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Definitions

• the epidermal growth factor family of receptors tyrosine kinases (ErbBs) consists of four members: EGFR/ErbB 1 /HER 1 , ErbB2/HER2/Neu, ErbB3/HER3, and ErbB4/HER4 (Wieduwilt and Moasser, C ell Mol Life Sci. (2008) 65(10): 1566-84). These receptors are widely expressed in epithelial, mesenchymal, and neuronal tissue and play critical roles in cell proliferation, differentiation, and development (Yano et al., Anticancer Res. (2003) 23(5A):3639-50).
• EGFR epidermal growth factor receptor
• EGFR epidermal growth factor receptor
• the extracellular region comprises four domains: Domains I and III are homologous ligand binding domains, and domains II and IV are cysteine rich domains (Ferguson, Annu Rev Biophys. (2008) 37:353-3).
• the structured Domain III of human EGFR is targeted by the FDA licensed monoclonal antibody cetuximab (Erbitux®). Separating the cetuximab-binding ability of EGFR from its biological activity by selective truncation of the receptor offers the potential for an inert, fully human cell surface marker (Li et al., Cancer Cell (2005) 7(4):301-11; Wang et al., Blood (2011) 118(5): 1255-63).
• a critical feature of a clinically useful cell surface marker is that the marker needs to be expressed at consistently high levels in the engineered cells such that the engineered cells can be sufficiently identified and targeted when needed.
• the present disclosure provides a recombinant polypeptide comprising an extracellular region, a transmembrane region, and an intracellular region, wherein the extracellular region comprises a human epidermal growth factor receptor (EGFR) Domain III sequence, and the intracellular region (i) comprises a juxtamembrane domain that is net-neutral or net-positively charged in the first at least three amino acids (ii) but lacks an active EGFR tyrosine kinase domain. In some embodiments, the polypeptide does not have any active tyrosine kinase domain.
• EGFR human epidermal growth factor receptor
• more than half of the amino acids of the juxtamembrane domain are glycine, serine, arginine, lysine, threonine, asparagine, glutamine, aspartic acid, glutamic acid, tyrosine, tryptophan, histidine, and/or proline.
• the amino acid at each position of the juxtamembrane domain is selected according to Table 1.
• the juxtamembrane domain comprises RRRHIVRKR (SEQ ID NO: 16), RRRHIVRK (SEQ ID NO: 17), RRRHIVR (SEQ ID NO: 18), RRRHIV (SEQ ID NO: 19), RRRHI (SEQ ID NO:20), RRRH (SEQ ID NO:21), RRR, RKR, or RR.
• the intracellular region does not contain any residue that is phosphorylated.
• the human EGFR Domain III sequence may comprise SEQ ID NO:2 or a functional variant thereof such as a sequence comprising at least 90% identity to SEQ ID NO:2.
• the extracellular region further comprises, C-terminal to the Domain III sequence, (i) a sequence derived from EGFR Domain IV, (ii) an artificial sequence, or (iii) both (i) and (ii).
• the extracellular region comprises amino acids 334-504, 334-525, or 334-645 of SEQ ID NO: 1.
• the transmembrane region is derived from a human EGFR transmembrane domain, optionally comprising SEQ ID NO: 5.
• the recombinant polypeptide comprises a signal peptide derived from human EGFR, human granulocyte-macrophage colony-stimulating factor (GM-CSF), human Ig kappa, mouse Ig kappa, or human CD33.
• the signal peptide may comprise SEQ ID NO:22, 23, 24, or 25.
• the recombinant polypeptide comprises SEQ ID NO:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40; or an amino acid sequence at least 90% identical thereto.
• the present disclosure provides a nucleic acid molecule, such as an expression construct, comprising a coding sequence for a recombinant polypeptide of the present disclosure.
• the nucleic acid molecule further comprises a coding sequence for a chimeric antigen receptor (CAR).
• the CAR may target, for example, a tumor antigen such as AFP, BCMA, CD 19, CD20, CD22, CD 123, EpCAM, GPC2, GPC3, HER2, MUC16, ROR1, or ROR2.
• the CAR may be bispecific, targeting, e.g., CD 19 and CD20 or CD 19 and CD22.
• the coding sequences for the recombinant polypeptide and the CAR are operably linked to the same promoter (e.g., a constitutive or inducible promoter; for example, an MND promoter) such that the two coding sequences are co-transcribed, and optionally the two coding sequences are separated by (i) an internal ribosome entry site (IRES) or (ii) a coding sequence for a self-cleaving peptide (e.g., a 2A peptide) wherein the coding sequences for the recombinant polypeptide, the CAR, and the self-cleaving peptide are in frame with each other.
• a constitutive or inducible promoter for example, an MND promoter
• the nucleic acid molecule further comprises a coding sequence for a third polypeptide, optionally wherein the third polypeptide is human c-Jun or a functional analog thereof.
• the coding sequences for the recombinant polypeptide, the CAR, and the human c-Jun are operably linked to the same promoter (e.g., a constitutive or inducible promoter; for example, an MND promoter) such that the three coding sequences are co-transcribed, and optionally the three coding sequences are separated from each other by (i) an IRES or (ii) a coding sequence for a self-cleaving peptide (e.g., a 2A peptide) wherein the coding sequences for the recombinant polypeptide, the CAR, the human c-Jun, and the self-cleaving peptide(s) are in frame with each other.
• a promoter e.g., a constitutive or inducible promoter; for
• the nucleic acid molecule is a viral vector, optionally a lentiviral or retroviral vector.
• the present disclosure provides a cell (e.g., autologous or allogeneic human T cells) comprising the nucleic acid molecule described herein; a recombinant virion comprising the nucleic acid molecule; and a pharmaceutical composition comprising the cell, the nucleic acid molecule, or the virion, and a pharmaceutically acceptable carrier.
• the present disclosure provides a method of treating a patient in need thereof, comprising administering the cell to the patient, optionally wherein the cell is autologous or allogeneic.
• the patient has cancer, and is given the T cell preparation described herein, where the T cells express a CAR, a T cell receptor (TCR), an engineered TCR, or a TCR mimic that is specific for a tumor antigen present in the cancer.
• TCR T cell receptor
• the method comprises administering to the patient an effective amount of an antibody specific for human EGFR once the patient has been treated (e.g., the cancer has regressed), wherein the antibody elicits cytotoxicity against T cells expressing the recombinant polypeptide, and optionally the antibody is IgGi or IgG2 (e.g., cetuximab).
• an antibody specific for human EGFR once the patient has been treated (e.g., the cancer has regressed)
• the antibody elicits cytotoxicity against T cells expressing the recombinant polypeptide
• the antibody is IgGi or IgG2 (e.g., cetuximab).
