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  • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
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  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
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  • A61K40/31—Chimeric antigen receptors [CAR]
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  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
  • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
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  • A61K40/46—Viral antigens
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/7051—T-cell receptor (TcR)-CD3 complex
• • C—CHEMISTRY; METALLURGY
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  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
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  • C12Y207/10—Protein-tyrosine kinases (2.7.10)
  • C12Y207/10001—Receptor protein-tyrosine kinase (2.7.10.1)

Definitions

• the present products and methods relate to the fields of immunology and purification of genetically modified cells, specifically to a truncated or otherwise modified receptor paired with a corresponding antibody, such as a polypeptide derived from human epidermal growth factor receptor (EGFR) paired with cetuximab, for use in cancer immunotherapy.
• EGFR human epidermal growth factor receptor
• Immune cell products with homogenous expression of tumor targeting chimeric antigen receptors are desirable for clinical evaluation of adoptive therapy strategies to eliminate the product-to-product variability of transgene expression otherwise intrinsic to transduction and other genetic modification procedures without subsequent selection.
• Immunotherapy using genetically redirected immune cells is an attractive approach for treating minimal residual disease in a variety of cancer patients.
• immunologic rejection of cell products expressing antibiotic selection proteins as part of the transduction strategy has impeded this strategy.
• a novel selection marker that is not expressed on human lymphocytes, does not contain endogenous signaling or trafficking function, and is recognized by a known, preferably commercially available, pharmaceutical grade antibody reagent that can be utilized for selection, in vivo tracking, and depletion of transduced cells would be a significant improvement in the art.
• the genetically modified cells may be modified by transduction, or any other process that adds, deletes, alters, or disrupts an endogenous nucleotide sequence.
• the genetically modified cells may be transduced T cells with altered activity, including altered immunoactivity.
• a non-immunogenic selection epitope compatible with immunomagnetic selection facilitates
• the non-immunogenic selection epitope is a gene encoding an endogenous cell-surface molecule that is modified or truncated to retain an extracellular epitope recognized by a known antibody or functional fragment thereof, and to remove any signaling or trafficking domains and/or any extracellular domains unrecognized by the known antibody.
• the removal of the signaling or trafficking domains and/or any extracellular domains unrecognized by the known antibody renders the endogenous cell-surface molecule inert, which is a desired property for the molecule.
• the non-immunogenic selection epitope may also be used for as a selection tool or tracking marker.
• the modified endogenous cell-surface molecule may be, but is not limited to, any cell-surface related receptor, ligand, glycoprotein, cell adhesion molecule, antigen, integrin or cluster of differentiation (CD) that is modified as described herein.
• the modified endogenous cell-surface molecule is a truncated tyrosine kinase receptor.
• the truncated tyrosine kinase receptor is a member of the epidermal growth factor receptor family (e.g., ErbB1 , ErbB2, ErbB3, ErbB4).
• Epidermal growth factor receptor also known as EGFR, ErbB1 and HER1
• EGFR human epidermal growth factor receptor
• HER1 HER1
• a gene encoding an EGFR polypeptide comprising human epidermal growth factor receptor (EGFR) that is constructed by removal of nucleic acid sequences that encode polypeptides including the membrane distal EGF- binding domain and the cytoplasmic signaling tail (a "truncated EGFR" or "EGFRt"), but retains the extracellular membrane proximal epitope recognized by an anti-EGFR antibody.
• the antibody is a known, commercially available anti-EGFR monoclonal antibody, such as cetuximab, matuzumab, necitumumab or
• EGFRt was demonstrated to have suicide gene potential through Erbitux® mediated antibody dependent cellular cytotoxicity (ADCC) pathways.
• ADCC antibody dependent cellular cytotoxicity
• EGFRt may be used as a non-immunogenic selection tool, tracking marker, and suicide gene for transduced T cells that have immunotherapeutic potential.
• the EGFRt nucleic acid may also be detected by means well known in the art.
• methods of discovering and designing modified, truncated or altered endogenous cell-surface molecules which bind to antibodies, preferably commercially available antibodies, as described herein are provided.
• the methods include modeling the protein of interest and truncating functional portions, while leaving the antibody-binding portions intact.
• the resulting modified receptor or ligand can be sorted using a labeled antibody and then enriched such that the concentration of the modified receptor or ligand is increased.
• Yet another embodiment provides a method of selecting transduced T cells comprising transducing T cells with a modified, truncated or altered
• the modified receptor sequence is EGFRt
• the antibody is preferably a biotinylated anti-EGFR monoclonal antibody.
• the T cells are then sorted by adding anti-biotin microbeads and selecting the T cells using
• modified ligand or receptor sequences such as the EGFRt sequence, may be contained in a suitable transfer vehicle such as a lentiviral vector.
