Modified monocytes / macrophages expressing chimeric antigen receptors and uses thereof

Abstract

The invention relates to a modified mononuclear cell / macrophage expressing a chimeric antigen receptor and application of the modified mononuclear cell / macrophage. The present invention includes methods and compositions for the treatment of cancer, whether a solid tumor or a hematological malignancy. By expressing chimeric antigen receptors in monocytes, macrophages or dendritic cells, the modified cells are recruited to the tumor microenvironment where they act as effective immune effectors by infiltrating the tumor and killing the target cells. One aspect includes modified cells and pharmaceutical compositions comprising the modified cells for adoptive cell therapy and treatment of diseases or conditions associated with immunosuppression.

Description

[0001] This application is a divisional application of patent application No. 201680054514.2 (PCT / US2016 / 044440) filed on July 28, 2016, entitled "Modified monocytes / macrophages expressing chimeric antigen receptors and their uses thereof" (which was filed on July 28, 2016, with application number 202211374453.1, entitled "Modified monocytes / macrophages expressing chimeric antigen receptors and their uses thereof").

[0002] Cross-references to related applications This application is entitled to priority under 35 USC § 119(e) to U.S. Provisional Patent Application No. 62 / 197,675, filed July 28, 2015, the entirety of which is incorporated herein by reference. Background of the Invention Cancer immunotherapy has demonstrated exciting clinical outcomes in a variety of solid tumors and hematologic malignancies. The endogenous immune system is generally unresponsive to malignant cells, or the body's response to the presence of malignant cells can be aggressively immunosuppressive. One way to enhance cancer treatment is through genetic modification of leukocytes to force the immune system to recognize tumors. T cells can be modified to express synthetic immune receptors containing extracellular target antibodies and intracellular signaling domains, known as chimeric antigen receptors (CARs). T cells expressing CARs targeting CD19 have been shown to have significant anti-leukemic efficacy, achieving complete remission in 90% of treated acute lymphoblastic leukemia patients (Maude, et al., NEJM, vol. 371:1507-17, 2014). These results are accompanied by robust T cell proliferation and well-documented T cell infiltration at tumor sites in such treated leukemia patients. Despite demonstrating high response rates in hematopoietic malignancies, CAR T-cell efficacy in solid tumors (and some lymphomas) may be limited. Possible explanations for this include a potentially weakened ability of T cells to infiltrate solid tumors, poor passage, an immunosuppressive tumor microenvironment, and minimal expression of tumor-specific antigens on solid tumor cells.

[0003] There is a need in the art for more effective compositions and methods for treating cancer by means of such compositions that improve specificity to tumor cells and increase invasion into tumor sites in solid tumors and hematological malignancies. The present invention fulfills this need. Invention Overview As disclosed herein, the present invention includes compositions and methods for using phagocytes with target effector activity.

[0004] On one hand, the present invention includes modified cells comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain of a stimulatory and / or costimulatory molecule, and wherein the cell is a monocyte, macrophage, or dendritic cell with target effector activity.

[0005] On the other hand, the present invention includes modified cells comprising nucleic acid sequences encoding chimeric antigen receptors (CARs), wherein the nucleic acid sequences comprise nucleic acid sequences encoding antigen-binding domains, nucleic acid sequences encoding transmembrane domains, and nucleic acid sequences encoding intracellular domains of stimulatory and / or costimulatory molecules, and wherein the cells are monocytes, macrophages, or dendritic cells that express CARs and have targeted effector activity.

[0006] On another front, the present invention includes a method for modifying cells, comprising introducing a chimeric antigen receptor (CAR) into monocytes, macrophages or dendritic cells, wherein the CAR comprises an antigen-binding domain, a transmembrane domain and an intracellular domain of a stimulatory and / or costimulatory molecule, and wherein the cells are monocytes, macrophages or dendritic cells that express the CAR and have target effector activity.

[0007] In another aspect, the present invention includes compositions comprising cells modified according to the methods described herein.

[0008] In various embodiments of the invention described herein, or any other aspect thereof, the antigen-binding domain of the CAR comprises an antibody selected from monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, single-domain antibodies, single-chain variable fragments, and antigen-binding fragments thereof. In another embodiment, the antigen-binding domain of the CAR is selected from anti-CD19 antibodies, anti-HER2 antibodies, and fragments thereof. In yet another embodiment, the intracellular domain of the CAR comprises a dual signal transduction domain.

[0009] In another embodiment, the targeted effector activity targets an antigen on a target cell that specifically binds to the antigen-binding domain of the CAR. In yet another embodiment, the targeted effector activity is selected from phagocytosis, targeted cellular cytotoxicity, antigen presentation, and cytokine secretion.

[0010] In another embodiment, the composition further comprises an agent selected from the following: nucleic acids, antibiotics, anti-inflammatory agents, antibodies or antibody fragments thereof, growth factors, cytokines, enzymes, proteins, peptides, fusion proteins, synthetic molecules, organic molecules, carbohydrates or analogs, lipids, hormones, microsomes, derivatives or variants thereof, and any combination thereof.

[0011] In another embodiment, the modified cells have at least one upregulated M1 marker and at least one downregulated M2 marker. In yet another embodiment, the modified cells are genetically modified to express CAR. In still another embodiment, the target effector activity is enhanced by inhibiting CD47 or SIRPa activity.

[0012] In another embodiment, introducing CAR into cells includes introducing a nucleic acid sequence encoding CAR, such as electroporation of CAR-encoding mRNA or transduction of cells with a viral vector containing a nucleic acid sequence encoding CAR.

[0013] In another embodiment, the targeted effector activity targets an antigen on a target cell that specifically binds to the antigen-binding domain of the CAR. In yet another embodiment, the targeted effector activity is selected from phagocytosis, cytotoxicity of the target cell, antigen presentation, and cytokine secretion.

[0014] In another embodiment, the method herein further includes inhibiting CD47 or SIRPα activity to enhance target effector activity, such as by contacting cells with blocking anti-CD47 or blocking anti-SIRPα antibodies. In yet another embodiment, the method further includes modifying cells to deliver an agent to a target, wherein the agent is selected from nucleic acids, antibiotics, anti-inflammatory agents, antibodies or antibody fragments thereof, growth factors, cytokines, enzymes, proteins, peptides, fusion proteins, synthetic molecules, organic molecules, carbohydrates or analogs, lipids, hormones, microsomes, derivatives or variants thereof, and any combination thereof.

[0015] On one hand, the present invention includes pharmaceutical compositions comprising the cells described herein.

[0016] On the other hand, the present invention includes the use of the modified cells described herein in the preparation of medicaments for treating immune responses in subjects in need. In yet another aspect, the present invention includes the use of the modified cells described herein in the preparation of medicaments for treating tumors or cancers in subjects in need.

[0017] On the other hand, the present invention includes a method of treating a disease or condition related to a tumor or cancer in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified cells described herein.

[0018] On another aspect, the present invention includes a method of treating a tumor of a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified cells described herein.

[0019] On the other hand, the present invention includes a method for stimulating an immune response in a subject to target tumor cells or tumor tissue, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified cells described herein. Brief description of the attached diagram The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. The drawings illustrate presently preferred embodiments for the purpose of illustrating the invention. However, it should be understood that the invention is not limited to the exact arrangement and means of the embodiments shown in the drawings.

[0020] Figure 1A This is a series of diagrams illustrating a concept of a chimeric antigen receptor (CAR) composed of genes / gene-products containing an extracellular domain, hinge domain, transmembrane domain, intracellular signal transduction domain (one or more), and / or a stoichiometric co-expression of an additional gene product, which may or may not be secreted. This additional gene product includes any gene / transcription / protein, including but not limited to cytokines, monoclonal antibodies, antibody fragments, single-chain variable fragments, enzymes, additional receptors, dominant / negative receptors, tumor-associated antigens (one or more), and any combination thereof. Furthermore, the CAR construct may include co-delivery of CRISPR / Cas9 gene-edited products or be introduced into the environment of CRISPR / Cas9 pre-edited cells.

[0021] Figure 1B It is a series of diagrams showing specific instances of CAR constructs, including CARMA-ζ, CARMA-γ, and CARMA-Dectin, which respectively contain antigen-specific scFv, CD8 hinge, CD8 transmembrane, and CD3 ζ, FcεRI universal γ subunit, or intracellular domain of Dectin-1.

[0022] Figure 2A shows CAR19z expression on the surface of myeloid cells after lentiviral transduction. CAR19z lentivirus was titrated at a three-fold dilution (dilutor) and used to transduce 1e5 / 0.1 mL mRFP+ THP1 cells. mRFP is a reporter gene (red fluorescent protein) expressed via lentiviral transduction of the myeloid cell line THP1. These cells can be induced to differentiate into macrophages after exposure to chemical PMA. THP1 cells were harvested 24 hours after transduction and stained with biotinylated protein L followed by streptavidin-APC to obtain CAR surface expression.

[0023] Figure 2B This is a diagram showing THP1 cells amplified and sorted by FACS to generate a 100% CAR19z-positive mRFP+ THP1 subline.

[0024] Figure 2CExpression of anti-CD19, anti-HER2, and anti-mesothelin lentiviral CAR constructs on THP1 macrophages is shown, with CAR(+) events in the upper right quadrant.

[0025] Figure 3A is a flowchart showing the CARMA subline generation using the THP1 macrophage model, the differentiation using 1 ng / mL phorbol 12-myristate 13-acetate (PMA), and the in vitro phagocytic assay.

[0026] Figure 3B shows a phagocytosis assay based on fluorescence microscopy demonstrating that anti-CD19 CAR macrophages (while wild-type (Wt) macrophages) phagocytose CD19-expressing K562 tumor cells.

[0027] Figure 3C shows a phagocytosis assay based on fluorescence microscopy demonstrating that anti-HER2 CAR macrophages (while wild-type (Wt) macrophages) phagocytose HER2-expressing K562 tumor cells.

[0028] Figure 3D shows a phagocytosis assay based on fluorescence microscopy demonstrating that anti-mesothelin CAR macrophages (while wild-type (Wt) macrophages) phagocytose K562 tumor cells expressing mesothelin.

[0029] Figure 3E The representative FACS plot shows that CARMA tumor phagocytosis was validated by a flow cytometry-based assay in which anti-CD19 mRFP+ CARMA cells were co-cultured with CD19+ GFP+ K562 cells and double-positive events were quantified.

[0030] Figure 3F is an image of mRFP shown in a standard 10x field of view used in the CARMA phagocytosis function list.

[0031] Figure 3G is an image of the overlay shown in the standard 10x field of view used in the CARMA swallowing function list.

[0032] Figure 3H shows a series of images demonstrating that mRFP / GFP double-positive events based on FACS were defined as erode events and verified by Amnis Imagestream FACS analysis. The events shown were gated based on double-positive events and sorted from high to low using the Amnis Imagestream erode algorithm.

[0033] Figure 3I shows a series of images demonstrating the phagocytosis of tumor cells by mRFP+CARMA in the THP-1 cell line model, further confirmed by confocal microscopy. These images confirm that GFP+ tumor cells were completely enclosed within the phagocytoids through three-dimensional confocal z-shaped stacking.

[0034] Figure 3J shows a series of images of a single CARMA cell exhibiting its encounter (fate) over time—where contact and immune synapse formation are the first steps leading to phagocytosis, tumor degradation with GFP loss as a marker of cell death, phagosome breakdown, and phagosome repair—encapsulating the survival of CARMA cells after phagocytosis by tumor cells.

[0035] Figure 3K This demonstrates CARMA's ability to engulf multiple tumor cells at once.

[0036] Figure 4A shows an in vitro phagocytosis assay of anti-CD19 CAR macrophages against CD19+ (target) or CD19- (control) GFP+ K562 tumor cells. Only antigen-loaded tumor cells were phagocytosed, demonstrating the antigen specificity of CARMA. To demonstrate the need for an intracellular signal transduction domain in CARMA function, a CAR19-Δζ construct (lacking an intracellular signal transduction domain) was used.

[0037] Figure 4B shows CAR19-Δζ macrophages that cannot engulf tumor cells.

[0038] Figure 4C shows that CAR19-Δζ macrophages have significantly reduced antitumor function through an in vitro luciferase-based specific dissolution assay.

[0039] Figure 4D is a diagram showing an in vitro CARMA phagocytosis assay performed in the presence of R406 (a Syk inhibitor). R406 independently eradicated CARMA phagocytic function, indicating that CAR signaling in macrophages is Syk-dependent and leads to actin polymerization and NMIIA-mediated phagocytosis.

[0040] Figure 4E is a diagram showing an in vitro CARMA phagocytosis assay in the presence of cytochalasin D (an inhibitor of actin polymerization). Cytochalasin D independently eradicated CARMA phagocytic function, indicating that CAR signaling in macrophages is Syk-dependent and leads to actin polymerization and NMIIA-mediated phagocytosis.

[0041] Figure 4FThis is a graph showing an in vitro CARMA phagocytosis assay performed in the presence of blebbistatin (a non-muscular myosin IIA inhibitor). Blebbistatin independently eradicated CARMA phagocytic function, indicating that CAR signaling in macrophages is Syk-dependent and leads to actin polymerization and NMIIA-mediated phagocytosis.

[0042] Figure 5A is a flow cytometry plot showing CD47 expression on the target tumor cell lines relative to the isotype control. These experiments used K562 and K562-CD19+ (K19), both high CD47 expression cell lines.

[0043] Figure 5B illustrates how the addition of anti-CD47 monoclonal antibody selectively enhances CAR (but not Wt) macrophage-mediated phagocytosis of tumor cells loaded with target antigens. Wt or CAR19ζ macrophages were cultured with CD19+ K562 tumor cells with 0, 0.01, 0.10, 1.00, or 10.0 mcg / mL anti-CD47 monoclonal antibody.

[0044] Figure 5C This is a diagram showing how the addition of anti-SIRPα monoclonal antibody selectively enhances CAR (but not Wt) macrophage-mediated phagocytosis of tumor cells loaded with target antigens. Wt or CAR19ζ macrophages were cultured with CD19+ K562 tumor cells with 0, 0.01, 0.10, 1.00, or 10.0 mcg / mL anti-SIRPα monoclonal antibody.

[0045] Figure 5D demonstrates how blocking the CD47 / SIRPα axis with an anti-SIRPα monoclonal antibody enhances the polyphagocytic activity of CAR macrophages (defined as macrophages that engulf two or more tumor cells at a time).

[0046] Figure 5E shows the in vitro phagocytosis assay. To control the opsonization effect of the CD47 / SIRPα blocking monoclonal antibody, a control anti-CD47 monoclonal antibody (clone 2D3) that binds to CD47 but does not block CD47 from reaching the SIRPα binding site was used in the in vitro phagocytosis assay. Only clones that block this binding site (anti-CD47, clone B6H12) or those that block the SIRPα receptor directly resulted in enhanced CARMA tumor phagocytosis.

[0047] Figure 5F shows in vitro phagocytosis of antigen-negative (CD19-negative) tumor cells. To test whether blocking the CD47 / SIRPα axis on CAR macrophages leads to loss of antigen specificity, in vitro phagocytosis of antigen-negative (CD19-negative) tumor cells was performed in the presence of anti-CD47 or anti-SIRPα monoclonal antibodies, and no phagocytosis was observed.

[0048] Figure 5G This graph shows the specificity of CARMA phagocytosis enhancement in the presence of SIRPα-blocking monoclonal antibodies by knocking out the SIRPα receptor on THP1 macrophages and comparing tumor phagocytosis of CARMA or SIRPα-KO CARMA in the absence or presence of SIRPα antibodies. CRISPR / Cas9 was used for SIRPα deletion, and cells were SIRPα-negatively sorted prior to functional assays. SIRPα knockout enhanced CARMA function, and adding anti-SIRPα back into the knockout cells did not further enhance phagocytosis.

[0049] Figure 6A shows the specific lysis of CD19+GFP+luciferase+ K562 cells induced by CAR19ζCARMA (but not Wt) macrophages (using a THP-1 macrophage model) in a dose-dependent manner at 48 hours in an in vitro luciferase-based killing assay.

[0050] Figure 6B shows the specific lysis of tumor cells induced by CAR19ζ or Wt THP-1 monocytes (undifferentiated, and therefore a model of monocytes rather than macrophages) in an in vitro luciferase-based killing assay at 48 hours in a dose-dependent manner.

[0051] Figure 6C shows a set of images of luciferase-driven bioluminescence derived from luciferase-positive CD19+ K562 tumor cells after 48 hours of in vitro co-culture with Wt or CAR19ζ macrophages in the absence or presence of 10 μg / mL anti-SIRPα monoclonal antibody.

[0052] Figure 6D This is a graph showing the specific lysis of Wt or CAR19ζ macrophages with + / - anti-SIRPα monoclonal antibodies.

[0053] Figure 7A This is a series of diagrams showing the generation of CAR constructs with intracellular domains of the FcεRI universal γ (CAR19γ, CARMA19γ) subunits, encapsulated in lentivirus, and used for transduction of THP-1 myeloid cells at lentiviral titers of three-fold serial dilutions. CAR19γ is expressed on THP-1 macrophages.

[0054] Figure 7B shows CAR19γ or CAR19ζ macrophages sorted at 100% CAR positivity and used for in vitro functional characterization. Both CAR19ζ and CAR19γ macrophages engulfed CD19+ tumor cells and showed synergistic effects with CD47 / SIRPα axis blockade induced by the addition of anti-SIRPα monoclonal antibody.

[0055] Figure 7C shows an in vitro phagocytosis assay demonstrating that both CAR19ζ and CAR19γ macrophages use Syk for signal transduction to drive tumor phagocytosis.

[0056] Figure 7D This is a graph showing that, under various E:T ratios, after 24 hours of co-culture, CAR19ζ and CAR19γ THP1 macrophages (while Wt THP1 macrophages) effectively killed CD19+ tumor cells in an in vitro luciferase-based specific lysis assay.

[0057] Figure 8A shows how macrophages respond to conserved molecular signals of infection, such as pathogen-associated molecular patterns, through constitutively expressed pathogen recognition receptors.

[0058] Figure 8B shows an in vitro phagocytic assay using CAR macrophages, which were independently pretreated with TLR1-9 ligands or a mediator control to enhance the tumor phagocytic function of CARMA. Ligands of TLR1, 2, 4, 5, and 6 enhanced the phagocytic function of CARMA.

[0059] Figure 8C This is a graph showing the differences in TLR ligand phagocytosis by CARMA in tumor cells between TLR3 or TLR6 ligand concentration ranges, indicating whether or not the phagocytosis is enhanced.

[0060] Figure 9A shows the binding of β-glucan (a yeast product) to Dectin-1 on the surface of macrophages, leading to activation and effector function. To test the ability of β-glucan to enhance CARMA function, in vitro tumor phagocytosis assays were performed with and without 5 mcg / mL β-glucan. β-glucan enhanced the phagocytic capacity of CAR (but not Wt) macrophages.

