (D6, top; D7, bottom). T cell lines confirmed as REEMEVHEL (SEQ ID NO:2)-specific are shown as black triangles; T cell lines specific for control epitopes shown as white triangles.
- C Flow images of CD8 + T cell clones stained with CBFP-MYHl l/HLA-B*40:0l peptide-HLA (pHLA) tetramer. Cells are gated on live single CD4 CD8 + cells.
- E Lysis, by high-avidity
- REEMEVHEL SEQ ID NO:2-specific clones D6.C6.1, D7.C8.1, D7.C24.1, and Dl 1.C5, of HLA-B*40:0l + LCL, HLA-B*40:02 + LCL, and HLA-B*40:0l7 B*40:02 LCL pulsed with 1 pg/mL peptide in a 4-hour 51 chromium release assay (CRA).
- Figures 5A-5H relate to experiments in which transgene constructs were used to generate epitope-bearing AML target cell lines and killing of these target cells by CBFP-MYHl l/B*40:0l-specific T cell clones was investigated. Specifically, vectors were used to transduce (1) the naturally HLA-B*40:0l + NB-4 cell line with coding sequence for the full-length CBFP-MYHl 1 type A fusion, and (2) the naturally CBFP- MYHl l + ME-l cell line with HLA-B*40:0l.
- FIG. 1 Schematic illustration of lentiviral constructs used to transduce HLA-B*40:0l + NB-4 cells with the full-length CBFP- MYH11 type A fusion (left) and CBFp-MYHl l + ME-l cells with HLA-B *40:01 (right). Both constructs included a purification/expression RQR8 tag separated from the antigenic or HLA coding sequence by a self-cleaving P2A peptide sequence.
- C Percent survival of NB-4 target cells (mock transduced or transduced to express CBFP-MYHl 1) at various timepoints during co culture with the indicated T cell clones.
- F Survival of mock-TD or HLA-B*40:0l-TD ME-l cells after the indicated number of hours (x-axis) of co-culture with each T cell clone. Viable cell numbers were assessed by flow cytometry.
- FIG. 1 Flow cytometry data showing peptide-HLA (REEMEVHEL (SEQ ID N0:2)-HLA-B*40:0l):tetramer- specific staining of the indicated CBFP:MYHl l-specific T cell clones.
- the x-axis shows staining with tetramer conjugated to phycoerythrin (PE) and the y-axis shows staining with tetramer conjugated to allophycocyanin (APC).
- H Lysis (CRA) of target cells pulsed with inv(l6) neoantigen peptide REEMEVHEL (SEQ ID NO:2) at the indicated concentrations by antigen-specific T cell clones of the present disclosure.
- T cell clones recognized target cells pulsed with peptide at concentrations as low as 0.1 ng/ml.
- FIG. 6A shows that CBFP:MYHl 1 -specific T cell clones of the present disclosure recognize an AML cell line pulsed with REEMEVHEL (SEQ ID NO:2) peptide and primary AML.
- Top row flow cytometry data for clone D6.C6.1.
- Middle row flow cytometry data for clone D7.C8.1.
- Bottom row flow cytometry data for clone D7.C24.1.
- T cell degranulation (CDl07a marker; y-axis) and cell size (FSC; x-axis) in response to: NB-4 control cells in the absence of peptide; NB-4 control cells pulsed with peptide; inv(l6) + HLA-B*40:0l primary AML cells; inv(l6) HLA-B*40:0l + primary AML cells; and (two right-hand most columns) inv(l6) + HLA-B*40:0l + primary AML cells.
- Figure 6B shows degranulation of the indicated T cell clones (left-hand side of figure) in response to primary AML or to antigen-pulsed target cells (positive controls), as determined by CD 107a expression (y-axis). Shown are representative flow plots from one experiment.
- Figure 6D shows lysis of primary AML by T cell clones in a 4-hour CRA with an effectontarget (E:T) ratio of 20: 1 (CBFP-MYHl U
- FIGS 7A-7G show that CBFP-MYHl l/B*40:0l-specific CD8 + T cells of the present disclosure recognize a unique epitope and have a naive phenotype in healthy donors.
- A Percent lysis, by REEMEVHEL (SEQ ID NO:2)-specific T cell clones, of HLA-B*40:0l + LCL pulsed with the SEQ ID NO:2 peptide or with variant peptides each having an alanine substitution at one position of SEQ ID NO:2 (see SEQ ID NOs:99-l07).
- CBFp-MYH l 1/HLA-B*40:01 is reproducibly immunogenic in three additional healthy donors (i)-(iii).
- CD8 + T cells were isolated from three additional HLA-B*40:0l + healthy donors and stimulated with autologous DC pulsed with CBFP-MYHl 1 peptide (SEQ ID NO:2).
- SEQ ID NO:2 CBFP-MYHl 1 peptide
- Figures 8A-8D show the presence of CBFP-MYHl l/B*40:0l-specific memory T cells in PBMC from HLA-B*40:0l + patients with CBFp-MYHl l + AML.
- A CBFp- MYH1 l/B*40:0l tetramer enrichment and staining of PBMC from one healthy donor (D3) and two individuals with CBFP-MYHl l + AML; all individuals were HLA- B*40:0l + .
- Sample from "AML1" was obtained at diagnosis.
- Samples from "AML2" were obtained at CR and post-HCT.
- Tetramer double-positive CD8 + T cells from each sample are shown gated in upper left of each scatter plot; other cells are tetramer- negative. Gating on viable single cells.
- B Frequency of CBFP-MYHl l/HLA- B*40:0l specific T cells (per million CD8 + T cells) isolated from HLA-B*40:0l + individuals with and without AML. Samples shown in Figure 8A are included here, as are additional patient samples. The absolute number of tetramer-positive
- CBFp:MYHl l/HLA-B*40:0l specific /HLA-B*40:0l specific CD8 + T cells was calculated from each tetramer-enriched PBMC sample and normalized to the total number of CD8 + T cells in the sample.
- C Phenotypic evaluation of the T cell subsets. Cells in dark gray indicate tetramer-positive CBFP-MYHl l/HLA-B*40:0l specific T cells as evaluated in (A); remaining cells are in light gray.
- inhibitory/activation receptors on CBFP:MYHl l/HLA-B*40:0l specific/B*40:0l- specific T cells shown by staining with PD-l versus CD45RO (left) and 2B4 versus CD45RO staining (right).
- FIGS 9A-9F show that TCRs specific for the CBFP-MYHl l/HLA-B*40:0l epitope confer anti-leukemic activity.
- A Expression of transgenic TCRs in CD8 + T cells transduced with D6.C6.1 B1A1, D7.C8.1 B1A1, D7.C24.1 B1A1, and Dl l.C5 Bl
- A2 TCR constructs is shown by staining with CBFP-MYHl l/HLA-B*40:0l tetramer.
- Al and “A2" refer to which of two encoded TCR a-chains (Al or A2) are present in the TCR.
- Figure 10A shows specific cytotoxicity (CRA) against target cells pulsed with varying concentrations of peptide by a CBFpMYHl l-specific HLA-A*020l -restricted T cell clone, D1.C27, of the present disclosure.
- the clone is specific for a
- Figure 10B shows specific lysis of unmodified OCI-AML3 AML cells (HLA- A*020l, CBFpMYHl , RUNXLRUNXlT ) or OCI-AML3 AML cells transduced to express either a variant CBFpXIYH l 1 or a variant RUNX1 : RUNX 1 T 1 neoantigen peptide, by T cell clone D1.C27, or by a T cell clone specific for the
- Figure 10C shows lysis (CRA) of target cells pulsed with CBFpMYHl 1 neoantigen peptide EEMEVHEL (SEQ ID NO:3) at the indicated concentrations by an HLA-B*40:0l/44:02-restricted clone of the present disclosure (D7.C24.1). An irrelevant control clone had no lytic activity.
- CRA lysis
- the present disclosure generally relates to binding proteins specific for
- AML CBFp YHl l + AML, modified immune cells expressing the same, polynucleotides that encode the binding proteins, and related uses.
- AML is a frequently lethal hematologic malignancy, with a survival rate of approximately 27%, and around 20,000 new AML diagnoses are made each year.
- Current therapies include vaccines to elicit an immune response against AML, the use of checkpoint inhibitors to prevent immune suppression, by AML, of an endogenous or therapeutic immune response, and T cell immunotherapy.
- T cell immunotherapy can be highly effective against hematologic malignancies, as exemplified by the results of clinical trials evaluating therapeutic T cells genetically modified with chimeric antigen receptors (CARs) targeting CD 19 for acute lymphoblastic leukemia (ALL). AML is also subject to T cell killing.
- CAR T cells typically target cell surface-expressed molecules, and candidate cell surface targets are also found on non-malignant myeloid progenitors and predicted to produce marrow aplasia.
- Alternative immunotherapy strategies include administration of T cells naturally expressing, or transduced to express, T cell receptors (TCR) specific for peptide antigens derived from endogenous cell proteins and presented on the cell surface in association with HLA molecules.
- TCR T cell receptors
- an appropriate antigen target is important for effective T cell immunotherapy.
- some antigens may have relatively low tumor-specific expression (e.g ., WT-l), and targeting such antigens may lead to undesirable killing of normal tissue and/or undesirably high levels of immune activity.
- ideal T cell antigen targets for immunotherapy are highly selectively expressed on tumors.
- Core binding factor (CBF) AMLs carry fusion gene mutations involving the alpha (CBFa, also known as RUNX1) or beta (CBFP) CBF components and respective fusion partners; i.e. RUNX1 RUNXTlin t(8:2l) and CBFpMYHl lin inv(l6) and t(l6: l6) (pl3;q22) cases, which produce amino acid sequences that are unique to tumor cells and may create T cell neoantigens.
- CBFa alpha
- CBFP beta
- the present disclosure provides binding proteins that are capable of specifically binding to a peptide containing a CBFpMYHl 1 neoantigen, and isolated
- modified immune cells that comprise polynucleotides encoding binding proteins that are capable of specifically binding to a peptide containing a CBFpMYHl 1 antigen, and methods of treating or preventing relapse of AML using such modified immune cells as described herein.
- any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
- any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness are to be understood to include any integer within the recited range, unless otherwise indicated.
- the term “about” means ⁇ 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms “a” and “an” as used herein refer to “one or more" of the enumerated components.
- a protein domain, region, or module e.g., a binding domain, hinge region, linker module
- a protein which may have one or more domains, regions, or modules
- 3%, 2% or 1% of the length of a domain, region, module, or protein and do not substantially affect (i.e., do not reduce the activity by more than 50%, such as no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% ) the activity of the domain(s), region(s), module(s), or protein (e.g, the target binding affinity of a binding protein).
- hematopoietic progenitor cell is a cell that can be derived from hematopoietic stem cells or fetal tissue and is capable of further differentiation into mature cells types (e.g, immune system cells).
- exemplary hematopoietic progenitor cells include those with a CD24 Lo Lin- CD117 + phenotype or those found in the thymus (referred to as progenitor thymocytes).
- an "immune system cell” means any cell of the immune system that originates from a hematopoietic stem cell in the bone marrow, which gives rise to two major lineages, a myeloid progenitor cell (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, meagakaryocytes and granulocytes) and a lymphoid progenitor cell (which give rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells).
- myeloid progenitor cell which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, meagakaryocytes and granulocytes
- lymphoid progenitor cell which give rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells.
- Exemplary immune system cells include a CD4 + T cell, a CD8 + T cell, a CD4 CD8 double negative T cell, a gd T cell, a regulatory T cell, a natural killer cell, and a dendritic cell.
- Macrophages and dendritic cells can be referred to as "antigen presenting cells” or "APCs,” which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with a TCR on the surface of a T cell.
- MHC major histocompatibility complex
- T cell or "T lymphocyte” is an immune system cell that matures in the thymus and produces T cell receptors (TCRs).
- T cells can be naive ("TN”; not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD 127, and CD45RA, and decreased or no expression of CD45RO as compared to T CM (described herein)), memory T cells (T M ) (antigen experienced and long-lived), including stem cell memory T cells, and effector cells (antigen-experienced, cytotoxic).
- T M can be further divided into subsets of central memory T cells (T CM expresses CD62L, CCR7, CD28, CD95, CD45RO, and CD127) and effector memory T cells (T EM express CD45RO, decreased expression of CD62L, CCR7, CD28, and CD45RA).
- Effector T cells (T E ) refers to antigen-experienced CD8 + cytotoxic T lymphocytes that express CD45RA, have decreased expression of CD62L, CCR7, and CD28 as compared to T EM , and are positive for granzyme and perforin.
- Helper T cells (T H ) are CD4 + cells that influence the activity of other immune cells by releasing cytokines.
- CD4 + T cells can activate and suppress an adaptive immune response, and which of those two functions is induced will depend on presence of other cells and signals.
- T cells can be collected using known techniques, and the various subpopulations or combinations thereof can be enriched or depleted by known techniques, such as by affinity binding to antibodies, flow cytometry, or immunomagnetic selection.
- Other exemplary T cells include regulatory T cells, such as CD4 + CD25 + (Foxp3 + ) regulatory T cells and Tregl7 cells, as well as Trl, Th3, CD8 + CD28 , and Qa-l restricted T cells.
- T cell receptor refers to an immunoglobulin superfamily member (having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail; see, e. g, Janeway el al ., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 433, 1997) capable of specifically binding to an antigen peptide bound to a MHC receptor.
- a TCR can be found on the surface of a cell or in soluble form and generally is comprised of a heterodimer having a and b chains (also known as TCR a and TCRP, respectively), or g and d chains (also known as TCRy and TCR5, respectively).
- a polynucleotide encoding a binding protein of this disclosure can be codon optimized to enhance expression in a particular host cell, such as a cell of the immune system, a hematopoietic stem cell, a T cell, a primary T cell, a T cell line, a NK cell, or a natural killer T cell (Scholten et al. , Clin. Immunol. 119: 135, 2006).
- exemplary T cells that can express binding proteins and TCRs of this disclosure include CD4 +
- T cells CD8 + T cells, and related subpopulations thereof (e.g, naive, central memory, stem cell memory, effector memory).
- TCR chains e.g, a-chain, b-chain
- a vailable domain e.g., a-chain variable domain or V a b-chain variable domain or Vp; typically amino acids 1 to 116 based on Kabat numbering (Kabat et al. , " Sequences of Proteins of Immunological Interest, US Dept.
- a-chain constant domain or C a typically 5 amino acids 117 to 259 based on Kabat
- b-chain constant domain or Cp typically amino acids 117 to 295 based on
- variable domains contain complementary determining regions (CDRs) separated by framework regions (FRs) (see, e.g., Jores et al., Proc. N at! Acad. Sci. USA 87:9138, 1990; Chothia et al, EMBO J. 7:3745, 1988; see also Lefranc et al, Dev. Comp. Immunol. 27:55, 2003).
- CDRs complementary determining regions
- FRs framework regions
- the source of a TCR as used in the present disclosure may be from various animal species, such as a human, mouse, rat, rabbit or other mammal.
- variable region refers to the domain of an immunoglobulin superfamily binding protein (e.g., a TCR a-chain or b-chain (or g chain and d chain for gd TCRs)) that is involved in binding of the immunoglobulin superfamily binding protein (e.g, TCR) to antigen.
- immunoglobulin superfamily binding protein e.g., a TCR a-chain or b-chain (or g chain and d chain for gd TCRs)
- the variable domains of the a-chain and b-chain (V a and Vp, respectively) of a native TCR generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs.
- V a domain is encoded by two separate DNA segments, the variable gene segment and the joining gene segment (V-J); the U b domain is encoded by three separate DNA segments, the variable gene segment, the diversity gene segment, and the joining gene segment (V-D-J).
- V-J variable gene segment
- V-D-J joining gene segment
- a single V a or Vp domain may be sufficient to confer antigen-binding specificity.
- TCRs that bind a particular antigen may be isolated using a V a or Vp domain from a TCR that binds the antigen to screen a library of complementary V a or Vp domains, respectively.
- CDR complementarity determining region
- HVR hypervariable region
- TCR immunoglobulin
- CDR3 is thought to be the main CDR responsible for recognizing processed antigen.
- CDR1 and CDR2 mainly interact with the MHC.
- CDR1 and CDR2 are encoded within the variable gene segment of a TCR variable region-coding sequence
- CDR3 is encoded by the region spanning the variable and joining segments for Va, or the region spanning variable, diversity, and joining segments for nb.
- the sequences of their corresponding CDR1 and CDR2 can be deduced.
- CDR3 is significantly more diverse because of the addition and loss of nucleotides during the recombination process.
- TCR variable domain sequences can be aligned to a numbering scheme (e.g ., Rabat, Chothia, Enhanced Chothia, and Aho), allowing equivalent residue positions to be annotated and for different molecules to be compared using ANARCI software tool (2016, Bioinformatics 15:298-300).
- a numbering scheme provides a standardized delineation of framework regions and CDRs in the TCR variable domains.
- CD8 co-receptor means the cell surface glycoprotein CD8, either as an alpha-alpha homodimer or an alpha-beta heterodimer.
- the CD8 co-receptor assists in the function of cytotoxic T cells (CD8 + ) and functions through signaling via its cytoplasmic tyrosine phosphorylation pathway (Gao and Jakobsen, Immunol. Today 21 :630-636, 2000; Cole and Gao, Cell. Mol. Immunol. 1 :81- 88, 2004).