• Figure 1 is a molecular model of EGFR vs. EGFRt proteins based on the crystal structure files.
• the EGFR structure on the left shows a full-length EGFR with the structure of the four extracellular domains (Domains l-IV).
• the middle structure shows the truncated EGFR (EGFRt), which is missing Domain I, Domain II, the Juxatmembrane Domain, and the Tyrosine Kinase Domain as compared to an unmodified EGFR.
• the EGFRt on the right shows truncated structure bound to Eribitux® Fab, comprised of VH-CM and V
• Figure 2 illustrates the selection of EGFRt + T cells using biotinylated cetuximab (referred to in the figure as Erbitux®).
• Figure 2a is a schematic of the cetuximab biotinylation and reformulation process.
• Figure 2b is a graph showing titration of biotinylated cetuximab.
• FIG. 2c depicts schematics of both the immunomagnetic (top) and the fluorescence activated cell sorting (bottom) EGFRt selection procedures.
• Figure 2d shows immunomagnetic selection of various T cell lines lentivirally transduced with CAR and EGFRt containing constructs. Schematics of the C D 19CAR-T2A- EG F Rt (left) and CD19CAR-T2A-EGFRt-IMPDH2dm (right) constructs contained in lentiviral vectors are shown above the corresponding pre- and post-selection flow cytometric analyses for surface EGFRt expression.
• Codon optimized sequence portions of the CD19-specific, CD28 co-stimulatory CAR, followed by the self-cleavable T2A, EGFRt and IMPDH2dm selection markers are indicated, along with the Elongation Factor 1 promoter sequences (EF-1 p), and the GCSFR alpha chain signal sequences (GCSFRss, which directs surface
• histograms represent staining with PE-conjugated anti-biotin antibody alone, and the percent positive cells are indicated in each histogram.
• CD19CAR + EGFRt + Line A occurred 3 days after transduction of T cell blasts.
• CD19CAR + EGFRt + Line B occurred after 3 REM stimulations of transduced CMVpp65-specific T C M-derived cells.
• Selection of CD19CAR + EGFRt + Line C occurred after 2 REM stimulations of transduced CD8 + TcM-derived cells.
• Selection of CD19CAR + EGFRt + Line D occurred after 1 REM stimulation of transduced T EM -derived cells.
• Selection of CD19CAR + EGFRt + IMPDH2dm + Line E occurred after 1 REM stimulation of transduced TcM-derived cells.
• FIG. 3 shows that the EGFRt expressed on selected T cells is inert.
• EGFRt expressed on T cells is not phosphorylated upon co-incubation with EGF.
• Negative control T cells, CD19CAR + EGFRt + Line A cells, or A431 cells were incubated for 5 minutes with or without either 100ng/mL EGF or cetuximab (referred to in the figure as Erbtx) and then lysed in the presence of phosphatase inhibitor. Lysates run on Western blots were then probed using antibodies specific for either ⁇ -actin, the cytoplasmic domain of EGFR, or the phosphorylated tyrosine at position 1068 of EGFR.
• Figure 3b shows that EGF does not bind to the surface of EGFRt expressing T cells.
• A431 , Line A, and negative control T cells were stained with PE-conjugated anti-EGFR, or either biotinylated cetuximab or biotinylated EGF followed by PE-conjugated streptavidin (black histogram) versus PE-conjugated isotype control Ab or streptavidin alone (open histogram) by flow cytometry. Percent positive staining is indicated in each histogram.
• Figure 4 illustrates that selected EGFRt + CD19R + T cells can be expanded with maintenance of effector phenotype.
• Figure 4a is a line graph showing expansion of EGFRt-selected T cells, Lines A-E, over 12 or more days after rapid expansion medium (REM) stimulation was initiated on the day of AutoMACSTM selection (day 0).
• REM rapid expansion medium
• T cells are magnetic activated cell sorting.
• REM rapid expansion medium
• 10 6 T cells with 30 ng/mL anti-CD38 (OKT3; Ortho Biotech, Raritan, NJ), 5 x 10 7 7-irradiated PBMCs (3500 cGy), and 10 7 T-irradiated LCLs (8000 cGy) in 50 mL CM; with addition of 50U/mL rhlL-2 and 10ng/ml rhlL-15 (CellGenix) every 48 hours, beginning on day 1 . T cells were re-stimulated in this manner every 14 days.
• Figure 4b shows histograms representing EGFRt-selected T cells (1 1 to 13 days after stimulation) that were phenotyped for surface EGFR (i.e., EGFRt, with biotinylated cetuximab), Fc (i.e., CAR), and T cell markers CD4 or CD8, (black histogram) vs. isotype control Ab (open histogram) by flow cytometry. Percent positive staining is indicated in each histogram. "N.D.” indicates no data.
