Extracorporeal transfection of immune effector cells

Abstract

Methods for extracorporeal transfection of an immune effector cell are provided. The method includes contacting blood, or a cell-containing fraction thereof, from a subject with a nanoparticle. The nanoparticle includes a surface-exposed immune effector binding moiety. Furthermore, the nanoparticle includes a nucleic acid encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR).

Description

Attorney Ref. No.: 123690-5005-WO EXTRACORPOREAL TRANSFECTION OF IMMUNE EFFECTOR CELLS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 551,954, filed February 9, 2024, which is herein incorporated by reference in its entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing that has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. The Sequence Listing for this application is labeled “123690-5005-WO Sequence Listing.xml”, which was created on February 10, 2025, and is 180,224 bytes in size. TECHNICAL FIELD

[0003] The present disclosure is directed extracorporeal transfection of immune effector cells. BACKGROUND

[0004] One of skill in the art will appreciate that CAR T cells are considered as “living drugs” as they have the capacity to migrate, proliferate and kill cognate-antigen expressing target cells in vivo. CAR T cell therapies have shown remarkable responses in patients with hematological cancers, that led to the regulatory approval of several CAR T products (Kymriah, Yescarta, Tecartus, Breyanzi, Abecma and Carvykti) in the market. See Salter, A.I., Pont, M.J. & Riddell, S.R. Chimeric antigen receptor-modified T cells: CD19 and the road beyond. Blood 131, 2621- 2629 (2018); Brudno, J.N. et al. T Cells Genetically Modified to Express an Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor Cause Remissions of Poor-Prognosis Relapsed Multiple Myeloma. J Clin Oncol 36, 2267-2280 (2018); Raje, N. et al. Anti-BCMA CAR T-Cell Therapy bb2121 in Relapsed or Refractory Multiple Myeloma. N Engl J Med 380, 1726-1737 (2019); Cohen, A.D. et al. B cell maturation antigen-specific CAR T cells are clinically active in multiple myeloma. J Clin Invest 129, 2210-2221 (2019); Park, J.H., Geyer, M.B. & Brentjens, R.J. CD19-targeted CAR T-cell therapeutics for hematologic malignancies: interpreting clinical 1 DB1 / 154850959.6Attorney Ref. No.: 123690-5005-WO outcomes to date. Blood 127, 3312-3320 (2016); Ceppi, F. & Gardner, R.A. Chimeric Antigen Receptor T Cells for B-Cell Acute Lymphoblastic Leukemia. Cancer J 25, 191-198 (2019); Kochenderfer, J.N. & Rosenberg, S.A. Treating B-cell cancer with T cells expressing anti-CD19 chimeric antigen receptors. Nat Rev Clin Oncol 10, 267-276 (2013); Maude, S.L. et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 371, 1507-1517 (2014); Lee, D.W. et al. T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. Lancet 385, 517-528 (2015); Turtle, C.J. et al. CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients. J Clin Invest 126, 2123-2138 (2016); Kalos, M. et al. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med 3, 95ra73 (2011), each of which is hereby incorporated by reference in its entirety for all purposes. The current standard CAR T manufacturing process involves isolation of mononuclear cells containing T cells from blood (apheresis) followed by T cell activation, gene transfer, ex vivo expansion and re-infusion typically requiring 7-14 days or even longer waiting times. See Park, J.H. et al. Long-Term Follow-up of CD19 CAR Therapy in Acute Lymphoblastic Leukemia. N Engl J Med 378, 449-459 (2018); Gardner, R.A. et al. Intent- to-treat leukemia remission by CD19 CAR T cells of defined formulation and dose in children and young adults. Blood 129, 3322-3331 (2017); Maude, S.L. et al. Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. N Engl J Med 378, 439-448 (2018); Tumaini, B. et al. Simplified process for the production of anti-CD19-CAR-engineered T cells. Cytotherapy 15, 1406-1415 (2013), each of which is hereby incorporated by reference in its entirety for all purposes.

[0005] Also, it has been shown that during the manufacturing process T cells are activated to rapidly proliferate and expand to sufficient numbers leading to differentiated effector cells which can compromise the potency of the product. See Ghassemi, S. et al. Reducing Ex Vivo Culture Improves the Antileukemic Activity of Chimeric Antigen Receptor (CAR) T Cells. Cancer Immunol Res 6, 1100-1109 (2018), which is hereby incorporated by reference in its entirety for all purposes. This intensive, lengthy manufacturing process can cause issues for patients, illustrated by reports describing that CAR T cells could not be infused in 20-30% of enrolled patients with B-cell acute lymphocytic leukemia (B-ALL), due to patients dying from disease progression before completion of the manufacturing protocol, or CAR T manufacturing failure. DB1 / 154850959.6 2Attorney Ref. No.: 123690-5005-WO See Park et al., 2018; Maude et al., 2018; Yang, J. et al. Next-day manufacture of a novel anti- CD19 CAR-T therapy for B-cell acute lymphoblastic leukemia: first-in-human clinical study. Blood Cancer J 12, 104 (2022), each of which is hereby incorporated by reference in its entirety for all purposes. In addition, currently the costs associated with manufacturing of CAR T cells are so high that it limits patient access to these novel therapies. Thus, shortening the duration of CAR T manufacturing process and in turn reducing the costs is to be crucial for patients.

[0006] Stable and safe genomic integration of CAR is a prerequisite for successful gene transfer in many applications. Integrating lentiviral (LV) or gamma-retroviral (RV) vectors were employed in manufacturing commercial CAR T products and in most of the CAR T clinical trials because of their ability to integrate a gene of interest into the host genome. However, both LV and RV vectors have concerns in terms of safety as they have the tendency to preferentially integrate into the genic regions, leading to insertional mutagenesis by activating oncogenes. See Hacein-Bey-Abina, S. et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302, 415-419 (2003); Gogol-Doring, A. et al. Genome-wide Profiling Reveals Remarkable Parallels Between Insertion Site Selection Properties of the MLV Retrovirus and the piggyBac Transposon in Primary Human CD4(+) T Cells. Mol Ther 24, 592- 606 (2016); Hacein-Bey-Abina, S. et al. Insertional oncogenesis in 4 patients after retrovirus- mediated gene therapy of SCID-X1. J Clin Invest 118, 3132-3142 (2008); Baum, C. et al. Chance or necessity? Insertional mutagenesis in gene therapy and its consequences. Mol Ther 9, 5-13 (2004); Deichmann, A. et al. Vector integration is nonrandom and clustered and influences the fate of lymphopoiesis in SCID-X1 gene therapy. J Clin Invest 117, 2225-2232 (2007); Kustikova, O. et al. Clonal dominance of hematopoietic stem cells triggered by retroviral gene marking. Science 308, 1171-1174 (2005), each of which is hereby incorporated by reference in its entirety for all purposes. SUMMARY

[0007] The present disclosure addresses the shortcomings disclosed above by providing extracorporeal transfection of immune effector cells.

[0008] Generation of sufficient number of CAR-T cells to combat liquid tumors represents a challenging area where safe, effective, and easily manufacturable delivery vectors are required. This includes but is not limited to retroviruses, lentiviruses and transposon / transposase system. DB1 / 154850959.6 3Attorney Ref. No.: 123690-5005-WO The latter, transposon / transposase system, has the advantage of being non-viral and potentially safer compared to viral vectors, and has the capacity to integrate the gene of interest (e.g., CAR) into the genome. In contrast to viral vectors, this system needs to be delivered into the cells using a delivery vehicle e.g. LNP. Sleeping Beauty (SB), TcBuster, Piggyback (and others) are DNA transposon systems that has been widely used in gene transfer applications because of their ability to integrate genes in a favorable, close-to-random integration pattern. See Kebriaei, P., Izsvak, Z., Narayanavari, S.A., Singh, H. & Ivics, Z. Gene Therapy with the Sleeping Beauty Transposon System. Trends Genet 33, 852-870 (2017), which is hereby incorporated by reference in its entirety for all purposes. The random, unbiased integration patterns are preferred as they seem to have lower risk related to carcinogenicity. Combination of transposon (delivered as DNA element) and transposase (delivered as DNA or RNA) by electroporation has been successfully used to generate CAR T cells ex vivo (CARAMBA clinical trial). See Prommerberger, S., et al., “CARAMBA: a first-in-human clinical trial with SLAMF7 CAR-T cells prepared by virus-free Sleeping Beauty gene transfer to treat multiple myeloma” Gene Therapy (2021) 28:560-571), which is hereby incorporated by reference in its entirety for all purposes. Although effective in ex vivo setting, electroporation is not amenable extracorporeal applications.

[0009] The development of ex vivo cell therapies is currently objected at reducing the time of manufacturing and at decentralization. This could allow major hospital centers to provide the manufacturing capabilities at the “bed side”. There is a number of developers, including but not limited to, Precigen (ultraCAR-T), Novartis (T-charge), AstraZeneca / GraCell (FasTCAR), Exuma Biotech (Rapid point of care rPOC), Sana Biotechnoilogy (extra coreporeal delivery), Galapagos / Cell Point (Lonza’s Cocoon platform), Umoja / Lupagen (VivoVec), BMS (Nex-T), Kyverna (Ingemui-T platform) that have generated rapid manufacturing protocols targeting decentralized, point of care manufacturing with times ranging from 6 h to 3 days. The point of care techniques include steps where patients cells undergo modifications and therefore they require quality control and release as they represent product manufacturing.

[0010] Given the above background, what is needed in the art are improved vein to vein time to absolute minimum, which are ideally sufficiently general to address the aforementioned limitations. DB1 / 154850959.6 4Attorney Ref. No.: 123690-5005-WO

[0011] To overcome some of these challenges and to provide extracorporeal transfection of immune effector cells, the present disclosure provides method of administration of targeted tLNP, containing DNA encoding CAR and mRNA encoding transposase. It provides a solution to the challenges associated with current CAR-T manufacturing, offering a streamlined, extracorporeal transfection approach that significantly reduces the time, cost, and logistical complexity of generating CAR-modified immune effector cells.

[0012] Traditional CAR-T therapies require lengthy and labor-intensive ex vivo manufacturing and manipulation that involve leukapheresis, cell activation, gene editing via viral or non-viral vectors, expansion, quality control, storage, transportation, and reinfusion, taking weeks and incurring substantial costs. These delays caused by the traditional CAR-T can be critical, especially for life-threatening for patients with aggressive malignancies, and manufacturing failures can prevent treatment entirely. The present disclosure overcomes these barriers by enabling direct, expedited extracorporeal delivery of CAR- or TCR- encoding nucleic acids into immune effector cells in whole blood or isolated cell fractions using lipid nanoparticles (LNPs) with surface-targeting moieties. This process eliminates the need for extensive manipulation of the cells, including complex cell culture, gene-editing, expansion, and centralized manufacturing, allowing for same-day (e.g., within 24 hours), point-of-care administration.

[0013] Additionally, by leveraging non-viral delivery methods, including but not limited to transposon-based integration systems, the invention mitigates the clinical risks associated with viral vectors, including insertional mutagenesis and genotoxicity. The ability to modify immune effector cells rapidly and safely within hours at point-of-care, rather than weeks, reduces costs, expands accessibility, and enables a broader range of treatment settings, making CAR-T and other immune cell therapies more widely available and clinically feasible.

[0014] One aspect of the present disclosure is directed to providing a method for extracorporeal transfection of an immune effector cell. The method includes contacting blood, or a cell- containing fraction thereof, from a subject with a nanoparticle. The nanoparticle includes a surface-exposed immune effector binding moiety. Moreover, the nanoparticle includes a nucleic acid encoding a first chimeric antigen receptor (CAR) or T-cell receptor (TCR).

[0015] In some embodiments, the contacting includes drawing blood from the subject and admixing a solution including the nanoparticle with the drawn blood. DB1 / 154850959.6 5Attorney Ref. No.: 123690-5005-WO

[0016] In some embodiments, the blood is drawn into an apheresis bag and the admixing includes adding the solution into the apheresis bag.

[0017] In some embodiments, an anticoagulant is added to the solution comprising the nanoparticle prior to, during, or after the admixing. In some embodiments, an anticoagulant is sodium citrate.

[0018] In some embodiments, the method further includes, after the admixing, returning the blood to the subject.

[0019] In some embodiments, the contacting includes apheresis of blood drawn from the subject, thereby generating a first blood fraction including immune effector cells and a second blood fraction free of immune effector cells, followed by admixing a solution including the nanoparticle with the first blood fraction.

[0020] In some embodiments, the contacting the contacting includes admixing a solution including the nanoparticle with blood drawn from the subject, followed by apheresis of the blood, thereby generating a first blood fraction including immune effector cells and a second blood fraction free of immune effector cells.

[0021] In some embodiments, the immune effector cells comprise peripheral blood mononuclear cells (PBMCs).

[0022] In some embodiments, the apheresis is leukopheresis.

[0023] In some embodiments, the first blood fraction is fractionated into an apheresis bag and the admixing includes adding the solution into the apheresis bag.

[0024] In some embodiments, after the contacting, cells in the first blood fraction are separated from free nanoparticles. In some embodiments, the separating includes washing the cells.

[0025] In some embodiments, the separating includes plasmapheresis, filtering with a spinning membrane filter, or centrifugation.

[0026] In some embodiments, the method further includes, after the admixing, returning the first blood fraction to the subject.

[0027] In some embodiments, the method described herein further includes washing the blood, or cells contained therein, prior to returning the blood, or cells contained therein, to the subject. DB1 / 154850959.6 6Attorney Ref. No.: 123690-5005-WO

[0028] In some embodiments, the contacting occurs in a closed vein-to-vein system.

[0029] In some embodiments, the contacting is performed at a cell density of at least 250,000 cells per mL or at least 1 million cells per mL.

[0030] In some embodiments, the contacting is performed at a cell density of at least 5 million cells per mL.

[0031] In some embodiments, the contacting is performed at a cell density of at least 10 million cells per mL.

[0032] In some embodiments, the contacting is performed at a cell density of from 1 million cells per mL to 20 million cells per mL.

[0033] In some embodiments, the contacting is performed at a ratio of DNA copy number to cells of at least 6×102DNA copies per cell, at least 8×102DNA copies per cell, at least 6×103DNA copies per cell, at least 8×103DNA copies per cell, at least 6×104DNA copies per cell, at least 8×104DNA copies per cell, at least 1×105DNA copies per cell, at least 2×105DNA copies per cell, at least 4×105DNA copies per cell, at least 6×105DNA copies per cell, at least 8×105DNA copies per cell, at least 1×106DNA copies per cell, 3×106DNA copies per cell, or at least 6×106DNA copies per cell.

[0034] In some embodiments, the contacting is performed at a nanoparticle dose of at least 2×10-6ng per cell, at least 5×10-6ng per cell, at least 2×10-5ng per cell, at least 5×10-5ng per cell, at least 2×10-4ng per cell, at least 5×10-4ng per cell, at least 0.00125 ng per cell, at least 0.0025 ng per cell, at least 0.005 ng per cell, at least 0.01 ng per cell, at least 0.02 ng per cell, or at least 0.04 ng per cell.

[0035] In some embodiments, the contacting includes admixing a solution comprising the nanoparticle with the drawn blood at a weight-to-volume ratio of nanoparticle dose to drawn blood of at least 0.1 ng / mL, at least 1 ng / mL, at least 5 ng / mL, at least 10 ng / mL, at least 25 ng / mL, at least 50 ng / mL, at least 100 ng / mL, at least 200 ng / mL, at least 400 ng / mL, at least 800 ng / mL, at least 1000 ng / mL, at least 1200 ng / mL, at least 1400 ng / mL, at least 1600 ng / mL, at least 1800 ng / mL, or at least 2000 ng / mL.

[0036] In some embodiments, the admixing occurs within twenty-four hours of drawing the blood from the subject. DB1 / 154850959.6 7Attorney Ref. No.: 123690-5005-WO

[0037] In some embodiments, the admixing occurs within four hours of drawing the blood from the subject.

[0038] In some embodiments, the admixing occurs within an hour of drawing the blood from the subject.

[0039] In some embodiments, the admixing occurs within fifteen minutes of drawing the blood from the subject.

[0040] In some embodiments, the admixing occurs within five minutes of drawing the blood from the subject.

[0041] In some embodiments, the blood, or cell-containing fraction thereof, is contacted with the nanoparticle for no more than six hours.

[0042] In some embodiments, the blood, or cell-containing fraction thereof, is contacted with the nanoparticle for no more than two hours.

[0043] In some embodiments, the blood, or cell-containing fraction thereof, is contacted with the nanoparticle for no more than one hour.

[0044] In some embodiments, the blood, or cell-containing fraction thereof, is contacted with the nanoparticle for no more than four hours.

[0045] In some embodiments, the blood, or cell-containing fraction thereof, is contacted with the nanoparticle for no more than 30 minutes.

[0046] In some embodiments, the blood, or cell-containing fraction thereof, is contacted with the nanoparticle for no more than 15 minutes.

[0047] In some embodiments, the blood, or cell-containing fraction thereof, is contacted with the nanoparticle for about ten minutes.

[0048] In some embodiments, the blood or first blood fraction is returned to the subject within twenty-four hours of drawing the blood from the subject.

[0049] In some embodiments, the blood or first blood fraction is returned to the subject within twelve hours of drawing the blood from the subject. DB1 / 154850959.6 8Attorney Ref. No.: 123690-5005-WO

[0050] In some embodiments, the blood or first blood fraction is returned to the subject within four hours of drawing the blood from the subject.

[0051] In some embodiments, the blood or first blood fraction is returned to the subject within one hour of drawing the blood from the subject.

[0052] In some embodiments, the blood or first blood fraction is returned to the subject within fifteen minutes of drawing the blood from the subject.

[0053] In some embodiments, the nanoparticle is a lipid nanoparticle (LNP).

[0054] In some embodiments, the immune effector cell is a T-cell.

[0055] In some embodiments, the immune effector cell is a B-cell.

[0056] In some embodiments, the immune effector cell is a monocyte.

[0057] In some embodiments, the immune effector cell is an NK-cell.

[0058] In some embodiments, the surface-exposed immune effector binding moiety binds CD2, CD4, CD3, CD5, CD7, CD8, CD25, CD127, CD152, CD11, or CD28.

[0059] In some embodiments, the CD7 binding moiety is an anti-CD7 antibody or antigen binding domain thereof.

[0060] In some embodiments, the CD7 binding moiety is an anti-CD7 VHH.

[0061] In some embodiments, the CD7 binding moiety includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 142, 146, 150, 154, 158, 162, 166, 170, and 174.

