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  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2809—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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Definitions

• the IL-13Ra2-binding domain includes an anti-IL-13Ra2 scFv or a ligand of IL-13Ra2.
• the ligand of IL-13Ra2 includes IL-13 or IL-13 zetakine, or an antigen-binding fragment thereof.
• the IL-13Ra2-binding domain includes the amino acid sequence of SEQ ID NO: 101 , or includes an amino acid sequence having at least 90% sequence identity (e.g., 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO: 101 .
• the invention features, in general, a polynucleotide encoding the CAR polypeptide and the BiTE of any one of the preceding aspects.
• the polynucleotide includes a CAR polypeptide encoding sequence and a BiTE encoding sequence, and wherein the CAR polypeptide encoding sequence and the BiTE encoding sequence are separated by a ribosome skipping moiety.
• the CAR polypeptide and/or the BiTE is expressed under a constitutive promoter, e.g., an elongation factor-1 alpha (EF1 a) promoter.
• EF1 a elongation factor-1 alpha
• the CAR polypeptide and/or the BiTE is expressed under an inducible promoter, e.g., wherein the inducible promoter is inducible by T cell receptor (TCR) or CAR signaling, e.g., a nuclear factor of activated T cells (NFAT) response element.
• TCR T cell receptor
• CAR signaling e.g., a nuclear factor of activated T cells (NFAT) response element.
• the CAR polypeptide and the BiTE are each expressed under a constitutive promoter.
• the CAR polypeptide is expressed under a constitutive promoter and the BiTE is expressed under an inducible promoter.
• the polynucleotide further includes a suicide gene.
• the polynucleotide includes a sequence encoding one or more signal sequences.
• the transmembrane domain includes a hinge/transmembrane domain, e.g., the hinge/transmembrane domain of an immunoglobulin-like protein (e.g., IgA, IgD, IgE, IgG, or IgM), CD28, CD8, or 4-1 BB.
• transmembrane domain of the CAR includes a CD8 hinge/transmembrane domain, which optionally includes the sequence of any one of SEQ ID NOs: 4, 10,
• the invention features a method of delivering a therapeutic agent to a tissue or organ in a patient to treat a disease or pathology, the method including administering to said patient a CAR T cell, genetically modified to secrete a therapeutic antibody, toxin, or agent, wherein the therapeutic antibody, toxin, or agent would, by itself, be unable to enter or penetrate the tissue or organ.
• a co-stimulatory ligand can include, but is not limited to, 4-1 BBL, 0X40 L, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1 , PD-L2, inducible COStimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll-like receptor and a ligand that specifically binds with B7-H3.
• 4-1 BBL 0X40 L
• Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites permitting ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide- directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations are well established and include, for example, those disclosed by Walder et al.
• viral vector refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle.
• the viral vector can contain a nucleic acid encoding a polypeptide as described herein in place of non-essential viral genes.
• the vector and/or particle may be utilized for the purpose of transferring nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.
• statically significant or“significantly” refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.
• Figs. 12A and 12B are flow cytometry histograms showing the expression of GARP (Fig. 12A) and LAP (Fig. 12B) by HUT78 cells.
• Figs. 16A-16C are photographs of western blots showing the presence of protein components of supernatants obtained from cultures of CART-EGFR-GARP T cells.
• Fig. 16A and 16B show the full gel, including molecular weight reference ladders.
• Fig. 16C is a longer exposure of the bottom region of the gel shown in Fig. 16B, in which a band between 10 and 15 kD is identified with an arrow, indicating the presence of a camelid antibody.
• Fig. 1 7 is a schematic drawing of CAR-EGFR-BiTE-(EGFR-CD3), an exemplary nucleic acid molecule encoding a CAR and a BiTE.
• Fig. 27A is a schematic drawing of BiTE-(CD19-CD3)-CAR-EGFR, an exemplary nucleic acid molecule encoding a CAR and an inducibly expressed BiTE.
• Fig. 33D shows a schematic representation of BiTE-EGFR and BiTE-CD19.
• Fig. 33H shows BiTE concentration in supernatant increases over time.
• Untransduced T cells UTD
• Fig. 34B shows Jurkat reporter T cells either untransduced (UTD) or transduced with CART- EGFRvlll. BiTE-CD19 or CART-EGFRvlll. BiTE-EGFR and co-cultured with U87 or U251 glioma cell lines for 18 hours at an E:T of 1 :1 . Activation is reflected by relative luminescence.
