Chimeric antigen receptors with enhanced signaling and activities and uses thereof

Abstract

Provided herein are recombinant antigen receptors, for example chimeric antigen receptors (CARs), that comprise modified cytoplasmic domains that provide improved signalling and thereby provide improved performance and safety. Also provided are polynucleotides encoding the recombinant antigen receptors, vectors comprising the polynucleotides, and engineered immune cells comprising the vectors and / or polynucleotides. The invention further provides methods for engineering immune cells to express the recombinant antigen receptors. Improved recombinant antigen receptor signalling is also provided by co-expressing a first recombinant antigen receptor and a second recombinant antigen receptor or co-expressing a recombinant antigen receptor and a protein involved in transducing the signal from the activated recombinant antigen receptor. Also provided are methods of treating a variety of conditions, including, but not limited to, blood cancers and cancers characterized by solid tumors, by administering the engineered cells to patients suffering from such a condition.

Description

[00189] In some embodiments, as non limiting examples, an RNA encodes a rare-cutting endonuclase, one monomer of the rare-cutting endonuclease such as half-TALE-nuclease, a CAR, at least one component of the multi-chain chimeric antigen receptor, a pTa or functional variant thereof, an exogenous nucleic acid, and / or one additional catalytic domain. Engineered immune cells

[00190] The invention also provides engineered immune cells comprising any of the polynucleotides that encode the recombinant antigen receptors e.g. CARs described herein. In some embodiments, such an encoding polynucleotide can be introduced into an immune cell as a transgene via a plasmid vector. In some embodiments, the plasmid vector can also contain, for example, a selection marker which provides for identification and / or selection of cells which received the vector.

[00191] Recombinant antigen receptor e.g. CAR polypeptides may be synthesized in situ in the cell after introduction of polynucleotides encoding the polypeptides into the cell. Alternatively, the polypeptides may be be produced outside of cells, and then introduced into cells. Methods for introducing a polynucleotide construct into cells are known in the art. In some embodiments, stable transformation methods can be used to integrate the polynucleotide construct into the genome of the cell. In other embodiments, transient transformation methods can be used to transiently express the polynucleotide construct, and the polynucleotide construct not integrated into the genome of the cell. In other embodiments, virus-mediated methods can be used. The polynucleotides may be introduced into a cell by any suitable means such as for example, recombinant viral vectors (e.g. retroviruses, adenoviruses), liposomes, and the like. Transient transformation methods include, for example without limitation, microinjection, electroporation or pa rticle bombardment. Polynucleotides may be included in vectors, such as for example plasmid vectors or viral vectors.

[00192] In some embodiments, the engineered immune cells disclosed herein further comprise one or more polynucleotides that express one or more polypepties that enhance the efficacy, activity, proliferation and / or persistence of the engineered immune cell, In certain embodiments, the engineered immune cells further express one or more chimeric cytokine receptors that provide the signal 3 of TCR signaling. In some embodiments, the chimeric cytokine receptors are inducible or constitutively active. Exemplary chimeric cytoline receptors are described in WO2020 / 180694, WO2020 / 180664, and WO2021 / 041806, all of which are incorporated herein by reference.

[00193] In some embodiments, the engineered immune cells disclosed herein further comprise one or more polynucleotides that express one or more polypepties that reduce reactivity of the allogeneic engineered immune cells in a recipient. In certain embodiments, the engineered immune cells further express one or more autologous / allogeneic immune defense receptors. Exemplary autologous / allogeneic immune defense receptors are described in WO2019 / 210081, which is herein incorporated by reference.

[00194] Also provided herein are isolated cells and cell lines obtained by the herein-described methods of engineering cells provided herein. In some embodiments, an isolated cell comprises at least one recombinant antigen receptor e.g. CAR as described herein. In some embodiments, an isolated cell comprises a population of different recombinant antigen receptors e.g. CARs, each species of which comprises different extracellular ligand-binding domains.

[00195] Also provided herein are isolated immune cells obtained according to any one of the methods described herein. Any immune cell capable of expressing heterologous DNAs can be used for the purpose of expressing the polypeptide of interest. In some embodiments, the immune cell used for expressing any one of the recombinant antigen receptors e.g. CARs described herein is a T cell. In some embodiments, an immune cell used for expressing CARs can be derived from, for example without limitation, a stem cell. The stem cells can be adult stem cells, non-human embryonic stem cells, more particularly non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells. Representative human stem cells are CD34+ cells.

[00196] In some embodiments, the engineered immune cells expressing at their cell surface membrane a recombinant antigen receptor e.g. CAR of the invention comprise a percentage of stem cell memory and central memory cells greater than 10%, 20%, 30%, 40%, 50%, or 60%. In some embodiments, the engineered immune cells expressing at their cell surface membrane a recombinant antigen receptor e.g. CAR of the invention comprise a percentage of stem cell memory and central memory cells of about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 15% to about 50%, about 15% to about 40%, about 20% to about 60%, or about 20% to about 70%.

[00197] The immune cell used for expressing any one of the recombinant antigen receptors e.g. CARs described herein can also be a dendritic cell, killer dendritic cell, a mast cell, a NK-cell, a B-cell or a T cell selected from the group consisting of inflammatory T-lymphocytes, cytotoxic T-lymphocytes, regulatory T-lymphocytes or helper T-lymphocytes. In some embodiments, the cell can be derived from the group consisting of CD4+ T-lymphocytes and CD8+ T-lymphocytes.

[00198] In one embodiment, the immune cell is an inflammatory T-lymphocyte that expresses any one of the CARs described herein. In one embodiment, the immune cell is a cytotoxic T-lymphocyte that expresses any one of the CARs described herein. In one embodiment, the immune cell is a regulatory T-lymphocyte that expresses any one of the CARs described herein. In one embodiment, the immune cell is a helper T-lymphocyte that expresses any one of the CARs described herein.

[00199] Also provided herein are cell lines obtained from a transformed T cell according to any of the herein-described methods. Also provided herein are modified cells resistant to an immunosuppressive treatment. In some embodiments, an isolated cell according to the invention comprises a polynucleotide encoding a CAR.

[00200] The immune cells of the invention can be activated and expanded, either prior to or after genetic modification of the T cells, using methods as generally described, for example without limitation, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005. T cells can be expanded in vitro or in vivo. Generally, the T cells of the invention can be expanded, for example, by contact with an agent that stimulates a CD3 TCR complex and a co-stimulatory molecule on the surface of the T cells to create an activation signal for the T cell. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins like phytohemagglutinin (PHA) can be used to create an activation signal for the T cell.

[00201] In some embodiments, T cell populations may be stimulated in vitro by contact with, for example, an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 5, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-y, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGFp, and TNF, or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl- cysteine and 2-mercaptoethanoi. Media can include RPMI 1640, A1M-V, DMEM, MEM, a- MEM, F-12, X-Vivo 1, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% CO2). T cells that have been exposed to varied stimulation times may exhibit different characteristics

[00202] In some embodiments, the cells of the invention can be expanded by co-culturing with tissue or cells. The cells can also be expanded in vivo, for example in the subject’s blood after administrating the cell into the subject.