• the present disclosure also provides the cell, the nucleic acid molecule, the recombinant virus, the pharmaceutical composition for use in the treatment methods, as well as the cell, the nucleic acid molecule, or the virus for the manufacture of a medicament for treating a patient as described herein.
• the present disclosure provides a method of making a genetically engineered human cell (e.g., engineered T cells), comprising providing an isolated human cell, and introducing the nucleic acid molecule or recombinant virus described herein into the human cell.
• a genetically engineered human cell e.g., engineered T cells
• FIGs. 1A and 1B show the binding of anti-EGFR antibody AY13 to live CAR+ T cells transduced with R12CAR-P2A-EGFRt or R12C AR-P2 A-EGFRt-DEARKAIAR, or to total live untransduced cells.
• FIG. 1A flow plot.
• FIG. 1B a bar graph quantitating the geometric mean fluorescence intensity (gMFI) data from FIG. 1A.
• “DEARKAIAR” a juxtamembrane sequence DE ARK AI ARVKRE SKRIVED AERLIRE A A A ASEKI SREAERLI (SEQ ID NO:41).
• R12CAR a CAR directed against ROR1.
• P2A a self-cleaving peptide.
• EGFRt a truncated human EGFR containing EGFR extracellular Domains III and IV and an EGFR transmembrane domain while lacking EGFR Domains I and II and EGFR intracellular sequence.
• FIG. 2A depicts the domain structure of human EGFR from NCBI Reference Sequence NP 005219.2.
• the transmembrane domain is SEQ ID NO:5
• the juxtamembrane domain is SEQ ID NO: 15 (full length sequence disclosed as SEQ ID NO:43).
• basic residues are indicated by red and “+,” and acidic residues by blue and Phosphorylated residues (green) are further described below.
• FIG. 2B depicts the design of certain embodiments of the present EGFR-derived polypeptides (with transmembrane domain SEQ ID NO:5 without or with a juxtamembrane domain of RRR, SEQ ID NO: 16, SEQ ID NO: 12, or SEQ ID NO: 13 (full length sequences disclosed as SEQ ID NOs:44-47, respectively, in order of appearance)).
• S. peptide signal peptide.
• GMCSF signal peptide derived from GM-CSF. Asterisks indicate the C-terminal end of the polypeptide.
• FIGs. 3A-C show the expression of a series of bi-cistronic expression constructs for EGFR-derived polypeptides in primary T cells obtained from two donors and transduced with the expression constructs.
• FIG. 3A depicts the basic structure of the constructs, which include coding sequences for R12 CAR.
• FIGs. 3B and 3C show the gMFIs for bound RORl-Fc fusion protein and AY13, respectively, in live R12 CAR+ transduced cells or live cells in the untransduced condition. Flow cytometry was performed eight days post transduction.
• FIGs. 3B and 3C disclose SEQ ID NOs:16, 12, 13, 16, 12, and 13, respectively, in order of appearance. [0023] FIG.
• FIG. 4A is a bar graph showing the percentages of transduced T cells as indicated by R12 CAR expression.
• the T cells were those shown in FIGs. 3A-C.
• Flow cytometry was performed five days post transduction.
• FIG. 4A discloses SEQ ID NOs:16, 12, 13, 16, 12, and 13, respectively, in order of appearance.
• FIG. 4B is a graph showing a comparison of EGFRt detection by Domain Ill-specific cetuximab and Domain Ill-specific AY13. Flow cytometry was performed five days post transduction. FIG. 4B discloses SEQ ID NOs:16, 12, and 13, respectively, in order of appearance.
• FIGs. 5A and 5B are graphs showing the effects of transduction efficiency on R12 CAR and EGFRt surface expression, respectively, in primary T cells transduced with the bi- cistronic expression constructs of FIGs. 3A-C. Flow cytometry was performed four days post transduction.
• FIGs. 5A and 5B disclose SEQ ID NOs:16, 12, and 13, respectively, in order of appearance.
• FIGs. 6A-C show the expression of a series of tri-cistronic expression constructs for EGFR-derived polypeptides in primary T cells obtained from two donors and transduced with the expression constructs.
• FIG. 6A depicts the basic structure of the constructs.
• FIGs. 6B and 6C show the gMFIs for bound RORl-Fc fusion protein and AY13, respectively, in live R12 CAR+ transduced cells or total live untransduced cells. Flow cytometry was performed eight days post transduction.
• FIGs. 6B and 6C each disclose SEQ ID NO: 16.
• FIG. 7 shows the antibody-dependent cellular cytotoxicity (ADCC) induced by cetuximab in CAR-T cells expressing EGFR-derived polypeptides from the bi-cistronic constructs of FIG. 3A. The figure shows the fraction of CAR-T cells remaining after four hours of cetuximab treatment, relative to CAR-T cells not treated with the antibody.
• Ritux rituximab.
• FIG. 8 is a graph showing cetuximab-induced cytotoxicity in CAR-T cells expressing EGFR-derived polypeptides from the tri-cistronic constructs of FIG. 6A. The figure shows the fraction of CAR-T cells remaining after four hours of cetuximab treatment, relative to CAR-T cells not treated with the antibody.
• FIG. 8 discloses SEQ ID NO: 16.
• FIG. 9 shows cetuximab-induced cytotoxicity in CAR-T cells expressing EGFR- derived polypeptides from the tri-cistronic constructs of FIG. 6A.
• the figure shows the fraction of CAR-T cells remaining after 24 hours of cetuximab treatment, relative to CAR-T cells not treated with the antibody.
• FIG. 9 discloses SEQ ID NO: 16.
• FIG. 10 is a bar graph quantitating the gMFI for anti-EGFR antibody binding in live ROR1 CAR+ cells transduced with EGFRt or variants thereof having additional intracellular juxtamembrane sequences (R, RR, RRR, or RKR) or in total live cells in the untransduced condition.
• FIGs. 11A and 11B show surface expression levels of EGFRt (white) or EGFR-RRR (EGFRt with an RRR juxtamembrane domain; grey) in mouse T cells transduced with MP71 retroviral constructs that were mono-cistronic (FIG. 11 A) or bi-cistronic (FIG. 11B). MFI: mean fluorescence intensity.
• FIG. 12A shows a schematic (top panel) of an in vivo study on the indicated CAR-T infusion products (middle panel) and expression levels of the EGFR polypeptides EGFRt or EGFR-RRR (bottom panel).
• FIG. 12B is a panel of graphs showing the kinetics of circulating EGFRt and EGFR- RRR CAR-T cells following cetuximab (Cetx) treatment (white circle) compared to rituximab (control; Ritx) (black circle). Grey line represents depletion time point.
