Compositions and methods for reducing therapeutic T cell toxicity

Abstract

Disclosed are immune effector cells engineered to express anti-CD3 antibodies disclosed herein that are configured to autoactivate pre-made immune effector cells and thereby reduce expression of T cell receptors (e.g., TCRαβ) that can result in GVHD. Methods are also disclosed for modifying donor immune effector cells to make them suitable for pre-made treatment of allogeneic subjects. These methods include engineering the cells to express anti-CD3 antibodies that are configured to activate the cells. In some embodiments, the antibody is a bispecific antibody that binds to the CD3 complex on immune effector cells. In other embodiments, the antibody is a membrane-bound anti-CD3 antibody that autoactivates the immune effector cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 159,222, filed March 10, 2021, U.S. Provisional Application No. 63 / 209,094, filed June 10, 2021, and U.S. Provisional Application No. 63 / 225,715, filed July 26, 2021, which are incorporated by reference in their entireties herein.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted in electronic format as an ASCII.txt file entitled "320803-2800 Sequence Listing_ST25", created on March 8, 2022, having 226,162 bytes. The contents of the Sequence Listing are incorporated herein in their entirety. [Background technology]

[0003] background Autologous T cells, such as chimeric antigen receptor (CAR) T cells, have changed the therapeutic landscape of hematological malignancies. Nevertheless, the use of donor-derived allogeneic T cells has many potential advantages over autologous approaches, such as the immediate availability of cryopreserved batches for patient treatment, possible standardization of T cell products, time for multiple cell modifications, re-administration or combination of T cells against different targets, and cost reduction using industrialized processes. However, allogeneic T cells can cause life-threatening graft-versus-host disease and can be rapidly eliminated by the host immune system. Thus, there is a need in the art for a method to modify allogeneic T cells with reduced potential for graft-versus-host disease. Summary of the Invention

[0004] overview Disclosed are pre-made immune effector cells engineered to express the anti-CD3 antibodies disclosed herein that are configured to reduce expression of T cell receptors (e.g., TCRαβ) that can autoactivate immune effector cells, thereby resulting in GVHD.

[0005] Methods are also disclosed for modifying donor immune effector cells to make them suitable for off-the-shelf treatment of allogeneic subjects. These methods include engineering the cells to express anti-CD3 antibodies that are configured to activate the cells. In some embodiments, the immune effector cells are further engineered to express chimeric antigen receptors (CARs). In other embodiments, the immune effector cells do not express CARs and instead rely on antibodies for targeting.

[0006] In some embodiments, the antibody is a bispecific antibody that crosslinks the CD3 complex on immune effector cells to another cell. In other embodiments, the antibody is a membrane-bound anti-CD3 antibody that autoactivates immune effector cells. In other embodiments, the antibody is a monospecific antibody, such as scFv.

[0007] Thus, also disclosed is a method for enhancing CAR-T cells for allogeneic cell transfer, comprising engineering CAR-T cells to secrete a monospecific anti-CD3 antibody.

[0008] In some embodiments, immune effector cells are obtained from allogeneic donors.Therefore, also disclosed is a method for treating cancer in a subject, comprising: obtaining immune effector cells from allogeneic donors; engineering immune effector cells to express anti-CD3 multispecific antibodies, wherein the antibodies are configured to bind to the CD3 complex on immune effector cells and to a second antigen on another cell in a manner sufficient to activate the CD3 complex; and administering the engineered immune effector cells to the subject in an amount effective to treat cancer.

[0009] In some embodiments, immune effector cells include any CD3-expressing lymphocytes involved in defending the body against infections and foreign substances.

[0010] Thus, bispecific molecules capable of cross-linking the CD3 complex on a pre-formed immune effector cell with an antigen on another cell are also disclosed. The bispecific molecules can be engineered from a fusion polypeptide comprising 1) a variable domain of an antibody that specifically binds to a target cell surface receptor and 2) a variable domain of an antibody that specifically binds to CD3. Either or both components of the bispecific molecule can also be engineered from non-antibody scaffolds, including but not limited to nanobodies, monobodies, cyclic peptides, small molecules, and engineered ankyrin repeat proteins (Darpins).

[0011] In some embodiments, the bispecific molecule is a bispecific T cell-engaging (BiTE) antibody (fusion polypeptide), for example having the following formula: SP-V L RV H R- / -V L 3-V H 3. SP-V H RV L R- / -V H 3-V L 3. SP-V L RV H R- / -V H 3-V L 3. SP-V H RV L R- / -V L 3-V H 3. SP-V L 3-V H 3- / -V L RV H R, SP-V H 3-V L 3- / -V H RV L R, SP-V L 3-V H3- / -V H RV L R, or SP-V H 3-V L 3- / -V L RV H R, where "SP" represents an optional signal peptide, During the ceremony, “V L R' is a light chain variable domain specific for a target cell surface receptor; During the ceremony, “V H 3" is a heavy chain variable domain specific for CD3; During the ceremony, “V L 3" is a light chain variable domain specific for CD3; During the ceremony, “V H R' is a heavy chain variable domain specific for a target cell surface receptor; where "-" consists of a peptide linker or a peptide bond; and where "- / -" consists of the peptide hinge sequence.

[0012] In some embodiments, the antibody is a bispecific antibody containing the complete heavy and light chain regions. In this embodiment, the antibody may be produced by methods described, such as the "knob and hole" format (Ridgway JB, et al, Protein Eng. 1996 9(7):617-21).

[0013] The target cell surface receptor may be any other cell surface receptor, channel or transporter expressed by the cell in some cases. In some embodiments, the receptor is a tumor-associated antigen (TAA). A tumor antigen is a protein produced by a tumor cell that induces an immune response, particularly a T-cell mediated immune response. The additional antigen binding domain may be an antibody or a natural ligand of the tumor antigen. The selection of the additional antigen binding domain depends on the specific type of cancer to be treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), EGFRvIII, IL-llRa, IL-13Ra, EGFR, CSPG4, FAP, B7H3, Kit, CA LX, CS-1, MUC1, BCMA, bcr-abl, HER2, β-human chorionic gonadotropin, alpha fetoprotein (AFP), ALK, CD19, CD123, cyclin Bl, lectin-reactive AFP, Fos-related antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RUL, RU2, SSX2, AKAP-4, LCK, OY-TESl, PAX5, SART3, CLL-1, Fucosyl GM1, GloboH, MN-CA IX, EPCAM, EVT6-AML, TGS5, human telomerase reverse transcriptase, polysialic acid (plysialic acid), PLAC1, RUL, RU2(AS), intestinal carboxylesterase, Lewis Y, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostase, prostate specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-Ia, LMP2, NCAM, p53, p53 mutant, Ras mutant, gplOO, prostein, OR51E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-beta, survivin and telomerase, legumain, HPV E6, E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1, prostate cancer tumor antigen-1 (PCTA-1), ML-IAP, MAGE, MAGE-A1, MAD-CT-1, MAD-CT-2, MelanA / MART1, XAGE1, ELF2M, ERG(TMPRSS2ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoint, NY-BR-1, ephnnB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD171, CD179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGFII, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRC5D, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, ROR1, Flt3, TAG72, TN Ag, Tie2, TEM1, TEM7R, CLDN6, TSHR, UPK2, and mesothelin. In a preferred embodiment, the tumor antigen is selected from the group consisting of folate receptor (FRa), mesothelin, EGFRvIII, IL-13Ra, CD123, CD19, CD33, BCMA, GD2, CLL-1, CA-IX, MUCl, HER2, and any combination thereof. Non-limiting examples of tumor antigens include: differentiation antigens such as tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi 5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; for example, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23H1, PSA, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCASl, SDCCAG1 6, TA-90\Mac-2 binding protein / cyclophilin C-related protein, TAAL6, TAG72, TLP, TPS, GPC3, MUC16, LMP1, EBMA-1, BARF-1, CS1, CD319, HER1, B7H6, L1CAM, IL6, and MET.

[0014] Also disclosed are isolated nucleic acids encoding the disclosed fusion polypeptides, as well as nucleic acid vectors containing the isolated nucleic acids operably linked to an expression control sequence. Also disclosed are cells transfected with these vectors, and the use of these cells to produce the disclosed fusion polypeptides.

[0015] Bispecific antigen-binding molecules can be formed from the dimerization of a heavy chain and a light chain. In these embodiments, L R is V H It dimerizes with R to form an antigen-binding site for target cell surface receptors, and V H 3 is V L 3 to form the antigen-binding site for CD3.

[0016] Also disclosed is a bispecific antibody that is a single polypeptide chain comprising a bispecific antibody having a first antigen-binding region and a second antigen-binding region. Optionally, the first antigen-binding region can specifically bind to a target receptor on a cell; the second antigen-binding region can specifically bind to CD3 on an immune effector cell.

[0017] Each of the first and second portions can comprise one, two, three or more antibody variable domains. In certain embodiments, each of the first and second portions comprises two variable domains, a variable heavy (V H ) domain and variable light (V L ) domain.

[0018] In some cases, the bispecific antibody is about 10 -7 M, 10 -8 M, 10 -9 M or less K D and has affinity for CD3.

[0019] Each of the first and second moieties can be derived from a natural antibody, such as a monoclonal antibody. In some cases, the antibody is human. In some cases, the bispecific antibody is modified to reduce immunogenicity when administered to humans. For example, the modification includes one or more techniques selected from the group consisting of chimerization, humanization, CDR grafting, deimmunization, and mutation of framework amino acids to correspond to the closest human germline sequence.

[0020] Also disclosed is a pharmaceutical composition comprising the molecule disclosed herein in a pharma-ceutically acceptable carrier.Also disclosed is a method for targeting ubiquitination of a target receptor in a subject, comprising administering to the subject a therapeutically effective amount of the disclosed pharmaceutical composition.Also disclosed is a kit comprising the bispecific antibody disclosed herein.