[0062] In some embodiments, the CD7 binding moiety includes: CDRs1-3 as set forth in SEQ ID NOs: 143-145, respectively, CDRs1-3 as set forth in SEQ ID NOs: 147-149, respectively, CDRs1-3 as set forth in SEQ ID NOs: 151-153, respectively, CDRs1-3 as set forth in SEQ ID NOs: 155-157, respectively, CDRs1-3 as set forth in SEQ ID NOs: 159-161, respectively, CDRs1-3 as set forth in SEQ ID NOs: 163-165, respectively, CDRs1-3 as set forth in SEQ ID NOs: 167-169, respectively, DB1 / 154850959.6 9Attorney Ref. No.: 123690-5005-WO CDRs1-3 as set forth in SEQ ID NOs: 171-173, respectively, or CDRs1-3 as set forth in SEQ ID NOs: 175-177, respectively.

[0063] In some embodiments, the nanoparticle includes a surface-exposed CD3 binding moiety.

[0064] In some embodiments, the CD3 binding moiety is an anti-CD3 antibody or antigen binding domain thereof.

[0065] In some embodiments, the CD3 binding moiety includes an anti-CD3 ScFv.

[0066] In some embodiments, the CD3 binding moiety includes a heavy chain variable region (VH) as set forth in SEQ ID NOS: 179, and a light chain variable region (VL) as set forth in SEQ ID NOS: 181; or a heavy chain variable region (VH) as set forth in SEQ ID NOS: 189, and a light chain variable region (VL) as set forth in SEQ ID NOS: 191.

[0067] In some embodiments, the CD3 binding moiety includes: VH-CDRs 1-3 as set forth in SEQ ID NOs: 182-184, respectively, and VL-CDRs 1-3 CDRs1 as set forth in SEQ ID NOs: 185-187, respectively; or VH-CDRs 1-3 as set forth in SEQ ID NOs: 192-194, respectively, and VL-CDRs 1-3 CDRs1 as set forth in SEQ ID NOs: 195-197, respectively.

[0068] In some embodiments, the deoxyribonucleic acid encoding the CAR or TCR is a plasmid, a minicircle DNA, a doggybone DNA, a miniplasmid DNA, Tiny plasmid, nanoplasmid or a close-ended DNA (ceDNA).

[0069] In some embodiments, the deoxyribonucleic acid encoding the CAR or TCR is a minicircle DNA.

[0070] In some embodiments, the deoxyribonucleic acid encoding the CAR or TCR is an open- ended DNA, or a single-stranded DNA (ssDNA).

[0071] In some embodiments, the deoxyribonucleic acid encoding the CAR or TCR includes a transposable element comprising a gene sequence encoding the CAR or TCR flanked by inverted terminal repeats (ITRs); and the nanoparticle further includes a nucleic acid encoding a transposase with specificity for the ITRs. DB1 / 154850959.6 10Attorney Ref. No.: 123690-5005-WO

[0072] In some embodiments, the transposable element is Sleeping Beauty (SB) 10 transposable element, a Sleeping Beauty 11 transposable element, a Sleeping Beauty 100X transposable element, a PiggyBac transposable element, a PiggyBat transposable element, a Mu transposable element, a TcBuster transposable element, a Mos1 transposable element, a Tol1 transposable element, a Tol2 transposable element, a Frog Prince transposable element, a spinON transposable element, a HimarI transposable element, a Passport transposable element, a Minos transposable element, a hAT transposable element, an Hsmar1 transposable element, a Harbinger transposable element, a Harbinger3-DR transposable element, an Ac / Ds transposable element, or a PIF transposable element.

[0073] In some embodiments, the transposable element is a Sleeping Beauty (SB) transposable element.

[0074] In some embodiments, the transposable element is a piggyBac (PB) transposable element.

[0075] In some embodiments, the nanoparticle includes one or more nucleic acids encoding a recombinase gene-editing system or a nuclease gene-editing system, wherein the recombinase gene-editing system or the nuclease gene-editing system has specificity for the deoxyribonucleic acid encoding the CAR or TCR.

[0076] In some embodiments, the recombinase gene-editing system is a Cre system, an FLP system, a lamda integrase system, a phiC31 integrase system, a Bxb1 integrase system, a gamma-delta resolvase system, a Tn3 resolvase system, or a Gin invertase system.

[0077] In some embodiments, the nuclease gene-editing system is a CRISPR / Cas system, an ARCUS system, a TALEN system, a meganuclease system, or a zinc finger nuclease system.

[0078] In some embodiments, the CAR or TCR has affinity for a cell-surface antigen selected from the group of genes consisting of TNFRSF17, IL3RA, SDC1, CD19, CD20, CD22, BCMA, MS4A1, CD7, CD123, CD135, CD38, CD138, CD269, TNFRSF8, CD33, CD38, CD5, NCAM1, CD70, ULBP1, ULBP2, IL1RAP, CEACAM5, MET, EGFR, EGFRvIII, EPCAM, EPHA2, ERBB2, GPC3, MSLN, Muc1, PDCD1, CD274, KDR, IL13RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, ROR2, GPRC5D, PSMA, CD23, WT1, CD44, CD44v6, CD174, SLAMF7, L1 CAM, FLT3, Sigle-6, GD2, PSCA, NY-ESO-1, GPNMB, CD276, CSPG4, MAGEA3, MAGEA4, CD133, TEM1, and combinations thereof. DB1 / 154850959.6 11Attorney Ref. No.: 123690-5005-WO

[0079] In some embodiments, the CAR or TCR has affinity for a single surface antigen.

[0080] In some embodiments, the CAR or TCR has affinity for two surface antigens.

[0081] In some embodiments, the CAR or TCR has affinity for three, or more, surface antigens.

[0082] In some embodiments, the CAR or TCR has affinity for CD19, CD20, CD22, BCMA, or combinations thereof.

[0083] In some embodiments, the CAR or TCR has affinity for a pair of cell-surface antigens including: CD19 and CD22; CD19 and CD20; CD20 and CD22; BCMA and CD19; BCMA and CD19; or BCMA and CD22.

[0084] In some embodiments, the CAR has affinity for CD19.

[0085] In some embodiments, the CAR has a polypeptide sequence of the CAR used in lisocabtagene maraleucel, tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, or obecabtagene autoleucel.

[0086] In some embodiments, the CAR has affinity for BCMA.

[0087] In some embodiments, the CAR has a polypeptide sequence of the CAR used in idecabtagene vicleucel or ciltacabtagene autoleucel.

[0088] In some embodiments, the CAR or TCR binds a T cell via interaction with one or more of the following: T-cell γ chains, T-cell β chains, T-cell δ chains, T-cell constant chains, CCR7, CD3, CD4, CD5, CD7, CD8, CD11b, CD11c, CD16, CD19, CD20, CD21, CD22, CD25, CD28, CD34, CD35, CD40, CD45RA, CD45RO, CD52, CD56, CD62L, CD68, CD80, CD95, CD117, CD127, CD133, CD137 (4-1BB), CD163, F4 / 80, IL-4Rα, Sca-1, CTLA-4, GITR, GARP, LAP, granzyme B, LFA-1, transferrin receptor, and combinations thereof.

[0089] In some embodiments, the method includes no treatment of the immune effector cell with an activation agent ex vivo. DB1 / 154850959.6 12Attorney Ref. No.: 123690-5005-WO

[0090] In some embodiments, the activation agent is a cytokine.

[0091] In some embodiments, the activation agent is CTS™ (Cell Therapy Systems).

[0092] In some embodiments, the activation agent is OKT-3.

[0093] In some embodiments, the activation agent is Dynabeads™ CD3 / CD28.

[0094] In some embodiments, the activation agent is MACS® GMP T Cell TransAct™.

[0095] In some embodiments, the subject has a disease or disorder, and the method further includes treating the disease or disorder in the subject including administering a therapeutically effective amount of the transfected immune effector cells to the subject.

[0096] In some embodiments, the disease or disorder is a cancer, an autoimmune disease, or an infectious disease.

[0097] In some embodiments, the subject has a cancer selected from the group consisting of lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, leukemia, liquid tumors, liver cancer, lung cancer, lymphoma, malignant mesothelioma, mastocytoma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, and urinary bladder cancer.

[0098] In some embodiments, the subject has an autoimmune disease selected from the group consisting of Arthritis, chronic obstructive pulmonary disease, ankylosing spondylitis, Crohn's disease (one of two types of idiopathic inflammatory bowel disease “IBD”), dermatomyositis, diabetes mellitus type 1, endometriosis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's disease, hidradenitis suppurativa, Kawasaki disease, IgA nephropathy, idiopathic thrombocytopenia purpura, interstitial cystitis, lupus erythematosus, DB1 / 154850959.6 13Attorney Ref. No.: 123690-5005-WO mixed connective tissue disease, morphea, myasthenia gravis, narcolepsy, neuromyotonia, pemphigus vulgaris, pernicious anaemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, relapsing polychondritis, rheumatoid arthritis, schizophrenia, scleroderma, Sjogren's syndrome, stiff person syndrome, temporal arteritis (also known as “giant cell arteritis”), ulcerative colitis (one of two types of idiopathic inflammatory bowel disease “IBD”), vasculitis, vitiligo, and Wegener's granulomatosis.

[0099] In some embodiments, the subject has an infectious disease selected from the group consisting of Acquired Immunodeficiency Syndrome (AIDS), cute or chronic bacterial infection, acute or chronic parasitic infection, acute or chronic viral infection, acute or chronic fungal infection, meningitis, hepatitis (A, B or C, or other viral hepatitis), peritonitis, pneumonia, epiglottitis, malaria, dengue hemorrhagic fever, leishmaniasis, streptococcal myositis, mycobacterium tuberculosis, mycobacterium avium intracellulare, Pneumocystis carinii pneumonia, orchitis / epidydimitis, legionella, Lyme disease, influenza A, epstein-barr virus, viral encephalitis / aseptic meningitis, and pelvic inflammatory disease.

[0100] Another aspect of the present disclosure provides a method for making a transfected T- cell, the method comprising contacting blood from a subject with a nanoparticle ex vivo, wherein the nanoparticle comprises: a surface-exposed CD7 binding moiety; a surface-exposed CD3 binding moiety; a deoxyribonucleic acid comprising a transposable element comprising a gene sequence encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR) flanked by inverted terminal repeats (ITRs); and an mRNA encoding a transposase with specificity for the ITRs.

[0101] Another aspect of the present disclosure provides a method for making a transfected T- cell, the method comprising isolating peripheral blood mononuclear cells (PBMCs) from blood from a subject and contacting the isolated PMBCs with a nanoparticle ex vivo, wherein the nanoparticle comprises: a surface-exposed CD7 binding moiety; a surface-exposed CD3 binding moiety; DB1 / 154850959.6 14Attorney Ref. No.: 123690-5005-WO a deoxyribonucleic acid comprising a transposable element comprising a gene sequence encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR) flanked by inverted terminal repeats (ITRs); and an mRNA encoding a transposase with specificity for the ITRs.

[0102] Another aspect of the present disclosure provides a method for treating a subject in need thereof, the method comprising administering to the subject a transfected immune effector cell prepared by contacting blood, or a cell-containing fraction thereof, from a subject with a nanoparticle ex vivo, wherein the nanoparticle comprises: a surface-exposed immune effector binding moiety; and a deoxyribonucleic acid encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR).

[0103] Another aspect of the present disclosure provides a method for treating a subject in need thereof, the method comprising administering to the subject a transfected T-cell prepared by contacting blood, or a T-cell-containing fraction thereof, from a subject with a nanoparticle ex vivo, wherein the nanoparticle comprises: a surface-exposed CD7 binding moiety; a surface-exposed CD3 binding moiety; a deoxyribonucleic acid comprising a transposable element comprising a gene sequence encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR) flanked by inverted terminal repeats (ITRs); and an mRNA encoding a transposase with specificity for the ITRs and a deoxyribonucleic acid encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR).

[0104] Another aspect of the present disclosure provides a nanoparticle for use in extracorporeal transfection of an immune effector cell, the transfection including contacting blood, or a cell-containing fraction thereof, from a subject with a nanoparticle, wherein the nanoparticle includes a surface-exposed immune effector binding moiety and a nucleic acid encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR).

[0105] In some embodiments, the nanoparticle described herein includes one or more features of any embodiment provided herein. DB1 / 154850959.6 15Attorney Ref. No.: 123690-5005-WO

[0106] Another aspect of the present disclosure provides a nanoparticle comprising a surface- exposed CD7 binding moiety, a surface-exposed CD3 binding moiety, and a deoxyribonucleic acid encoding a polypeptide or functional RNA.

[0107] In some embodiments, the nanoparticle is a lipid nanoparticle (LNP).

[0108] In some embodiments, the CD7 binding moiety is an anti-CD7 antibody or antigen binding domain thereof.

[0109] In some embodiments, the CD7 binding moiety is an anti-CD7 VHH.

[0110] In some embodiments, the CD7 binding moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 142, 146, 150, 154, 158, 162, 166, 170, and 174.

[0111] In some embodiments, the CD7 binding moiety comprises: CDRs1-3 as set forth in SEQ ID NOs: 143-145, respectively, CDRs1-3 as set forth in SEQ ID NOs: 147-149, respectively, CDRs1-3 as set forth in SEQ ID NOs: 151-153, respectively, CDRs1-3 as set forth in SEQ ID NOs: 155-157, respectively, CDRs1-3 as set forth in SEQ ID NOs: 159-161, respectively, CDRs1-3 as set forth in SEQ ID NOs: 163-165, respectively, CDRs1-3 as set forth in SEQ ID NOs: 167-169, respectively, CDRs1-3 as set forth in SEQ ID NOs: 171-173, respectively, or CDRs1-3 as set forth in SEQ ID NOs: 175-177, respectively.

[0112] In some embodiments, the nanoparticle is an LNP and the CD7 binding moiety is conjugated to a surface-exposed lipid of the LNP.

[0113] In some embodiments, the CD3 binding moiety is an anti-CD3 antibody or antigen binding domain thereof.

[0114] In some embodiments, the CD3 binding moiety comprises an anti-CD3 ScFv. DB1 / 154850959.6 16Attorney Ref. No.: 123690-5005-WO

[0115] In some embodiments, the CD3 binding moiety comprises a heavy chain variable region (VH) as set forth in SEQ ID NOS: 179, and a light chain variable region (VL) as set forth in SEQ ID NOS: 181, or a heavy chain variable region (VH) as set forth in SEQ ID NOS: 189, and a light chain variable region (VL) as set forth in SEQ ID NOS: 191.

[0116] In some embodiments, the CD3 binding moiety comprises VH-CDRs 1-3 as set forth in SEQ ID NOs: 182-184, respectively, and VL-CDRs 1-3 CDRs1 as set forth in SEQ ID NOs: 185-187, respectively, or VH-CDRs 1-3 as set forth in SEQ ID NOs: 192-194, respectively, and VL-CDRs 1-3 CDRs1 as set forth in SEQ ID NOs: 195-197, respectively.

[0117] In some embodiments, the nanoparticle is an LNP and the CD3 binding moiety is conjugated to a surface-exposed lipid of the LNP.

[0118] In some embodiments, the deoxyribonucleic acid encodes a chimeric antigen receptor (CAR) or T-cell receptor (TCR).

[0119] In some embodiments, the deoxyribonucleic acid is a plasmid, a minicircle DNA, a doggybone DNA, a miniplasmid DNA, a Tiny plasmid, a nanoplasmid or a close-ended DNA (ceDNA).

[0120] In some embodiments, the deoxyribonucleic acid is a minicircle DNA.

[0121] In some embodiments, the deoxyribonucleic acid is an open-ended DNA or a single- stranded DNA (ssDNA).

[0122] In some embodiments, the deoxyribonucleic acid comprises a transposable element comprising a gene sequence encoding the polypeptide or functional RNA flanked by inverted terminal repeats (ITRs), and the nanoparticle further comprises a nucleic acid encoding a transposase with specificity for the ITRs.

[0123] In some embodiments, the transposable element is selected from the group consisting of a Sleeping Beauty (SB) 10 transposable element, a Sleeping Beauty 11 transposable element, a Sleeping Beauty 100X transposable element, a PiggyBac transposable element, a PiggyBat transposable element, a Mu transposable element, a TcBuster transposable element, a Mos1 transposable element, a Tol1 transposable element, a Tol2 transposable element, a Frog Prince transposable element, a spinON transposable element, a HimarI transposable element, a Passport transposable element, a Minos transposable element, a hAT transposable element, an Hsmar1 DB1 / 154850959.6 17Attorney Ref. No.: 123690-5005-WO transposable element, a Harbinger transposable element, a Harbinger3-DR transposable element, an Ac / Ds transposable element, and a PIF transposable element.

[0124] In some embodiments, the transposable element is a Sleeping Beauty (SB) transposable element.

[0125] In some embodiments, the transposable element is a piggyBac (PB) transposable element.

[0126] In some embodiments, the nanoparticle comprises one or more nucleic acids encoding a recombinase gene-editing system or a nuclease gene-editing system, wherein the recombinase gene-editing system or the nuclease gene-editing system has specificity for the deoxyribonucleic acid encoding the polypeptide or functional RNA.

[0127] In some embodiments, the recombinase gene-editing system is selected from the group consisting of a Cre system, an FLP system, a lamda integrase system, a phiC31 integrase system, a Bxb1 integrase system, a gamma-delta resolvase system, a Tn3 resolvase system, and a Gin invertase system.

[0128] In some embodiments, the nuclease gene-editing system is select from the group consisting of a CRISPR / Cas system, an ARCUS system, a TALEN system, a meganuclease system, and a zinc finger nuclease system.

[0129] In some embodiments, the CAR or TCR has affinity for a cell-surface antigen selected from the group of genes consisting of TNFRSF17, IL3RA, SDC1, CD19, CD20, CD22, BCMA, MS4A1, CD7, CD123, CD135, CD38, CD138, CD269, TNFRSF8, CD33, CD38, CD5, NCAM1, CD70, ULBP1, ULBP2, IL1RAP, CEACAM5, MET, EGFR, EGFRvIII, EPCAM, EPHA2, ERBB2, GPC3, MSLN, Muc1, PDCD1, CD274, KDR, IL13RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, ROR2, GPRC5D, PSMA, CD23, WT1, CD44, CD44v6, CD174, SLAMF7, L1 CAM, FLT3, Sigle-6, GD2, PSCA, NY-ESO-1, GPNMB, CD276, CSPG4, MAGEA3, MAGEA4, CD133, TEM1, and combinations thereof.

[0130] In some embodiments, the CAR or TCR has affinity for a single surface antigen.

[0131] In some embodiments, the CAR or TCR has affinity for two surface antigens.

[0132] In some embodiments, the CAR or TCR has affinity for three, or more, surface antigens. DB1 / 154850959.6 18Attorney Ref. No.: 123690-5005-WO

[0133] In some embodiments, the CAR or TCR has affinity for CD19, CD20, CD22, BCMA, or a combination thereof.

[0134] In some embodiments, the CAR or TCR has affinity for a pair of cell-surface antigens selected from CD19 and CD22; CD19 and CD20; CD20 and CD22; BCMA and CD19; BCMA and CD19; and BCMA and CD22.

[0135] In some embodiments, the CAR has affinity for CD19.