• FIG. 12C shows the results of an in vivo study on the depletion kinetics of circulating EGFRt and EGFR-RRR transduced T cells.
• Top panel a schematic showing the adoptive transfer of EGFR + T cells.
• Bottom panel graphs showing kinetics of circulating EGFR + T cells following treatment with cetuximab (white circle). Grey line represents depletion time point.
• FIG. 13A shows the results of an in vivo study on the depletion kinetics of circulating EGFRt (left) and EGFR-RRR (right) CAR-T cells following treatment with cetuximab (white circle) or vehicle (black circle). Shaded area represents post-depletion window.
• FIG. 13B shows rebound kinetics of circulating B cells in the study shown in FIG. 13A. Two different doses of cetuximab were used as indicated.
• FIG. 13C shows the frequency of B-cell-aplastic animals (below 3% CD19 + out of total CD45) following high (lmg) or low (O.lmg) dose cetuximab administration.
• An important component of cell therapy is a compact, functionally inert cell surface marker that can be used for detecting, selecting, and enriching engineered cells, and for in vivo cell ablation.
• the present disclosure provides novel EGFR-derived proteins that can be used for these purposes. These proteins lack the ligand-binding and/or signal transduction functions of wildtype EGFR, but can still be recognized by common anti-EGFR antibodies.
• EGFR-derived proteins can be expressed at high levels on cell surface and therefore are particularly useful as a safety switch (suicide gene) in cell therapy.
• a pharmaceutical grade anti-EGFR antibody such as cetuximab, panitumumab, nimotuzumab, or necitumumab can be administered to the patient, thereby removing the engineered cells through antibody-dependent cellular cytotoxicity (ADCC), complement- dependent cytotoxicity (CDC), and/or antibody-dependent cellular phagocytosis (ADCP).
• ADCC antibody-dependent cellular cytotoxicity
• CDC complement- dependent cytotoxicity
• ADCP antibody-dependent cellular phagocytosis
• the signal peptide spans amino acids 1-24.
• the extracellular sequence spans amino acids 25-645, wherein Domain I, Domain II, Domain III, and Domain IV span amino acids 25-188, 189-333, 334-504, and 505-645, respectively.
• the transmembrane domain spans amino acids 646-668.
• the intracellular domain spans amino acids 669-1,210, where the juxtamembrane domain spans amino acids 669-703 and the tyrosine kinase domain spans amino acids 704-1,210.
• an EGFR amino acid position recited herein refers to the position in SEQ ID NO: 1 or a corresponding position in a variant of SEQ ID NO: 1 (e.g., a naturally occurring polymorphic variant or a genetically engineered variant).
• polypeptides of the present disclosure are derived from EGFR but contain only a partial, rather than entire, sequence of EGFR. These polypeptides are cell surface proteins when expressed in mammalian cells. The polypeptides’ extracellular, transmembrane, and intracellular regions are described below. A. Extracellular Region
• the extracellular region of the present EGFR-derived polypeptides comprises the epitope bound by an anti-EGFR antibody such as cetuximab.
• the region may comprise Domain III of EGFR, such as the following Domain III sequence, or a functional variant thereof:
• “functional variant” is meant a sequence having sequence variations, such as deletions, insertions, and/or substitutions (e.g., conservative substitutions), that do not affect the sequence’s desired biological function.
• a functional variant of SEQ ID NO:2 can be still bound by cetuximab.
• the extracellular region may further comprise additional EGFR sequences such as those that help stabilize disulfide bonds in the Domain III structure.
• the extracellular region may comprise a Domain
• a Domain IV-derived sequence may comprise the following Domain IV sequence:
• a Domain IV-derived sequence may alternatively comprise a functional variant of SEQ ID NO:3.
• a functional variant can help maintain Domain Ill’s tertiary structure to allow the binding of the polypeptide by an anti-EGFR antibody such as cetuximab.
• the functional variant may contain just a portion of a natural EGFR Domain IV, with or without additional sequences heterologous to EGFR (i.e., sequences that are not part of a natural EGFR sequence).
• a Domain IV-derived sequence includes a portion of a natural EGFR domain IV sequence, which portion includes amino acid residues involved in maintaining the structural fold of Domain III.
• Such amino acid residues include the W492 residue of mature EGFR (corresponding to W516 of SEQ ID NO:l and W12 of SEQ ID NO:3; boxed in the sequences above) and optionally one or more residues adjacent to it.
• Structural analysis shows that W492 is important to the folding of EGFR Domain III, as this residue points into the core of Domain III and makes important side chain packing interactions.
• Examples of Domain IV- derived sequences are those including residues 492-496 of mature EGFR (corresponding to residues 516-520 of SEQ ID NO:l and residues 12-16 of SEQ ID NO:3).
• a Domain IV-derived sequence is (SEQ ID NO:4), in which natural Domain IV’s residues 482-491 and 497-621 are removed,
• V481 is connected to W492 through a synthetic four-residue linker (boxed above), and P496 is followed by a G/S linker (underlined above) linking it to the transmembrane region of the present polypeptide.
• the extracellular region of the present EGFR-derived polypeptides lacks the EGFR extracellular portion that binds ligands such as EGF and TGF- alpha.
• the extracellular region does not include any sequence of Domain I and/or Domain II of EGFR or includes only partial sequences from either or both Domains.
• the extracellular region of the present polypeptides includes additional sequences.
• the extracellular region may comprise a stalk region immediately N-terminal to the transmembrane domain.
• the stalk region may be, for example, a flexible stalk such as a G/S rich peptide linker or a structured stalk such as the CH2-CH3 domains from an antibody constant region or an extracellular domain from another protein.
• the extracellular region also may comprise an additional functional domain, such as antigen-binding domains (e.g., an scFv or a designed ankyrin repeat protein (DARPin)).
• antigen-binding domains e.g., an scFv or a designed ankyrin repeat protein (DARPin)
• the transmembrane region of the present polypeptides contains a hydrophobic sequence.
• This region may comprise an artificial sequence or may be derived from any transmembrane protein, which may be, for example, ERBB1 (EGFR), ERBB2 (HER2), ERBB3 (HER3), ERBB4 (HER4), INSR, IGF1R, INSRR, PGFRA, PGFRB, KIT, CSF1R, FLT3, VGFR1, VGFR2, VGFR3, FGFR1, FGFR2, FGFR3, FGFR4, PTK7, NTRK1, NTRK2, NTRK3, ROR1, ROR2, MUSK, MET, RON, UFO, TYR03, MERTK, TIE1, TIE2, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHAA, EPHB1, EPHB2, EPHB3, EPHB4, EPHB6, RET, RYK, DDR1, DDR2, ROS1,
• transmembrane region is derived from EGFR, with the sequence of IATGMVGALLLLLVVALGIGLFM (SEQ ID NO:5), or a functional variant thereof.