[00203] In some embodiments, an isolated cell according to the present invention comprises one inactivated gene selected from the group consisting of CD52, GR, PD-1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, HLA, TCRa and TCRP and / or expresses a CAR, a multi-chain CAR and / or a pTa transgene. In some embodiments, an isolated cell comprises polynucleotides encoding polypeptides comprising a multi-chain CAR. In some embodiments, the isolated cell according to the present invention comprises two inactivated genes selected from the group consisting of: CD52 and GR, CD52 and TCRa, CDR52 and TCRp, GR and TCRa, GR and TCRp, TCRa and TCRp, PD-1 and TCRa, PD-1 and TCRp, CTLA-4 and TCRa, CTLA-4 and TCRp, LAG3 and TCRa, LAG3 and TCRp, Tim3 and TCRa, Tim3 and TCRp, BTLA and TCRa, BTLA and TCRp, BY55 and TCRa, BY55 and TCRp, TIGIT and TCRa, TIGIT and TCRp, B7H5 and TCRa, B7H5 and TCRp, LAIR1 and TCRa, LAIR1 and TCRp, SIGLEC10 and TCRa, SIGLEC10 and TCRp, 2B4 and TCRa, 2B4 and TCRp and / or expresses a CAR, a multi-chain CAR and a pTa transgene.

[00204] In some embodiments, TCR is rendered not functional in the cells according to the invention by inactivating TCRa gene and / or TCRP gene(s). In some embodiments, a method to obtain modified cells derived from an individual is provided, wherein the cells can proliferate independently of the major histocompatibility complex (MHC) signaling pathway. Modified cells, which can proliferate independently of the MHC signaling pathway, susceptible to be obtained by this method are encompassed in the scope of the present invention. Modified cells disclosed herein can be used in for treating individuals in need thereof against Host versus Graft (HvG) rejection and Graft versus Host Disease (GvHD); therefore in the scope of the present invention is a method of treating individuals in need thereof against Host versus Graft (HvG) rejection and Graft versus Host Disease (GvHD) comprising treating said individual by administering to said individual an effective amount of modified cells comprising inactivated TCRa and / or TCRp genes.

[00205] In some embodiments, the immune cells are engineered to be resistant to one or more chemotherapy drugs. The chemotherapy drug can be, for example, a purine nucleotide analogue (PNA), thus making the immune cell suitable for cancer treatment combining adoptive immunotherapy and chemotherapy. Exemplary PNAs include, for example, clofarabine, fludarabine, and cytarabine, alone or in combination. PNAs are metabolized by deoxycytidine kinase (dCK) into mono-, di-, and tri-phosphate PNA. Their tri-phosphate forms compete with ATP for DNA synthesis, act as pro-apoptotic agents, and are potent inhibitors of ribonucleotide reductase (RNR), which is involved in trinucleotide production. Provided herein are target-specific CAR-T cells comprising an inactivated dCK gene. In some embodiments, the dCK knockout cells are made by transfection of T cells using polynucleotides encoding specific TAL-nulcease directed against dCK genes by, for example, electroporation of mRNA. The dCK knockout target-specific CAR-T cells are resistant to PNAs, including for example clorofarabine and / or fludarabine, and maintain T cell cytotoxic activity toward FLT3-expressing cells.

[00206] In some embodiments, isolated cells or cell lines of the invention can comprise a pTa or a functional variant thereof. In some embodiments, an isolated cell or cell line can be further genetically modified by inactivating the TCRa gene.

[00207] In some embodiments, the CAR-T cell comprises a polynucleotide encoding a suicide polypeptide, such as for example RQR8. See, e.g., WO2013153391 A, which is hereby incorporated by reference in its entirety. In CAR-T cells comprising the polynucleotide, the suicide polypeptide is expressed at the surface of a CAR-T cell. In some embodiments, the suicide polypeptide comprises the amino acid sequence shown in SEQ ID NO: 79. CPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSP APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLL S LVITLYCNHRNRRRVCKCPRPVV (SEQ ID NO: 79).

[00208] The suicide polypeptide may also comprise a signal peptide at the amino terminus. In some embodiments, the suicide polypeptide comprises the amino acid sequence shown in SEQ ID NO: 80. MGTSLLCWMALCLLGADHADACPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAK PTTTACPYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD FACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV (SEQ ID NO: 80).

[00209] When the suicide polypeptide is expressed at the surface of a CAR-T cell, binding of rituximab to the rituximab epitopes of the polypeptide causes lysis of the cell. More than one molecule of rituximab may bind per polypeptide expressed at the cell surface. Each rituximab epitope of the polypeptide may bind a separate molecule of rituximab. Deletion of target-specific CAR-T cells may occur in vivo, for example by administering rituximab to a subject. The decision to delete the transferred cells may arise from undesirable effects being detected in the subject which are attributable to the transferred cells, such as for example, when unacceptable levels of toxicity are detected.

[00210] In some embodiments, upon administration to a patient, engineered immune cells expressing at their cell surface any one of the recombinant antigen receptors e.g. CARs described herein may reduce, kill or lyse endogenous cells of the patient that express the target antigen (e.g. DLL3, BCMA, EGFRvIII, Flt-3, WT-1, CD20, CD23, CD30, CD38, CD70, CD33, CD133, LeY, NKG2D, CS1, CD44v6, ROR1, CD19, Claudin-18.2 (Claudin-18A2, or Claudinl8 isoform 2), DLL3 (Delta-like protein 3, Drosophila Delta homolog 3, Delta3 ), Mucl6, Mucl7 (Mucinl7, Muc3), FAP alpha (Fibroblast Activation Protein alpha), Ly6G6D (Lymphocyte antigen 6 complex locus protein G6d, c6orf23, G6D, MEGT1, NG25), and / or RNF43 (E3 ubiquitin-protein ligase RNF43, RING finger protein 43)) of the recombinant antigen receptor e.g. CAR. In one embodiment, a percentage reduction or lysis of such endogenous cells or cells of a cell line expressing the target antigen by engineered immune cells expressing any one of the recombinant antigen receptors e.g. CARs described herein is at least about or greater than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In one embodiment, a percentage reduction or lysis of such target antigen-expressing endogenous cells or cells of a cell line expressing the target antigen by engineered immune cells expressing any one of the target-specific CARs, such as DLL3 CAR described herein, is about 5% to about 95%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 25% to about 75%, or about 25% to about 60%. Method for sorting recombinant antigen receptor e.g. CAR-positive immune cells

[00211] In one aspect, provided are methods for in vitro sorting of a population of immune cells, wherein a subset of the population of immune cells comprises engineered immune cells expressing any one of the recombinant antigen receptors e.g. CARs comprising epitopes specific for monoclonal antibodies described herein. The method comprises contacting the population of immune cells with a monoclonal antibody specific for the epitopes and selecting the immune cells that bind to the monoclonal antibody to obtain a population of cells enriched in engineered immune cells expressing the recombinant antigen receptor e.g. CAR.