[0061] Figure 9BThis is a series of graphs showing in vitro luciferase-based specific lysis assays performed at various effector (E):target (T) ratios in the presence of 0, 0.5, 5, or 50 μg / mL β-glucan to test the ability of β-glucan to enhance CARMA tumor-killing activity. β-glucan enhances the specific lysis of antigen-loaded tumor cells induced by CAR (but not by Wt) THP-1 macrophages.

[0062] Figure 10A is a series of diagrams showing the generation of CAR constructs consisting of intracellular signal transduction domains of Dectin-1 cells. These constructs were encapsulated in lentivirus and used to transduce THP-1 myeloid cells at lentivirus titers of three-fold serial dilutions.

[0063] Figure 10B shows the detection of CAR on the surface of macrophages expressing the CD8TM-Dectin1 CAR construct.

[0064] Figure 10C is a diagram showing the detection of CAR on the surface of macrophages expressing the DectinTM-Dectin1 CAR construct.

[0065] Figure 10D This image shows the results of testing CD8TM-Dectin1 CAR and DectinTM-Dectin1 CAR macrophages in an in vitro luciferase killing assay. Both constructs showed specific lysis of tumor cells.

[0066] Figure 10E shows Dectin1-CAR macrophages tested against K562 (control) or K19 (target) tumor cells in an in vitro tumor phagocytosis assay. Dectin1-CAR macrophages selectively phagocytose tumor cells loaded with homologous antigens.

[0067] Figure 10F is a series of graphs showing the ability of Dectin-1 CAR macrophages to engulf a variety of tumor cells.

[0068] Figure 10G This is a graph showing an in vitro tumor phagocytosis assay. Dectin1-CAR macrophages showed synergistic effects with SIRPα blockade or with TLR ligand pretreatment.

[0069] Figure 11A is a series of graphs showing the levels of cadherin in three different CD19+ target cell lines relative to the isotype control.

[0070] Figure 11B is a graph showing the normalized average fluorescence intensity of cadherin expression in three different CD19+ target cell lines.

[0071] Figure 11CThis figure shows that low levels of cadherin moderately protect target cells (particularly the Nalm6 and JEKO cell lines) from phagocytosis by CAR19z macrophages. These data suggest that the exploitation of cadherin deposition / induction could be used as an additional means to enhance CARMA effector function.

[0072] Figure 12A This is a series of diagrams showing the anti-HER2 CAR construct being cloned into an mRNA expression plasmid, transcribed in vitro, and the mRNA being directly electroporated into native human mononuclear cells.

[0073] Figure 12B The series of figures shows that the efficiency of anti-HER2 CAR mRNA electroporation into primary human monocyte-derived macrophages (fully differentiated) is 79.7%.

[0074] Figure 12C The following figure shows that although mRNA electroporation leads to high CAR transfection efficiency in monocytes and macrophages, CAR expression is transient due to mRNA degradation, peaking on day 2 after in vitro electroporation and disappearing by day 7.

[0075] Figure 13A shows NSGS mice injected with 1E6 SKOV3 CBG / GFP+ human ovarian cancer cells (a model of metastatic intraperitoneal cancer spread in HER2+ ovarian cancer) via IP injection. Mice were co-injected with native human macrophages electroporated with either a mock or anti-HER2 CAR mRNA (1:1 E:T ratio), and tumor burden was imaged. CAR macrophages showed a marginal reduction in tumor growth after approximately two weeks. The first time point at which tumor burden was quantified by bioluminescence was 24 hours post-treatment, indicating that CAR monocytes and macrophages were active during this first 24 hours.

[0076] Figure 13B This image shows an NSGS mouse model of metastatic intraperitoneal carcinoma of HER2+ ovarian cancer, injected via IP with 1E6 SKOV3 CBG / GFP+ human ovarian cancer cells. Mice were co-injected with either mimicry-electroporated or anti-HER2 CAR mRNA-electroporated native human monocytes (1:1 E:T ratio), and tumor burden was imaged. CAR monocytes showed a marginal reduction in tumor growth after approximately two weeks. The first time point at which tumor burden was quantified by bioluminescence was 24 hours post-treatment, indicating activity of CAR monocytes and macrophages within this first 24 hours.

[0077] Figure 14AThis is a series of figures showing the lentiviral delivery of CAR transgenes to native human monocyte-derived macrophages using various CAR constructs. CAR19 was delivered to human macrophages via lentiviral transduction, showing transduction efficiencies of 4.27% and 38.9% in the control vs. CAR19 lentivirus (MOI 10) groups.

[0078] Figure 14B This is a series of representative FACS plots showing the expression of anti-HER2 CAR in native human macrophages, with transduction efficiencies of 1.47% and 18.1% in control and MOI 10 CAR LV conditions, respectively.

[0079] Figure 15A This is a series of graphs showing that the transduction efficiency of anti-CD19 peaks at the midpoint of transduction (day 4). Monocyte-derived macrophages were generated by differentiating CD14+ selected cells (derived from apheresis products of normal donor blood) in GM-CSF conditioned medium for 7 days. To optimize CAR delivery via lentiviral transduction, macrophages were transduced with anti-CD19 lentivirus at different time points during the monocyte-to-macrophage differentiation process.

[0080] Figure 15B is a series of graphs showing that the transduction efficiency of anti-HER2 reached its peak at the midpoint of transduction (day 4). Monocyte-derived macrophages were generated by differentiating CD14+ selected cells (products of normal donor blood separation) in GM-CSF conditioned medium for 7 days. To optimize CAR delivery via lentiviral transduction, macrophages were transduced with anti-HER2 lentivirus at different time points in the monocyte-to-macrophage differentiation process.

[0081] Figure 15C These are a series of graphs showing the trend of phagocytic efficiency in accordance with CAR transduction efficiency, with the peak value of macrophages transduced on day 4 during differentiation.

[0082] Figure 16A is a series of diagrams showing a series of alternative transduction methods for delivering transgenes to native human macrophages, considering that mRNA electroporation is transient and lentiviruses are only moderately effective and require high titers. Adenovirus (recombinant, replication-deficient) was identified as an effective method for native human macrophage transduction. On native human macrophages, expression of the Coxackie adenovirus receptor (a stopping protein of Ad5) and CD46 (a stopping protein of Ad35) was tested relative to the isotype control, with CD46 (but not the Coxackie adenovirus receptor) being highly expressed. Therefore, a chimeric Ad5f35 adenovirus was used for native human macrophage transduction and was engineered using standard molecular biology techniques to express a chimeric antigen receptor against HER2 (GFP and blank Ad5f35 virus were used as controls).

[0083] Figure 16B shows that Ad5f35 efficiently delivered the transgene (GFP as a model transgene) into human macrophages at an MOI of 1000, and that the expression increased over time by quantitatively monitoring the GFP signal on the IVIS spectrum.

[0084] Figure 16C This is a graph showing a comparison of transduction dynamics of native human macrophages at different time points across a wide range of MOIs up to 10,000.

[0085] Figure 16D is a series of representative FACS plots of anti-HER2 CAR expression on Ad5f35-transduced human macrophages 48 hours post-transduction under a wide range of viral MOIs.

[0086] Figure 16E These are a series of representative fluorescence microscopy images of native human macrophages transduced with Ad5f35-GFP, with the highest transduction efficiency shown at MOI 1000.

[0087] Figure 17A This is a series of graphs showing native human CARMA as tested in an in vitro phagocytosis assay by FACS analysis. Macrophages (untransduced, or anti-HER2 CAR) were stained with DiI before co-culturing with GFP+ SKOV3 ovarian cancer cells. Phagocytosis, defined by a DiI / GFP double-positive event, was measured at 26.6% in the CAR group and 4.55% in the control group.

[0088] Figure 17B is a visual representation of a series of double-positive events that represent phagocytosis. To verify that the DiI / GFP double-positive events are phagocytic events and not doublets, cytochalasin D (a phagocytosis inhibitor) was added to the experimental group, and CAR-mediated phagocytosis was fully eradicated to a level of 1.74%. To further verify that native human CAR macrophages can phagocytose tumor cells, double-positive events were gated using Amnis Imagestream FACS and sorted from high to low using the Amnis phagocytosis-erosion algorithm.

[0089] Figure 17C This is a series of confocal microscopy images showing Dil-stained CAR-HER2 macrophages co-cultured with SKOV3-GFP.

[0090] Figure 18This image shows CAR (but not UTD) human macrophages phagocytosing breast cancer cells. Anti-HER2 CAR native human macrophages were generated using Ad5f35-CAR transduction from monocyte-derived macrophages. These cells (or untransduced control cells) were used as effectors in an in vitro FACS-based phagocytosis assay of SKBR3 human breast cancer cells. Furthermore, the addition of an anti-SIRPα monoclonal antibody enhanced CARMA (but not UTD macrophages) phagocytosis of breast cancer cells. These results demonstrate that the synergistic effect between CD47 / SIRPα axis blockade and CARMA observed in the THP-1 model has been transferred to native human macrophage studies.

[0091] Figure 19 This is a series of representative FACS plots showing that CARMA exhibits complete phagocytosis of pH-Rodo Green E. coli particles. To demonstrate that CAR macrophages remain functional innate immune cells in the antimicrobial sense and have not lost their ability to respond to infectious stimuli, untransduced control or CAR macrophages were used in the FACS-based E. coli phagocytosis assay.

[0092] Figure 20A shows native human anti-HER2 CARMA as an effector cell assay in an in vitro luciferase-based killing assay. After 48 hours of co-culture, anti-HER2 CARMA (but not control UTD macrophages) caused specific lysis of HER2+ K562 cells (but not control K562 cells lacking HER2 expression).

[0093] Figure 20B shows an in vitro luciferase-based killing assay using SKBR3 breast cancer cells as a target. CARMA (but not control UTD or control blank Ad5f35 transduced macrophages) showed significant antitumor activity in both models after co-culturing for 48 hours.

[0094] Figure 20C This is a graph showing an in vitro luciferase-based killing assay using SKOV3 ovarian cancer cells as a target. CARMA (but not control UTD or control blank Ad5f35 transduced macrophages) showed significant antitumor activity in both models after co-culturing for 48 hours.

[0095] Figure 20D illustrates the synergistic effect between CD47 / SIRPα axis blockade in the killing assay. SKOV3 ovarian cancer cells were co-cultured with culture medium, control untransduced macrophages, anti-HER2 CARMA, anti-HER2 CARMA + anti-CD47 mAB (10 mcg / mL), or anti-HER2 CARMA + anti-SIRPα (10 mcg / mL), and luciferase signaling was continuously measured. CARMA led to complete tumor eradication by day 13, and the kinetics of tumor eradication were even faster in the presence of CD47 / SIRPα axis blockade.

[0096] Figure 20E shows the synergistic effect with β-glucan, which was confirmed in the THP-1 macrophage CARMA model, and β-glucan pretreatment of CARMA led to enhanced tumor killing dynamics.

[0097] Figure 20F This is a graph showing the regulation of the antitumor effect caused by CARMA exposure to LPS (TLR-4 ligand) or polyIC (TLR-3 ligand).

[0098] Figure 21 This is a series of graphs showing the ability of native human CARMA to clear tumors in an in vitro luciferase assay. GFP+ SKOV3 ovarian cancer cells were co-cultured with control UTD macrophages, control UTD macrophages + 10 mcg / mL trastuzumab, control blank Ad5f35 virus-transduced macrophages, or anti-HER2 native human CARMA. CARMA (but not under control conditions) was able to clear tumor cells.

[0099] Figure 22A shows the dose-dependent upregulation of the M1 marker CD80 / CD86 and the dose-dependent downregulation of the M2 marker CD163 as measured by FACS. Macrophages are phenotypically plastic cells capable of receiving different functional characteristics and are generally classified into M1 and M2 macrophages—M1 being inflammatory / activated and M2 being immunosuppressive / tumor-promoting. M1 and M2 markers were measured 48 hours after transduction of native human macrophages with Ad5f35 CAR virus.

[0100] Figure 22B This is a series of graphs showing the effects of M1 and M2 markers on CAR expression or Ad5f35 transduction. Macrophages were not transduced, transduced with blank Ad5f35, or transduced with anti-HER2 Ad5f35, and blank / CAR Ad5f35 showed the same phenotypic conversion pattern.

[0101] Figure 22C shows that CARMA exposed to inhibitory cytokines maintained its cytotoxic activity in an in vitro specific lysis assay based on luciferase at 48 hours. Control UTD macrophages treated with inhibitory cytokines showed enhanced tumor growth.

[0102] Figure 22D This is a set of graphs showing the resistance of human CAR macrophages to immunosuppression. Control UTD, blank Ad5f35, or anti-HER2 CAR Ad5f35-transduced macrophages were exposed to 10 ng / mL IL-4 (a typical M2-inducing cytokine) or cancer cells (SKOV3, ovarian cancer cell line; HDLM2, Hodgkin lymphoma cell line) that had previously shown to subvert macrophages to M2 phenotype during co-culture. Control UTD macrophages upregulated CD206 (an M2 marker that responds specifically to IL-4 stimulation via STAT6 phosphorylation). Blank Ad5f35, and even more so CAR-Ad5f35-transduced macrophages, showed resistance to IL-4 and tumor-induced M2 phenotype subversion.

[0103] Figure 22E is a graph showing the metabolic phenotype and oxygen consumption rate of control UTD or anti-HER2 CAR macrophages exposed to IL-4 for 24 hours to polarize to M2 (or not).

[0104] Figure 22F This is a graph showing that CARMA is resistant to M2 downconversion, based on phenotypic, metabolic, and functional assays.

[0105] Figure 23A This is a graph showing native human normal donor monocytes (purified via CD14-positive selection) transduced with Ad5f35-CAR-HER2 at an MOI ranging from 0 (UTD) to 1000. CAR expression was measured by FACS at 48 hours post-transduction. Ad5f35 efficiently generated CAR monocytes, and expression peaked at an MOI of 1000.

[0106] Figure 23B shows the transduction efficiency of native monocytes.

[0107] Figure 23C shows the high viability of monocytes at MOIs up to 1000 (measured by FACS Live / DeadAqua analysis).

[0108] Figure 23D is a series of graphs showing the upregulation of M1 activation markers in human monocytes with CAR (but not in untransduced (UTD) cells).

[0109] Figure 23EThis is a series of graphs showing the downregulation of M2 markers due to CAR (but not transduced (UTD)) human monocytes.

[0110] Figure 24A shows the anti-HER2 CAR monocyte killing effect of HER2+ SKBR3 cells (human breast cancer) as evaluated by an in vitro luciferase-based killing assay.

[0111] Figure 24B This is a graph showing the anti-HER2 CAR mononuclear cell killing effect of HER2+ SKOV3 cells (human ovarian cancer) as assessed by an in vitro luciferase-based killing assay.

[0112] Figure 25A is a schematic diagram of NOD-scid IL2Rg-null-IL3 / GM / SF and NSG-SGM3 (NSGS) mice used for in vivo modeling of human HER2(+) ovarian cancer xenografts. On day 0, mice were intraperitoneally (IP) injected with 7.5E5 click beetle green luciferase (CBG luc)-positive / green fluorescent protein (GFP)-positive SKOV3 ovarian cancer cells—serving as a model of intraperitoneal cancer spread, an invasive and inherently metastatic model of solid malignancy. Mice were either untreated (tumor only) or injected on day 0 with a single dose of 4E6 untransduced human macrophages (UTD) or CAR-HER2 human macrophages (CARMA) via IP.

[0113] Figure 25B shows a mouse model using bioluminescence (total flux; photons / second) as a representation of tumor burden for continuous imaging.

[0114] Figure 25C This is a graph showing the survival percentage of mice treated with CARMA. The tumor burden in CARMA-treated mice was reduced by approximately two orders of magnitude.

[0115] Figure 25D is a set of images showing that mice treated with CARMA had a 30-day survival gain (p = 0.018) compared to untreated mice or mice treated with UTD macrophages.

[0116] Figure 25E This is a set of images showing tumors obtained from mice that died on day 36 and assessed by human CD45 expression based on FACS analysis of the presence of adoptive metastatic human macrophages.

[0117] Figure 26A is a diagram showing the surface CAR expression of human macrophages transduced at a fold increase of 1000 fold 48 hours after transduction, either untransduced (UTD) or transduced with blank Ad5f35 virions (blank / empty) or Ad5f35-CAR-HER2-ζ (CARMA), as verified by FACS analysis.

[0118] Figure 26B is a set of graphs showing the surface markers of M1 macrophage polarization as evaluated in cells transduced via blank Ad5f35 or CAR-HER2-ζ Ad5f35. M1 markers (HLA DR, CD86, CD80, PDL1) were upregulated, while M2 markers (CD206, CD163) were downregulated.

[0119] Figure 26C This is a schematic diagram of NSGS mice used in an IP model of HER2+ metastatic ovarian cancer, divided into four treatment groups (n=5 per group). The mice were either untreated or injected with IP on day 0 with untransduced 1E7 macrophages, blank Ad5f35-transduced macrophages, or CAR-HER2-ζ-transduced macrophages.

[0120] Figure 26D This is a set of images showing tumor burden monitored by continuous bioluminescence imaging, with representative data displayed on day 27 post-tumor implantation.

[0121] Figure 26E This is a graph showing tumor burden monitored by continuous bioluminescence imaging, with representative data displayed on day 27 post-tumor implantation.

[0122] Figure 27A is a schematic diagram of NSGS mice used for the IP model of HER2+ metastatic ovarian cancer and divided into 4 treatment groups (n=5 per group). The treatment groups include untreated mice and mice given 3E6, 1E7 or 2E7 CAR-HER2-ζ human macrophages via IP on day 0.

[0123] Figure 27B is a graph showing tumor burden monitored by continuous bioluminescence imaging. A dose-dependent response to macrophage number was observed in this model.

[0124] Figure 27C This is a graph showing the dose-dependent tumor eradication (relative to untreated mice) on day 36 post-implantation resulting from a single dose of CAR-HER2 macrophages (3E6, 1E7, or 2E7 macrophages per mouse).

[0125] Figure 28This is an example of a treatment method proposed by CARMA. In short, patient monocytes are selected from peripheral blood, differentiated and transduced in vitro to express CAR, co-stimulated (or not) with a synergistic compound, and then injected back into the patient intravenously, intraperitoneally, intratumorally, via interventional radiology procedures, or through other routes. Note that the differentiation process can be skipped, and the monocytes can be transduced and infused back into the patient. The monocyte source can also be an HLA-matched donor. Detailed description definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice of testing the invention, preferred materials and methods are described herein. The following terms will be used in the description and assertion of the invention.

[0126] It should also be understood that the terminology used in this document is for the purpose of describing particular implementations only and is not intended to be limiting.

[0127] The articles “a” and “a kind” are used in this text to refer to one or more (i.e., at least one) of the grammatical objects of the article. For example, “an element” means one element or more.

[0128] When referring to measurable values ​​such as quantity or duration, the term “about” as used herein means a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1%, of the specified value, because such variation is suitable for implementing the methods of this disclosure.