• Figure 4C are five lines graphs, one for each of Lines A-E, of EGFRt-selected T cells (within 1 1 to 15 days after REM stimulation) incubated for 4 hours with 5 Cr-labled NS0, U251 T, CD19t-expressing NS0, CMV pp65-expressing U251 T, CD19-expressing Daudi or SupB15, or OKT3-expressing LCL cells as targets at the indicated E:T ratios. Chromium release was measured to determine cytotoxic activity.
• Figure 4d is a graph showing MPA resistance of the CD19CAR + EGFRt + IMPDH2dm + Line E. Control T cells that do not express IMPDH2dm and EGFRt-selected IMPDH2dm-expressing Line E cells were cultured either with or without 1 ⁇ MPA and total cell numbers were monitored.
• FIG. 5 shows EGFRt expression can be used as a tracking marker for in vivo T cell engraftment.
• Day 36 bone marrow harvested from a control mouse or from a mouse that had received 10 7 CD19CAR + EGFRt + Line C at day 0 was stained using PerCP-conjugated anti-human CD45 and biotinylated cetuximab ("Bio- Erb") followed by PE-conjugated streptavidin. Quadrants were created based on isotype control staining, and percent positive staining in each quadrant is indicated in each histogram.
• Figure 6 is a graph showing EGFRt expression targets T cells for cetuximab (referred to in the figure as Erbitux®) mediated ADCC.
• 5 Cr-labeled Line A cells were pre-incubated either with or without up to 2C ⁇ g/mL of cetuximab or the CD20-specific mAb Rituxan as a negative control prior to addition of human PBMC as effectors.
• Figure 7 is the nucleotide (sense strand is SEQ ID NO: 1 , antisense strand is SEQ ID NO: 2) and amino acid (SEQ ID NO: 3) sequences of GMCSFR alpha chain signal sequence linked to EGFRt.
• the GMCSFR alpha chain signal sequence, which directs surface expression, is encoded by nucleotides 1 -66.
• EGFRt is encoded by nucleotides 67-1071 .
• Figure 8 is the nucleotide (sense strand is SEQ ID NO: 4, antisense strand is SEQ ID NO: 5) and amino acid (SEQ ID NO: 6) sequences of CD19R- CD28gg-Zeta(CO)-T2A-EGFRt.
• CD19R-CD28gg-Zeta(CO) is encoded by nucleotides 1 -2040;
• T2A is encoded by nucleotides 2041 -21 12;
• GMCSFR is encoded by nucleotides 21 13-2178;
• EGFRt is encoded by nucleotides 2179-3186.
• FIG. 9 is a graph showing CD19R-CD28gg-Zeta(CO)-T2A-EGFRt expression.
• Transduction of anti-CD3/anti-CD28 bead stimulated primary T cell blasts with the CD19R-CD28gg-Zeta(CO)-T2A-EGFRt_epHIV7 lentiviral vector (MOI 3) results in surface detection of both the CAR (using a biotinylated anti-Fc Ab and streptavidin-PE) and the truncated EGFR molecule (using a biotinylated cetuximab Ab and streptavidin-PE) by flow cytometry on day 4. The white peak in each panel is non-transduced control T cell blasts.
• Figure 10 is a schema showing a possible process flow for clinical trials for testing products of the present disclosure.
• Erbitux® is a registered trademark for the anti-EGFR monoclonal antibody cetuximab and is intended to independently include the trade name product formulation, the generic drug, and the active pharmaceutical ingredient(s) of the trade name product.
• genetic modification means any process that adds, deletes, alters, or disrupts an endogenous nucleotide sequence and includes, but is not limited to viral mediated gene transfer, liposome mediated transfer, transformation, transfection and transduction, e.g., viral mediated gene transfer such as the use of vectors based on DNA viruses such as lentivirus, adenovirus, retroviruses, adeno- associated virus and herpes virus.
• antibody includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies and antibody fragments that may be human, mouse, humanized, chimeric, or derived from another species.
• a "monoclonal antibody” is an antibody obtained from a population of substantially homogeneous antibodies that is being directed against a specific antigenic site.
• Variant refers to polypeptides having amino acid sequences that differ to some extent from a native sequence polypeptide. Ordinarily, amino acid sequence variants will possess at least about 80% sequence identity, more preferably, at least about 90% homologous by sequence. The amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the reference amino acid sequence.
• Percentage identity or “percent identity” is defined as the percentage of residues in the amino acid sequence variant that are identical after best aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
• Methods and computer programs for the alignment are well known in the art. Such programs include GAP, BESTFIT, FASTA, BLAST or Align 2.
• ADCC antibody-dependent cell-mediated cytotoxicity
• nonspecific cytotoxic cells that express Fc receptors, such as natural killer cells, neutrophils, and macrophages, recognize bound antibody on a target cell and cause lysis of the target cell. ADCC activity may be assessed using methods, such as those described in U.S. Pat. No. 5,821 ,337.