[0136] In some embodiments, the CAR has a polypeptide sequence of the CAR used in lisocabtagene maraleucel, tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, or obecabtagene autoleucel.

[0137] In some embodiments, the CAR has affinity for BCMA.

[0138] In some embodiments, the CAR has a polypeptide sequence of the CAR used in idecabtagene vicleucel or ciltacabtagene autoleucel.

[0139] In some embodiments, the CAR or TCR binds a T cell via interaction with one or more of the following: T-cell γ chains, T-cell β chains, T-cell δ chains, T-cell constant chains, CCR7, CD3, CD4, CD5, CD7, CD8, CD11b, CD11c, CD16, CD19, CD20, CD21, CD22, CD25, CD28, CD34, CD35, CD40, CD45RA, CD45RO, CD52, CD56, CD62L, CD68, CD80, CD95, CD117, CD127, CD133, CD137 (4-1BB), CD163, F4 / 80, IL-4R?, Sca-1, CTLA-4, GITR, GARP, LAP, granzyme B, LFA-1, transferrin receptor, and combinations thereof.

[0140] Another aspect of the present disclosure provides the use of a nanoparticle in the manufacture of a medicament for extracorporeal transfection of an immune effector cell, the transfection including contacting blood, or a cell-containing fraction thereof, from a subject with a nanoparticle, wherein the nanoparticle includes:

[0141] a surface-exposed immune effector binding moiety;

[0142] and a nucleic acid encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR).

[0143] In some embodiments, the use of the nanoparticle described herein includes one or more features of any embodiment provided herein. DB1 / 154850959.6 19Attorney Ref. No.: 123690-5005-WO

[0144] The present disclosure has other features and advantages that will be apparent from, or are set forth in more detail in, the accompanying drawings, which are incorporated herein, and the following Detailed Description, which together serve to explain certain principles of exemplary embodiments of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0145] Figure 1 illustrates a comparison of point of care manufacturing to extracorporeal administration of tLNP, in accordance with an exemplary embodiment of the present disclosure;

[0146] Figures 2A and 2B collectively illustrate an example of tLNP transfection of T-cells in whole blood after short incubation times, in accordance with an exemplary embodiment of the present disclosure;

[0147] Figures 3A and 3B collectively illustrate flow plots of GFP expression in T cells (3A) and B cells (3B) following incubation of whole blood with tLNP comprising minicircle DNA encoding a GFP gene, in accordance with an exemplary embodiment of the present disclosure;

[0148] Figure 4 illustrates T and NK cell specificity targeting with CD7 targeted LNPs, in accordance with an exemplary embodiment of the present disclosure;

[0149] Figure 5 illustrates efficient generation of proliferative CAR-T cells in whole blood at a range of LNP doses within 6 hours, in accordance with an exemplary embodiment of the present disclosure;

[0150] Figures 6A, 6B, 6C, 6D, and 6E collectively illustrate efficient LNP mediated T cell transfection within 1 hour in whole blood leading to generation of proliferative CAR-T cells, in accordance with an exemplary embodiment of the present disclosure;

[0151] Figure 7 illustrates efficient LNP mediated T cell transfection within 4 hours in isolated PBMCs leading to generation of CAR-T cells at varying cell densities and particle doses, in accordance with an exemplary embodiment of the present disclosure; and

[0152] Figure 8 illustrates and compared stable CAR expression induced by LNP mediated T cell transfection in isolated PBMCs using 10 min incubation or within 4 hours in whole blood., in accordance with an exemplary embodiment of the present disclosure. DB1 / 154850959.6 20Attorney Ref. No.: 123690-5005-WO

[0153] It should be understood that the appended drawings are not necessarily to scale, presenting a somewhat simplified representation of various features illustrative of the basic principles of the invention. DETAILED DESCRIPTION

[0154] The present disclosure is directed to providing methods for extracorporeal transfection of an immune effector cell.

[0155] Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings. In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the present disclosure. However, it will be apparent to one of ordinary skill in the art that the present disclosure may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to unnecessarily obscure aspects of the embodiments.

[0156] It will also be understood that, although the terms first, second, etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. For instance, a first subject could be termed a second subject, and, similarly, a second subject could be termed a first subject, without departing from the scope of the present disclosure. The first subject and the second subject are both subjects, but they are not the same subject.

[0157] The terminology used in the present disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will also be understood that the term “and / or” as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items. It will be further understood that the terms “comprises” and / or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. DB1 / 154850959.6 21Attorney Ref. No.: 123690-5005-WO

[0158] The foregoing description included example systems, methods, techniques, instruction sequences, and computing machine program products that embody illustrative implementations. For purposes of explanation, numerous specific details are set forth in order to provide an understanding of various implementations of the inventive subject matter. It will be evident, however, to those skilled in the art that implementations of the inventive subject matter may be practiced without these specific details. In general, well-known instruction instances, protocols, structures, and techniques have not been shown in detail.

[0159] The foregoing description, for purpose of explanation, has been described with reference to specific implementations. However, the illustrative discussions below are not intended to be exhaustive or to limit the implementations to the precise forms disclosed. Many modifications and variations are possible in view of the above teachings. The implementations are chosen and described in order to best explain the principles and their practical applications, to thereby enable others skilled in the art to best utilize the implementations and various implementations with various modifications as are suited to the particular use contemplated.

[0160] In the interest of clarity, not all of the routine features of the implementations described herein are shown and described. It will be appreciated that, in the development of any such actual implementation, numerous implementation-specific decisions are made in order to achieve the designer’s specific goals, such as compliance with use case- and business-related constraints, and that these specific goals will vary from one implementation to another and from one designer to another. Moreover, it will be appreciated that such a design effort might be complex and time- consuming, but nevertheless be a routine undertaking of engineering for those of ordering skill in the art having the benefit of the present disclosure.

[0161] As used herein, the term “if” may be construed to mean “when” or “upon” or “in response to determining” or “in response to detecting,” depending on the context. Similarly, the phrase “if it is determined” or “if [a stated condition or event] is detected” may be construed to mean “upon determining” or “in response to determining” or “upon detecting [the stated condition or event]” or “in response to detecting [the stated condition or event],” depending on the context.

[0162] As used herein, the term “about” or “approximately” can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which can DB1 / 154850959.6 22Attorney Ref. No.: 123690-5005-WO depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. “About” can mean a range of ± 20%, ± 10%, ± 5%, or ± 1% of a given value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” means within an acceptable error range for the particular value. The term “about” can have the meaning as commonly understood by one of ordinary skill in the art. The term “about” can refer to ± 10%. The term “about” can refer to ± 5%.

[0163] In some embodiments, the present disclosure provides methods for extracorporeal transfection of an immune effector cell.

[0164] In some embodiments, a method includes for extracorporeal transfection of an immune effector cell contacting blood, or a cell-containing fraction thereof, from a subject with a nanoparticle. The nanoparticle includes a surface-exposed immune effector binding moiety. Moreover, the nanoparticle includes a nucleic acid encoding a first chimeric antigen receptor (CAR) or T-cell receptor (TCR).

[0165] In some embodiments, the contacting includes drawing blood from the subject and admixing a solution including the nanoparticle with the drawn blood.

[0166] In some embodiments, the blood is drawn into a pheresis bag and the admixing includes adding the solution into the pheresis bag.

[0167] In some embodiments, the method further includes, after the admixing, returning the blood to the subject.

[0168] In some embodiments, the contacting includes apheresis of blood drawn from the subject, thereby generating a first blood fraction including immune effector cells and a second blood fraction free of immune effector cells, followed by admixing a solution including the nanoparticle with the first blood fraction.

[0169] In some embodiments, the first blood fraction is fractionated into an apheresis bag and the admixing includes adding the solution into the pheresis bag.

[0170] In some embodiments, the method further includes, after the admixing, returning the first blood fraction to the subject. DB1 / 154850959.6 23Attorney Ref. No.: 123690-5005-WO

[0171] In some embodiments, the admixing occurs within twenty-four hours of drawing the blood from the subject.

[0172] In some embodiments, the admixing occurs within four hours of drawing the blood from the subject.

[0173] In some embodiments, the admixing occurs within an hour of drawing the blood from the subject.

[0174] In some embodiments, the admixing occurs within fifteen minutes of drawing the blood from the subject.

[0175] In some embodiments, the blood or first blood fraction is returned to the subject within twenty-four hours of drawing the blood from the subject.

[0176] In some embodiments, the blood or first blood fraction is returned to the subject within four hours of drawing the blood from the subject.

[0177] In some embodiments, the blood or first blood fraction is returned to the subject within one hour of drawing the blood from the subject.

[0178] In some embodiments, the blood or first blood fraction is returned to the subject within fifteen minutes of drawing the blood from the subject.

[0179] In some embodiments, the nanoparticle is a lipid nanoparticle (LNP).

[0180] In some embodiments, the immune effector cell is a T-cell.

[0181] In some embodiments, the immune effector cell is a B-cell.

[0182] In some embodiments, the immune effector cell is a monocyte.

[0183] In some embodiments, the immune effector cell is an NK-cell.

[0184] In some embodiments, the nucleic acid encoding the CAR or TCR is a DNA .

[0185] In some embodiments, the DNA includes a transposable element comprising a gene sequence flanked by inverted terminal repeats (ITRs); and a nucleic acid encoding a transposase with specificity for the ITRs.

[0186] In some embodiments, the gene sequence of the transposable element encodes a CAR or TCR. DB1 / 154850959.6 24Attorney Ref. No.: 123690-5005-WO

[0187] In some embodiments, the nucleic acid (e.g.) encoding the CAR or TCR is a transposable element including a gene sequence flanked by inverted terminal repeats (ITRs) and the nanoparticle further includes a nucleic acid encoding a transposase with specificity for the ITRs.

[0188] In some embodiments, the CAR or TCR has affinity for a cell-surface antigen selected from the group of genes TNFRSF17, IL3RA, SDC1, CD19, CD20, BCMA, MS4A1, CD7, CD22, CD123, CD135, CD38, CD138, CD269, TNFRSF8, CD33, CD38, CD5, NCAM1, CD70, ULBP1, ULBP2, IL1RAP, CEACAM5, MET, EGFR, EGFRvIII, EPCAM, EPHA2, ERBB2, GPC3, MSLN, Muc1, PDCD1, CD274, KDR, IL13RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, ROR2, GPRC5D, PSMA, CD23, WT1, CD44, CD44v6, CD174, SLAMF7, L1 CAM, FLT3, Sigle-6, GD2, PSCA, NY-ESO-1, GPNMB, CD276, CSPG4, MAGEA3, MAGEA4, CD133, and TEM1.

[0189] In some embodiments, the transposable element is carried on a plasmid, a minicircle DNA, a doggybone DNA, a miniplasmid DNA, Tiny plasmid, nanoplasmid or a close-ended DNA (ceDNA).

[0190] In some embodiments, the transposable element is carried on an open-ended DNA, or a single-stranded DNA (ssDNA).

[0191] In some embodiments, the nucleic acid encoding the transposase is mRNA.

[0192] In some embodiments, the nucleic acid encoding the transposase is DNA.

[0193] In some embodiments, the present disclosure provides a method of administration of targeted tLNP, containing DNA encoding CAR and mRNA encoding transposase. The patient blood is drawn and tLNP (injectable) is sterile mixed with the patient blood (which includes anticoagulants) and after a brief incubation (1min – 8h) is returned to the patient without any additional modifications, see Fig.1. The extracorporeal administration procedure, where patient derived material is mixed with tLNP (fully released injectable) but undergoes no additional modification steps before re-infusion, will avoid the need for quality control testing and release of the CAR-T product before infusion. This results in short vein-to-vein time (reduced to the time needed to incubate the tLNP with the patient blood). These CAR transduced cells are expanded and functionalized, inside the patient’s own body, to subsequently find and fight the DB1 / 154850959.6 25Attorney Ref. No.: 123690-5005-WO tumor. Elimination of the need to modify the patient cells removes the requirement for cell manufacturing and release and therefore solves the major logistic issue that arises at a point of manufacturing or classical ex vivo CAR T manufacturing procedures.

[0194] Combination of the tLNP particle that can activate T-cells, deliver DNA and RNA to permanently modify T-cells in whole blood and the novel route of administration (extracorporeal) provides a novel solution for patients. It removes the current need for complex manufacturing and release, long waiting time and a need for specialize hospital centers. The mixing of tLNP with patient blood, incubation and transfusion can be potentially performed in many hospitals. I. Lipid nanoparticle

[0195] In some embodiments, the nanoparticle described herein is a lipid nanoparticle. In some embodiments, the nanoparticle is selected from the group consisting of a liposome, a micelle, a solid-lipid nanoparticle, a cyclodextrin nanoparticle, a dendrimer, a polymeric nanoparticle, a micro / nano-emulsion, and any combinations thereof.

[0196] As used herein, the term “lipid nanoparticle” or “LNP” refers to a cell, or model cell, comprising a lipid layer. In some embodiments, lipid nanoparticles described herein include a cationic lipid. As used herein, a “cationic lipid” refers to a lipid having a net positive charge. In some embodiments, lipid nanoparticles described herein are made of ionizable lipids. In addition to ionizable lipids, lipid nanoparticles may contain a helper lipid to promote cell binding, cholesterol to fill the gaps between the lipids, and / or a polyethylene glycol (PEG) to reduce opsonization by serum proteins and reticuloendothelial clearance. In some embodiments, lipid nanoparticles comprise one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic, or zwitterionic lipids); one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to a polymer; one or more sterols (e.g., cholesterol); and, optionally, one or more targeting molecules (e.g., conjugated receptors, receptor ligands, antibodies); or combinations of the foregoing.

[0197] In some instances, the process of incorporation of a desired nucleic acid into a lipid nanoparticle is referred to as “loading.” Exemplary methods are described in Lasic et al., FEBS Lett. (1992) 312:255-8, incorporated herein by reference in its entirety. In some embodiments, the lipid nanoparticles-incorporated nucleic acids may be completely or partially located in the DB1 / 154850959.6 26Attorney Ref. No.: 123690-5005-WO interior space of the lipid nanoparticle, within the bilayer membrane of the lipid nanoparticle, or associated with the exterior surface of the lipid nanoparticle membrane. The incorporation of nucleic acids into lipid nanoparticles is also referred to herein as “encapsulation” wherein the nucleic acid is entirely or substantially contained within the interior space of the lipid nanoparticle.

[0198] In some embodiments, the nucleic acids described herein can be formulated in lipid nanoparticles using microfluidic technology (see Whitesides, George M., “The Origins and the Future of Microfluidics,” Nature 442: 368-373 (2006); and Abraham et al., “Chaotic Mixer for Microchannels,” Science 295: 647-651 (2002); each of which is herein incorporated by reference in its entirety). In some embodiments, the nucleic acids can be formulated in lipid nanoparticles using a micromixer chip such as, but not limited to, those from Harvard Apparatus (Holliston, MA) or Dolomite Microfluidics (Royston, UK). A micromixer chip can be used for rapid mixing of two or more fluid streams with a split and recombine mechanism. II. Immune effector binding moiety binding moiety

[0199] As discussed herein, the nanoparticles, e.g., lipid nanoparticles, of the present disclosure may be targeted to a particular cell type to facilitate its extracorporeal transfection. In some embodiments, the targeting is achieved through the incorporation of specific binding moieties on the nanoparticle surface, which selectively interact with markers or receptors expressed on the desired cell type. In some embodiments, the targeted cell is an immune effector cell, e.g., a T cell, a natural killer (NK) cell, a B cell, or a combination thereof. This can be accomplished by using one or more of the lipids described herein.

[0200] With regard to surface-exposed immune effector cell binding moieties (or immune effector binding moieties as the terms are referred to herein interchangeably), any suitable binding moieties can be contemplated. In some embodiments, the binding moieties are designed to selectively recognize and bind to immune effector cells without inducing activation, thereby preserving the resting state of the targeted T cells. This approach may be advantageous in applications where maintaining immune effector cell quiescence during extracorporeal transfection is preferred to prevent unintended immune activation.

[0201] In other embodiments, one or more activation-inducing immune effector-cell binding moieties may be included alongside non-activating binding moieties. In such configurations, the DB1 / 154850959.6 27Attorney Ref. No.: 123690-5005-WO non-activating moieties serve to specifically recognize and target immune effector cells, while the activation-inducing moieties can facilitate binding as well as controlled immune effector-cell stimulation when needed. The combination of both types of moieties may enable precise modulation of immune effector cell activity, allowing for tailored transfection strategies depending on the therapeutic application.

[0202] At least in some embodiments, an advantage of the disclosure is the recognition that activation of resting immune effector cells, such as T-cells, reorganizes the nuclear membrane, facilitating importation of recombinant DNA transfected in the cell by the disclosed LNPs for stable integration into the genome. In some embodiments, this is achieved by stimulating a cell- surface receptor via a non-activating immune effector binding moiety. In some embodiments, this is achieved by stimulating a cell-surface receptor such as CD3, e.g., by functionalizing the LNP with an anti-CD3 immune effector binding moiety. In some embodiments, this is achieved by including an mRNA encoding a CAR / TCR in the LNP to promote early expression of the CAR / TCR, facilitating activation of the T-cell. In some embodiments, a non-activating immune effector binding moiety, an mRNA encoding a CAR / TCR in the LNP, or both, are utilized in the LNP to promote genomic integration as well as stable expression of CAR / TCR in the immune effector cell after transfection. Accordingly, the LNP in turn facilitates the rapid preparation of CAR-T cells allowing the process to be performed bedside / same day and eliminating the need for lengthy CAR-T production processes.

[0203] A immune effector cell binding moiety that may be highly advantageously used includes a binding moiety which binds with CD7 on the surface of a T cell. Suitable anti-CD7 immune effector binding moieties are listed in Tables 1a below. As shown herein, targeting CD7 was highly useful in that resting T cells remained in their resting state. Hence, in some embodiments, binding moieties that do not activate immune effector cells may be used, such as T-cell binding moieties binding with CD7, or the like.

[0204] In some embodiments, targeting can be enhanced by including one or more binding moieties at a solvent accessible surface of an LNP particle.

[0205] An immune effector binding moiety includes any naturally occurring, synthetic, semi- synthetic, or recombinantly produced binding partner for a biological molecule or another target of interest. Exemplary binding moieties of this disclosure include an antibody, a Fab′, F(ab′)2, DB1 / 154850959.6 28Attorney Ref. No.: 123690-5005-WO Fab, Fv, rIgG, scFv, hcAb (heavy chain antibody), a single domain antibody, VHH, VNAR, sdAb, nanobody, receptor ectodomain or ligand-binding portions thereof, or ligand (e.g., cytokines, chemokines). In some embodiments, a binding moiety may include a member of a specific binding pair, e.g., an antibody-antigen pair, a ligand-receptor pair, etc. In certain embodiments, the binding moiety is an antibody. Cell-specific targeting / binding can be implemented, for example, by using lipid conjugated-immune cell binding moiety conjugates described herein.