[0021] Also disclosed is an expression vector comprising an isolated nucleic acid encoding the bispecific antibody disclosed herein operably linked to an expression control sequence. Also disclosed is a cell comprising the disclosed expression vector. The cell can be a primary cell, a transformed cell, a cell line, etc. In some cases, the cell is a mammalian cell line. In some cases, the cell is a non-mammalian cell line. For example, the cell can be a bacterial or insect cell line.

[0022] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will become apparent from the description and drawings, and from the claims. [Brief description of the drawings]

[0023] [Figure 1-1]Figures 1A-1C show CD3 / TCRαβ expression in vitro after treatment with CD19 BiTE or Her2 BiTE (Figure 1A); Her2 BITE+αHer2BB, Her2 CAR+αCD3εIL15RA, or Her2 CAR+αCD3ε scFV (Figure 1B); and CD19 BiTE+41BBLCD80, CD19 TriKE1(aCD28), or CD19 TriKE2(41BBL) (Figure 1C). [Figure 1-2] Same as above. [Figure 1-3] Same as above.

[0024] [Figure 2-1] Figure 2A and Figure 2B show GvDH model test. Figure 2A shows NSG mice treated with total body irradiation and 1e7 Her2 BiTE-T or CD19 BiTE-T cells. Figure 2B shows NSG mice treated with chemotherapy and 1e7 CTX+CAR-T or CTX+BiTE-T cells. [Figure 2-2] Same as above.

[0025] [Diagram 3] Figure 3 shows CD3 / TCRαβ expression in vivo without antigen. NSG mice were irradiated and then received T cells. Donor T cells in peripheral blood were assessed at specific time points. D12, day 12 after T cell injection.

[0026] [Figure 4] Figure 4 shows CD3 / TCRαβ expression in vivo with antigen. NSG mice were injected with tumor cells and then received T cell treatment. Donor T cells in the peripheral blood or tumor were assessed at specific time points.

[0027] [Figure 5-1]Figures 5A-5L show that BiTE-T cells produce less cytokines and are comparable or better than CAR-T in terms of efficacy in the presence of host T cells. Cytotoxicity (Figure 5A) and cytokine production (Figure 5B) of CD19 BiTE-T cells or CAR-T cells against 3T3.hCD19 cells. Cytotoxicity (Figure 5C) and cytokine production (Figure 5D) of Her2 BiTE-T cells or CAR-T cells against A375.Her2 cells. Figures 5E-5H show a comparative study of CD19 BiTE-T cells and CAR-T cells in vivo in the presence of host T cells. Figure 5E shows the study design. Male NSG mice were subjected to 1.2 Gy total body irradiation followed 1 day later by transfer of 7 million HLA-A2+ activated T cells. On day 0, Nalm6GL cells were given. On day 3, mice were treated with HLA-A2-CD19 BiTE-T cells or CAR-T cells. Survival (Figure 5F), donor T cells (Figure 5G), and recipient T cells (Figure 6H) in peripheral blood were monitored over time. Figures 5I-5L show a comparative study of Her2 BiTE-T cells and CAR-T cells in vivo in the presence of host T cells. Figure 5I shows the study design. Male NSG mice were subjected to 1.2 Gy total body irradiation followed 1 day later by transplantation of 5 million HLA-A2+ activated T cells. On day 0, A375.Her2 cells were given. On day 8, mice were treated with HLA-A2-Her2 BiTE-T cells or CAR-T cells. Tumor growth (Figure 5J), donor T cells (Figure 5K), and recipient T cells (Figure 5L) in peripheral blood were monitored over time. DTC, donor T cells; RTC, recipient T cells. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 5-4] Same as above. [Figure 5-5] Same as above. [Figure 5-6] Same as above. [Figure 5-7] Same as above. [Figure 5-8] Same as above.

[0028] [Figure 6-1]Figures 6A-6O show efficacy of costimulation or cytokine-enhanced BiTE-T cells comparable to or exceeding that of CAR-T cells. Figures 6A-6C show sequential killing assays on A375.Her2 cells. Figure 6A shows T cell proliferation over time. Figure 6B shows TCRαβ expression over time. Figure 6C shows CD3 expression over time. Figures 6D-6H show efficacy comparison of costimulation-enhanced Her2 BiTE-T cells to CAR-T cells. Figure 6D shows study design. Figure 6E shows tumor growth. Figure 6F shows percentage of tumor-infiltrating T cells. Figure 6G shows donor T cells in peripheral blood over time. Figure 7H shows weight change over time. Figures 6I-6M show data from a comparative study of costimulation-enhanced CD19 BiTE-T cells and CAR-T cells. Figure 6I shows study design. Figure 6J shows survival. Small arrows indicate death events unrelated to leukemia or GvHD. Figure 6K shows body weight changes. Figure 6L shows donor T persistence in peripheral blood. Figure 6M shows CD3 / TCRαβ expression on WK 4 blood T cells. Figure 6N (Her2) and Figure 6O (CD19) show evaluation of BiTE-T cells overexpressing IL7 and IL15 in an in vitro sequential killing assay. [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above. [Figure 6-5] Same as above. [Figure 6-6] Same as above. [Figure 6-7] Same as above. [Figure 6-8] Same as above. [Figure 6-9] Same as above.

[0029] [Figure 7-1]Figures 7A-7I show CD3 engagement-induced TCRαβ / CD3 downregulation and reduced alloreactivity of BiTE engineered T cells. Figure 7A shows mouse TCR / CD3 expression on mouse T cells after 24 hours of treatment with mCD3ε antibody coated on plates. Figure 7B shows a schematic of the BiTE and CAR constructs used in the study. Figure 7C shows TCRαβ evaluation on UT, BiTE-T or CAR-T cells using two different clones of TCRαβ antibody. Figure 7D shows TCRαβ / CD3 expression on CAR-T cells engineered with membrane-bound or soluble secreted CD3ε scFv. Figure 7E shows cytotoxicity of CAR-T cells engineered with membrane-bound or soluble secreted CD3ε scFv. Figure 7F shows immunophenotype of BiTE-T cells and CAR-T cells. Figures 7G-7I show GvHD risk study with chemotherapy. Mice were treated with 250 mg / kg cyclophosphamide (CTX) and then given 10 million transduced or untransduced T cells (UT). Figure 7G shows survival. Figure 7H shows body weight change. Figure 7I shows long-term leukemia protection by BiTE-T cells. In the same study, surviving mice were challenged with NALM6GL cells 4.5 months after BiTE-T cells. Leukemia cells in peripheral blood were evaluated 3 weeks after challenge. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 7-4] Same as above. [Figure 7-5] Same as above. [Figure 7-6] Same as above. [Figure 7-7] Same as above.

[0030] [Figure 8-1]Figure 8A shows a representative flow plot of CD19 BiTE-T cells inducing CD3 / TCRαβ downregulation on HLA-mismatched donor T cells. Figure 8B shows that Her2 BiTE-T cells reduced CD3 / TCRαβ expression on allogeneic T cells. HLA-A2+ Her2 BiTE-T cells were co-cultured with HLA-A2- PBMCs for 5 days without cytokines. All cells were subjected to flow analysis. [Figure 8-2] Same as above.

[0031] [Figure 9-1] Figures 9A-9H show that BiTE-T cells are not as durable as CAR-T cells. Figure 10A shows T cell expansion in a CD19 sequential killing assay. Figure 9B shows NALM6GL cell killing in a sequential killing assay. Figure 9C shows T cell expansion in an A375.Her2 sequential killing assay. Figure 9D shows A375.Her2 cell killing in a sequential killing assay. Figure 10E (survival) and Figure 9F (T cell persistence) show in vivo efficacy comparison of CD19 BiTE-T cells and CAR-T cells. Figure 9G (tumor growth) and Figure 9H (T cell persistence) show in vivo efficacy comparison of Her2 BiTE-T cells and CAR-T cells. [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 9-4] Same as above.

[0032] [Figure 10] FIG. 10 shows bioluminescence data of costimulation-enhanced CD19 BiTE-T cells compared to CD19 CAR-T cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Detailed Description Before describing the present disclosure in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0034] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, and any other stated or intervening value within that stated range, unless the context clearly dictates otherwise, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, which are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0035] 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, the preferred methods and materials are described herein.

[0036] All publications and patents cited herein are incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by prior disclosure. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0037] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual components and features which may be readily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the disclosure. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0038] The embodiments of the present disclosure employ, unless otherwise indicated, chemical, biological and other techniques within the skill of those in the art.

[0039] The following examples are provided to provide those skilled in the art with a complete disclosure and description of how to carry out the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless otherwise specified, parts are parts by weight, temperature is in ° C., and pressure is at or near atmospheric pressure. Standard temperature and pressure are defined as 20° C. and 1 atmosphere.

[0040] Before describing embodiments of the present disclosure in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, etc., and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The present disclosure also allows steps to be carried out in differing order where this is logically possible.

[0041] Please note that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0042] The term "antibody" refers to immunoglobulins, derivatives thereof that maintain specific binding ability, and proteins having binding domains that are homologous or nearly homologous to immunoglobulin binding domains. These proteins may be derived from natural sources or may be partially or wholly synthetically produced. Antibodies may be monoclonal or polyclonal. Antibodies may be members of any immunoglobulin class from any species, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. In an exemplary embodiment, the antibodies used in the methods and compositions described herein are derivatives of the IgG class.

[0043] The term "antibody fragment" refers to any derivative of an antibody that is less than full length. In an exemplary embodiment, an antibody fragment retains at least a significant portion of the specific binding ability of the full length antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv diabody, Fc and Fd fragments. Antibody fragments may be produced by any means. For example, antibody fragments may be produced enzymatically or chemically by fragmentation of an intact antibody, recombinantly produced from a gene encoding a partial antibody sequence, or wholly or partially synthetically produced. An antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragment may comprise multiple chains linked together, for example, by disulfide bonds. The fragment may optionally be a multimolecular complex. A functional antibody fragment typically comprises at least about 50 amino acids, more typically at least about 200 amino acids.

[0044] The term "antigen-binding site" refers to the region of an antibody that specifically binds to an epitope on an antigen.