• Effector cells are leukocytes which express one or more constant region receptors and perform effector functions.
• terapéuticaally effective amount refers to an amount of a compound or molecule effective to treat a disease or disorder.
• Cancer refers to cells undergoing uncontrolled cellular growth.
• cancer examples include colorectal cancer and head and neck cancer.
• chemotherapeutic agent is a chemical compound useful in the treatment of cancer.
• a "cytokine” is a protein released by one cell to act on another cell as an intercellular mediator.
• Non-immunogenic refers to a material that does not initiate, provoke or enhance an immune response where the immune response includes the adaptive and/or innate immune responses.
• the term "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). Some genes may be developed which lack, in whole or in part, introns. Some leader sequences may enhance translation of the nucleic acid into polypeptides.
• isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
• a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
• Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
• a "vector” may be any agent capable of delivering or maintaining nucleic acid in a host cell, and includes viral vectors (e.g. retroviral vectors, lentiviral vectors, adenoviral vectors, or adeno-associated viral vectors), plasmids, naked nucleic acids, nucleic acids complexed with polypeptide or other molecules and nucleic acids immobilized onto solid phase particles.
• viral vectors e.g. retroviral vectors, lentiviral vectors, adenoviral vectors, or adeno-associated viral vectors
• plasmids naked nucleic acids
• nucleic acids complexed with polypeptide or other molecules e.g. retroviral vectors, lentiviral vectors, or adeno-associated viral vectors
• naked nucleic acids naked nucleic acids
• nucleic acids complexed with polypeptide or other molecules e.g., adenoviral vectors, or adeno-associated viral
• Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
• Receptor means a polypeptide that is capable of specific binding to a molecule. Whereas many receptors may typically operate on the surface of a cell, some receptors may bind ligands when located inside the cell (and prior to transport to the surface) or may reside predominantly intra-cellularly and bind ligand therein.
• Antibody or functional fragment thereof means an immunoglobulin molecule that specifically binds to, or is immunologically reactive with a particular antigen or epitope, and includes both polyclonal and monoclonal antibodies.
• the term antibody includes genetically engineered or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, and tetrabodies).
• the term functional antibody fragment includes antigen binding fragments of antibodies, including e.g., Fab', F(ab').sub.2, Fab, Fv, rlgG, and scFv fragments.
• the term scFv refers to a single chain Fv antibody in which the variable domains of the heavy chain and of the light chain of a traditional two chain antibody have been joined to form one chain.
• a gene encoding a modified endogenous cell- surface molecule that may be used as a non-immunogenic selection epitope compatible with immunomagnetic selection is provided.
• a non-immunogenic selection epitope may facilitate immunotherapy in cancer patients without undesirable immunologic rejection of cell products.
• the endogenous cell surface molecule may be modified or truncated to retain an extracellular epitope recognized by a known antibody or functional fragment thereof, and to remove any signaling or trafficking domains and/or any extracellular domains unrecognized by said known antibody.
• a modified endogenous cell surface molecule which lacks a signaling or trafficking domain and/or any extracellular domains unrecognized by said known antibody is rendered inert.
• the modified endogenous cell-surface molecule may be, but is not limited to, any non-immunogenic cell-surface related receptor, glycoprotein, cell adhesion molecule, antigen, integrin or cluster of differentiation (CD) that is modified as described herein. Modification of such cell-surface molecules is accomplished by keeping an epitope that is recognized by a known antibody or functional fragment thereof; and removing any signaling or trafficking domains and/or any extracellular domains unrecognized by a known antibody. Removal of the signaling or trafficking domains and/or any extracellular domains unrecognized by a known antibody renders the endogenous cell-surface molecule non-immunogenic and/or inert.
• CD cluster of differentiation
• Examples of endogenous cell-surface molecules that may be modified or truncated according to the embodiments described herein include, but are not limited to EpCAM, VEGFR, integrins (e.g., integrins ⁇ 3, ⁇ 4, ⁇ 3 ⁇ 4 ⁇ 3, ⁇ 4 ⁇ 7, ⁇ 5 ⁇ 1, ⁇ 3, ⁇ ), TNF receptor superfamily (e.g., TRAIL-R1 , TRAIL-R2), PDGF Receptor, interferon receptor, folate receptor, GPNMB, ICAM-1 , HLA-DR, CEA, CA-125, MUC1 , TAG-72, IL-6 receptor, 5T4, GD2, GD3, or clusters of differentiation (e.g., CD2, CD3, CD4, CD5, CD1 1 , CD1 1 a/LFA-1 , CD15, CD18/ITGB2, CD19, CD20, CD22, CD23/lgE Receptor, CD25, CD28, CD30, CD33, CD38, CD
• the modified endogenous cell-surface molecule is encoded by a modified or truncated tyrosine kinase receptor gene.