• a juxtamembrane domain refers to an intracellular portion of a cell surface protein immediately C-terminal to the transmembrane domain.
• a high cell surface expression level ensures that the cell expressing the protein is recognized by an anti-EGFR antibody and thus ensures the eradication of the cell through, e.g., ADCC, CDC, and/or ADCP.
• the juxtamembrane domain in the present polypeptide may be from 1 to 20 (e.g., 2-20, 3-20, 4-20, 5-20, 2-18, 3-18, 4-18, or 5-18) amino acids long. They also can be longer than 20 amino acids.
• the first 1 or more (e.g., first 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 ,14, 15, 16, 17, 18, 19, or 20) amino acids of the intracellular region of the present polypeptide is a net-neutral or net-positively charged sequence (e.g., the number of arginine and lysine residues is greater than or equal to the number of aspartic acid and glutamic acid residues).
• those first amino acids contain more than 30% (e.g., more than 40, 50, 60, 70, 80, or 90%) hydrophilic amino acids.
• amino acid choices at each position of the sequence appended to the C-terminus of the transmembrane domain are shown in Table 1 below.
• the present juxtamembrane domain may be derived from the juxtamembrane region of a natural cell surface protein, such as a juxtamembrane region (e.g., the entire or partial sequence of the first 20 juxtamembrane amino acids) of a human receptor tyrosine kinase that interacts with phosphatidylcholine (PC), phosphatidylserine (PS), or phosphatidylinositol-4,5- bisphosphate (PIP2) (see, e.g., Hedger et al., Sci Rep. (2015) 5: 9198).
• a juxtamembrane region e.g., the entire or partial sequence of the first 20 juxtamembrane amino acids
• PC phosphatidylcholine
• PS phosphatidylserine
• PIP2 phosphatidylinositol-4,5- bisphosphate
• receptor tyrosine kinases examples include ERBB1 (EGFR), ERBB2 (HER2), ERBB3 (HER3), ERBB4 (HER4), INSR, IGF1R, INSRR, PGFRA, PGFRB, KIT, CSF1R, FLT3, VGFR1, VGFR2, VGFR3, FGFR1, FGFR2, FGFR3, FGFR4, PTK7, NTRK1, NTRK2, NTRK3, ROR1, ROR2, MUSK, MET,
• the derived sequence may contain mutations (e.g., substitutions or deletions) that remove residues known to be phosphorylated so as to circumvent any unintended signal transducing ability of the present protein.
• the juxtamembrane domain of the present polypeptide comprises a juxtamembrane region of EGFR, such as:
• an EGFR-derived juxtamembrane domain is derived from the first 19 amino acids of a natural EGFR juxtamembrane region (e.g., SEQ NO ID: 15) and does not include the entirety of the remaining portion of the natural juxtamembrane region, so as to avoid dimerization of the present polypeptide.
• the residues known to be phosphorylated (boxed above, corresponding to T678, T693, and S695 of SEQ ID NO:l) are deleted or substituted.
• Nonlimiting examples of EGFR-derived juxtamembrane domains comprise one of the following sequences:
• the intracellular region also includes an additional sequence C- terminal to the juxtamembrane domain, e.g., a functional domain (e.g., a switch receptor).
• a functional domain e.g., a switch receptor
• the present EGFR-derived protein lacks a functional tyrosine kinase domain of EGFR such that the protein lacks signal transducing ability.
• the protein lacks the entirety of a region that corresponds to amino acids 704-1,210 of SEQ ID NO: 1.
• the intracellular region does not contain any potential phosphorylation motif.
• the coding sequence for the present polypeptide includes a coding sequence for a signal peptide.
• the signal peptide may facilitate the cell surface expression of the polypeptide and is cleaved from the mature polypeptide.
• the signal peptide may be derived from that of any cell surface protein or secreted protein.
• the signal peptide may be a signal peptide shown below:
• the present polypeptide comprises, consists of, or consists essentially of EGF Domain III (italicized), Domain IV (underlined), and transmembrane domain, with a juxtamembrane domain (not shown) appended to the C-terminus of the transmembrane domain, with or without a signal peptide (not shown):
• the present polypeptide comprises, consists of, or consists essentially of EGFR Domain III (italicized), modified portion of EGFR Domain IV (boldfaced and underlined), EGFR transmembrane domain, and a juxtamembrane domain (not shown) appended to the C-terminus of the transmembrane domain, with or without a signal peptide (not shown):
• the present polypeptide comprises, consists of, or consists essentially of EGFR Domain III (italicized), synthetic sequence (boldfaced and underlined),
• EGFR transmembrane domain and a juxtamembrane domain (not shown) appended to the C- terminus of the transmembrane domain, with or without a signal peptide (not shown):
• the present polypeptide comprises, consists of, or consists essentially of a GM-CSF signal peptide (boldfaced), EGFR Domain III (italicized), EGFR domain IV (underlined), EGFR transmembrane domain (boldfaced and italicized) and a juxtamembrane domain having the sequence of RRR:
• the present polypeptide comprises, consists of, or consists essentially of a GM-CSF signal peptide (boldfaced), EGFR Domain III (italicized), EGFR
• the present polypeptide comprises, consists of, or consists essentially of a GM-CSF signal peptide (boldfaced), EGFR Domain III (italicized), EGFR
• the present polypeptide comprises, consists of, or consists essentially of a GM-CSF signal peptide (boldfaced), EGFR Domain III (italicized), EGFR
• the present polypeptide comprises, consists of, or consists essentially of a GM-CSF signal peptide (boldfaced), EGFR Domain III (italicized), modified portion of EGFR Domain IV (underlined), EGFR transmembrane domain (boldfaced and italicized), and a juxtamembrane domain having the sequence of RRR:
• the present polypeptide comprises, consists of, or consists essentially of a GM-CSF signal peptide (boldfaced), EGFR Domain III (italicized), modified portion of EGFR Domain IV (underlined), EGFR transmembrane domain (boldfaced and italicized), and a juxtamembrane domain having the sequence of RRRHIVRKR (SEQ ID NO: 16) NO : 34 )
• the present polypeptide comprises, consists of, or consists essentially of a GM-CSF signal peptide (boldfaced), EGFR Domain III (italicized), modified portion of EGFR Domain IV (underlined), EGFR transmembrane domain (boldfaced and italicized), and a juxtamembrane domain having the sequence of RRRSGGGGSGGGGS (SEQ ID NO: 12):
• the present polypeptide comprises, consists of, or consists essentially of a GM-CSF signal peptide (boldfaced), EGFR Domain III (italicized), modified portion of EGFR Domain IV (underlined), EGFR transmembrane domain (boldfaced and italicized), and a juxtamembrane domain having the sequence of SGGGGSGGGGS (SEQ ID NO:13):
• the present polypeptide comprises, consists of, or consists essentially of a GM-CSF signal peptide (boldfaced), EGFR Domain III (italicized), synthetic sequence (underlined), EGFR transmembrane domain (boldfaced and italicized), and a juxtamembrane domain having the sequence of RRR:
• the present polypeptide comprises, consists of, or consists essentially of a GM-CSF signal peptide (boldfaced), EGFR Domain III (italicized), synthetic sequence (underlined), EGFR transmembrane domain (boldfaced and italicized), and a juxtamembrane domain having the sequence of RRRHIVRKR (SEQ ID NO: 16):
• the present polypeptide comprises, consists of, or consists essentially of a GM-CSF signal peptide (boldfaced), EGFR Domain III (italicized), synthetic sequence (underlined), EGFR transmembrane domain (boldfaced and italicized), and a juxtamembrane domain having the sequence of RRRSGGGGSGGGGS (SEQ ID NO: 12):
• the present polypeptide comprises, consists of, or consists essentially of a GM-CSF signal peptide (boldfaced), EGFR Domain III (italicized), synthetic sequence (underlined), EGFR transmembrane domain (boldfaced and italicized), and a juxtamembrane domain having the sequence of SGGGGSGGGGS (SEQ ID NO: 13):
• EGFR-derived polypeptides that are at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical in sequence to the above exemplified sequences.