- CD8 beta chains see UniProtKB identifier P 10966
- a single CD8 alpha chain see UniProtKB identifier P01732
- CD4 is an immunoglobulin co-receptor glycoprotein that assists the TCR in communicating with antigen-presenting cells (see, Campbell & Reece, Biology 909 (Benjamin Cummings, Sixth Ed., 2002)). CD4 is found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells, and includes four immunoglobulin domains (Dl to D4) that are expressed at the cell surface. During antigen presentation, CD4 is recruited, along with the TCR complex, to bind to different regions of the MHCII molecule (CD4 binds MHCII b2, while the TCR complex binds MHCII a ⁇ /b ⁇ ). Without wishing to be bound by theory, it is believed that close proximity to the TCR complex allows CD4-associated kinase molecules to
- TAMs immunoreceptor tyrosine activation motifs
- a TCR is found on the surface of T cells (or T lymphocytes) and associates with a CD3 complex.
- CD3 is a multi-protein complex of six chains (see, Abbas and Lichtman, 2003; Janeway et al, p. 172 and 178, 1999) that is associated with antigen signaling in T cells.
- the complex comprises a CD3y chain, a CD35 chain, two CD3e chains, and a homodimer of CD3z chains.
- the CD3y, CD3P, and CD3e chains are related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain.
- the transmembrane regions of the CD3y, CD3p, and CD3e chains are negatively charged, which is believed to allow these chains to associate with the positively charged T cell receptor chains.
- the intracellular tails of the CD3y, CD3P, and CD3e chains each contain a single conserved motif known as an immunoreceptor tyrosine based activation motif or IT AM, whereas each 003z chain has three. Without wishing to be bound by theory, it is believed that the ITAMs are important for the signaling capacity of a TCR complex.
- CD3 as used in the present disclosure may be from various animal species, including human, mouse, rat, or other mammals.
- TCR complex refers to a complex formed by the association of CD3 with TCR.
- a TCR complex can be composed of a CD3y chain, a CD3P chain, two CD3e chains, a homodimer of CD3z chains, a TCRa chain, and a TCRP chain.
- a TCR complex can be composed of a CD3y chain, a CD3P chain, two CD3e chains, a homodimer of 003z chains, a TCRy chain, and a TCRP chain.
- a “component of a TCR complex”, as used herein, refers to a TCR chain (; i.e ., TCRa, TCRp, TCRy or TCR5), a CD3 chain (i.e., CD3y, CD35, CD3e or CD3C), or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCRa and TCRP, a complex of TCRy and TCR5, a complex of CD3e and CD35, a complex of CD3y and CD3e, or a sub-TCR complex of TCRa, TCRP, CD3y, CD35, and two CD3e chains).
- CAR Chimeric antigen receptor
- CARs can include an extracellular portion comprising an antigen-binding domain (e.g ., obtained or derived from an immunoglobulin or immunoglobulin-like molecule, such as a TCR binding domain derived or obtained from a TCR specific for a cancer antigen, a scFv derived or obtained from an antibody, or an antigen-binding domain derived or obtained from a killer immunoreceptor from an NK cell) linked to a transmembrane domain and one or more intracellular signaling domains (optionally containing co- stimulatory domain(s)) (see, e.g., Sadelain et al., Cancer Discov., 3(4):388 (2013); see also Harris and Kranz, Trends Pharmacol.
- an antigen-binding domain e.g ., obtained or derived from an immunoglobulin or immunoglobulin-like molecule, such as a TCR binding domain derived or obtained from a TCR specific for a cancer antigen, a s
- CARs of the present disclosure that specifically bind to a Core Binding Factor subunit b: Myosin Heavy chain 11 (CBFP:MYHl 1) antigen (e.g, in the context of a peptide:HLA complex) comprise a TCR Va domain and a nb domain.
- CBFP:MYHl 1 antigen e.g, in the context of a peptide:HLA complex
- Antigen refers to an immunogenic molecule that provokes an immune response. This immune response may involve antibody production, activation of specific immunologically-competent cells (e.g., T cells), or both.
- An antigen immunologically-competent cell
- An antigen immunologically-competent molecule
- An antigen immunologically-competent molecule
- An antigen immunologically-competent molecule
- An antigen immunologically-competent cells
- glycopeptide polypeptide, glycopolypeptide, polynucleotide, polysaccharide, lipid or the like. It is readily apparent that an antigen can be synthesized, produced
- exemplary biological samples that can contain one or more antigens include tissue samples, tumor samples, cells, biological fluids, or combinations thereof.
- Antigens can be produced by cells that have been modified or genetically engineered to express an antigen, or that endogenously (e.g., without modification or genetic engineering by human intervention) express a mutation or polymorphism that is immunogenic.
- a “neoantigen,” as used herein, refers to a host cellular product containing a structural change, alteration, or mutation that creates a new antigen or antigenic epitope that has not previously been observed in the subject’s genome (i.e., in a sample of healthy tissue from the subject) or been "seen” or recognized by the host's immune system, which: (a) is processed by the cell’s antigen- processing and transport mechanisms and presented on the cell surface in association with an MHC (e.g, HLA) molecule; and (b) elicits an immune response (e.g, a cellular (T cell) response).
- MHC e.g, HLA
- Neoantigens may originate, for example, from coding polynucleotides having alterations (substitution, addition, deletion) that result in an altered or mutated product, or from the insertion of an exogenous nucleic acid molecule or protein into a cell, or from exposure to environmental factors (e.g, chemical, radiological) resulting in a genetic change. Neoantigens may arise separately from a tumor antigen, or may arise from or be associated with a tumor antigen. "Tumor neoantigen” (or “tumor-specific neoantigen”) refers to a protein comprising a neoantigenic determinant associated with, arising from, or arising within a tumor cell or plurality of cells within a tumor.
- Tumor neoantigenic determinants are found on, for example, antigenic tumor proteins or peptides that contain one or more somatic mutations or chromosomal rearrangements encoded by the DNA of tumor cells (e.g, AML), as well as proteins or peptides from viral open reading frames associated with virus-associated tumors (e.g, cervical cancers, some head and neck cancers).
- AML tumor cells
- proteins or peptides from viral open reading frames associated with virus-associated tumors e.g, cervical cancers, some head and neck cancers.
- epitope includes any molecule, structure, amino acid sequence or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, T cell receptor (TCR), chimeric antigen receptor, or other binding molecule, domain or protein.
- a cognate binding molecule such as an immunoglobulin, T cell receptor (TCR), chimeric antigen receptor, or other binding molecule, domain or protein.
- Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three dimensional structural characteristics, as well as specific charge characteristics.
- Core Binding Factor subunit b: Myosin Heavy chain 11 antigen (or neoantigen) or "Core Binding Factor subunit b: Myosin Heavy chain 11 peptide antigen (or neoantigen)” refers to a naturally or synthetically produced peptide portion of a EBRb:MUH11 fusion protein ranging in length from about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, up to about 20 amino acids, and comprising at least one amino acid alteration caused by a inv(l6) chromosomal inversion event, which peptide can form a complex with a MHC (e.g, HLA) molecule, and a binding protein of this disclosure specific for a CBFb:MYHl 1 peptide:MHC (e.g,
- An exemplary CBFb:MYHl 1 antigen comprises a peptide having the amino acid sequence REEMEVHEL (SEQ ID NO:2); or EEMEVHEL (SEQ ID NO:3); or (Q)LLAVTVHEL (SEQ ID NO: l).
- MHC Major histocompatibility complex
- MHC class I molecules are heterodimers having a membrane spanning a chain (with three a domains) and a non- covalently associated b2 microglobulin.
- MHC class II molecules are composed of two transmembrane glycoproteins, a and b, both of which span the membrane. Each chain comprises two domains.
- MHC class I molecules deliver peptides originating in the
- MHC class II molecules deliver peptides originating in the vesicular system to
- HLAs corresponding to "class I" MHC present peptides from inside the cell and include, for example, HLA-A, HLA-B, and HLA-C. Alleles include, for example, HLA A*02:0l; HLA-A*03:0l; HLA-A*l l0l; HLA- B*40:0l; HLA-B *40: 02; HLA-B *44: 02; or HLA-B *44: 03.
- HLAs corresponding to "class II" MHC present peptides from outside the cell and include, for example, HLA- DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR.
- APC antigen presenting cells
- MHC major histocompatibility complex
- processed antigen peptides originating in the cytosol are generally from about 7 amino acids to about 11 amino acids in length and will associate with class I MHC (HLA) molecules
- peptides processed in the vesicular system e.g, bacterial, viral
- HLA class I MHC
- peptides processed in the vesicular system will vary in length from about 10 amino acids to about 25 amino acids and associate with class II MHC (HLA) molecules.
- CBFP:MYHl l-specific binding protein refers to a protein or polypeptide, such as, for example, a TCR, a scTCR, or CAR, that specifically binds to a CBFP:MYHl lpeptide antigen (or to a CBFP:MYHl lpeptide antigemHLA complex, e.g., on a cell surface), and does not bind a peptide that does not contain the CBFP:MYHl 1 peptide and does not bind to an HLA complex containing such a peptide.
- a CBFP:MYHl l-specific binding protein refers to a protein or polypeptide, such as, for example, a TCR, a scTCR, or CAR, that specifically binds to a CBFP:MYHl lpeptide antigen (or to a CBFP:MYHl lpeptide antigemHLA complex, e.g
- Binding proteins of this disclosure will contain a binding domain specific for a target.
- a "binding domain” also referred to as a “binding region” or “binding moiety” refers to a molecule or portion thereof (e.g, peptide, oligopeptide, polypeptide, protein) that possesses the ability to specifically and non-covalently associate, unite, or combine with a target (e.g, CBFP:MYHl 1 peptide or CBFP:MYHl 1 peptide:MHC complex).
- a binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule, a molecular complex (i.e. complex comprising two or more biological molecules), or other target of interest.
- exemplary binding domains include immunoglobulin variable regions or single chain constructs comprising the same (e.g., single chain TCR (scTCR)).
- a CBFP:MYHl 1 -specific binding protein specifically binds to a CBFP:MYHl 1- containing peptide (or an CBFP:MYHl lpeptide:HLA complex) with a 3 ⁇ 4 of less than about 10 8 M, less than about 10 9 M, less than about 10 10 M, less than about 10 11 M, less than about 10 12 M, or less than about 10 13 M, or with an affinity that is about the same as, at least about the same as, or is greater than at or about the affinity exhibited by an exemplary CBFP:MYHl 1 -specific binding protein provided herein, such as any of the CBFP:MYHl 1 -specific TCRs provided herein, for example, as measured by the same assay.
- a CBFP:MYHl 1 -specific binding protein comprises a CBFP:MYHl 1 -specific immunoglobulin superfamily binding protein or binding portion thereof.
- binding protein e.g ., TCR receptor
- binding domain or fusion protein thereof
- K a i.e., an equilibrium association constant of a particular binding interaction with units of l/M
- Binding proteins or binding domains may be classified as “high affinity” binding proteins or binding domains (or fusion proteins thereof) or as “low affinity” binding proteins or binding domains (or fusion proteins thereof). "High affinity” binding proteins or binding domains refer to those binding proteins or binding domains having a K a of at least
- Low affinity binding proteins or binding domains refer to those binding proteins or binding domains having a K a of up to 10 7 M 1 , up to
- affinity can be defined as an equilibrium dissociation constant (3 ⁇ 4) of a particular binding interaction with units of M (e.g.,
- an CBFP:MYHl 1 -specific binding domain alone i.e., without any other portion of a CBFP:MYHl 1 -specific binding protein
- an CBFP:MYHl 1 -specific binding domain includes an CBFP:MYHl 1 -specific scTCR (e.g, single chain a TCR proteins such as Va-L-nb, nb-L-Va, Va-Ca-L-Va, or Va-L-Vb-Cb, wherein Va and nb are TCRa and b variable domains respectively, Ca and Cb are TCRa and b constant domains, respectively, and L is a linker, such as a linker described herein).
- CBFP:MYHl 1 -specific scTCR e.g, single chain a TCR proteins such as Va-L-nb, nb-L-Va, Va-Ca-L-Va, or Va-L-Vb-Cb, wherein Va and nb are TCRa and b variable domains respectively, Ca and Cb are TCRa and b constant domains, respectively, and L is a linker, such
- fusion proteins comprising a scTCR of the present disclosure linked to the constant domain of an antibody (e.g ., IgG (1, 2, 3, 4), IgE, IgD, IgA, IgM, and variants thereof) or a fragment thereof (e.g. , a fragment that, in some embodiments, retains binding to one or more Fc receptors, to Clq, to Protein A, to Protein G, or any combination thereof), and including immunoglobulin heavy chain monomers and multimers, such as Fc dimers; see, e.g., Wong el al., ./. Immunol. 198: 1 Supp. (2017).
- Variant Fc polypeptides comprising mutations that enhance, reduce, or abrogate binding to or by, e.g, FcRn or other Fc receptors, are known and are contemplated within this disclosure.
- a receptor or binding domain may have "enhanced affinity," which refers to selected or engineered receptors or binding domains with stronger binding to a target antigen than a wild type (or parent) binding domain.
- enhanced affinity may be due to a K a (equilibrium association constant) for the target antigen that is higher than the wild type binding domain, due to a 3 ⁇ 4
- enhanced affinity TCRs can be codon optimized to enhance expression in a particular host cell, such as a cell of the immune system, a hematopoietic stem cell, a T cell, a primary T cell, a T cell line, a NK cell, or a natural killer T cell (Scholten el al, Clin. Immunol. 119: 135, 2006).
- the T cell can be a CD4 + or a CD8 + T cell.
- binding domains of the present disclosure that specifically bind a particular target, as well as determining binding domain or fusion protein affinities, such as multimer/tetramer staining, Western blot, ELISA, analytical ultracentrifugation, spectroscopy and surface plasmon resonance (Biacore®) analysis
- Biacore® surface plasmon resonance
- altered domain refers to a motif, region, domain, peptide, polypeptide, or protein with a non-identical sequence identity to a wild type motif, region, domain, peptide, polypeptide, or protein (e.g ., a wild type TCRa chain, TCRP chain, TCRa constant domain, TCRP constant domain) of at least 85% (e.g., a wild type TCRa chain, TCRP chain, TCRa constant domain, TCRP constant domain) of at least 85% (e.g.,
- Altered domains or altered proteins or derivatives can include those based on all possible codon choices for the same amino acid and codon choices based on
- conservative amino acid substitutions For example, the following six group's each contain amino acids that are conservative substitutions for one another: 1) alanine (ala; A), serine (ser; S), threonine (thr; T); 2) aspartic acid (asp; D), glutamic acid (glu; E); 3) asparagine (asn; N), glutamine (gln; Q); 4) arginine (arg; R), lysine (lys; K); 5)
- nucleic acid or “nucleic acid molecule” refers to any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides,
- nucleic acids of the present disclosure are produced by PCR.
- Nucleic acids can be composed of monomers that are naturally occurring nucleotides (such as
- deoxyribonucleotides and ribonucleotides can have modifications in or replacement of sugar moieties, or pyrimidine or purine base moieties.
- Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate,
- Nucleic acid molecules can be either single- stranded or double-stranded.
- isolated means that the material is removed from its original environment (e.g, the natural environment if it is naturally occurring).
- a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated.
- Such a nucleic acid could be part of a vector and/or such nucleic acid or polypeptide could be part of a composition (e.g, a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide.
- gene means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region ("leader and trailer") as well as intervening sequences (introns) between individual coding segments (exons).
- the terms “recombinant”, “engineered”, and “modified” refer to a cell, microorganism, nucleic acid molecule, polypeptide, protein, plasmid, or vector that has been modified by introduction of an exogenous nucleic acid molecule, or refers to a cell or microorganism that has been genetically engineered by human
- heterologous nucleic acid molecule refers to a cell or microorganism that has been altered such that expression of an endogenous nucleic acid molecule or gene is controlled, deregulated or constitutive, where such alterations or modifications can be introduced by genetic engineering.
- Human-generated genetic alterations can include, for example, modifications introducing nucleic acid molecules (which may include an expression control element, such as a promoter) encoding one or more proteins or enzymes, or other nucleic acid molecule additions, deletions, substitutions, or other functional disruption of or addition to a cell's genetic material.
- Exemplary modifications include those in coding regions or functional fragments thereof of heterologous or homologous polypeptides from a reference or parent molecule.
- mutation refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively.
- a mutation can result in several different types of change in sequence, including substitution, insertion or deletion of nucleotide(s) or amino acid(s).
- a mutation is a substitution of one or three codons or amino acids, a deletion of one to about 5 codons or amino acids, or a combination thereof.
- a "conservative substitution” is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties.
- Exemplary conservative substitutions are well known in the art (see, e.g. , WO 97/09433 at page 10; Lehninger, Biochemistry, 2 nd Edition; Worth Publishers, Inc. NY, NY, pp.71-77, 1975; Lewin, Genes IV, Oxford ETniversity Press, NY and Cell Press, Cambridge, MA, p. 8, 1990).
- proteins e.g., binding protein, immunogenic peptide
- proteins comprise a variant sequence as compared to a reference sequence (e.g, a variant TCR CDR3P as compared to a reference TCR CDR3P disclosed herein).
- a "variant" amino acid sequence, peptide, or polypeptide refers to a an amino acid sequence (or peptide or polypeptide) having one or two amino acid substitutions, deletions, or insertions as compared to a reference amino acid sequence.