[00212] In some embodiments, said monoclonal antibody specific for said epitope is optionally conjugated to a fluorophore. In this embodiment, the step of selecting the cells that bind to the monoclonal antibody can be done by Fluorescence Activated Cell Sorting (FACS). In some embodiments, said monoclonal antibody specific for said epitope is optionally conjugated to a magnetic particle. In this embodiment, the step of selecting the cells that bind to the monoclonal antibody can be done by Magnetic Activated Cell Sorting (MACS).

[00213] In some embodiments, the population of recombinant antigen receptor-expressing e.g. CAR-expressing immune cells obtained when using the method for in vitro sorting of immune cells described herein, comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the recombinant antigen receptor-expressing e.g. CAR-expressing immune cells. In some embodiments, the population of expressing immune cells obtained when using the method for in vitro sorting of CAR-expressing immune cells described herein, comprises at least 85% of recombinant antigen receptor-expressing e.g. CAR-expressing immune cells.

[00214] In some embodiments, the mAbs used in the in vitro sorting method are previously bound onto a support such as a column or on beads such as routinely realized by the skilled in the art. In some embodiments, immune cells expressing CARs are T-cells.

[00215] According to the invention, cells to be administered to the recipient may be enriched in vitro from the source population. Methods of expanding source populations are well known in the art, and may include selecting cells that express an antigen such as CD34 antigen, using combinations of density centrifugation, immuno-magnetic bead purification, affinity chromatography, and fluorescent activated cell sorting, known to those skilled in the art.

[00216] Flow cytometry is widely used in the art and is a method well known to one of ordinary skill to sort and quantify specific cell types within a population of cells. In general, flow cytometry is a method for quantitating components or structural features of cells primarily by optical means. Since different cell types can be distinguished by quantitating structural features, flow cytometry and cell sorting can be used to count and sort cells of different phenotypes in a mixture.

[00217] A flow cytometric analysis involves two basic steps: 1) labeling selected cell types with one or more labeled markers, and 2) determining the number of labeled cells relative to the total number of cells in the population.

[00218] The primary method of labeling cell types is by binding labeled antibodies to markers expressed by the specific cell type. The antibodies are either directly labeled with a fluorescent compound or indirectly labeled using, for example, a fluorescent- labeled second antibody which recognizes the first antibody.

[00219] In some embodiments, the method used for sorting immune cells expressing a CAR is the Magnetic- Activated Cell Sorting (MACS).

[00220] Magnetic-activated cell sorting (MACS) is a method for separation of various cell populations depending on their surface antigens (CD molecules) by using superparamagnetic nanoparticles and columns. It takes a few simple steps to get pure cell populations. Cells in a single-cell suspension are magnetically labeled with microbeads. The sample is applied to a column composed of ferromagnetic spheres, which are covered with a cell-friendly coating allowing fast and gentle separation of cells. The unlabeled cells pass through while the magnetically labeled cells are retained within the column. The flowthrough can be collected as the unlabeled cell fraction. After a short washing step, the column is removed from the separator, and the magnetically labeled cells are eluted from the column.

[00221] In some embodiments, the mAb used in the method for sorting immune cells expressing the CAR is chosen from alemtuzumab, ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, QBEND-10 and / or ustekinumab. In some embodiments, said mAb is rituximab. In another embodiment, said mAb is QBEND-10. Therapeutic applications

[00222] Isolated cells obtained by the methods described herein, or cell lines derived from such isolated cells, expressing recombinant antigen receptors of the invention e.g. CARs of the invention can be used as a medicament. In some embodiments, such a medicament can be used for treating a disease or a condition associated with the target antigen of the recombinant antigen receptor e.g CAR of the invention. Because the target specificity of the recombinant antigen receptors e.g. CARs of the present invention can be engineered toward the target of choice, conditions treatable with the recombinant antigen receptors e.g. CARs of the invention are diverse. These include but are not limited to various forms of cancer, such as, for example, cancers associated with the expression of DLL3.

[00223] In some embodiments, an isolated cell according to the invention, or cell line derived from the isolated cells, or an antibody can be used in the manufacture of a medicament for treatment of a cancer in a subject in need thereof.

[00224] In some embodiments, immune cells containing a recombinant antigen receptor, e.g., a CAR, of the disclosure can be used to treat such malignancies as small cell lung cancer, melanoma, low grade gliomas, glioma, glioblastoma, medullary thyroid cancer, carcinoids, dispersed neuroendocrine tumors in the pancreas, bladder and prostate, testicular cancer, lymphoma, leukemia, Renal Cell Carcinoma (RCC), Non-Hodgkin’s Lymphoma, Hodgkin’s Disease (HD), Waldenstrom’s macroglobulinemia, Acute Myeloid Leukemia, Multiple Myeloma, diffuse large-cell lymphoma, follicular lymphoma, and lung adenocarcinomas with neuroendocrine features. In exemplary embodiments, the CARcontaining immune cells, e.g., the anti-DLL3 CAR-T cells of the disclosure, are used to treat small cell lung cancer.

[00225] Also provided herein are methods for treating subjects. In some embodiments, the method comprises providing an immune cell of the invention to a subject in need thereof. In some embodiments, the method comprises a step of administering transformed immune cells of the invention to a subject in need thereof.

[00226] In some embodiments, T cells of the invention can undergo robust in vivo T cell expansion and can persist for an extended amount of time.

[00227] Methods of treatment of the invention can be ameliorating, curative or prophylactic. The method of the invention may be either part of an autologous immunotherapy or part of an allogeneic immunotherapy treatment. The invention is particularly suitable for allogeneic immunotherapy. T cells from donors can be transformed into non-alloreactive cells using standard protocols and reproduced as needed, thereby producing CAR-T cells which may be administered to one or several subjects. Such CAR-T cell therapy can be made available as an “off the shelf’ therapeutic product.

[00228] Cells that can be used with the disclosed methods are described in the previous section. Treatment can be used to treat subjects diagnosed with, for example, cancer. Cancers that may be treated include, for example without limitation, adult tumors / cancers and pediatric tumors / cancers. In some embodiments, the treatment can be in combination with one or more therapies against cancer selected from the group of antibodies therapy, chemotherapy, cytokines therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy and radiation therapy.

[00229] In some embodiments, treatment can be adminstered into subjects undergoing an immunosuppressive treatment. Indeed, embodiments of the invention rely on cells or a population of cells, which have been made resistant to at least one immunosuppressive agent due to the inactivation of a gene encoding a receptor for such immunosuppressive agent. In this aspect, the immunosuppressive treatment should help the selection and expansion of the T cells according to the invention within the subject. The administration of the cells or population of cells according to the invention may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a subject subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In some embodiments, the cell compositions of the invention are administered by intravenous injection.