• the present disclosure provides expression constructs suitable for expressing the EGFR-derived proteins in cells that are used in cell therapy.
• An expression construct of the present disclosure includes an expression cassette comprising a coding sequence for the EGFR- derived polypeptide (preferably including a signal peptide) linked operably to one or more transcriptional regulatory elements.
• transcriptional regulatory elements refer to nucleotide sequences in the expression construct that control expression of the coding sequence, for example, by regulating the tissue-specific expression patterns and transcription efficiency of the EGFR-derived polypeptide coding sequence, the stability of the RNA transcripts, and the translation efficiency of the RNA transcripts. These elements may be one or more of a promoter, a Kozak sequence, an enhancer, an RNA-stabilizing element (e.g., a WPRE sequence), a polyadenylation signal, and any combination thereof.
• the expression cassette contains a mammalian promoter that is constitutively active or inducible in the target cells.
• useful promoters are, without limitation, a Moloney murine leukemia virus (MoMuLV) LTR, an MND (a synthetic promoter containing the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer), a Rous sarcoma virus (RSV) LTR, a cytomegalovirus (CMV) promoter, a CMV immediate early promoter, a simian virus 40 (SV40) promoter, a dihydrofolate reductase (DHFR) promoter, a b-actin promoter, a phosphoglycerate kinase (PGK) promoter, an EFla promoter, a thymidine kinase (TK) promoter, a tetracycline responsive promoter (TRE), an E
• the expression cassette also comprises additional regulatory sequences, for example, an internal ribosome entry site (IRES) or a sequence encoding a self cleaving peptide to allow co-expression of another polypeptide in addition to the EGFR-derived polypeptide.
• IRS internal ribosome entry site
• self-cleaving peptides also known as ribosomal skipping peptides
• 2A peptides which are viral derived peptides with a typical length of 18-22 amino acids and include T2A, P2A, E2A, and F2A (Liu et al., Sci Rep. (2017) 7:2193).
• the present expression construct also expresses an antigen receptor and/or another additional polypeptide.
• the antigen receptor may be, for example, an antibody, an engineered antibody such as an scFv, a CAR, an engineered TCR, a TCR mimic (e.g., an antibody-T cell receptor (abTCR) or a chimeric antibody-T cell receptor (caTCR)), or a chimeric signaling receptor (CSR).
• an abTCR may comprise an engineered TCR in which the antigen-binding domain of a TCR (e.g., an alpha/beta TCR or a gamma/delta TCR) has been replaced by that of an antibody (with or without the antibody’s constant domains); the engineered TCR then becomes specific for the antibody’s antigen while retaining the TCR’s signaling functions.
• a TCR e.g., an alpha/beta TCR or a gamma/delta TCR
• a CSR may comprise (1) an extracellular binding domain (e.g., natural/modified receptor extracellular domain, natural/modified ligand extracellular domain, scFv, nanobody, Fab, DARPin, and affibody), (2) a transmembrane domain, and (3) an intracellular signaling domain (e.g., a domain that activates transcription factors, or recruits and/or activates JAK/STAT, kinases, phosphatases, and ubiquitin; SH3; SH2; and PDZ).
• an extracellular binding domain e.g., natural/modified receptor extracellular domain, natural/modified ligand extracellular domain, scFv, nanobody, Fab, DARPin, and affibody
• an intracellular signaling domain e.g., a domain that activates transcription factors, or recruits and/or activates JAK/STAT, kinases, phosphatases, and ubiquitin
• SH3; SH2; and PDZ
• the antigen receptor may target an antigen of interest (e.g., a tumor antigen or an antigen of a pathogen).
• the antigens may include, without limitation, AFP (alpha-fetoprotein), anb6 or another integrin, BCMA, B7-H3, B7-H6, CA9 (carbonic anhydrase 9), CCL-1 (C-C motif chemokine ligand 1), CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7/8, CD45, CD47, CD56, CD66e, CD70, CD74, CD79a, CD79b, CD98, CD 123, CD 138, CD171, CD352, CEA (carcinoembryonic antigen), Claudin 18.2, Claudin 6, c-MET, DLL3 (delta-like protein 3), DLL4, ENPP3 (ectonucleotide pyrophosphata
• the antigen receptor may be bispecific and target two different antigens, such as two of the antigens listed above.
• the antigen receptor such as a CAR, targets CD 19 and CD20, or CD 19 and CD22.
• the additional polypeptide may be, for example, a cytokine (e.g., IL-2, IL-7, IL-12, IL- 15, IL-23, and engineered variants thereof), a cytokine receptor (e.g., IL-12R, IL-7R, and engineered variants thereof), a chemokine, a transcription factor (e.g., c-Jun or c-fos; see, e.g., WO 2019/118902), functional analogs thereof, other engineered receptors (e.g. TGFBetaR), and other engineered effectors (e.g., secretory secondary effector; see, e.g., WO 2018/200585).
• a cytokine e.g., IL-2, IL-7, IL-12, IL- 15, IL-23, and engineered variants thereof
• a cytokine receptor e.g., IL-12R, IL-7R, and engineered variants
• the coding sequences of these additional polypeptides may be under the control of different promoters from the EGFR-derived polypeptide coding sequence. Alternatively, they may be under the control of the same promoter as the EGFR-derived coding sequence but are separated from each other through an IRES or an in-frame coding sequence for a 2A peptide, such that the coding sequences can be co-expressed under the same promoter.