- a variant amino acid sequence, peptide, or polypeptide retains substantially a same functionality (e.g, binding specificity and affinity for a peptide:HLA complex) as the reference molecule; for example, a variant TCR fragment as disclosed herein retains about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or 100% of the antigen-binding specificity and affinity as compared to a reference TCR binding fragment.
- construct refers to any polynucleotide that contains a recombinant nucleic acid molecule.
- a "transgene” or “transgene construct” refers to a construct that contains two or more genes operably linked in an arrangement that is not found in nature.
- the term “operably-linked” refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other.
- a promoter is operably- linked with a coding sequence when it can affect the expression of that coding sequence ( i.e ., the coding sequence is under the transcriptional control of the promoter).
- Unlinked means that the associated genetic elements are not closely associated with one another and the function of one does not affect the other.
- the genes present in a transgene are operably linked to an expression control sequence (e.g ., a promoter).
- a construct e.g., a transgene
- a vector e.g, a bacterial vector, a viral vector
- a "vector” is a nucleic acid molecule that is capable of transporting another nucleic acid molecule.
- Vectors can be, for example, plasmids, cosmids, viruses, a RNA vector or a linear or circular DNA or RNA molecule that can include chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acid molecules.
- Exemplary vectors are those capable of autonomous replication (episomal vector) or expression of nucleic acid molecules to which they are linked (expression vectors). Vectors useful in the compostions and methods of this disclosure are described further herein.
- expression refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene.
- the process can include transcription, post-transcriptional control, post- transcriptional modification, translation, post-translational control, post translational modification, or any combination thereof.
- the term "introduced” in the context of inserting a nucleic acid molecule into a cell means “transfection", or “transformation”, or “transduction” and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell wherein the nucleic acid molecule can be incorporated into the genome of a cell (e.g, a chromosome, a plasmid, a plastid, or a mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
- a cell e.g, a chromosome, a plasmid, a plastid, or a mitochondrial DNA
- transiently expressed e.g., transfected mRNA
- heterologous or exogenous nucleic acid molecule, construct or sequence refers to a nucleic acid molecule or portion of a nucleic acid molecule that is not native to a host cell, but can be homologous to a nucleic acid molecule or portion of a nucleic acid molecule from the host cell.
- the source of the heterologous or exogenous nucleic acid molecule, construct or sequence can be from a different genus or species.
- a heterologous or exogenous nucleic acid molecule is added (i.e., not endogenous or native) to a host cell or host genome by, for example, conjugation, transformation, transfection, transduction, electroporation, or the like, wherein the added molecule can integrate into the host genome or exist as extra- chromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), and can be present in multiple copies.
- heterologous refers to a non-native enzyme, protein or other activity encoded by an exogenous nucleic acid molecule introduced into the host cell, even if the host cell encodes a homologous protein or activity.
- more than one heterologous or exogenous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof.
- two or more exogenous nucleic acid molecules are introduced into a host cell, it is understood that the two or more exogenous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into the host chromosome at a single site or multiple sites, or any combination thereof.
- the number of referenced heterologous nucleic acid molecules or protein activities refers to the number of encoding nucleic acid molecules or the number of protein activities, not the number of separate nucleic acid molecules introduced into a host cel 1.
- endogenous refers to a gene, protein, or activity that is normally present in a host cell. Moreover, a gene, protein or activity that is mutated, overexpressed, shuffled, duplicated or otherwise altered as compared to a parent gene, protein or activity is still considered to be endogenous or native to that particular host cell.
- an endogenous control sequence from a first gene e.g ., a promoter, translational attenuation sequences
- a second native gene or nucleic acid molecule can be used to alter or regulate expression of a second native gene or nucleic acid molecule, wherein the expression or regulation of the second native gene or nucleic acid molecule differs from normal expression or regulation in a parent cell.
- homologous refers to a molecule or activity found in or derived from a host cell, species or strain.
- a heterologous or exogenous nucleic acid molecule can be homologous to a native host cell gene, and can optionally have an altered expression level, a different sequence, an altered activity, or any combination thereof.
- Sequence identity refers to the percentage of amino acid residues in one sequence that are identical with the amino acid residues in another reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
- the percentage sequence identity values can be generated using the NCBI BLAST 2.0 software as defined by Altschul et al. (1997), Nucl. Acids Res. 25:3389-3402, with the parameters set to default values.
- the present disclosure provides an isolated polynucleotide encoding a binding protein that comprises a T cell receptor (TCR) a chain variable (Va) domain and a TCR b chain variable (nb) domain, wherein the encoded binding protein is capable of specifically binding to a peptide comprising a Core Binding Factor subunit bi ⁇ n ⁇ ob ⁇ h Heavy chain 11 (EBRb:MUH11) antigen.
- TCR T cell receptor
- Va chain variable
- nb TCR b chain variable
- EBRb:MUH11 Core Binding Factor subunit bi ⁇ n ⁇ ob ⁇ h Heavy chain 11
- the EBRb:MUH11 antigen comprises or consists of the amino acid sequence REEMEVHEL (SEQ ID NO:2) or EEMEVHEL (SEQ ID NO:3), wherein the heterologous polynucleotide is codon optimized for expression in a host cell.
- the antigen comprises or consists of the amino acid sequence REEMEVHEL (SEQ ID NO:2).
- the CBFP:MYHl 1 antigen comprises or consists of the amino acid sequence (Q)LLAVTVHEL (SEQ ID NO: l)
- the encoded binding protein is capable of specifically binding to a CBFP:MYHl l-HLA complex.
- the HLA comprises HLA-A*02:0l; HLA-A*03:0l; HLA-A*l 1 :01; HLA-B *40:01; HLA-B*44:02; HLA-B*40:02; or HLA-B*44:03.
- the HLA comprises HLA-B*40:0l.
- Va exons are assembled from different variable and joining gene segments (V-J), and nb exons are assembled from different variable, diversity, and joining gene segments (V-D-J).
- the TCRa chromosomal locus has 70-80 variable gene segments and 61 joining gene segments.
- the TCRj3 chromosomal locus has 52 variable gene segments, and two separate clusters of each containing a single diversity gene segment, together with six or seven joining gene segments.
- Functional Va and nb gene exons are generated by the recombination of a variable gene segment with a joining gene segment for Va, and a variable gene segment with a diversity gene segment and a joining gene segment for nb.
- the polynucleotide encoding the nb domain comprises: (i) a TCRBV05-0l*0l gene segment, a TCRBD02-0l*02 gene segment, and a
- TCRBJ02-02*0l gene segment (ii) a TCRBV04-0l*0l gene segment, a TCRBD01- 01*01 gene segment, and a TCRBJ02-0l*0l gene segment; (iii) a TCRBV04-0l*0l gene segment, a TCRBD02-0l*0l gene segment, and a TCRBJ01-01*01 gene segment; (iv) a TCRBV07-02*0l gene segment, a TCRBD01-01*01 gene segment, and a TCRBJ02-0l*0l gene segment; (v) a TCRBV06-0l*0l gene segment, a TCRBD01- 01*01 gene segment, and a TCRBJ02-0l*0l gene segment; or (vi) a TCRBV04-02*0l gene segment, a TCRBD gene segment, and a TCRBJ01-04*01 gene segment.
- the polynucleotide encoding the Va domain comprises: (i) a TCRAV20-01 gene segment, a TCRDD01-01 gene segment, and a TCRAJ45- 01*01 gene segment; (ii) a TCRAV08-06 gene segment, a TCRAD gene segment, and a TCRAJ52-0l*0l gene segment; or (iii) a TCRAV38-01 gene segment, a TCRBD01- 01*01 gene segment, and a TCRAJ50-0l*0l gene segment.
- the polynucleotide encoding the Va domain comprises a TCRAV20-01 gene segment, a TCRDD01-01 gene segment and a
- the polynucleotide encoding the nb domain comprises a TCRBV07-02*0l gene segment, a TCRBDO 1-01*01 gene segment, and a TCRBJ02-0l*0l gene segment;
- the polynucleotide encoding the Va domain comprises a TCRAV08-06 gene segment, a TCRAD gene segment, and a TCRAJ52- 01*01 gene segment, and the polynucleotide encoding the nb domain comprises a TCRBV06-0l*0l gene segment, a TCRBDO 1-01*01 gene segment, and a TCRBJ02- 01*01 gene segment;
- the polynucleotide encoding the Va domain comprises a TCRAV38-01 gene segment, a TCRD gene segment, and a TCRAJ50-0l*0l gene segment, and the polynucleotide encoding the nb domain
- the encoded nb domain comprises or consists of an amino acid sequence having at least 90% (i.e ., at least 90%, 91%, 92%, 93%, 94%,
- the encoded Va domain comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs:2l-24.
- the encoded nb domain comprises a Complementarity Determining Region 3 (CDR3b) comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs:4-8 or 12, or a variant thereof.
- the encoded Va domain comprises a Complementarity Determining Region 3 (CDR3a) comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 13-16, or a variant thereof.
- an encoded nb domain or an encoded Va domain has at least 90% identity to a reference nb or Va amino acid sequence (respectively) as disclosed herein, provided that: (a) at least three or four of the CDRs have no mutations; (b) the CDRs that do have mutations ( e.g ., a variant of a CDR3 sequence as disclosed herein) have only up to two amino acid substitutions, up to a contiguous five amino acid deletion, or a combination thereof; and (c) the encoded binding protein retains its ability to bind to a peptide comprising a CBFP:MYHl 1 antigen.
- the encoded Va domain comprises no change in amino acid sequence of CDR1
- the encoded nb domain comprises no change in amino acid sequence of CDR1
- the CDR1 of the encoded Va domain and the CDR1 of the encoded nb domain comprise no change in amino acid sequence.
- the encoded Va domain comprises no change in amino acid sequence of CDR2
- the encoded nb domain comprises no change in amino acid sequence of CDR2
- the CDR2 of the encoded Va domain and the CDR2 of the encoded nb domain comprise no change in amino acid sequence.
- the encoded CDR3b comprises or consists of the amino acid sequence set forth in SEQ ID NO:4, and the encoded CDR3a comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16.
- the encoded nb domain has at least 90% identity to the amino acid sequence set forth in SEQ ID NO:20
- the encoded Va domain has at least 90% identity to the amino acid sequence set forth in SEQ ID NO:24.
- the encoded nb domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:20
- the encoded Va domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:24.
- the encoded O ⁇ E3b comprises or consists of the amino acid sequence set forth in SEQ ID NO:6, and the encoded CDR3a comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15.
- the encoded nb domain has at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 19, and the encoded Va domain has at least 90% identity to the amino acid sequence set forth in SEQ ID NO:23.
- the encoded nb domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19, and the encoded Va domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:23.
- the encoded CDR3P comprises or consists of the amino acid sequence set forth in SEQ ID NO:7
- the encoded CDR3a comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13.
- the encoded nb domain has at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 17, and the encoded Va domain has at least 90% identity to the amino acid sequence set forth in SEQ ID NO:2l.
- the encoded nb domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17, and the encoded Va domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:2l.
- the encoded O ⁇ E3b comprises or consists of the amino acid sequence set forth in SEQ ID NO:8, and the encoded CDR3a comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14.
- the encoded nb domain has at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 18, and the encoded Va domain has at least 90% identity to the amino acid sequence set forth in SEQ ID NO:22.
- the encoded nb domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18, and the encoded Va domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:22.
- a binding protein-encoding polynucleotide of the present disclosure may, in some embodiments, comprise a polynucleotide that encodes a TCR constant domain.
- a TCR constant domain is modified to enhance pairing of desired TCR chains. For example, enhanced pairing in a host T cell between a heterologous TCR a-chain and a heterologous TCR b-chain due to a modification results in the preferential assembly of a TCR comprising two heterologous chains over an undesired mispairing of a heterologous TCR chain with an endogenous TCR chain (see, e.g., Govers et al, Trends Mol. Med. 16(2):P (2010), the TCR modifications of which are herein incorporated by reference).
- Exemplary modifications to enhance pairing of heterologous TCR chains include the introduction of complementary cysteine residues in each of the heterologous TCR a-chain and b-chain.
- a polynucleotide encoding a heterologous TCR a-chain encodes a cysteine at amino acid position 48 (corresponding to the full-length, mature human TCR a-chain sequence) and a polynucleotide encoding a heterologous TCR b-chain encodes a cysteine at amino acid position 57 (corresponding to the full-length mature human TCR b-chain sequence).
- the encoded Cb comprises or consists of an amino acid sequence having at least 90% identity to any one of SEQ ID NOs:25-28.
- the polynucleotide further comprises a polynucleotide encoding a TCR a-chain constant domain (Ca).
- the encoded Ca comprises or consists of an amino acid sequence having at least 90% identity to any one of SEQ ID NOs:29-32.
- the encoded binding protein comprises a Va domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:24, a Ca domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:32; a nb domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:20; and a Ob domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:28.
- the encoded binding protein comprises a TCR a-chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:40, and a TCR b-chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:39.
- the encoded binding protein comprises a Va domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:23, a Ca domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:3 l; a nb domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 19; and a Ob domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:27.
- the encoded binding protein comprises a TCR a-chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:38, and a TCR b-chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:37.
- the encoded binding protein comprises a Va domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:2l, a Ca domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:29; a nb domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17; and a Ob domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:25.
- the encoded binding protein comprises a TCR a-chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:34, and a TCR b-chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:33.
- the encoded binding protein comprises a Va domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:22, a Ca domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:30; a nb domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:l8; and a Ob domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:26.
- the encoded binding protein comprises a TCR a-chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:36, and a TCR b-chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:35.
- the encoded binding protein can comprise a TCR, a chimeric antigen receptor (CAR), a single-chain TCR (scTCR), or any combination thereof.
- CAR chimeric antigen receptor
- scTCR single-chain TCR
- an encoded binding protein binds to residues 2, 3, 5, 6, 7, and 9 of SEQ ID NO:2.
- an encoded binding protein of the present disclosure is capable of binding to a peptide comprising or consisting of the consensus amino acid sequence set forth in SEQ ID NO: 109, wherein the peptide is optionally in complex with a HLA molecule, such as, for example, a HLA-B *40:01 or a HLA-B*40:02 molecule.
- a peptide bound by a binding protein of the present disclosure comprises or consists of the amino acid sequence set forth in SEQ ID NO: 119.
- a peptide bound by a binding protein of the present disclosure comprises or consists of the amino acid sequence set forth in SEQ ID NO: 110.
- alanine mutagenesis of any one or more of residues 2, 3, 5, 6, 7, or 9 of SEQ ID NO:2 reduces or abrogates binding by the binding protein (e.g ., to a SEQ ID NO:2-HLA complex, such as a SEQ ID N0:2-HLA-B*40:0l complex).
- an encoded binding protein of the present disclosure does not bind to a variant of SEQ ID NO:2 that does not contain an Arg at a position corresponding to position 2 of SEQ ID NO:2; a Glu at a position corresponding to position 3 of SEQ ID NO:2; a a Glu at a position
- an encoded binding protein of the present disclosure does not bind to a variant of SEQ ID NO:2 that does not contain an Arg at a position corresponding to position 2 of SEQ ID NO:2; a Glu at a position corresponding to position 3 of SEQ ID NO:2; a a Glu at a position corresponding to position 5 of SEQ ID NO:2; a Val at a position corresponding to position 6 of SEQ ID NO:2; a His at a position corresponding to position 7 of SEQ ID NO:2; and a Leu at a position corresponding to position 9 of SEQ ID NO :2.
- a polynucleotide can be codon- optimized for expression by a T cell (e.g., a human T cell).
- a polynucleotide can introduce a cysteine modification in the constant domain of the TCR alpha and/or TCR beta chains in order to improve pairing and avoid mispairing with the native TCR.
- a binding protein-encoding polynucleotide comprises a CDR3P-encoding polynucleotide that comprises a nucleotide sequence set forth in any one of SEQ ID NOs:50-53, and/or a CDR3a-encoding polynucleotide set forth in any one of SEQ ID NOs:54-57.
- a binding protein-encoding polynucleotide comprises a CDR3P-encoding polynucleotide as set forth in SEQ ID NO: 52, and a CDR3a-encoding polynucleotide as set forth in SEQ ID NO:56.
- a binding protein-encoding polynucleotide comprises a CDRjp-encoding polynucleotide as set forth in SEQ ID NO:50, and a CDR3a-encoding polynucleotide as set forth in SEQ ID NO:54.
- a binding protein-encoding polynucleotide comprises a CDRjp-encoding polynucleotide as set forth in SEQ ID NO:5l, and a CDR3a-encoding polynucleotide as set forth in SEQ ID NO: 55.
- a binding protein-encoding polynucleotide comprises a CDRjp-encoding polynucleotide as set forth in SEQ ID NO:53, and a CDR3a-encoding polynucleotide as set forth in SEQ ID NO:57.
- a binding protein-encoding polynucleotide comprises a nb-encoding polynucleotide that has at least 80% (i.e., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identity to the nucleotide sequence set forth in any one of SEQ ID NOs:64, 69, 74, or 79, and a Va-encoding polynucleotide that has at least 80% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 66, 71, 76, or 81.
- a binding protein-encoding polynucleotide comprises a nb-encoding polynucleotide that has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO:69, and a Va-encoding polynucleotide that has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 71.
- a binding protein-encoding polynucleotide comprises a nb-encoding polynucleotide that has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO:79, and a Va-encoding polynucleotide that has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 81.
- a binding protein-encoding polynucleotide comprises a nb-encoding polynucleotide that has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO:64, and a Va-encoding polynucleotide that has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO:66.