[0129] As used herein, “activation” refers to the state of monocytes / macrophages that have been adequately stimulated to induce detectable cell proliferation or to exert their effector function. Activation can also be associated with induced cytokine production, phagocytosis, cell signaling, target cell killing, or antigen processing and presentation. The term “activated monocytes / macrophages” refers, among other things, monocytes / macrophages that are undergoing cell division or exerting their effector function.

[0130] As used herein, the terms “application,” “biological agent,” or “therapeutic agent” refer to molecules that can be expressed, released, secreted, or delivered to a target by modified cells as described herein. Such applications include, but are not limited to, nucleic acids, antibiotics, anti-inflammatory agents, antibodies or antibody fragments thereof, growth factors, cytokines, enzymes, proteins, peptides, fusion proteins, synthetic molecules, organic molecules (e.g., small molecules), carbohydrates or analogs, lipids, hormones, microsomes, their derivatives or variants, and any combination thereof. The application may bind to the target or any cellular part present on the target cell, such as receptors, antigenic determinants, or other binding sites. The application may diffuse or be transported into the cell, where it can exert its effects within the cell.

[0131] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be complete immunoglobulins derived from natural or recombinant sources, and can be the immunoreactive portion of a complete immunoglobulin. Antibodies are generally tetramers of immunoglobulin molecules. The antibodies of this invention can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies (scFv) and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0132] The term "antibody fragment" refers to a portion of a complete antibody and specifically refers to the antigenic determination variable region of the complete antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0133] As used in this article, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in the native conformation of all antibody molecules.

[0134] As used in this article, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in the native conformation of all antibody molecules. α and β light chains refer to the two main antibody light chains of the same type.

[0135] As used herein, the term "synthetic antibody" refers to an antibody generated using recombinant DNA technology, such as, for example, the phage-expressed antibody described herein. The term should also be interpreted as meaning an antibody generated by synthesizing a DNA molecule encoding an antibody (and such DNA molecule expressing an antibody protein) or defining an amino acid sequence of the antibody, wherein such DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence techniques available and known in the art.

[0136] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response may involve antibody production, or activation of specific immunocompetent cells, or both. Those skilled in the art will understand that any macromolecule, including substantially all proteins or peptides, can act as an antigen. Furthermore, antigens can be derived from recombinant DNA or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or a portion of a protein encoding an immune response therefore encodes the term "antigen" as used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is apparent that the invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, those skilled in the art will understand that an antigen need not be encoded entirely by a "gene." It is apparent that antigens can be synthesized or can be derived from biological samples. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0137] As used herein, the term "antitumor effect" refers to biological effects that can be manifested through tumor volume reduction, tumor cell count reduction, metastasis reduction, increased life expectancy, or relief of various physiological symptoms associated with cancerous conditions. The "antitumor effect" can also be demonstrated by the ability of the peptides, polynucleotides, cells, and antibodies of this invention to prevent the initial development of tumors.

[0138] According to the present invention, the term "autoantigen" refers to any autoantigen recognized as foreign by the immune system. Autoantigens include, but are not limited to, cellular proteins, phosphoproteins, cell surface proteins, cellular lipids, nucleic acids, and glycoproteins, including cell surface receptors.

[0139] The term "autoimmune disease" as used in this article is defined as a disorder caused by an autoimmune response. Autoimmune diseases are the result of an inappropriate and excessive reaction to autoantigens. Examples of autoimmune diseases include, but are not limited to, Addision disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, type I diabetes, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthritis, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis.

[0140] As used in this article, the term "self" means any material that originates from the same individual and is later reintroduced into that individual.

[0141] "Same species" refers to grafts from different animals of the same species.

[0142] "Heterogeneous" refers to grafts originating from animals of different species.

[0143] As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and similar cancers. In some embodiments, the cancer is medullary thyroid carcinoma.

[0144] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial T-cell surface receptor modified to be expressed on immune effector cells and specifically bind to antigens. CARs can be used as a treatment via adoptive cell transfer. Monocytes are removed from a patient (blood, tumor, or ascites) and modified to express a specific receptor for a particular antigen form. In some embodiments, for example, a CAR specific to tumor-associated antigens has been expressed. CARs may also include an intracellular activation domain, a transmembrane domain, and an extracellular domain containing a tumor-associated antigen-binding region. In some aspects, a CAR comprises a fusion of a single-chain variable fragment (scFv)-derived monoclonal antibody fused to both the CD3-ζ transmembrane and intracellular domains. The specificity of the CAR design may be derived from a ligand of the receptor (e.g., a peptide). In some embodiments, a CAR can target cancer by redirecting the expression of the CAR to monocytes / macrophages specific for tumor-associated antigens.

[0145] The term "chimeric intracellular signaling molecule" refers to a recombinant receptor comprising one or more intracellular domains of one or more stimulatory and / or costimulatory molecules. Chimeric intracellular signaling molecules substantially lack extracellular domains. In some embodiments, chimeric intracellular signaling molecules include additional domains, such as transmembrane domains, detectable tags, and spacer domains.

[0146] As used herein, the term "conserved sequence modification" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing that amino acid sequence. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conserved amino acid substitution is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues within the CDR region of an antibody can be substituted with other amino acid residues from the same side chain family, and the antigen-binding capacity of the altered antibody can be tested using the functional assays described herein.

[0147] As used herein, the term "co-stimulatory ligand" includes molecules on antigen-presenting cells (e.g., aAPCs, dendritic cells, B cells, and similar cells) that specifically bind to homologous co-stimulatory molecules on monocytes / macrophages, thereby providing signals that mediate monocyte / macrophage responses—including, but not limited to, proliferation, activation, differentiation, and similar responses. Co-stimulatory ligands may include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin β receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll ligand receptors and ligands that specifically bind to B7-H3. Costimulatory ligands also include antibodies that specifically bind to costimulatory molecules present on monocytes / macrophages, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0148] "Co-stimulatory molecules" refer to molecules on innate immune cells that enhance or diminish the initial stimulus. For example, pathogen-associated pattern recognition receptors, such as TLRs (enhanced) or the CD47 / SIRPα axis (diminished), are molecules on innate immune cells. Co-stimulatory molecules include, but are not limited to, TCR, CD3ζ, CD3γ, CD3δ, CD3ε, CD86, universal FcRγ, FcRβ (FcεR1b), CD79a, CD79b, and Fcγ. RIIa, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligand specifically binding to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19 , CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD 103. ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, other costimulatory molecules described herein, any derivatives, variants or fragments thereof, any synthetic sequence of costimulatory molecules with the same activity, and any combination thereof.

[0149] As used in this article, "co-stimulatory signal" refers to a signal that binds to a master signal, such as the activation of CAR on macrophages, leading to macrophage activation.

[0150] The term "cytotoxic" or "cytotoxic" refers to cells that kill or destroy. In one implementation, the cytotoxicity of metabolically enhanced cells is increased, for example, by increasing the cytolytic activity of macrophages.

[0151] "Disease" is a state of health in an animal in which the animal is unable to maintain homeostasis, and in which the animal's health continues to deteriorate if the disease does not improve. In contrast, an "impairment" is a state of health in which the animal is able to maintain homeostasis, but in which the animal's health is not as good as it would be without the impairment. An impairment does not necessarily lead to a further decline in the animal's health if left untreated.

[0152] The terms "effective amount" or "therapeutic effective amount" are used interchangeably herein and refer to the amount of a compound, formulation, material, or composition that effectively achieves a specific biological effect or provides therapeutic or preventative benefit as described herein. Such an effect may include, but is not limited to, antitumor activity determined by any suitable means in the art.

[0153] "Encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, acting as a template for other polymers (polymers) and macromolecules with defined nucleotide (i.e., rRNA, tRNA, and mRNA) sequences or defined amino acid sequences used in the synthesis of biological processes, and the resulting biological properties. Thus, a gene encodes a protein—if the transcription and translation of the mRNA corresponding to that gene produces that protein in a cell or other biological system. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing) and the non-coding strand that serve as the transcription template for a gene or cDNA can be referred to as the protein or other product encoding that gene or cDNA.

[0154] As used herein, “endogenous” means any material that originates from or is produced within an organism, cell, tissue, or system.

[0155] As used herein, the term “exogenous” means any material introduced from or produced outside an organism, cell, tissue, or system.

[0156] As used herein, the term "amplification" refers to an increase in quantity, such as an increase in the number of monocytes / macrophages. In one embodiment, the number of monocytes / macrophages expanded in vitro is increased relative to the number originally present in the culture. In another embodiment, the number of monocytes / macrophages expanded in vitro is increased relative to other cell types in the culture. As used herein, the term "in vitro" refers to cells that have been removed from a living organism (e.g., a human) and multiplied outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0157] As used herein, the term “expression” is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0158] "Expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operatively linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or by an in vitro expression system. Expression vectors include all those known in the art, such as viscera, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) containing the recombinant polynucleotide.

[0159] As used herein, “homology” refers to the subunit sequence identity between two polymer molecules, such as two nucleic acid molecules—like two DNA molecules or two RNA molecules—or two polypeptide molecules. Two molecules are homologous when the subunit positions are occupied by the same monomeric subunit; for example, if one position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. Homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half the positions in two sequences (e.g., five positions in a polymer of ten subunit length) are homologous, then the two sequences are 50% homologous; if 90% of the positions (e.g., nine out of ten) are matching or homologous, then the two sequences are 90% homologous. When applied to nucleic acids or proteins, “homology” as used herein refers to sequences having approximately 50% sequence identity. More preferably, the homologous sequences have about 75% sequence identity, and even more preferably, they have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0160] The “humanized” form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequence of the antibody) containing a minimal sequence derived from a non-human immunoglobulin. Humanized antibodies are mostly human immunoglobulins (receptor antibodies) in which residues from the complementarity-determining region (CDR) of the receptor are replaced by residues from the CDR of a non-human species (donor antibody) with the desired specificity, affinity, and capability, such as mouse, rat, or rabbit. In some cases, Fv frame region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in either the receptor antibody or the introduced CDR or frame sequence. These modifications are performed to further improve and optimize antibody performance. Generally, humanized antibodies will contain substantially all of at least one variable domain, typically two variable domains, where all or substantially all of the CDR regions correspond to the CDR regions of the non-human immunoglobulin, and all or substantially all of the FR regions are the FR regions of the human immunoglobulin sequence. Humanized antibodies will ideally also contain at least a portion of the immunoglobulin constant region (Fc), typically human immunoglobulin. For more details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0161] "Whole human" refers to immunoglobulins in which the entire molecule is of human origin or is composed of the same amino acid sequence as human antibodies, such as antibodies.

[0162] As used herein, “identity” refers to the subunit sequence identity between two polymer molecules, and more particularly between two amino acid molecules, such as two polypeptide molecules. Two amino acid sequences are identical when they have the same residues at the same positions; for example, if one position in each of two polypeptide molecules is occupied by arginine, then they are identical at that position. Identity, or the degree to which two amino acid sequences have the same residues at the same positions in an alignment, is usually expressed as a percentage. Identity between two amino acid sequences is a direct function of the number of matches or identical positions; for example, if half of the positions in two sequences (e.g., 5 positions in a polymer of 10 amino acids in length) are the same, then the two sequences have 50% identity; if 90% of the positions (e.g., 9 out of 10) are matches or identical, then the two amino acid sequences have 90% identity.

[0163] "Substantially identical" means that the polypeptide or nucleic acid molecule exhibits at least 50% identity with a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Preferably, such a sequence has at least 60%, more preferably 80% or 85%, and even more preferably 90%, 95%, or even 99% identity with the sequence used for comparison at the amino acid or nucleic acid level.

[0164] A guide nucleic acid sequence is complementary to one strand (nucleotide sequence) of a target site in a double-stranded DNA sequence. The percentage of complementarity between the guide nucleic acid sequence and the target sequence can be at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The guide nucleic acid sequence can be at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more nucleotides long. In some embodiments, the guide nucleic acid sequence comprises a continuous segment of 10 to 40 nucleotides. The variable targeting domain can consist of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence (see, for example, the modifications described herein), or any combination thereof.

[0165] Sequence identity is typically determined using sequence analysis software (e.g., the Sequence Analysis Software Package from Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705), BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning a degree of homology to various substitutions, deletions, and / or other modifications. Conserved substitutions generally include substitutions in the following group: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method for determining the degree of identity, the BLAST program can be used, where e -3 and e -100 The probability scores between them indicate closely related sequences.

[0166] As used herein, the term "immunoglobulin" or "Ig" is defined as a class of proteins that act as antibodies. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. This class of proteins includes five members: IgA, IgG, IgM, IgD, and IgE. IgA is a primary antibody found in bodily secretions such as saliva, tears, breast milk, gastrointestinal secretions, and respiratory and genitourinary mucus. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response of most subjects. It is the most effective immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in the defense against bacteria and viruses. IgD is an immunoglobulin without known antibody function but can act as an antigen receptor. IgE is an immunoglobulin that mediates an immediate hypersensitivity reaction by inducing the release of mediators from mast cells and basophils upon exposure to an allergen.

[0167] As used herein, the term “immune response” is defined as the cellular response to an antigen that occurs when lymphocytes recognize an antigen molecule as foreign and induce antibody formation and / or activate lymphocytes to eradicate the antigen.

[0168] As used herein, “illustrating material” includes publications, records, figures, or any other medium of expression that can be used to convey the usefulness of the compositions and methods of the present invention. For example, illustrating material for a kit of the present invention may be, for example, attached to or shipped with a container containing the nucleic acids, peptides, and / or compositions of the present invention. Alternatively, illustrating material may be shipped separately from the container to illustrate the appropriate use of the material and compounds by the recipient.

[0169] "Separated" means altered or removed from its native state. For example, nucleic acids or peptides naturally present in living animals are not "separated," but the same nucleic acids or peptides that are partially or completely separated from their native counterparts are "separated." Separated nucleic acids or proteins can exist in a substantially purified form or in non-native environments such as, for example, host cells.

[0170] As used in this article, "lentivirus" refers to a genus within the family Retroviridae. Lentivirals are unique among retroviruses in their ability to infect non-dividing cells; they can transfer significant amounts of genetic information into the host cell's DNA, making them one of the most efficient gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Lentiviral-derived vectors provide a means to achieve significant levels of gene transfer in vivo.

[0171] As used herein, the term "modification" means an alteration in the state or structure of the molecules or cells of this invention. Molecules can be modified in a variety of ways, including chemical, structural, and functional modifications. Cells can be modified through the introduction of nucleic acids.

[0172] As used herein, the term "modulation" refers to the mediating of a detectable increase or decrease in the level of response in a subject compared to the level of response in a subject without treatment or the compound, and / or compared to the level of response in a otherwise identical but untreated subject. This term includes interference with and / or influence on natural signals or responses, thereby mediating a beneficial therapeutic response in the subject (preferably a human).

[0173] In the context of this invention, the following abbreviations are used for commonly used nucleic acid bases. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0174] Unless otherwise specified, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. A nucleotide sequence encoding a protein or RNA phrase may also include introns, such that the nucleotide sequence encoding that protein may contain one or more introns in some forms.

[0175] The term "operably linked" refers to a functional link between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when they are in a functional relationship. Similarly, a promoter is operably linked to a coding sequence if it affects transcription or expression of that sequence. Generally, operably linked DNA sequences are contiguous and located within the same reading frame when linking two protein-coding regions is required.

[0176] The term "overexpressed" tumor antigen, or "overexpression" of a tumor antigen, is intended to indicate an abnormal level of expression of a tumor antigen in cells of a diseased region, such as a solid tumor, within a specific tissue or organ of a patient, relative to the expression level in normal cells of that tissue or organ. Patients with solid tumors or hematologic malignancies characterized by tumor antigen overexpression can be identified by standard tests known in the art.

[0177] "Parenteral" administration of the immunogenic composition includes techniques such as subcutaneous (sc), intravenous (iv), intramuscular (im), intratumoral (it), intraperitoneal (ip), or intrasternal injection or infusion.

[0178] As used herein, the term "polynucleotide" is defined as a nucleotide chain. Furthermore, nucleic acids are polymers of nucleotides. Therefore, nucleic acids and polynucleotides, as used herein, are interchangeable. Those skilled in the art will know that nucleic acids are polynucleotides, which can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, those obtained by any means available in the art (non-limitingly including recombination methods, i.e., utilizing common cloning techniques and PCR). TM Nucleic acid sequences obtained from recombinant libraries or cell genome clones, as well as all nucleic acid sequences obtained through synthetic means.

[0179] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, the term refers both to short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers) and longer chains (which are generally referred to in the art as proteins, of which there are many types). “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and others. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0180] As used herein, the term “promoter” is defined as a DNA sequence that is recognized by the cell’s synthetic apparatus or an introduced synthetic apparatus required to initiate the specific transcription of a polynucleotide sequence.

[0181] As used herein, the term "promoter / regulatory sequence" means the nucleic acid sequence required for the expression of a gene product operatively linked to a promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in others, it may include enhancer sequences and other regulatory elements required for gene product expression. A promoter / regulatory sequence may, for example, be a promoter / regulatory sequence for expressing a gene product in a tissue-specific manner.

[0182] A "constitutive" promoter is a nucleotide sequence that, under most or all physiological conditions of the cell, results in the production of a gene product in the cell when operatively linked to a polynucleotide that encodes or defines the gene product.

[0183] An "inducible" promoter is a nucleotide sequence that, essentially, only leads to the production of a gene product in the cell when it is operatively linked to a polynucleotide that encodes or defines the gene product, in the presence of an inducer corresponding to the promoter in the cell.

[0184] A "tissue-specific" promoter is a nucleotide sequence that results in the production of a gene product in the cell only when the cell is a cell of the tissue type corresponding to the promoter, and when operatively linked to a polynucleotide encoding or defining the gene.

[0185] The term "immunosuppressive resistance" refers to the absence or reduction of inhibition of the activity or activation of the immune system.

[0186] "Signal transduction pathways" refer to the biochemical relationships between various signal transduction molecules that influence the transmission of signals from one part of the cell to another. The phrase "cell surface receptors" includes molecules and molecular complexes that can receive signals and transmit them across the cell membrane.

[0187] "Single-chain antibody" refers to an antibody formed through recombinant DNA technology, in which segments of the immunoglobulin heavy and light chains are linked to the Fv region via an engineered span of amino acids. Various methods for generating single-chain antibodies are known, including those described in U.S. Patent Nos. 4,694,778; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; and Skerra et al. (1988) Science 242:1038-1041.

[0188] As used herein with respect to antibodies, the term "specific binding" means an antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in the sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, this cross-species reactivity itself does not alter the antibody's classification as specific. In another instance, an antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, this cross-reactivity itself does not alter the antibody's classification as specific. In some cases, the terms "specific binding" or "specific binding" may be used when referring to the interaction of an antibody, protein, or peptide with a second chemical species, meaning that the interaction depends on the presence of a specific structure on the chemical species (e.g., an antigenic determinant or epitope); for example, the antibody recognizes and binds to a specific protein structure rather than recognizing and binding to proteins in general. If an antibody is specific for epitope "A," the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody in a reaction involving labeled "A" and the antibody.