• Fig. 41 B shows a schematic representation of panels shown in Fig. 41 A; CART-EGFR (top), CART-EGFRvlll.BiTE-CD19 (middle), and CART-EGFRvlll.BiTE-EGFR (bottom).
• Fig. 41 C shows CD25 and CD69 expression on CAR T cells and CART.BiTE cells (mCherry- positive) as well as bystander T cells (mCherry-negative) after coculture with EGFR-expressing tumor, U87.
• Fig. 43A shows a schematic representation of experimental design in which a heterogeneous population (10% EGFRvlll-positive, 90% wild-type) of U87 glioma cells (5 x 10 3 ) was implanted orthotopically into the brains of NSG mice. Both U87 and U87vl II cells were modified with CBG-luc so that total intracranial tumor burden could be visualized by bioluminescent imaging. Mice were treated intraventricularly on day 2 post-implantation with untransduced T cells (UTD), CART-EGFRvlll.BiTE- CD19 cells, or CART-EGFRvlll.BiTE-EGFR cells.
• UTD untransduced T cells
• CART-EGFRvlll.BiTE- CD19 cells CART-EGFRvlll.BiTE-EGFR cells
• 44C shows a schematic diagram of an exemplary tandem anti-IL- 13Ra2/anti-EGFRvlll CAR construct, which includes an EF1 a promoter, an IL-13 ligand (IL-13 zetakine), an anti-EGFRvlll scFv, a CD8 transmembrane domain, a 4-1 BB co-stimulatory domain, a ⁇ 3z domain, a T2A peptide sequence, and a reporter gene (mCherry).
• Fig. 45A is a series of graphs showing the results of flow cytometry analysis to assess expression of IL-13Ra2 in U87 human glioblastoma cells and U87 cells transduced to express EGFRvlll (U87vlll).
• Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.
• the antibody reagent is an anti-EGFR or anti-EGFRvlll antibody reagent and includes the sequence of SEQ ID NO: 21 , 27, 33, 36, 42, 45, 55, 57, 65, or 103, or includes a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to the sequence of SEQ ID NO: 21 , 27, 33, 36, 42, 45, 55, 57, 65, or 103.
• the anti-EGFRvlll scFv corresponds to the sequence of SEQ ID NO: 27, 36, 45, 57, 65, or 1 03; includes the sequence of SEQ ID NO: 27, 36, 45, 57, 65, or 1 03, or includes a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to the sequence of SEQ ID NO: 27, 36, 45, 57, 65, or 103.
• An immune cell including a CAR polypeptide including an extracellular target binding domain including an anti-EGFRvlll scFv may secrete an anti-EGFR BiTE as described below.
• the anti-EGFRvlll scFv includes a VL corresponding to the amino acid sequence of SEQ ID NO: 1 12 or 1 14, including the amino acid sequence of SEQ ID NO: 1 12 or 1 14, or including an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO: 1 12 or 1 14.
• the VH may be positioned N-terminal to the VL, or the VL may be positioned N-terminal to the VH.
• any cell-surface moiety can be targeted by a CAR.
• the target will be a cell-surface polypeptide that may be differentially or preferentially expressed on a cell that one wishes to target for a T cell response.
• antibody reagents can be targeted against, e.g., Glycoprotein A
• GITR GITR, 0X40, 4-1 BB, and LAG-3.
• antibody domains can be targeted against, e.g., EGFR or EGFRvlll, as described herein.
• Targeting tumor antigens or tumor-associated antigens that are specific to the tumors can provide a means to target tumor cells while avoiding or at least limiting collateral damage to non-tumor cells or tissues.
• additional tumor antigens, tumor-associated antigens, or other antigen of interest include CD19, CD37, BCMA (tumor necrosis factor receptor superfamily member 17 (TNFRSF17); NCBI Gene ID: 608; NCBI Ref Seq:
• CD8 is an antigen preferentially found on the cell surface of cytotoxic T lymphocytes. CD8 mediates cell-cell interactions within the immune system, and acts as a T cell co-receptor.
• CD8 consists of an alpha (CD8a or CD8a) and beta (CD8p or CD8b) chain.