- a binding protein-encoding polynucleotide comprises a nb-encoding polynucleotide that comprises the nucleotide sequence set forth in SEQ ID NO:72, and a Va-encoding polynucleotide that comprises a nucleotide sequence set forth in SEQ ID NO:74.
- a binding protein-encoding polynucleotide can comprise a CP-encoding polynucleotide that has at least 80% identity to the nucleotide sequence set forth in any one of SEQ ID NOs:65, 70, 75, or 80, and a Ca-encoding polynucleotide that that has at least 80% identity to the nucleotide sequence set forth in any one of SEQ ID NOs:67, 72, 77, or 82.
- a single polynucleotide encodes a binding protein as described herein, or, alternatively, the binding protein may be encoded by more than one polynucleotide.
- components or portions of a binding protein may be encoded by two or more polynucleotides, which may be contained on a single nucleic acid molecule or may be contained on two or more nucleic acid molecules.
- a polynucleotide encoding two or more components or portions of a binding protein of the present disclosure comprises the two or more coding sequences operatively associated in a single open reading frame.
- desired gene products such as, for example, contemporaneous expression of alpha- and beta-chains of a TCR, such that they are produced in about a 1 : 1 ratio.
- two or more substituent gene products of a binding protein of this disclosure such as a TCR (e.g ., alpha- and beta-chains) or CAR, are expressed as separate molecules and associate post- translationally.
- two or more substituent gene products of a binding protein of this disclosure are expressed as a single peptide with the parts separated by a cleavable or removable segment.
- self-cleaving peptides useful for expression of separable polypeptides encoded by a single polynucleotide or vector are known in the art and include, for example, a Porcine teschovirus-l 2 A (P2A) peptide, a Thoseaasigna virus 2A (T2A) peptide, an Equine rhinitis A virus (ERAV) 2A (E2A) peptide, and a Foot-and-Mouth disease virus 2A (F2A) peptide.
- P2A Porcine teschovirus-l 2 A
- T2A Thoseaasigna virus 2A
- E2A Equine rhinitis A virus
- F2A Foot-and-Mouth disease virus
- a heterologous polynucleotide encoding a TCR a-chain and a heterologous polynucleotide encoding a TCR b-chain are contained in a single open reading frame comprised in the engineered immune cell, wherein the single open reading frame further comprises a polynucleotide encoding a self-cleaving peptide disposed between the a-chain-encoding polynucleotide and the b-chain- encoding polynucleotide.
- the encoded self-cleaving peptide comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs:4l-44.
- the polynucleotide encoding the self-cleaving peptide comprises or consists of the nucleotide sequence set forth in any one of SEQ ID NOs: 58-62.
- an encoded binding protein of the present disclosure comprises one or more junction amino acids.
- Junction amino acids or “junction amino acid residues” refer to one or more ( e.g ., 2 to about 10) amino acid residues between two adjacent motifs, regions or domains of a polypeptide, such as between a binding domain and an adjacent constant domain or between a TCR chain and an adjacent self-cleaving peptide.
- Junction amino acids can result from the design of a construct that encodes a fusion protein (e.g., amino acid residues resulting from the use of a restriction enzyme site during the construction of a nucleic acid molecule encoding a fusion protein), or from cleavage of, for example, a self-cleaving peptide adjacent one or more domains of an encoded binding protein of this disclosure (e.g, a P2A peptide disposed between a TCR a-chain and a TCR b-chain, the self-cleavage of which can leave one or more junction amino acids in the a-chain, the TCR b-chain, or both).
- a fusion protein e.g., amino acid residues resulting from the use of a restriction enzyme site during the construction of a nucleic acid molecule encoding a fusion protein
- cleavage of, for example, a self-cleaving peptide adjacent one or more domains of an encoded binding protein of this disclosure e.g
- a binding protein is expressed as part of a transgene construct that encodes, and/or a modified immune cell can encode: one or more additional accessory protein, such as a safety switch protein; a tag, a selection marker; a CD8 co-receptor b-chain; a CD8 co-receptor a-chain or both; or any combination thereof.
- additional accessory protein such as a safety switch protein; a tag, a selection marker; a CD8 co-receptor b-chain; a CD8 co-receptor a-chain or both; or any combination thereof.
- polynucleotides and transgene constructs useful for encoding and expressing binding proteins and accessory components are described in PCT application PCT/US2017/053112, the polynucleotides, transgene constructs, and accessory components, including the nucleotide and amino acid sequences, of which are hereby incorporated by reference.
- any or all of a binding protein of the present disclosure, a safety switch protein, a tag, a selection marker, a CD8 co-receptor b-chain, or a CD8 co-receptor a-chain may be encoded by a single nucleic acid molecule or may be encoded by polynucleotide sequences that are, or are present on, seprate nucleic acid molecules.
- Exemplary safety switch proteins include, for example, a truncated EGF receptor polypeptide (huEGFRt) that is devoid of extracellular N-terminal ligand binding domains and intracellular receptor tyrosine kinase activity, but that retains its native amino acid sequence, has type I transmembrane cell surface localization, and has a conformationally intact binding epitope for pharmaceutical-grade anti-EGFR monoclonal antibody, cetuximab (Erbitux) tEGF receptor (tEGFr; Wang et al. , Blood 118: 1255-1263, 2011); a caspase polypeptide (e.g ., iCasp9; Straathof et al.
- huEGFRt truncated EGF receptor polypeptide
- accessory components useful for modified immune cells of the present disclosure comprise a tag or selection marker that allows the cells to be identified, sorted, isolated, enriched, or tracked.
- marked immune cells having desired characteristics e.g, an antigen-specific TCR and a safety switch protein
- selection marker comprises a nucleic acid construct (and the encoded gene product) that confers an identifiable change to a cell permitting detection and positive selection of immune cells transduced with a polynucleotide comprising a selection marker.
- RQR is a selection marker that comprises a major extracellular loop of CD20 and two minimal CD34 binding sites.
- an RQR-encoding polynucleotide comprises a polynucleotide that encodes the 16-amino-acid CD34 minimal epitope.
- the CD34 minimal epitope is incorporated at the amino terminal position of a CD8 co-receptor stalk domain (Q8).
- the CD34 minimal binding site sequence can be combined with a target epitope for CD20 to form a compact marker/suicide gene for T cells (RQR8) (Philip et al. , 2014, incorporated by reference herein).
- This construct allows for the selection of immune cells expressing the construct, with for example, CD34 specific antibody bound to magnetic beads (Miltenyi) and that utilizes clinically accepted pharmaceutical antibody, rituximab, that allows for the selective deletion of a transgene expressing engineered T cell (Philip et al ., 2014).
- selection markers also include several truncated type I transmembrane proteins normally not expressed on T cells: the truncated low-affinity nerve growth factor, truncated CD 19, and truncated CD34 (see for example, Di Stasi et al, N. Engl. J Med. 365:1673-1683, 2011; Mavilio et al, Blood 83: 1988-1997, 1994; Fehse et al, Mol. Ther. 7:448-456, 2000; each incorporated herein in their entirety).
- a useful feature of CD 19 and CD34 is the availability of the off-the-shelf Miltenyi CliniMACsTM selection system that can target these markers for clinical-grade sorting.
- CD 19 and CD34 are relatively large surface proteins that may tax the vector packaging capacity and transcriptional efficiency of an integrating vector.
- Surface markers containing the extracellular, non-signaling domains or various proteins e.g., CD 19, CD34, LNGFR
- Any selection marker may be employed and should be acceptable for Good Manufacturing Practices.
- selection markers are expressed with a polynucleotide that encodes a gene product of interest (e.g, a binding protein of the present disclosure, such as a TCR or CAR).
- selection markers include, for example, reporters such as GFP, EGFP, b-gal or chloramphenicol acetyltransferase (CAT).
- a selection marker such as, for example, CD34 is expressed by a cell and the CD34 can be used to select enrich for, or isolate (e.g, by immunomagnetic selection) the transduced cells of interest for use in the methods described herein.
- a CD34 marker is distinguished from an anti-CD34 antibody, or, for example, a scFv, TCR, or other antigen recognition moiety that binds to CD34.
- a selection marker comprises an RQR polypeptide, a truncated low-affinity nerve growth factor (tNGFR), a truncated CD 19 (tCDl9), a truncated CD34 (tCD34), or any combination thereof.
- CD4 + T cells in an immunotherapy cell product can provide antigen-induced IL-2 secretion and augment persistence and function of introduced cytotoxic CD8 + T cells (see, e.g., Kennedy et al, Immunol. Rev. 222 129 (2008); Nakanishi et ah, Nature 4 ⁇ 52(7272):5l0 (2009)).
- CD8 + T cells see, e.g., Kennedy et al, Immunol. Rev. 222 129 (2008); Nakanishi et ah, Nature 4 ⁇ 52(7272):5l0 (2009).
- a class I restricted TCR in CD4 + T cells may require the transfer of a CD8 co-receptor to enhance sensitivity of the TCR to class I HLA peptide complexes.
- CD4 co-receptors differ in structure to CD8 and have been shown to be ineffective substitutes for CD8 co-receptors (see, e.g, Stone & Kranz, Front. Immunol. 4:244 (2013); see also Cole et al, Immunology 737(2): 139 (2012).
- another accessory protein for use in the compositions and methods of this disclosure comprises a CD8 co- receptor or component thereof.
- Engineered immune cells comprising a heterologous polynucleotide encoding a binding protein of the present disclosure may, in certain embodiments, further comprise a heterologous polynucleotide encoding a CD8 co-receptor protein, or a beta-chain or alpha-chain component thereof.
- An encoded CD8 co-receptor includes, in some embodiments, an a-chain or a fragment or variant thereof.
- An amino acid sequence of the human CD8 co-receptor a - chain precursor is known and is provided at, for example, UniProtKB -P30433 (see also UniProtKB - P31783; -P10732; and -P10731).
- An encoded CD8 co-receptor includes, in some embodiments, a b-chain or a fragment or variant thereof.
- An amino acid sequence of the human CD8 co-receptor b- chain precursor is known and is provided at, for example, UniProtKB -P 10966 (see also UniProtKB - Q9UQ56; -E9PD41; Q8TD28; and -P30434; and -P05541).
- the encoded RQR polypeptide is contained in a b-chain, an a-chain, or both, or a fragment or variant of either or both, of the encoded CD8 co-receptor.
- a modified immune cell comprises a heterologous polynucleotide encoding iCasp9 and a heterologous polynucleotide encoding a recombinant CD8 co-receptor protein that comprises a b-chain containing a RQR polypeptide and further comprises a CD8 a- chain.
- An isolated polynucleotide of this disclosure may further comprise a polynucleotide encoding a safety switch protein, a selection marker, a CD8 co-receptor beta chain, or a CD8 co-receptor alpha chain as disclosed herein, or may comprise a polynucleotide encoding any combination thereof.
- a polynucleotide encoding a binding protein further comprises a polynucleotide that encodes a selection marker, wherein the encoded selection marker can comprise or consist of the amino acid sequence set forth in SEQ ID NO:45.
- the polynucleotide encoding the selection marker comprises or consists of the nucleotide sequence set forth in SEQ ID NO:63.
- a polynucleotide is codon- optimized for expression in an immune cell, such as, for example, a T cell.
- an isolated polynucleotide of the present disclosure encodes a binding protein, a self-cleaving peptide, and a selection marker, and has at least 80% (/.e.,at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identity to the nucleotide sequence set forth in SEQ ID NO:73.
- the isolated polynucleotide comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 73.
- an isolated polynucleotide of the present disclosure encodes a binding protein, a self-cleaving peptide, and a selection marker, and has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 83.
- the isolated polynucleotide comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 83.
- an isolated polynucleotide of the present disclosure encodes a binding protein, a self-cleaving peptide, and a selection marker, and has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 68.
- the isolated polynucleotide comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 68.
- an isolated polynucleotide of the present disclosure encodes a binding protein, a self-cleaving peptide, and a selection marker, and has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO:78.
- the isolated polynucleotide comprises or consists of the nucleotide sequence set forth in SEQ ID NO:78.
- an encoded polypeptide of this disclosure can comprise a "signal peptide” (also known as a leader sequence, leader peptide, or transit peptide).
- Signal peptides target newly synthesized polypeptides to their appropriate location inside or outside the cell.
- a signal peptide may be removed from the polypeptide during or once localization or secretion is completed.
- polypeptides that have a signal peptide are referred to herein as a "pre-protein” and polypeptides having their signal peptide removed are referred to herein as “mature” proteins or polypeptides.
- a “linker” refers to an amino acid sequence that connects two proteins, polypeptides, peptides, domains, regions, or motifs and may provide a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity (e.g ., scTCR) to a target molecule or retains signaling activity (e.g., TCR complex).
- a linker is comprised of about two to about 35 amino acids, for instance, or about four to about 20 amino acids or about eight to about 15 amino acids or about 15 to about 25 amino acids.
- Exemplary linkers include glycine-serine linkers, such as those comprising any one or more of the amino acid sequences set forth in SEQ ID NOs:95-98.
- vectors that comprise a polynucleotide or transgene construct of the instant disclosure.
- Some examples of vectors include plasmids, viral vectors, cosmids, and others.
- Some vectors may be capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors), whereas other vectors may be integrated into the genome of a host cell or promote integration of the polynucleotide insert upon introduction into the host cell and thereby replicate along with the host genome (e.g ., lentiviral vector, retroviral vector).
- vectors are capable of directing the expression of genes to which they are operatively linked (these vectors may be referred to as "expression vectors").
- expression vectors e.g., polynucleotides encoding binding proteins as described herein
- agents e.g., polynucleotides encoding binding proteins as described herein
- each agent may reside in separate or the same vectors, and multiple vectors (each containing a different agent or the same agent) may be introduced to a cell or cell population or administered to a subject.
- polynucleotides of the present disclosure may be operatively linked to certain elements of a vector.
- polynucleotide sequences that are needed to effect the expression and processing of coding sequences to which they are ligated may be operatively linked.
- Expression control sequences may include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and possibly sequences that enhance protein secretion.
- Expression control sequences may be operatively linked if they are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
- the vector comprises a plasmid vector or a viral vector (e.g, a vector selected from lentiviral vector or a g-retroviral vector).
- Viral vectors include retrovirus, adenovirus, parvovirus (e.g, adeno-associated viruses), coronavirus, negative strand RNA viruses such as ortho-myxovirus (e.g, influenza virus), rhabdovirus (e.g, rabies and vesicular stomatitis virus), paramyxovirus (e.g, measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g, Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox).
- herpesvirus e.g, Herpe
- viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavims, and hepatitis virus, for example.
- retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, and spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).
- “Retroviruses” are viruses having an RNA genome, which is reverse-transcribed into DNA using a reverse transcriptase enzyme, the reverse-transcribed DNA is then incorporated into the host cell genome.
- “Gammaretrovirus” refers to a genus of the retroviridae family. Examples of gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses.
- "Lentiviral vector,” as used herein, means HIV-based lentiviral vectors for gene delivery, which can be integrative or non-integrative, have relatively large packaging capacity, and can transduce a range of different cell types. Lentiviral vectors are usually generated following transient transfection of three (packaging, envelope and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter the target cell through the interaction of viral surface
- the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex.
- the product of reverse transcription is a double-stranded linear viral DNA, which is the substrate for viral integration into the DNA of infected cells.
- the viral vector can be a gammaretrovirus, e.g., Moloney murine leukemia virus (MLV)-derived vectors.
- the viral vector can be a more complex retrovirus-derived vector, e.g., a lentivirus-derived vector. HIV- 1 -derived vectors belong to this category.
- Other examples include lentivirus vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (ovine lentivirus).
- Retroviral and lentiviral vector constructs and expression systems are also commercially available.
- Other viral vectors also can be used for polynucleotide delivery including DNA viral vectors, including, for example adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex viruses (HSVs), including amplicon vectors, replication-defective HSV and attenuated HSV (Krisky et al., Gene Ther. 5: 1517, 1998).
- HSVs herpes simplex viruses
- vectors recently developed for gene therapy uses can also be used with the compositions and methods of this disclosure.
- Such vectors include those derived from baculoviruses and a- viruses. (Jolly, D J. 1999. Emerging Viral Vectors pp 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as Sleeping Beauty or other transposon vectors).
- the viral vector may also comprise additional sequences between the two (or more) transcripts allowing for bicistronic or multi cistronic expression.
- additional sequences used in viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptide, or any combination thereof.
- a vector is capable of delivering the polynucleotide or transgene construct to a host cell (e.g ., a hematopoietic progenitor cell or a human immune system cell).
- a vector is capable of delivering a polynucleotide or transgene construct to human immune system cell, such as, for example, a CD4 + T cell, a CD8 + T cell, a CD4 CD8 double negative T cell, a stem cell memory T cell, a gd T cell, a natural killer cell, a dendritic cell, or any combination thereof.
- a vector is capable of delivering a transgene construct to a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.
- a vector that encodes a polynucleotide or transgene construct of the present disclosure may further comprise a polynucleotide that encodes a nuclease that can be used to perform a chromosomal knockout in a host cell (e.g., a CRISPR-Cas endonuclease or another endonuclease as disclosed herein) or that can be used to to deliver a therapeutic polynucleotide or transgene or portion thereof to a host cell in a gene therapy replacement or gene repair therapy.