[0045] The term "bispecific antibody" refers to an antibody that has two different antigen-binding regions defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes in one target.

[0046] The term "carrier" refers to a compound, composition, substance, or structure that, when combined with a compound or composition, aids or facilitates the preparation, storage, administration, delivery, efficacy, selectivity, or any other characteristic of the compound or composition for its intended use or purpose. For example, the carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0047] The term "engineered antibody" refers to a recombinant molecule comprising at least an antibody fragment comprising an antigen-binding site derived from the variable domain of an antibody heavy and / or light chain, and optionally comprising all or a portion of the variable and / or constant domains of an antibody from any of the Ig classes (e.g., IgA, IgD, IgE, IgG, IgM and IgY).

[0048] The term "epitope" refers to the region of an antigen to which an antibody binds preferentially and specifically. A monoclonal antibody preferentially binds to a single specific epitope of a molecule that can be molecularly defined. In the present invention, multiple epitopes can be recognized by multispecific antibodies.

[0049] A "fusion protein" or "fusion polypeptide" refers to a hybrid polypeptide that contains polypeptide moieties from at least two different polypeptides. The moieties may be derived from proteins of the same organism, in which case the fusion protein is said to be "intraspecies," "intragenic," etc. In various embodiments, the fusion polypeptide may contain one or more amino acid sequences linked to a first polypeptide. When more than one amino acid sequence is fused to the first polypeptide, the fusion sequences may be multiple copies of the same sequence or may be different amino acid sequences. The first polypeptide may be fused to the N-terminus, C-terminus, or N-terminus and C-terminus of the second polypeptide. Additionally, the first polypeptide may be inserted within the sequence of the second polypeptide.

[0050] The term "Fab fragment" refers to a fragment of an antibody containing an antigen-binding site generated by cleavage of an antibody with the enzyme papain, which cleaves N-terminal to the inter-heavy chain disulfide bond in the hinge region to generate two Fab fragments from one antibody molecule.

[0051] The term "F(ab')2 fragment" refers to a fragment of an antibody containing two antigen-binding sites produced by cleavage of an antibody molecule with the enzyme pepsin, which cleaves C-terminal to the inter-heavy chain disulfide bond in the hinge region.

[0052] The term "Fc fragment" refers to a fragment of an antibody containing the constant domain of its heavy chain.

[0053] The term "Fv fragment" refers to a fragment of an antibody comprising the variable domains of its heavy and light chains.

[0054] A "genetic construct" refers to a nucleic acid, e.g., a vector, plasmid, viral genome, etc., that contains a "coding sequence" for a polypeptide or is otherwise transcribable into biologically active RNA (e.g., antisense, decoy, ribozyme, etc.) and that can be transfected into a cell, e.g., a mammalian cell in certain embodiments, and cause expression of the coding sequence in a cell transfected with the construct. A genetic construct can include one or more regulatory elements operably linked to the coding sequence, as well as intron sequences, polyadenylation sites, origins of replication, marker genes, etc.

[0055] The term "isolated polypeptide" refers to a polypeptide that may be prepared from recombinant DNA or RNA, or may be of synthetic origin, any combination thereof, or may be a naturally occurring polypeptide, and that is (1) not associated with proteins with which it is normally associated in nature, (2) isolated from the cell in which it normally resides, (3) essentially free from other proteins from the same cellular source, (4) expressed by cells from a different species, or (5) not found in nature.

[0056] The term "isolated nucleic acid" refers to a polynucleotide of genomic, cDNA, synthetic, or natural origin, or some combination thereof, that is (1) not associated with cells in which the "isolated nucleic acid" is found in nature, or (2) operably linked to a polynucleotide with which it is not linked in nature.

[0057] The term "linker" is art-recognized and refers to a molecule or group of molecules that connects two compounds, such as two polypeptides. A linker may consist of a single linking molecule or may include a linking molecule and a spacer molecule intended to separate the linking molecule and the compounds by a specific distance.

[0058] The term "multivalent antibody" refers to an antibody or engineered antibody that contains more than one antigen recognition site. For example, a "bivalent" antibody has two antigen recognition sites, while a "tetravalent" antibody has four antigen recognition sites. Terms such as "monospecific," "bispecific," "trispecific," and "tetraspecific" refer to the number of different antigen recognition site specificities present in a multivalent antibody (as opposed to the number of antigen recognition sites). For example, the antigen recognition sites of a "monospecific" antibody all bind to the same epitope. A "bispecific" antibody has at least one antigen recognition site that binds to a first epitope and at least one antigen recognition site that binds to a second epitope that is different from the first epitope. A "multivalent monospecific" antibody has multiple antigen recognition sites that all bind to the same epitope. A "multivalent bispecific" antibody has multiple antigen recognition sites, some of which bind to a first epitope and some of which bind to a second epitope that is different from the first epitope.

[0059] The term "nucleic acid" refers to a polymeric form of nucleotides, typically ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide. This term should also be understood to include, as equivalents, either RNA or DNA analogs made from nucleotide analogs, as well as single-stranded (such as sense or antisense) and double-stranded polynucleotides, as applicable to the described embodiment.

[0060] The term "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0061] As used herein, "peptidomimetic" refers to a mimic of a peptide that includes some modification of normal peptide chemistry. Peptidomimetics typically enhance some properties of the original peptide (e.g., increased stability, increased efficacy, enhanced delivery, extended half-life, etc.). Methods for making peptidomimetics based on known polypeptide sequences are described, for example, in U.S. Patent Nos. 5,631,280; 5,612,895; and 5,579,250. The use of peptidomimetics may include the incorporation of non-amino acid residues with non-amide bonds at given positions. One embodiment of the present invention is a peptidomimetic, where the compound has bonds, peptide backbones, or amino acid components replaced with appropriate mimetics. Some non-limiting examples of unnatural amino acids that may be suitable amino acid mimetics include β-alanine, L-α-aminobutyric acid, L-γ-aminobutyric acid, L-α-aminoisobutyric acid, L-ε-aminocaproic acid, 7-aminoheptanoic acid, L-aspartic acid, L-glutamic acid, N-ε-Boc-N-α-CBZ-L-lysine, N-ε-Boc-N-α-Fmoc-L-lysine, L-methionine sulfone, L-norleucine, L-norvaline, N-α-Boc-N-δCBZ-L-ornithine, N-δ-Boc-N-α-CBZ-L-ornithine, Boc-p-nitro-L-phenylalanine, Boc-hydroxyproline, and Boc-L-thioproline.

[0062] The terms "protein" (if single chain), "polypeptide" and "peptide" are used interchangeably herein when referring to a gene product, for example as may be encoded by a coding sequence. When referring to a "polypeptide" herein, those skilled in the art will recognize that protein may be used instead, unless the context clearly indicates otherwise. "Protein" may also refer to an association of one or more polypeptides. "Gene product" refers to a molecule produced as a result of transcription of a gene. Gene products include RNA molecules transcribed from a gene, as well as proteins translated from such transcripts.

[0063] The term "polypeptide fragment" or "fragment", when used in reference to a particular polypeptide, refers to a polypeptide that has a deletion of amino acid residues compared to the reference polypeptide itself, but the remaining amino acid sequence is usually identical to the amino acid sequence of the reference polypeptide. Such deletions can occur at the amino or carboxy terminus of the reference polypeptide, or both. A fragment is typically at least about 5, 6, 8, or 10 amino acids in length, at least about 14 amino acids in length, at least about 20, 30, 40, or 50 amino acids in length, at least about 75 amino acids in length, or at least about 100, 150, 200, 300, 500, or more amino acids in length. A fragment can retain one or more of the biological activities of the reference polypeptide. In various embodiments, the fragment can include the enzymatic activity and / or interaction site of the reference polypeptide. In another embodiment, the fragment can be immunogenic.

[0064] The term "single chain variable fragment or scFv" refers to an Fv fragment in which the heavy and light chain domains are linked. One or more scFv fragments can be linked to other antibody fragments (such as heavy or light chain constant domains) to form antibody constructs with one or more antigen recognition sites.

[0065] As used herein, the term "specifically binds" when referring to a polypeptide (including antibodies) or receptor refers to a binding reaction that determines the presence of a protein or polypeptide or receptor in a heterogeneous population of proteins and other biologics. Thus, under specified conditions (e.g., immunoassay conditions in the case of antibodies), a particular ligand or antibody "specifically binds" to its particular "target" if it does not bind in significant amounts to other proteins present in the sample or to other proteins that the ligand or antibody may contact in an organism (e.g., an antibody specifically binds to an endothelial antigen). Generally, a first molecule that "specifically binds" to a second molecule will have a binding affinity of about 10 times that of the second molecule. 5 M -1 (For example, 10 6 M -1 , 107 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , and 10 12 M -1 The affinity constant (Ka) of the α-terminal end of the α-terminal lipoprotein is greater than or equal to 1.

[0066] The term "specifically deliver" as used herein refers to the preferential association of a molecule with cells or tissues that have a particular target molecule or marker, and not with cells or tissues that lack the target molecule. Of course, it is recognized that some degree of non-specific interaction may occur between a molecule and non-target cells or tissues. Nevertheless, specific delivery can be distinguished as being mediated through specific recognition of a target molecule. Typically, specific delivery results in a much stronger association between the delivered molecule and cells that have the target molecule than between the delivered molecule and cells that lack the target molecule.

[0067] The term "subject" refers to any individual who is the target of administration or treatment. A subject may be a vertebrate, e.g., a mammal. Thus, a subject may be a human or veterinary patient. The term "patient" refers to a subject receiving treatment from a clinician, e.g., a physician.

[0068] The term "therapeutically effective" refers to an amount of the composition used that is sufficient to ameliorate one or more causes or symptoms of a disease or disorder. Such an amelioration does not necessarily require elimination, but only a reduction or alteration.