• the expression constructs of the present disclosure may be delivered to target cells in vitro , ex vivo or in vivo by suitable means such as electroporation, sonoporation, viral transduction, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, and nanoparticles (e.g., polymeric or lipid nanoparticles).
• the expression constructs may be viral vectors and are delivered to the target cells through recombinant viruses containing the constructs.
• the viral vectors contain the EGFR-derived polypeptide expression cassette and minimal viral sequences required for packaging and subsequent integration into a host (if applicable).
• the missing viral functions are supplied in trans by the packaging cell line used to package the recombinant virus.
• the viral vector may be, for example, vaccinia vectors, adenoviral vectors, lentiviral vectors, poxyviral vectors, herpes simplex viral vectors, adeno- associated viral vectors, retroviral vectors, and hybrid viral vectors.
• the EGFR-derived polypeptide expression cassette may be stably integrated into the genome of the target cells, or remain in the cells episomally. Integration into the host genome is possible with retrovirus and lentivirus.
• the present expression constructs may be introduced into cells used in cell therapy.
• These cells are, for example, multipotent cells such as hematopoietic stem cells, various progenitor or precursor cells of hematopoietic lineages, and various immune cells (e.g., human autologous or allogeneic T, natural killer (NK), dendritic, or B cells).
• These cells may also be pluripotent stem cells (PSCs) such as human embryonic stem cells and induced PSCs, which can be used to generate therapeutic cell populations.
• pluripotent and multipotent cells are differentiated into a desired cell type in vitro before being implanted into the patient.
• the present disclosure provides engineered T lymphocytes that express the EGFR-derived protein and from the same construct or from a separate construct, one or more additional polypeptides.
• the one or more additional polypeptides may be an antigen receptor such as an antibody, an engineered antibody such as an scFv, a CAR, an engineered TCR, a TCR mimic (e.g., an abTCR or caTCR), or a CSR, as described above.
• the antigen receptor may target, for example, the antigens described above.
• the additional polypeptide also may be, for example, a cytokine, a cytokine receptor, a chemokine, a transcription factor, a functional analog of the foregoing, another engineered receptor, or an engineered effector as described above.
• the coding sequences of these additional polypeptides may be under the control of different promoters from the EGFR-derived polypeptide coding sequence.
• the present disclosure provides engineered autologous or allogeneic NK cells expressing engineered receptors, and engineered B lymphocytes expressing an antibody, an engineered antibody, or an engineered tissue-specific cell expressing a therapeutic protein.
• the genetically engineered cells described herein may be provided in a pharmaceutical composition containing the cells and a pharmaceutically acceptable carrier.
• the pharmaceutically acceptable carrier may be cell culture medium that optionally does not contain any animal-derived component.
• the cells may be cryopreserved. Prior to use, the cells may be thawed, and diluted in a sterile cell medium.
• the cells may be administered into the patient systemically (e.g., through intravenous injection or infusion), or locally (e.g., through direct injection to a local tissue, e.g., at the site of a solid tumor).
• a therapeutically effective number of engineered cells are administered to the patient.
• the term “therapeutically effective” refers to a number of cells or amount of pharmaceutical composition that is sufficient, when administered to a human subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, prevent, and/or delay the onset or progression of the symptom(s) of the disease, disorder, and/or condition.
• a therapeutically effective amount of engineered CAR-T cells is an amount that is sufficient to cause tumor growth arrest, tumor regression, prevention of tumor metastasis, or prevention of tumor recurrence.
• an anti-EGFR antibody may be administered to the patient at an amount that is sufficient to cause antibody-mediated killing of the cells.
• cetuximab e.g., Erbitux ®
• cetuximab can be administered through infusion at one or more doses determined as appropriate for the number of engineered cells remaining in the patient.
• Example 1 Surface Expression of EGFR-Derived Polypeptides
• This example describes studies analyzing the effects of various juxtamembrane domains on the cell surface expression levels of EGFR-derived polypeptides.
• Lentiviral constructs were generated with bi-cistronic or tri-cistronic expression cassettes.
• the coding sequences for (i) a RORl-specific R12 CAR, (ii) a P2A self-cleaving peptide, and (iii) EGFRt (a truncated EGFR having only Domains III and IV and the transmembrane domain; SEQ ID NO:26) or a variant having additionally an intracellular juxtamembrane domain were linked in frame and placed under the control of an MND promoter.
• the coding sequences for (i) c-Jun, (ii) a P2A peptide, (iii) a ROR1 -specific R12 CAR, (iv) a P2A peptide; and (v) EGFRt or a variant having additionally an intracellular juxtamembrane domain were linked in frame and placed under the control of an MND promoter.
• the R12 CAR was derived from the R12 anti-RORl antibody (Yang et al., PLoS One. (2011) 6:e21018) and contains a CD28-derived transmembrane domain, a 4- IBB costimulatory domain, and a CD3 zeta signaling domain.
• Jurkat cells were obtained from American Type Culture Collection (ATCC; Manassas VA). For lentiviral transduction, the cells were fed with fresh media 4-16 hours before transduction, followed by incubation with lentivirus in complete media + LentiBOOSTTM at the manufacturer’s recommended concentration (Sirion Biotech). Eighteen hours after transduction, lentivirus and LentiBOOSTTM were diluted by addition of 1 volume of fresh media.
• Pre-selected, cryopreserved primary human CD4+ and CD8+ T cells from normal donors were obtained from Bloodworks (Seattle WA).
• Human T cells were cultured in OpTmizer medium (Thermo Fisher) supplemented with Immune Cell Serum Replacement (Thermo Fisher), 2 mM L-glutamine (Gibco), 2 mM Glutamax (Gibco), 200 IU/ml IL-2 (R&D systems), 120 IU/ml IL-7 (R&D systems), and 20 IU/ml IL-15 (R&D systems).
• the T cells were stimulated with a 1 :100 dilution of T cell TransAct (Miltenyi) for 30 hours. Virus was then added to the T cells for 18-24 hours. Stimulation and viral infection were then terminated by addition of 7 volumes of fresh media without TransAct, and cells were cultured for 3-7 additional days before analysis.
• Flow cytometry was performed on a Ze5 cytometer (Bio-Rad Laboratories). To determine expression of cell surface markers, about lxlO 5 to 2xl0 5 total cells were transferred to a V bottom 96 well culture dish (Coming). Cells were washed twice with flow cytometry staining buffer (eBioscience), and then stained with the relevant reagents in a total volume of 50 m ⁇ flow cytometry staining buffer for 30 minutes on ice. After staining, the cells were washed twice with flow cytometry staining buffer, fixed in FluoroFix Buffer (BioLegend), and kept at 4°C in the dark until analysis.