- a nuclease used for a chromosomal knockout or a gene replacement or gene repair therapy can be delivered to a host cell independent of a vector that encodes a
- host cells that comprise a polynucleotide of the present disclosure (i.e., encoding a binding protein and optionally encoding one or more additional proteins, such as a selection marker or a self-cleaving peptide).
- a host cell may be generated by, for example, transfection or transduction with a vector of the present disclosure.
- a host cell comprises an immune system cell, which in certain embodiments may be a human immune system cell.
- the host cell comprises a T cell, a NK cell, a NK-T cell, or any combination thereof.
- the T cell comprises a CD8 + T cell, CD4 T cells, or any combination of T cell subset thereof.
- the present disclosure provides a modified immune cell comprising a heterologous polynucleotide that encodes a binding protein that specifically binds to a CBFP:MYHl 1 antigen, wherein the encoded binding protein is expressed at the surface ( e.g ., is anchored in or tethered to the extracellular membrane and has an extracellular portion that specifically binds to a CBFpAIYH l 1 antigen) of the modified immune cell.
- the encoded binding protein comprises a TCR nb domain and a TCR Va domain
- the encoded nb domain comprises or consists of an amino acid sequence having at least 90% (i.e., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or more) identity to the amino acid sequence set forth in any one of SEQ ID NOs: 17-20
- the encoded Va domain comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs:2l-24.
- a modified immune cell of the present disclosure comprises a heterologous polynucleotide encoding a binding protein that includes: (a) a T cell receptor (TCR) a chain variable (Va) domain comprising a CDR3a comprising the amino acid sequence set forth in any one of SEQ ID NOs: 13-16, and a TCR b chain variable (nb) domain; (b) a TCR nb domain comprising a CDR3a comprising the amino acid sequence set forth in any one of SEQ ID NOs:4-8 or 12, and a TCR Va domain; or (c) a TCR Va domain of (a) and a TCR nb domain of (b), wherein the encoded binding protein is expressed at the cell surface of the modified immune cell and wherein the encoded binding protein is capable of specifically binding to a peptide containing an a Core Binding Factor subunit bi ⁇ n ⁇ ob ⁇ h Heavy chain 11 (OBRb:MUH 1 1) antigen that comprises or consists
- an encoded binding protein of the present disclosure is capable of binding to a peptide comprising or consisting of the consensus amino acid sequence set forth in SEQ ID NO: 109, wherein the peptide is optionally in complex with a HLA molecule, such as, for example, an HLA-B *40:01 or a HLA-B*40:02 molecule.
- a peptide bound by a binding protein of the present disclosure comprises or consists of the amino acid sequence set forth in SEQ ID NO: 109
- a peptide bound by a binding protein of the present disclosure comprises or consists of the amino acid sequence set forth in SEQ ID NO: 119.
- alanine mutagenesis of any one or more of residues 2, 3, 5, 6, 7, or 9 of SEQ ID NO:2 reduces or abrogates binding by the binding protein (e.g., to a SEQ ID NO:2-HLA complex, such as a SEQ ID N0:2-HLA-B*40:0l complex).
- an encoded binding protein of the present disclosure does not bind to a variant of SEQ ID NO:2 that does not contain an Arg at a position corresponding to position 2 of SEQ ID NO:2; a Glu at a position corresponding to position 3 of SEQ ID NO:2; a Glu at a position corresponding to position 5 of SEQ ID NO:2; a Val at a position corresponding to position 6 of SEQ ID NO:2; a His at a position corresponding to position 7 of SEQ ID NO:2; or a Leu at a position
- an encoded binding protein of the present disclosure does not bind to a variant of SEQ ID NO:2 that does not contain an Arg at a position corresponding to position 2 of SEQ ID NO:2; a Glu at a position corresponding to position 3 of SEQ ID NO:2; a Glu at a position corresponding to position 5 of SEQ ID NO:2; a Val at a position corresponding to position 6 of SEQ ID NO:2; a His at a position corresponding to position 7 of SEQ ID NO:2; and a Leu at a position corresponding to position 9 of SEQ ID NO:2.
- the encoded CDR3P comprises or consists of the amino acid sequence set forth in SEQ ID NO:6, and the encoded CDR3a comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15.
- the encoded CDR3P comprises or consists of the amino acid sequence set forth in SEQ ID NO:6
- the encoded CDR3a comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15.
- the encoded nb domain has at least 90% (z.e.,at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identity to the amino acid sequence set forth in SEQ ID NO: 19, and the encoded Va domain has at least 90% identity to the amino acid sequence set forth in SEQ ID NO:23.
- the encoded nb domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19
- the encoded Va domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:23.
- the encoded CDIE ⁇ comprises or consists of the amino acid sequence set forth in SEQ ID NO:4, and the encoded CDR3a comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16.
- the encoded nb domain has at least 90% identity to the amino acid sequence set forth in SEQ ID NO:20
- the encoded Va domain has at least 90% identity to the amino acid sequence set forth in SEQ ID NO:24.
- the encoded nb domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:20
- the encoded Va domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:24.
- the encoded O ⁇ E3b comprises or consists of the amino acid sequence set forth in SEQ ID NO:7
- the encoded CDR3a comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13.
- the encoded nb domain has at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 17, and the Va domain has at least 90% identity to the amino acid sequence set forth in SEQ ID NO:2l.
- the encoded nb domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17, and the encoded Va domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:2l.
- the encoded CDR3P comprises or consists of the amino acid sequence set forth in SEQ ID NO:8, and the CDR3a comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14.
- the encoded nb domain has at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 18, and the encoded Va domain has at least 90% identity to the amino acid sequence set forth in SEQ ID NO:22.
- the encoded nb domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18, and the encoded Va domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:22.
- a modified immune cell can further comprise a polynucleotide encoding a TCR b-chain constant domain (Ob), wherein the encoded Ob is expressed as a component of the binding protein.
- the encoded Ob comprises or consists of an amino acid sequence having at least 90% identity to any one of SEQ ID NOs:25-28.
- a modified immune cell can further comprise a polynucleotide encoding a TCR a-chain constant domain (Ca), wherein the encoded Ca is expressed as a component of the binding protein.
- the Ca comprises or consists of an amino acid sequence having at least 90% identity to any one of SEQ ID NOs:29-32.
- a modified immune cell of the present disclosure expresses a binding protein, wherein the binding protein comprises a Va domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:23, a Ca domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:3 l; a nb domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 19; and a Ob domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:27.
- the binding protein comprises a TCR a-chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:38, and a TCR b-chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:37.
- a modified immune cell of the present disclosure expresses a binding protein, wherein the binding protein comprises a Va domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:24, a Ca domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:32; a nb domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:20; and a Ob domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:28.
- the binding protein comprises a TCR a-chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:40, and a TCR b-chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:39.
- a modified immune cell of the present disclosure expresses a binding protein, wherein the binding protein comprises a Va domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:2l, a Ca domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:29; a nb domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17; and a Ob domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:25.
- the binding protein comprises a TCR a-chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:34, and a TCR b-chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:33.
- a modified immune cell of the present disclosure expresses a binding protein, wherein the binding protein comprises a Va domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:22, a Ca domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:30; a nb domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:l8; and a Ob domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:26.
- the binding protein comprises a TCR a-chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:36, and a TCR b-chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:35.
- a binding protein expressed by a modified immune cell is capable of specifically binding to a CBFP:MYHl l-HLA complex.
- the ELLA comprises HLA-A*02:0l; HLA-A*03:0l; HLA-A* 11 :01; HLA-B *40:01; HLA-B*44:02; or HLA-B*44:03.
- the ELLA comprises HLA-B*40:0l.
- a binding protein expressed by a modified immune cell can comprise a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or any combination thereof.
- an antigen-binding fragment of a TCR comprises a single chain TCR (scTCR), which comprises both the TCR V a and TCR Vp domains, but only a single TCR constant domain (C a or Cp).
- an antigen-binding fragment of a TCR or a chimeric antigen receptor is chimeric (e.g ., comprises amino acid residues or motifs from more than one donor or species), humanized (e.g., comprises residues from a non human organism that are altered or substituted so as to reduce the risk of
- any suitable immune cell may be modified to include a heterologous polynucleotide encoding a binding protein of this disclosure, including, for example, a T cell, a NK cell, or a NK-T cell.
- a modified immune cell comprises a CD4 + T cell, a CD8 + T cell, or both.
- Any appropriate method can be used to transfect or transduce the cells, for example, the T cells, or to administer the polynucleotides or compositions of the present methods.
- Known methods for delivering polynucleotides to host cells include, for example, use of cationic polymers, lipid-like molecules, and certain commercial products such as, for example, IN-VIVO-JET PEI.
- Other methods include ex vivo transduction, injection, electroporation, DEAE-dextran, sonication loading, liposome- mediated transfection, receptor-mediated transduction, microprojectile bombardment, transposon-mediated transfer, and the like.
- Still further methods of transfecting or transducing host cells employ vectors, described in further detail herein.
- a modified immune cell expressing a binding protein of the present disclosure is capable, after about 4-hours of co-culture with HLA-B*40:0l + cells that are pulsed with a peptide comprising the amino acid sequence of SEQ ID NO:2 at a concentration of about 1 ng/mL or less (e.g, about 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 ng/mL, or less), of killing about 50% or more of the pulsed cells.
- a modified immune cell expressing a binding protein of the present disclosure is capable specifically killing a cell that expresses on its cell surface a SEQ ID NO:2:HLA-B*40:02 complex.
- an engineered immune cell may modified to reduce or eliminate expression of one or more endogenous genes that encode a polypeptide involved in immune signaling or other related activities.
- exemplary gene knockouts include those that encode PD-l, LAG-3, CTLA4, TIM3, an HLA molecule, a TCR molecule, or the like.
- endogenously expressed immune cell proteins may be recognized as foreign by an allogeneic host receiving the modified immune cells, which may result in elimination of the modified immune cells (e.g ., an HLA allele), or may downregulate the immune activity of the modified immune cells (e.g., PD-l, LAG-3, CTLA4), or may interfere with the binding activity of a heterologously expressed binding protein of the present disclosure (e.g, an endogenous TCR of a modified T cell that binds a non- CBFpAIYH l 1 antigen and thereby interferes with the modified immune cell binding a cell that expresses CBFP:MYHl 1 antigen).
- a heterologously expressed binding protein of the present disclosure e.g, an endogenous TCR of a modified T cell that binds a non- CBFpAIYH l 1 antigen and thereby interferes with the modified immune cell binding a cell that expresses CBFP:MYHl 1 antigen).
- a modified immune cell is a donor cell (e.g, allogeneic) or an autologous cell.
- a modified immune cell of this disclosure comprises a chromosomal gene knockout of one or more of a gene that encodes PD-l, LAG-3, CTLA4, TIM3, TIGIT, an HLA component (e.g, a gene that encodes an al macroglobulin, an a2 macroglobulin, an a3 macroglobulin, a b ⁇ microglobulin, or a b2 microglobulin), or a TCR component (e.g., a gene that encodes a TCR variable region or a TCR constant region) (see, e.g., Torikai el al., Nature Sci. Rep.
- HLA component e.g, a gene that encodes an al macroglobulin, an a2 macroglobulin, an a3 macroglobulin, a b ⁇ microglobulin, or a b2 microglobulin
- TCR component e.g., a gene that encodes a TCR variable region or a TCR
- chromosomal gene knockout refers to a genetic alteration or introduced inhibitory agent in a host cell that prevents (e.g, reduces, delays, suppresses, or abrogates) production, by the host cell, of a functionally active endogenous polypeptide product. Alterations resulting in a chromosomal gene knockout can include, for example, introduced nonsense mutations (including the formation of premature stop codons), missense mutations, gene deletion, and strand breaks, as well as the heterologous expression of inhibitory nucleic acid molecules that inhibit endogenous gene expression in the host cell.
- a chromosomal gene knock-out or gene knock-in is made by chromosomal editing of a host cell.
- Chromosomal editing can be performed using, for example, endonucleases.
- endonucleases refers to an enzyme capable of catalyzing cleavage of a phosphodiester bond within a polynucleotide chain.
- an endonuclease is capable of cleaving a targeted gene thereby inactivating or "knocking out" the targeted gene.
- An endonuclease may be a naturally occurring, recombinant, genetically modified, or fusion endonuclease.
- the nucleic acid strand breaks caused by the endonuclease are commonly repaired through the distinct mechanisms of homologous recombination or non-homologous end joining (NHEJ).
- NHEJ non-homologous end joining
- a donor nucleic acid molecule may be used for a donor gene "knock-in”, for target gene "knock-out”, and optionally to inactivate a target gene through a donor gene knock in or target gene knock out event.
- NHEJ is an error- prone repair process that often results in changes to the DNA sequence at the site of the cleavage, e.g., a substitution, deletion, or addition of at least one nucleotide.
- NHEJ may be used to "knock-out" a target gene.
- Examples of endonucleases include zinc finger nucleases, TALE-nucleases, CRISPR-Cas nucleases, meganucleases, and megaTALs.
- a "zinc finger nuclease” refers to a fusion protein comprising a zinc finger DNA-binding domain fused to a non-specific DNA cleavage domain, such as a Fokl endonuclease.
- ZFN zinc finger nuclease
- Each zinc finger motif of about 30 amino acids binds to about 3 base pairs of DNA, and amino acids at certain residues can be changed to alter triplet sequence specificity (see, e.g., Desjarlais et al., Proc. Natl. Acad. Sci.
- ZFNs mediate genome editing by catalyzing the formation of a site-specific DNA double strand break (DSB) in the genome, and targeted integration of a transgene comprising flanking sequences homologous to the genome at the site of DSB is facilitated by homology directed repair.
- DSB DNA double strand break
- a DSB generated by a ZFN can result in knock out of target gene via repair by non-homologous end joining (NHEJ), which is an error-prone cellular repair pathway that results in the insertion or deletion of nucleotides at the cleavage site.
- NHEJ non-homologous end joining
- a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, made using a ZFN molecule.
- TALEN transcription activator-like effector nuclease
- a "TALE DNA binding domain” or “TALE” is composed of one or more TALE repeat domains/units, each generally having a highly conserved 33-35 amino acid sequence with divergent l2th and l3th amino acids.
- the TALE repeat domains are involved in binding of the TALE to a target DNA sequence.
- the divergent amino acid residues referred to as the Repeat Variable Diresidue (RVD), correlate with specific nucleotide recognition.
- RVD Repeat Variable Diresidue
- the natural (canonical) code for DNA recognition of these TALEs has been determined such that an HD (histine-aspartic acid) sequence at positions 12 and 13 of the TALE leads to the TALE binding to cytosine (C), NG (asparagine-glycine) binds to a T nucleotide, NI (asparagine-isoleucine) to A, NN (asparagine-asparagine) binds to a G or A nucleotide, and NG (asparagine-glycine) binds to a T nucleotide.
- Non-canonical (atypical) RVDs are also known (see, e.g., ET.S. Patent Publication No.
- TALENs can be used to direct site-specific double-strand breaks (DSB) in the genome of T cells.
- Non- homologous end joining (NHEJ) ligates DNA from both sides of a double-strand break in which there is little or no sequence overlap for annealing, thereby introducing errors that knock out gene expression.
- homology directed repair can introduce a transgene at the site of DSB providing homologous flanking sequences are present in the transgene.
- a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, and made using a TALEN molecule.
- CRISPR/Cas nuclease system refers to a system that employs a CRISPR RNA (crRNA)-guided Cas nuclease to recognize target sites within a genome (known as protospacers) via base-pairing complementarity and then to cleave the DNA if a short, conserved protospacer associated motif (PAM) immediately follows 3’ of the complementary target sequence.
- CRISPR/Cas systems are classified into three types (i.e., type I, type II, and type III) based on the sequence and structure of the Cas nucleases.
- the crRNA-guided surveillance complexes in types I and III need multiple Cas subunits.
- Type II system the most studied, comprises at least three components: an RNA-guided Cas9 nuclease, a crRNA, and a trans-acting crRNA (tracrRNA).
- the tracrRNA comprises a duplex forming region.
- a crRNA and a tracrRNA form a duplex that is capable of interacting with a Cas9 nuclease and guiding the
- Cas9/crRNA:tracrRNA complex to a specific site on the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA upstream from a PAM.
- Cas9 nuclease cleaves a double-stranded break within a region defined by the crRNA spacer. Repair by NHEJ results in insertions and/or deletions which disrupt expression of the targeted locus.
- a transgene with homologous flanking sequences can be introduced at the site of DSB via homology directed repair.
- the crRNA and tracrRNA can be engineered into a single guide RNA (sgRNA or gRNA) (see, e.g., Jinek et al., Science 337:816-21, 2012). Further, the region of the guide RNA complementary to the target site can be altered or programed to target a desired sequence (Xie et al, PLOS One 9:el00448, 2014; U.S. Pat. Appl. Pub. No. US 2014/0068797, U.S. Pat. Appl. Pub. No. US 2014/0186843; U.S. Pat. No. 8,697,359, and PCT Publication No. WO 2015/071474; each of which is incorporated by reference).
- a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, and made using a CRISPR/Cas nuclease system.
- gRNA sequences and methods of using the same to knock out endogenous genes that encode immune cell proteins include those described in Ren et al., Clin. Cancer Res. 23(9):2255-2266 (2017), the gRNAs, CAS9 DNAs, vectors, and gene knockout techniques of which are hereby incorporated by reference in their entirety.