• tyrosine kinase receptors that may be modified or truncated according to the embodiments described herein include, but are not limited to, members of the endothelial growth factor receptor family (EGRF/ErbB1/HER1 ; ErbB2/HER2/neu; ErbB3/HER3; ErbB4/HER4), hepatocyte growth factor receptor (HGFR/c-MET) and insulin-like growth factor receptor-1 (IGF-1 R).
• modified tyrosine kinase receptors retain an extracellular epitope recognized by a known antibody or functional fragment thereof, and lack at least a tyrosine kinase domain.
• a modified tyrosine kinase receptor which lacks at least a tyrosine kinase domain renders the receptor inert.
• tyrosine kinase receptor Commercial antibodies that may be used to recognize a modified tyrosine kinase receptor include, but are not limited to AMG-102, AMG-479, BIIB022OA-5D5, CP-751 ,871 , IMC-A12, R1507, cetuximab, cixutumumab, ertumaxomab, figitumumab, matuzumab, necitumumab, panitumumab, pertuzumab, nimotuzumab, robatumumab, trastuzumab, zalutumumab.
• the modified endogenous cell surface molecule is a truncated EGFR (tEGFR) that lacks the membrane distal EGF-binding domain and the cytoplasmic signaling tail, but retains the extracellular membrane proximal epitope recognized by a known antibody or functional fragment thereof (e.g., cetuximab, matuzumab, necitumumab or panitumumab).
• tEGFR truncated EGFR
• the tEGFR is missing Domain I, Domain I I, the Juxtamembrane Domain and the Tyrosine Kinase Domain as compared to an unmodified EGFR ( Figure 1 ).
• a gene encoding a modified endogenous cell surface molecule may be used as a cell selection or enrichment marker for a genetically modified population of immune cells (e.g., T cells).
• the gene encoding a modified endogenous cell surface molecule may be coupled to a gene encoding a tumor targeting chimeric antigen receptor (CAR). These genes may be inserted into a vector to transduce the population of T cells to be genetically modified.
• CAR tumor targeting chimeric antigen receptor
• the cells that are successfully transduced and express the CAR and modified endogenous cell-surface molecule are enriched by any suitable purification method, such as immunomagnetic purification with anti-biotin microbeads or fluorochrome-conjugated anti-biotin for fluorescence activated cell sorting, using a commercial antibody that recognizes the modified endogenous cell-surface molecule expressed by the transduced cell.
• suitable purification method such as immunomagnetic purification with anti-biotin microbeads or fluorochrome-conjugated anti-biotin for fluorescence activated cell sorting, using a commercial antibody that recognizes the modified endogenous cell-surface molecule expressed by the transduced cell.
• a gene encoding a truncated human epidermal growth factor receptor (EGFRt) that lacks the membrane distal EGF-binding domain and the cytoplasmic signaling tail, but retains the extracellular membrane proximal epitope recognized by the FDA-approved anti-EGFR monoclonal antibody (mAb) cetuximab or another anti-EGFR antibody, is constructed and described herein.
• the EGFRt may be coupled with chimeric antigen receptors specific for a tumor associated antigen.
• the tumor associated antigen may be CD19, CD20, or CD22, or any other tumor associated antigen, but is preferably CD19 (CD19CAR).
• the tumor associated antigen is followed by a C-terminal 2A cleavable linker and the coding sequence for EGFRt.
• the biotinylated-cetuximab may be used in conjunction with commercially available anti-biotin microbeads for the purpose of immunomagnetic purification of the tumor associated antigen/CAR-expressing transductants. In the instance where the tumor associated antigen is CD19 the product is CD19CAR- expressing transductants.
• the biotinylated-cetuximab may be used in conjunction with Fluorochrome-conjugated anti-biotin for fluorescence activated cell sorting.
• a modified endogenous cell-surface molecule may be used as a marker for in vivo T cell engraftment.
• the modified endogenous cell-surface molecule is EGFRt
• the EGFRt may be used to track the uptake of the T cells to which it is attached in vivo without affecting cellular function of the T cells or the cells to which the T cells are targeted, such as bone marrow cells in a transplant situation.
• cetuximab conjugated to probes or reporter genes such as sr39TK may be used to improve the tracking potential of EGFRt-expressing cells to patients via PET imaging techniques.
• a modified endogenous cell-surface molecule may be used to induce cell suicide.
• EGFRt may be used as a suicide gene via cetuximab mediated complement and/or antibody dependent cell mediated cytotoxicity (ADCC) pathways.
• ADCC antibody dependent cell mediated cytotoxicity
• the truncated epidermal growth factor receptor (EGFRt) selection epitope or other modified cell-surface molecule is attached to other sequences.
• One exemplar sequence is the GMCSFR alpha chain signal sequence, which directs surface expression, attached to EGFRt.