[0189] The term "stimulation" refers to a primary response mediated by the binding of a stimulating molecule (e.g., the TCR / CD3 complex) to its homologous ligand, thereby mediating a signal transduction event (such as, but not limited to, signal transduction via Fc receptor mechanisms or via synthetic CARs). Stimulation can mediate alterations in the expression of certain molecules, such as TGF-β downregulation and / or cytoskeleton remodeling and similar events.

[0190] The term "stimulatory molecule" as used in this article refers to a molecule on monocytes / macrophages that specifically binds to homologous stimulatory ligands present on antigen-presenting cells.

[0191] As used herein, “stimulatory ligand” means a ligand that, when present on antigen-presenting cells (e.g., aAPCs, dendritic cells, B cells, and similar cells) or tumor cells, can specifically bind to homologous binding partners (referred to herein as “stimulatory molecules”) on monocytes / macrophages to mediate immune cell responses (including, but not limited to, activation, initiation of immune responses, proliferation, and similar responses). Stimulatory ligands are well known in the art and include Toll-like receptor (TLR) ligands, anti-Toll-like receptor antibodies, agonists, and monocyte / macrophage receptor antibodies. Additionally, cytokines, such as interferon-γ, are effective stimulants of macrophages.

[0192] The term "object" is intended to include any living organism (e.g., a mammal) in which an immune response can be elicited. As used herein, "object" or "patient" can be a human or a non-human mammal. Non-human mammals include, for example, livestock and pets, such as mammals belonging to the families Sheep, Bovidae, Suidae, Canidae, Felidae, and Muridae. Preferably, the object is a human.

[0193] As used herein, “substantially purified” cells are cells that are substantially free of other cell types. Substantially purified cells also refer to cells that have been isolated from other cell types that are normally associated with them in their natural state. In some cases, a substantially purified cell population refers to a homogeneous cell population. In other cases, this term refers only to cells that have been isolated from cells that are naturally associated with them in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0194] "Target site" or "target sequence" refers to the genomic nucleic acid sequence that defines the portion of the nucleic acid that binds to which the binding molecule can specifically bind under conditions sufficient to allow binding to occur.

[0195] "Target" refers to a cell, organ, or site in the body that needs treatment.

[0196] As used herein, the term "T cell receptor" or "TCR" refers to a membrane protein complex that participates in T cell activation in response to antigen presentation. The TCR is responsible for recognizing antigens that bind to the major histocompatibility complex molecule. The TCR consists of heterodimers of α (a) and β (β) chains, although in some cells it consists of γ and δ (γ / δ) chains. The TCR can exist in both α / β and γ / δ forms, which are structurally similar but have different anatomical locations and functions. Each chain consists of two extracellular domains and one variable and constant domain. In some embodiments, the TCR on any cell containing a TCR can be modified, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and γδ T cells.

[0197] As used herein, the term "therapeutic" means treatment and / or prevention. Therapeutic effects are achieved through the suppression, relief, or eradication of the disease state.

[0198] As used herein, the terms “transfection,” “transformation,” or “transduction” refer to the process of transferring or introducing exogenous nucleic acids into host cells. Cells that are “transfected,” “transformed,” or “transduced” are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. These cells include the original target cell and its progeny.

[0199] In this article, “treatment” means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by the subject.

[0200] As used in this article, the term "tumor" refers to the abnormal growth of tissue, which can be benign, precancerous, malignant, or metastatic.

[0201] As used in this article, the phrase “under transcriptional control” or “operably linked” means that the promoter is in the proper position and orientation relative to the polynucleotide to control the initiation of transcription by RNA polymerase and the expression of the polynucleotide.

[0202] A “vector” is a composition of substances containing isolated nucleic acids and capable of being used to deliver those isolated nucleic acids into the cell. Various vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term “vector” includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and analogues. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and similar vectors.

[0203] Scope: Throughout this disclosure, various aspects of the invention can be shown in the form of a scope. It should be understood that the scope description is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Therefore, the scope description should be considered as specifically disclosing all possible sub-ranges and individual values ​​within that range. For example, a scope description such as 1 to 6 should be considered as specifically disclosing sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual values ​​within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the scope.

[0204] describe Mounting evidence suggests that macrophages are abundant in the tumor microenvironment of various cancers, where they can be classically activated (M1, anti-tumor) or optionally activated (M2, pro-tumor) phenotypes. Macrophages are potent effectors of the innate immune system and are capable of at least three distinct anti-tumor functions: phagocytosis, cytotoxicity, and antigen presentation to orchestrate adaptive immune responses. While T cells require antigen-dependent activation via T cell receptors or chimeric immune receptors, macrophages can be activated in a variety of ways. Direct macrophage activation is antigen-independent, relying on mechanisms such as pathogen-associated molecular pattern recognition via Toll-like receptors (TLRs). Immune complex-mediated activation is antigen-dependent but requires the presence of antigen-specific antibodies and the absence of inhibitory CD47-SIRPα interactions.

[0205] Tumor-associated macrophages have been shown to undergo genetic rearrangement through the tumor microenvironment, becoming key immunosuppressive participants within it. Therefore, the ability to genetically engineer macrophages to prevent immunosuppressive genetic rearrangements would be a vertical advancement in this field.

[0206] This invention includes compositions and methods for treating malignant diseases. The invention includes expressing chimeric antigen receptors on monocytes, macrophages, or dendritic cells. These modified cells are recruited to the tumor microenvironment, where they act as effective immune effectors by infiltrating the tumor and killing target cells.

[0207] Chimeric antigen receptor (CAR) In one aspect of the invention, modified monocytes, macrophages, or dendritic cells are generated by expressing a CAR therein. Therefore, the invention includes a CAR and a nucleic acid construct encoding the CAR, wherein the CAR includes an antigen-binding domain, a transmembrane domain, and an intracellular domain.

[0208] On one hand, the present invention includes modified cells comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and a co-stimulatory molecule intracellular domain, and wherein the cell is a monocyte, macrophage, or dendritic cell with targeted effector activity. On the other hand, the present invention includes modified cells comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the nucleic acid sequence comprises a nucleic acid sequence encoding an antigen-binding domain, a nucleic acid sequence encoding a transmembrane domain, and a nucleic acid sequence encoding a co-stimulatory molecule intracellular domain, and wherein the cell is a monocyte, macrophage, or dendritic cell expressing the CAR and having targeted effector activity. In one embodiment, the targeted effector activity is directed against an antigen on a target cell that specifically binds to the antigen-binding domain of the CAR. In another embodiment, the targeted effector activity is selected from phagocytosis, cytotoxicity of the target cell, antigen presentation, and cytokine secretion.

[0209] Antigen-binding domain In one embodiment, the CAR of the present invention comprises an antigen-binding domain that binds to antigens on target cells. Examples of cell surface markers that can serve as antigens binding to the antigen-binding domain of the CAR include those associated with viral, bacterial and parasitic infections, autoimmune diseases, and cancer cells.

[0210] The selection of antigen-binding domains depends on the type and quantity of antigens present on the surface of the target cell. For example, antigen-binding domains may be selected to recognize antigens that act as cell surface markers on the target cell that are associated with a specific disease state.

[0211] In one embodiment, the antigen-binding domain binds to a tumor antigen, such as a specific antigen of a target tumor or cancer. In one embodiment, the tumor antigen of the present invention comprises one or more antigenic cancer epitopes. Non-limiting examples of tumor-associated antigens include CD19; CD123; CD22; CD30; CD171; CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn (Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD 117); Interleukin-13 receptor subunit α-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin-11 receptor α (IL-11Ra); Prostate stem cell antigen (PSCA); Protease serine 21 (Testisin or PRSS21); Vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; Platelet-derived growth factor receptor β (PDGFR-β); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Folate receptor α; Receptor tyrosine protein kinase ERBB2 (Her2 / neu); Mucin 1, Cell surface associated compound (MUC1); Epidermal growth factor receptor (EGFR); Neural cell adhesion molecule (NCAM); Prostase; Prostatic acid phosphatase (PAP); Mutated elongation factor 2 (ELF2M); Ephrin B2; Fibroblast activating protein α (FAP); Insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); Proteasome (Macropain) subunit β-type 9 (LMP2); Glycoprotein 100 (gp100); Oncogene fusion protein (bcr-abl) composed of the breakpoint cluster region (BCR) and Abelson murine leukemia virus oncogene homologue 1 (Abl); Tyrosinase; Ephrin type A receptor 2 (EphA2); Fucose GM1; Sialic acid Lewis adhesion molecule (sLe).Ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor β; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7 related compound (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C5 group D member (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); globoH (the hexasaccharide moiety of glycoceramide); breast differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cell receptor 1 (HAVCR1); Adrenaline receptor β3 (ADRB3); Pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); Lymphocyte antigen 6 complex, locus K9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCRγ alternating reading frame protein (TARP); Wilms tumor protein (WT1); Cancer / testis antigen 1 (NY-ESO-1); Cancer / testis antigen 2 (LAGE-1a); Melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene 6 located on chromosome 12p (ETV6-AML); Sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); Angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma testis antigen-1 (MAD-CT-1); melanoma testis antigen-2 (MAD-CT-2); Fos-associated antigen 1; tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen 1 recognized by T cells (MelanA or MART1); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma apoptosis inhibitor (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosamine transferase V (NA17); pairing box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myeloma virus oncogene neuroblastoma-derived homologue (MYCN); Ras Member of the same family, C (RhoC);Tyrosinase-associated protein 2 (TRP-2); Cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Sites), T cell-recognized squamous cell carcinoma antigen 3 (SART3); Pairing box protein Pax-5 (PAX5); Proacrosin-binding protein sp32 (OY-TES1); Lymphocyte-specific protein tyrosine kinase (LCK); Kinase-anchored protein 4 (AKAP-4); Synovial sarcoma X breakpoint 2 (SSX2); Receptor to advanced glycation end products (RAGE-1); Renal ubiquitous protein 1 (RU1); Renal ubiquitous protein 2 (RU2); Legumain; Human papillomavirus E6 (UPVE6); Human papillomavirus E7 (UPV E7); Intestinal carboxylesterase; Mutant heat shock protein 70-2 (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecular-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); mucin-like hormone receptor-like protein 2 containing EGF-like modules (EMR2); lymphocyte antigen 75 (LY75); phosphatidylinositol proteoglycan-3 (Glypican-3, GPC3); Fc receptor-like protein 5 (FCRL5); and immunoglobulin λ-like polypeptide 1 (IGLL1).

[0212] The antigen-binding domain may include any domain that binds to an antigen, and may include, but is not limited to, monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, non-human antibodies, and any fragments thereof. Thus, in one embodiment, the antigen-binding domain partially comprises a mammalian antibody or a fragment thereof. In another embodiment, the antigen-binding domain of the CAR is selected from anti-CD19 antibodies, anti-HER2 antibodies, and fragments thereof.

[0213] In some cases, the antigen-binding domain is derived from the same species in which the CAR will ultimately be used. For example, for use in humans, the antigen-binding domain of the CAR contains a human antibody, a humanized antibody, or a fragment thereof.

[0214] In some aspects of the invention, the antigen-binding domain is operatively linked to another domain of the CAR, such as a transmembrane domain or an intracellular domain, for expression in cells. In one embodiment, the nucleic acid encoding the antigen-binding domain is operatively linked to both a nucleic acid encoding the transmembrane domain and a nucleic acid encoding the intracellular domain.

[0215] Transmembrane domain Regarding transmembrane domains, CARs can be designed to include transmembrane domains that link the antigen-binding domain of the CAR to intracellular domains. In one embodiment, the transmembrane domain is naturally associated with one or more domains in the CAR. In some cases, the transmembrane domain may be selectively modified, or modified by amino acid substitution, to prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0216] The transmembrane domain can be derived from natural or synthetic sources. In the case of a natural source, the domain can originate from any membrane-binding or transmembrane protein. The transmembrane region specifically used in this invention can be derived from (i.e., comprising at least one or more of the following transmembrane regions): the α, β, or ζ chain of a T-cell receptor; CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154; Toll-like receptor 1 (TLR1); TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In some cases, various human hinges, including human Ig (immunoglobulin) hinges, can also be used.

[0217] In one embodiment, the transmembrane domain may be synthetic, in which case it will primarily contain hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain.

[0218] Intracellular domains The intracellular or cytoplasmic domains of CARs include intracellular domains similar to or identical to those of chimeric intracellular signaling molecules described elsewhere in this document, and are responsible for the activation of cells expressing CARs.

[0219] In one implementation, the intracellular domains of the CAR include domains responsible for signal activation and / or transduction.

[0220] Examples of intracellular domains used in this invention include, but are not limited to, cytoplasmic portions of surface receptors, co-stimulatory molecules, and any molecules that work together to initiate signal transduction in monocytes, macrophages, or dendritic cells, as well as any derivatives or variants of these elements and any synthetic sequences having the same functional capabilities.

[0221] Examples of intracellular domains include fragments or domains from one or more molecules or receptors, including but not limited to TCR, CD3ζ, CD3γ, CD3δ, CD3ε, CD86, universal FcRγ, FcRβ (FcεR1b), CD79a, CD79b, FcγRI1a, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that specifically bind to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, and NKp80. (KLRF1), CD127, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD 11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other costimulatory molecules described herein, any derivatives, variants or fragments thereof, any synthetic sequence of costimulatory molecules with the same activity, and any combination thereof.

[0222] In one embodiment, the intracellular domain of the CAR comprises dual signal transduction domains, such as 41BB, CD28, ICOS, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, CD116 receptor β chain, CSF1-R, LRP1 / CD91, SR-A1, SR-A2, MARCO, SR-CL1, SR-CL2, SR-C, SR-E, CR1, CR3, CR4, dectin1, DEC-205, DC-SIGN, CD14, CD36, LOX-1, CD11b, together with any combination of any of the signal transduction domains listed in the foregoing paragraphs. In another embodiment, the intracellular domain of the CAR comprises any portion of one or more co-stimulatory molecules, such as at least one signal transduction domain from CD3, the FcεRIγ chain, any derivative or variant thereof, any synthetic sequence thereof having the same functional capacity, and any combination thereof.

[0223] A spacer domain may be incorporated between the antigen-binding domain and the transmembrane domain of the CAR, or between the intracellular domain and the transmembrane domain of the CAR. As used herein, the term "spacer domain" generally means any oligopeptide or polypeptide that functions to link the transmembrane domain to the antigen-binding domain or the intracellular domain in a polypeptide chain. In one embodiment, the spacer domain may contain up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. In another embodiment, short oligopeptide or polypeptide linkers, preferably between 2 and 10 amino acids in length, may form a link between the transmembrane domain and the intracellular domain of the CAR. Examples of linkers include glycine-serine duplexes.

[0224] Human antibodies Human antibodies or fragments thereof may be preferred when utilizing the antigen-binding domain of a CAR. Fully human antibodies are particularly desirable for therapeutic treatment of human subjects. Human antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries obtained from human immunoglobulin sequences, including improvements to these techniques. See also U.S. Patents 4,444,887 and 4,716,111; and PCT disclosures WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741; each of which is incorporated herein by reference in its entirety.

[0225] Human antibodies can also be generated using transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes. For example, human heavy chain and light chain immunoglobulin gene complexes can be randomly or through homologous recombination introduced into mouse embryonic stem cells. Optionally, in addition to human heavy chain and light chain genes, human variable regions, constant regions, and diversity regions can also be introduced into mouse embryonic stem cells. The mouse heavy chain and light chain immunoglobulin genes can be rendered inactive, separately or simultaneously, by introducing human immunoglobulin gene loci through homologous recombination. For example, homozygous deletion of the antibody heavy chain linker (JH) gene in chimeric and germline mutant mice has been described as leading to complete suppression of endogenous antibody production. Modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then bred to produce homozygous offspring expressing human antibodies. The transgenic mice are immunized in a conventional manner with a selected antigen (e.g., all or part of the polypeptide of the present invention). Antibodies targeting selected targets can be obtained from immunized transgenic mice using conventional hybridoma techniques. Human immunoglobulin transgenes obtained from transgenic mice undergo rearrangement during B cell differentiation and subsequently experience class switching and somatic mutations. Therefore, this technique can be used to generate therapeutically useful IgG, IgA, IgM, and IgE antibodies, including but not limited to IgG1 (γ1) and IgG3. For an overview of this technique for generating human antibodies, see Lonberg and Huszar (Int. Rev. Immunol., 13:65-93 (1995)). For a detailed discussion of the techniques used to generate human antibodies and human monoclonal antibodies, and the protocols for producing such antibodies, see, for example, PCT Publications WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735; and U.S. Patents 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598, all of which are incorporated herein by reference in their entirety. Furthermore, companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) may engage in the use of techniques similar to those described above to provide human antibodies against selected antigens.For a specific discussion of transferring human germline immunoglobulin gene arrays in germline mutant mice that produce human antibodies upon antigen challenge, see, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993); and Duchosalet et al., Nature, 355:258 (1992).

[0226] Human antibodies can also be obtained from phage display libraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); Vaughan et al., Nature Biotech., 14:309 (1996)). Phage display technology (McCafferty et al., Nature, 348:552-553 (1990)) can be used to generate human antibodies and antibody fragments in vitro from a gene library of immunoglobulin variable (V) domains from unimmunized donors. According to this technology, antibody V domain genes are cloned in-frame into the major or minor coat protein genes of filamentous phages such as M13 or fd, and displayed as functional antibody fragments on the surface of phage particles. Since filamentous particles contain a single-stranded DNA copy of the phage genome, the selection based on the functional characteristics of the antibody also leads to the selection of the coding genes for antibodies that exhibit these characteristics. Therefore, phages mimic some characteristics of B cells. Phage display can be performed in various forms; for a review, see, for example, Johnson, Kevin S, and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of the V gene segment can be used for phage display. (Clackson et al., Nature, ...) 352:624-628 (1991) isolated multiple antibodies from a small randomized library of the V gene derived from the spleen of non-immunized mice. Zyrazole antibodies. A V gene library derived from non-immunized human donors can be constructed, and antibodies against a variety of antigens, including autoantigens, can be isolated substantially according to the techniques described in Marks et al., J. Mol. Biol., 222:581-597 (1991) or Griffith et al., EMBO J., 12:725-734 (1993). See also U.S. Patent Nos. 5,565,332 and 5,573,905, both of which are incorporated herein by reference in their entirety.

[0227] Human antibodies can also be produced in vitro by activating B cells (see U.S. Patent Nos. 5,567,610 and 5,229,275, both of which are incorporated herein by reference in their entirety). Human antibodies can also be produced in vitro using hybridoma techniques, such as, but not limited to, those described by Roder et al. (Methods Enzymol., 121:140-167 (1986)).

[0228] Humanized antibodies Optionally, in some embodiments, non-human antibodies may be humanized, wherein specific sequences or regions of the antibody are modified to increase similarity to naturally occurring human antibodies. For example, in this invention, the antibody or a fragment thereof may comprise a non-human mammalian scFv. In one embodiment, the antigen-binding domain is partially humanized.