[00230] In some embodiments, the administration of the cells or population of cells can comprise administration of, for example, about 104 to about 109 cells per kg body weight including all integer values of cell numbers within those ranges. In some embodiments the administration of the cells or population of cells can comprise administration of about 105 to 106 cells per kg body weight including all integer values of cell numbers within those ranges. The cells or population of cells can be administered in one or more doses. In some embodiments, said effective amount of cells can be administered as a single dose. In some embodiments, said effective amount of cells can be administered as more than one dose over a period time. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the subject. The cells or population of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of optimal ranges of effective amounts of a given cell type for a particular disease or conditions within the skill of the art. An effective amount means an amount which provides a therapeutic or prophylactic benefit. The dosage administered will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired. In some embodimetns, an effective amount of cells or composition comprising those cells are administered parenterally. In some embodiments, administration can be an intravenous administration. In some embodimetns, administration can be directly done by injection within a tumor.

[00231] In some embodiments of the invention, cells are administered to a subject in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to treatment with agents such as monoclonal antibody therapy, CCR2 antagonist (e.g., INC-8761), antiviral therapy, cidofovir and interleukin-2, Cytarabine (also known as ARA-C) or nataliziimab treatment for MS patients or efaliztimab treatment for psoriasis patients or other treatments for PML patients. In some embodiments, target-specific CAR-T cells described herein with enhanced signaling, such as DLL3 CAR T cells described herein are administered to a subject in conjunction with one or more of the following: an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, or PF-06801591), an anti-PD-Ll antibody (e.g., avelumab, atezolizumab, or durvalumab), an anti-OX40 antibody (e.g., PF-04518600), an anti-4-lBB antibody (e.g., PF-05082566), an anti-MCSF antibody (e.g., PD-0360324), an anti-GITR antibody, and / or an anti-TIGIT antibody. In some embodiments, DLL3-specific CAR-T cells with enhanced signaling comprising the amino acid sequence shown in SEQ ID NOs: 43, 46 or 49, or other target-specific CAR-T cells, are administered to a subject in conjunction with anti-PD-Ll antibody avelumab. In further embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and / or irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin) (Henderson, Naya et al. 1991; Liu, Albers et al. 1992; Bierer, Hollander et al. 1993). In further embodiments, the T cells of the invention may be used in combination with Receptor Tyrosine Kinase inhibitors such as Midostaurin and Sunitinib, mTOR inhibitors such as Rapamacyn and Everolimus, epigenetic modulators such as Vormostat, proteasome inhibitors such as Bortezomib, immunomodulatory agents such as lenalidomide, Hedgehog inhibitors such as Erismodegib and PF-04449913 or Isocitrate Dehydrogenase (IDH) inhibitors such as AG-120 and AG-221. In a further embodiment, the cell compositions of the invention are administered to a subject in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH, In some embodiments, the cell compositions of the invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. For example, In some embodiments, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the expanded immune cells of the invention. In some embodiments, expanded cells are administered before or following surgery.

[00232] In some embodiments, provided are methods for depleting engineered immune cells of the invention as described herein from a subject adminstered with said cells. Depletion can be by inhibition or elimination.

[00233] In one aspect, a method for depleting engineered immune cells expressing a recombinant antigen receptor of the invention e.g. CAR of the invention comprising an epitope specific for a monoclonal antibody comprises contacting said engineered immune cell with a monoclonal antibody specific for the epitope.

[00234] In some embodiments, a method for depleting from a subject administered with engineered immune cells of the invention comprising an epitope specific for a monoclonal antibody comprises administering to the subject a monoclonal antibody specific for the epitope. In these embodiments, administration of the monoclonal antibody specific for the epitope present in the extracellular domain of the recombinant antigen receptor e.g. CAR to the subject eliminates or inhibits the activity of engineered recombinant antigen receptorexpressing e.g. CAR-expressing immune cells from the subject. In one aspect, depletion of engineered immune cells allows for recovery of an endogenous population of cells that express the target antigen of the recombinant antigen receptor of the invention.

[00235] In one aspect, the invention relates to a method for promoting recovery of endogenous target antigen-expressing cells in a subject administered with engineered immune cells expressing at cell surface a recombinant antigen receptor e.g. CAR of the invention comprising an epitope specific for a monoclonal antibody, the method comprising administering a monoclonal antibody specific for the epitope to the subject. In one aspect, the term “recovery” refers to increasing the number of endogenous target antigenexpressing cells. The number of endogenous target antigen-expressing cells may increase due to increase in proliferation of endogenous target-expressing cells and / or due to reduction in elimination of such endogenous cells by the engineered immune cells. In some embodiments, administration of the monoclonal antibody to the subject depletes the engineered immune cells and increases the number of endogenous target antigen-expressing cells in the subject. In one embodiment, administration of the monoclonal antibody to the subject increases the number of endogenous target antigen-expressing cells by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, compared to the number of endogenous target antigen-expressing cells prior to administration of the monoclonal antibody.

[00236] In one aspect, provided is a method for treating a target antigen-mediated condition in a subject, the method comprising: (a) administering to the subject engineered immune cells expressing at cell surface recombinant antigen receptors e.g. CARs of the invention comprising one or more epitopes specific for one or more monoclonal antibodies; and (b) subsequently depleting the engineered immune cells from the subject by administering one or more monoclonal antibodies specific for the epitope to the subject.

[00237] In some embodiments, the mAbs used in the method for depleting CARexpressing engineered immune cells are selected from alemtuzumab, ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, QBEND-10, ustekinumab, and combinations thereof.

[00238] In some embodiments, the step of administering a monoclonal antibody to the subject comprises infusing the subject with the monoclonal antibody. In some embodiments, the amount of epitope-specific mAb administered to the subject is sufficient to eliminate at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the engineered immune cell in the subject.

[00239] In some embodiments, the step of administering a monoclonal antibody to the subject comprises infusing the subject with 375mg / m2 of rituximab, once or several times weekly.

[00240] In some embodiments, when immune cells expressing a CAR comprising an mAb-specific epitope (CAR-expressing immune cells) are depleted in a CDC assay using epitopespecific mAb, the amount of viable engineered immune cells decreases, e.g. by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

[00241] In some embodiments, a cytotoxic drug is coupled to the epitope-specific mAbs which are used to deplete the engineered immune cells. By combining targeting capabilities of monoclonal antibodies with the cell-killing ability of cytotoxic drugs, antibody-drug conjugate (ADC) allows a sensitive discrimination between healthy and diseased tissue when compared to the use of the drug alone. Market approvals were received for several ADCs; the technology for making them -particularly on linkers- is abundantly presented in the following prior art (Payne, G. (2003) Cancer Cell 3:207-212; Trail et al (2003) Cancer Immunol. Immunother. 52:328-337; Syrigos andEpenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278).