[0069] The term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, i.e., treatment specifically directed to ameliorate a disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment directed to remove the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure a disease, pathological condition, or disorder; preventive treatment, i.e., treatment aimed at minimizing or partially or completely suppressing the onset of the associated disease, pathological condition, or disorder; and supportive treatment, i.e., treatment used to supplement another specific therapy aimed at ameliorating the associated disease, pathological condition, or disorder. Off-the-shelf immune effector cells

[0070] Disclosed are pre-made immune effector cells engineered to express anti-CD3 antibodies disclosed herein, which are configured to reduce the expression of T cell receptors (e.g., TCRαβ) that can autoactivate immune effector cells and thereby result in GVHD. Thus, these cells are preferably obtained from healthy donor subjects. Immune effector cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. Immune effector cells can be obtained from blood drawn from a subject using any number of techniques known to those skilled in the art, such as Ficoll™ separation. For example, cells from an individual's circulating blood can be obtained by apheresis. In some embodiments, immune effector cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example by centrifugation through a PERCOLL™ gradient or by countercurrent centrifugation elution. Specific subpopulations of immune effector cells can be further isolated by positive or negative selection techniques.For example, immune effector cells can be isolated by, for example, incubating antibody-conjugated beads with a time sufficient for positive selection of desired immune effector cells using a combination of antibodies against surface markers specific to positively selected cells.Alternatively, enrichment of immune effector cell populations can be achieved by negative selection using a combination of antibodies against surface markers specific to negatively selected cells.

[0071] In some embodiments, immune effector cells include any white blood cells involved in defending the body against infections and foreign substances.For example, immune effector cells can include lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combination thereof.For example, immune effector cells can include T lymphocytes, preferably cytotoxic T lymphocytes (CTLs).

[0072] T cells or T lymphocytes can be distinguished from other lymphocytes such as B cells and natural killer cells (NK cells) by the presence of a T cell receptor (TCR) on the cell surface. They are called T cells because they mature in the thymus gland (although some also mature in the tonsils). There are several subsets of T cells, each with different functions.

[0073] T helper cells (T H Helper T cells (T cells) assist other white blood cells in immunological processes including the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells are activated when presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist an active immune response. These cells are classified as T cells, which secrete different cytokines to promote different types of immune responses. H 1. T H 2. T H 3. T H 17, T H 9, or T FH They can be differentiated into one of several subtypes, including:

[0074] Cytotoxic T cells (T C CTLs (cells that inhibit immune cells) destroy virus-infected and tumor cells and are also involved in transplant rejection. These cells express the CD8 glycoprotein on their surface and are therefore known as CD8 + Also known as T cells, these cells recognize their targets by binding to antigens associated with MHC class I molecules present on the surface of all nucleated cells. Through IL-10, adenosine and other molecules secreted by regulatory T cells, they can inactivate CD8+ cells into an anergic state, thus preventing autoimmune diseases.

[0075] Memory T cells are a subset of antigen-specific T cells that persist for long periods after an infection has resolved. They expand into large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with a "memory" against past infections. Memory cells are CD4 + or CD8 + Memory T cells typically express the cell surface protein CD45RO.

[0076] Regulatory T cells (T cells), formerly known as suppressor T cells, reg CD4 T cells are crucial for the maintenance of immune tolerance. Their main role is to shut off T cell-mediated immunity towards the end of the immune response and to suppress autoreactive T cells that have escaped the process of negative selection in the thymus. + T reg Two major classes of cells exist: naturally occurring T reg Cells and adaptive T reg Cells are described.

[0077] Natural killer T (NKT) cells (not to be confused with natural killer (NK) cells) bridge the adaptive and innate immune systems. Unlike conventional T cells, which recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigens presented by a molecule called CD1d.

[0078] In some embodiments, the T cells comprise a mixture of CD4+ cells. In other embodiments, the T cells are enriched for one or more subsets based on cell surface expression. For example, in some cases, the T cells are enriched for cytotoxic CD8 + In some embodiments, the T cells include γδ T cells that have distinct T cell receptors (TCRs) with one γ and one δ chain instead of α and β chains.

[0079] Natural killer (NK) cells are CD56 receptor mediated NK cell proliferation and metabolism, and they are capable of killing virally infected and transformed cells and constitute an important cell subset of the innate immune system. + CD3 - It is a large granular lymphocyte (Godfrey J, et al. Leuk Lymphoma 2012 53:1666-1676). cytotoxic CD8 + Unlike T lymphocytes, NK cells can initiate cytotoxicity against tumor cells without the need for prior sensitization and can eradicate MHC-I negative cells as well (Narni-Mancinelli E, et al. Int Immunol 2011 23:427-431). NK cells are safer effector cells because they can avoid the potentially fatal complications of cytokine storm (Morgan RA, et al. Mol Ther 2010 18:843-851), tumor lysis syndrome (Porter DL, et al. N Engl J Med 2011 365:725-733), and on-target, off-tumor effects. Although NK cells have a well-known role as killer cells for cancer cells and NK cell dysfunction has been widely documented as crucial to the progression of MM (Godfrey J, et al. Leuk Lymphoma 2012 53:1666-1676; Fauriat C, et al. Leukemia 2006 20:732-733), means by which NK cell-mediated anti-MM activity may be enhanced have been largely unexplored prior to the disclosed CARs.

[0080] Epstein-Barr virus (EBV)-induced lymphoproliferative disease (EBV-LPD) and other EBV-associated cancers are significant causes of morbidity and mortality for recipients of allogeneic hematopoietic cell transplants (HCT) or solid organ transplants (SOT), especially those who have received specific T cell-reactive Abs to prevent or treat GVHD. Prevention and treatment by adoptive transfer of autologous or allogeneic EBV-specific cytotoxic T cells, and subsequent long-term restoration of immunity to EBV-associated lymphoproliferation, have yielded positive results in the management of these uniformly fatal complications of allogeneic tissue transfer. Thus, in some embodiments, the disclosed immune effector cells comprising one or more of the CAR polypeptides of the invention are allogeneic or autologous EBV-specific cytotoxic T lymphocytes (CTLs). For example, generation of EBV-specific cytotoxic T cells can include isolating PBMCs from EBV-seropositive autologous or allogeneic donors and enriching them for T cells by depleting monocytes and NK cells. EBV-specific cytotoxic T cells can also be generated by contacting donor PBMCs or purified donor T cells with "stimulator" cells that express one or more EBV antigens and present the EBV antigen(s) to unstimulated T cells, thereby causing stimulation and expansion of EBV-specific CTLs. EBV antigens include, for example, latent membrane proteins (LMPs) and EBV nuclear antigen (EBNA) proteins, such as LMP-1, LMP-2A and LMP-2B, as well as EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C and EBNA-LP. Cytotoxic T cells that contain a T cell receptor(s) that recognize one or more EBV-specific antigens are considered to be "sensitized" to these EBV antigen(s) and are therefore referred to herein as "EBV-sensitized cytotoxic T cells."Known methods for generating allogeneic or autologous EBV-specific cytotoxic T cell populations, which may comprise one or more of the CAR polypeptides of the invention, are described, for example, in Barker et al., Blood 2010 116(23):5045-49; Doubrovina, et al., Blood 2012 119(11):2644-56; Koehne, et al. Blood 2002 99(5):1730-40; and Smith et al. Cancer Res 2012 72(5):1116-25, which are incorporated by reference for their teachings. bispecific antibody

[0081] Bispecific antibodies may comprise a heavy chain containing one or more variable regions and / or a light chain containing one or more variable regions. Bispecific antibodies can be constructed using only antibody variable domains. A fairly efficient and relatively simple method is to use V H Domain and V L The key to successful engineering of engineered antibodies is to make the linker sequences between the domains so short that they cannot fold back and bind to each other. Reducing the linker length to 3-12 residues prevents the monomeric organization of the scFv molecule and promotes intermolecular VH-VL pairing with the formation of 60 kDa non-covalent scFv dimeric "diabodies". The diabody format can also be used to generate recombinant bispecific antibodies obtained by non-covalent binding of two single-chain fusion products consisting of a VH domain from one antibody linked by a short linker to the VL domain of another antibody. Further reduction of the linker length to less than three residues can result in the formation of trimers ("triabodies", approx. 90 kDa) or tetramers ("tetrabodies", approx. 120 kDa). For a review of engineered antibodies, particularly single domain fragments, see Holliger and Hudson, 2005, Nature Biotechnology, 23:1126-1136. All such engineered antibodies can be used in the fusion polypeptides provided herein.

[0082] Peptide linkers (-) suitable for the production of scFv antibodies are Kumada Y, et al.Biochemical Engineering Journal.2007 35(2):158-165;Albrecht H,et al.J Immunol Methods.2006 310(1-2):100-16;Feng J,et al.J Immunol Methods.2003 282(1-2):33-43;Griffiths AD,et al.Curr Opin Biotechnol.1998 9(1):102-8;Huston JS,et al.Methods Enzymol.1991 203:46-88;Bird RE,et al.Science.1988 242(4877):423-6;Takkinen K,et al.Protein Eng.1991 4(7):837-41; Smallshaw JE, et al. Protein Eng. 1999 12(7):623-30; Argos PJ Mol Biol. 1990 211(4):943-58; and Whitlow M, et al. Protein Eng. 1993 6(8):989-95, the teachings of which are incorporated herein by reference, as well as methods for using a variety of linkers to produce scFv antibodies against different targets.

[0083] Tetravalent Tandab® may be prepared substantially as described in WO 1999 / 057150 A3 or US2006 / 0233787, which are incorporated by reference for their teachings on how to make Tandab® molecules.

[0084] The antigen recognition site or the entire variable region of the engineered antibody may be derived from one or more parent antibodies against any antigen of interest (e.g., target receptor ECD or TMUL ECD). Parent antibodies may include naturally occurring antibodies or antibody fragments, antibodies or antibody fragments adapted from naturally occurring antibodies, or antibodies constructed de novo using sequences of antibodies or antibody fragments known to be specific for an antigen of interest. Sequences that may be derived from parent antibodies include heavy and / or light chain variable regions and / or CDRs, framework regions, or other portions.

[0085] In some cases V L 3 is the amino acid sequence [ka] or a fragment or variant thereof capable of binding to CD3 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 1. H 3 is the amino acid sequence [ka] or a fragment or variant thereof capable of binding to CD3 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:2.