[0229] Humanized antibodies can be generated using a variety of techniques known in the art, including but not limited to CDR transplantation (see, for example, European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and US Patent Nos. 5,225,539, 5,530,101 and 5,585,089, all of which are incorporated herein by reference in their entirety), overlay or resurfacing (see, for example, European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS, 91:969-973, all of which are incorporated herein by reference in their entirety), chain substitution (chainshuffling) (see, for example, U.S. Patent No. 5,565,332, which is incorporated herein by reference in its entirety), and in, for example, U.S. Patent Application Publication No. US2005 / 0042664, U.S. Patent Application Publication No. US2005 / 0048617, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84 The techniques disclosed in (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu JS, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994) are all incorporated herein by reference in their entirety. Typically, the framework residues in the framework region are replaced by corresponding residues from the CDR donor antibody to alter, preferably improve, antigen binding.These framework substitutions are determined using methods well-known in the art, such as modeling the interaction between CDRs and framework residues to identify framework residues important for antigen binding and sequence comparison to identify aberrant framework residues at specific locations. (See, for example, Queen et al., U.S. Patent No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, both of which are incorporated herein by reference.) Humanized antibodies contain one or more amino acid residues introduced from non-human sources. These non-human amino acid residues are often referred to as "input" residues and are generally derived from "input" variable domains. Therefore, humanized antibodies contain one or more CDRs from non-human immunoglobulin molecules and a framework region from humans. Humanization of antibodies is well known in the art and can be performed largely according to the methods of Winter and his colleagues (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence, i.e., CDR transplantation (EP 239,400; PCT Publication No. WO 91 / 09967; and U.S. Patent Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640; the contents of which are incorporated herein by reference in their entirety). In such humanized chimeric antibodies, sequences significantly smaller than the full-body human variable domain have been replaced by corresponding sequences from non-human species. In practice, humanized antibodies are generally human antibodies in which some CDR residues and possibly some framework (FR) residues have been replaced by residues from similar sites in rodent antibodies. Antibody humanization can also be achieved by overlay and surface repair (EP592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) or chain substitution (US Patent No. 5,565,332), the contents of which are incorporated herein by reference in their entirety.

[0230] The selection of human variable domains (light and heavy chains) used for preparing humanized antibodies aims to reduce antigenicity. Following a so-called "best-fit" approach, sequences for rodent antibody variable domains are screened against known human variable domain sequences throughout the library. The human sequence closest to the rodent sequence is then accepted as the human frame (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987), the entire contents of which are incorporated herein by reference). Another approach utilizes specific frames obtained from common sequences of all human antibodies with specific subgroups of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993), the contents of which are incorporated herein by reference in their entirety).

[0231] Antibodies can be humanized, retaining high affinity for target antigens and possessing other advantageous biological properties. According to one aspect of the invention, humanized antibodies are prepared by analyzing the parent sequence and various conceptual humanized products using a three-dimensional model of the parent sequence and the humanized sequence. Three-dimensional immunoglobulin models are commonly used and well-known to those skilled in the art. Computer programs are available that exemplify and demonstrate possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. These demonstrations allow for the analysis of the potential influence of residues on the function of the candidate immunoglobulin sequence, i.e., the analysis of residues affecting the ability of the candidate immunoglobulin to bind to the target antigen. In this way, FR residues can be selected and combined from the receptor sequence and the input sequence to obtain desired antibody characteristics, such as increased affinity for the target antigen. Overall, CDR residues are directly and most substantially involved in influencing antigen binding.

[0232] Humanized antibodies retain similar antigen specificity to the original antibodies. However, using certain humanization methods, the affinity and / or specificity of the antibody binding to the target antigen can be increased using "directed evolution" methods, as described in Wu et al., J. Mol. Biol., 294:151 (1999), the entire contents of which are incorporated herein by reference.

[0233] carrier As described elsewhere in this document, CARs can be introduced into monocytes, macrophages, or dendritic cells using vectors. In one aspect, the present invention includes vectors comprising nucleic acid sequences encoding the CARs described herein. In one embodiment, the vectors include plasmid vectors, viral vectors, retrotransposons (e.g., piggyback, Sleeping Beauty), site-directed insertion vectors (e.g., CRISPR, Zn finger nucleases, TALEN), or suicide expression vectors, or other vectors known in the art.

[0234] All of the above constructs can be used with third-generation lentiviral vector plasmids approved for human cells, other viral vectors, or RNA. In one embodiment, the vector is a viral vector, such as a lentiviral vector. In another embodiment, the vector is an RNA vector.

[0235] The generation of any molecule described herein can be verified by sequencing. Expression of full-length proteins can be verified using immunoblotting, immunohistochemistry, flow cytometry, or other techniques known and available in the art.

[0236] The present invention also provides vectors in which the DNA of the present invention is inserted. Vectors, including those derived from retroviruses such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for the long-term stable integration of transgenes and their spread in daughter cells. Lentiviral vectors have the added advantage of being able to transduce non-proliferating cells, such as hepatocytes, compared to onco-retroviruses. They also have the added advantage of producing low immunogenicity in the target organism.

[0237] Expression of natural or synthetic nucleic acids is generally achieved by operatively linking the nucleic acid or a portion thereof to a promoter and incorporating the construct into an expression vector. A vector is a generally capable entity that can replicate in mammalian cells and / or integrate into the mammalian cellular genome. A typical vector contains transcription and translation terminators, a start sequence, and a promoter that can be used to regulate the expression of the desired nucleic acid sequence.

[0238] Nucleic acids can be cloned into any number of different types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phage particles, phage derivatives, animal viruses, and entrapments. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0239] Expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art and has been described in, for example, Sambrook et al., 2012, *MOLECULAR CLONING: A LABORATORY MANUAL*, Volumes 1-4, Cold Spring Harbor Press, NY, and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more optional markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0240] Other promoter elements, such as enhancers, regulate the frequency of transcription initiation. These are typically located 30–110 bp upstream of the start site, although recent studies have shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, allowing promoter function to be maintained even when elements are inverted or moved relative to each other. In the thymidine kinase (TK) promoter, the spacing between promoter elements can increase to 50 bp before activity begins to decline. Depending on the promoter, individual elements can act collectively or independently to activate transcription.

[0241] An example of a promoter is the immediate early cell cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked to it. However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, elongation factor-1α promoter, and human gene promoters—such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the invention is not limited to the use of constitutive promoters. Inducible promoters are also considered as part of the invention. The use of inducible promoters provides a molecular switch capable of turning on the expression of the polynucleotide sequence operatively linked to it (when such expression is desired) or turning off such expression (when expression is not desired). Examples of inducible promoters include, but are not limited to, metallo-thione promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0242] To assess the expression of a peptide or its fraction, the expression vector to be introduced into cells may also contain a selective marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells intended to be transfected or infected via a viral vector. In other respects, selective markers may be carried on a separate segment of DNA and used in co-transfection procedures. Both selective markers and reporter genes may have appropriate regulatory sequences flanking them to enable expression in host cells. Useful selective markers include, for example, antibiotic resistance genes such as neo and analogues.

[0243] Reporter genes are used to identify potentially transfected cells and evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by a recipient organism or tissue and encodes a polypeptide whose expression is manifested by a readily detectable property, such as enzymatic activity. Reporter gene expression is assessed at an appropriate time after DNA has been introduced into recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Teiet al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well-known and can be prepared using known techniques or are commercially available. Generally, a construct with a minimal 5' flanking region that indicates the highest expression level of the reporter gene is identified as the promoter. Such promoter regions can be linked to the reporter gene and used to evaluate the ability of the agent to regulate promoter-driven transcription.

[0244] Introduction of nucleic acids On one hand, the present invention includes a method for modifying cells, comprising introducing a chimeric antigen receptor (CAR) into monocytes, macrophages, or dendritic cells, wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and a co-stimulatory molecule intracellular domain, and wherein the cells are monocytes, macrophages, or dendritic cells expressing the CAR and possessing targeted effector activity. In one embodiment, introducing the CAR into the cells comprises introducing a nucleic acid sequence encoding the CAR. In another embodiment, introducing the nucleic acid sequence comprises electroporating mRNA encoding the CAR.

[0245] Methods for introducing and expressing genes such as CARs in cells are known in the art. In the case of expression vectors, the vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0246] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, and similar methods. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Volumes 1–4, Cold Spring Harbor Press, NY). Nucleic acids can be introduced into target cells using commercially available methods, including electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), and multiporator (Eppendort, Hamburg, Germany). Nucleic acids can also be introduced into cells using cationic liposome-mediated transfection, lipid transfection, polymer encapsulation, peptide-mediated transfection, or biological projectile particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum GeneTher., 12(8):861-70 (2001)).

[0247] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. RNA vectors include vectors having an RNA promoter and / or other relevant domains for producing RNA transcripts. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex viruses, adenoviruses, and adeno-associated viruses and analogues. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0248] Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems—including oil-in-water emulsions, microcells, hybrid microcells, and liposomes. An exemplary colloidal system used as a delivery medium in vitro and in vivo is the liposome (e.g., an artificial membrane vesicle).

[0249] In the case of using non-viral delivery systems, an exemplary delivery medium is liposomes. The use of lipid formulations is considered for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). Alternatively, nucleic acids can be associated with lipids. Lipid-associated nucleic acids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linkers that bind to both liposomes and oligonucleotides, entrapped within liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in lipids as a suspension, contained or complexed within microparticles, or otherwise associated with lipids. Lipid, lipid / DNA, or lipid / expression vector-associated compositions are not limited to any specific structure in solution. For example, they can exist in a bilayer structure, such as microparticles, or have a “collapsed” structure. They can also be simply dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids can be naturally occurring fatty substances or synthetic lipids. For example, lipids include fat droplets that are naturally present in the cytoplasm, as well as compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0250] Suitable lipids are available from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) is available from Sigma, St. Louis, MO; dihexadecanyl phosphate (“DCP”) is available from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) is available from Calbiochem-Behring; and dimyristyl phosphatidylglycerol (“DMPG”) and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, AL.). The lipid stock solution in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. “Liposome” is a general concept encompassing various monolayer and multilayer lipid media formed by the formation of closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure—a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. It forms spontaneously when phospholipids are suspended in excess aqueous solution. The lipid components undergo self-rearrangement before the formation of the closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions with solution structures different from typical vesicle structures are also included. For example, lipids may present as clusters or simply as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also considered.

[0251] Regardless of the method used to introduce exogenous nucleic acids into host cells or otherwise expose cells to the molecules described herein, a variety of assays can be performed to confirm the presence of nucleic acids in host cells. These assays include, for example, "molecular biology" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; and "biochemical" assays, such as assays to detect the presence or absence of a specific peptide—e.g., by immunological means (ELISA and Western blotting) or by assays described herein to identify agents falling within the scope of this invention.

[0252] In one embodiment, one or more nucleic acid sequences are introduced by a method selected from: transduced cell populations, transfected cell populations, and electroporated cell populations. In one embodiment, the cell population comprises one or more of the nucleic acid sequences described herein.

[0253] In one embodiment, the nucleic acid introduced into the cell is RNA. In another embodiment, the RNA is mRNA, including in vitro transcribed RNA or synthetic RNA. RNA is produced by in vitro transcription using a template generated by polymerase chain reaction (PCR). The target DNA from any source can be directly converted into a template by PCR for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The DNA source can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequences, or any other suitable DNA source. The desired template for in vitro transcription is CAR.

[0254] PCR can be used to generate templates for in vitro transcription of mRNA, which is then introduced into cells. Methods for performing PCR are well known in the art. Primers used for PCR are designed to have regions substantially complementary to the DNA region used as the PCR template. As used herein, “substantially complementary” means a nucleotide sequence in which most or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary or mismatched. Substantially complementary sequences are capable of annealing or hybridizing with the target DNA under annealing conditions used for PCR. Primers can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify gene portions (open reading frames) that are normally transcribed in cells—including the 5' and 3' UTRs. Primers can also be designed to amplify gene portions encoding specific target domains. In one embodiment, primers are designed to amplify the coding region of human cDNA—including all or part of the 5' and 3' UTRs. Primers that can be used for PCR are generated by synthetic methods well known in the art. A “forward primer” is a primer containing a nucleotide region substantially complementary to the nucleotides upstream of the DNA sequence to be amplified on the DNA template. "Upstream" in this document refers to the 5' position of the DNA sequence to be amplified relative to the coding strand. "Reverse primer" is a primer containing a nucleotide region substantially complementary to the double-stranded DNA template downstream of the DNA sequence to be amplified. "Downstream" in this document refers to the 3' position of the DNA sequence to be amplified relative to the coding strand.

[0255] Chemical structures capable of promoting RNA stability and / or translation efficiency can also be utilized. The RNA preferably has 5' and 3' UTRs. In one embodiment, the length of the 5' UTR is between 0 and 3000 nucleotides. The lengths of the 5' and 3' UTR sequences to be added to the coding region can be altered by various methods, including, but not limited to, designing PCR primers that anneal to different regions of the UTR. Using this method, those skilled in the art can modify the 5' and 3' UTR lengths required for optimal translation efficiency after transfection of the transcribed RNA.

[0256] The 5' and 3' UTRs can be naturally occurring endogenous 5' and 3' UTRs of the target gene. Alternatively, non-endogenous UTR sequences for the target gene can be added by incorporating the UTR sequence into forward and reverse primers or through any other modification of the template. The use of non-endogenous UTR sequences for the target gene can be effectively used to improve RNA stability and / or translation efficiency. For example, it is known that AU-rich elements in the 3' UTR sequence can reduce mRNA stability. Therefore, the 3' UTR can be selected or designed based on UTR properties known in the art to increase the stability of transcribed RNA.

[0257] In one embodiment, the 5'UTR may contain the Kozak sequence of an endogenous gene. Alternatively, when a non-endogenous 5'UTR for the target gene is added via PCR as described above, a shared Kozak sequence can be redesigned by adding a 5'UTR sequence. Kozak sequences can improve the translation efficiency of some RNA transcripts, but it appears that not all RNAs require them for efficient translation. The need for Kozak sequences in various mRNAs is known in the art. In other embodiments, the 5'UTR may be derived from an RNA virus whose RNA genome is stable in the cell. In other embodiments, various nucleotide analogs may be applied in the 3' or 5'UTR to inhibit exonuclease degradation of the mRNA.

[0258] To achieve RNA synthesis from a DNA template without gene cloning, a transcription promoter should be attached to the DNA template upstream of the sequence to be transcribed. When the sequence acting as an RNA polymerase promoter is added to the 5' end of the forward primer, the RNA polymerase promoter will be incorporated into the PCR product upstream of the open reading frame to be transcribed. In one embodiment, the promoter is the T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. The common nucleotide sequences of the T7, T3, and SP6 promoters are known in the art.

[0259] In one implementation, the mRNA has caps at both the 5' end and the 3' poly(A) tail, which determine ribosome binding, translation initiation, and mRNA stability in the cell. On a circular DNA template (e.g., plasmid DNA), RNA polymerase produces a long, cascaded product unsuitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the 3' UTR end produces a normal-sized mRNA that is ineffective for eukaryotic transfection—even if it is polyadenylated post-transcriptionally.

[0260] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003)).

[0261] The conventional method for integrating poly-A / T segments into DNA templates is molecular cloning. However, poly-A / T sequences integrated into plasmid DNA can lead to plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes the cloning process not only laborious and time-consuming but also often unreliable. Therefore, there is a strong desire for methods that allow the construction of DNA templates using poly-A / T 3' segments without the need for cloning.

[0262] Poly(A) / T segments of the transcribed DNA template can be generated during PCR using reverse primers containing poly(T) tails, such as 100T tails (sizes can be 50-5000T), or generated after PCR by any other method, including but not limited to DNA ligation or in vitro recombination. The poly(A) tail also contributes to RNA stability and reduces its degradation. Generally, the length of the poly(A) tail is positively correlated with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosine.

[0263] Following in vitro transcription using a poly(A) polymerase such as E. coli poly(A) polymerase (E-PAP), the poly(A) tail of RNA can be further extended. In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in approximately a two-fold increase in RNA translation efficiency. Additionally, the attachment of different chemical groups to the 3' end can increase mRNA stability. This attachment can include modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using a poly(A) polymerase. ATP analogs can further increase RNA stability.

[0264] The 5' cap also provides stability to the RNA molecule. In a preferred embodiment, the RNA produced by the methods disclosed herein includes a 5' cap. The 5' cap is provided using techniques known in the art and described herein (Cougot, et al., Trendsin Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0265] The RNA produced by the methods disclosed herein may also contain an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA and promotes translation initiation. Any solute suitable for cell electroporation may be included, and it may contain factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

[0266] Several in vitro transcribed RNA (IVT-RNA) vectors are known in the literature, which are used as templates for in vitro transcription in a standardized manner and have been genetically modified to produce stable RNA transcripts. The current approach in this field is based on plasmid vectors with the following structure: a 5' RNA polymerase promoter capable of RNA transcription, a target gene—with its 3' and / or 5' flanking regions being untranslated regions (UTRs), and a 3' polyadenylated cassette containing 50–70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenylated cassette by a type II restriction enzyme (the recognition sequence corresponds to the cleavage site). The polyadenylated cassette thus corresponds to the subsequent poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain part of the enzyme cleavage site after linearization, extending or masking the poly(A) sequence at the 3' end. It is unclear whether this non-physiological overhang affects the amount of protein produced intracellularly by this construct.

[0267] On one hand, RNA constructs are delivered into cells via electroporation. See, for example, formulations and methods for electroporating nucleic acid constructs into mammalian cells taught in US 2004 / 0014645, US 2005 / 0052630A1, US 2005 / 0070841A1, US 2004 / 0059285A1, and US 2004 / 0092907A1. Various parameters, including electric field strength, required for electroporation of any known cell type are generally known in relevant research literature in the art and in numerous patents and applications. See, for example, US Patent Nos. 6,678,556, 7,171,264, and 7,173,116. Devices for therapeutic applications of electroporation are commercially available, such as the MedPulser™ DNA Electroporation Therapeutic System (Inovio / Genetronics, San Diego, Calif.), and are described in patents such as U.S. Patent Nos. 6,567,694, 6,516,223, 5,993,434, 6,181,964, 6,241,701, and 6,233,482. Electroporation can also be used for in vitro cell transfection, as described in US20070128708A1. Electroporation can also be used for the in vitro delivery of nucleic acids into cells. Therefore, electroporation-mediated delivery of nucleic acids in cells, including expression constructs, using any of the various available devices and electroporation systems known to those skilled in the art presents exciting new means of delivering target RNA to target cells.