• CD8a sequences are known for a number of species, e.g., human CD8a, (NCBI Gene ID: 925) polypeptide (e.g., NCBI Ref Seq NP_001 139345.1 ) and mRNA (e.g., NCBI Ref Seq NM_ 000002.12).
• CD8 can refer to human CD8, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any of the aspects, e.g., in veterinary applications, CD8 can refer to the CD8 of, e.g., dog, cat, cow, horse, pig, and the like.
• Homologs and/or orthologs of human CD8 are readily identified for such species by one of skill in the art, e.g., using the NCBI ortholog search function or searching available sequence data for a given species for sequence similar to a reference CD8 sequence.
• immunoreceptor tyrosine-based activation motif (ITAM)- containing intracellular signaling domains include those derived from K z, FcRy, FcRp, CD3y, CD30, CD36, CD3r
• Such an inducible promoter can be used, e.g., to ensure that the antibody is expressed only upon T cell activation, and thus only, e.g., when the CAR T cell is within the tumor microenvironment, to which locale it may be advantageous to have antibody production limited.
• the CAR coding sequences can be 5’ or 3’ to the therapeutic agent (e.g., an antibody reagent or cytokine) coding sequences in various vector designs within the invention.
• the antibody reagent is an anti-CD19 antibody reagent and includes the sequence of SEQ ID NO: 51 or 63, or includes a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to the sequence of SEQ ID NO: 51 or 63.
• the antibody reagent is an anti-CD3 antibody reagent and includes the sequence of SEQ ID NO: 34, 43, 52, 56, or 64, or includes a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to the sequence of SEQ ID NO: 34, 43, 52, 56, or 64.
• the antibody reagent can include C225, 3C10, Cetuximab, or 2173, or an antigen-binding fragment thereof.
• Antibody reagents can also be targeted against any other antigens described herein or known in the art.
• the CAR T cells of the invention can be used to deliver other therapeutic agents including, but not limited to, cytokines and toxins.
• the CAR T cell secreted BiTEs may, e.g., stimulate the CAR T cell itself, or operate in a paracrine fashion by redirecting nonspecific bystander T cells against tumors and therefore enhance the anti-tumor effects of CAR T cell immunotherapy.
• CAR T cell-mediated BiTE secretion may allow for the reduction of risk of undesired BiTE activity in systemic tissues by directing BiTE secretion to the tumor microenvironment.
• Exemplary BiTE constructs are provided below; however, BiTEs other than those described herein may also be useful for the invention.
• BiTE is an anti-CD19 BiTE including an anti-CD19 scFv and an anti-CD3 scFv (also referred to herein as BiTE-CD19).
• the anti-CD19 scFv may be arranged in the VH-VL orientation, or in the VL-VH orientation.
• One aspect of the technology described herein relates to a mammalian cell including any of the CAR polypeptides described herein (optionally together with another therapeutic agent (e.g., an antibody reagent (e.g., a scFv, a camelid antibody, or a BiTE) or a cytokine)); or a nucleic acid encoding any of the CAR polypeptides described herein (optionally together with another therapeutic agent (e.g., an antibody reagent (e.g., a scFv, a camelid antibody, or a cytokine)).
• another therapeutic agent e.g., an antibody reagent (e.g., a scFv, a camelid antibody, or a cytokine)
• another therapeutic agent e.g., an antibody reagent (e.g., a scFv, a camelid antibody, or a cytokine)
• another therapeutic agent e.g.,
• CART.BiTEs described herein e.g., a CART-EGFRvlll.BiTE-EGFR, can be used to treat a glioblastoma having reduced EGFRvlll expression.
• Non-limiting examples of lymphoma include diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphomas, Burkitt’s lymphoma, hairy cell leukemia (HCL), and T cell lymphoma (e.g., peripheral T cell lymphoma (PTCL), including cutaneous T cell lymphoma (CTCL) and anaplastic large cell lymphoma (ALCL)).
• the cancer is DLBCL or follicular lymphoma.
• a mammalian cell e.g., a T cell
• a mammalian cell can be engineered to include any of the CAR polypeptides described herein (including CAR polypeptides that are cleavably linked to antibody reagents or cytokines, as described herein); or a nucleic acid encoding any of the CAR polypeptides (and optionally also a genetically encoded antibody reagent or cytokine) described herein.