[0086] Other anti-CD3 antibody sequences (eg, OKT3) are known in the art and can be used in the disclosed system.

[0087] In some cases, the target cell surface receptor is Her2. H R is the amino acid sequence [ka] or a fragment or variant thereof capable of binding to Her2 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:3. L R is the amino acid sequence [ka] [ka] or a fragment or variant thereof capable of binding to Her2 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:4.

[0088] In some cases, the target cell surface receptor is EGFR. H R is the amino acid sequence [ka] or a fragment or variant thereof capable of binding to EGFR having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:5. L R is the amino acid sequence [ka] or a fragment or variant thereof capable of binding to EGFR having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:6.

[0089] In some cases, the target cell surface receptor is CSPG4. H R is the amino acid sequence [ka] or a fragment or variant thereof capable of binding to a CSPG4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:7. L R is the amino acid sequence [ka] or a fragment or variant thereof capable of binding to a CSPG4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:8.

[0090] In some cases, the target cell surface receptor is CD20. H R is the amino acid sequence [ka] or a fragment or variant thereof capable of binding to a CSPG4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:9. L R is the amino acid sequence [ka] or a fragment or variant thereof capable of binding to CSPG4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:10.

[0091] In some cases, the target cell surface receptor is CD20. H R is the amino acid sequence [ka] or a fragment or variant thereof capable of binding to a CSPG4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 11. L R is the amino acid sequence [ka] or a fragment or variant thereof capable of binding to CSPG4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:12.

[0092] In some cases, the target cell surface receptor is PSMA. H R is the amino acid sequence [ka] or a fragment or variant thereof capable of binding to a CSPG4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 13. L R is the amino acid sequence [ka] or a fragment or variant thereof capable of binding to a CSPG4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:14.

[0093] In some cases, the target cell surface receptor is BCMA. H R is the amino acid sequence [ka] or a fragment or variant thereof capable of binding to a CSPG4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 15. L R is the amino acid sequence [ka] or a fragment or variant thereof capable of binding to a CSPG4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:16.

[0094] In some cases, the target cell surface receptor is mesothelin. H R is the amino acid sequence [ka] or a fragment or variant thereof capable of binding to a CSPG4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 17. L R is the amino acid sequence [ka] or a fragment or variant thereof capable of binding to a CSPG4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:18.

[0095] In some cases, the target cell surface receptor is GPC3. H R is the amino acid sequence [ka] or a fragment or variant thereof capable of binding to CSPG4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 19. L R is the amino acid sequence [ka] or a fragment or variant thereof capable of binding to CSPG4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:20.

[0096] In some cases, the target cell surface receptor is EpCAM. H R is the amino acid sequence [ka] or a fragment or variant thereof capable of binding to CSPG4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 21. L R is the amino acid sequence [ka] or a fragment or variant thereof capable of binding to CSPG4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:22.

[0097] In some cases, the target cell surface receptor is GD2. H R is the amino acid sequence [ka] [ka] or a fragment or variant thereof capable of binding to CSPG4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 23. L R is the amino acid sequence [ka] or a fragment or variant thereof capable of binding to CSPG4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:24.

[0098] The particular length of the peptide linker (--) used to link the scFv molecules together is important in determining the half-life, immunogenicity and activity of the entire construct. In some embodiments, the linker sequence (--) is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids long. In some embodiments, the linker sequence (--) comprises GGGGS (SEQ ID NO: 25). In some cases, the linker comprises a sequence of 2, 3, 4, 5 or more GGGGS (SEQ ID NO: 25).

[0099] In some embodiments, the hinge sequence (- / -) comprises EPKSCDKTHTCP (SEQ ID NO: 26). H -V L It is long enough so as not to interfere with proper folding and association of the chains, but not so long as to cause additional immunogenicity.

[0100] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and Her2 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:27.

[0101] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] [ka] or a fragment or variant thereof capable of binding to CD3 and Her2 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:28.

[0102] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and EGFR having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:29.

[0103] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] [ka] or a fragment or variant thereof capable of binding to CD3 and EGFR having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:30.

[0104] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and CSPG4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:31.

[0105] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and CSPG4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:32.

[0106] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] [ka] or a fragment or variant thereof capable of binding to CD3 and CD20 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:33.

[0107] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and CD20 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:34.

[0108] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and CD20 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:35.

[0109] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and CD20 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:36.

[0110] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and PSMA having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:37.

[0111] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] [ka] or a fragment or variant thereof capable of binding to CD3 and PSMA having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:38.

[0112] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and BCMA having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:39.

[0113] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and BCMA having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:40.

[0114] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and mesothelin having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:41.

[0115] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and mesothelin having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:42.

[0116] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] [ka] or a fragment or variant thereof capable of binding to CD3 and GPC3 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:43.

[0117] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and GPC3 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:44.

[0118] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and EpCAM having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:45.

[0119] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and EpCAM having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:46.

[0120] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and GD2 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:47.

[0121] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] [ka] or a fragment or variant thereof capable of binding to CD3 and GD2 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:48.

[0122] In some cases, the encoded bispecific antibody further comprises a signal peptide. In some embodiments, the signal peptide comprises the following amino acid sequence: MDFQVQIFSFLLISASVIMSRG (SEQ ID NO: 49).

[0123] Thus, in some embodiments, the encoded bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and Her2 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:50.

[0124] Thus, in some embodiments, the encoded bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and Her2 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:51.

[0125] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and EGFR having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:52.

[0126] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and EGFR having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:53.

[0127] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] [ka] or a fragment or variant thereof capable of binding to CD3 and CSPG4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:54.

[0128] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and CSPG4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:55.

[0129] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and CD20 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:56.

[0130] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and CD20 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:57.

[0131] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and CD20 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:58.

[0132] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] [ka] or a fragment or variant thereof capable of binding to CD3 and CD20 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:59.

[0133] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and PSMA having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:60.

[0134] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and PSMA having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:61.

[0135] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] [ka] or a fragment or variant thereof capable of binding to CD3 and BCMA having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:62.

[0136] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and BCMA having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 63.

[0137] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and mesothelin having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:64.

[0138] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and mesothelin having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:65.

[0139] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and GPC3 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:66.

[0140] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] [ka] or a fragment or variant thereof capable of binding to CD3 and GPC3 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:67.

[0141] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and EpCAM having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:68.

[0142] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and EpCAM having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:69.

[0143] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and GD2 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:70.

[0144] Thus, in some embodiments, the bispecific antibody has the following amino acid sequence: [ka] or a fragment or variant thereof capable of binding to CD3 and GD2 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:71.

[0145] Candidate engineered antibodies or fusion polypeptides themselves for inclusion in fusion polypeptides can be screened for activity using various known assays.For example, screening assays for determining binding specificity are well known and routinely performed in the art.For a comprehensive discussion of such assays, see Harlow et al. (Eds.), ANTIBODIES: A LABORATORY MANUAL; Cold Spring Harbor Laboratory; Cold Spring Harbor, NY, 1988, Chapter 6.

[0146] In some embodiments, bispecific antibodies may be subjected to modifications to reduce immunogenicity when administered to humans. Such modifications may include one or more of the techniques commonly known as chimerization, humanization, CDR grafting, deimmunization, and / or mutation of framework region amino acids to correspond to the closest human germline sequence (germlinization). Thus, modified bispecific antibodies remain administrable for longer periods with reduced or no immune response-related side effects than corresponding bispecific antibodies that have not undergone such modification(s). Those skilled in the art will understand how to determine the extent to which an antibody must be modified to prevent it from eliciting an unwanted host immune response. Pharmaceutical Compositions

[0147] Also disclosed are pharmaceutical compositions comprising the disclosed molecules in a pharma- ceutically acceptable carrier. Pharmaceutical carriers are known to those skilled in the art. These are most typically standard carriers for administering drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. For example, suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (21 ed.) ed. PP. Gerbino, Lippincott Williams & Wilkins, Philadelphia, PA. 2005. Typically, an appropriate amount of a pharma- ceutically acceptable salt is used in the formulation to make the formulation isotonic. Pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. The solution should be RNAse-free. Additional carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, the matrices being in the form of shaped articles, e.g., films, liposomes, or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending on, for instance, the route of administration and concentration of composition being administered.

[0148] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants and absorption delaying agents that are physiologically compatible with the bispecific antibodies of the present invention. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils, carboxymethylcellulose colloidal solutions, tragacanth gum, and injectable organic esters such as ethyl oleate, and / or various buffers. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Treatment

[0149] Adoptive transfer of the disclosed pre-made T cells can be used to treat a variety of diseases and conditions in allogeneic subjects, such as cancer, autoimmune diseases, diabetes, neurological disorders, chronic viral infections, bacterial infections, parasitic infections, Alzheimer's disease, heart disease, etc. The disclosed pre-made T cells can also be engineered to express one or more CARs.

[0150] The disclosed pre-made T cells can be administered as a pharmaceutical composition alone or in combination with other components such as diluents and / or IL-2, IL-15 or other cytokines or cell populations. Briefly, a pharmaceutical composition can include a target cell population as described herein in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents or excipients. Such compositions can include a buffer, such as neutral buffered saline, phosphate buffered saline, etc.; a carbohydrate, such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or amino acid, such as glycine; an antioxidant; a chelating agent, such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The compositions for use in the disclosed methods are, in some embodiments, formulated for intravenous administration. The pharmaceutical composition can be administered in any manner that adequately treats MM. The amount and frequency of administration will be determined by factors such as the patient's condition and the severity of the patient's disease, but the appropriate dosage may be determined by clinical trials.