- Meganucleases can be divided into five families based on sequence and structure motifs: LAGLIDADG (SEQ ID NO: 159), GIY-YIG (SEQ ID NO: 160), HNH, His-Cys box and PD-(D/E)XK (SEQ ID NO:l6l).
- Exemplary meganucleases include I-Scel, I-Ceul, PI-PspI, RI-Sce, I- ScelV, I-Csml, I-Panl, I-Scell, I-Ppol, I-SceIII, I-Crel, I-Tevl, I-TevII and I-TevIII, whose recognition sequences are known (see, e.g., Ei.S. Patent Nos. 5,420,032 and 6,833,252; Belfort et al., Nucleic Acids Res .
- naturally-occurring meganucleases may be used to promote site-specific genome modification of a target selected from PD-l, LAG3,
- TIM3, CTLA4, TIGIT an HLA-encoding gene, or a TCR component-encoding gene.
- an engineered meganuclease having a novel binding specificity for a target gene is used for site-specific genome modification (see, e.g., Porteus et al., Nat. Biotechnol. 23:967-73, 2005; Sussman et al., J. Mol. Biol. 342: 31-41, 2004; Epinat et al., Nucleic Acids Res. 37:2952-62, 2003; Chevalier et al., Molec. Cell 70:895-905, 2002; Ashworth et al., Nature 441:656-659, 2006; Paques et al., Curr. Gene Ther. 7:49- 66, 2007; U.S.
- a chromosomal gene knockout is generated using a homing endonuclease that has been modified with modular DNA binding domains of TALENs to make a fusion protein known as a megaTAL.
- MegaTALs can be utilized to not only knock-out one or more target genes, but to also introduce (knock in) heterologous or exogenous
- polynucleotides when used in combination with an exogenous donor template encoding a polypeptide of interest.
- a chromosomal gene knockout comprises an inhibitory nucleic acid molecule that is introduced into a host cell (e.g, an immune cell) comprising a heterologous polynucleotide encoding an antigen-specific receptor that specifically binds to a tumor associated antigen, wherein the inhibitory nucleic acid molecule encodes a target-specific inhibitor and wherein the encoded target-specific inhibitor inhibits endogenous gene expression ⁇ i.e., of PD-l, TIM3, LAG3, CTLA4, TIGIT, an HLA component, or a TCR component, or any combination thereof) in the host immune cell.
- a host cell e.g, an immune cell
- a heterologous polynucleotide encoding an antigen-specific receptor that specifically binds to a tumor associated antigen
- the inhibitory nucleic acid molecule encodes a target-specific inhibitor and wherein the encoded target-specific inhibitor inhibits endogenous gene expression ⁇ i.e., of PD-
- a chromosomal gene knockout can be confirmed directly by DNA sequencing of the host immune cell following use of the knockout procedure or agent.
- Chromosomal gene knockouts can also be inferred from the absence of gene expression e.g ., the absence of an mRNA or polypeptide product encoded by the gene) following the knockout.
- compositions are provided herein that comprise a modified immune cell of the present disclosure and a pharmaceutically acceptable carrier, diluent, or excipient.
- unit doses that comprise an effective amount of a modified immune cell or of a composition comprising the modified immune cell.
- a unit dose comprises (i) a composition comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD4 + T cells, combined with (ii) a composition comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD8 + T cells, in about a 1 :1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells ⁇ i.e., has less than about 50%, less than about 40%, less than about 30%, less then about 20%, less than about 10%, less than about 5%, or less then about 1% the population of naive T cells present in a unit dose as compared to a patient sample having a comparable number of PBMCs).
- a unit dose comprises (i) a composition comprising at least about 50% modified CD4 + T cells, combined with (ii) a composition comprising at least about 50% modified CD8 + T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells.
- a unit dose comprises (i) a composition comprising at least about 60% modified CD4 + T cells, combined with (ii) a composition comprising at least about 60% modified CD8 + T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells.
- a unit dose comprises (i) a composition comprising at least about 70% engineered CD4 + T cells, combined with (ii) a composition comprising at least about 70% engineered CD8 + T cells, in about a 1 :1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells.
- a unit dose comprises (i) a composition comprising at least about 80% modified CD4 + T cells, combined with (ii) a composition comprising at least about 80% modified CD8 + T cells, in about a 1 :1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells.
- a unit dose comprises (i) a composition comprising at least about 85% modified CD4 + T cells, combined with (ii) a composition comprising at least about 85% modified CD8 + T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells.
- a unit dose comprises (i) a composition comprising at least about 90% modified CD4 + T cells, combined with (ii) a composition comprising at least about 90% modified CD8 + T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells.
- a unit dose of the present disclosure may comprise a modified immune cell as described herein (i.e., expressing a binding protein specific for a CBFP:MYHl 1 antigen) and a modified immune cell expressing a binding protein specific for a different antigen (e.g ., a different CBFP:MYHl 1 antigen, or an antigen from a different protein or target, such as, for example, CD 19, CD20, CD22, BCMA,
- a unit dose can comprise modified CD8 + T cells expressing a binding protein that specifically binds to a SEQ ID N0:2-HLA-B*40:0l complex, and modified CD4 + T cells (and/or modified CD8 + T cells) expressing a binding protein (e.g., a CAR) that specifically binds to a CD 19 antigen.
- a binding protein e.g., a CAR
- a unit dose comprises equal, or approximately equal numbers of engineered CD45RA CD3 + CD8 + and modified CD45RA CD3 + CD4 + T M cells.
- the present disclosure provides methods for treating or for preventing a relapse of AML in a subject, the method comprising administering to the subject a unit dose comprising a modified immune cell of this disclosure (or a composition comprising a modified immune cell), thereby treating or preventing relapse of AML.
- Treatment refers to medical management of a disease, disorder, or condition of a subject (e.g ., a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat).
- a subject e.g ., a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat.
- an appropriate dose or treatment regimen comprising a modified immune cell of the present disclosure, and optionally an adjuvant, is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit.
- Therapeutic or prophylactic/preventive benefit includes improved clinical outcome; lessening or alleviation of symptoms associated with a disease;
- a “therapeutically effective amount” or “effective amount”, as used herein, refers to an amount of modified immune cells sufficient to result in a therapeutic effect, including improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease, stabilization of disease state; delay of disease progression; remission; survival; or prolonged survival in a statistically significant manner.
- a therapeutically effective amount refers to the effects of that ingredient or cell expressing that ingredient alone.
- a therapeutically effective amount refers to the combined amounts of active ingredients or combined adjunctive active ingredient with a cell expressing an active ingredient that results in a therapeutic effect, whether administered serially or simultaneously.
- a combination may also be a cell expressing more than one active ingredient.
- pharmaceutically acceptable excipient or carrier or “physiologically acceptable excipient or carrier” refer to biologically compatible vehicles, e.g., physiological saline, which are described in greater detail herein, that are suitable for administration to a human or other non-human mammalian subject and generally recognized as safe or not causing a serious adverse event.
- statically significant refers to a p value of 0.050 or less when calculated using the Students t-test and indicates that it is unlikely that a particular event or result being measured has arisen by chance.
- Subjects that can be treated by the present invention are, in general, human and other primate subjects, such as monkeys and apes for veterinary medicine purposes.
- the subject may be a human subject.
- the subjects can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.
- Cells according to the present disclosure may be administered in a manner appropriate to the disease, condition, or disorder to be treated as determined by persons skilled in the medical art.
- a modified immune cell or unit dose as described herein is administered intravenously, intraperitoneally, intratumorally, into the bone marrow, into a lymph node, or into the cerebrospinal fluid so as to encounter target cells (e.g., leukemia cells).
- target cells e.g., leukemia cells.
- An appropriate dose, suitable duration, and frequency of administration of the compositions will be determined by such factors as a condition of the patient; size, type, and severity of the disease, condition, or disorder; the particular form of the active ingredient; and the method of administration.
- immunotherapy refers to administration of naturally occurring or genetically engineered, disease- or antigen-specific immune cells (e.g, T cells).
- Adoptive cellular immunotherapy may be autologous (immune cells are from the recipient), allogeneic (immune cells are from a donor of the same species) or syngeneic (immune cells are from a donor genetically identical to the recipient).
- the subject expresses a CBFP:MYHl 1 antigen comprising or consisting of the amino acid sequence (Q)LLAVTVHEL (SEQ ID NO: 1); REEMEVHEL (SEQ ID NO:2); or EEMEVHEL (SEQ ID NO:3).
- the subject is HLA-A* 02:01 + ; HLA-A*03:0l + ; HLA-A*l l :0l + ; HLA- B*40:0l + ; HLA-B*40:03 + ; HLA-B*44:02 + ; or HLA-B*44:03 + .
- the amount of cells in a composition or unit dose is at least one cell (for example, one modified CD8 + T cell subpopulation (e.g, optionally comprising memory and/or naive CD8 + T cells); one modified CD4 + T cell subpopulation (e.g, optionally comprising memory and/or naive CD4 + T cells)) or is more typically greater than 10 2 cells, for example, up to 10 4 , up to 10 5 , up to 10 6 , up to 10 7 , up to 10 8 , up to 10 9 , or more than 10 10 cells.
- the cells are administered in a range from about 10 4 to about 10 10 cells/m 2 , preferably in a range of about 10 5 to about 10 9 cells/m 2 .
- an administered dose comprises up to about 3.3 x 10 5 cells/kg. In some embodiments, an administered dose comprises up to about 1 x 10 6 cells/kg. In some embodiments, an administered dose comprises up to about 3.3 x 10 6 cells/kg. In some embodiments, an administered dose comprises up to about 1 x 10 7 cells/kg. In certain embodiments, a modified immune cell is administered to a subject at a dose comprising up to about 5 x 10 4 cells/kg, 5 x 10 5 cells/kg, 5 x 10 6 cells/kg, or up to about 5 x 10 7 cells/kg.
- a modified immune cell is administered to a subject at a dose comprising at least about 5 x 10 4 cells/kg, 5 x 10 5 cells/kg, 5 x 10 6 cells/kg, or up to about 5 x 10 7 cells/kg.
- the number of cells will depend upon the ultimate use for which the composition is intended as well the type of cells included therein.
- cells modified to contain a binding protein will comprise a cell population containing at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of such cells.
- cells are generally in a volume of a liter or less, 500 mls or less, 250 mls or less, or 100 mls or less.
- the density of the desired cells is typically greater than 10 4 cells/ml and generally is greater than 10 7 cells/ml, generally 10 8 cells/ml or greater.
- the cells may be administered as a single infusion or in multiple infusions over a range of time.
- a clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 cells.
- a unit dose of the modified immune cells can be co- administered with ( e.g ., simultaneously or contemporaneously with) hematopoietic stem cells from an allogeneic donor (e.g., a donor that is CBFP:MYHl 1 -negative, HLA-B *40:01 -negative, or both).
- an allogeneic donor e.g., a donor that is CBFP:MYHl 1 -negative, HLA-B *40:01 -negative, or both.
- one or more of the modified immune cells comprised in the unit dose is autologous to the subject.
- the subject receiving the modified immune cell has previously received lymphodepleting chemotherapy.
- the lymphodepleting chemotherapy comprises cyclophosphamide, fludarabine, anti thymocyte globulin, or a combination thereof.
- the method further comprises administering an inhibitor of an immune checkpoint molecule to the subject.
- compositions that comprise a modified immune cell as disclosed herein and a pharmaceutically acceptable carrier, diluents, or excipient.
- Suitable excipients include water, saline, dextrose, glycerol, or the like and combinations thereof.
- compositions comprising fusion proteins or host cells as disclosed herein further comprise a suitable infusion media.
- suitable infusion media can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), 5% dextrose in water, Ringer's lactate can be utilized.
- An infusion medium can be supplemented with human serum albumin or other human serum components.
- compositions may be administered in a manner appropriate to the disease or condition to be treated (or prevented) as determined by persons skilled in the medical art.
- An appropriate dose and a suitable duration and frequency of administration of the compositions will be determined by such factors as the health condition of the patient, size of the patient (i.e., weight, mass, or body area), the type and severity of the patient's condition, the particular form of the active ingredient, and the method of administration.
- an appropriate dose and treatment regimen provide the composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (such as described herein, including an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and/or overall survival, or a lessening of symptom severity).
- An effective amount of a pharmaceutical composition refers to an amount sufficient, at dosages and for periods of time needed, to achieve the desired clinical results or beneficial treatment, as described herein.
- An effective amount may be delivered in one or more administrations. If the administration is to a subject already known or confirmed to have a disease or disease-state, the term "therapeutic amount” may be used in reference to treatment, whereas “prophylactically effective amount” may be used to describe administrating an effective amount to a subject that is susceptible or at risk of developing a disease or disease-state (e.g., recurrence) as a preventative course.
- a disease or disease-state e.g., recurrence
- compositions described herein may be presented in unit- dose or multi-dose containers, such as sealed ampoules or vials. Such containers may be frozen to preserve the stability of the formulation until infusion into the patient.
- Doses will vary, but a preferred dose for administration of a modified immune cell as described herein is about 10 cells/m , about 5 x 10 cells/m , about 10 cells/m , about 5 x 10 5 cells/m 2 , about 10 6 cells/m 2 , about 5 x 10 6 cells/m 2 , about 10 7 cells/m 2 , about 5 x 10 7 cells/m 2 , about 10 8 cells/m 2 , about 5 x 10 8 cells/m 2 , about 10 9 cells/m 2 , about 5 x 10 9 cells/m 2 , about 10 10 cells/m 2 , about 5 x 10 10 cells/m 2 , or about 10 11 cells/m 2 .
- a unit dose comprises a modified immune cell as described herein at a dose of about 10 4 cells/m 2 to about 10 11 cells/m 2 .
- the composition may also include sterile aqueous or oleaginous solution or suspension.
- suitable non-toxic parenterally acceptable diluents or solvents include water, Ringer’s solution, isotonic salt solution, l,3-butanediol, ethanol, propylene glycol or polythethylene glycols in mixtures with water.
- Aqueous solutions or suspensions may further comprise one or more buffering agents, such as sodium acetate, sodium citrate, sodium borate or sodium tartrate.
- any material used in preparing any dosage unit formulation should be pharmaceutically pure and substantially non-toxic in the amounts employed.
- the active compounds may be incorporated into sustained-release preparation and formulations. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit may contain a predetermined quantity of engineered immune cells or active compound calculated to produce the desired effect in association with an appropriate pharmaceutical carrier.
- an appropriate dosage and treatment regimen provides the active molecules or cells in an amount sufficient to provide a benefit.
- a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated subjects as compared to non-treated subjects.
- Increases in preexisting immune responses to a tumor protein generally correlate with an improved clinical outcome.
- Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which are routine.
- a dose should be sufficient to prevent, delay the onset of, or diminish the severity of a disease associated with disease or disorder.
- Prophylactic benefit of the immunogenic compositions administered according to the methods described herein can be determined by performing pre-clinical (including in vitro and in vivo animal studies) and clinical studies and analyzing data obtained therefrom by appropriate statistical, biological, and clinical methods and techniques, all of which can readily be practiced by a person skilled in the art.
- administration of a composition refers to delivering the same to a subject, regardless of the route or mode of delivery. Administration may be effected continuously or intermittently, and parenterally. Administration may be for treating a subject already confirmed as having a recognized condition, disease or disease state, or for treating a subject susceptible to or at risk of developing such a condition, disease or disease state.
- Co-administration with an adjunctive therapy may include simultaneous and/or sequential delivery of multiple agents in any order and on any dosing schedule (e.g., modified immune cells with one or more cytokines; immunosuppressive therapy such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low dose of a mycophenolic acid prodrug, or any combination thereof).
- a plurality of doses of a modified immune cell described herein is administered to the subject, which may be administered at intervals between administrations of about two to about four weeks.
- Treatment or prevention methods of this disclosure may be administered to a subject as part of a treatment course or regimen, which may comprise additional treatments prior to, or after, administration of the instantly disclosed unit doses, cells, or compositions.
- a subject receiving a unit dose of the modified immune cell is receiving or had previously received a hematopoietic cell transplant (HCT; including myeloablative and non-myeloablative HCT).
- HCT hematopoietic cell transplant
- a modified immune cell of the present disclosure can be administered with or shortly after hematopoietic stem cells in a modified HCT therapy.
- the HCT comprises a donor hematopoieitic cell comprising a chromosomal knockout of a gene that encodes an HLA component, a chromosomal knockout of a gene that encodes a TCR component, or both.
- a lymphodepleting chemotherapy comprises a conditioning regimen comprising cyclophosphamide, fludarabine, anti -thymocyte globulin, or a combination thereof.
- Methods according to this disclosure may further include administering one or more additional agents to treat the disease or disorder in a combination therapy.
- a combination therapy comprises administering a modified immune cell with (concurrently, simultaneously, or sequentially) an immune checkpoint inhibitor.
- a combination therapy comprises administering a modified immune cell with an agonist of a stimulatory immune checkpoint agent.
- a combination therapy comprises administering a modified immune cell with a secondary therapy, such as
- chemotherapeutic agent a radiation therapy, a surgery, an antibody, or any combination thereof.
- immune suppression agent refers to one or more cells, proteins, molecules, compounds or complexes providing inhibitory signals to assist in controlling or suppressing an immune response.
- immune suppression agents include those molecules that partially or totally block immune stimulation; decrease, prevent or delay immune activation; or increase, activate, or up regulate immune suppression.