[0268] Cell source In one embodiment, phagocytes are used in the compositions and methods described herein. The phagocytes, such as monocytes, macrophages, and / or dendritic cells, are obtained from the subject. Non-limiting examples of the subject include humans, dogs, cats, mice, rats, and their transgenic species. Preferably, the subject is a human. Cells can be obtained from a variety of sources, including peripheral blood monocytes, bone marrow, lymph node tissue, spleen tissue, umbilical cord, and tumors. In some embodiments, any number of monocytes, macrophages, dendritic cells, or progenitor cell lines available in the art can be used. In some embodiments, cells can be obtained from blood units collected from the subject using any number of techniques known to those skilled in the art, such as Ficoll separation. In one embodiment, cells from an individual's circulating blood are obtained by blood separation or leukocyte extraction. Blood separation products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells (white blood cells), red blood cells (red blood cells), and platelets. Cells collected via blood separation can be washed to remove plasma components and placed in appropriate buffers or media, such as phosphate-buffered saline (PBS) or wash buffers—which are calcium-deficient and may be magnesium-deficient or lack multiple (if not all) divalent cations—for subsequent processing steps. After washing, the cells can be resuspended in various biocompatible buffers, such as calcium- and magnesium-free PBS. Alternatively, unwanted components in the blood separation sample can be removed, and the cells can be directly resuspended in a culture medium.

[0269] In another embodiment, cells are separated from peripheral blood by lysing blood red blood cells and depleting lymphocytes and blood red blood cells (e.g., by PERCOLL™ gradient centrifugation). Optionally, cells can be separated from the umbilical cord. In any case, specific subpopulations of monocytes, macrophages, and / or dendritic cells can be further separated using positive or negative selection techniques.

[0270] The mononuclear cells isolated in this way may not contain cells expressing certain antigens, including but not limited to CD34, CD3, CD4, CD8, CD14, CD19, or CD20. The depletion of these cells can be accomplished using isolated antibodies, biological samples containing antibodies such as ascites, antibodies bound to physical supports, and antibodies bound to cells.

[0271] Enrichment of monocyte, macrophage, and / or dendritic cell populations resulting from negative selection can be achieved using antibody combinations targeting specific surface markers of the negatively selected cells. Preferred methods include cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry, which utilizes a mixture of monoclonal antibodies against surface markers present on the negatively selected cells. For example, enrichment of monocyte, macrophage, and / or dendritic cell populations resulting from negative selection can be achieved using a mixture of monoclonal antibodies, typically including antibodies against CD34, CD3, CD4, CD8, CD14, CD19, or CD20.

[0272] During the separation of the desired cell population via positive or negative selection, the concentrations of cells and surfaces (e.g., particles, such as beads) can vary. In some embodiments, it is desirable to significantly reduce the volume in which beads and cells are mixed together (i.e., increase the cell concentration) to ensure maximum contact between cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In another embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, a concentration greater than 100 million cells / ml is used. In a further embodiment, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells / ml are used. In yet another embodiment, cell concentrations of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells / ml are used. In a further embodiment, a concentration of 125 million or 150 million cells / ml may be used. Using high concentrations of cells can lead to increased cell yield, cell activation, and cell proliferation.

[0273] In one embodiment, the cell population includes the monocytes, macrophages, or dendritic cells of the present invention. Examples of cell populations include, but are not limited to, purified populations of peripheral blood mononuclear cells, umbilical cord blood cells, monocytes, macrophages, or dendritic cells, as well as cell lines. In another embodiment, the peripheral blood mononuclear cells include a population of monocytes, macrophages, or dendritic cells. In yet another embodiment, the purified cells include a population of monocytes, macrophages, or dendritic cells.

[0274] In another embodiment, the cells have upregulated an M1 marker and downregulated an M2 marker. For example, at least one M1 marker, such as HLA DR, CD86, CD80, and PDL1, is upregulated in phagocytes. In another instance, at least one M2 marker, such as CD206 and CD163, is downregulated in phagocytes. In one embodiment, the cells have at least one upregulated M1 marker and at least one downregulated M2 marker.

[0275] In another embodiment, the target effector activity in phagocytes is enhanced by inhibiting CD47 or SIRPα activity. CD47 and / or SIRPα activity can be inhibited by treating phagocytes with anti-CD47 or anti-SIRPα antibodies. Alternatively, CD47 or SIRPα activity can be inhibited by any method known to those skilled in the art.

[0276] Cell expansion In one embodiment, cells or cell populations comprising monocytes, macrophages, or dendritic cells are cultured for expansion. In another embodiment, cells or cell populations comprising progenitor cells are cultured for differentiation and expansion of monocytes, macrophages, or dendritic cells. This invention includes expanding populations of monocytes, macrophages, or dendritic cells containing chimeric antigen receptors as described herein.

[0277] The data disclosed herein demonstrate that cells can be amplified by the methods disclosed herein by approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 100,000, 1,000,000, 10,000,000 or more, and any and all whole or partial integers therebetween. In one embodiment, cell amplification is in the range of approximately 20 to approximately 50 times.

[0278] After culturing, cells can be cultured in cell culture medium in a culture device for a certain period of time or until the cells reach a high cell density of confluence or optimal passage, and then transferred to another culture device. This culture device can be any culture device commonly used for in vitro cell culture. Preferably, the confluence level is 70% or higher before transferring the cells to another culture device. More preferably, the confluence level is 90% or higher. The time period can be any time suitable for in vitro cell culture. The culture medium can be changed at any time during cell culture. Preferably, the culture medium is changed approximately every 2 to 3 days. The cells are then harvested from the culture device, after which they can be used immediately or stored for later use.

[0279] The culture steps described herein (in contact with the agents described herein) can be very short, for example, less than 24 hours, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culture steps further described herein (in contact with the agents described herein) can be longer, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

[0280] In one embodiment, cells may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. Suitable conditions for cell culture include appropriate culture media (e.g., macrophage complete medium, DMEM / F12, DMEM / F12-10 (Invitrogen)) which may contain factors required for proliferation and viability, including serum (e.g., fetal bovine or human serum), L-glutamine, insulin, M-CSF, GM-CSF, IL-10, IL-12, IL-15, TGF-β, and TNF-α, or any other additives known to those skilled in the art for cell growth. Other additives for cell growth include, but are not limited to, surfactants, plasma protein powder, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. Culture media may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, and supplemented with amino acids, sodium pyruvate, and vitamins, serum-free or supplemented with adequate serum (or plasma) or a defined group of hormones, and / or sufficient amounts of cytokines (one or more) for cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are included only in the experimental culture and not in the cell culture to be perfused into the target. Target cells are maintained under conditions necessary for growth, such as a suitable temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2).

[0281] Culture media used for culturing cells may include agents that can activate cells. For example, agents that activate monocytes, macrophages, or dendritic cells are known in the art to be included in the culture medium.

[0282] treat The modified cells described herein can be included in compositions for use in therapeutic subjects. In one aspect, the composition comprises modified cells containing the chimeric antigen receptor described herein. The composition may include a pharmaceutical composition and further include a pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition containing the modified cells may be administered.

[0283] In one aspect, the present invention includes a method for treating a subject with a tumor or cancer-related disease or condition, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified cells described herein. In another aspect, the present invention includes a method for treating a subject with a solid tumor, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified cells described herein. In yet another aspect, the present invention includes a method for stimulating an immune response in a subject against target tumor cells or tumor tissue, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified cells described herein. In yet another aspect, the present invention includes the use of the modified cells described herein in the preparation of a medicament for treating an immune response in a subject in need. In yet another aspect, the present invention includes the use of the modified cells described herein in the preparation of a medicament for treating a tumor or cancer in a subject in need.

[0284] The modified cells generated as described herein possess targeted effector activity. In one embodiment, the modified cells possess targeted effector activity against antigens on target cells, such as through specific binding to the antigen-binding domain of a CAR. In another embodiment, the targeted effector activity includes, but is not limited to, phagocytosis, cytotoxicity of the target cell, antigen presentation, and cytokine secretion.

[0285] In another embodiment, the modified cells described herein have the ability to deliver an agent, biological agent, or therapeutic agent to a target. Cells may be modified or engineered to deliver an agent to a target, wherein the agent is selected from nucleic acids, antibiotics, anti-inflammatory agents, antibodies or antibody fragments thereof, growth factors, cytokines, enzymes, proteins, peptides, fusion proteins, synthetic molecules, organic molecules, carbohydrates or analogs, lipids, hormones, microsomes, derivatives or variants thereof, and any combination thereof. As a non-limiting example, macrophages modified with a CAR targeting a tumor antigen are capable of secreting agents such as cytokines or antibodies to assist macrophage function. Antibodies, such as anti-CD47 / anti-SIRPα mAB, may also assist macrophage function. In yet another example, macrophages modified with a CAR targeting a tumor antigen are engineered to encode siRNA that assists macrophage function by downregulating a repressor gene (i.e., SIRPα). In yet another example, CAR macrophages are engineered to express a dominant-negative (or otherwise mutated) form of a receptor or enzyme that assists macrophage function.

[0286] In one embodiment, the macrophages are modified with multiple genes, including at least one gene comprising a CAR and at least one other gene containing genetic elements that enhance the function of the CAR macrophages. In another embodiment, the macrophages are modified with multiple genes, including at least one gene comprising a CAR and at least one other gene assisting or programmatically rearranging the function of other immune cells, such as T cells within the tumor microenvironment.

[0287] Furthermore, the modified cells can be administered to animals, preferably mammals, and even more preferably humans, to suppress immune responses, such as those common in autoimmune diseases like diabetes, psoriasis, rheumatoid arthritis, multiple sclerosis, GVHD, enhanced allograft tolerance induction, graft rejection, and similar diseases. Additionally, the cells of the present invention can be used to treat any condition in which a reduced or otherwise suppressed immune response, particularly a cell-mediated immune response, is desired for the treatment or alleviation of the disease. In one aspect, the present invention includes conditions in the treatment subject, such as autoimmune diseases, including administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the cell populations described herein. Furthermore, the cells of the present invention can be administered as pretreatment or conditioning prior to treatment with alternative anticancer immunotherapies (including, but not limited to, CAR T cells, tumor-infiltrating lymphocytes, or checkpoint inhibitors).

[0288] Examples of autoimmune diseases include, but are not limited to, acquired immunodeficiency syndrome (AIDS, a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, and celiac sprue-dermatitis. Hepetiformis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, Crest syndrome, Crohn's disease, Degos disease, juvenile dermatomyositis, discoid lupus, idiopathic mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Graves-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia. (anemia), polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Rett syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive systemic sclerosis (PSS), also known as systemic sclerosis (SS)), Sjogren's syndrome, stiff-person syndrome, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vitiligo, and Wegener's granulomatosis.

[0289] These cells can also be used to treat inflammatory diseases. Examples of inflammatory diseases include, but are not limited to, chronic and acute inflammatory conditions. Examples of inflammatory diseases include Alzheimer's disease, asthma, atopic dermatitis, allergies, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft-versus-host disease, hemolytic anemia, osteoarthritis, sepsis, stroke, tissue and organ transplantation, vasculitis, diabetic retinopathy, and ventilator-induced lung injury.

[0290] The cells of this invention can be used to treat cancer. Cancer includes tumors that are not vascularized or have not yet become substantially vascularized, as well as vascularized tumors. Cancer can include non-solid tumors (such as hematologic malignancies, such as leukemia and lymphoma) or can include solid tumors. Types of cancer treated with the cells of this invention include, but are not limited to, carcinomas, germ cell tumors, and sarcomas, as well as certain leukemias or lymphomas, benign and malignant tumors, and malignancies such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and childhood tumors / cancers are also included.

[0291] Solid tumors are abnormal masses of tissue that do not typically contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors (such as sarcoma and carcinoma) include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoma, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, Wiltoma, etc. MS tumors, cervical cancer, testicular tumors, seminoma, bladder cancer, melanoma, and CNS tumors (such as gliomas (e.g., brainstem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germ cell tumor, medulloblastoma, schwannoma, craniopharyngioma, ependymoma, pineal tumor, angioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma, and brain metastases).

[0292] Blood cancers are cancers of the blood or bone marrow. Examples of blood (or hematogenous) cancers include leukemia—including acute leukemia (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic leukemia, and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (painless and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and myelodysplastic syndromes.

[0293] The cells of the present invention can be administered at doses, routes, and frequencies determined in appropriate preclinical and clinical trials and experiments. The cell compositions can be administered multiple times at doses within these ranges. Administration of the cells of the present invention can be combined with other methods determined by those skilled in the art for the treatment of desired diseases or conditions.

[0294] The cells of the present invention to be given can be autologous, allogeneic, or xenogeneic relative to the object of treatment.

[0295] The cells of this invention can be administered in any convenient manner known to those skilled in the art. The cells of this invention can be administered to the recipient via aerosol inhalation, injection, ingestion, transfer, implantation, or transplantation. The compositions described herein can be administered via artery, subcutaneous, intradermal, intratumoral, intranodular, intramedullary, intramuscular, or intravenous routes. iv .) Administered by injection or intraperitoneal administration. In other cases, the cells of the present invention are injected directly into sites of inflammation, localized disease sites, lymph nodes, organs, tumors, and similar locations in the subject.

[0296] Pharmaceutical Composition The pharmaceutical compositions of the present invention may comprise the cells described herein—in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0297] The pharmaceutical compositions of the present invention can be administered in a manner suitable for treating (or preventing) a disease. The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the disease, although appropriate dosage can be determined through clinical trials.

[0298] When referring to "immunely effective dose," "anti-immune response effective dose," "immune response suppression effective dose," or "therapeutic dose," the exact amount of the composition of the present invention to be administered can be determined by a physician taking into account individual differences in the patient's (subject's) age, weight, immune response, and condition. Generally speaking, a pharmaceutical composition comprising the cells described herein can be administered in doses of 10... 4 Up to 10 9 Cells / kg body weight, preferably 10 5 Up to 10 6A dose of cells per kg body weight—including all integer values ​​within these ranges—is administered. The cell compositions described herein can also be administered multiple times at these doses. Cells can be administered using infusion techniques well-known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a specific patient can be readily determined by those skilled in the art through monitoring the patient's disease signs and adjusting treatment accordingly.

[0299] In some embodiments, it is desirable to administer monocytes, macrophages, or dendritic cells to the subject, followed by a subsequent re-drawing of blood (or blood separation) according to the invention to activate the monocytes, macrophages, or dendritic cells therefrom, and to re-infuse the patient with these activated cells. This process can be performed multiple times every few weeks. In some embodiments, cells can be activated from blood draws ranging from 10 ml to 400 ml. In some embodiments, cells are activated from blood draws of 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, or 100 ml. Without being bound by theory, this multiple blood draw / re-infusion protocol allows for the selection of specific cell populations.

[0300] In some embodiments of the invention, cells are modified using the methods described herein or other methods known in the art, wherein the cells are expanded to therapeutic levels in conjunction with (e.g., before, during, or after) any number of relevant treatment modalities administered to the patient, including but not limited to pharmaceutical treatments such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab treatment—for MS patients—or for PML patients. In a further embodiment, the cells of the present invention can be used in combination with CAR-T cell therapy, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies or other immunoablative agents such as the anti-CD52 antibody alemtuzumab (CAM PATH), anti-CD3 antibodies or other antibody therapies, cytotoxins, fludaribine, cyclosporine, FK506, rapamycin, mycophenolate mofetil, steroids, FR901228, cytokines, and irradiation. These drugs inhibit the calcium-dependent phosphatase troponin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important for growth factor-induced signaling. (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). In a further embodiment, the cell composition of the present invention is administered to the patient in conjunction with (e.g., before, during, or after) bone marrow transplantation, lymphocyte ablation therapy—using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, Rituxan, or antibodies such as OKT3 or CAMPATH. For example, in one embodiment, the subject may undergo standard treatment of high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives an infusion of the cells of the present invention after transplantation. In other embodiments, the cells may be administered before or after surgery.

[0301] The dosage of the above treatment to be administered will vary depending on the condition to be treated and the exact attributes of the treatment recipient. Dosage scaling for humans can be done according to practices accepted in the art. For example, for adult patients, the dosage of CAMPATH is generally in the range of 1 to 100 mg, usually administered daily for 1 to 30 days. The preferred daily dose is 1 to 10 mg, although in some cases larger doses of up to 40 mg per day may be used (described in U.S. Patent No. 6,120,766).

[0302] It should be understood that the methods and compositions used in this invention are not limited to the specific formulations presented in the examples. The examples below are presented to provide those skilled in the art with a complete disclosure and description of how to prepare and use the cells, amplification and culture methods, and therapeutic methods of this invention, and are not intended to limit the scope of what the inventors consider to be their invention.

[0303] Unless otherwise stated, the practice of this invention utilizes conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, all of which are well within the knowledge of those skilled in the art. Such techniques are well explained in the literature, such as "Molecular Cloning: A Laboratory Manual", fourthedition (Sambrook, 2012); "Oligonucleotide Synthesis" (Gait, 1984); "Culture of Animal Cells" (Freshney, 2010); "Methods in Enzymology"; "Handbook of Experimental Immunology" (Weir, 1997); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Short Protocols in Molecular Biology" (Ausubel, 2002); "Polymerase Chain Reaction: Principles, Applications and Troubleshooting" (Babar, 2011); and "Current Protocols in Immunology" (Coligan, 2002). These techniques are applicable to the generation of the polynucleotides and peptides of this invention and can therefore be considered in the manufacture and practice of this invention. The following sections will discuss techniques that are particularly effective for specific implementations.

[0304] Experimental Examples The invention is further described in detail with reference to the following experimental embodiments. These embodiments are provided for illustrative purposes only and are not intended to be limiting, unless otherwise stated. Therefore, the invention should not be construed as limited to the following embodiments, but should be interpreted as encompassing any and all modifications that may be apparent from the teachings provided herein.

[0305] Unless further described, it is believed that those skilled in the art will be able to prepare and apply the compounds of the present invention and practice the claimed methods using the foregoing description and the exemplary embodiments described below. Therefore, the working examples below specifically point out preferred embodiments of the invention and are not to be construed as limiting the remainder of this disclosure in any way.

[0306] The materials and methods used in these experiments are now described.

[0307] Cell Culture: THP1, K562, SKOV3, SKBR3, HDLM2, MD468, and all cell lines were cultured in RPMI 1640 supplemented with 10% fetal bovine serum and penicillin / streptomycin at 37°C and 5% CO2. The THP1 mRFP+ subline (Wt) was generated through lentiviral transduction and FACS purification of the mRFP+ cell line. THP1 mRFP+CAR19z+ (CAR19z; CARMA19z), THP1 mRFP+CAR19Δz+ (CAR19Δz; CARMA19Δz), THP1 mRFP+ MesoZ+, and THP1 mRFP+ CARHer2z+ (CARHer2z; CARMAHer2z) sublines were generated from the THP1 mRFP+ subline. Monocyte differentiation was induced by culturing cells in medium with 1 ng / mL phorbol 1,2-myristate 1,3-acetate for 48 hours.

[0308] Native human macrophages: Native human monocytes were purified from the products of normal donor blood separation using Miltenyi CD14 MicroBeads (Miltenyi, 130-050-201). Monocytes were cultured for 7 days in MACS GMP cell differentiation bags (Miltenyi, 170-076-400) in X-Vivo medium supplemented with 5% human AB serum or RPMI 1640 supplemented with 10% fetal bovine serum, penicillin / streptomycin, glutamax, and 10 ng / mL recombinant human GM-CSF (PeproTech, 300-03). Macrophages were harvested on day 7 and cryopreserved in FBS + 10% DMSO for later use.