[0151] When an "immunologically effective amount," "antitumor effective amount," "tumor inhibiting effective amount," or "therapeutic amount" is indicated, 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 age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject). Pharmaceutical compositions comprising the T cells described herein can be administered in doses of 10 4 ~10 9 Dosage of cells / kg body weight, e.g., 10 5 ~10 6It can be generally stated that the T cell composition can be administered at a dose of cells / kg body weight (including all integer values ​​within these ranges). The T cell composition can also be administered multiple times at these doses. The cells can be administered by using injection techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be easily determined by those skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0152] Administration of the disclosed compositions may be performed in any convenient manner, including injection, transfusion, or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramuscularly, intravenously (iv) or intraperitoneally. In some embodiments, the disclosed compositions are administered to a patient by intradermal or subcutaneous injection. In some embodiments, the disclosed compositions are administered by iv injection. The compositions may also be injected directly into a tumor, lymph node, or site of infection.

[0153] In certain embodiments, the disclosed pre-made T cells are administered to patients in combination with (e.g., before, simultaneously with, or after) any number of relevant treatment modalities, including, but not limited to, thalidomide, dexamethasone, bortezomib, and lenalidomide. In further embodiments, the CAR-modified immune effector cells may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytotoxins, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. In some embodiments, the CAR-modified immune effector cells are administered to the patient in combination with (e.g., before, simultaneously with, or after) bone marrow transplantation, T-cell depleting therapy using either chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cell composition of the present invention is administered after B-cell depleting therapy, such as agents that react with CD20, e.g., Rituxan. For example, in some embodiments, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the present invention. In further embodiments, the expanded cells are administered before or after surgery.

[0154] In some embodiments, the method is used to treat cancer in an allogeneic subject. In some aspects, the cancer may be a sarcoma, lymphoma, leukemia, carcinoma, blastoma, or germ cell tumor. A representative, but non-limiting list of cancers that can be treated using the disclosed compositions includes lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of the head and neck, kidney cancer, lung cancer, including small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinoma of the mouth, throat, larynx and lung, endometrial cancer, cervical cancer, cervical cancer, breast cancer, epithelial cancer, kidney cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck cancer, colon cancer, hematopoietic cancer; testicular cancer; colon and rectal cancer, prostate cancer, and pancreatic cancer.

[0155] The disclosed pre-made T cells can be used in combination with any compound, moiety or group that has a cytotoxic or cytostatic effect. Drug moieties include chemotherapeutic agents that can function as microtubulin inhibitors, mitotic inhibitors, topoisomerase inhibitors or DNA intercalators, particularly those used in cancer treatment.

[0156] The disclosed off-the-shelf T cells can be used in combination with checkpoint inhibitors. Two known inhibitory checkpoint pathways include signaling through the cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed death 1 (PD-1) receptors. These proteins are members of the CD28-B7 family of co-signaling molecules that play key roles across all stages of T-cell function. The PD-1 receptor (also known as CD279) is expressed on the surface of activated T cells. Its ligands, PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273), are expressed on the surface of APCs such as dendritic cells or macrophages. While PD-L1 is the predominant ligand, PD-L2 has a much more restricted expression pattern. Binding of the ligand to PD-1 transmits an inhibitory signal to the T cell, reducing cytokine production and suppressing T-cell proliferation. Checkpoint inhibitors include, but are not limited to, antibodies that block: PD-1 (nivolumab (BMS-936558 or MDX1106), CT-011, MK-3475), PD-L1 (MDX-1105 (BMS-936559), MPDL3280A, MSB0010718C), PD-L2 (rHIgM12B7), CTLA-4 (ipilimumab (MDX-010), tremelimumab (CP-675, 206)), IDO, B7-H3 (MGA271), B7-H4, TIM3, LAG-3 (BMS-986016).

[0157] Human monoclonal antibodies against programmed death 1 (PD-1) and methods of using anti-PD-1 antibodies alone or in combination with other immunotherapeutic agents to treat cancer are described in U.S. Patent No. 8,008,449, which is incorporated by reference for these antibodies. Anti-PD-L1 antibodies and their uses are described in U.S. Patent No. 8,552,154, which is incorporated by reference for these antibodies. Anti-cancer agents comprising anti-PD-1 or anti-PD-L1 antibodies are described in U.S. Patent No. 8,617,546, which is incorporated by reference for these antibodies.

[0158] In some embodiments, the PDL1 inhibitor comprises an antibody that specifically binds to PDL1, such as BMS-936559 (Bristol-Myers Squibb) or MPDL3280A (Roche). In some embodiments, the PD1 inhibitor comprises an antibody that specifically binds to PD1, such as lambrolizumab (Merck), nivolumab (Bristol-Myers Squibb), or MEDI4736 (AstraZeneca). Human monoclonal antibodies against PD-1 and methods of using anti-PD-1 antibodies alone or in combination with other immunotherapeutic agents to treat cancer are described in U.S. Patent No. 8,008,449, which is incorporated by reference for these antibodies. Anti-PD-L1 antibodies and their uses are described in U.S. Patent No. 8,552,154, which is incorporated by reference for these antibodies. Anti-cancer agents that include anti-PD-1 or anti-PD-L1 antibodies are described in U.S. Patent No. 8,617,546, which is incorporated by reference for these antibodies.

[0159] The disclosed off-the-shelf T cells can be used in combination with other cancer immunotherapies. There are two different types of immunotherapy: passive immunotherapy uses components of the immune system to direct targeted cytotoxic activity against cancer cells without necessarily initiating an immune response in the patient, whereas active immunotherapy actively induces an endogenous immune response. Passive strategies include the use of monoclonal antibodies (mAbs), which are produced by B cells in response to specific antigens. The development of hybridoma technology and the identification of tumor-specific antigens in the 1970s enabled the pharmaceutical development of mAbs that can specifically target tumor cells for destruction by the immune system. So far, mAbs have been the biggest success story of immunotherapy; the top three best-selling anticancer drugs in 2012 were mAbs. Among them is rituximab (Rituxan, Genentech), which binds to the CD20 protein, which is highly expressed on the surface of B-cell malignancies such as non-Hodgkin's lymphoma (NHL). Rituximab has been approved by the FDA for the treatment of NHL and chronic lymphocytic leukemia (CLL) in combination with chemotherapy. Another important mAb is trastuzumab (Herceptin; Genentech), which has revolutionized the treatment of HER2 (human epidermal growth factor receptor 2)-positive breast cancer by targeting the expression of HER2.

[0160] To generate optimal "killer" CD8 T cell responses, costimulation is required in addition to T cell receptor activation, which can be provided by ligation of tumor necrosis factor receptor family members including OX40 (CD134) and 4-1BB (CD137). OX40 is of particular interest because treatment with activating (agonist) anti-OX40 mAb enhances T cell differentiation and cytolytic function, resulting in enhanced antitumor immunity against a variety of tumors.

[0161] In some embodiments, such additional therapeutic agents may be selected from antimetabolites such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine.

[0162] In some embodiments, such additional therapeutic agents may be selected from mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, alkylating agents such as cisplatin, and other platinum derivatives such as carboplatin.

[0163] In some embodiments, such additional therapeutic agents may be selected from antimitotic agents, such as taxanes, eg, docetaxel and paclitaxel, and vinca alkaloids, eg, vindesine, vincristine, vinblastine, and vinorelbine.

[0164] In some embodiments, such additional therapeutic agents may be selected from topoisomerase inhibitors, such as topotecan or irinotecan, or cytostatic agents, such as etoposide and teniposide.

[0165] In some embodiments, such additional therapeutic agents may be selected from a growth factor inhibitor, for example an inhibitor of ErbB1 (EGFR) (e.g., an EGFR antibody, for example, zalutumumab, cetuximab, panitumumab, or nimotuzumab, or other EGFR inhibitors, for example, gefitinib or erlotinib), another inhibitor of ErbB2 (HER2 / neu) (e.g., a HER2 antibody, for example, trastuzumab, trastuzumab-DM1, or pertuzumab), or an inhibitor of both EGFR and HER2, for example, lapatinib).

[0166] In some embodiments, such additional therapeutic agents may be selected from tyrosine kinase inhibitors such as imatinib (Glivec, Gleevec STI571) or lapatinib.

[0167] Thus, in some embodiments, the disclosed antibodies are used in combination with ofatumumab, zanolimumab, daratumumab, ranibizumab, nimotuzumab, panitumumab, hu806, daclizumab (Zenapax), basiliximab (Simulect), infliximab (Remicade), adalimumab (Humira), natalizumab (Tysabri), omalizumab (Xolair), efalizumab (Raptiva), and / or rituximab.

[0168] In some embodiments, the therapeutic agent for use in combination with pre-made T cells to treat the above disorders may be an anti-cancer cytokine, chemokine, or combination thereof. Examples of suitable cytokines and growth factors include IFNy, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa (e.g., INFa2b), IFN, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFa. Suitable chemokines may include Glu-Leu-Arg (ELR) negative chemokines from the human CXC and CC chemokine families, such as IP-10, MCP-3, MIG, and SDF-Ia. Suitable cytokines include cytokine derivatives, cytokine variants, cytokine fragments, and cytokine fusion proteins.

[0169] In some embodiments, the therapeutic agent for use in the commercially available T cell combinations for treating the above disorders may be a cell cycle control / apoptosis regulator "modulator." Cell cycle control / apoptosis regulators may include molecules that target and modulate cell cycle control / apoptosis regulators, such as (i) cdc-25 (e.g., NSC 663284), (ii) cyclin-dependent kinases that overstimulate the cell cycle (e.g., flavopiridol (L868275, HMR1275), 7-hydroxystaurosporine (UCN-01, KW-2401), and roscovitine (R-roscovitine, CYC202)), and (iii) telomerase modulators (e.g., BIBR1532, SOT-095, GRN163, and compositions described in, for example, U.S. Pat. Nos. 6,440,735 and 6,713,055). Non-limiting examples of molecules that interfere with apoptotic pathways include TNF-related apoptosis-inducing ligand (TRAIL) / apoptosis-2 ligand (Apo-2L), antibodies that activate the TRAIL receptor, IFN, and antisense Bcl-2.