• GMCSFR is encoded by nucleotides 1 -66 and EGFRt is encoded by nucleotides 67-1071 of SEQ ID NO: 1. See Figure 7.
• Figure 7 Also in Figure 7 is the antisense strand (SEQ ID NO: 2) and amino acid (SEQ ID NO: 3) sequences of GMCSFR alpha chain signal sequence linked to EGFRt.
• Another such sequence is a codon-optimized cDNA sequence encoding an anti-CD19 costimulatory chimeric antigen receptor (CD19R- CD28gg-Zeta(CO)), and a cleavable T2A linker.
• Cytotoxic T lymphocytes (CTLs) modified to express a CD19-specific chimeric antigen receptor (CAR) that signals via a cytoplasmic costimulatory (CD28) domain fused to the cytoplasmic ⁇ 3- ⁇ domain exhibits superior anti-tumor potency that can be attributed to CD28-mediated survival and enhanced cytokine production.
• This construct may be further modified to incorporate a C-terminal 2A cleavable linker followed by the coding sequence for a truncated human EGFR (EGFRt) for the purpose of immunomagnetic purification of CAR-expressing transductants using cetuximab-biotin/anti-biotin microbeads.
• EGFRt truncated human EGFR
• SEQ ID NOS: 4 nucleotide sense strand
• 5 nucleotide anti-sense strand
• 6 protein
• EGFRt is a truncated human epidermal growth factor receptor that lacks the membrane distal EGF-binding domain and the ectoplasmic signaling tail, but retains the extracellular membrane proximal epitope recognized by the commercial anti-EGFR mAb cetuximab. See Figure 1.
• Biotinylated-cetuximab is applied to immunomagnetic selection in combination with anti-biotin microbeads (Miltenyi).
• Human OKT3 blasts that had been lentivirally transduced with CD19R- CD28gg-Zeta(CO)-T2A-EGFRt were subjected to immunomagnetic selection using the Miltenyi AutoMACS device, and the frequency of EGFRt+CAR+ T cells was enriched from 22% (pre-selection) to 99% (post-selection) without observable toxicity to the cell preparation (Fig. 3).
• the EGFRt can be purified using fluorescence-based cell sorting techniques.
• EGFRt Due to the absence of the EGF-binding domains and intracellular signaling domains, EGFRt is inactive when expressed by T cells. Importantly, the EGFRt-selected T cells maintain their desired effector phenotype - including antitumor cyotoxic activity mediated by the chimeric antigen receptor that is coordinately expressed with the EGFRt - and remain amenable to established expansion protocols.
• this EGFRt has various advantages for immunotherapeutic cell products compared to other selection markers that have been previously reported. Specifically, unlike truncated CD4 and CD19, it is not endogenously expressed by subpopulations of lymphocytes. Furthermore, in contrast to truncated CD34 and low affinity nerve growth factor receptor, it does not have any activity that might negatively affect the immune cell product (i.e., in terms of signaling or trafficking). Lastly, it alone can be bound/recognized by a known, preferably commercially available, pharmaceutical grade antibody reagent, i.e., cetuximab. Together, these attributes make EGFRt a superior selection marker for any transfection/transduction system that can be applied to the generation of cell products for adoptive
• EGFRt is well suited to be used as a selection marker for lentivirally transduced T cells of immunotherapeutic relevance.
• FITC-, PE- and PerCP-conjugated isotype controls PerCP-conjugated anti-CD8, FITC conjugated anti-CD4, PE-conjugated anti-IFNy, PerCP-conjugated anti-CD45 and PE-conjugated streptavidin were obtained from BD Biosciences (San Jose, CA). Biotinylated anti-Fc was purchased from Jackson ImmunoResearch Laboratories, Inc. (Westgrove, PA). PE-conjugated anti-Biotin was purchased from Miltenyi Biotec (Auburn, CA). Biotinylated EGF was purchased from Molecular Probes® Invitrogen (Carlsbad, CA).
• PE-conjugated anti-EGFR was purchased from Abeam Inc. (Cambridge, MA). All antibodies and biotin-EGF were used according to the manufacturer's instructions. Flow cytometric data acquisition was performed on a FACScalibur (BD Biosciences), and the percentage of cells in a region of analysis was calculated using FCS Express V3 (De Novo Software, Los Angeles, CA).
• CM culture media
• PBMC blood cells
• washed PBMC were first autoMACSTM depleted using anti-CD45RA beads (Miltenyi Biotec) per the manufacturer's protocol, and in some cases also depleted with PE-conjugated anti-CD4 (BD Biosciences) with anti-PE beads (Miltenyi Biotec).