[0206] In some embodiments, one or more binding moieties described herein may be a surface- bound antibody or surface bound antigen binding fragment thereof, which can permit tuning of cell targeting specificity. This is especially useful since highly specific antibodies can be raised against an epitope of interest for the desired targeting site. In one embodiment, multiple different antibodies can be incorporated into, and presented at the surface of an LNP, where each antibody binds to different epitopes on the same antigen or different epitopes on different antigens. Such approaches can increase the avidity and specificity of targeting interactions to a particular target cell.

[0207] In some embodiments, a binding moiety or combination of binding moieties can be selected based on the desired localization, function, or structural features of a given target cell. For example, in order to target a T-cell, T-cell population or T-cell subpopulation, one or more antibodies or antigen binding fragments or antigen binding derivatives thereof may be selected that target a T-cell, such as via a T-cell surface antigen. Exemplary T-cell surface antigens include, but are not limited to, for example, CD2, CD3, CD4, CD5, CD7, CD8, CD11, CD11a, CD25, CD127, CD152, CD28, CD39, CD69, CD103, CD137, CD45, T-cell receptor (TCR) β, TCR-α, TCR-α / β, TCR-γ / δ, PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, GL7, TLR2, TLR4, TLR5 and IL-15 receptor. In order to target an NK cell, or NK cell population, one or more antibodies, antigen binding fragments or antigen binding derivatives thereof maybe selected that target an NK cell such as via a NK cell surface antigen. Exemplary NK cell surface antigens include, but are not limited to, CD48, CD56, CD85a, CD85c, CD85d, CD85e, CD85f, CD85i, CD85j, CD158b2, CD161, CD244, CD16a, CD16b, IL-2 receptor, CD27, CD28, CD48, CD69, CD70, CD86, CD112, CD122, CD155, CD161, CD244, CD266, CD314 / NKG2D, CD336 / NKP44, CD337 / NKP30. In order to target a B cell or B cell population, one or more antibodies, antigen binding fragments or antigen binding derivatives thereof maybe selected that DB1 / 154850959.6 29Attorney Ref. No.: 123690-5005-WO target a B cell such as via a B cell antigen. Exemplary B cell antigens include, but are not limited to, CD19 for all B cells except plasma cells, CD19, CD25, and CD30 for activated B cells, CD27, CD38, CD78, CD138, and CD319 for plasma cells, CD20, CD27, CD40, CD80 and PDL- 2 for memory cells, Notch2, CD1, CD21, and CD27 for marginal zone B cells, CD21, CD22, and CD23 for follicular B cells, and CD1, CD5, CD21, CD24, and TLR4 for regulatory B cells.

[0208] In some embodiments, the binding moiety is covalently coupled to a lipid in the lipid blend via a polyethylene glycol (PEG) containing linker. In some embodiments, the lipid covalently coupled to the immune cell binding moiety via a PEG containing linker is distearoyl- phosphatidylethanolamine (DSPE), distearoylglycerol (DSG), dimyrstoyl- phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), dimyristoyl- glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DEPPE), dipalmitoyl-glycerol (DPG), or ceramide. In some embodiments, the PEG is PEG 2000.

[0209] The coupling / conjugation of the binding moiety to LNPs can be achieved via well- established approaches to decorate LNPs with ligands that are stabilized by hydrophobic interactions. Two general techniques include “post-insertion” technique, that is, insertion of a lipid-conjugate into, for example, a pre-formed LNP, and the “standard” technique, wherein the lipid-conjugate is included in the lipid mixture during, for example, the LNP formation steps. The post-insertion technique results in LNP having lipid-conjugates mainly in the external face of the LNP bilayer membrane, whereas standard techniques provide LNP having lipid-conjugates on both internal and external faces. The method is especially useful for vesicles made from phospholipids (which can contain cholesterol) and also for vesicles containing PEG-lipids (such as PEG-DAAs and PEG-DAGs). Methods of making LNP-conjugate, are taught, for example, in U.S. Patent Nos.5,705,385; 6,586,410; 5,981,501; 6,534,484; and 6,852,334; U.S. Patent Publication No.20020072121; PCT Publication No. WO 00 / 62813, as well as Ishida, et al., FEBS Lett.1999, 460, 129-133, the disclosures of which are herein incorporated by reference in their entireties for all purposes.

[0210] In some embodiments, the LNPs disclosed herein comprises a binding moiety that specifically targets CD7 localized on the surface of a T cell. In some embodiments, the T-cell binding moiety targeting CD7 comprises an antigen binding domain that specifically binds CD7. In some embodiments, the antigen binding domain is a VHH anti-CD7 antibody. In some DB1 / 154850959.6 30Attorney Ref. No.: 123690-5005-WO embodiments, the antigen binding domain is covalently conjugated to DSPE in the LNP via a PEG containing linker. In one embodiments, the T-cell binding moiety comprises DSPE-PEG- VHH-CD7.

[0211] In some embodiments, the LNP is functionalized with a CD7-binding moiety comprising one or more domains having amino acid sequences as set forth in Table 1a below, as well as fragments or variants thereof. SEQ IDName Organism SequenceNO. 142 H7 LamaEVQLVESGGG LVQAGGSLRL SCAASGSNLD glama YYAIGWVRQA PGKEREGVSC ISSSGSLTYY ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TAVYYCAADC RPRLGDYGPY EYDYWGQGTQ VTVSS 143 H7Lama GSNLDYYA CDR1 glama 144 H7Lama ISSSGSLT CDR2 glama 145 CD7VLama AADCRPRLGDYGPYEYDY HH glama CDR3 146 2A-C6 LlamaEVQLVESGGGLVQAGGSLRLSCAGSGGNFGLYAM Glama AWFRQAPGKEREFVAAISWSGDTTRYVDSVKGRF TISRDNAKSTTYLQMDSLKPEDTAVYYCAAKSNFG SGVYDTNWYTSRYERWGQGTQVTVSS 147 2A-C6Llama GGNFGLYA CDR1 Glama 148 2A-C6Llama ISWSGDTT CDR2 Glama 149 2A-C6Llama AAKSNFGSGVYDTNWYTSRYER CDR3 Glama DB1 / 154850959.6 31Attorney Ref. No.: 123690-5005-WO 150 2A-Llama EVQLVESGGGLVQAGNSLRLSCAFSGRTFSGFTMG H11 Glama WFRQAPGKEREFVAYITGSGGMTDYSNSVKGRFAI SRDNANDTVNLEMNSLKAEDTAVYYCAAGWILIS RMWRQYSYWGQGTQVTVSS 151 2A-Llama GRTFSGFT H11 Glama CDR1 152 2A-Llama ITGSGGMT H11 Glama CDR2 153 2A-Llama AAGWILISRMWRQYSY H11 Glama CDR3 154 3B-E9 LlamaEVQLVESGGGLVQAGNSLRLSCAFSGRTFSGYTM Glama GWFRQAPGKEREFVGLITGSGSMTDYSDSVKGRF AISRDNAKNTVNLEMNSLKAEDTAVYYCAAGWV LISRMWRQYSSWGQGTQVTVSS 155 3B-E9Llama GRTFSGYT CDR1 Glama 156 3B-E9Llama ITGSGSMT CDR2 Glama 157 3B-E9Llama AAGWVLISRMWRQYSS CDR3 Glama 158 4C-D9 LlamaEVQLVESGGGLGQSGGSLRLSCVVSGGIFSFKVMG Glama WYRQAPGTQREVVATITPGGRTNYADSVKGRFTIS RDNAKNTVDLQMNSLKPEDTAVYYCNVFSDVPGS WGQGTQVTVSS 159 4C-D9Llama GGIFSFKV CDR1 Glama 160 4C-D9Llama TPGGRT CDR2 Glama DB1 / 154850959.6 32Attorney Ref. No.: 123690-5005-WO 161 4C-D9Llama NVFSDVPGS CDR3 Glama 162 4D-C7 LlamaEVQLVESGGGLVQAGDSLTLSCAASGRTLSMYFM Glama GWFRQAPGKEREFVASITRFAESTYYTDSVKGRFTI SRDNVKEVVYLQMSSLQPEDTATYYCAAKFGWG LPPANEYEYDNWGQGTQVTVSS 163 4D-C7Llama GRTLSMYF CDR1 Glama 164 4D-C7Llama ITRFAEST CDR2 Glama 165 4D-C7Llama AKFGWGLPPANEYEYDN CDR3 Glama 166 4D-Llama EVQLVESGGGLVQAGNSLRLSCAFSGRTFSGFTMG D11 Glama WFRQAPGKEREFVAYITGSGGMTDYSDSVKGRFAI SRDNAKNTVNLEMNSLIAEDTAVYYCAAGWILISR MWRQYSYWGQGTQVTVSS 167 4D-Llama GRTFSGFT D11 Glama CDR1 168 4D-Llama ITGSGGMT D11 Glama CDR2 169 4D-Llama AAGWILISRMWRQYSY D11 Glama CDR3 170 4D-E5 LlamaEVQLVESGGGLVQPGGSLMLSCVVSGIPFVFKVMG Glama WYRQAPGKQREVVATITSGGRTNYNDAVKGRFTI SRDNDKNTVYLQMTSLKPEDTAMYYCNVFSDIPR SWGQGTQVTVSS 171 4D-E5Llama GIPFVFKV CDR1 Glama DB1 / 154850959.6 33Attorney Ref. No.: 123690-5005-WO 172 4D-E5Llama ITSGGRT CDR2 Glama 173 4D-E5Llama NVFSDIPRS CDR3 Glama 174 5A-C6 LlamaEVQLVESGGGLVQPGGSLRLSCVVSGSIFGSAFNFK Glama TMGWYRQPPGKQREVVATITPGGRTNYADSVKGR FTISRDNAKNSVYLQMNSVKPEDTAVYYCNVFSDI PGSWGQGTQVTVSS 175 5A-C6Llama GSIFGSAFNFKT CDR1 Glama 176 5A-C6Llama ITPGGRT CDR2 Glama 177 5A-C6Llama NVFSDIPGS CDR3 Glama

[0212] In some embodiments, the CD7 binding moiety comprises a CD7 antibody or antigen binding domain thereof selected from the group of consisting of 2A-C6, 4D-C7, 4C-D9, 5A-C6, 3B-E9, 2A-H11, 4D-D11, H7, and 4D-E5.

[0213] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the antigen binding domain of H7. In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the amino acid sequence set forth in SEQ ID NO: 142.

[0214] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the CDRs1-3 as set forth in SEQ ID NOs: 143-145, respectively.

[0215] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the antigen binding domain of 2A-C6. In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the amino acid sequence set forth in SEQ ID NO: 146.

[0216] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the CDRs1-3 as set forth in SEQ ID NOs: 147-149, respectively. DB1 / 154850959.6 34Attorney Ref. No.: 123690-5005-WO

[0217] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the antigen binding domain of 2A-H11. In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the amino acid sequence set forth in SEQ ID NO: 150.

[0218] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the CDRs1-3 as set forth in SEQ ID NOs: 151-153, respectively.

[0219] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the antigen binding domain of 3B-E9. In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the amino acid sequence set forth in SEQ ID NO: 154.

[0220] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the CDRs1-3 as set forth in SEQ ID NOs: 155-157, respectively.

[0221] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the antigen binding domain of 4C-D9. In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the amino acid sequence set forth in SEQ ID NO: 158.

[0222] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the CDRs1-3 as set forth in SEQ ID NOs: 159-161, respectively.

[0223] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the antigen binding domain of 4D-C7. In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the amino acid sequence set forth in SEQ ID NO: 162.

[0224] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the CDRs1-3 as set forth in SEQ ID NOs: 163-165, respectively.

[0225] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the antigen binding domain of 4D-D11. In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the amino acid sequence set forth in SEQ ID NO: 166.

[0226] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the CDRs1-3 as set forth in SEQ ID NOs: 167-169, respectively.

[0227] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the antigen binding domain of 4D-E5. In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the amino acid sequence set forth in SEQ ID NO: 170. DB1 / 154850959.6 35Attorney Ref. No.: 123690-5005-WO

[0228] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the CDRs1-3 as set forth in SEQ ID NOs: 171-173, respectively.

[0229] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the antigen binding domain of 5A-C6. In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the amino acid sequence set forth in SEQ ID NO: 174.

[0230] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the CDRs1-3 as set forth in SEQ ID NOs: 175-177, respectively.

[0231] In some embodiments, the LNPs disclosed herein further comprises a second binding moiety, in addition to the CD7 binding moiety.

[0232] In some embodiments, the LNP is functionalized with a second, CD3-binding moiety. In some embodiments, the CD3-binding moiety contains one or more domains having amino acid sequences as set forth in Table 1b below, as well as fragments or variants thereof. SEQ IDName SequenceNO. 178 UCHT1EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKG anti-CD3 LEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAE DTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGGSGGGGS GGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKP GKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYY CQQGNTLPWTFGQGTKVEIK 179 UCHT1EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKG anti-CD3 LEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAE heavy chain DTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS variable region 180 UCHT1GGGGSGGGGSGGGGS anti-CD3 linker 181 UCHT1DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKL anti-CD3 LIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNT light chain LPWTFGQGTKVEIK DB1 / 154850959.6 36Attorney Ref. No.: 123690-5005-WO variable region 182 UCHT1GYSFTGYT anti-CD3 heavy chain CDR1 183 UCHT1INPYKGVS anti-CD3 heavy chain CDR2 184 UCHT1ARSGYYGDSDWYFDV anti-CD3 heavy chain CDR3 185 UCHT1QDIRNY anti-CD3 light chain CDR1 186 UCHT1YTS anti-CD3 light chain CDR2 187 UCHT1QQGNTLPWT anti-CD3 light chain CDR3 188 SP34 anti-EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKG CD3 LEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLR AEDTAVYYCVRHGNFGNSYVSYFAYWGQGTLVTVSSGGGGSGG GGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANW VQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTISGAQPE DEADYYCALWYSNLWVFGGGTKLTVL 189 SP34 anti- EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGDB1 / 154850959.6 37Attorney Ref. No.: 123690-5005-WO CD3 heavy LEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLR chain AEDTAVYYCVRHGNFGNSYVSYFAYWGQGTLVTVSS variable region 190 SP34 anti-GGGGSGGGGSGGGGS CD3 linker 191 SP34 anti-QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQ CD3 light APRGLIGGTNKRAPGVPARFSGSLLGGKAALTISGAQPEDEADYYC chain ALWYSNLWVFGGGTKLTVL variable region 192 SP34 anti-AASGFTFNTYAMN CD3 heavy chain CDR1 193 SP34 anti-RIRSKYNNYATY CD3 heavy chain CDR2 194 SP34 anti-VRHGNFGNSYVSYFAY CD3 heavy chain CDR3 195 SP34 anti-GSSTGAVTTSNYAN CD3 light chain CDR1 196 SP34 anti-GGTNKRAP CD3 light chain CDR2 197 SP34 anti-ALWYSNLWV CD3 light DB1 / 154850959.6 38Attorney Ref. No.: 123690-5005-WO chain CDR3

[0233] In some embodiments, the CD3 binding moiety comprises a CD3 antibody or antigen binding domain thereof comprises an ScFv. In some embodiments, the CD3 binding moiety comprises a CD3 antibody or antigen binding domain thereof selected from the group of consisting of UCHT1-CD3, and SP34-CD3.

[0234] In some embodiments, the CD3 antibody or antigen binding domain thereof comprises the antigen binding domain of UCHT1. In some embodiments, the CD3 antibody or antigen binding domain thereof comprises the amino acid sequence set forth in SEQ ID NO: 178.

[0235] In some embodiments, the CD3 antibody or antigen binding domain thereof comprises the heavy chain variable region set forth in SEQ ID NO: 179, and the light chain variable region as set forth in SEQ ID NO:181.

[0236] In some embodiments, the CD3 antibody or antigen binding domain thereof comprises the heavy chain variable region as set forth in SEQ ID NO:179 and the light chain variable region as set forth in SEQ ID NO: 181, linked by the linker as set forth in SEQ ID NO:180.

[0237] In some embodiments, the CD3 antibody or antigen binding domain thereof comprises the heavy chain CDRs1-3 as set forth in SEQ ID NOs: 182-184, respectively, and the light chain CDRs1-3 as set forth in SEQ ID NO: 185-187.

[0238] In some embodiments, the CD3 antibody or antigen binding domain thereof comprises the antigen binding domain of SP34. In some embodiments, the CD3 antibody or antigen binding domain thereof comprises the amino acid sequence set forth in SEQ ID NO: 188.

[0239] In some embodiments, the CD3 antibody or antigen binding domain thereof comprises the heavy chain variable region set forth in SEQ ID NO: 189, and the light chain variable region as set forth in SEQ ID NO:191.

[0240] In some embodiments, the CD3 antibody or antigen binding domain thereof comprises the heavy chain variable region as set forth in SEQ ID NO:189 and the light chain variable region as set forth in SEQ ID NO: 191, linked by the linker as set forth in SEQ ID NO:190. DB1 / 154850959.6 39Attorney Ref. No.: 123690-5005-WO

[0241] In some embodiments, the CD3 antibody or antigen binding domain thereof comprises the heavy chain CDRs1-3 as set forth in SEQ ID NOs: 192-194, respectively, and the light chain CDRs1-3 as set forth in SEQ ID NO: 195-197.

[0242] In some embodiments, the LNP of the present disclosure comprises an anti-CD7 binding moiety and an anti-CD3 binding moiety, each of which is described herein. In some embodiments, the surface-exposed immune effector binding moieties on the LNP are an anti- CD7 VHH and an anti-CD3 scFv, or any equivalent variant thereof, as described herein.

[0243] Two general techniques include “post-insertion” technique, that is, insertion of a CPL into, for example, a pre-formed LNP, and the “standard” technique, wherein the CPL is included in the lipid mixture during, for example, the LNP formation steps. The post-insertion technique results in LNP having CPLs mainly in the external face of the LNP bilayer membrane, whereas standard techniques provide LNP having CPLs on both internal and external faces. The method is especially useful for vesicles made from phospholipids (which can contain cholesterol) and also for vesicles containing PEG-lipids (such as PEG-DAAs and PEG-DAGs). Methods of making LNP-CPL, are taught, for example, in U.S. Patent Nos.5,705,385; 6,586,410; 5,981,501; 6,534,484; and 6,852,334; U.S. Patent Publication No.20020072121; and PCT Publication No. WO 2000062813 A2, the disclosures of which are herein incorporated by reference in their entirety for all purposes. III. Integration system

[0244] In some embodiments, the nanoparticle described herein contains deoxyribonucleic acid (DNA) that contains a sequence encoding a chimeric antigen receptor (CAR) or a T-cell receptor (TCR). In some embodiments, the sequence encoding a CAR or TCR is operably linked to a promoter that, upon transfection into an immune effector cell, facilitates the expression of CAR or TCR. In some embodiments, the sequence encoding a CAR or TCR is operably linked to a promoter that enables its expression in the cytoplasm of the immune effector cell.