[0170] In some embodiments, therapeutic agents for use in combination with pre-made T cells to treat the above disorders may be hormone modulating agents, such as agents useful in anti-androgenic and anti-estrogen therapy. Examples of such hormone regulators are tamoxifen, idoxifene, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol / ethinyl, antiandrogens (such as flutamide / eurexin), progestins (such as hydroxyprogesterone caproate, medroxy-progesterone / provera, megestrol acepate / megas), corticosteroids (such as hydrocortisone, prednisone), luteinizing hormone releasing hormone (and its analogs, as well as other LHRH agonists such as buserelin and goserelin), aromatase inhibitors (such as anastrazole / arimidex, aminoglutethimide / citraden, exemestane) or hormone inhibitors (such as octreotide / sandostatin).

[0171] In some embodiments, therapeutic agents for use in combination with pre-made T cells to treat the above disorders may be anti-cancer nucleic acids or anti-cancer inhibitory RNA molecules.

[0172] Such co-administration may be simultaneous, separate or sequential. For simultaneous administration, the agents may be administered as one composition or as separate compositions, as appropriate.

[0173] In some embodiments, the disclosed pre-made T cells are administered in combination with radiation therapy. Radiation therapy can include radiation or provide associated administration of radiopharmaceuticals to the patient. The source of radiation can be either external or internal to the patient being treated (radiation treatment can be, for example, in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that can be used in carrying out such methods include, for example, radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodine-123, iodine-131 and indium-111.

[0174] In some embodiments, the disclosed pre-made T cells are administered in combination with surgery.

[0175] Although several embodiments of the invention have been described, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. EXAMPLES

[0176] Example 1: Figures 1A-1C show CD3 / TCRαβ expression in vitro after treatment with CD19 BiTE or Her2 BiTE (Figure 1A); Her2 BITE+αHer2BB, Her2 CAR+αCD3εIL15RA, or Her2 CAR+αCD3ε scFV (Figure 1B); and CD19 BiTE+41BBLCD80, CD19 TriKE1(aCD28), or CD19 TriKE2(41BBL) (Figure 1C).

[0177] Figure 2A and Figure 2B show GvDH model test. Figure 2A shows NSG mice treated with total body irradiation and 1e7 Her2 BiTE-T or CD19 BiTE-T cells. Figure 2B shows NSG mice treated with chemotherapy and 1e7 CTX+CAR-T or CTX+BiTE-T cells.

[0178] Figure 3 shows CD3 / TCRαβ expression in vivo without antigen. NSG mice were irradiated and then received T cells. Donor T cells in peripheral blood were assessed at specific time points. D12, day 12 after T cell injection.

[0179] Figure 4 shows CD3 / TCRαβ expression in vivo with antigen. NSG mice were injected with tumor cells and then received T cell treatment. Donor T cells in the peripheral blood or tumor were assessed at specific time points.

[0180] Example 2: Next, an in vitro serial killing assay was set up to evaluate TCRαβ and CD3 on BiTE-T cells in the presence of target antigen. T cells were repeatedly challenged with target cells until they could no longer kill the target cells. Expression of TCRαβ and CD3ε was evaluated every 48 h during this period. For both CD19 and Her2 targets, BiTE-T cells maintained low TCRαβ / CD3 throughout the challenge assay, in contrast to CAR-T and UT cells. In vivo, Her2 BiTE-T cells were evaluated using a Her2-expressing solid tumor model. NSG mice were first inoculated with Her2-expressing human melanoma cells A375 (A375.Her2) and then subjected to BiTE-T or UT treatment. Blood T cells and tumor-infiltrating T cells were evaluated by flow cytometry. In peripheral blood, Her2 BiTE-T cells showed limited presence with low CD3ε. Tumor-infiltrating Her2 BiTE-T cells also display lower TCRαβ / CD3 than CAR-T cells. These results indicate that BiTE-T cells maintain low CD3ε and TCRαβ in the presence of target antigen.

[0181] Allogeneic OTS T cells are expected to overcome host rejection to persist longer and maximize their efficacy. One important mechanism of host rejection is host T cells recognizing non-self antigens via TCR. We co-cultured CD19 BiTE-T cells with HLA-mismatched T cells for 72 h and found that BiTE-T reduced CD3 and TCRαβ on recipient T cells. Similar results were observed when alloMLR assays were set up by co-culturing CD19 or Her2 BiTE-T cells with HLA-mismatched PBMCs (Figure 8). These data suggest that donor BiTE-T cells have the potential to overcome host rejection by releasing BiTEs that bind to TCRαβ / CD3 on host T cells.

[0182] The anti-cancer activity of BiTE-T cells has been demonstrated by other groups (Iwahori, K. et al. Mol Ther 2015 23:171-178; Choi, BD et al. Nat Biotechnol 2019 37:1049-1058; Liu, X. et al. Protein Cell 2017 8:514-526). Here, we performed a side-by-side comparison between BiTE-T and second-generation CAR-T cells for their anti-cancer activity. CD19 or Her2 BiTE-T and CAR-T cells sharing the same scFv were evaluated. For both targets, BiTE-T cells show comparable, if not better, killing than CAR-T cells in vitro (Figure 5A, Figure 5C). BiTE-T cells produced only a fraction of the cytokines produced by CAR-T cells, including IFNγ, IL2, IL6 and TNFα (Figure 5B, 5D). This suggests that BiTE-T cells may be safer than CAR-T cells. However, CAR-T cells outperformed BiTE-T cells in sequential killing assays (Figures 9A-9D) and also provided better survival, persistence and efficacy in NSG models engrafted with NALM6 or A375.Her2 cells (Figures 9E-9H). This is not surprising, since second generation CAR-T cells have an integrated costimulatory domain.

[0183] One potential advantage of BiTE-T cells is their ability to recruit bystander or host T cells. Therefore, we employed a mouse model to mimic an environment containing pre-existing host T cells (Mo, F. et al. Nat Biotechnol 2021 39:56-63). NSG mice were irradiated and then transplanted with activated HLA-A2+ human T cells. The mice were then injected with tumor cells and later treated with HLA-A2-BiTE-T or CAR-T cells (Figure 5E). In the NSG NALM6 model with pre-existing T cells, both CD19-targeted BiTE-T and CAR-T cells showed comparable survival benefits compared to UT cells (Figure 5F). The expansion of donor CD19 CAR-T cells was also suppressed in this model, whereas host T cells showed expansion in the CAR-T-treated group (Figure 5G, 5H). In a Her2+ tumor model with pre-existing host T cells (Figure 5I), CAR-T cells were unable to inhibit tumor growth, whereas BiTE-T cells still could (Figure 5J). In peripheral blood, both Her2-targeted BiTE-T cells and CAR-T cells showed very limited persistence (Figure 5K). However, Her2 BiTE-T cells induced greater host T cell expansion than CAR-T cells (Figure 5L).

[0184] To enhance efficacy, costimulation was added to BiTE-T cells. T cells were co-transduced with BiTE and a combination of 4-1BBL and CD80 (Velasquez, MP et al. Cancer Immunol Res 2017 5:860-870) to generate next-generation BiTE-T cells. These enhanced Her2 BiTE-T cells show better proliferation than Her2 CAR-T cells in serial killing assays while maintaining low levels of CD3 / TCRαβ (Figure 6A-6C). In vivo (Figure 6D), Her2 BiTE-T cells show comparable tumor suppression, blood persistence, tumor infiltration and body weight as CAR-T cells (Figure 6E-6H). Similarly, in the NALM6 model (Figure 6I), enhanced CD19 BiTE-T cells have comparable efficacy and possibly better blood persistence as CAR-T cells (Figure 6J-6L, Figure 10). Enhanced CD19 BiTE-T cells maintained low CD3 / TCRαβ in vivo (Figure 6M). BiTE-T cells were also enhanced by engineering with a combination of IL7 and IL15 genes. Cytokine-engineered BiTE T cells outperformed CAR-T cells in in vitro sequential killing assays (Figures 6N, 6O).

[0185] This study proposes a new strategy for allogeneic OTS T cell therapy. CAR-T cell therapy is currently one of the most powerful cancer therapeutics approved by the FDA. Recent clinical trials have shown that allogeneic CAR-T cells could potentially replace their autologous counterparts, albeit at some sacrifice in potency. BiTE-T cells may have several advantages over allogeneic OTS CAR-T cells. First, BiTE-T cells are less complicated to produce and are a natural, "pure" product. BiTE-T cell production is essentially the same as autologous CAR-T production, whereas allogeneic CAR-T cells require gene editing and TCRαβ negative purification. Unlike current CAR-T products that have untransduced "dormant" T cells, BiTE-T cell products fully comprise BiTEs, whether transduced or not, so the final product has 100% activity. Second, BiTE-T cells have the potential to overcome host rejection without lymphodepletion or further manipulation. Current allogeneic CAR-T cells require stringent lymphodepletion regimens for longer therapeutic window or more manipulations such as autoimmune defense receptors to kill host T cells (Mo,F.et al.Nat Biotechnol 2021 39:56-63). In contrast, BiTE-T cells have the potential to expand and retarget host T cells, perhaps not requiring lymphodepletion or requiring mild lymphodepletion, providing additional killing power. Third, BiTE-T cells are safer because they release fewer cytokines when killing targets. One concern with BiTE-T cell products without TCRαβ negative purification is that contaminated untransduced T cells may cause GvHD. However, high doses of human BiTE-T cells with appropriate untransduced T cell contamination did not induce GvHD in an immunodeficient NSG mouse model. If used in settings with no or little lymphodepletion, the risk of BiTE-T causing GvHD would be even lower.Another potential drawback of BiTE-T cells is that they are less potent than CAR-T cells.This can be largely fixed by adding costimulatory or cytokines.

[0186] In summary, BiTE-T cells offer a novel and facile method for "off-the-shelf" allogeneic T-cell therapy that warrants further evaluation in clinical trials.