[00242] In some embodiments, the epitope-specific mAb to be infused is conjugated beforehand with a molecule able to promote complement dependent cytotoxicity (CDC). Therefore, the complement system helps or complements the ability of antibodies to clear pathogens from the organism. When stimulated by one of several, is triggered an activation cascade as a massive amplification of the response and activation of the cell-killing membrane attack complex. Different molecules may be used to conjugate the mAb, such as glycans (Courtois, A, Gac-Breton, S., Berthou, C, Guezennec, J., Bordron, A. and Boisset, C. (2012), Complement dependent cytotoxicity activity of therapeutic antibody fragments is acquired by immunogenic glycan coupling, Electronic Journal of Biotechnology ISSN: 0717-3458; http: / / www.ejbiotechnology.info DOI: 10.2225 / voll5-issue5). Kits

[00243] The invention also provides kits for use in the instant methods. Kits of the invention include one or more containers comprising a polynucleotide encoding a recombinant antigen receptor e.g. CAR of the present invention or an engineered immune cell comprising a polynucleotide encoding the recombinant antigen receptor e.g. CAR of the invention, and instructions for use in accordance with any of the methods of the invention described herein. Generally, these instructions comprise a description of administration of the engineered immune cell for the herein-described therapeutic treatments.

[00244] The instructions relating to the use of the engineered immune cells or antibodies as described herein generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[00245] The kits of this invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. A kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a recombinant antigen receptor e.g. CAR of the present invention. The container may further comprise a second pharmaceutically active agent.

[00246] Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container.

[00247] The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. EXAMPLES Example 1: Altering the quality and quantity of CD3£ ITAMs as a strategy to improve CAR signaling

[00248] A recombinant antigen receptor e.g. a CAR comprising a wildtype CD3(^ ITAM-containing domain has only 3 ITAMs, all of which derive from CD3(^, whereas the naturally occurring TCR / CD3 holocomplex has 10 ITAMs comprising contributions from all four CD3 chains (delta, epsilon, gamma, and zeta). Reducing or increasing the number or diversity of IT AMs modulates both TCR and CAR function, underscoring the significance of these parameters. See, e.g., Bettini, M. L. et al. Cutting Edge: CD3ITAM Diversity Is Required for Optimal TCR Signaling and Thymocyte Development. J. Immunol. 199, 15551560 (2017); Feucht, J. et al. Calibration of CAR activation potential directs alternative T cell fates and therapeutic potency. Nature Medicine 25, 82-88 (2019); Majzner, R. G. et al. Low CD 19 Antigen Density Diminishes Efficacy of CD 19 CAR T Cells and Can be Overcome By Rational Redesign of CAR Signaling Domains. Blood 132, 963 (2018). Thus, the quantity and quality of IT AMs and the complement of proximate signaling mediators differ between CARs and TCRs.

[00249] To test whether the sensitivity of CAR signaling can be improved by more closely mimicking TCR signaling, we altered the quality and quantity of CD3(^ IT AMs in a DLL3 CAR (10G1-K). In one set of constructs, the number of IT AMs affiliated with the CAR was decreased or increased (e.g. by truncating CAR after the first ITAM or by concatenating two copies of the CD3^ cytoplasmic tail) (FIG. 1A). In addition, the character of the IT AMs in these constructs was varied by replacing them in the CD3(^ framework with IT AMs from other CD3 chains in various configurations (FIG. 1A). In a second set of constructs, the residue in the second position in the canonical YXX(L / I) ITAM motif was mutated to Ala, a mutation previously reported to reduce the degree of receptor clustering necessary to initiate signaling (FIG. IB, Table 1). See, e.g., Sunder-Plassmann, R. et al. Functional analysis of immunoreceptor tyrosine-based activation motif (ITAM)-mediated signal transduction: The two YxxL segments within a single CD3C-ITAM are functionally distinct. Eur. J. Immunol. 27, 2001-2009 (1997). We reasoned that this mutation may therefore facilitate signaling in response to low density tumor antigens. Constructs that combine the strategies described for FIG. 1A & IB were also created (FIG. IC). Example 2: Overexpression of downstream T cell signaling mediators to improve CAR signaling

[00250] CARs employing the 4-IBB costimulatory domain inefficiently recruit downstream mediators of T cell signaling (e.g. ZAP70) (see Gudipati, V. et al. Inefficient CAR-proximal signaling blunts antigen sensitivity. Nat. Immunol. (2020). doi:10.1038 / s41590-020-0719-0) and overexpression of these mediators (e.g. Lek) has been shown to boost CAR function (see Sun, C. et al. THEMIS-SHP1 Recruitment by 4-IBB Tunes LCK-Mediated Priming of Chimeric Antigen Receptor-Redirected T Cells. Cancer Cell 37, 216-225.e6 (2020)).We designed a series of constructs that employ an intervening 2A ribosomal skip sequence to couple CAR expression with overexpression of one of multiple downstream mediators of T cell signaling (see, e.g.. Table 1, listing the following: SEQ ID NO: 1 (ZAP70), 2 (Lek), 4 (Fyn), 6 (Syk), 8 (LAT), and 9 (Unci 19), SEQ ID NOs: 3 (Truncated Lek), 5 (Truncated Fyn), and 7 (Truncated Syk)). See FIGs.3 A-B. Example 3: Comparison of CAR constructs with different quality and quantity of IT AMs in vitro

[00251] In this example, the constructs described in Example 1 were transduced in primary human T cells and tested for in vitro cytotoxic activity.

[00252] To make lentivirus expression constructs described in Example 1, HEK-293T cells were plated at 1.5 million cells per mL in 2mL of DMEM (Gibco) supplemented with 10% FBS (Hyclone) per well of a 6-well plate on Day -1. On Day 0, the lentivirus was prepared by mixing together with lentiviral packaging vectors 1.5ug psPAX2, 0.5ug pMD2G, and 0.5ug of the appropriate transfer CAR added to the DNA mix. The DLL3-specific CAR clone 10G1-K was used in this experiment. See WO2020 / 180591.

[00253] The mixture was incubated at room temperature for 20 minutes and the total volume of 500uL was slowly added to the sides of the wells containing HEK-293T. Purified T cells were activated in X-Vivo-15 medium (Lonza) supplemented with lOOIU / mL human IL-2 (Miltenyi Biotec), 10% FBS (Hyclone), and human T TransAct (Miltenyi Biotec, Cat# 130-111-160, 1:100 dilution). On Day 1, the media from each well of the 6-well plate was replaced with 2mL per well of T cell transduction media, i.e., X-Vivo-15 supplemented with 10% FBS. On Day 2, T cells were resuspended at 0.4 million cells per mL in 1.5 mL of T cell transduction media per well of a Grex-24 plate (Wilson Wolf, cat# 80192M). The lentiviral supernatants from HEK293T cells (about 1.5ml) were harvested and passed through a 0.45 micron filter (EMD Millipore) to remove cell debris, and then added to the T cells along with lOOIU / mL human IL-2. On Day 5, 4.5 mL of T cell expansion media, i.e., X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio) was added to each well of a Grex-24 plate. On Day 9 and Day 13, transduction efficiency was determined by detecting the percentage of T cells that express BFP and recognize recombinant DLL3 (Adipogen) using flow cytometry. Cells were expanded into larger flasks or G-Rex vessels (Wilson Wolf) as needed using T cell expansion media. On Day 14, DLL3 CAR-T cells were cryopreserved. Percentage of cells stained with recombinant DLL3 was normalized across clones right before cryopreservation.