[0309] Phagocytosis assay: Wt or CARMA mRFP+ THP1 subline was differentiated for 48 hours with phorbol 1,2-myristate,3-acetate. The GFP+ antigen-loaded tumor subline, K562 CD19+GFP+ cells, was added to differentiated THP1 macrophages at a 1:1 ratio after PMA washing. Macrophages were co-cultured with target tumor cells for 4 hours, and phagocytosis was quantitatively analyzed by fluorescence microscopy using the EVOS FL automated cell imaging system. The mean of three fields of view was considered n, and all cases were quantified in triplicate. Phagocytosis was analyzed on BD LSR-Fortessa using FACS. Flow cytometry data were analyzed using FlowJo (Treestar, Inc.). Live, singlets-gated mRFP / GFP double-positive events were considered phagocytosis. CD47 / SIRPα axis blockade was achieved by adding blocking monoclonal antibodies (mouse anti-human CD47 clone B6H12, eBioscience #14-0479-82; mouse anti-human CD47 clone 2D3 as a negative control, eBioscience #14-0478-82; mouse anti-human SIRPα clone SE5A5, BioLegend #323802) at the start of co-culture at specified concentrations. TLR co-stimulation was achieved by adding a TLR1-9 agonist (human TLR1-9 agonist kit; Invivogen #tlrl-kit1hw) during co-culture.

[0310] In vitro killing assay: Wt or CAR-loaded macrophages were co-cultured with antigen-loaded or control click beetle green luciferase (CBG) / green fluorescent protein (GFP) positive target tumor cells at different effector-to-target ratios (starting at 30:1 and reduced with three-fold dilutions). Tumor burden was determined using bioluminescence imaging with an IVIS spectral imaging system (Perkin Elmer). Specific lysis percentage was calculated as follows: % Specific dissolution = ((treated pores - pores with tumor only) / (maximum killing - pores with tumor only) * 100) Time-lapse microscopy: CAR-mediated phagocytosis fluorescence time-lapse video microscopy was performed using the EVOS FL automated cell imaging system. Images were captured every 40 seconds for 18 hours. Image analysis was performed using FIJI imaging software.

[0311] Lentiviral generation and transfection: A chimeric antigen receptor construct was synthesized de novo via GeneArt (Life Technologies) and cloned into a lentiviral vector as previously described. Concentrated lentiviruses were generated using HEK293T cells as previously described.

[0312] Adenovirus production and transfection: Ad5f35 chimeric adenovirus vectors encoding GFP, CAR, or not encoding a transgene were generated and titrated under the CMV promoter according to standard molecular biology procedures. Native human macrophages were transduced at different fold increases and serially imaged using the EVOS FL automated cell imaging system to obtain GFP expression and viability. CAR expression was assessed by FACS analysis of surface CAR expression using His-labeled antigen and anti-His-APC secondary antibody (R&D Biosystems Clone AD1.1.10).

[0313] Flow cytometry: FACS was performed on a BD LSR Fortessa. Surface CAR expression was detected using biotinylated protein L (GenScriptM00097) and anti-streptolysin APC (BioLegend, #405207) or His-labeled antigen and anti-His-APC secondary antibody (R&D Biosystems Clone AD1.1.10). Fc receptors were blocked with human Trustain FcX (BioLegend, #422301) prior to staining. Background determination was performed using mouse IgG1κ APC isotype control, and CD47 expression was determined using mouse anti-human CD47 APC (eBioscience #17-0479-41). Caloretin expression was determined using mouse anti-caloretin PE clone FMC75 (Abcam #ab83220). All flow cytometry results were gated based on live / dead aqua fixable dead cell stain (Life Technologies L34957).

[0314] Imagestream cytometry: FACS and single-cell fluorescence imaging were performed on an ImageStream Mark II imaging flow cytometer (EMD Millipore). In short, mRFP+ or DiI-stained macrophages (CAR or control) were co-cultured with GFP+ tumor cells for 4 hours prior to fixation and ImageStream data acquisition. Data were analyzed using ImageStream software (EMD Millipore).

[0315] RNA electroporation: Using standard molecular biology techniques, the CAR construct was cloned into an in vitro transcription plasmid under the control of the T7 promoter. The CAR mRNA was transcribed in vitro using the mMessage mMachine T7 Ultra in vitro transcription kit (Thermo Fisher), purified using the RNEasy RNA purification kit (Qiagen), and electroporated into human macrophages using a BTX ECM850 electroporator (BTX Harvard Apparatus). CAR expression at different time points after electroporation was determined using FACS analysis.

[0316] TLR / Dectin-1 Pretreatment: TLR or Dectin-1 pretreatment in Wt or CAR macrophages prior to in vitro phagocytosis or killing assays was performed as follows: Cells were pre-cultured for 30 minutes with either a recommended dose of a TLR 1-9 agonist (Human TLR1-9 Agonist Kit, Invivogen) or β-glucan (MP Biomedicals, LLC) prior to co-culture. The in vitro effects of Wt or CAR macrophages were compared between the untreated and pretreated cases.

[0317] Macrophage / Monocyte Phenotyping: The following surface markers were evaluated as part of a macrophage / monocyte immunophenotyping FACS kit to obtain M1 / M2 differentiation: CD80, CD86, CD163, CD206, CD11B, HLA-DR, HLA-A / B / C, PDL1, and PDL2 (BioLegend). TruStain FcX was used for Fc receptor blockade prior to immunostaining. Macrophages / monocytes were exposed to or not exposed to an activation condition, i.e., 48 hours of Ad5f35 transduction, prior to phenotypic assessment.

[0318] Hippocampal assay: The metabolic phenotype and oxygen consumption of macrophages were determined using the Seahorse XF assay (Agilent). Control or CAR macrophages were exposed to a medium containing either a control or immunosuppressive cytokines for 24 hours prior to analysis. Throughout the hippocampal assay, cells were successively treated with oligomycin, FCCP, and retenone. Each condition was repeated six times.

[0319] In vivo experiments: NOD-scid IL2Rg-null-IL3 / GM / SF and NSG-SGM3 (NSGS) mice were used as a human xenograft model. Mice implanted with CBG-luciferase-positive human SKOV3 ovarian cancer cells were either untreated or treated with different doses of untransduced, blank Ad5f35-transduced, or Ad5f35 CAR-HER2-transduced human macrophages. Tandem bioluminescence imaging (IVIS Spectrum, Perkin Elmer) was performed to monitor tumor burden. Organs and tumors were harvested after sacrifice for FACS analysis. Kaplan-Meier analysis was used to monitor and compare overall survival.

[0320] The experimental results are described below.

[0321] Figure 1A This is a schematic diagram of a chimeric antigen receptor (CAR), which consists of a gene / gene-product containing an extracellular domain, a hinge domain, a transmembrane domain, an intracellular signal transduction domain (one or more), and / or a stoichiometric 2A (P2A, T2A) for co-expression of an additional gene product, which may or may not be secreted. This additional gene product includes any gene / transcription / protein, including but not limited to cytokines, monoclonal antibodies, antibody fragments, single-chain variable fragments, enzymes, additional receptors, dominant / negative receptors, tumor-associated antigens (one or more), and any combination thereof. Furthermore, the CAR construct may include co-delivery of CRISPR / Cas9 gene-edited materials or be introduced into the environment of CRISPR / Cas9 pre-edited cells. Specific examples of CAR constructs are provided in [the document / section / etc.]. Figure 1B The model includes CARMA-ζ, CARMA-γ, and CARMA-Dectin, which respectively contain antigen-specific scFv, CD8 hinge, CD8 transmembrane, and CD3 ζ, FcεRI universal γ subunit, or intracellular domains of Dectin-1.

[0322] Figure 2A shows CAR19z expression on the surface of myeloid cells after lentiviral transduction. CAR19z lentivirus was titrated at a three-fold dilution and used to transduce 1e5 / 0.1 mL mRFP+ THP1 cells. mRFP is a reporter gene (red fluorescent protein) expressed via lentiviral transduction of the myeloid cell line THP1. These cells can be induced to differentiate into macrophages after exposure to chemical PMA. THP1 cells were harvested 24 hours after transduction and stained with biotinylated protein L followed by streptavidin-APC staining to obtain CAR surface expression. Transduced THP1 cells were expanded and sorted by FACS to generate a 100% CAR19z-positive mRFP+ THP1 subline (…). Figure 2B ). Figure 2C Expression of anti-CD19, anti-HER2 and anti-mesothelin lentiviral CAR constructs on THP1 macrophages, with CAR(+) events in the upper right quadrant.

[0323] Figure 3A is a flowchart illustrating the CARMA subline generation using a THP1 macrophage model, differentiation, and in vitro phagocytosis assays with 1 ng / mL phorbol 12-myristate 13-acetate (PMA). Anti-CD19, anti-HER2, and anti-mesothelin CAR macrophages (unlike wild-type (Wt) macrophages) phagocytosed K562 tumor cells expressing CD19, HER2, or mesothelin, respectively, as demonstrated by fluorescence microscopy-based phagocytosis assays (Figures 3B-3D). CARMA tumor phagocytosis was further validated by flow cytometry-based assays in which anti-CD19 mRFP+ CARMA were co-cultured with CD19+ GFP+ K562 cells, and double-positive events were quantified (represented by representative FACS plots). Figure 3EThe standard 10x field of view used in the CARMA phagocytic function list is shown, with individual mRFP (Fig. 3F) or coverage (Fig. 3G). FACS-based mRFP / GFP double-positive events are defined as phagocytic events, and this is verified by Amnis Imagestream FACS analysis. The events shown are gated based on double-positive events and sorted from high to low using the Amnis Imagestream phagocytosis-erosion algorithm (Fig. 3H). Phagocytosis of tumor cells by mRFP + CARMA in the THP-1 cell line model was further demonstrated by confocal microscopy, confirming that GFP + tumor cells were completely enclosed within the phagosomes through three-dimensional confocal z-shaped stacking reconstruction (Fig. 3I). Fig. 3J shows the encounters of a single CARMA cell over time—where contact and immune synapse formation are the first steps leading to phagocytosis, tumor degradation with GFP loss as a marker of cell death, phagosome breakdown, and phagosome repair—demonstrating CARMA survival after tumor cell phagocytosis. Figure 3K This demonstrates CARMA's ability to engulf multiple tumor cells at once.

[0324] Anti-CD19 CAR macrophages were tested using an in vitro phagocytosis assay targeting CD19+ (target) or CD19- (control) GFP+ K562 tumor cells. The antigen specificity of CARMA was demonstrated by the fact that only antigen-loaded tumor cells were phagocytosed (Fig. 4A). To demonstrate the requirement of intracellular signaling domains for CARMA function, a CAR19-Δζ construct (lacking intracellular signaling domains) was used. CAR19-Δζ macrophages failed to phagocytose tumor cells and exhibited significantly reduced antitumor function by an in vitro luciferase-based specific lysis assay (Figs. 4B and 4C). In vitro CARMA phagocytosis assays were performed in the presence of R406 (a Syk inhibitor), cytochalasin D (an actin polymerization inhibitor), or blebbistatin (a non-muscular myosin IIA inhibitor). R406, cytochalasin D, and blebbistatin independently eradicated CARMA's phagocytic function, indicating that CAR signaling in macrophages is Syk-dependent and leads to actin polymerization and NMIIA-mediated phagocytosis (Figs. 4D-4F).

[0325] Figure 5A is a flow cytometry plot showing CD47 expression on the target tumor cell lines relative to the isotype control. K562 and K562-CD19+ (K19), both high CD47 expression cell lines, were used in these experiments.

[0326] Figure 5B illustrates how the addition of anti-CD47 monoclonal antibody selectively enhances CAR (but not Wt) macrophage-mediated phagocytosis of tumor cells carrying target antigens. Wt or CAR19ζ macrophages were cultured with CD19+ K562 tumor cells, along with 0, 0.01, 0.10, 1.00, or 10.0 mcg / mL of anti-CD47 monoclonal antibody.

[0327] Figure 5C This is a diagram showing how the addition of anti-SIRPα monoclonal antibody selectively enhances CAR (but not Wt) macrophage-mediated phagocytosis of tumor cells carrying target antigens. Wt or CAR19ζ macrophages were cultured with CD19+ K562 tumor cells, with anti-SIRPα monoclonal antibody at concentrations of 0, 0.01, 0.10, 1.00, or 10.0 mcg / mL.

[0328] Figure 5D demonstrates how blocking the CD47 / SIRPα axis with an anti-SIRPα monoclonal antibody enhances the multiphagocytic activity of CAR macrophages (defined as macrophages that engulf two or more tumor cells at a time).

[0329] To control the opsonization effect of CD47 / SIRPα blocking monoclonal antibodies, a control anti-CD47 monoclonal antibody (clone 2D3) that binds to CD47 but does not block CD47 from reaching the SIRPα binding site was used in the in vitro phagocytosis assay. Only clones that block this binding site (anti-CD47, clone B6H12) or those that block the SIRPα receptor directly resulted in enhanced CARMA tumor phagocytosis (Figure 5E).

[0330] To test whether blocking the CD47 / SIRPα axis on CAR macrophages leads to loss of antigen specificity, in vitro phagocytosis of antigen-negative (CD19-negative) tumor cells was performed in the presence of anti-CD47 or anti-SIRPα monoclonal antibodies, and no phagocytosis was observed (Fig. 5F).

[0331] The specificity of enhanced CARMA phagocytosis in the presence of SIRPα-blocking monoclonal antibodies was tested by knocking out the SIRPα receptor on THP1 macrophages and comparing tumor phagocytosis of CARMA or SIRPα-KO CARMA in the absence or presence of SIRPα antibodies. CRISPR / Cas9 was used for SIRPα deletion, and cells were SIRPα-negatively sorted prior to functional assays. SIRPα knockout enhanced CARMA function, and adding anti-SIRPα back into the knockout cells did not further enhance phagocytosis. Figure 5G ).

[0332] Figure 6A shows the specific lysis of CD19+GFP+luciferase+ K562 cells induced by CAR19ζCARMA (but not Wt) macrophages (using a THP-1 macrophage model) in a dose-dependent manner at 48 hours in an in vitro luciferase-based killing assay.

[0333] Figure 6B shows the specific lysis of tumor cells induced by CAR19ζ or Wt THP-1 monocytes (undifferentiated, and therefore a model of monocytes rather than macrophages) in an in vitro luciferase-based killing assay at 48 hours in a dose-dependent manner.

[0334] Figure 6C shows a set of images of luciferase-driven bioluminescence derived from luciferase-positive CD19+ K562 tumor cells after 48 hours of in vitro co-culture with Wt or CAR19ζ macrophages in the absence or presence of 10 mcg / mL anti-SIRPα monoclonal antibody. Figure 6D This is a graph showing the specific lysis of Wt or CAR19ζ macrophages with + / - anti-SIRPα monoclonal antibodies.

[0335] CAR constructs with intracellular domains of the FcεRI universal γ (CAR19γ, CARMA19γ) subunit were generated, encapsulated in lentivirus, and used to transduce THP-1 myeloid cells in three-fold sequential viral dilutions. CAR19γ was expressed on THP-1 macrophages. Figure 7A ).

[0336] CAR19γ or CAR19ζ macrophages were sorted for 100% CAR positivity and used for in vitro functional characterization. Both CAR19ζ and CAR19γ macrophages phagocytosed CD19+ tumor cells and showed synergistic effects with CD47 / SIRPα axis blockade induced by the addition of anti-SIRPα monoclonal antibody (Figure 7B).

[0337] In an in vitro phagocytosis assay, R406 Syk demonstrated that both CAR19ζ and CAR19γ macrophages use Syk for signal transduction to drive tumor phagocytosis (Figure 7C).

[0338] At various E:T ratios, after 24 hours of co-culture, in an in vitro luciferase-based specific lysis assay, both CAR19ζ and CAR19γ THP1 macrophages (while Wt THP1 macrophages) effectively killed CD19+ tumor cells. Figure 7D ).

[0339] As leukocytes of the innate immune system, macrophages respond to conserved molecular signals of infection, such as pathogen-associated molecular patterns, through constitutively expressed pathogen recognition receptors. Toll-like receptors are the most well-characterized pathogen recognition receptors and are known to activate macrophages.

[0340] To enhance the tumor phagocytic function of CARMA, in vitro phagocytosis assays were performed using CAR macrophages pretreated independently with TLR1-9 ligands or mediator controls. Ligands of TLR1, 2, 4, 5, and 6 enhanced the phagocytic function of CARMA (Figure 8A). This suggests that TLR ligands can be used to pretreat CARMA during production, or that TLR signaling domains can be encoded into CAR constructs to enhance CAR signaling and downstream effector effects—as novel second-generation / progenitor CARMA constructs.

[0341] Figures 8B and 8C show the differences in TLR ligands between those that enhance or do not enhance CARMA phagocytosis of tumor cells across a wide range of TLR3 or TLR6 ligand concentrations.

[0342] β-glucan (a yeast product) binds to Dectin-1 on the surface of macrophages and leads to activation and effector function. To test the ability of β-glucan to enhance CARMA function, in vitro tumor phagocytosis assays were performed in the absence or presence of 5 mcg / mL β-glucan. β-glucan enhances the phagocytic capacity of CAR (but not Wt) macrophages (Figure 9A).

[0343] To test the ability of β-glucan to enhance the tumor-killing ability of CARMA, in vitro luciferase-based specific lysis assays were performed at various E:T ratios in the presence of 0, 0.5, 5, or 50 mcg / mL β-glucan. β-glucan enhanced the specific lysis of antigen-loaded tumor cells induced by CAR (but not by Wt) THP-1 macrophages. Figure 9B These results suggest that β-glucan can be used as an adjuvant in the preparation of CARMA, or that the intracellular signal transduction domain of Dectin-1 can be encoded into CAR transgenes.

[0344] Given that β-glucan enhances the function of CARMA, CAR constructs consisting of intracellular signal transduction domains of Dectin-1 were generated (Fig. 10A). These constructs were encapsulated in lentivirus and used to transduce THP-1 myeloid cells at three-fold serially diluted lentiviral titers. CAR was detected on the surface in both the CD8TM-Dectin1 CAR and DectinTM-Dectin1 CAR constructs (Figs. 10B and 10C). Cells were 100% positively sorted and used for downstream in vitro functional experiments.

[0345] CD8TM-Dectin1 CAR and DectinTM-Dectin1 CAR macrophages were tested in an in vitro luciferase killing assay. Both constructs showed specific lysis of tumor cells. Figure 10D ).

[0346] Dectin1-CAR macrophages were tested in an in vitro tumor phagocytosis assay against K562 (control) or K19 (target) tumor cells, and Dectin1-CAR macrophages selectively phagocytosed tumor cells loaded with homologous antigens (Fig. 10E). Dectin-1 CAR macrophages demonstrated the ability to phagocytose a variety of tumor cells (Fig. 10F).

[0347] In in vitro tumor phagocytosis assays, Dectin1-CAR macrophages showed synergistic effects with SIRPα blockade or pretreatment with TLR ligands. Figure 10G ).