• T cells can be obtained from a subject using standard techniques known in the field. For example, T cells can be isolated from peripheral blood taken from a donor or patient. T cells can be isolated from a mammal. Preferably, T cells are isolated from a human.
• the CAR polypeptide (and optionally the antibody reagent or cytokine) of any of the CARs described herein is expressed in a mammalian cell via transfection or electroporation of an expression vector including a nucleic acid encoding the CAR.
• a“condition” refers to a cancer, a plasma cell disease or disorder, or an autoimmune disease or disorder.
• Subjects having a condition can be identified by a physician using current methods of diagnosing the condition. Symptoms and/or complications of the condition, which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to, fatigue, persistent infections, and persistent bleeding. Tests that may aid in a diagnosis of, e.g., the condition, but are not limited to, blood screening and bone marrow testing, and are known in the art for a given condition. A family history for a condition, or exposure to risk factors for a condition can also aid in determining if a subject is likely to have the condition or in making a diagnosis of the condition.
• the activated CAR T cells described herein are administered as a monotherapy, i.e., another treatment for the condition is not concurrently administered to the subject.
• the administered amount or dosage of the activated CAR T cells, the additional agent (e.g., second or third agent), or all is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually.
• the amount or dosage of the activated CAR T cells, the additional agent (e.g., second or third agent), or all, that results in a desired effect (e.g., treatment of cancer) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amount or dosage of each agent individually required to achieve the same therapeutic effect.
• the activated CAR T cells described herein can be used in a treatment regimen in combination with surgery, chemotherapy, radiation, an mTOR pathway inhibitor,
• the activated CAR T cells described herein can be used in combination with a checkpoint inhibitor.
• checkpoint inhibitors include anti-PD-1 inhibitors (Nivolumab, MK-3475, Pembrolizumab, Pidilizumab, AMP-224, AMP-514), anti-CTLA4 inhibitors (Ipilimumab and
• WO 03/064383 everolimus (Afinitor® or RADOOI); rapamycin (AY22989, Sirolimus®); simapimod (CAS 164301 -51 -3); emsirolimus, (5- ⁇ 2,4- Bis[(35,)-3-methylmorpholin-4-yl]pyrido[2,3-(i]pyrimidin-7-yl ⁇ -2- methoxyphenyl)methanol (AZD8055); 2- Amino-8-[iraw5,-4-(2-hydroxyethoxy)cyclohexyl]-6- (6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3- JJpyrimidin-7(8H)-one (PF04691 502, CAS 1013101 -36-4); and N2-[l,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-l- benzopyran-2- yl)morpholinium-4-yl]me
• EGFRvlll CAR T cells mediate antitumor activity in vitro.
• EGFRvlll CAR was designed and synthesized, which was used with initial tests in vitro. In vitro characterization of this CAR demonstrates that the EGFRvlll CAR mediates significant and specific cytotoxicity against the human glioma U87vlll cell line (Fig. 1 ; EGFRvlll CAR transduced T cells potently and specifically mediate cytotoxicity against the U87vl II human glioma cell line). This effect was observed in a subcutaneous models of human GBM xenograft, where even established, bulky tumors responded to CART-EGFRvlll (Figs.
• Treg-targeting constructs include two LAP- targeting CARs (CAR-LAP-L-H (Fig. 8A) and CAR-LAP-H-L (Fig. 8B); in which each anti-LAP scFv contains a reversal in heavy (H) and light (L) chain arrangement), a GARP-targeting CAR construct (CAR- GARP; Fig. 8C), and an EGFR-targeting CAR construct further encoding an anti-GARP camelid antibody (CAR-EGFR-GARP; Fig. 8D). Transduction efficiencies of each construct were assessed using flow cytometry by measuring the percentage of mCherry-positive cells and are provided below.
• CART-LAP-H-L was more effective at killing non-activated Tregs in comparison to CART-LAP-L-H.
• LAP-targeted CAR T cells were then compared to GARP-targeted CART cells in an analogous Treg killing assay across two different donors at a CAR T cell-to-Treg ratio of 1 :1 for four days (Figs. 10A and 10B).
• Figs. 1 1 A and 1 1 B characterize non-activated and activated Treg killing by LAP-targeted CAR T cells, relative to untransduced controls, by the number of target Tregs remaining at the end of a three-day coculture as a function of CAR T cell-to-Treg cell ratio.