[00254] To determine the percentage of T cells that were successfully transduced with DLL3 CAR, T cells were first incubated with lug / ml Flag tagged recombinant DLL3 (Adipogen) in PBS+1%BSA for 20 minutes at 4°C. Then cells were washed with PBS+1%BSA, stained with PE labelled anti-Flag antibodies (Biolegend, Cat# 637310) and analyzed using flow cytometry. Examples of CAR T cells are shown in FIG.2A-bottom panels. The results in FIG. 2A-bottom panels show that conventional (comprising wildtype CD3(^ ITAM-containing domain) or modified CARs (comprising a non-wildtype CD3(^ ITAM-containing domain) were expressed on the surface of primary T-cells. These results showed strong correlation between BFP expression and recombinant DLL3 staining, suggesting these constructs expressed properly and the proteins expressed had no major issues of folding or surface localization. The plots were gated on live CD3+ cells. The numbers on the plots are the percentage of cells that expressed each CAR construct. FIG. 2E shows the detection of conventional or modified CARs in a second human donor.

[00255] To compare the efficacy of conventional and modified CARs in a short-term kinetic killing assay, CAR T cells were incubated with DLL3 positive DMS273 (low antigen density) or WM266.4 (high antigen density) cells expressing nuclear GFP at indicated effectortarget (E:T) ratio in T cell expansion media, i.e., X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio). The tissue culture plates were placed in Incucyte and the number of GFP positive target cells was counted every 6 hours. FIGs. 2B-2D show in one human T cell donor, several constructs described in Example 1 improved CAR T cytotoxicity against both DMS273 and WM266.4 target cells (relative to CAR comprising a wildtype CD3(^ ITAM-containing domain). FIG. 2F-2G show that in CAR T cells derived from a second human donor, several constructs described in Example 1 also improved CAR T cell cytotoxicity (relative to CAR comprising a wildtype CD3(^ ITAM-containing domain).

[00256] To compare the efficacy of CARs described in Example 1 and Example 2 in a long-term cytotoxicity assay, CAR-T cells were exposed repeatedly to the target DLL3 every 2 to 3 days to promote CAR-T cells proliferation. On the first day of the assay, 5,000 firefly luciferase labelled WM266.4 or DMS273 cells were seeded in 96-well plates with black wall and flat clear bottom in lOOul X-Vivo-15 medium with 5% of human serum. After target cells attached to the bottom of the plates, CAR T cells were thawed and added to plated target cells in X-VIVO medium with 5% of human serum. Every 2 to 3 days thereafter, 100 pl medium containing CAR T cells were transferred to freshly plated target cells and percentage lysis of previously plated target cells were determined using one-glo assay system (Promega). Each condition was assayed in 3 replicates. Average percentage of lysis and standard deviation were plotted in FIGs. 3A-3B. The experimental data of serial killing assay shows that after repeated exposure of CAR T cells to DLL3 positive target cells, some of the constructs performed better than the conventional CD3(^WT construct. Example 4: Insertion of Lek recruitment motifs (LRM) as a strategy to enable recruitment of Lek to CAR synapses in a more TCR-like manner

[00257] Synapse formation for CARs does not require co-receptor (CD8 or CD4) involvement. As a result, the co-receptor-associated Lek kinase that is central to TCR signaling is coordinated with target recognition for the TCR synapse but is not for the CAR synapse (FIG. 4A). See Davenport, A. J. et al. Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity. Proc. Natl. Acad. Sci. U. S. A. 115, E2068-E2076 (2018).

[00258] To facilitate Lck-mediated signaling events in CAR T cells, constructs were designed to deliver a CAR with a cytoplasmic domain modified to include a co-receptor-derived sequence motif that recruits T cell signaling mediators (FIG. 4B). In this example, a Lek recruitment motif (LRM) (derived, for example, from CD8, CD4, or CD28) was inserted between cytoplasmic domains and the intracellular domain of the CAR, between the co-stimulatory domain and the IT AM containing domain, or at the C-terminus of the CAR cytoplasmic tail, enabling directed recruitment of Lek to the CAR receptor (FIG. 5A). We hypothesized that the constructs will promote more efficient phosphorylation of the CAR IT AMs and ZAP70, and thus more efficient signaling of the CAR upon antigen binding and synapse formation. Exemplary LRM amino acid sequences of SEQ ID NOs: 55 (CD8LRM-1), 56 (2XCD8LRM-1), 57 (CD8LRM-2), 58 (CD28LRM), 59 (CD28LRMY3), 64 (CD4LRM)) are shown in Table 1. Example 5: Comparison of CAR constructs with LRM in an in vitro cytotoxicity assay

[00259] In this example, the constructs described in Example 4 were transduced in primary human T cells tested for in vitro cytotoxic activity. Lentivirus encoding constructs described in Example 4 and primary T cells transduced with these lentiviruses were produced using methods described in Example 3.

[00260] The results in FIG. 5B show that conventional CAR or CARs with an LRM insertion were expressed on the surface of primary T-cells. These T cells showed strong correlation between BFP expression and recombinant DLL3 staining, suggesting these constructs expressed properly and the proteins expressed had no major issues of folding or surface localization. The plots were gated on live CD3+ cells. The numbers on the plots are the percentage of cells expressing each CAR construct.

[00261] To compare the efficacy of conventional and modified CARs in a short-term kinetic killing assay, CAR T cells were incubated with DLL3 positive DMS273 (low antigen density) or WM266.4 (high antigen density) cells expressing nuclear GFP at indicated effector Target (E:T) ratio in T cell expansion media, i.e., X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio). The tissue culture plates were placed in Incucyte and the number of GFP positive target cells was counted every 6 hours. FIG. 5C shows constructs with an LRM inserted between transmembrane domain and 4-1BB cytoplasmic domain did not perform as well as the conventional CAR construct. On the other hand, FIGs. 5D-5E show that several CAR constructs with an LRM inserted at the C-terminus of the CAR intracellular domain demonstrated comparable or better cytotoxicity against both DMS273 and WM266.4 target cells. The DLL3-specific CAR clone 10G1-K was used in this experiment. Example 6: LckCARs as a strategy to boost recruitment of Lek to the CAR synapse and to enable combinatorial CAR targeting

[00262] A major obstacle to applying CAR T therapies to solid tumors has been the lack of suitable targets. Ideally, solid tumor targets can be identified that are expressed at high and relatively uniform levels across tumor cells but that are not expressed or expressed only at low levels on healthy tissues (as CD 19 is in non-solid tumors). If solid tumor antigens cannot be identified with this auspicious confluence of characteristics, CAR design should accommodate the shortcomings of the less than ideal solid tumor target antigens. For instance, CAR-mediated targeting of solid tumor antigens that are also expressed in normal tissues may require combinatorial targeting (e.g. via logic gates) to de-risk these liabilities.