• the resulting cells then underwent autoMACSTM positive selection using biotinylated DREG56 (anti-CD62L) and anti-biotin beads (Miltenyi Biotec) to produce purified CD62L + CD45RO + TCM- CD8 + cells were further selected in some cases using AutoMACSTM (Miltenyi Biotec) per the manufacturer's protocol.
• CMV-specific cells were generated by stimulating T cells with 5U/ml rhlL-2 (Chiron, Emeryville, CA) and autologous irradiated viral antigen presenting cells at a 4:1
• the viral antigen presenting cells were derived from PBMC that had been genetically modified to express CMVpp65 antigen.
• PBMC peripheral blood mononuclear cells
• PBMC peripheral blood mononuclear cells
• 5 x 10 7 cells were aliquoted into 0.2-cm cuvettes containing 1 C ⁇ g HygroR-pp65_pEK (or pmaxGFP from Amaxa Inc., as a transfection control) in a final volume of 100 ⁇ -Jcuvette, and electroporated using the Amaxa Nucleofector I (Amaxa Inc.), program U-14, after which cells were allowed to recover for 6 hours at 37°C prior to ' 7-irradiation (1200 cGy).
• Amaxa Nucleofector I Amaxa Inc.
• CD19CAR-T2A-EGFRt_epHIV7 pJ02104
• CD19CAR-T2A-EGFRt_epHIV7 pJ02104
• CD19CAR-T2A-EGFRt_epHIV7 pJ02104
• EGFRt-T2A-IMPDH2dm_epHIV7 (pJ021 1 1 ) lentiviral constructs contain a) the chimeric antigen receptor (CAR) sequences consisting of the VH and V
• _ gene segments of the CD19-specific FmC63 mAb, an lgG 1 hinge-CH2-CH3, the CAR chimeric antigen receptor
• Lentiviral transduction was carried out on T cells that were stimulated with either 30 ng/mL anti-CD38 (OKT3; Ortho Biotech, Raritan, NJ) (i.e., for Line A) or human CD3/CD28Dynal beads at a 1 : 10 ratio (i.e., for Lines B, C, D and E) and 25U IL2/ml.
• Cells were cultured for up to 2 hours at 37°C on RetroNectin® (50ug/ml) coated plates prior to addition of the lentivirus at an MOI of 3 and 5 ⁇ g ml polyybrene. After 4 hours, warm medium was added to triple to volume, and the cells were then washed and plated in fresh media after 48hours.
• LCLs lymphoblastoid cell lines
• mice myeloma line NS0 (gift from Andrew Raubitschek, City of Hope National Medical Center, Duarte, CA) was resuspended in nucleofection solution using the Nucleofector kit T (Amaxa Inc., Gaithersberg, MD), CD19t- DHFRdm-2A-IL12_pEK (pJ01607) or GFP-IMPDH2dm-2A-IL15_pcDNA3.1 (+) (pJ01043) plasmid was added at 5 ⁇ g 5x10 6 cells, and cells were electroporated using the Amaxa Nucleofector I, program T-27.
• NS0 - CD19t- DHFRdm-2A-IL12_pEK (cJ03935) and NS0 - GFP:IMPDH2- IL15(IL2ss)_pcDNA3.1 (+) (cJ02096) were grown in DMEM (Irvine Scientific, Santa Ana, CA) supplemented with 10% heat-inactivated FCS, 25mM HEPES, and 2 mM L-glutamine in the presence of either 0.05uM methotrexate (MTX) or 6 ⁇ mycophenolic acid (MPA).
• MTX methotrexate
• MPA mycophenolic acid
• U251 T-pp65 were generated by lentiviral transduction of U251T with pp65-2A-eGFP-ffluc_epHIV7 (pJ01928) at an MOI of 1 .
• the resulting U251 T - pp65-2A-eGFP-ffluc_epHIV7 were then FACS sorted for the GFP + population (cJ05058).
• the Daudi lymphoma line was purchased from ATCC and grown in media consisting of RPMI 1640 (Irvine Scientific), 2 mM L- Glutamine (Irvine Scientific), 10% heat-inactivated FCS (Hyclone).
• SupB15 acute lymphoblastic leukemia cells and A431 epidermoid carcinoma cells were purchased from ATCC.