• Figs. 1 1 C and 1 1 D show analogous data from the same donor, in which cytotoxicity is measured by luciferase expression.
• the BiTE is flanked by cleavable linkers P2A and T2A to enable separate secretion of the BiTE, while the CAR is targeted to the cell surface.
• Other exemplary BiTE-encoding CAR constructs e.g., encoding a BiTE targeting CD19 are depicted in Figs. 26A and 26B.
• BiTE secretion by CART-EGFR-BiTE-(EGFR-CD3) cells was confirmed by isolating supernatant from cultures containing SupT1 cells transduced with CAR-EGFR-BiTE-(EGFR-CD3), calculating the concentration of BiTE in the supernatant based on OD450, and performing western blot analysis.
• the concentration of BiTE in the supernatant was 0.604 ng/mL.
• Results of a western blot experiment are shown in Fig. 19. A band in lane two at about 50-60 kD was observed, indicating the presence of BiTE molecules in the supernatant.
• Human T cells were purified from anonymous human healthy donor leukapheresis product (Stem Cell Technologies) purchased from the MGH blood bank under an IND-exempt protocol. Cells were transduced with lentivirus corresponding to various second-generation CAR T-cell constructs. In brief, bulk human T cells were activated on day 0 using CD3/CD28 Dynabeads (Life Technologies) and cultured in RPMI 1640 medium with GlutaMAX and HEPES supplemented with 10% FBS and 20 lU/mL of recombinant human IL-2. Lentiviral transduction of cells was performed on day 1 and unless otherwise indicated, cells were permitted to expand until day 10 and subsequently transferred to storage in liquid nitrogen prior to functional assays.
• mice treated with intravenous (IV) untransduced (UTD) T cells demonstrated outgrowth of EGFRvlll-positive tumor
• those treated with CART-EGFRvlll cells showed varying degrees of tumor growth, reflected by abrogated bioluminescent signal in some mice (Fig. 32B). Nevertheless, palpable, measurable tumors progressed in these mice (Fig. 32C).
• BiTE-EGFR has been tested for toxicity in Cynomolgus monkeys and was safe at dose equivalents of approximately 800 pg/d for a 70 kg patient (Lutterbuese et al., Proc. Natl. Acad. Sci. U S A 107:12065-12610, 2010); this is calculated to be 5 orders of magnitude greater than the projected BiTE secretion that would result from a systemic infusion of CART-EGFRvlll. BiTE-EGFR cells in humans.
• CAR-transduced T cells can be engineered to both translate and secrete BiTEs
• the functional capacity of the CART.BiTE cells in mediating antitumor immune responses was next determined.
• CART-EGFRvlll.BiTE-EGFR cells were found to mediate rapid reduction in target cell viability against multiple glioma cell lines and at varied effector-to-target (E:T) ratios (Fig. 36A). When displayed as percent cytotoxicity at several time points, CART. BiTE cells were significantly more efficacious against GBM cells even when compared to positive control CART-EGFR (Fig. 36B).
• BT74 reliably demonstrated heterogeneous expression of both EGFR and EGFRvlll (Fig. 37A). Highlighting this heterogeneity, Jurkat reporter T cells transduced with CART-EGFRvlll.BiTE-CD19 were activated in the presence of BT74— albeit to a significantly lesser degree than those transduced with CART- EGFRvlll.BiTE-EGFR— consistent with CAR-mediated recognition of EGFRvlll-expressing cells in culture (Fig. 37B).
• mice treated with CART-EGFRvlll. BiTE-CD19 cells also eventually demonstrated treatment effect, this occurred late in the course of the experiment and, in retrospect, was consistent with reports that BT74 may have the ability to upregulate EGFRvlll when passaged in vivo over time (Pandita et al., Genes Chromosomes Cancer 39:29-36, 2004).
• BiTE-EGFR was dependent on CAR recognition of its cognate antigen, EGFRvlll, or if secreted BiTEs in the absence of CAR engagement were sufficient to detect measurable antitumor responses in vivo.
• human glioma cells U251
• U251 is considered one of the most stringent glioma models in which to test efficacy, given its lack of EGFRvlll expression and relatively decreased surface expression of EGFR (Fig. 36C), as well as greater resistance to cell death from CART.BiTE cells in vitro (Figs. 36A and 36B). Furthermore, compared to other cell lines, U251 has specifically been cited for its ability to most closely reflect the salient pathobiological features of human GBM when implanted in mice (Radaelli et al. Histol. Histopathol. 24:879-891 , 2009).