- immunosuppression agents to target include PD-l, PD-L1, PD- L2, LAG3, CTLA4, B7-H3, B7-H4, CD244/2B4, HVEM, BTLA, CD160, TIM3, GAL9, KIR, PVR1G (CD112R), PVRL2, adenosine, A2aR, immunosuppressive cytokines (e.g., IL-10, IL-4, IL-1RA, IL-35), IDO, arginase, VISTA, TIGIT, LAIR1, CEACAM-l, CEACAM-3, CEACAM-5, Treg cells, or any combination thereof.
- immunosuppression agents to target include PD-l, PD-L1, PD- L2, LAG3, CTLA4, B7-H3, B7-H4, CD244/2B4, HVEM, BTLA, CD160, TIM3, GAL9, KIR, PVR1G (CD112R), PVRL2, adenosine
- An immune suppression agent inhibitor may be a compound, an antibody, an antibody fragment or fusion polypeptide (e.g., Fc fusion, such as CTLA4-Fc or LAG3-Fc), an antisense molecule, a ribozyme or RNAi molecule, or a low molecular weight organic molecule.
- a method may comprise a modifiedimmune cell with one or more inhibitor of any one of the following immune suppression components, singly or in any combination.
- a modified immune cell is used in combination with a PD-l inhibitor, for example a PD-l -specific antibody or binding fragment thereof, such as pidilizumab, nivolumab, pembrolizumab, MEDI0680 (formerly AMP-514), AMP- 224, BMS-936558 or any combination thereof.
- a PD-l inhibitor for example a PD-l -specific antibody or binding fragment thereof, such as pidilizumab, nivolumab, pembrolizumab, MEDI0680 (formerly AMP-514), AMP- 224, BMS-936558 or any combination thereof.
- a modified immune cell of the present disclosure (or a modified host cell expressing the same) is used in combination with a PD-L1 specific antibody or binding fragment thereof, such as BMS-936559, durvalumab (MEDI4736), atezolizumab (RG7446), avelumab (MSB0010718C), MPDL3280A, or any combination thereof.
- a modifiedimmune cell of the present disclosure is used in combination with a LAG3 inhibitor, such as LAG525, IMP321, IMP701, 9H12, BMS-986016, or any combination thereof.
- a modifiedimmune cell is used in combination with an inhibitor of CTLA4.
- a modified immune cell is used in combination with a CTLA4 specific antibody or binding fragment thereof, such as ipilimumab, tremelimumab, CTLA4-Ig fusion proteins (e.g., abatacept, belatacept), or any combination thereof.
- a modifiedimmune cell is used in combination with a B7-H3 specific antibody or binding fragment thereof, such as enoblituzumab
- a B7-H4 antibody binding fragment may be a scFv or fusion protein thereof, as described in, for example, Dangaj et al, Cancer Res. 73:4820, 2013, as well as those described in U.S. Patent No. 9,574,000 and PCT Patent
- a modified immune cell is used in combination with an inhibitor of CD244.
- a modified immune cell is used in combination with an inhibitor of BLTA, HVEM, CD 160, or any combination thereof.
- Anti CD- 160 antibodies are described in, for example, PCT Publication No. WO 2010/084158.
- a modified immune cell is used in combination with an inhibitor of TIM3.
- a modified immune cell is used in combination with an inhibitor of Gal9.
- a modified immune cell is used in combination with an inhibitor of adenosine signaling, such as a decoy adenosine receptor.
- a modified immune cell is used in combination with an inhibitor of A2aR.
- a modified immune cell is used in combination with an inhibitor of KIR, such as lirilumab (BMS-986015).
- a modified immune cell is used in combination with an inhibitor of an inhibitory cytokine (typically, a cytokine other than TGFP) or Treg development or activity.
- a modified immune cell is used in combination with an IDO inhibitor, such as levo-l-methyl tryptophan, epacadostat (INCB024360; Liu et al ., Blood 775:3520-30, 2010), ebselen (Terentis et al. , Biochem. ⁇ 9:591-600, 2010), indoximod, NLG919 (Mautino et al., American Association for Cancer Research l04th Annual Meeting 2013; Apr 6-10, 2013), 1 -methyl-tryptophan (l-MT)-tira-pazamine, or any combination thereof.
- a modified immune cell is used in combination with an arginase inhibitor, such as N(omega)-Nitro-L-arginine methyl ester (L-NAME), N- omega-hydroxy-nor-l-arginine (nor-NOHA), L-NOHA, 2(S)-amino-6-boronohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine (BEC), or any combination thereof.
- an arginase inhibitor such as N(omega)-Nitro-L-arginine methyl ester (L-NAME), N- omega-hydroxy-nor-l-arginine (nor-NOHA), L-NOHA, 2(S)-amino-6-boronohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine (BEC), or any combination thereof.
- a modified immune cell is used in combination with an inhibitor of VISTA, such as CA-170 (Curis, Lexington, Mass.).
- a modifiedimmune cell is used in combination with an inhibitor of TIGIT such as, for example, COM902 (Compugen, Toronto, Ontario Canada), an inhibitor of CD155, such as, for example, COM701 (Compugen), or both.
- an inhibitor of TIGIT such as, for example, COM902 (Compugen, Toronto, Ontario Canada)
- an inhibitor of CD155 such as, for example, COM701 (Compugen)
- COM701 Compugen
- a modified immune cell is used in combination with an inhibitor of PVRIG, PVRL2, or both.
- Anti-PVRIG antibodies are described in, for example, PCT Publication No. WO 2016/134333.
- Anti-PVRL2 antibodies are described in, for example, PCT Publication No. WO 2017/021526.
- an engineered immune cell is used in combination with a LAIR1 inhibitor.
- a modified immune cell is used in combination with an inhibitor of CEACAM-l, CEACAM-3, CEACAM-5, or any combination thereof.
- a modified immune cell is used in combination with an agent that increases the activity (i.e., is an agonist) of a stimulatory immune checkpoint molecule.
- an engineered immune cell can be used in combination with a CD137 (4-1BB) agonist (such as, for example, urelumab), a CD134 (OX-40) agonist (such as, for example, MEDI6469, MEDI6383, or MEDI0562), lenalidomide, pomalidomide, a CD27 agonist (such as, for example, CDX-l 127), a CD28 agonist (such as, for example, TGN1412, CD80, or CD86), a CD40 agonist (such as, for example, CP-870,893, rhuCD40L, or SGN-40), a CD122 agonist (such as, for example, IL-2) an agonist of GITR (such as, for example, humanized monoclonal antibodies described in PCT Patent Publication No.
- a method may comprise administering a modified immune cell with one or more agonist of a stimulatory immune checkpoint molecule, including any of the foregoing, singly or in any combination.
- a combination therapy comprises a modified immune cell and a secondary therapy comprising one or more of: an antibody or antigen binding-fragment thereof that is specific for a cancer antigen expressed by the non- inflamed solid tumor, a radiation treatment, a surgery, a chemotherapeutic agent, a cytokine, RNAi, or any combination thereof.
- a combination therapy method comprises administering a modified immune cell and further administering a radiation treatment or a surgery.
- Radiation therapy is well-known in the art and includes X-ray therapies, such as gamma-irradiation, and radiopharmaceutical therapies.
- Surgeries and surgical techniques appropriate to treating a given cancer in a subject are well-known to those of ordinary skill in the art.
- a combination therapy method comprises administering a modified immune cell and further administering a chemotherapeutic agent.
- a chemotherapeutic agent includes, but is not limited to, an inhibitor of chromatin function, a topoisomerase inhibitor, a microtubule inhibiting drug, a DNA damaging agent, an antimetabolite (such as folate antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), a DNA synthesis inhibitor, a DNA interactive agent (such as an intercalating agent), and a DNA repair inhibitor.
- Illustrative chemotherapeutic agents include, without limitation, the following groups: anti-metabolites/anti-cancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2- chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busul
- daunorubicin doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents;
- antiproliferative/antimitotic alkylating agents such as nitrogen mustards
- Cytokines may be used to manipulate host immune response towards anticancer activity. See, e.g., Floros & Tarhini, Semin. Oncol. 42( 4):539-548, 2015. Cytokines useful for promoting immune anticancer or antitumor response include, for example, IFN-a, IL-2, IL-3, IL-4, IL-10, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-24, and GM-CSF, singly or in any combination with a modified immune cell of this disclosure.
- Also provided herein are methods for modulating an adoptive immunotherapy wherein the methods comprise administering, to a subject who has previously received a modified immune cell of the present disclosure that comprises a heterologous polynucleotide encoding a safety switch protein, a cognate compound of the safety switch protein in an amount effective to ablate in the subject the previously
- the safety switch protein comprises tEGFR and the cognate compound is cetuximab, or the safety switch protein comprises iCasp9 and the cognate compound is AP1903 (e.g., dimerized AP1903), or the safety switch protein comprises a RQR polypeptide and the cognate compound is rituximab, or the safety switch protein comprises a myc binding domain and the cognate compound is an antibody specific for the myc binding domain.
- methods are provided for manufacturing a composition, or a unit dose of the present disclosure.
- the methods comprise combining (i) an aliquot of a host cell transduced with a vector of the present disclosure with (ii) a pharmaceutically acceptable carrier.
- vectors of the present disclosure are used to transfect/transduce a host cell (e.g ., a T cell) for use in adoptive transfer therapy (e.g., targeting a cancer antigen).
- the methods further comprise, prior to the aliquotting, culturing the transduced host cell and selecting the transduced cell as having incorporated (i.e., expressing) the vector.
- the methods comprise, following the culturing and selection and prior to the aliquotting, expanding the transduced host cell.
- the manufactured composition or unit dose may be frozen for later use. Any appropriate host cell can be used for manufacturing a composition or unit dose according to the instant methods, including, for example, a hematopoietic stem cell, a T cell, a primary T cell, a T cell line, a NK cell, or a NK-T cell.
- the methods comprise a host cell which is a CD8 + T cell, a CD4 + T cell, or both.
- CBF Core Binding Transcription Factor
- RUNX 1 CBFa subunit
- CBFP CBFP subunit
- FOG. 1 A Core Binding Transcription Factor
- Two mutant forms of CBF ((i) a CBFP:MYHl 1 fusion generated by an inversion (inv(l6)); (ii) a RUNXFRUNXTl fusion generated by a t(8;2l) translocation (FIG. 1B)) are associated with Acute Myeloid Leukemia and repress transcription of target genes.
- IBF Core Binding Transcription Factor
- Variants of the inv(l6) (CBFP-MYHl 1) fusion gene are created by different chromosomal breakpoints.
- type A is the most common ( Thomasger et al. Leukemia 27:725-731 (2007)), occurring in 80-90% of patients.
- the type A fusion (FIG. 3 A), is produced by joining exon 5 of CBFP, located in 16r13, and exon 34 of MYH11, which is located in l6q22.
- the 20 HLA class I molecules evaluated for predicted binding were HLA-A*0l :0l, -A*02:0l, -A*03:0l, -A*l l :0l, -A*24:02, -B*07:02, -B*08:0l, -B*35:0l, -B*40:0l, -B*44:02, - B*44:03, -C*03:03, -C*03:04, -C*04:0l, -C*05:0l, -C*06:02, -C*07:0l, -C*07:02, and -C* 12:03.
- Predicted peptides were defined as candidate epitopes if they had predicted IC50 ⁇ 500 nM for any HLA by >1 algorithm. Table 1. _ HLA predictions of potential CBFB-MYH11 type A fusion epitopes
- HLA-binding prediction algorithms can identify peptides with a high probability of binding to a particular HLA molecule. However, not all predicted peptides actually bind to the predicted HLA, and not all that bind will be immunogenic (see Robbins et al. Nat. Med. 79:747-752 (2013); see also Yadav, et al, Nature 575:572-576 (2014)). Therefore, the immunogenicity of candidate CBFB-MYH11 epitopes was directly tested in vitro.
- peptides comprising the candidate CBFB-MYH11 epitopes were synthesized using standard Fmoc chemistry (Genscript), reconstituted to a stock concentration of 10 mg/mL in DMSO, and stored at -20°C in aliquots until use.
- PBMCs peripheral blood mononuclear cells
- PBMCs peripheral blood mononuclear cells
- RPMI 1640 RPMI 1640 supplemented with 20% human serum and 10% dimethylsulfoxide (DMSO) in vapor-phase liquid nitrogen in aliquots until use.
- DMSO dimethylsulfoxide
- CD8 + T cells were obtained by immunomagnetic bead depletion of CD8 cells from HLA-typed volunteer PBMC (CD8 + T cell isolation kit, Miltenyi Biotec).
- Autologous DC generated from monocytes by a modified fast DC protocol were used as antigen-presenting cells (APCs) as described by Bleakley et al. ( Blood 775:4923-4933(2010)).
- T cells were maintained in RPMI 1640, 10% human serum, 1% penicillin/streptomycin, 3mM L-glutamine, and 50mM b-mercaptoethanol (CTL medium).
- the CD8 + T cells isolated from HLA-typed healthy donors were stimulated with autologous mature monocyte-derived dendritic cells (DC) that had been pulsed with a pool of candidate CBFP-MYHl 1 peptide epitopes, to identify immunogenic CBFP- MYH11 epitopes and epitope-specific T cells.
- DC autologous mature monocyte-derived dendritic cells
- the CD8 + T cells were plated in >360 replicate wells of 96-well plates at 3 to 6 c 10 4 per well, with a T cell to DC ratio of 30: 1 in CTL medium.
- Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (B-LCL) were prepared and maintained in RPMI 1640, 10% fetal calf serum and 1% penicillin/streptomycin (LCL medium), as described by Bleakley et al. ( Blood
- Cytotoxicity was measured in short-term (4-hour) CRA, using 51 Cr-labeled target cells. Briefly, target cells were labeled with 51 Cr overnight (cell lines) at 37°C and 5% C0 2. Effector cells were added to labeled target cells, and incubated for 4 hours. After co-incubation, supernatant was harvested for g-counting. Specific lysis was calculated using a standard formula (Riddell et al, ./. Immunol. 746:2795-2804 (1991)). Targets used included autologous and allogeneic LCL with and without peptides in varying concentrations.
- Split-well CRA with LCL with or without peptide were performed on day 12- 13. A well was scored positive if it exhibited more than 20% specific lysis of peptide- pulsed LCL and lysis of peptide-pulsed LCL >2 times higher than that of LCLs without peptide. Peptide-specific T cells were cloned by limiting dilution using OKT3, IL-2, and feeder cells, and screened by split-well CRA on day 11-13. A clone was scored positive if it exhibited more than 20% specific lysis of peptide-pulsed LCL and lysis of peptide-pulsed LCL >5 times higher than LCL without peptide. Positive clones were expanded using OKT3, IL-2 and feeder cells (Riddell and Greenberg, J Immunol.
- Flow cytometry was performed on a 5-laser (355 nm, 405 nm, 488 nm, 552 or 532 nm and 628 or 640 nm) Fortessa X50 or Symphony instrument (BD). Cell sorting was performed on a 3-laser (405 nm, 488 nm, 633 nm) or 5-laser (355 nm, 408 nm, 488 nm, 561 nm, 635 nm) Aria II device (BD). All data was analyzed with FlowJo software (Tree Star). Fluorochrome-conjugated pHLA tetramers were produced in-house by the Fred Hutchinson Cancer Research Center Immune Monitoring Lab. All mAh used for flow cytometry were mouse anti-human, except for rituximab, which is an engineered chimeric anti -human mAh. Antibodies used in the flow cytometry experiments described herein are shown in Table 2, below.
- Table 3 provides candidate CBFpMYH 1 1 neoantigen peptides and their HLA restrictions, along with half-maximal lysis values; functional avidity values; and recognition, which refer to T cell activity against target cells pulsed with the CBFpAIYH l 1 neoantigen and T cell recognition of primary AML or AML cell lines ( see Examples 3 and 4 herein) expressing the indicated CBFp YHl 1 neoantigen.
- Table 4 provides the amino acid sequences of the candidate CBFpMYHl 1 neoantigen peptides produced by the fusion/indels described in Table 3, and identifies exemplary CBFp YHl 1 -responding T cell clones of the present disclosure.
- HLA typing of the healthy donors was performed. Briefly, genomic DNA was isolated from donor PBMC (QIAamp DNA Blood Kit: Qiagen) and used for HLA typing by PCR (Allset Gold Low-Resolution ABC Kit: One Lambda) or by next- generation sequencing (NGS) using the ScisGo HLA v6 typing kit (Scisco Genetics Inc., Seattle, WA). Briefly, the NGS employs an amplicon-based 2-stage PCR, followed by sample pooling and sequencing using a MiSeq v2 PE500 (Illumina, San Diego, CA). Table 5 shows HLA-typing of healthy donors used in immunogenicity screening experiments.
- REEMEVHEL SEQ ID NO:2
- HLA-B*40:0l HLA -B*40:0l
- the SEQ ID NO:2 peptide was predicted to bind to HLA-B*40:0l but not HLA-A*02:0l or -C*03:04. All six clones stained with SEQ ID N0:2/HLA-B*40:0l peptide/HLA (pHLA) tetramer (FIG. 4C).
- HLA-diverse LCL target cells pulsed with SEQ ID NO:2 peptide were tested for lysis. Only HLA- B*40:0l + LCL were lysed by the six clones (FIG. 4D).
- HLA-C*03:04 + B*40:0 LCL were not lysed, indicating that clones did not recognize the epitope presented on HLA- C*03:04.
- REEMEVHEL SEQ ID NO:2
- HLA-binding predictions for the SEQ ID NO:2 peptide and HLA-B*40:0l are included on the first line for comparison.