[0245] In some embodiments, the DNA encoding a CAR or TCR includes a genomic integration system that can integrate the CAR / TCR-encoding sequence into the genome of the host immune effector cell.

[0246] In some embodiments, the deoxyribonucleic acid encoding the CAR or TCR is a plasmid, a minicircle DNA, a doggybone DNA, a linear DNA, a miniplasmid DNA, Tiny DB1 / 154850959.6 40Attorney Ref. No.: 123690-5005-WO plasmid, nanoplasmid, or a close-ended DNA (ceDNA). In a preferred embodiment, the DNA is a minicircle DNA.

[0247] In some embodiments, the deoxyribonucleic acid encoding the CAR or TCR is an open- ended DNA, or a single-stranded DNA (ssDNA).

[0248] In some embodiments, “transposons”, or referred to interchangeably herein as “transposable elements” (TEs), or “transposon system”, is used. A transposon comprises DNA sequences that move from one location on the genome to another. “Transposon mutagenesis”, or “transposition mutagenesis”, is a biological process that allows genes to be transferred to a host organism's chromosome, interrupting or modifying the function of an extant gene on the chromosome and causing mutation.

[0249] A number of transposon systems that are able to also transpose in cells e.g. vertebrates have been isolated or designed, such as Sleeping Beauty (Izsvak and Ivics Molecular Therapy (2004) 9, 147-156), Sleeping Beauty 10 (Izsvak and Ivics Molecular Therapy (2004) 9, 147- 156), Sleeping Beauty 11 (Izsvak and Ivics Molecular Therapy (2004) 9, 147-156), Sleeping Beauty 100X (Izsvak and Ivics Molecular Therapy (2004) 9, 147-156; Mates et al., Nat. Gen. 2009), PiggyBac (Wilson et al. Molecular Therapy (2007) 15, 139-145), PiggyBat (Mitra et al. Proc Natl Acad Sci USA, 110(1), 234-239; Ray, et al. (2008)), Mu (WO / 2021 / 041922; Mizuuchi, K., Cell, 35: 785, 1983; Savilahti, H, et al, EMBO J., 14: 4893, 1995), TcBuster (Woodard et al. “Comparative Analysis of the Recently Discovered hAT Transposon TcBuster in Human Cells,” PLOS ONE, 7(11): e42666 (November 2012)), Mos1 (Tosi and Beverley, 2000), Tol1 (Koga et al. J. Human Genetics 2007, 52(7):628-635), Tol2 (Kawakami et al. PNAS (2000) 97 (21): 11403-11408), Frog Prince (Miskey et al. Nucleic Acids Res. December 1, (2003) 31 (23): 6873-6881), spinON (Pace et al. (2008) Proc. Natl. Acad. Sci. USA.2008; 105(44):17023- 17028), Himar1 (Lampe et al., 1996), Passport (Clar et al., Nucleic Acids Research, 2009, 37(4): 1239-1247), Minos (Klinakis et al. EMBO Rep 2000, 1(5):416-421; Franz and Savakis Nucleic Acids Res 1991, 19(23):6646), hAT (Arensburger et al. (2011) Genetics 188(1): 45-57), Hsmar1 (Miskey et al. Mol Cell. Biol.2007, 27(12):4589-4600), Harbinger (Sinzelle et al. Proc. Natl. Acad. Sci. USA 2008, 105(12):4715-4720), Harbinger3-DR (Sinzelle et al. Proc. Natl. Acad. Sci. USA 2008, 105(12):4715-4720), Ac / Ds (Emelyanov et al. Genetics 2006, 174(3):1095-1104), and PIF (Zhang et al., Genetics.2004 Feb;166(2):971-86). DB1 / 154850959.6 41Attorney Ref. No.: 123690-5005-WO

[0250] Generally, DNA transposons translocate from one DNA site to another in a simple, cut- and-paste manner. Each of these elements has their own advantages, for example, Sleeping Beauty is particularly useful in region-specific mutagenesis, whereas Tol2 has the highest tendency to integrate into expressed genes. Hyperactive systems are available for Sleeping Beauty and piggyBac (PB). Most importantly, these transposons have distinct target site preferences, and can therefore introduce sequence alterations in overlapping, but distinct sets of genes.

[0251] In some embodiments, a synthetic DNA transposon (e.g. “Sleeping Beauty” (SB) transposon system) designed to introduce precisely defined DNA sequences into the chromosome of vertebrate animals is adapted to genetically modify the T cells (Cooper et al., Blood 105:1622-31, (2005), the disclosures of which are incorporated by reference herein).

[0252] In some embodiments, the Sleeping Beauty transposon system is composed of a Sleeping Beauty (SB) transposase and a transposon that was designed to insert specific sequences of DNA into genomes of vertebrate animals. In some embodiments, the SB system described herein contains a two inverted terminal repeats sequences (ITRs) surrounding the gene of interest (GOI) and its promoter (CAG). ITRs were excised from the transposon plasmid and randomly inserted into the genome by the SB transposase, allowing stable expression in cells. Description of the SB transposon system and ITR sequences can be found, for example, in U.S. Patent Nos.9,228,180; 6,489,458; and 8,227,432, and U. S. Patent Publication Nos. 20220340898; and 20150152406, each of which is incorporated herein by reference in its entirety.

[0253] In some embodiments, the PiggyBac (PB) transposon is a mobile genetic element that efficiently transposes between nucleic acids and chromosomes via a “cut and paste” mechanism. During transposition, the PB transposase recognizes transposon-specific inverted terminal repeat sequences (ITRs) located on both ends of the transposon vector and efficiently moves the content gene of interest from the original sites and efficiently integrates it into TTAA chromosomal sites. The powerful activity of the PiggyBac transposon system enables genes of interest between the two ITRs in the PB vector to be easily mobilized into target genomes. Description of the PB transposon system can be found in, for example, Woltjen et al. (Nature 458:766-770, 2009), incorporated herein by reference in its entirety. DB1 / 154850959.6 42Attorney Ref. No.: 123690-5005-WO

[0254] Transposition may be a precise process, in which a defined DNA segment may be excised from one DNA molecule and moved to another site in the same or different DNA molecule or genome. In some embodiments, a Sleeping Beauty transposon system of the present disclosure is used for expressing the GOI in T cells. In some embodiments, the SB transposon system comprises a Sleeping Beauty (SB) transposase and a SB transposon. In some embodiments, the SB transposon is composed of a two inverted terminal repeats sequences (ITRs) surrounding the gene of interest (GOI) and its promoter (CAG). ITRs were excised from the transposon plasmid and randomly inserted into the genome by the SB transposase, allowing stable expression in cells. As do other Tc1 / mariner-type transposases, SB transposase inserts a transposon into a TA dinucleotide base pair in a recipient DNA sequence. The insertion site can be elsewhere in the same DNA molecule, or in another DNA molecule (or chromosome).

[0255] In mammalian genomes, including humans, there are approximately 200 million TA sites. The TA insertion site may be duplicated in the process of transposon integration. This duplication of the TA sequence may be a hallmark of transposition and used to ascertain the mechanism in some experiments. The transposase can be encoded either within the transposon or the transposase can be supplied by another source, in which case the transposon becomes a non- autonomous element. Non-autonomous transposons may be useful as genetic tools because after insertion they cannot independently continue to excise and re-insert. SB transposons envisaged to be used as non-viral vectors for introduction of genes into genomes of vertebrate animals and for gene therapy.

[0256] In some embodiments, the nanoparticle further encapsulates one or more nucleic acids encoding additional components of the genomic integration system, as required by the specific genomic integration system used. In some embodiments, a transposon system is used and the nanoparticle encapsulates a nucleic acid encoding a transposase that specifically recognizes the ITR sequences in the encapsulated DNA encoding the corresponding transposon.

[0257] In addition to transposon systems, other integration systems known in the art can also be employed in connection with the method described herein, such as recombinase gene-editing systems or nuclease gene-editing system.

[0258] Exemplary recombinase gene-editing systems include, but are not limited to, site- specific recombination systems that use recombinase enzymes to mediate DNA rearrangements DB1 / 154850959.6 43Attorney Ref. No.: 123690-5005-WO at specific recognition sequences. Such systems provide efficient and targeted genomic modifications with minimal off-target effects. Recombinase gene-editing systems suitable for use in the present methods include, but are not limited to, the following: a Cre system, an FLP system, a lambda integrase system, a phiC31 integrase system, a Bxb1 integrase system, a gamma-delta resolvase system, a Tn3 resolvase system, and a Gin invertase system. Recombinase gene-editing systems are well known to those skilled in the art. Descriptions of the exemplary recombinase gene-editing systems are present in Xu et al., BMC Biotechnol.2013 Oct.20; 13: 87; Innis et al., Biotechnol. Bioeng.2017 August; 114(8): 1837-46; Yang et al., Nat. Methods.2014 December; 11(12): 1261-66; Fuhrmann-Benzakein, et al., Proc. Natd. Acad. Sci. USA (2000); Lyznik et al., Plant Cell Rep, 21:925-932 (2003); Methods Mol. Biol.2012; 859:203-28; International Patent Publication No. WO 1999025821; U.S. Patent Publication No. 20150140665; and U.S. Patent No.10,975,392, the disclosure of each of which is hereby incorporated by reference in its entireties.

[0259] Exemplary nuclease gene-editing systems include, but are not limited to, genome- editing tools that rely on programmable nucleases to introduce double-strand breaks at specific DNA sequences, thereby enabling targeted gene modifications through DNA repair pathways. Such systems include, but are not limited to, a CRISPR / Cas system, an ARCUS system, a TALEN system, a meganuclease system, and a zinc finger nuclease system. Nuclease gene- editing systems are well known to those skilled in the art. Exemplary disclosures of them can be found in Gal et al., Trends Biotechnol.2013 May 9;31(7):397–405, Platt et. al. Cell; 159 (2): 440-455 (2014), and Shoop et al., Nat Metab.2023 Nov 30;5(12):2169–2183, the disclosure of each of which is hereby incorporated by reference in its entireties. IV. CAR and TCR

[0260] In some embodiments, the nanoparticle described herein contains deoxyribonucleic acid (DNA) that contains a sequence encoding a chimeric antigen receptor (CAR) or a T-cell receptor (TCR). In some embodiments, the sequence encoding a CAR or TCR is operably linked to a promoter that, upon transfection into an immune effector cell, facilitates the expression of CAR or TCR. In some embodiments, the sequence encoding a CAR or TCR is operably linked to a promoter that enables its expression in the cytoplasm of the immune effector cell. In other embodiments, the DNA encoding a CAR or TCR includes a genomic integration system, such as DB1 / 154850959.6 44Attorney Ref. No.: 123690-5005-WO a transposon system, to integrate the CAR- or TCR-encoding sequence into the genome of the host immune effector cell and facilitate durable expression of the CAR or TCR.

[0261] In some embodiments, the nanoparticle described herein encapsulates one CAR / TCR- encoding nucleic acid. In some embodiments, the nanoparticle described herein encapsulates more than one CAR / TCR-encoding nucleic acid.

[0262] Exemplary CAR techniques for use in accordance with the methods provided herein are known in the art. CAR techniques suitable for use in accordance with the methods provided herein include any described in Marofi et al., Stem Cell Res Ther 12: 81 (2021); Townsend et al., J Exp Clin Cancer Res 37: 163 (2018); Ma et al., Int J Biol Sci 15(12): 2548-2560 (2019); Zhao and Cao, Front Immunol 10: 2250 (2019); and Han et al., J Cancer 12(2): 326-334 (2021), the contents of each of which are incorporated by reference herein in their entireties. Exemplary TCR techniques for use in accordance with the methods provided herein are known in the art. TCR techniques suitable for use in accordance with the methods provided herein include any described in Zhao and Cao, Front Immunol 10: 2250 (2019); Ping et al., Protein Cell 9(3): 254- 266 (2018); and Zhang and Wang, Technol Cancer Res Treat 18: 1533033819831068 (2019), the contents of each of which are incorporated by reference herein in their entireties.

[0263] With respect to the structure of a CAR, in some embodiments, the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the antigen binding domain binds to an antigen on a target cell, e.g., a cancer cell. The antigen binding domain (also referred to as an extracellular domain) can bind antigens as is known in the art. In some embodiments, the antigen binding domain can be a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, a nanobody, a single-chain variable fragment (scFv), F(ab')2, Fab', Fab, Fv, and the like.

[0264] In some embodiments, the CAR comprises a hinge domain. A hinge domain may be derived from a protein selected from the group consisting of the CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8.alpha., CD8.beta., CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11 d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAMI), CD66b (CEACAM8), CD66c DB1 / 154850959.6 45Attorney Ref. No.: 123690-5005-WO (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B-cell antigen receptor complex-associated alpha chain), CD79B (B-cell antigen receptor complex- associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-zeta), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), inducible T cell co-stimulator (ICOS), LFA-1 (CD11 a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R beta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, a CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, activating NK cell receptors, or Toll ligand receptor, IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM or fragment or combination thereof.

[0265] In some embodiments, the CAR further comprises a transmembrane domain derived from a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD45, CD4, CD5, CD7, CD8 alpha, CD8 beta, CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD18, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, ICOS (CD278), 4- 1BB(CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD19, CD19a, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1A, CD1B, CD1C, CD1D, CD1E, ITGAE, CD103, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, CD29, ITGB2 (LFA-1, CD18), ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (TACTILE), CEACAMI, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C activating NK cell receptors, an Immunoglobulin protein, BTLA, CD247, CD276 (B7-H3), CD30, CD84, CDS, cytokine receptor, Fc gamma receptor, GADS, ICAM-1, Ig alpha (CD79a), integrins, LAT, a ligand that DB1 / 154850959.6 46Attorney Ref. No.: 123690-5005-WO binds with CD83, LIGHT, MHC class 1 molecule, PAG / Cbp, TNFSF14, a Toll ligand receptor, TRANCE / RANKL, or a fragment, truncation, or a combination thereof.

[0266] In some embodiments, the intracellular domain(s) of the CAR comprises a costimulatory domain(s). For instance, a signaling domain can contain a costimulatory domain. Or, a signaling domain can contain one or more costimulatory domains. In certain embodiments, the signaling domain comprises a costimulatory domain. In other embodiments, the signaling domains comprise costimulatory domains. In some cases, when the CAR comprises two or more costimulatory domains, two costimulatory domains are not the same. In some embodiments, the costimulatory domains comprise two costimulatory domains that are not the same. In some embodiments, the costimulatory domain enhances cytokine production, CAR-T cell proliferation, and / or CAR-T cell persistence during T cell activation. In some embodiments, the costimulatory domains enhance cytokine production, CAR-T cell proliferation, and / or CAR-T cell persistence during T cell activation. In some embodiments, the intracellular domain of a CAR comprises a primary signaling domain, a costimulatory domain, or both of a primary signaling domain and a costimulatory domain. In some embodiments, the primary signaling domain comprises a functional signaling domain of one or more proteins selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCERIG), FcR beta (Fc Epsilon Rlb), CD79a, CD79b, Fcgamma RIIa, DAP10, and DAP124-1BB / CD137, activating NK cell receptors, an Immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8alpha, CD8beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAMI, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrins, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, ligand that binds with CD83, LIGHT, LIGHT, LTBR, Ly9 (CD229), Ly108), lymphocyte function-associated antigen-1 (LFA-1; CD1-1a / CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), Signaling Lymphocytic Activation Molecules (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; DB1 / 154850959.6 47Attorney Ref. No.: 123690-5005-WO 2B4), SLAMF6 (NTB-A, SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or a fragment, truncation, or a combination thereof.

[0267] In some embodiments, the costimulatory domain of a CAR comprises a functional domain of one or more proteins selected from the group consisting of CD27, CD28, 4- 1BB(CD137), OX40, CD30, CD40, PD-1, ICOS, CD2, CD7, LIGHT, NKG2C, lymphocyte function-associated antigen-1 (LFA-1), MYD88, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, ITGAE, CD103, ITGAL, CD1A (NCBI Gene ID: 909), CD1B (NCBI Gene ID: 910), CD1C (NCBI Gene ID: 911), CD1D (NCBI Gene ID: 912), CD1E (NCBI Gene ID: 913), ITGAM, ITGAX, ITGB1, CD29, ITGB2 (CD18, LFA- 1), ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAMI, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, and NKG2D.

[0268] As described herein, a fourth generation CAR can contain an antigen binding domain, a transmembrane domain, three or four signaling domains, and a domain which upon successful signaling of the CAR induces expression of a cytokine gene. In some instances, the cytokine gene is an endogenous or exogenous cytokine gene of the hypoimmunogenic cells. In some cases, the cytokine gene encodes a pro-inflammatory cytokine. In some embodiments, the pro- inflammatory cytokine is selected from a group that includes IL-1, IL-2, IL-9, IL-12, IL-18, TNF, IFN-gamma, and a functional fragment thereof. In some embodiments, the domain which upon successful signaling of the CAR induces expression of the cytokine gene comprises a transcription factor or functional domain or fragment thereof.

[0269] In some embodiments, the CAR comprises a CD3 zeta (CD3ζ) domain or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; and (ii) a CD28 domain, or a 4-1BB DB1 / 154850959.6 48Attorney Ref. No.: 123690-5005-WO domain, or functional variant thereof. In other embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; and (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof. In certain embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof; and (iv) a cytokine or costimulatory ligand transgene. In some embodiments, the CAR comprises a (i) an anti-CD19 scFv; (ii) a CD8α hinge and transmembrane domain or functional variant thereof; (iii) a 4-1BB costimulatory domain or functional variant thereof; and (iv) a CD3ζ signaling domain or functional variant thereof.

[0270] In some embodiments, the CAR used in the methods and compositions described herein comprises a polypeptide sequence of a CAR used in a known cell-based CAR-T therapy. In some embodiments In some embodiments, the CAR-T therapy has been approved for marketing by one or more national medicines regulatory agency. Non-limiting examples of such CAR-T therapies include lisocabtagene maraleucel (e.g., sold under the tradename Breyanzi®), tisagenlecleucel (e.g., as sold under the tradename Kymriah®), axicabtagene ciloleucel (e.g., as sold under the tradename Yescarta®), brexucabtagene autoleucel (e.g., as sold under the tradename Tecartus®), idecabtagene vicleucel (e.g., as sold under the tradename Abecma®), ciltacabtagene autoleucel (e.g., as sold under the tradename Carvykti®), and obecabtagene autoleucel (e.g., as sold under the tradename Aucatzyl®). In some embodiments, a nucleic acid encoding a CAR used in a known cell-based CAR-T therapy, as used in the compositions and methods described herein, has a same polynucleotide sequence as a polynucleotide sequence encoding the CAR in the known CAR-T therapy. However, because of the degeneracy of the nucleic acid code, other polynucleotide sequences encoding the same CAR may be used. For example, in some embodiments, a polynucleotide sequence encoding a known CAR is codon- optimized to improve expression in human cells.