• mice treated with cells expressing CART- EGFRvlll and BiTE-CD19 demonstrated progressive tumor burden that was comparable to those receiving UTD control.
• BiTE-EGFR was necessary and sufficient to mediate antitumor efficacy against GBM, even in the absence of EGFRvlll, one potential concern could be that CART.BiTE might also result in significant on-target, off-tumor toxicity in normal human tissues that express wild-type EGFR.
• TILs tumor-infiltrating lymphocytes
• confocal microscopy was first used to visualize the distribution of EGFR-specific BiTEs on both CAR T cells and untransduced T cells in culture. As before, transduced cells were identified by expression of the mCherry fluorescent protein (Fig. 33A). In addition, biotinylated EGFR was used for sensitive detection of the anti- EGFR scFv, which in this case could be expressed either as a transmembrane protein in the form of a CAR, or as the unbound arm of a BiTE opposite to its binding site for CD3.
• T cells exist in various states of differentiation, each with unique functional capabilities.
• BiTEs have been shown to selectively promote expansion of well-differentiated effector memory cells (TEM) (Bargou et al. , Science 321 :974-977, 2008); however, superior outcomes for CARTs have been achieved using less differentiated stem cell memory (TSCM) or central memory (TCM) subtypes (Sadelain et al., Nature 545:423-431 , 2017).
• TSCM differentiated stem cell memory
• TCM central memory
• These less differentiated phenotypes are associated with enhanced expansion and persistence, the capacity for self-renewal, and the ability to generate shorter- lived TEM.
• each CAR T cell population induced cytotoxicity of the target cell population (a 1 :1 ratio of U87 and U87vlll glioblastoma cells), with the tandem anti-IL-13Ra2/anti-EGFRvlll CAR T cells showing the highest efficacy in specific lysis compared to the CAR T cells targeting the single antigens at the effectontarget (E:T) ratios of 10:1 and 3:1 .
• E:T effectontarget
• tandem CARs targeting two or more distinct antigens, e.g., EGFRvlll and IL-13Ra2, which can be engineered to secrete bispecific antibodies (e.g., BiTEs) targeting an additional antigen, e.g., EGFR or HER2.
• BiTEs bispecific antibodies
• This technique can be extended to other tandem CARs or BiTEs targeting other surface tumor antigens, e.g., EGFR, EGFRvlll, CD19, CD79b, CD37, PSMA,
• a tandem CAR can be designed to target EGFR and EGFRvlll, PSMA and PSCA; CD1 9 and CD79b; CD79b and CD37; CD19 and CD37; EphA1 and Her2; EphA1 and mesothelin; Her2 and mesothelin, MUC1 and MUC16; as well as other combinations of the aforementioned tumor antigens.
• CD8 signal sequence (amino acids 1 -21 of SEQ ID NO: 1 )
• KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 5) O ⁇ 3z (amino acids 240-351 of SEQ ID NO: 1 )
• MALPVTALLLPLALLLHAARP (SEQ ID NO: 8)
• P2A amino acids 470-491 of SEQ ID NO: 44
• GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 49)
• IgK signal sequence (amino acids 494-514 of SEQ ID NO: 44)
• IgK signal sequence amino acids 1 -21 of SEQ ID NO: 53
• Anti-CD3 scFv amino acids 268-510 of SEQ ID NO: 53
• RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 60) Construct 9 - (NFAT response element) - IgK signal sequence - CD19 scFv - CD3 scFv - His-tag - (EF1a Promoter) - 2173 (anti-EGFRvlll) scFv - CD8 Hinge/TM - 4-1 BB ICD - O ⁇ 3z (SEQ ID NO: 61 ) including (NFAT response element), IgK signal sequence (amino acids 1 -21 of SEQ ID NO: 61 ; SEQ ID NO: 62), CD19 scFv (amino acids 22-271 of SEQ ID NO: 61 ; SEQ ID NO: 63), CD3 sc
• Anti-CD19 scFv (amino acids 22-271 of SEQ ID NO: 61 )
• SEQ ID NO: 69 including CD8 signal sequence (amino acids 1 -21 of SEQ ID NO: 69; SEQ ID NO: 70), anti-GARP scFv (H-L) (amino acids 22-274 of SEQ ID NO: 69; SEQ ID NO: 71 ), CD8 hinge/TM (amino acids 275-343 of SEQ ID NO: 69; SEQ ID NO: 72), 4-1 BB ICD (amino acids 344-385 of SEQ ID NO: 69; SEQ ID NO: 73), O ⁇ 3z (amino acids 386-497 of SEQ ID NO: 69; SEQ ID NO: 74).