• Flow cytometry data was analyzed using FlowJo 10 (Tree Star).
• AY13 antibody labeled with fluorochrome BV421 BioLegend
• Purified recombinant ROR1 fused to human Ig Fc was produced in-house and conjugated to Alexa 647 dye for detecting R12 CAR.
• eFluor 780 Fixable Viability dye (eBioscience) was included during primary antibody stain at a 1:8000 dilution.
• glycine may disrupt a-helical structure and is enriched in the juxtamembrane region of other receptor tyrosine kinase proteins
• intracellular domains containing unstructured glycine/serine-rich linkers SGGGGSGGGGS; SEQ ID NO: 13
• RRRSGGGGSGGGGS gly cine/ serine-rich linker
• the surface gMFI of the R12 CAR was uniform across all constructs at a matching transduction frequency.
• the surface gMFI of all EGFRt variants containing an intracellular sequence was about three to five-fold higher than that of the EGFRt marker lacking this sequence (FIGs. 5A and 5B).
• the EGFRt modules contained either no intracellular domain or an intracellular domain comprising amino acids 669-671 or 669-677 from the human EGFR sequence.
• R12 CAR expression was similar across all constructs.
• the surface expression of EGFRt increased by about four folds with the addition of a juxtamembrane sequence (FIGs. 6B and 6C).
• Example 2 Cetuximab-Mediated ADCC of CAR-T cells Expressing EGFRt Variants
• This example describes studies analyzing the efficiency of the EGFR-derived proteins described herein as a safety switch in cell therapy. Altered surface expression of EGFRt could impact the utility of this marker as a selection marker and safety switch in vivo.
• Cetuximab induces ADCC of tumor cells in an EGFR-dependent manner (Kimura et al., Cancer Sci. (2007) 98(8): 1275-80).
• NK cells Primary human natural killer (NK) cells were used as effector cells in ADCC assays. Natural killer cells were isolated from cryopreserved, T cell depleted (CD4-/CD8-) PBMC (AllCells) by negative selection using the EasySep Human NK Cell Kit (StemCell) according to manufacturer’s protocol. To activate their cytolytic function, isolated NK cells were cultured in RPMI-10 supplemented with 10 ng/ml human IL-15 overnight before use (Wagner et al., J Clin Invest. (2017) 127(11):4042-58; Derer 2012, J Immunol. (2012) 189(11):5230-9).
• Cryopreserved, transduced primary T cells were thawed and pre-cultured overnight in OpTmizer medium plus cytokines as described above. The cells were then counted, resuspended in RPMI-10, and added to a V bottom 96 well plate in a 100 m ⁇ volume and incubated with (i) a cetuximab biosimilar at the indicated final concentration, (ii) no antibody (0), or (iii) 2,000 ng/ml rituximab biosimilar (R&D Systems) for 15 minutes at 37°C.
• IL-15 primed NK cells were then added at a 10:1 ratio of NK:CAR-T cells and the V bottom plate was gently centrifuged (lOOxg, 30 sec) to bring effector and target cells together. After 4 hours of co-culture, remaining CAR+
• T cells were identified by FACS. Samples were stained with anti-CD3, anti-CD56, RORl-Fc, and FVD780, fixed, and acquired on the Ze5 cytometer under volumetric counting mode. Antibody specific ADCC of T cells was assessed by comparing the total live CD56-CD3+ROR1- Fc+ populations treated or not treated with the antibody.
• ROR1 -specific CAR and (iii) an EGFR-derived polypeptide were designed as described in Example 1.
• FIG. 10 shows gMFI for anti -EGFR antibody binding in ROR1 CAR+ transduced cells or total live cells in the untransduced condition.
• an EGFR-derived polypeptide containing the RKR juxtamembrane sequence maintained strong cell surface expression.
• Comparison of cell surface expression levels of EGFR-derived polypeptides containing juxtamembrane sequences with one, two, or three arginine residues (R, RR, or RRR) shows that truncation of the intracellular juxtamembrane domain from RRR to RR reduced surface expression by 2.7%. In contrast, truncation of the intracellular juxtamembrane domain from RRR to R reduced surface expression by 22%.
• the MP71 retroviral vector was used to generate the constructs used in these studies.
• the vector was modified to incorporate coding sequences for human EGFRt (MP71 -EGFRt), a variant having the juxtamembrane domain RRR (MP71-EGFR-RRR), a bi-cistronic CAR expression cassette encoding the mCD19scFv.28z CAR (also annotated as ml9.28z or mCD19.28z) and EGFRt or EGFR-RRR (MP71-mCD19scFv.28z.EGFRt/EGFR-RRR; also annotated herein as MP71_ml9.28z.P2A.EGFRt/EGFR-RRR), or a tri-cistronic CAR expression cassette encoding c-Jun, mCD19scFv.28z, and EGFRt or EGFR-RRR (MP71- cJun.mCD19scFv.
• the bi-cistronic CAR constructs included a coding sequence for a CAR (mCD19.28z CAR), which included a murine CD8a signal peptide (UniProt P01731 amino acids 1-27), a murine CD19-specific scFv derived from the ID3 hybridoma (Davila et al., PLoS One (2013) 8(4):e61338), murine CD8a hinge and transmembrane regions (UniProt P01731 amino acids 151-219), a murine CD28 intracellular region (UniProt P31041 amino acids 177-218), and a murine CD3z intracellular domain (UniProt P24161 amino acids 52-164).
• mCD19.28z CAR included a murine CD8a signal peptide (UniProt P01731 amino acids 1-27), a murine CD19-specific scFv derived from the ID3 hybridoma (Davila et al
• This CAR-coding sequence was linked by a coding sequence for a P2A self-cleaving peptide sequence to the coding sequence for the human EGFR polypeptide (UniProt P00533 amino acids 334-668 for human EGFRt).
• a coding sequence for murine c-Jun (UniProt P05627 amino acids 1-334) was cloned upstream of the mCD19.28z CAR coding sequence and linked by a T2A peptide coding sequence.
• Plat-E cells (Cell Biolabs) were transiently transfected using calcium phosphate (Takara). Supernatants were collected 48 hours later, filtered through 0.45 ⁇ m filters, and snap frozen on dry ice prior to storage at -80°C. C57BL/6J and B6.SJL (CD45.1) donor mice were acquired from Jackson Laboratory.
• Murine CD8 T cells were enriched using negative selection (StemCell) and stimulated with 1 pg/ml plate-bound anti-CD3 (145-2C11) and anti-CD28 (37.51) for 20 hours at 37°C and 5% CO2 in complete RPMI (RPMI 1640, 10% heat-inactivated FBS, 1 mM HEPES, 100 U/ml penicillin/streptomycin, 1 mM sodium pyruvate, and 50 pM b-mercaptoethanol) supplemented with 50 U/ml murine IL-2 (PeproTech).