[00263] To facilitate combinatorial targeting, a non-standard “CAR” or a second recombinant antigen receptor was designed, in which the intracellular domain of the non standard CAR comprises, instead of domains that provide signal 1 and / or signal 2, a downstream mediator of T cell signaling or a functional variant thereof. As an example, such a non-standard “LckCAR” is shown in FIG. 6A. The LckCAR fuses an antigen recognition domain via the CD8 (or alternative) transmembrane domain to a protein comprising a Lck-recruitment motif (e.g. CD8 cytoplasmic tail) or directly to the Lek protein itself. Targeting of this LckCAR to the same target as a target-specific CAR may increase Lek recruitment to the synapse, juxtaposing Lek with IT AM substrates and thereby augmenting signaling efficiency (FIG. 6A). Targeting of the LckCAR to a second tumor-associated target antigen distinct from that recognized by the co-expressed CAR may enable AND-gated combinatorial targeting of solid tumor targets, thereby improving safety (FIG. 6B). There are many potential variations on this theme employing fusions of an extracellular antigen binding domain to different downstream T cell signaling mediators, such as ZAP70 and LAT. Example 7: Comparison of CAR constructs with different IT AMs or LRM in the context of DLL3 CAR 4H8-R2S

[00264] In this example, the constructs described in FIG. 1 were evaluated in the context of DLL3 CAR clone 4H8-R2S (see WO2020 / 180591) to demonstrate that the designs are broadly applicable.

[00265] To make lentivirus encoding constructs described in Example 1, HEK-293T cells were plated at 1.5 million cells per mL in 2mL of DMEM (Gibco) supplemented with 10% FBS (Hyclone) per well of a 6-well plate on Day -1. On Day 0, the lentivirus was prepared by mixing together with lentiviral packaging vectors 1.5ug psPAX2, 0.5ug pMD2G, and 0.5ug of the appropriate transfer CAR added to the DNA mix. The mixture was incubated at room temperature for 20 minutes and the total volume of 500uL was slowly added to the sides of the wells containing HEK-293T. Purified T cells were activated in X-Vivo-15 medium (Lonza) supplemented with lOOIU / mL human IL-2 (Miltenyi Biotec), 10% FBS (Hyclone), and human T TransAct (Miltenyi Biotec, Cat# 130-111-160, 1:100 dilution). On Day 1, the media from each well of the 6-well plate was replaced with 2mL per well of T cell transduction media, i.e., X-Vivo-15 supplemented with 10% FBS. On Day 2, T cells were resuspended at 0.4 million cells per mL in 1.5 mL of T cell transduction media per well of a Grex-24 plate (Wilson Wolf, cat# 80192M). The lentiviral supernatants from HEK293T cells (about 1.5ml) were harvested and passed through a 0.45 micron filter (EMD Millipore) to remove cell debris, and then added to the T cells along with lOOIU / mL human IL-2. On Day 5, 4.5 mL of T cell expansion media, i.e., X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio) was added to each well of a Grex-24 plate. On Day 9 and Day 13, transduction efficiency was determined by detecting the percentage of T cells that recognize recombinant Flag-DLL3 (Adipogen) using flow cytometry. Cells were expanded into larger flasks or G-Rex vessels (Wilson Wolf) as needed using T cell expansion media. On Day 14 or Day 16, DLL3 CAR-T cells were cryopreserved. Percentage of cells stained with recombinant DLL3 was normalized across clones right before cry opreservation.

[00266] To determine the percentage of T cells that were successfully transduced with the DLL3 CAR, T cells were first incubated with lug / ml Flag tagged recombinant DLL3 (Adipogen) in PBS+1%BSA for 20 minutes at 4°C. The cells were then washed with PBS+1%BSA, stained with PE labelled anti-Flag antibodies (Biolegend, Cat# 637310) and analyzed using flow cytometry.

[00267] FIGs. 7A-B, FIGs. 8A-E and FIGs. 9A-B show series of results of CAR T cells produced from three different human donors, respectively. FIGs. 7A, 8A and 9A show flow cytometry data exhibiting expression of various DLL3 CAR 4H8-R2S constructs on the surface of CAR T cells generated from three separate human donors. The plots are gated on live CD3+ cells. The numbers indicate the percentage of cells expressing each CAR construct. Most of the modified CAR T cells have similar percentage of CAR T cells compared to control CAR T cells. The activity data were normalized against transduction efficiency.

[00268] To compare the efficacy of various DLL3 CAR 4H8-R2S in a short-term kinetic killing assay, CAR T cells were incubated with DLL3 positive DMS273 cells (low antigen densitycell line) or WM266.4 cells (high antigen density cell line) expressing nuclear GFP at indicated effectortarget (E:T) ratio in RPMI (Gibco) supplemented with 10% FBS (Hyclone). The tissue culture plates were placed in Incucyte and the number of GFP positive target cells was counted every 6 hours. FIG. 7B shows CD3^(zdzezg) construct in the context of DLL3 CAR 4H8-R2S performed better than the DLL3 CAR 4H8-R2S CD3(^WT construct, while CARs with the CD3^(YAYAYA) and CD3^(zdzezg-6xYA) modifications were comparable to CD3(^WT. FIG. 8B shows results of cytotoxicity assay where CAR T were prepared using primary T cells from a separate human donor. The results show that CD3^(zdzezg) and CD3^ constructs were superior to CD3(^WT in cytotoxic activity.

[00269] To compare the efficacy of CARs in a long-term cytotoxicity assay, CAR-T cells were exposed repeatedly to their targets every 2 to 3 days causing the CAR-T cells to undergo proliferation and in certain cases, differentiation and, in certain cases, exhaustion. PD-L1 / PD-1 pathway has been shown to downregulate TCR and CAR signaling. To test if modified CAR constructs can resist the inhibition from PD-L1 / PD-1 axis, 5,000 firefly luciferase labelled parental or PD-L1 overexpressed WM266.4 and DMS273 cells were seeded in 96-well plates with black wall and flat clear bottom in 50ul RPMI (Gibco) supplemented with 10% FBS (Hyclone). After target cells attached to the bottom of the plates, CAR T cells were thawed and added to plated target cells. Every 2 to 3 days thereafter, 100 pl medium containing CAR T cells were transferred to freshly plated target cells and percentage lysis of previously plated target cells were determined using one-glo assay system (Promega). Each condition was assayed in 5 replicates. Average percentage of lysis and standard deviation were plotted. FIG. 8C and FIG. 9B show data of CAR T cells from two separate human donors. The results show that after repeated exposure of CAR T cells to DLL3 positive target cells, CD3^(zdzezg) construct performed better than the CD3(^WT construct in the context of DLL3 CAR 4H8-R2S, against both parental DLL3-expressing target cells or target cells further overexpressing PD-L1. The CD3^ construct was superior to the CD3^WT construct in some settings and comparable to CD3(^WT in other settings.