• CD8 signal sequence amino acids 1 -21 of SEQ ID NO: 69; SEQ ID NO: 70
• anti-GARP scFv H-L
• CD8 hinge/TM amino acids 275-343 of SEQ ID NO: 69; SEQ ID NO: 72
• 4-1 BB ICD amino acids 344-385 of SEQ ID NO
• Anti-GARP scFv H-L (amino acids 22-274 of SEQ ID NO: 69)
• CD8 signal sequence (amino acids 1 -21 of SEQ ID NO: 75)
• CD8 hinge/TM amino acids 275-343 of SEQ ID NO: 75
• Construct 12 (Tandem CAR) - IL-13 zetakine - linker - EGFRvlll scFv - CD8 hinge/TM - 4-1 BB ICD - O ⁇ 3z (SEQ ID NO: 100) including IL-13 zetakine (SEQ ID NO: 101 (amino acids 1 -1 12 of SEQ ID NO: 100)); linker (SEQ ID NO: 1 02 (amino acids 1 13-132 of SEQ ID NO: 1 00)); EGFRvlll scFv (SEQ ID NO:
• SEQ ID NO: 103 amino acids 133-378 of SEQ ID NO: 100
• CD8 hinge/TM amino acids 379-447 of SEQ ID NO: 100
• 4-1 BB ICD amino acids 448-489 of SEQ ID NO: 100
• O ⁇ 3z amino acids 490-601 of SEQ ID NO: 100
• CD8 hinge/TM amino acids 379-447 of SEQ ID NO: 100
• a chimeric antigen receptor (CAR) polypeptide comprising an extracellular domain comprising a first antigen-binding domain that binds to a first antigen and a second antigen-binding domain that binds to a second antigen;
• BiTE bispecific T cell engager
• the first and second antigens are independently selected from epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvlll), CD19, CD79b, CD37, prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), interleukin-13 receptor alpha 2 (IL-13Ra2), ephrin type-A receptor 1 (EphA1 ), human epidermal growth factor receptor 2 (HER2), mesothelin, mucin 1 , cell surface associated (MUC1 ), or mucin 16, cell surface associated (MUC16).
• EGFR epidermal growth factor receptor
• EGFRvlll epidermal growth factor receptor variant III
• CD19 CD79b
• CD37 prostate-specific membrane antigen
• PSCA prostate stem cell antigen
• IL-13Ra2 interleukin-13 receptor alpha 2
• EphA1 ephrin type-A receptor 1
• HER2 human epidermal growth factor receptor 2
• the antigen-binding fragment of the antibody comprises a single domain antibody or a single chain variable fragment (scFv).
• linker comprises an amino acid having at least 90% sequence identity to the linker of SEQ ID NO: 102, 107, 108, 109, or 1 10.
• transmembrane domain comprises a hinge/transmembrane domain.
• hinge/transmembrane domain comprises the hinge/transmembrane domain of an immunoglobulin-like protein, CD28, CD8, or 4-1 BB.
• the intracellular signaling domain comprises the intracellular signaling domain of TCF3 ⁇ 4 FcRy, FcRp, CD3y, CD30, CD3e, CD3q, O ⁇ 3z, CD22, CD79a, CD79b, or CD66d.
• intracellular signaling domain comprises the intracellular signaling domain of O ⁇ 3z, optionally comprising the amino acid sequence of SEQ ID NO:
• co-stimulatory domain comprises the co-stimulatory domain of 4-1 BB, CD27, CD28, or OX-40.
• the co-stimulatory domain comprises the co stimulatory domain of 4-1 BB, optionally comprising the amino acid sequence of SEQ ID NO: 5, 1 1 , 17, 23, 29, 38, 47, 59, 67, 73, 79, or 105, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 5, 1 1 , 17, 23, 29, 38, 47, 59, 67, 73, 79, or 105.

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