[0271] Accordingly, in some embodiments, the disclosure provides a nanoparticle comprising a surface-exposed immune effector binding moiety, and a DNA encoding a CAR, where the CAR has a polypeptide sequence of the CAR used in lisocabtagene maraleucel, tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, idecabtagene vicleucel, ciltacabtagene DB1 / 154850959.6 49Attorney Ref. No.: 123690-5005-WO autoleucel, or obecabtagene autoleucel. In some embodiments, the nanoparticle further comprises one or more nucleic acids encoding an additional CAR or CARs, such as another, second CAR. In some such embodiments, the surface-exposed immune effector binding moiety binds to CD7 and / or CD3. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP).

[0272] In some embodiments, the disclosure provides a nanoparticle comprising a surface- exposed immune effector binding moiety, and a DNA encoding a CAR, where the CAR has affinity for CD19 and has a polypeptide sequence of the CAR used in lisocabtagene maraleucel. In some such embodiments, the surface-exposed immune effector binding moiety binds to CD7 and / or CD3. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the DNA encoding the CAR having affinity for CD19 comprises a transposable element having a sequence encoding the CAR that is flanked by inverted terminal repeats (ITRs) and the nanoparticle further comprises a nucleic acid (e.g., a DNA or an mRNA) encoding a transposase with specificity for the ITRs. In some embodiments, the transposase is a Sleeping Beauty transposase.

[0273] In some embodiments, the disclosure provides a nanoparticle comprising a surface- exposed immune effector binding moiety, and a DNA encoding a CAR, where the CAR has affinity for CD19 and has a polypeptide sequence of the CAR used in tisagenlecleucel. In some such embodiments, the surface-exposed immune effector binding moiety binds to CD7 and / or CD3. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the DNA encoding the CAR having affinity for CD19 comprises a transposable element having a sequence encoding the CAR that is flanked by inverted terminal repeats (ITRs) and the nanoparticle further comprises a nucleic acid (e.g., a DNA or an mRNA) encoding a transposase with specificity for the ITRs. In some embodiments, the transposase is a Sleeping Beauty transposase.

[0274] In some embodiments, the disclosure provides a nanoparticle comprising a surface- exposed immune effector binding moiety, and a DNA encoding a CAR, where the CAR has affinity for CD19 and has a polypeptide sequence of the CAR used in axicabtagene ciloleucel. In some such embodiments, the surface-exposed immune effector binding moiety binds to CD7 and / or CD3. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the DNA encoding the CAR having affinity for CD19 comprises a transposable DB1 / 154850959.6 50Attorney Ref. No.: 123690-5005-WO element having a sequence encoding the CAR that is flanked by inverted terminal repeats (ITRs) and the nanoparticle further comprises a nucleic acid (e.g., a DNA or an mRNA) encoding a transposase with specificity for the ITRs. In some embodiments, the transposase is a Sleeping Beauty transposase.

[0275] In some embodiments, the disclosure provides a nanoparticle comprising a surface- exposed immune effector binding moiety, and a DNA encoding a CAR, where the CAR has affinity for CD19 and has a polypeptide sequence of the CAR used in brexucabtagene autoleucel. In some such embodiments, the surface-exposed immune effector binding moiety binds to CD7 and / or CD3. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the DNA encoding the CAR having affinity for CD19 comprises a transposable element having a sequence encoding the CAR that is flanked by inverted terminal repeats (ITRs) and the nanoparticle further comprises a nucleic acid (e.g., a DNA or an mRNA) encoding a transposase with specificity for the ITRs. In some embodiments, the transposase is a Sleeping Beauty transposase.

[0276] In some embodiments, the disclosure provides a nanoparticle comprising a surface- exposed immune effector binding moiety, and a DNA encoding a CAR, where the CAR has affinity for CD19 and has a polypeptide sequence of the CAR used in idecabtagene vicleucel. In some such embodiments, the surface-exposed immune effector binding moiety binds to CD7 and / or CD3. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the DNA encoding the CAR having affinity for CD19 comprises a transposable element having a sequence encoding the CAR that is flanked by inverted terminal repeats (ITRs) and the nanoparticle further comprises a nucleic acid (e.g., a DNA or an mRNA) encoding a transposase with specificity for the ITRs. In some embodiments, the transposase is a Sleeping Beauty transposase.

[0277] In some embodiments, the disclosure provides a nanoparticle comprising a surface- exposed immune effector binding moiety, and a DNA encoding a CAR, where the CAR has affinity for CD19 and has a polypeptide sequence of the CAR used in ciltacabtagene autoleucel. In some such embodiments, the surface-exposed immune effector binding moiety binds to CD7 and / or CD3. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the DNA encoding the CAR having affinity for CD19 comprises a transposable DB1 / 154850959.6 51Attorney Ref. No.: 123690-5005-WO element having a sequence encoding the CAR that is flanked by inverted terminal repeats (ITRs) and the nanoparticle further comprises a nucleic acid (e.g., a DNA or an mRNA) encoding a transposase with specificity for the ITRs. In some embodiments, the transposase is a Sleeping Beauty transposase.

[0278] In some embodiments, the disclosure provides a nanoparticle comprising a surface- exposed immune effector binding moiety, and a DNA encoding a CAR, where the CAR has affinity for CD19 and has a polypeptide sequence of the CAR used in obecabtagene autoleucel. In some such embodiments, the surface-exposed immune effector binding moiety binds to CD7 and / or CD3. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the DNA encoding the CAR having affinity for CD19 comprises a transposable element having a sequence encoding the CAR that is flanked by inverted terminal repeats (ITRs) and the nanoparticle further comprises a nucleic acid (e.g., a DNA or an mRNA) encoding a transposase with specificity for the ITRs. In some embodiments, the transposase is a Sleeping Beauty transposase.

[0279] In some embodiments, the disclosure provides a nanoparticle comprising a surface- exposed immune effector binding moiety, and a DNA encoding a CAR, where the CAR has affinity for BCMA and has a polypeptide sequence of the CAR used in idecabtagene vicleucel. In some such embodiments, the surface-exposed immune effector binding moiety binds to CD7 and / or CD3. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the DNA encoding the CAR having affinity for BCMA comprises a transposable element having a sequence encoding the CAR that is flanked by inverted terminal repeats (ITRs) and the nanoparticle further comprises a nucleic acid (e.g., a DNA or an mRNA) encoding a transposase with specificity for the ITRs. In some embodiments, the transposase is a Sleeping Beauty transposase.

[0280] In some embodiments, the disclosure provides a nanoparticle comprising a surface- exposed immune effector binding moiety, and a DNA encoding a CAR, where the CAR has affinity for BCMA and has a polypeptide sequence of the CAR used in ciltacabtagene autoleucel. . In some such embodiments, the surface-exposed immune effector binding moiety binds to CD7 and / or CD3. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the DNA encoding the CAR having affinity for BCMA comprises a transposable DB1 / 154850959.6 52Attorney Ref. No.: 123690-5005-WO element having a sequence encoding the CAR that is flanked by inverted terminal repeats (ITRs) and the nanoparticle further comprises a nucleic acid (e.g., a DNA or an mRNA) encoding a transposase with specificity for the ITRs. In some embodiments, the transposase is a Sleeping Beauty transposase.