[00270] To measure cytokines secreted from DLL3 CAR-T cells, CAR-T cells were incubated with WM266.4 and DMS273 at effectortarget (E:T) ratio of 1:1 in RPMI (Gibco) supplemented 10% FBS (Hyclone). 24 hours later, tissue culture supernatant was collected and the levels of 3 cytokines [interferon gamma (IFN-y), tumor necrosis factor alpha (TNF-a), and IL-2] in the supernatants were measured using human proinflammatory tissue culture 9-plex assay (MSD) following manufacturer’s protocol.

[00271] FIG. 8D shows CD3^(zdzezg) and CD3^ CAR T cells secreted higher levels of IFN-y, TNF-a and IL-2 than CD3(^WT CAR T cells when co-cultured with DMS273 (low DLL3 density) cell line, n=3.

[00272] FIG. 8E shows CD3^(zdzezg),CD3^, CD3^(YAYAYA), and CD3£WT-CD8LRM secret comparable or higher levels of cytokines than CD3(^WT CAR T cells when co-cultured with WM266.4 (high DLL3 density) cell line, n=3. Example 8: Comparison of CAR constructs with different quality and quantity of ITAMs in the context of DLL3 CAR 2G1-RSR

[00273] In this example, the constructs described in FIG. 1 were evaluated in the context of DLL3 CAR clone 2G1-RSR (see WO2020 / 180591) to demonstrate the designs are broadly applicable.

[00274] 2G1-RSR DLL3 CAR T cells were produced as described and detected in Example 7. To compare the efficacy of CARs in a long-term cytotoxicity assay, CAR-T cells were exposed repeatedly to their targets every 2 to 3 days causing the CAR-T cells to undergo proliferation and in certain cases, differentiation and exhaustion. On the first day of the assay, 5,000 firefly luciferase labelled parental DMS273 or DMS273 overexpressing PD-L1 were seeded in 96-well plates with black wall and flat clear bottom in 50ul RPMI (Gibco) supplemented with 10% FBS (Hyclone). After target cells attached to the bottom of the plates, CAR T cells were thawed and added to plated target cells. Every 2 to 3 days thereafter, 100 pl medium containing CAR T cells were transferred to freshly plated target cells and percentage lysis of previously plated target cells were determined using one-glo assay system (Promega). Each condition was assayed in 3 replicates. FIG. 10 shows data of long-term cytotoxicity assay, the CD3^(zdzezg) construct performed better than the CD3(^WT construct in the context of DLL3 CAR 2G1-RSR on both parental and PD-L1-overexpressing DMS273 cells. References:

[00275] Bettini, M. L. et al. Cutting Edge: CD3 ITAM Diversity Is Required for Optimal TCR Signaling and Thymocyte Development. J. Immunol. 199, 1555-1560 (2017).

[00276] Feucht, J. et al. Calibration of CAR activation potential directs alternative T cell fates and therapeutic potency. Nature Medicine 25, 82-88 (2019).

[00277] Majzner, R. G. et al. Low CD 19 Antigen Density Diminishes Efficacy of CD 19 CAR T Cells and Can be Overcome By Rational Redesign of CAR Signaling Domains. Blood 132, 963 (2018).

[00278] Sunder-Plassmann, R., Lialios, P., Madsen, M., Koyasu, S. & Reinherz, E. L. Functional analysis of immunoreceptor tyrosine-based activation motif (ITAM)-mediated signal transduction: The two YxxL segments within a single CD3^-ITAM are functionally distinct. Eur. J. Immunol. 27, 2001-2009 (1997).

[00279] Gudipati, V. et al. Inefficient CAR-proximal signaling blunts antigen sensitivity. Nat. Immunol. (2020). doi:10.1038 / s41590-020-0719-0

[00280] Sun, C. et al. THEMIS-SHP1 Recruitment by 4-IBB Tunes LCK-Mediated Priming of Chimeric Antigen Receptor-Redirected T Cells. Cancer Cell 37, 216-225.e6 5    (2020).

[00281] Davenport, A. J. et al. Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity. Proc. Natl. Acad. Set. U. S. A. 115, E2068-E2076 (2018).

Claims

2021312871  29 Sep 2025WHAT IS CLAIMED IS1.     A recombinant antigen receptor comprising an extracellular antigen binding domain,a transmembrane domain, and an intracellular domain that comprises a costimulatory domain and an ITAM-containing domain, whereinthe ITAM-containing domain comprises from N-terminus to C-terminus (a) CD3z1 ITAM, CD3d ITAM, CD3z2 ITAM, CD3e ITAM, CD3z3 ITAM, CD3g ITAM, (b) CD3z1 (YAEL (SEQ ID NO: 152)) ITAM, CD3z2 (YAEL (SEQ ID NO: 152)) ITAM, CD3z3 (YAGL (SEQ ID NO: 153)) ITAM , or (c) CD3z1 (YAEL (SEQ ID NO: 152)) ITAM, CD3d (YAPL (SEQ ID NO: 154)) ITAM, CD3z2 (YAEL (SEQ ID NO: 152)) ITAM, CD3e (YAPI (SEQ ID NO: 155)) ITAM, CD3z3 (YAGL (SEQ ID NO: 153)) ITAM, CD3g (YAPL (SEQ ID NO: 154)) ITAM.

2. The recombinant antigen receptor of claim 1, wherein the recombinant antigenreceptor is a chimeric antigen receptor (CAR).

3. The recombinant antigen receptor of claim 1 or claim 2, wherein the antigen bindingdomain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL).

4. The recombinant antigen receptor of any one of claims 1-3, wherein the costimulatory domain comprises 4-1BB co-stimulatory domain.

5. The recombinant antigen receptor of any one of claims 1-4, wherein the intracellulardomain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 35 and 37.

6. The recombinant antigen receptor of claim 5, wherein the intracellular domaincomprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 30 and 37.

7. The recombinant antigen receptor of claim 5, wherein the intracellular domaincomprises an amino acid sequence of SEQ ID NO:35.

8. A polynucleotide comprising a DNA sequence encoding the recombinant antigenreceptor of any one of claims 1-7.

9. A vector comprising the polynucleotide of claim 8.

10. An engineered immune cell comprising the recombinant antigen receptor of any one of claims 1-7.2021312871  29 Sep 202511. A pharmaceutical composition comprising the engineered immune cell of claim 10.

12. A recombinant antigen receptor comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular domain, wherein the intracellular domain comprises a 4-1BB co-stimulatory domain, a CD3z ITAM-containing domain, and further comprises an additional Lck recruiting motif (LRM), wherein the additional LRM is a CD8 LRM, and wherein the CD8 LRM comprises, consists of or consists essentially of the amino acid sequence of SEQ ID NO: 56.

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