- lentiviral vectors were used to transduce the naturally B*40:0l + NB-4 cell line with the full length CBFB-MYH11 type A fusion, and to transduce the naturally CBFB-MYH11 + ME-l cell line with HLA-B*40:0l (FIG. 5A).
- NB-4, and ME-l AML cell lines were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and maintained in LCL medium with 20% FCS.
- NB-4 was confirmed to express HLA-B*40:0l by HLA typing and was transduced to express the CBFP-MYHl 1 fusion sequence and an RQR8 selection marker (SEQ ID NO:45; Dossa et al., Blood 737: 108-120 (2016); Philip et al., Blood 124: 1277-1287 (2014)) for selection.
- ME-l cells expresse the type A CBFP- MYH11 fusion sequence ( see Nielsen and Andreatta, Genome Med.
- HLA-B *40:01 :02 The sequence for HLA-B *40:01 :02 was obtained from IPD-IMGT/HLA (www.ebi.ac.uk/cgi-bin/ipd/imgt/hla/get_allele.cgi?B*40:0l :02:0l) and synthesized as a synthetic nucleotide block (GeneArt, Life Technologies), transferred into a Topo vector (LifeTechnologies), and then cloned into the LV vector pRRLSIN.cPPT.MSCV.WPRE with an upstream RQR8 selection marker, as described above.
- LentiX-293T cells (Clontech) were transfected with the LV backbone plasmids along with PAX2 and VSVg packaging plasmids using the CalPhos transfection system (Clontech) per the manufacturer’s protocol. Virus particles were harvested after 48 hours and filtered through a 0.45 pm filter prior to use. AML cell lines were transduced by the addition of LV supernatant and lpg/mL of polybrene followed by 90 minutes of centrifugation at 800xg and returned to the incubator at 37°C, 5% C0 2.
- Transduction efficiency of AML cell lines was assessed by flow cytometry 72 hours after spin inoculation based on staining for the CD34 epitope (Q component of RQR8) or CD20 epitope (R component of RQR8). Transduced cell lines were then enriched to >95% purity by flow sorting, expanded, and cryopreserved until use.
- Lenti-X 293T cells were used, and were maintained in DMEM (Invitrogen) supplemented with 10% FCS, 25mM HEPES, 2mM L-glutamine, 1% penicillin/streptomycin, and detached for passage using 0.05% trypsin-EDTA (Invitrogen).
- NB-4 cells were transduced with the CBFP-MYHl 1 type A fusion coding sequence (Liu et al ., Science 261 : 1041-1044 (1993); Cancer Genome Atlas Research Network, 2013; N Engl J Med 368, 2059-2074; Claxton et al., Blood 83: 1750- 1756(1994)).
- a coding sequence for the selection marker RQR8 was included in the construct upstream of the fusion for tracking and selection of transduced cells.
- the transgene components were separated by 2A elements from the porcine teschovirus (P2A) (SEQ ID NO:4l).
- transgenes were codon-optimized and synthesized by GeneArt (Life Technologies), cloned into the LV vector pRRLSIN.cPPT.MSCV.WPRE by restriction digestion and ligation, and confirmed by Sanger sequencing.
- T cell clones The ability of T cell clones to recognize and eliminate the HLA-B*40:0l + CBFP-MYHl 1 -expressing cell lines was then tested in a flow cytometry -based cytotoxicity assay. Briefly, cytotoxicity of T cells against the AML cell lines was measured in co-culture up to 120 hours by assessing survival of targets. Effector and targets cells were plated in a 1 :1 E:T ratio (5 x 10 5 each) in six replicate wells in LCL medium in a 96 well plate, and co-cultured at 37°C in 5% C0 2. Wells with only effectors and only targets were included as controls.
- the cultures were centrifuged and the cell pellets stained with antibodies against CD33, CD8, DAPI (0.002 pg/mL; Sigma-Aldrich), and, in some assays, CD34 and/or CD20 mAh to distinguish target cells expressing transgene constructs.
- Fluorescent CountBright counting beads (Invitrogen) were used to calculate absolute numbers of live (DAPI negative) target cells. Percent survival was calculated as [(absolute number of live targets with effector)/(ab solute number of live targets without effector) *100]
- REEMEVHEL SEQ ID NO:2
- cytotoxicity assays were performed. Briefly, high-avidity REEMVEHEL (SEQ ID NO:2)-specific clones were tested for CD 107 A expression in response to AML target cells.
- CD 107a cytotoxic degranulation assays, effector T cells and target cells (primary AML) were washed and plated in a 1 :2 E:T ratio in LCL medium with GolgiStop transport inhibitor (BD
- CBFP-MYHl l/B*40:0l- specific T cell clones were also tested for lysis of the AML samples using a
- target cells were labeled with 51 Cr overnight (cell lines) or for 6 hours (primary leukemia) at 37°C and 5% C0 2
- leukemia cells were thawed, washed, suspended in LCL medium supplemented with 500 U/mL interferon-g, and incubated for 24 hours at 37°C prior to CRA initiation.
- positions 2 and 9 of SEQ ID NO:2 appear to be necessary for the peptide to bind HLA-B*40:0l, as expected for anchor residues (Falk el al ., Immunogenetics 41 : 165-168 (1995)).
- the glutamic acid residues at positions 3 and 5 and the valine, histidine and leucine residues at positions 6, 7, and 9 were essential for T cell recognition, but did not affect predicted HLA binding.
- the SEQ ID NO:2 epitope was searched against the non-redundant BLAST database of Homo sapiens protein sequences.
- REEMEVHEL SEQ ID NO:2
- HLA- B*40:0l -restricted microbial T cell epitopes in the Immune Epitope Database (IEDB) were examined. Briefly, known microbial T cell epitopes were identified by searching IEDB (www.iedb.org), restricting to all human infectious disease, class I-restricted targets with positive immune assays.
- REEMEVHEL (SEQ ID NO:2) was then used as the query sequence and aligned to the identified known microbial epitopes using protein BLAST with a BLOSUN62 matrix and default parameters (gap existence cost 11, gap extension cost 1, conditional compositional score matrix for compositional adjustment) and automatic adjustment of parameters for short input sequences. Sequences were compared visually using WebLogo (Crooks et al. , Genome Res. 14: 1188-1190 (2004). No overall similarity was revealed when aligning REEMEVHEL (SEQ ID NO:2) with known HLA-B*40:0l -restricted microbial T cell epitopes, except (as expected) at anchor positions 2 and 9 (FIG. 7D).
- REEMEVHEL SEQ ID Non specific T cells represent cross-reactive memory T cells
- pHLA tetramer enrichment was used to isolate unmanipulated REEMEVHEL (SEQ ID NO:2)-specific HLA- B*40:0l CD8 + T cells.
- T cells were enriched from PBMC from healthy donors or individuals with active or treated AML by tetramer enrichment, using methods adapted from Moon and co-workers (Moon et ah, Nat. Protoc. 4:565-581 (2009)).
- cryopreserved PBMC from healthy donors or individuals with active or treated AML were thawed and rested in CTL medium with 0.25 units/mL benzonase (EMD Millipore) and 1 ng/mL IL-15, at 37°C for 2 hours.
- Cells were then pre incubated in 500 pL IX PBS/5% FBS with 50 nM dasatinib and incubated for 15 minutes at 37°C to enhance TCR expression (Lissina et al., J. Immunol. Methods 340: 11-24 (2009)). Cells were then washed to removed dasatinib, and a single-cell suspension was prepared in 200 m ⁇ of IX PBS/5% FBS.
- APC-conjugated CBFP- MYH1 l/B*40:0l tetramer was added to a concentration of 30 nM and incubated at room temperature for 15 minutes, followed by a wash in 15 ml of ice-cold MACS buffer (PBS + 1% FBS and 2 mM EDTA). Tetramer-stained cells were then
- Labeled cells were eluted by flushing the unmagnetized column twice with 5 mL of MACS buffer. After centrifugation, cell pellets from the enriched and column flow through fractions were resuspended in PBS/5% FBS. Cell suspensions were incubated with PE-conjugated CBFP-MYHl l/B*40:0l tetramer for 30 minutes on ice, then with surface antibodies for 15 minutes on ice. Cells were washed with MACS buffer twice and resuspended in MACS buffer with DAPI prior to sorting.
- BV421 -conjugated anti-CD4, -CD14, -CD16, -CD123, and -CD117 were also included on a single channel with DAPI (dump).
- Gating strategy to identify antigen-specific CD8+ T cells was as follows: single cells, BV421/DAPI negative, CD8 + APC + PE + .
- PBMC from HLA-B*40:0l + individuals with AML two with active CBFB-MYH1 G AML at diagnosis (Dx), two with active CBFB-MYH11 + AML (Dx), and one with CBFB-MYH11 + AML in complete molecular remission before (CR) and after HCT (post-HCT)) were assayed for the presence of REEMEVHEL(SEQ ID NO:2) -specific CD8 + T cells.
- pHLA tetramer enrichment of PBMC (as described in Example 5), was performed, and cell surface markers were evaluated.
- epitope-specific CD8 + T cells were detected in all CBFP-MYHl l + patient samples, as well as in a sample from a healthy donor (D3; FIGS. 8A and 8B).
- the absolute number of epitope-specific tetramer-positive or total CD8 + T cells was calculated as follows: [(cell events/bead events)*(bead concentration)* (bead volume/sample volume)* (total cell volume)].
- Cell events for total CD8 + T cells were from the single cell/dump7CD8 + gate and epitope-specific tetramer-positive cells were from the single cell/dump
- CD8 + cell number was calculated by adding the absolute number of CD8 + cells in the flow-through and in the tetramer-enriched eluate. Total epitope-specific cell number was calculated from the eluate only.
- CD45RO CCR7 in leukemia patients (AML1 and AML2 CR, FIG. 8C).
- epitope-specific CD8 + T cells in the post-HCT sample were predominantly
- CD45RA + CD45RO CCR7 + similar to healthy donors (FIG. 8C, see also FIGS. 7F and 7G).
- Epitope-specific T cells post-HCT were presumably donor-derived (CD3 chimerism was 98% donor), and exposed to minimal, if any, CBFP-MYHl l + AML during HCT because the patient was in a deep remission, explaining the relative paucity of CBFP-MYHl 1 -specific memory T cells.
- epitope-specific memory T cells exposed to CBFP-MYHl l + AML in vivo had a higher frequency of 2B4 expression (AML1 Dx, 29%; AML2 CR 58%; FIG. 8D) than did epitope-specific T cells exposed to minimal or no antigen in vivo (AML2 post-HCT, 9%; D3, 0%; FIG. 8D).
- High-avidity REEMEVHEL (SEQ ID NO:2)-specific TCRs were sequenced and cloned to see whether AML-induced dysfunction of CBFB-MYH1 l/B*40:0l-specific T cells could be circumvented by heterologous expression of TCRs.
- the b chains from all six T cell clones were sequenced, and a diverse set of REEMEVHEL (SEQ ID NO:2)-specific TCRs was identified (Table 8).
- TCRBV04-0l *0l was shared by more than one clone.
- TCR a chains of the four clones with the highest functional avidity D6.C6.1, D7.C8.1, D7.C24.1, D11.C5 were also sequenced.
- sequencing was performed by next-generation sequencing (Adaptive Biotechnologies) and rapid amplification of complementary DNA ends (RACE) polymerase chain reaction (PCR).
- RNA was extracted from each CBFP- MYH1 l/HLA-B*40:0l-specific T cell clone.
- cDNA 5' first-strand complementary DNA
- RACE-PCR rapid amplification of cDNA ends polymerase chain reaction
- cDNA was synthesized from RNA using 5' CDS Primer A, SMARTer IIA oligo, and SMARTScribe Reverse Transcriptase. The cDNA was then used to perform a RACE-PCR reaction using Phusion High-Fidelity DNA Polymerase and gene-specific primers for the TCR a (hTCR_Calpha-R 5'- CAGCCGCAGCGTCATGAGCAGATTA-3'(SEQ ID NO: 115)) or TCR b chain (hTCR_Cbl-R 5'- CC ACTTCCAGGGCTGCCTTCAGAAATC-3 ' (SEQ ID NO: 116) and hTCR_Cb2-R 5'- TGGGATGGTTTTGGAGCTAGCCTCTGG-3 ' (SEQ ID NO:
- RACE-PCR products were purified and sequenced to identify TCR a and b chains.
- TCR variable, diversity, and joining regions were defined using IMGT/V- QEIEST software.
- TCRs were constructed by pairing the TRA and TRB sequences encoding the dominant chains in each OBRb-MUHI l/B*40:0l-specific T cell clone.
- Two TCRs each were assembled from D6.C6.1 and Dl l. C5 (TCR B1A1 and TCR BlA2; two a chains encoded by a single clone has been previously described ( see Padovan et al. , Science 262:422-424 (1993)).
- One TCR each was assembled from D7.C8.1 and D7.C24.1 (TCR B1A1).
- TRA V-J-encoding sequences and TRB V-D-J-encoding sequences were fused to sequences encoding human constant TRA and TRB chains, respectively.
- TRA and TRB sequences were confirmed by PCR using a forward primer from the 5’ end of the appropriate V region and reverse primers from the TRA or TRB constant region(s), followed by Sanger sequencing.
- Complementary cysteine residues at positions 48 (Thr to Cys) and 57 (Ser to Cys) were incorporated into the TRA and TRB constant domains to increase exogenous TCR pairing and decrease mispairing with endogenous TCR (see, e.g., Dossa et al., Blood 131 : 108-120 (2016)).
- TCR chains were separated by codon-diversified 2A elements from the porcine teschovirus (P2A).
- Transgenes were codon-optimized to enhance expression, synthesized by Gene Art (Life Technologies), and cloned into the pRRLSIN.cPPT.MSCV.WPRE LV by restriction digestion and ligation.
- T cells immunomagnetically purified from normal donor PBMC were activated with Dynabeads Human T-Activator CD3/CD28 (ThermoFisher Scientific) in 50 IU/mL IL- 2 for 24 hours. T cells were then transduced with LV supernatant. Lenti viral vectors were produced and cells were transduced as described in Example 3.
- T cells were stained with CBFP-MYH l 1/HLA-B*40:01 pHLA tetramer and anti-CD8 mAh.
- CBFP-MYHl 1 tetramer + CD8 + T cells were sorted to >95% purity and expanded using OKT3, IL-2 and feeder cells. After 10 days, T cells were evaluated by flow cytometry and functional assays.
- TCRs of interest Five of the six TCRs of interest were codon-optimized and cloned into LV, and healthy donor CD8 + T cells were transduced.
- Four TCRs had strong expression in transduced donor cells, as determined by pHLA tetramer staining (FIG. 9A). All four TCRs conferred specificity for REEMEVHEL (SEQ ID NO:2) (FIGS. 9B and 9C).
- T cells transduced with the D7.C24.1 TCR showed particularly high functional avidity in a peptide titration CRA (FIG.
- CD8 + T cells isolated from a healthy HLA-typed donor were stimulated with autologous dendritic cells pulsed with peptides including (Q)LLAVTVHEL (SEQ ID NO: l) and (Q)LLAVTVHEL-specific T cell lines were identified using CRA, single-cell cloned by limiting dilution, and expanded. Four clones (D1.C27, D2.C1, D10.C8, D10.C78) specific for (Q)LLAVTVHEL (SEQ ID NO: 1) were identified. Clone Dl .C27 showed high functional avidity for
- (Q)LLAVTVHEL SEQ ID NO: 1
- the naturally HLA-A*02:0l + AML cell line OCI-AML3 was transduced with LV constructs encoding the 22 amino acids spanning the splice variant CBFB- MYH11 fusion and including (Q)LLAVTVHEL (SEQ ID NO: 1) and separately with a construct encoding an amino acid sequence spanning a RUNX1-RUNX1T1 fusion.
- the LV constructs were similar to those shown in Figure 5A and included an RQR8 tag for selection/expression separated from the sequence encoding the epitope by a P2A element.
- Transduced cells were sorted to >95% purity on the RQR8 tag prior to use as target cells.
- 0CI-AML3 cells without transduction or with transduction of either the splice variant CBFB-MYH11 fusion or the RUNX1-RUNX1T1 constructs were used as target cells in a CRA with D1.C27 T cells as effectors.
- EEMEVHEL SEQ ID NO:3
- REEMEVHEL SEQ ID NO:2
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Abstract
La présente invention concerne des compositions et des procédés pour cibler un facteur de liaison du noyau, de sous-unité β : un antigène de chaîne lourde de myosine 11 (CBFβ : MYH11) pour, par exemple, prévenir ou gérer une rechute d'une leucémie myéloïde aiguë (AML). L'invention concerne également des polynucléotides et des constructions transgéniques codant pour des protéines de liaison, telles qu'un récepteur de lymphocytes T ou un récepteur antigénique chimérique. De telles constructions transgéniques peuvent être transduites dans une cellule immunitaire, par exemple un lymphocyte T, et peuvent être utilisées en immunothérapie chez un sujet soufrant ou présentant un risque de récidive d'une AML.
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WO2024148212A3 (fr) * | 2023-01-06 | 2024-08-08 | The Trustees Of The University Of Pennsylvania | Récepteurs de lymphocytes t dirigés contre la protéine sox2 |
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WO2024148212A3 (fr) * | 2023-01-06 | 2024-08-08 | The Trustees Of The University Of Pennsylvania | Récepteurs de lymphocytes t dirigés contre la protéine sox2 |
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