[0281] In some embodiments, the CAR described herein comprises one or more domains having amino acid sequences as set forth in Table 2 below, as well as fragments or variants thereof. Table 2. Exemplary amino acid sequences of CAR domains SEQ ID NO Amino acid sequence Description1 MALPVTALLLPLALLLHAARP CD8Alpha signalpeptide 2METDTLLLWVLLLWVPGSTG IgK signal peptide3 MLLLVTSLLLCELPHPAFLLIP GMCSFR-Alpha(CSF2RA) signal peptide 4TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVCD8Alpha hinge HTRGLDFACD domain 5IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGCD28 hinge domain PSKP 6AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPCD28 hinge domain LFPGPSKP 7ESKYGPPCPPCP IgG4 hinge domain8 ESKYGPPCPSCP IgG4 hinge domain9 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISIgG4 hinge-CH2- RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN CH3 domain AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK 10 IYIWAPLAGTCGVLLLSLVITLYC CD8Alphatransmembrane domain 11 FWVLVVVGGVLACYSLLVTVAFIIFWV CD28 transmembranedomain 12 MFWVLVVVGGVLACYSLLVTVAFIIFWV CD28 transmembranedomain 13 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE4-1BB costimulatory EEEGGCEL domain 14 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPCD28 costimulatory RDFAAYRS domain DB1 / 154850959.6 53Attorney Ref. No.: 123690-5005-WO 15 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDCD3Zeta signaling VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD domain KMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPR 16 RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDCD3Zeta signaling VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD domain (with Q to K KMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT mutation at position KDTYDALHMQALPPR 14) 17 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNAnti-CD19 FMC63 WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSG scFv entire sequence, TDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGT with Whitlow linker KLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGL VAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGL EWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVF LKMNSLQTDDTAIYYCAKHYYYGGSYAMDYW GQGTSVTVSS 18 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNAnti-CD19 FMC63 WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSG scFv light chain TDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGT variable region KLEIT 19 QDISKY Anti-CD19 FMC63scFv light chain CDR1 20 HTS Anti-CD19 FMC63scFv light chain CDR2 21 QQGNTLPYT Anti-CD19 FMC63scFv light chain CDR3 22 GSTSGSGKPGSGEGSTKG Whitlow linker23 EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVAnti-CD19 FMC63 SWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLT scFv heavy chain IIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYY variable region GGSYAMDYWGQGTSVTVSS 24 GVSLPDYG Anti-CD19 FMC63scFv heavy chain CDR1 25 IWGSETT Anti-CD19 FMC63scFv heavy chain CDR2 26 AKHYYYGGSYAMDY Anti-CD19 FMC63scFv heavy chain CDR3 27 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNAnti-CD19 FMC63 WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSG scFv entire sequence, TDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGT with 3xG4S linker DB1 / 154850959.6 54Attorney Ref. No.: 123690-5005-WO KLEITGGGGSGGGGSGGGGSEVKLQESGPGLVA PSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEW LGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLK MNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQ GTSVTVSS 28 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAExemplary CD19 SLGDRVTISCRASQDISKYLNWYQQKPDGTVKLL CAR amino acid IYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDI sequence ATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPG SGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSG VSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYY NSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIY YCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPA PRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL DFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG CELRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR 29 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSATisagenlecleucel SLGDRVTISCRASQDISKYLNWYQQKPDGTVKLL CD19 CAR amino IYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDI acid sequence ATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGG SGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVS LPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNS ALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYC AKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF ACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE LRVKFSRSADAPAYKQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR 30 MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLisocabtagene LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLI maraleucel CD19 YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDI CAR amino acid ATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPG sequence SGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSG VSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYY NSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIY YCAKHYYYGGSYAMDYWGQGTSVTVSSESKYG PPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFW VKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP EEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL DB1 / 154850959.6 55Attorney Ref. No.: 123690-5005-WO NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDG LYQGLSTATKDTYDALHMQALPPR 31 MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASAxicabtagene LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLI ciloleucel CD19 YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDI CAR amino acid ATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPG sequence SGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSG VSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYY NSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIY YCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIE VMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPS KPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRS RLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAA YRSRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR 32 DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWAnti-CD20 Leu16 YQKKPGSSPKPWIYATSNLASGVPARFSGSGSGT scFv entire sequence, SYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTK with Whitlow linker LEIKGSTSGSGKPGSGEGSTKGEVQLQQSGAELV KPGASVKMSCKASGYTFTSYNMHWVKQTPGQG LEWIGAIYPGNGDTSYNQKFKGKATLTADKSSST AYMQLSSLTSEDSADYYCARSNYYGSSYWFFDV WGAGTTVTVSS 33 DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWAnti-CD20 Leu16 YQKKPGSSPKPWIYATSNLASGVPARFSGSGSGT scFv light chain SYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTK variable region LEIK 34 RASSSVNYMD Anti-CD20 Leu16scFv light chain CDR1 35 ATSNLAS Anti-CD20 Leu16scFv light chain CDR2 36 QQWSFNPPT Anti-CD20 Leu16scFv light chain CDR3 37 EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNAnti-CD20 Leu16 MHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFK scFv heavy chain GKATLTADKSSSTAYMQLSSLTSEDSADYYCAR SNYYGSSYWFFDVWGAGTTVTVSS 38 SYNMH Anti-CD20 Leu16scFv heavy chain CDR1 DB1 / 154850959.6 56Attorney Ref. No.: 123690-5005-WO 39 AIYPGNGDTSYNQKFKG Anti-CD20 Leu16scFv heavy chain CDR2 40 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAnti-CD22 m971 AWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVS scFv entire sequence, VKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCA with 3xG4S linker REVTGDLEDAFDIWGQGTMVTVSSGGGGSGGG GSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ TIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSR FSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQ TFGQGTKLEIK 41 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAnti-CD22 m971 AWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVS scFv heavy chain VKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCA variable region REVTGDLEDAFDIWGQGTMVTVSS 42 GDSVSSNSAA Anti-CD22 m971scFv heavy chain CDR1 43 TYYRSKWYN Anti-CD22 m971scFv heavy chain CDR2 44 AREVTGDLEDAFDI Anti-CD22 m971scFv heavy chain CDR3 45 DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNAnti-CD22 m971 WYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSG scFv light chain TDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTK LEIK 46 QTIWSY Anti-CD22 m971scFv light chain CDR1 47 AAS Anti-CD22 m971scFv light chain CDR2 48 QQSYSIPQT Anti-CD22 m971scFv light chain CDR3 49 QVQLQQSGPGMVKPSQTLSLTCAISGDSVSSNSVAnti-CD22 m971-L7 AWNWIRQSPSRGLEWLGRTYYRSTWYNDYAVS scFv entire sequence, MKSRITINPDTNKNQFSLQLNSVTPEDTAVYYCA with 3xG4S linker REVTGDLEDAFDIWGQGTMVTVSSGGGGSGGG GSGGGGSDIQMIQSPSSLSASVGDRVTITCRASQT IWSYLNWYRQRPGEAPNLLIYAASSLQSGVPSRF SGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQT FGQGTKLEIK 50 QVQLQQSGPGMVKPSQTLSLTCAISGDSVSSNSV Anti-CD22 m971-L7DB1 / 154850959.6 57Attorney Ref. No.: 123690-5005-WO AWNWIRQSPSRGLEWLGRTYYRSTWYNDYAVS scFv heavy chain MKSRITINPDTNKNQFSLQLNSVTPEDTAVYYCA variable region REVTGDLEDAFDIWGQGTMVTVSS 51 GDSVSSNSVA Anti-CD22 m971-L7scFv heavy chain CDR1 52 TYYRSTWYN Anti-CD22 m971-L7scFv heavy chain CDR2 53 AREVTGDLEDAFDI Anti-CD22 m971-L7scFv heavy chain CDR3 54 DIQMIQSPSSLSASVGDRVTITCRASQTIWSYLNWAnti-CD22 m971-L7 YRQRPGEAPNLLIYAASSLQSGVPSRFSGRGSGT scFv light chain DFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKL variable region EIK 55 QTIWSY Anti-CD22 m971-L7scFv light chain CDR1 56 AAS Anti-CD22 m971-L7scFv light chain CDR2 57 QQSYSIPQT Anti-CD22 m971-L7scFv light chain CDR3 58 DIVLTQSPASLAMSLGKRATISCRASESVSVIGAHAnti-BCMA LIHWYQQKPGQPPKLLIYLASNLETGVPARFSGS C11D5.3 scFv entire GSGTDFTLTIDPVEEDDVAIYSCLQSRIFPRTFGG sequence, with GTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPE Whitlow linker LKKPGETVKISCKASGYTFTDYSINWVKRAPGKG LKWMGWINTETREPAYAYDFRGRFAFSLETSAS TAYLQINNLKYEDTATYFCALDYSYAMDYWGQ GTSVTVSS 59 DIVLTQSPASLAMSLGKRATISCRASESVSVIGAHAnti-BCMA LIHWYQQKPGQPPKLLIYLASNLETGVPARFSGS C11D5.3 scFv light GSGTDFTLTIDPVEEDDVAIYSCLQSRIFPRTFGG chain variable region GTKLEIK 60 RASESVSVIGAHLIH Anti-BCMAC11D5.3 scFv light chain CDR1 61 LASNLET Anti-BCMAC11D5.3 scFv light chain CDR2 62 LQSRIFPRT Anti-BCMAC11D5.3 scFv light chain CDR3 DB1 / 154850959.6 58Attorney Ref. No.: 123690-5005-WO 63 QIQLVQSGPELKKPGETVKISCKASGYTFTDYSINAnti-BCMA WVKRAPGKGLKWMGWINTETREPAYAYDFRGR C11D5.3 scFv heavy FAFSLETSASTAYLQINNLKYEDTATYFCALDYS chain variable region YAMDYWGQGTSVTVSS 64 DYSIN Anti-BCMAC11D5.3 scFv heavy chain CDR1 65 WINTETREPAYAYDFRG Anti-BCMAC11D5.3 scFv heavy chain CDR2 66 DYSYAMDY Anti-BCMAC11D5.3 scFv heavy chain CDR3 67 DIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLAnti-BCMA IYWYQQKPGQPPTLLIQLASNVQTGVPARFSGSG C12A3.2 scFv entire SRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGG sequence, with TKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPEL Whitlow linker KKPGETVKISCKASGYTFRHYSMNWVKQAPGK GLKWMGRINTESGVPIYADDFKGRFAFSVETSAS TAYLVINNLKDEDTASYFCSNDYLYSLDFWGQG TALTVSS 68 DIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLAnti-BCMA IYWYQQKPGQPPTLLIQLASNVQTGVPARFSGSG C12A3.2 scFv light SRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGG chain variable region TKLEIK 69 RASESVTILGSHLIY Anti-BCMAC12A3.2 scFv light chain CDR1 70 LASNVQT Anti-BCMAC12A3.2 scFv light chain CDR2 71 LQSRTIPRT Anti-BCMAC12A3.2 scFv light chain CDR3 72 QIQLVQSGPELKKPGETVKISCKASGYTFRHYSMAnti-BCMA NWVKQAPGKGLKWMGRINTESGVPIYADDFKG C12A3.2 scFv heavy RFAFSVETSASTAYLVINNLKDEDTASYFCSNDY chain variable region LYSLDFWGQGTALTVSS 73 HYSMN Anti-BCMAC12A3.2 scFv heavy chain CDR1 74 RINTESGVPIYADDFKG Anti-BCMAC12A3.2 scFv heavy chain CDR2 75 DYLYSLDF Anti-BCMAC12A3.2 scFv heavy DB1 / 154850959.6 59Attorney Ref. No.: 123690-5005-WO chain CDR3 76 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMAnti-BCMA SWVRQAPGKGLEWVSSISGSGDYIYYADSVKGR FHVH33 entire FTISRDISKNTLYLQMNSLRAEDTAVYYCAKEGT sequence GANSSLADYRGQGTLVTVSS 77 GFTFSSYA Anti-BCMAFHVH33 CDR1 78 ISGSGDYI Anti-BCMAFHVH33 CDR2 79 AKEGTGANSSLADY Anti-BCMAFHVH33 CDR3 80 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWAnti-BCMA CT103A YQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGT scFv entire sequence, DFTLTISSLQPEDFATYYCQQKYDLLTFGGGTKV with Whitlow linker EIKGSTSGSGKPGSGEGSTKGQLQLQESGPGLVK PSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLE WIGSISYSGSTYYNPSLKSRVTISVDTSKNQFSLK LSSVTAADTAVYYCARDRGDTILDVWGQGTMV TVSS 81 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWAnti-BCMA CT103A YQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGT scFv light chain DFTLTISSLQPEDFATYYCQQKYDLLTFGGGTKV variable region EIK 82 QSISSY Anti-BCMA CT103AscFv light chain CDR1 83 AAS Anti-BCMA CT103AscFv light chain CDR2 84 QQKYDLLT Anti-BCMA CT103AscFv light chain CDR3 85 QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYAnti-BCMA CT103A WGWIRQPPGKGLEWIGSISYSGSTYYNPSLKSRV scFv heavy chain TISVDTSKNQFSLKLSSVTAADTAVYYCARDRGD variable region TILDVWGQGTMVTVSS 86 GGSISSSSYY Anti-BCMA CT103AscFv heavy chain CDR1 87 ISYSGST Anti-BCMA CT103AscFv heavy chain CDR2 88 ARDRGDTILDV Anti-BCMA CT103AscFv heavy chain CDR3 89 MALPVTALLLPLALLLHAARPDIQMTQSPSSLSA Exemplary BCMADB1 / 154850959.6 60Attorney Ref. No.: 123690-5005-WO SVGDRVTITCRASQSISSYLNWYQQKPGKAPKLL CAR amino acid IYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDF sequence ATYYCQQKYDLLTFGGGTKVEIKGSTSGSGKPGS GEGSTKGQLQLQESGPGLVKPSETLSLTCTVSGG SISSSSYYWGWIRQPPGKGLEWIGSISYSGSTYYN PSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYY CARDRGDTILDVWGQGTMVTVSSFVPVFLPAKP TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC NHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEEGGCELRVKFSRSADAPAYQQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR 90 ACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVCD8 Transmembrane LLLSLVITLYC 91 TTTPAPRPPTPAPTIASQPLSLRPE CD8 Hinge92 GGGGSGGGGSGGGGS linker93 SNYYGSSYWFFDV Anti-CD20 Leu16scFv heavy chain CDR3 94 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNAnti-CD19 FMC63 WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSG scFv entire sequence, TDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGT with Whitlow linker KLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGL VAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGL EWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVF LKMNSLQTDDTAIYYCAKHYYYGGSYAMDYW GQGTSVTVSS 95 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNAnti-CD19 FMC63 WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSG scFv light chain TDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGT variable region KLEIT 96 QDISKY Anti-CD19 FMC63scFv light chain CDR1 97 HTS Anti-CD19 FMC63scFv light chain CDR2 98 QQGNTLPYT Anti-CD19 FMC63scFv light chain CDR3 99 GSTSGSGKPGSGEGSTKG Whitlow linker100 EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVAnti-CD19 FMC63 SWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLT scFv heavy chain IIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYY variable region DB1 / 154850959.6 61Attorney Ref. No.: 123690-5005-WO GGSYAMDYWGQGTSVTVSS 101 GVSLPDYG Anti-CD19 FMC63scFv heavy chain CDR1 102 IWGSETT Anti-CD19 FMC63scFv heavy chain CDR2 103 AKHYYYGGSYAMDY Anti-CD19 FMC63scFv heavy chain CDR3 104 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNAnti-CD19 FMC63 WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSG scFv entire sequence, TDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGT with 3xG4S linker KLEITGGGGSGGGGSGGGGSEVKLQESGPGLVA PSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEW LGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLK MNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQ GTSVTVSS 105 GGGGSGGGGSGGGGS 3xG4S linker106 MALPVTALLLPPLALLLHAARPDIQMTQTTSSLAExemplary CD19 SLGDRVTISCRASQDISKYLNWYQQKPDGTVKLL CAR amino acid IYHTSRLHSVVPSRFSGSGSGTDYSLTISNLEQEDI sequence ATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPG SGEGSTKGEVKLQESGPGVLVAPSQSLSVCTVSG SLPDYGVSWIRQPPGKGLEWIGWVSGETTYNSA LYKSRLTIIKDNSKSQVFLKMNSLQTTDTAIYYC AKHYYYQGGSYAMDYWGQGTSVTSSSTTTPAR PTPTPAPTIASQPLSLRPEACRPAAGGAVHTRGGL DFACDIYWLAPLAGTCGVLLLVITYLCKRGRRKL LYIFKQPFRVPVQTQQEEDGCSCRFPFEEEGEGCE LRVKFSRSADAPAYQGQCNQLYNELNLGREEYD VLDKRRGRDEGMSGPKRPRKNPEQGLYNELQK DKMAEAYSIEGMGKGERGRKGHGDLGYQGLST ATKDTYDALHMQALPPR 107 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSATisagenlecleucel SLGDRVTISCRASQDISKYLNWYQQKPDGTVKLL CD19 CAR amino IYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDI acid sequence ATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGG SGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVS LPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNS ALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYC AKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF ACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE LRVKFSRSADAPAYKQGQNQLYNELNLGRREEY DB1 / 154850959.6 62Attorney Ref. No.: 123690-5005-WO DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR 108 MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLisocabtagene LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLI maraleucel CD19 YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDI CAR amino acid ATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPG sequence SGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSG VSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYY NSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIY YCAKHYYYGGSYAMDYWGQGTSVTVSSESKYG PPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFW VKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP EEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDG LYQGLSTATKDTYDALHMQALPPR 109 MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASAxicabtagene LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLI ciloleucel CD19 YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDI CAR amino acid ATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPG sequence SGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSG VSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYY NSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIY YCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIE VMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPS KPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRS RLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAA YRSRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR 110 DIVLTQSPASLAMSLGKRATISCRASESVSVIGAHAnti-BCMA LIHWYQQKPGQPPKLLIYLASNLETGVPARFSGS C11D5.3 scFv entire GSGTDFTLTIDPVEEDDVAIYSCLQSRIFPRTFGG sequence, with GTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPE Whitlow linker LKKPGETVKISCKASGYTFTDYSINWVKRAPGKG LKWMGWINTETREPAYAYDFRGRFAFSLETSAS TAYLQINNLKYEDTATYFCALDYSYAMDYWGQ GTSVTVSS 111 DIVLTQSPASLAMSLGKRATISCRASESVSVIGAHAnti-BCMA LIHWYQQKPGQPPKLLIYLASNLETGVPARFSGS C11D5.3 scFv light GSGTDFTLTIDPVEEDDVAIYSCLQSRIFPRTFGG chain variable region GTKLEIK 112 RASESVSVIGAHLIH Anti-BCMAC11D5.3 scFv light DB1 / 154850959.6 63Attorney Ref. No.: 123690-5005-WO chain CDR1 113 LASNLET Anti-BCMAC11D5.3 scFv light chain CDR2 114 LQSRIFPRT Anti-BCMAC11D5.3 scFv light chain CDR3 115 QIQLVQSGPELKKPGETVKISCKASGYTFTDYSINAnti-BCMA WVKRAPGKGLKWMGWINTETREPAYAYDFRGR C11D5.3 scFv heavy FAFSLETSASTAYLQINNLKYEDTATYFCALDYS chain variable region YAMDYWGQGTSVTVSS 116 DYSIN Anti-BCMAC11D5.3 scFv heavy chain CDR1 117 WINTETREPAYAYDFRG Anti-BCMAC11D5.3 scFv heavy chain CDR2 118 DYSYAMDY Anti-BCMAC11D5.3 scFv heavy chain CDR3 119 DIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLAnti-BCMA IYWYQQKPGQPPTLLIQLASNVQTGVPARFSGSG C12A3.2 scFv entire SRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGT sequence, with KLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELK Whitlow linker KPGETVKISCKASYTFRHYSMNWVKQAPGKGLK WMGRINTESGVPIYADDFKGRFAFSVETSASTAY LVINNLKDEDTASYFCSNDYLYSLDFWGQGTAL TVSS 120 DIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLAnti-BCMA IYWYQQKPGQPPTLLIQLASNVQTGVPARFSGSG C12A3.2 scFv light SRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGT chain variable region KLEIK 121 RASESVTILGSHLIY Anti-BCMAC12A3.2 scFv light chain CDR1 122 LASNVQT Anti-BCMAC12A3.2 scFv light chain CDR2 123 LQSRTIPRT Anti-BCMAC12A3.2 scFv light chain CDR3 124 QIQLVQSGPELKKPGETVKISCKASYTFRHYSMNAnti-BCMA WVKQAPGKGLKWMGRINTESGVPIYADDFKGR C12A3.2 scFv heavy FAFSVETSASTAYLVINNLKDEDTASYFCSNDYL chain variable region YSLDFWGQGTALTVSS 125 HYSMN Anti-BCMADB1 / 154850959.6 64Attorney Ref. No.: 123690-5005-WO C12A3.2 scFv heavy chain CDR1 126 RINTESGVPIYADDFKG Anti-BCMAC12A3.2 scFv heavy chain CDR2 127 DYLYSLDF Anti-BCMAC12A3.2 scFv heavy chain CDR3 128 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMAnti-BCMA SWVRQAPGKGLEWVSSISGSGDYIYYADSVKGR FHVH33 entire FTISRDISKNTLYLQMNSLRAEDTAVYYCAKEGT sequence GANSSLADYRGQGTLVTVSS 129 GFTFSSYA Anti-BCMAFHVH33 CDR1 130 ISGSGDYI Anti-BCMAFHVH33 CDR2 131 AKEGTGANSSLADY Anti-BCMAFHVH33 CDR3 132 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWAnti-BCMA CT103A YQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGT scFv entire sequence, DFTLTISSLQPEDFATYYCQQKYDLLTFGGGTKV with Whitlow linker EIKGSTSGSGKPGSGEGSTKGQLQLQESGPGLVK PSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLE WIGSISYSGSTYYNPSLKSRVTISVDTSKNQFSLK LSSVTAADTAVYYCARDRGDTILDVWGQGTMV TVSS 133 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWAnti-BCMA CT103A YQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGT scFv light chain DFTLTISSLQPEDFATYYCQQKYDLLTFGGGTKV variable region EIK 134 QSISSY Anti-BCMA CT103AscFv light chain CDR1 135 AAS Anti-BCMA CT103AscFv light chain CDR2 136 QQKYDLLT Anti-BCMA CT103AscFv light chain CDR3 137 QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYAnti-BCMA CT103A WGWIRQPPGKGLEWIGSISYSGSTYYNPSLKSRV scFv heavy chain TISVDTSKNQFSLKLSSVTAADTAVYYCARDRGD variable region TILDVWGQGTMVTVSS 138 GGSISSSSYY Anti-BCMA CT103AscFv heavy chain CDR1 DB1 / 154850959.6 65Attorney Ref. No.: 123690-5005-WO 139 ISYSGST Anti-BCMA CT103AscFv heavy chain CDR2 140 ARDRGDTILDV Anti-BCMA CT103AscFv heavy chain CDR3 141 MALPVTALLLPLALLLHAARPDIQMTQSPSSLSAExemplary BCMA SVGDRVTITCRASQSISSYLNWYQQKPGKAPKLL CAR amino acid IYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDF sequence ATYYCQQKYDLLTFGGGTKVEIKGSTSGSGKPGS GEGSTKGQLQLQESGPGLVKPSETLSLTCTVSGG SISSSSYYWGWIRQPPGKGLEWIGSISYSGSTYYN PSLKSRVTISVDTSKNQFSLKLSSVTAADTAAVY YCARDRGDTILDVWGQGTMVTVSSFVPVFLPAK PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA VHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLY CNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGC SCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAFAYSEIGMKGERRGKG HDGLYQGLSTATKDTYDALHMQALPPR

[0282] In some embodiments, the antigen binding domain of the CAR described herein binds to CD19 and comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NOs: 24, 25, and 26, respectively, and a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NOs: 19, 20, and 21, respectively; (b) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 23, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 18; and / or the amino acid sequence set forth in SEQ ID NO:17 or 27.

[0283] In some embodiments, the antigen binding domain of the CAR described herein comprises the amino acid sequence set forth in SEQ ID NO:28, 29, 30, or 31.

[0284] In some embodiments, the extracellular binding domain of the CD19 CAR comprises an scFv derived from the FMC63 monoclonal antibody (FMC63), which comprises the heavy chain variable region (VH) and the light chain variable region (VL) of FMC63 connected by a linker. FMC63 and the derived scFv have been described in Nicholson et al., Mol. Immun.34(16- 17):1157-1165 (1997) and PCT Application Publication No. WO2018 / 213337, the entire contents of each of which are incorporated by reference herein. In some embodiments, the DB1 / 154850959.6 66Attorney Ref. No.: 123690-5005-WO amino acid sequences of the entire FMC63-derived scFv (also referred to as FMC63 scFv) and its different portions are provided in Table 2.

[0285] In some embodiments, the CD19-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO:94, 95, or 100, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 94, 95, or 100.

[0286] In some embodiments, the CD19-specific scFv may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 96-98 and 101-103. In some embodiments, the CD19-specific scFv may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 96-98. In some embodiments, the CD19-specific scFv may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 101-103. In any of these embodiments, the CD19-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the CD19 CAR comprises or consists of the one or more CDRs as described herein.

[0287] In some embodiments, the linker linking the VH and the VL portions of the scFv is a Whitlow linker having an amino acid sequence set forth in SEQ ID NO:99. In some embodiments, the Whitlow linker may be replaced by a different linker, for example, a 3xG4S linker having an amino acid sequence set forth in SEQ ID NO:105, which gives rise to a different FMC63-derived scFv having an amino acid sequence set forth in SEQ ID NO:104. In certain of these embodiments, the CD19-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO:29 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:104.

[0288] In some embodiments, the extracellular binding domain of the CD19 CAR is derived from an antibody specific to CD19, including, for example, SJ25C1 (Bejcek et al., Cancer Res.55:2346-2351 (1995)), HD37 (Pezutto et al., J. Immunol.138(9):2793-2799 (1987)), 4G7 DB1 / 154850959.6 67Attorney Ref. No.: 123690-5005-WO (Meeker et al., Hybridoma 3:305-320 (1984)), B43 (Bejcek (1995)), BLY3 (Bejcek (1995)), B4 (Freedman et al., 70:418-427 (1987)), B4 HB12b (Kansas & Tedder, J. Immunol.147:4094-4102 (1991); Yazawa et al., Proc. Natl. Acad. Sci. USA 102:15178- 15183 (2005); Herbst et al., J. Pharmacol. Exp. Ther.335:213-222 (2010)), BU12 (Callard et al., J. Immunology, 148(10): 2983-2987 (1992)), and CLB-CD19 (De Rie Cell. Immunol.118:368-381(1989)). In any of these embodiments, the extracellular binding domain of the CD19 CAR can comprise or consist of the VH, the VL, and / or one or more CDRs of any of the antibodies.

[0289] Non-limiting examples of commercially available embodiments of CD19 CARs of the present disclosure include tisagenlecleucel, lisocabtagene maraleucel, axicabtagene ciloleucel, and brexucabtagene autoleucel.

[0290] In some embodiments, the CAR described herein is tisagenlecleucel or portions thereof. Tisagenlecleucel comprises a CD19 CAR with the following components: CD8α signal peptide, FMC63 scFv (VL-3xG4S linker-VH), CD8α hinge domain, CD8α transmembrane domain, 4- 1BB costimulatory domain, and CD3ζ signaling domain. The nucleotide and amino acid sequence of the CD19 CAR in tisagenlecleucel are provided in Table 2.

[0291] In some embodiments, the CAR described herein is lisocabtagene maraleucel or portions thereof. Lisocabtagene maraleucel comprises a CD19 CAR with the following components: GMCSFR-α or CSF2RA signal peptide, FMC63 scFv (VL-Whitlow linker-VH), IgG4 hinge domain, CD28 transmembrane domain, 4-1BB costimulatory domain, and CD3ζ signaling domain. The nucleotide and amino acid sequence of the CD19 CAR in lisocabtagene maraleucel are provided in Table 2.

[0292] In some embodiments, the CAR described herein is axicabtagene ciloleucel or portions thereof. Axicabtagene ciloleucel comprises a CD19 CAR with the following components: GMCSFR- α or CSF2RA signal peptide, FMC63 scFv (VL-Whitlow linker-VH), CD28 hinge domain, CD28 transmembrane domain, CD28 costimulatory domain, and CD3ζ signaling domain. The nucleotide and amino acid sequence of the CD19 CAR in axicabtagene ciloleucel are provided in Table 2.

[0293] In some embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding brexucabtagene autoleucel or portions thereof. Brexucabtagene autoleucel comprises a CD19 CAR with the following components: GMCSFR- DB1 / 154850959.6 68Attorney Ref. No.: 123690-5005-WO α signal peptide, FMC63 scFv, CD28 hinge domain, CD28 transmembrane domain, CD28 costimulatory domain, and CD3ζ signaling domain.

[0294] In some embodiments, the CAR described herein has a corresponding amino acid sequence set forth in SEQ ID NO: 107, 108, or 109, respectively, or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 107, 108, or 109, respectively.

[0295] Additional exemplary amino acid sequences of the antigen binding domain of the CAR disclosed herein can be present in PCT / US2021 / 065157, PCT / US2022 / 011877, each of which is incorporated herein by reference in their entireties.

[0296] In some embodiments, the TCR disclosed herein refers to a TCR complex formed by the association of CD3 with TCR. In some embodiments, the TCR complex comprises two heterodimeric TCR chains, TCR α, TCR β (in αβ T cells) or TCR γ, TCR δ (in γδ T cells) and six CD3 chains which for a multiprotein complex that recognizes peptide antigens presented on MHC. The TCR complex forms the ligand binding site, and CD3 complex proteins mediate signaling and subsequent T cell activation. The TCRα is encoded by TRA gene, β chain encoded by TRB, γ chain encoded by TRG and δ chain encoded by TRD.

[0297] A component of a physiologic TCR complex refers to a TCR chain (i.e., TCR α, TCR β, TCR γ or TCR δ), a CD3 chain (i.e., CD3 γ, CD3 β, CD3 ε or CD3 ζ), or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCR α and TCR β, a complex of TCR γ and TCR δ, a complex of CD3 ε and CD3 δ, a complex of CD3 γ and CD3 ε, or a sub- TCR complex of TCR α, TCR β, CD3 γ, CD3 δ, and two CD3 ε chains). In some embodiments, the complex may be formed by two or more TCR chains or CD3 chains (e.g., a complex of TCR α and TCR β, a complex of TCR γ and TCR δ, a complex of CD3ε and CD3 δ, a complex of CD3 γ and CD3 ε, or a sub-TCR complex of TCR α, TCR β, CD3 γ, CD3 δ, and two CD3 ε chains).