[0348] Figure 11A shows the cadherin levels in three different CD19+ target cell lines relative to the isotype control. Figure 11B shows the normalized mean fluorescence intensity of cadherin expression in the three different CD19+ target cell lines.

[0349] Figure 11C This figure shows that low levels of cadherin moderately protect target cells (particularly the Nalm6 and JEKO cell lines) from phagocytosis by CAR19z macrophages. These data suggest that the exploitation of cadherin deposition / induction could be used as an additional means to enhance CARMA effector function.

[0350] To validate and test the role of CAR in native human monocyte-derived macrophages, several gene delivery methods were tested. Figure 12A In this study, the anti-HER2 CAR construct was cloned into an mRNA expression plasmid, transcribed in vitro, and the mRNA was directly electroporated into native human monocytes. Figure 13A shows the gating strategy, viability, and transfection efficiency of cells electroporated relative to the mimic. Figure 12B The efficiency of anti-HER2 CAR mRNA electroporation into native human monocyte-derived macrophages (fully differentiated) was shown to be 79.7%.

[0351] Figure 12C The following figure shows that although mRNA electroporation leads to high CAR transfection efficiency in monocytes and macrophages, CAR expression is transient due to mRNA degradation, peaking on day 2 after in vitro electroporation and disappearing by day 7.

[0352] NSGS mice were injected via IP with 1E6 SKOV3 CBG / GFP+ human ovarian cancer cells (a model of metastatic intraperitoneal cancer spread in HER2+ ovarian cancer). Mice were co-injected with either mimicked electroporated or anti-HER2 CAR mRNA-electroplated native human monocytes or native human macrophages (1:1 E:T ratio), and tumor burden was imaged. CAR macrophages (Fig. 13A) and CAR monocytes ( Figure 13B The results showed a marginal reduction in tumor growth after approximately two weeks. The first time point at which tumor burden was quantified by bioluminescence was 24 hours post-treatment, indicating that CAR monocytes and macrophages were active during this first 24 hours.

[0353] Lentiviral delivery of CAR transgenes to native human monocyte-derived macrophages was tested using various CAR constructs. Figure 14A In this study, CAR19 was delivered to human macrophages via lentiviral transduction, showing transduction efficiencies of 4.27% and 38.9% in the control vs. CAR19 lentivirus (MOI10) groups, respectively. This demonstrates the FACS gating strategy.

[0354] Figure 14B The image is a representative FACS plot showing the expression of anti-HER2 CAR in native human macrophages, with transduction efficiencies of 1.47% and 18.1% in the control and MOI 10 CAR LV conditions, respectively.

[0355] Monocyte-derived macrophages were generated by differentiating CD14+ selected cells (products of normal donor blood separation) in GM-CSF conditioned medium for 7 days. To optimize CAR delivery via lentiviral transduction, macrophages were transduced with anti-CD19 and anti-HER2 lentiviruses at different time points during the monocyte-to-macrophage differentiation process. For both anti-CD19 and anti-HER2 CAR constructs, transduction efficiency peaked at the midpoint of transduction (day 4). Figure 15A And 15B). Anti-CD19 CAR native human macrophages were used in an in vitro FACS-based phagocytosis assay against CD19+GFP+ K562 tumor cells, where the CD11b+ / GFP+ event was defined as a phagocytic event. In this assay, as shown in [example missing], [missing information]. Figure 15A Macrophages transduced at different time points. Figure 15C A series of graphs showing the trend of phagocytic efficiency in accordance with CAR transduction efficiency, with the peak of macrophages transduced on day 4 during differentiation.

[0356] Given that mRNA electroporation is transient and lentiviruses are only moderately effective and require high titers, alternative transduction methods for delivering transgenes to native human macrophages were tested. Adenovirus (recombinant, replication-deficient) was identified as an effective method for native human macrophage transduction. Expression of the Coxackie adenovirus receptor (a stopping protein of Ad5) and CD46 (a stopping protein of Ad35) was tested on native human macrophages relative to the isotype control, with CD46 (but not the Coxackie adenovirus receptor) being highly expressed (Figure 16A). Therefore, a chimeric Ad5f35 adenovirus was used for native human macrophage transduction and was engineered using standard molecular biology techniques to express a chimeric antigen receptor against HER2 (GFP and blank Ad5f35 virus were used as controls).

[0357] Figure 16B shows that Ad5f35 efficiently delivered the transgene (GFP as a model transgene) into human macrophages at an MOI of 1000, and the expression increased over time as quantitatively monitored by the GFP signal on the IVIS spectrum. Figure 16C Transduction kinetics of native human macrophages at different time points across a wide range of MOIs up to 10,000 were compared.

[0358] Figure 16C A series of representative FACS plots showing anti-HER2 CAR expression on Ad5f35-transduced human macrophages 48 hours post-transduction under a wide range of viral MOIs.

[0359] Figure 16D shows representative fluorescence microscopy images of native human macrophages transduced with Ad5f35-GFP, with the highest transduction efficiency shown at an MOI of 1000.

[0360] Native human CARMA was tested in an in vitro phagocytosis assay using FACS analysis. Macrophages (untransduced or anti-HER2 CAR) were stained with DiI before co-culturing with GFP+ SKOV3 ovarian cancer cells. Phagocytosis, defined as a DiI / GFP double-positive event, was measured at 26.6% in the CAR group and 4.55% in the control group. Figure 17ATo verify that the DiI / GFP double-positive event was a phagocytic event and not a dual event, cytochalasin D (a phagocytosis inhibitor) was added to the experimental group, and CAR-mediated phagocytosis was fully eradicated to a level of 1.74%. To further verify that native human CAR macrophages could phagocytose tumor cells, the double-positive events were gated using Amnis Imagestream FACS and sorted from high to low using the Amnis phagocytosis-erosion algorithm, visually demonstrating that these double-positive events represented phagocytosis (Fig. 17B). Furthermore, DiI-stained CAR-HER2 macrophages were co-cultured with SKOV3-GFP and imaged using confocal microscopy, and phagocytosis was verified.

[0361] Anti-HER2 CAR native human macrophages were generated by transducing monocyte-derived macrophages with Ad5f35-CAR. These cells (or untransduced control cells) were used as effectors in an in vitro FACS-based phagocytosis assay of SKBR3 human breast cancer cells. Figure 18 This study demonstrated that CAR (but not UTD) human macrophages engulfed breast cancer cells. Furthermore, the addition of an anti-SIRPα monoclonal antibody enhanced the phagocytosis of breast cancer cells by CARMA (but not by UTD macrophages). These results demonstrate that the synergistic effect between CD47 / SIRPα axis blockade and CARMA observed in the THP-1 model has been transferred to native human macrophage studies.

[0362] Macrophages are white blood cells of the innate immune system and therefore possess sentinel antimicrobial properties. To demonstrate that CAR macrophages remain functional innate immune cells in an antimicrobial sense and have not lost their ability to respond to infectious stimuli, control untransduced or CAR macrophages were used in a FACS-based E. coli phagocytosis assay. Figure 19 The representative FACS plot shows that CARMA demonstrates complete phagocytosis of pH-Rodo Green E. coli particles.

[0363] Native anti-HER2 CARMA was tested as effector cells in an in vitro luciferase-based killing assay. After 48 hours of co-culture, anti-HER2 CARMA (but not control UTD macrophages) caused specific lysis of HER2+ K562 cells (but not control K562 cells lacking HER2 expression) (Fig. 20A). To demonstrate that CARMA killing can physiologically translate to HER2-expressing tumor cells (as opposed to K562-HER2 cells overexpressing HER2 via lentiviral transduction), SKBR3 breast cancer cells and SKOV3 ovarian cancer cells were used as targets. CARMA (but not control UTD or control blank Ad5f35-transduced macrophages) showed significant antitumor activity in both models after 48 hours of co-culture (Figs. 20B and 20C). To test the synergistic effect between CD47 / SIRPα axis blockade in a killing assay, SKOV3 ovarian cancer cells were co-cultured with culture medium, untransduced control macrophages, anti-HER2 CARMA, anti-HER2 CARMA + anti-CD47 mAB (10 mcg / mL), or anti-HER2 CARMA + anti-SIRPα (10 mcg / mL), and luciferase signaling was continuously measured. CARMA led to complete tumor eradication by day 13, and the kinetics of tumor eradication were even faster in the presence of CD47 / SIRPα axis blockade (Fig. 20D). The synergistic effect with β-glucan, as demonstrated in the THP-1 macrophage CARMA model, was tested in similar experiments, and β-glucan pretreatment with CARMA resulted in enhanced tumor-killing kinetics (Fig. 20E). CARMA exposure to LPS (TLR-4 ligand) or Poly-IC (TLR-3 ligand) modulated the antitumor effect. Figure 20F ).

[0364] The luciferase assays shown in Figures 20A-20F demonstrated the ability of native human CARMA to clear tumors in vitro. To validate these results, GFP+ SKOV3 ovarian cancer cells were co-cultured with control UTD macrophages, control UTD macrophages + 10 mcg / mL trastuzumab, control blank Ad5f35 virus-transduced macrophages, or anti-HER2 native human CARMA. CARMA (but not under control conditions) was able to clear tumor cells (…). Figure 21 ).

[0365] Macrophages are phenotypic cells capable of accepting different functional characteristics and are generally classified into M1 and M2 macrophages—M1 being inflammatory / activated and M2 being immunosuppressive / tumor-promoting. Forty-eight hours after transduction of native human macrophages with Ad5f35 CAR virus, dose-dependent upregulation of the M1 marker CD80 / CD86 and dose-dependent downregulation of the M2 marker CD163 were measured by FACS (Figure 22A). To test whether this effect was a result of CAR expression or Ad5f35 transduction, macrophages were not transduced, transduced with blank Ad5f35, or transduced with anti-HER2 Ad5f35, and blank / CARAd5f35 showed the same phenotypic transformation pattern. Figure 22B ).

[0366] The solid tumor microenvironment is generally immunosuppressive and can lead to macrophage polarization to the M2 state. To test whether CARMA (which is M1 polarized due to viral transduction) is resistant to M2 downconversion mediated by immunosuppressive cytokines, control untransduced or anti-HER2 CAR human macrophages were exposed to IL-4, IL-10, or IL-13 for 24 hours before co-culturing with SKOV3 ovarian cancer cells. Control UTD macrophages treated with inhibitory cytokines resulted in enhanced tumor growth, while CARMA exposed to inhibitory cytokines maintained its cytotoxic activity at 48 hours in a luciferase-based in vitro specific lysis assay (Figure 22C).

[0367] To further test the resistance of human CAR macrophages to immunosuppression, control UTD, blank Ad5f35, or anti-HER2 CAR Ad5f35-transduced macrophages were exposed to 10 ng / mL IL-4 (a typical M2-inducing cytokine) or cancer cells (SKOV3, an ovarian cancer cell line; HDLM2, a Hodgkin lymphoma cell line) that had previously shown to convert macrophages to M2 during co-culture. Control UTD macrophages upregulated CD206 (an M2 marker that responds specifically to IL-4 stimulation via STAT6 phosphorylation). Blank Ad5f35, and even more so CAR-Ad5f35-transduced macrophages, showed resistance to IL-4 and tumor-induced M2 phenotype deconversion. Figure 22D ).

[0368] To further characterize the phenotype of CAR macrophages, oxygen consumption was measured using a hippocampal assay to probe metabolic phenotype. M2 macrophages exhibited a higher basal oxygen consumption rate than M0 or M1 macrophages because ATP production was more dependent on oxidative phosphorylation. Control UTD or anti-HER2 CAR macrophages were exposed to IL-4 for 24 hours to polarize to M2 (or not), and oxygen consumption rates were measured. Control UTD macrophages showed the characteristic increased basal oxygen consumption of M2 macrophages, while CARMA did not respond to IL-4, indicating resistance to M2 downconversion (Figure 22 E). These data, combined with phenotypic, metabolic, and functional assays, demonstrate that CARMA resists M2 downconversion.

[0369] Native human normal donor monocytes (purified via CD14-positive selection) were transduced with Ad5f35-CAR-HER2 at an MOI ranging from 0 (UTD) to 1000. CAR expression was measured by FACS 48 hours post-transduction. CAR monocytes were efficiently generated via Ad5f35, and expression peaked at an MOI of 1000. Figure 23A And 23B). At MOIs up to 1000, monocytes maintained high viability (measured by FACS Live / Dead Aqua analysis) (Fig. 23C). As analyzed by FACS, CAR (but not UTD) human monocytes upregulated the M1 activation marker (Fig. 23D) and downregulated the M2 marker ( Figure 23E This confirmed the M1 monocyte phenotype 48 hours after transduction.

[0370] Anti-HER2 CAR monocyte killing was evaluated using an in vitro luciferase-based killing assay at a range of effector:target (E:T) ratios. Untransduced (UTD) or CAR-HER2-ζ (CAR) monocytes were co-cultured with HER2+SKBR3 (human breast cancer) or HER2+SKOV3 (human ovarian cancer) cells in vitro. Specific lysis at 24, 48, and 96 hours after the start of co-culture was calculated and determined. CAR (but not UTD) monocytes lysed breast and ovarian cancer cells in vitro (Figures 24A and 24B).

[0371] NOD-scid IL2Rg-null-IL3 / GM / SF and NSG-SGM3 (NSGS) mice were used to model human HER2(+) ovarian cancer xenografts in vivo. On day 0, mice were intraperitoneally (IP) injected with 7.5E5 click beetle green luciferase (CBG luc)-positive / green fluorescent protein (GFP)-positive SKOV3 ovarian cancer cells—serving as a model of intraperitoneal cancer spread, an invasive and inherently metastatic model of solid malignancy. Mice were either untreated (tumor only) or injected on day 0 with a single dose of 4E6 untransduced or CAR-HER2 (CARMA) human macrophages via IP (illustrated, Figure 25A). Serial imaging of mice was performed using bioluminescence (total flux; photons / second) as a representative of tumor burden. Mice treated with CARMA showed a tumor burden reduction of approximately two orders of magnitude (Figures 25B and 25C). Mice treated with CARMA showed a 30-day survival benefit compared to untreated or UTD-treated mice (p = 0.018) (Fig. 25D). To demonstrate the accessibility of macrophages to solid tumor nodules, tumors were harvested from mice that died on day 36, and the presence of adoptive metastatic human macrophages was assessed via human CD45 expression based on FACS analysis. Figure 25E ).

[0372] Human macrophages were transduced without transduction (UTD) or with transgenic-free blank Ad5f35 virions (blank) or Ad5f35-CAR-HER2-ζ (CARMA) at an infection fold of 1000. Surface CAR expression was validated by FACS analysis 48 hours post-transduction (Fig. 26A). Surface markers were assessed to confirm M1 macrophage polarization in blank Ad5f35 or CAR-HER2-ζ Ad5f35 transduced cells. M1 markers (HLA DR, CD86, CD80, PDL1) were upregulated, while M2 markers (CD206, CD163) were downregulated (Fig. 26B). NSGS mice were again used in the IP model of HER2+ metastatic ovarian cancer and were divided into four treatment groups (n=5 per group). Mice were either untreated or administered an IP injection on day 0 of untransduced 1E7 macrophages, blank Ad5f35-transduced macrophages, or CAR-HER2-ζ-transduced macrophages. Figure 26C Tumor burden was monitored using continuous bioluminescence imaging, with representative data showing a tumor burden on day 27 post-implantation (…). Figure 26D and 26E On day 20 post-treatment, the tumor burden in CARMA-treated mice was approximately 2400 times smaller than that in untreated mice.

[0373] NSGS mice were used to establish an IP model of HER2+ metastatic ovarian cancer and were divided into four treatment groups (n=5 per group), including untreated mice and mice receiving CAR-HER2-ζ human macrophages (3E6, 1E7, or 2E7) via IP on day 0 (Fig. 27A). Tumor burden was monitored by serial bioluminescence imaging, and a macrophage number-dependent dose-response was observed in this model (Fig. 27B). A single dose of CAR-HER2 macrophages (3E6, 1E7, or 2E7 macrophages per mouse) resulted in dose-dependent tumor eradication on day 36 post-implantation (compared to untreated mice). Figure 27C ).

[0374] Figure 28 An example of the treatment method proposed by CARMA. In short, patient monocytes are selected from peripheral blood, differentiated and transduced in vitro to express CAR, co-stimulated (or not) with a synergistic compound, and injected back into the patient intravenously, intraperitoneally, intratumorally, via interventional radiology procedures, or through other routes. Note that the differentiation process can be skipped, and the monocytes can be transduced and infused back into the patient. The monocyte source can also be an HLA-matched donor. Other implementation methods

[0375] The listing of elements in any definition of a variable herein includes the variable being defined as any single element or combination (or sub-combination) of the listed elements. The description of embodiments herein includes embodiments as any single embodiment or in combination with any other embodiment or parts thereof.

[0376] The disclosure of each patent, patent application, and publication cited herein is incorporated herein by reference in its entirety. While the invention has been disclosed with reference to specific embodiments, it will be apparent to those skilled in the art that other embodiments and variations of the invention will occur to them without departing from the actual spirit and scope thereof. The appended claims are intended to be construed as encompassing all such embodiments and equivalent variations.

Claims

1. A modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain of a stimulatory and / or costimulatory molecule, and wherein the cell is a monocyte, macrophage, or dendritic cell with target effector activity.

2. A modified cell comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the nucleic acid sequence comprises a nucleic acid sequence encoding an antigen-binding domain, a nucleic acid sequence encoding a transmembrane domain, and a nucleic acid sequence encoding an intracellular domain of a stimulatory and / or costimulatory molecule, and wherein the cell is a monocyte, macrophage, or dendritic cell expressing CAR and having targeted effector activity.

3. The modified cell of claim 1 or 2, wherein the antigen-binding domain of the CAR comprises an antibody selected from monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, single-domain antibodies, single-chain variable fragments, and their antigen-binding fragments.

4. The modified cell of claim 1 or 2, wherein the antigen-binding domain of the CAR is selected from anti-CD19 antibody, anti-HER2 antibody and fragments thereof.

5. The modified cell of claim 1 or 2, wherein the intracellular domain of the CAR comprises a dual signal transduction domain.

6. The modified cell of claim 1 or 2, wherein the targeted effector activity is directed against an antigen on a target cell that specifically binds to the antigen-binding domain of the CAR.

7. The modified cell of claim 1 or 2, wherein the target effector activity is selected from phagocytosis, cytotoxicity of the target cell, antigen presentation, and cytokine secretion.

8. The modified cell of claim 1 or 2 further comprises an agent selected from the following: nucleic acid, antibiotic, anti-inflammatory agent, antibody or antibody fragment thereof, growth factor, cytokine, enzyme, protein, peptide, fusion protein, synthetic molecule, organic molecule, carbohydrate or analogue, lipid, hormone, microsome, derivative or variant thereof, and any combination thereof.

9. The modified cell of claim 1 or claim 2, wherein the modified cell has at least one upregulated M1 marker and at least one downregulated M2 marker.

10. The modified cell of claim 1 or 2, wherein the modified cell is genetically modified to express the CAR.

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