• RPMI 1640 10% heat-inactivated FBS
• 1 mM HEPES 100 U/ml penicillin/streptomycin, 1 mM sodium pyruvate
• 50 pM b-mercaptoethanol supplemented with 50 U/ml murine IL-2 (PeproTech).
• RetroNectin® (Takara) and captured by centrifugation for 2 hours at 2560 ref at 32°C.
• Stimulated CD8 + T cells were harvested and resuspended at lxlO 6 cells/ml in complete RPMI supplemented with 50 U/ml JL-2 and anti-CD3/28 mouse T- activator DynabeadsTM (Thermo Fisher) at a 1:1 ratio.
• Virus-coated wells were aspirated and rinsed with PBS, followed by addition of the T cells, centrifugation at 800 ref for 30 min at 32°C, and incubation at 37°C in 5% CO2.
• T cells were incubated for an additional 24 hours. T cells were harvested, resuspended at lxlO 6 cells/ml in complete RPMI supplemented with 50 U/ml murine IL-15 (PeproTech), and incubated for an additional 48 hours. Magnetic activator beads were subsequently removed and T cell transduction efficiency (40-60% EGFR + ) was confirmed by flow cytometry. Transduced cells were then prepared for adoptive transfer by resuspending CD8 T cells at 3xl0 6 EGFR + /100 ⁇ l in serum-free RPMI 1640 and kept on ice prior to adoptive transfer.
• CAR-T cell adoptive transfer 6- to 8-week old C57BL/6J mice were pre conditioned with intraperitoneal injection of 200 mg/kg cyclophosphamide and were injected intravenously by retro-orbital injection with 3xl0 6 EGFR + CAR-T cells after 6 hours.
• 100 ⁇ l blood samples were collected by retro-orbital bleeding into EDTA-coated tubes on the indicated days post CAR-T cell transfer and the blood samples were treated with two rounds of ACK lysis buffer prior to surface staining. Samples were stained using LIVE/DEADTM Fixable Aqua Dead Cell stain kit (Invitrogen) at 4°C for 15 minutes.
• Cells were also stained in the dark at 4°C for 30 minutes in flow buffer (PBS, 1 mM EDTA, and 2% FBS) with anti-CD8a FITC (53-6.7, BioLegend unless stated otherwise), anti-CD19 PerCP-CyTM 5.5 (1D3), anti-CD4 PE-CyTM 7 (RM4-5), anti-CD45.2 APC/FireTM 750 (104), anti-CD45.1 Brilliant Violet 421TM (A20), hEGFR APC or PE (AY13), and acquired on BD FACSCelestaTM cell analyzer.
• flow buffer PBS, 1 mM EDTA, and 2% FBS
• anti-CD8a FITC 53-6.7, BioLegend unless stated otherwise
• anti-CD19 PerCP-CyTM 5.5 (1D3) anti-CD4 PE-CyTM 7 (RM4-5), anti-CD45.2 APC/FireTM 750 (104), anti-CD45.1 Brilliant Violet 421TM (A20), hEGFR A
• cetuximab was infused at 1 mg or 0.1 mg per mouse on day 8. Expansion and depletion of CAR-T cells were monitored in blood samples by flow cytometry. The mice were shown to exhibit B-cell aplasia when the frequency of CD19 + B cells was maintained below 3% of the total circulating endogenous CD45.2 + cells. Results
• EGFR-RRR Exhibits Superior Surface Expression Levels in the Infusion Product
• T cells were transduced at similar levels (50-56%), EGFR + T cells exhibited >3-fold increase in EGFR-RRR surface expression compared to EGFRt (FIG. 11 A).
• a coding sequence for an EGFR polypeptide was linked by a coding sequence for a P2A self-cleaving peptide to the 3’ end of a coding sequence for a CAR targeting mouse CD 19.
• CAR-T cells transduced with the EGFR-RRR construct displayed high levels of EGFR staining than CAR-T cells transduced with the EGFRt construct (FIG. 11B).
• EGFR-RRR exhibited increased surface expression levels on the CAR T cell infusion product.
• EGFR-RRR Is a Stable Target for Antibody-Mediated Depletion in vivo
• Stably expressed EGFRt can be targeted for depletion with the EGFR-targeting antibody cetuximab (Paszkiewicz et al., J Clin Invest. (2016) 126(11):4262-72).
• cetuximab the EGFR-targeting antibody cetuximab
• EGFRt or EGFR-RRR were expressed in a bi-cistronic (downstream of mCD19 CAR) or tri- cistronic construct (downstream of cJun.mCD19 CAR) on congenically marked CD45.1 + donor CD8 + T cells.
• FIG. 12A The data show that EGFR-RRR exhibited higher levels of surface expression in the infusion product (FIG. 12A, bottom).
• Bulk T cell infusions containing 3xl0 6 EGFR + CAR- T cells were adoptively transferred into lymphodepleted mice. Circulating CAR-T cell levels were tracked in the blood as indicated (FIG. 12A, top).
• EGFR + CAR-T cells underwent an expansion peak one to two weeks post infusion and declined thereafter.
• EGFR-RRR Mediates More Rapid Rebound of B Cells Following T Cell Depletion by Cetuximab
• EGFR-RRR Mediates More Rapid Rebound of B Cells Following T Cell Depletion by Cetuximab
• congenically marked mCD19.28z CAR-T cells expressing EGFRt or EGFR-RRR were adoptively transferred into lymphodepleted mice. T cell engraftment and B cell aplasia were tracked in the blood over time (FIG. 13A).
• mice that received mCD19.28z CAR T cells with or without subsequent depletion.
• mice treated with mCD19.28z CAR T cells exhibited sustained B cell aplasia
• B cells in cetuximab-treated mice rebounded within 3 weeks of antibody administration.
• Cetuximab administration in mice previously infused with EGFR-RRR CAR-T cells resulted in more rapid rebound of B cells, including at the lower administered dose (FIG. 13B), and a more rapid resolution of B cell aplasia (FIG. 13C). Therefore, EGFR-RRR mediates more efficient depletion of CAR-T cells, resulting in more rapid shutdown kinetics of the CAR-T cell response following cetuximab.
• mCD19.28z.EGFR-RRR CAR-T cells exhibit higher expression levels of EGFR. This expression was maintained in vivo and was efficiently targeted for depletion with cetuximab. In contrast to EGFRt, targeting EGFR-RRR with cetuximab resulted in complete depletion of mCD19.28z.CAR T cells and more rapid rebound of B cells in vivo.

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