• the cytolytic activity of T cells was determined by 4-hour chromium- release assay (CRA), where effector cells were seeded into triplicate wells of V- bottom 96-well micro-plates containing 5x10 3 5 Cr-labeled targeT cells (Na 2 5 Cr0 4 ; (5mCi/mL); Amersham Pharmacia, Piscataway, NJ) at various E:T ratios in 200 uL of CM and incubated for 4 hours at 5% CO 2 , 37°C. Plates were centrifuged, and 100 ⁇ of supernatant was removed from each well to assess chromium release using a ⁇ - counter (Packard Cobra II, Downer's Grove, IL). The percent specific lysis was calculated as follows: 100 x (experimental release - spontaneous
• Antibody dependent cell mediated cytotoxicity was determined by chromium release as above using 5x10 3 5 Cr-labeled targeT cells that had been pre- incubated for 90 min with up to 10 ⁇ g mL of either cetuximab or rituximab (a CD20- specific mAb), washed and then co-incubated with 5x10 5 freshly isolated PBMC. T cell engraftment and cetuximab mediated suicide in vivo
• mice For T cell engraftment, six- to ten-week old NOD/Scid IL-2RyC nu " mice are injected i.v. on day 0 with 10 7 T cells (Line C). 2x10 7 irradiated (8000 rads) NSO - GFP:IMPDH2-IL15(IL2ss)_pcDNA3.1 (+) (cJ02096) cells are administered i.p. 3 times a week starting on day 0 to provide a systemic supply of human IL-15 in vivo. Bone marrow was harvested from euthanized animals and analyzed by flow cytometry. Antibody dependent cell mediated cytotoxicity assays are performed to determine the activity of cetuximab against EGFRt + T cells.
• EGFRt truncated human EGFR
• transmembrane domain and extracellular domains III and IV of the full length EGFR was generated as a non-immunogenic selection epitope compatible with
• the EGFRt retains the ability to be bound by cetuximab, but not have any signaling capacity due to the absence of the intracellular domains. Furthermore, it lacks the N-terminal domain required for EGF-binding.
• biotinylated-cetuximab was generated (Fig. 2a, b) to be used in conjunction with commercially available anti-biotin microbeads and an AutoMACSTM separator (Miltenyi Biotec) (Fig. 2c).
• Lentiviral transduction of various T cell lines with EGFRt- containing constructs, where the EGFRt gene was separated from other genes of interest on either one or both ends with the self-cleaving T2A sequence consistently resulted in surface detection of the EGFRt molecule on less than 40% of the cells (Fig. 2d). Surface detection may also be accomplished with a EGFRt-sr39TK fusion.
• Immunomagnetic selection allowed for recovery of EGFRt + T cell populations with greater than 90% purity.
• T cell populations that underwent this transduction and selection procedure included anti-CD3/anti-CD28 bead stimulated T cell blasts (for Line A), central memory (CD45RO + CD62L + T CM ) derived T cells (for Lines B, C and E), which in some cases were also pre-selected for CMV specificity (via the endogenous TCR; for Line B) or CD8 expression (for Line C), as well as effector memory (CD62L " CD45RO + TEM) derived T cells (for line D).
• CD45RO + CD62L + T CM central memory
• CD45RO + CD62L + T CM central memory
• CD45RO + CD62L + T CM central memory
• CD45RO + CD62L + T CM central memory
• CD45RO + CD62L + T CM central memory
• CD45RO + CD62L + T CM central memory
• CD45RO + CD62L + T CM central memory
• CD45RO + CD62L + T CM central memory
• CD8 expression
• the selected T cells were expanded 30-fold or greater within 12 days after REM stimulation with OKT3, irradiated PBMC feeders and LCL, IL-2 and IL-15 (Fig. 4a).
• Flow cytometric analysis of the resulting expanded EGFRt + T cells further confirmed that that they express the CD19CAR and T cell markers such as CD8, TCR, CD3, perforin, granzyme, etc. (Fig. 4b).
• CD19CAR-directed cytotoxic activity of these EGFRt- selected lines is evident in chromium release assays using CD19-expressing tumor targets (Fig. 4c).
• Cetuximab mediated cytotoxicity of EGFRt + T cells
• cetuximab is known to lyse EGFR-expressing cells via antibody dependent cell mediated cytotoxicity (ADCC), assays were performed to determine the ADCC activity of cetuximab against EGFRt + T cells (Fig. 6). Using 5 Cr-labeled Line A cells as targeted and freshly isolated human PBMC as effectors, cetuximab was found to significantly mediate chromium-release above that seen when using the CD20-specific humanized mAb Rituxan.
• ADCC antibody dependent cell mediated cytotoxicity
• RituximabTM is not given as part of the Salvage/Priming chemotherapy regimen, research participants may receive a single intravenous infusion of RituximabTM (chimeric anti- CD20 antibody) at 375 mg/m 2 within 4-weeks of the planned auto-HSCT procedure.
• RituximabTM infusion would be carried out per standard practice including premedication with diphenhydramine and acetaminophen and hydrocortisone.
• the autologous cryopreserved CD19R + CD8 + EGFRt + T cell product will be transported, thawed and infused at the patient's bedside.
• CD34 splice variant an attractive marker for selection of gene-modified cells. Mol Ther V. 448-56.
• plRES-CD4t a dicistronic expression vector for MACS- or FACS-based selection of transfected cells. Biotechniques 26: 683-8.
• CD34 modulates the trafficking behavior of hematopoietic cells in vivo.

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