[0187] method Study design The aim of this study was to develop a novel allogeneic "off-the-shelf" T cell therapy. CD19 and Her2 targeted BiTE engineered T cells were evaluated for CD3 / TCRαβ expression. GvHD risk of BiTE-T cells was assessed using chemically conditioned or total body irradiated NSG mice. Side-by-side comparisons were also performed between BiTE-T cells and CAR-T cells for cytotoxicity, cytokines, sequential killing, and in vivo efficacy with or without host T cells. Two xenograft mouse models were used to compare efficacy between BiTE-T cells and CAR-T cells, including hematological and solid tumors. Various healthy donors were used to generate engineered T cells. All experiments were independently repeated at least twice.

[0188] Cells and media The NALM6GL cell line, which expresses both GFP and firefly luciferase, was purchased from ATCC and cultured in RPMI1640 medium supplemented with 10% FBS, L-glutamine and Pen / Strep (RPMI10). The human melanoma A375 cell line was kindly donated by Dr. Cecilia Ramello and cultured in DMEM medium supplemented with 10% FBS, L-glutamine and Pen / Strep. Her2-expressing A375 (A375.Her2) was generated by transducing A375 cells with a retrovirus expressing a truncated Her2. Human T cells were cultured in RPMI10 supplemented with 10 ng / ml IL7 and 5 ng / ml IL15.

[0189] Retroviral constructs, γ-retrovirus production and T cell transduction DNA for human Her2 CAR, human CD19 BiTE, Her2 BiTE, 4-1BBL-CD80, and other constructs were synthesized and subcloned into SFG retroviral vector by Genewiz (South Plainfield, NJ). Human CD19 CAR was previously described (Li, G. et al. JCI Insight 2018 3). Her2 CAR and Her2 BiTE use the same scFv from trastuzumab. CD19 CAR and CD19 BiTE use the same scFv from FMC63 clone. Recombinant gamma-retrovirus production was previously described (Li, G., et al. Methods Mol Biol 2017 1514:111-118). Briefly, retroviral constructs containing CAR or BiTE were transiently transfected into H29 cells. Supernatant from transfected H29 was used to transduce RD114 cells to generate stable producer cell lines. Retroviral supernatant from RD114 cells was harvested, 0.45 μm filtered, and cryopreserved for future use. For T cell transduction, healthy donor leukocyte packs were purchased from Stemcell Technologies (Vancouver, Canada) and ALLCELLS (Alameda, CA). PBMCs were isolated using standard Ficoll methods and cryopreserved for future use. On day 0, T cells were isolated from cryopreserved PBMCs using a human T cell isolation kit (Stemcell Technologies). T cells were then activated using human CD3 / CD28 dynabeads (ThermoFisher Scientific, Waltham, MA). On days 1 and 2, T cells were transduced with fresh BiTE or CAR retroviruses in RetroNectin (Takara Bio, Mountain View, CA)-coated plates at 2000g and 32°C for 1 h. On day 3, fresh medium containing cytokines was added. Dynabeads were removed on day 6 and T cells were further expanded in complete medium containing 10 ng / ml human IL7 and 5 ng / ml human IL15. Medium and cytokines were replenished every 2-3 days.T cells from days 8 to 14 were used for in vivo studies.

[0190] Flow cytometry The following antibody clones were used for flow cytometry: from BD: CD3 (clone SK7), CD8 (clone RPA-T8), CD45 (clone HI30), TCRαβ (clone T10B9.1A-31), CD45RA (HI100), PD1 (clone EH12.2H7), CD69 (clone FN50), CD25 (clone 2A3). From Biolegend: CD4 (clone OKT4), TCRαβ (clone IP26), CCR7 (clone G043H7), CD62L (clone DREG-56). Fc receptor binding inhibitors (ThermoFisher) were routinely used for staining. Whole blood samples were stained and lysed using BD FACS lysis solution. CountBright absolute counting beads (ThermoFisher) were added to measure cell number. Flow data were acquired on a BD LSRII or BD FACSymphony flow cytometer. Data were analyzed using FlowJo version 10.

[0191] ELISA An Ella machine (ProteinSimple, San Jose) was used for ELISA. 24-72 h supernatants from cytotoxicity or TCR stimulation assays were added to ELLA cartridges for cytokine measurements.

[0192] TCR / CD3 stimulation assay For human T cell stimulation assays, non-tissue culture treated 24-well plates were coated overnight with 1 μg / ml human TCRαβ antibody (clone IP26). T cells were added and cultured in the plates. For activation markers, T cells were subjected to flow analysis after 5 h of stimulation. Supernatants were collected after 24 h of stimulation and cytokines were measured by ELISA. For BiTE stimulation, human CD19 BiTE (Blinatumomab) was purchased from BPS Bioscience (San Diego, CA). T cells were co-cultured with 3T3.hCD19 or 3T3.mCD19 cells with increasing concentrations of Blinatumomab. For mouse T cell stimulation, T cells were isolated from spleens and added to 24-well plates coated with mouse CD3ε antibody (clone 145-2C11). After 5 h, cells were analyzed by flow cytometry.

[0193] Cytotoxicity and sequential killing assays The xCELLigence RTCA instrument (Agilent Technologies, Santa Clara, CA) was used for the cytotoxicity assay. T cells were co-cultured with target cells at various E:T ratios in RTCA E-plates, and real-time cell killing was recorded by the instrument. For the sequential killing assay, T cells were co-cultured with target cells at an E:T ratio of 5:1 in non-tissue culture treated 24-well plates. Every 2 days or longer, a small fraction of T cells was analyzed by flow cytometry for phenotype and cell number, and the remaining cells were transferred to a new plate containing fresh medium and challenged with target cells.

[0194] Mouse studies All mouse protocols were approved by the University of South Florida Animal Care and Use Committee. NSG mice were purchased from Jackson Lab and housed in the USF animal facility. Male NSG mice aged 8–12 weeks were used for in vivo studies. For GvHD assessment, NSG mice received 2.5 Gy total body irradiation or 250 mg / kg cyclophosphamide (CTX) and then received T cells at a dose of 10 million the following day. Mice were monitored for survival and any signs of GvHD (e.g., hunched posture, slow motility, weight loss). Mice with more than 20% weight loss and other GvHD symptoms were sacrificed. For efficacy testing against hematological tumor models, NSG mice were intravenously injected with 500,000 NALM6GL cells. Three days later, mice were intravenously injected with 10 million CAR-T cells, BiTE-T cells or UT cells. Survival was monitored twice weekly. For solid tumor models, NSG mice were subcutaneously injected with 500,000 A375.Her2 cells in the right flank. Seven to 10 days later, mice were injected intravenously with 10 million CAR-T cells, BiTE-T cells or UT cells. Blood was collected weekly and tumor tissue was harvested at the end point. For mice with pre-existing T cells, NSG mice were subjected to 1.2 Gy total body irradiation and then transferred with activated T cells. Mice were injected with tumor cells and treated with HLA-mismatched T cells. See timeline in figure for details. Tumor tissue was treated with gentlMACS Octo Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) and digested with Liberase and DNase I (Roche, Mannheim, Germany). Isolated tumor cells were subjected to flow analysis.

[0195] statistical methods GraphPad Prism 8 was used for statistical analysis as indicated in the figure legends. Data are presented as mean ± sd. Unpaired parametric t-tests were used for two-group comparisons. One-way ANOVA was used for multiple group comparisons. Survival was compared using the log-rank test.

[0196] 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 the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0197] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the scope of the following claims.

Claims

1. 1. A composition for use in a method for treating cancer in a subject, the composition comprising engineered immune effector cells, the method comprising: Obtaining immune effector cells from an allogeneic donor; engineering the immune effector cells to express an anti-CD3 multispecific antibody, the antibody configured to bind to a CD3 complex on the immune effector cell and a second antigen on another cell in a manner sufficient to activate the CD3 complex; and administering said composition to said subject.

2. The composition of claim 1 , wherein the antibody is a bispecific antibody.

3. The composition of claim 1 , wherein the second antigen is a tumor antigen.

4. The second antigen is EpCAM, CCR5, CD19, HER-2 neu, HER-3, HER-4, EGFR, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5. Ａ c, MUC5 Ｂ , MUC7, DhCG, Lewis-Y, CD20, CD33, CD30, ganglioside GD3, 9-O-acetyl-GD3, GM2, Globo H, fucosyl GM1, polySA, GD2, carboanhydrase IX (MN / CA IX), CD44v6, sonic hedgehog (Shh), Wue-1, plasma cell antigen, (membrane bound) IgE, melanoma chondroitin sulfate proteoglycan (MCSP), CCR8, TNF-alpha precursor, STEAP, mesothelin, A33 antigen, prostate stem cell antigen (PSCA), Ly-6; desmoglein 4, E-cadherin neoepitope, fetal acetylcholine receptor, CD25, CA19-9 marker, CA-125 marker and Mullerian inhibitory substance (MIS) receptor type II, sTn (sialylated Tn antigen; TAG-72), FAP (fibroblast activation antigen), endosialin, EGFRvIII, LG, SAS and CD63.

5. 5. The composition of any one of claims 1 to 4, wherein the immune effector cells are further engineered to express a chimeric antigen receptor (CAR).

6. 6. The composition of any one of claims 1 to 5, wherein the immune effector cells are selected from the group consisting of αβ T cells, γδ T cells, natural killer (NK) cells, natural killer T (NKT) cells, B cells, innate lymphoid cells (ILCs), cytokine-induced killer (CIK) cells, cytotoxic T lymphocytes (CTLs), lymphokine-activated killer (LAK) cells, regulatory T cells, or any combination thereof.

7. 1. An in vitro or ex vivo method for enhancing CAR-T cells for allogeneic cell transfer, comprising engineering said CAR-T cells to secrete a monospecific anti-CD3 antibody.

8. The method of claim 7, wherein the monospecific anti-CD3 antibody is a single-chain variable fragment (scFv).

9. CAR-T cells engineered to express a monospecific anti-CD3 antibody.

10. 1. An in vitro or ex vivo method for enhancing immune effector cells for allogeneic cell transfer, comprising: engineering the immune effector cells to express a membrane-bound anti-CD3 antibody; The method, wherein the anti-CD3 antibody is configured to bind to a CD3 complex on the immune effector cell in a manner sufficient to autoactivate the CD3 complex.

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