[0298] In some embodiments, the TCR disclosed herein is comprised of a heterodimeric TCR that are joined by disulfide bonds (α / β or γ / δ TCR) and forms a non-covalent multiprotein complex with the CD3 chains. Particularly, the TCR chains are type I proteins consisting of an extracellular region, transmembrane region and a short cytoplasmic tail. The extracellular DB1 / 154850959.6 69Attorney Ref. No.: 123690-5005-WO domain contains a hypervariable V region responsible for antigen recognition and a constant C region that is membrane proximal. The transmembrane and cytoplasmic domains form non- covalent interactions with the CD3 chains to stabilize the TCR complex and mediate downstream signaling (Kuhns et al., Immunol Rev, 250 (2012); Wucherpfennig et al., Cold Spring Harb Perspect Biol, 2: a005140 (2010)). A review of TCRs and their design is provided in Blankenstein et al., Curr Opin Immunol, 33: 112-9 (2015) (herein incorporated by reference for the subject matter and purpose referenced herein). Methods for producing engineered TCRs are described in, e.g., Bowerman et al., Mol Immunol, 46: 3000-8 (2009), which is herein incorporated by reference in its entirety. Furthermore, TCRs that bind a particular antigen may be isolated using a Vα or Vβ domain from a TCR that binds the antigen to screen a library of complementary Vα or Vβ domains, respectively. In further embodiments, a TCR is found on the surface of T cells and associates with the CD3 complex. The source of a TCR as used in the present disclosure may be from various animal species, such as a human, mouse, rat, rabbit or other mammal.

[0299] In some embodiments, the TCR complex disclosed herein further comprises an (a) extracellular domain; (b) transmembrane domain; and (c) Cytoplasmic domain. The extracellular domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any protein, but a membrane-bound or transmembrane protein. In one aspect the extracellular domain is capable of associating with the transmembrane domain. Non-limiting examples of an extracellular domain of particular use in this present disclosure may include that of α, β, γ, or δ chain of the TCR, or CD3 ε, CD3 γ, or CD3 δ, or in alternative embodiments, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

[0300] In some embodiments, the antigen binding domain of the TCR described herein can comprise one member of an interacting pair, e.g., the antigen binding domain may be one member, or a fragment thereof, of an interacting pair comprising a receptor and a ligand. Either the receptor or ligand, or fragments thereof, may be referred to as the antigen binding domain. The other member which is not referred to as the antigen binding domain can comprise the epitope to which the antigen binding domain specifically binds. The second antigen binding domain can be linked to any member of the TCR complex, and the TCR may be an α / β or γ / δ TCR. The second antigen binding domain can be linked to at least one of a TCR chain, a cluster DB1 / 154850959.6 70Attorney Ref. No.: 123690-5005-WO of differentiation 3 (CD3) chain, or CD3 z chain. The second antigen binding domain can be linked to transmembrane receptor of a TCR, e.g., TCR δ, TCR γ, TCR α, or TCR β. The second antigen binding domain can be linked to a CD3 chain, e.g., CD3 ε, CD3 δ, or CD3 γ. The second antigen binding domain can be linked to CD3 z chain.

[0301] In some embodiments, the antigen binding domain of the TCR described herein can include one or more of the following: T-cell γ chains, T-cell β chains, T-cell δ chains, T-cell constant chains, CCR7, CD3, CD4, CD5, CD7, CD8, CD11b, CD11c, CD16, CD19, CD20, CD21, CD22, CD25, CD28, CD34, CD35, CD40, CD45RA, CD45RO, CD52, CD56, CD62L, CD68, CD80, CD95, CD117, CD127, CD133, CD137 (4-1BB), CD163, F4 / 80, IL-4Rα, Sca-1, CTLA-4, GITR, GARP, LAP, granzyme B, LFA-1, transferrin receptor, and combinations thereof.

[0302] In some embodiments, the transmembrane domain of the TCR complex may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one aspect the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the TCR complex has bound to a target (i.e., peptide-MHC). Non-limiting examples of a transmembrane domain of particular use in this present disclosure may include α, β, γ or δ chain of the TCR, CD28, CD3 ε, CD3 γ, CD3 δ CD3 z, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. The transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived. In one aspect, the transmembrane domain is one that is associated with one of the other domains of the TCR used. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex.

[0303] In some embodiments, an extracellular domain and a transmembrane domain may be encoded by a single genomic sequence. In alternative embodiments, the sequence can be designed to comprise a transmembrane domain that is heterologous to the extracellular domain. Optionally, a short oligo- or polypeptide linker, between 2 and 10 amino acids in length may DB1 / 154850959.6 71Attorney Ref. No.: 123690-5005-WO form the linkage between the transmembrane domain and the cytoplasmic region of the TCR polypeptide. For example, a glycine-serine doublet is used to provide a particularly suitable linker.

[0304] In some embodiments, the cytoplasmic domain of the TCR comprises an intracellular domain. In some embodiments, the intracellular domain is from CD3 γ, CD3 δ, CD3 ε, TCR α, TCR β, TCR γ or TCR S. In some embodiments, the intracellular domain comprises a signaling domain, if the TCR complex contains CD3 γ, δ, ε polypeptides; TCR α, TCR β, TCR γ, and TCR δ subunits generally have short (e.g., 1-19 amino acids in length) intracellular domains and are generally lacking in a signaling domain. An intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the TCRs has been introduced. While the intracellular domains of TCR α, TCR β, TCR γ, and TCR δ do not have signaling domains, they are able to recruit proteins having a primary intracellular signaling domain described herein, e.g., CD3 z, which functions as an intracellular signaling domain.

[0305] In some embodiments, the TCR subunit comprises (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit. In some embodiments, the TCR extracellular domain comprises an extracellular domain or portion thereof of a TCR α chain, a TCR β chain, a TCR γ chain, a TCR δ chain, a CD3 ε TCR subunit, a CD3 γ TCR subunit, a CD3 δ TCR subunit and functional fragments thereof. In some embodiments, the TCR subunit comprises a transmembrane domain comprising a transmembrane domain of a TCR α chain, a TCR β chain, a TCR γ chain, a TCR δ chain, a CD3 z TCR subunit, a CD3 ε TCR subunit, a CD3 γ TCR subunit, a CD3 δ TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and functional fragments thereof. In some embodiments, the TCR subunit comprises a TCR intracellular domain of CD3 ε, CD3 γ, CD3 δ, TCR α, TCR β, TCR γ, or TCR δ. In some embodiments, the intracellular domain comprises a stimulatory domain of a protein comprising an intracellular signaling domain of CD3 ε, CD3 γ, CD3 δ and functional fragments thereof.

[0306] In some embodiments, the TCR described herein includes but is not limited to Tecelra (afamitresgene autoleucel) and Kimmtrak (tebentafusp-tebn). DB1 / 154850959.6 72Attorney Ref. No.: 123690-5005-WO

[0307] In some embodiments, the CAR and / or TCR of the present disclosure comprises antigen binding domain that has affinity for one or more cell-surface antigens, for example tumor- associated antigens (TAAs). In some embodiments, the tumor-associated antigens are selected from the group consisting of: TNFRSF17, IL3RA, SDC1, CD19, CD20, CD22, BCMA, MS4A1, CD7, CD123, CD135, CD38, CD138, CD269, TNFRSF8, CD33, CD38, CD5, NCAM1, CD70, ULBP1, ULBP2, IL1RAP, CEACAM5, MET, EGFR, EGFRvIII, EPCAM, EPHA2, ERBB2, GPC3, MSLN, Muc1, PDCD1, CD274, KDR, IL13RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, ROR2, GPRC5D, PSMA, CD23, WT1, CD44, CD44v6, CD174, SLAMF7, L1 CAM, FLT3, Sigle-6, GD2, PSCA, NY-ESO-1, GPNMB, CD276, CSPG4, MAGEA3, MAGEA4, CD133, TEM1, and combinations thereof.

[0308] In some further embodiments, the tumor-associated antigens targeted by the CAR and / or TCR described herein are selected from the group consisting of: CD19; CD123; CD22; CD30; CD171; CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECLI); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (αNeuSAc(2-8)αNeuSAc(2-3)βDGaip(1- 4)bDGIcp(1-1)Cer); ganglioside GM3 (αNeuSAc(2-3)βDGalp(1-4)βDGlcp(1-1)Cer); GM-CSF receptor; TNF receptor superfamily member 17 (TNFRSF17, BCMA); B-lymphocyte cell adhesion molecule; Tn antigen ((Tn Ag) or (GaINAcu-Ser / Thr)); prostate-specific membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (RORI); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); Protease Serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); HLA class I antigen A-2 alpha; HLA antigen; Lewis(Y)antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; delta like 3 (DLL3); Folate receptor alpha; Folate receptor beta, GDNF alpha 4 receptor, Receptor tyrosine-protein kinase, ERBB2 (Her2 / neu); Mucin 1, cell surface associated (MUC1); APRIL receptor; ADP ribosyl cyclase-1; Ephb4 tyrosine kinase receptor, DCAMKL1 serine threonine kinase, Aspartate beta-hydroxylase, epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2; fibroblast activation DB1 / 154850959.6 73Attorney Ref. No.: 123690-5005-WO protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2 (EphA2); ephrin type-A receptor 3 (EphA3), Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); transglutaminase 5 (TGS5); high molecular weight-melanoma associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); six transmembrane epithelial antigen of the prostate I (STEAP1); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRCSD); IL-15 receptor (IL-15); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2 (ORS IE2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer / testis antigen 1 (NY-ESO- 1); Cancer / testis antigen 2 (LAGE-la); Melanoma associated antigen 1 (MAGE-A1); Melanoma associated antigen 3 (MAGE-A3); Melanoma associated antigen 4 (MAGE-A4); T cell receptor beta 2 chain C; ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MADCT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53, (p53); p53 mutant; prostein; survivin; telomerase; prostate carcinoma tumor antigen-1 (PCTA-1 or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MARTI); Rat sarcoma (Ras) mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin-A1; Cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P4501B1(CYP IBI); CCCTC-Binding Factor DB1 / 154850959.6 74Attorney Ref. No.: 123690-5005-WO (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites), Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY-TES I); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); Peptidoglycan recognition protein, synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced Glycation Endproducts (RAGE-I); renal ubiquitous 1 (RUI); renal ubiquitous 2 (RU2); legumain; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIRI); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin- like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-2 (GPC2); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1) , and combinations thereof. In some embodiments, the target is an epitope of the tumor associated antigen presented in an MHC.

[0309] In yet some further embodiments, the tumor-associated antigen is selected from CD150, 5T4, ActRIIA, B7, TNF receptor superfamily member 17 (TNFRSF17, BCMA), CA-125, CCNA1, CD123, CD126, CD138, CD14, CD148, CD15, CD19, CD20, CD200, CD21, CD22, CD23, CD24, CD25, CD26, CD261, CD262, CD30, CD33, CD362, CD37, CD38, CD4, CD40, CD40L, CD44, CD46, CD5, CD52, CD53, CD54, CD56, CD66a-d, CD74, CD8, CD80, CD92, CE7, CS-1, CSPG4, ED-B fibronectin, EGFR, EGFRvIII, EGP-2, EGP-4, EPHa2, ErbB2, ErbB3, ErbB4, FBP, HER1-HER2 in combination, HER2-HER3 in combination, HERV-K, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, HLA-DR, HLA class I antigen alpha G, HM1.24, K-Ras GTPase, HMW-MAA, Her2, Her2 / neu, IGF-1R, IL-11 Ralpha, IL-13R-alpha2, IL-2, IL-22R-alpha, IL-6, IL-6R, Ia, Ii, L1-CAM, L1-cell adhesion molecule, Lewis Y, Ll-CAM, MAGE A3, MAGE-A1, MART-1, MUC1, NKG2C ligands, NKG2D Ligands, NYESO-1, OEPHa2, PIGF, PSCA, PSMA, ROR1, T101, TAC, TAG72, TIM-3, TRAIL-R1, TRAIL-R1 (DR4), TRAIL-R2 (DR5), VEGF, VEGFR2, WT-I, a G-protein coupled receptor, alphafetoprotein (AFP), an angiogenesis factor, an exogenous cognate binding molecule (ExoCBM), oncogene product, anti-folate receptor, c-Met, carcinoembryonic antigen DB1 / 154850959.6 75Attorney Ref. No.: 123690-5005-WO (CEA), cyclin (D 1), ephrinB2, epithelial tumor antigen, estrogen receptor, fetal acetylcholine e receptor, folate binding protein, gp100, hepatitis B surface antigen, Epstein-Barr nuclear antigen 1, Latent membrane protein 1, Secreted protein BARF1, P2X7 purinoceptor, Syndecan-1, kappa chain, kappa light chain, kdr, lambda chain, livin, melanoma-associated antigen, mesothelin, mouse double minute 2 homolog (MDM2), mucin 16 (MUC16), mutated p53, mutated ras, necrosis antigens, oncofetal antigen, ROR2, progesterone receptor, prostate specific antigen, tEGFR, tenascin, P2-Microgiobuiin, Fc Receptor-like 5 (FcRL5), and combinations thereof.

[0310] In some embodiments, the CAR or TCR has affinity for TNFRSF17. In some embodiments, the CAR or TCR has affinity for IL3RA. In some embodiments, the CAR or TCR has affinity for SDC1. In some embodiments, the CAR or TCR has affinity for CD19. In some embodiments, the CAR or TCR has affinity for CD20. In some embodiments, the CAR or TCR has affinity for CD22. In some embodiments, the CAR or TCR has affinity for BCMA. In some embodiments, the CAR or TCR has affinity for MS4A1. In some embodiments, the CAR or TCR has affinity for CD7. In some embodiments, the CAR or TCR has affinity for CD123. In some embodiments, the CAR or TCR has affinity for CD135. In some embodiments, the CAR or TCR has affinity for CD38. In some embodiments, the CAR or TCR has affinity for CD138. In some embodiments, the CAR or TCR has affinity for CD269. In some embodiments, the CAR or TCR has affinity for TNFRSF8. In some embodiments, the CAR or TCR has affinity for CD33. In some embodiments, the CAR or TCR has affinity for CD5. In some embodiments, the CAR or TCR has affinity for NCAM1. In some embodiments, the CAR or TCR has affinity for CD70. In some embodiments, the CAR or TCR has affinity for ULBP1. In some embodiments, the CAR or TCR has affinity for ULBP2. In some embodiments, the CAR or TCR has affinity for IL1RAP. In some embodiments, the CAR or TCR has affinity for CEACAM5. In some embodiments, the CAR or TCR has affinity for MET. In some embodiments, the CAR or TCR has affinity for EGFR. In some embodiments, the CAR or TCR has affinity for EGFRvIII. In some embodiments, the CAR or TCR has affinity for EPCAM. In some embodiments, the CAR or TCR has affinity for EPHA2. In some embodiments, the CAR or TCR has affinity for ERBB2. In some embodiments, the CAR or TCR has affinity for GPC3. In some embodiments, the CAR or TCR has affinity for MSLN. In some embodiments, the CAR or TCR has affinity for Muc1. In some embodiments, the CAR or TCR has affinity for PDCD1. In some embodiments, the CAR or TCR has affinity for CD274. In some embodiments, the CAR or TCR has affinity for KDR. In DB1 / 154850959.6 76Attorney Ref. No.: 123690-5005-WO some embodiments, the CAR or TCR has affinity for IL13RA2. In some embodiments, the CAR or TCR has affinity for FOLH1. In some embodiments, the CAR or TCR has affinity for FAP. In some embodiments, the CAR or TCR has affinity for CA9. In some embodiments, the CAR or TCR has affinity for FOLR1. In some embodiments, the CAR or TCR has affinity for L1CAM. In some embodiments, the CAR or TCR has affinity for ROR1. In some embodiments, the CAR or TCR has affinity for ROR2. In some embodiments, the CAR or TCR has affinity for GPRC5D. In some embodiments, the CAR or TCR has affinity for PSMA. In some embodiments, the CAR or TCR has affinity for CD23. In some embodiments, the CAR or TCR has affinity for WT1. In some embodiments, the CAR or TCR has affinity for CD44. In some embodiments, the CAR or TCR has affinity for CD44v6. In some embodiments, the CAR or TCR has affinity for CD174. In some embodiments, the CAR or TCR has affinity for SLAMF7. In some embodiments, the CAR or TCR has affinity for FLT3. In some embodiments, the CAR or TCR has affinity for Sigle-6. In some embodiments, the CAR or TCR has affinity for GD2. In some embodiments, the CAR or TCR has affinity for PSCA. In some embodiments, the CAR or TCR has affinity for NY-ESO-1. In some embodiments, the CAR or TCR has affinity for GPNMB. In some embodiments, the CAR or TCR has affinity for CD276. In some embodiments, the CAR or TCR has affinity for CSPG4. In some embodiments, the CAR or TCR has affinity for MAGEA3. In some embodiments, the CAR or TCR has affinity for MAGEA4. In some embodiments, the CAR or TCR has affinity for CD133. In some embodiments, the CAR or TCR has affinity for TEM1.

[0311] In some embodiments, the CAR or TCR is bispecific and has affinity for a combination selected from the group consisting of: TNFRSF17 and IL3RA, TNFRSF17 and SDC1, TNFRSF17 and CD19, TNFRSF17 and CD20, TNFRSF17 and CD22, TNFRSF17 and BCMA, TNFRSF17 and MS4A1, TNFRSF17 and CD7, TNFRSF17 and CD123, TNFRSF17 and CD135, TNFRSF17 and CD38, TNFRSF17 and CD138, TNFRSF17 and CD269, TNFRSF17 and TNFRSF8, TNFRSF17 and CD33, TNFRSF17 and CD5, TNFRSF17 and NCAM1, TNFRSF17 and CD70, TNFRSF17 and ULBP1, TNFRSF17 and ULBP2, TNFRSF17 and IL1RAP, TNFRSF17 and CEACAM5, TNFRSF17 and MET, TNFRSF17 and EGFR, TNFRSF17 and EGFRvIII, TNFRSF17 and EPCAM, TNFRSF17 and EPHA2, TNFRSF17 and ERBB2, TNFRSF17 and GPC3, TNFRSF17 and MSLN, TNFRSF17 and Muc1, TNFRSF17 and PDCD1, TNFRSF17 and CD274, TNFRSF17 and KDR, TNFRSF17 and IL13RA2, TNFRSF17 DB1 / 154850959.6 77Attorney Ref. No.: 123690-5005-WO and FOLH1, TNFRSF17 and FAP, TNFRSF17 and CA9, TNFRSF17 and FOLR1, TNFRSF17 and L1CAM, TNFRSF17 and ROR1, TNFRSF17 and ROR2, TNFRSF17 and GPRC5D, TNFRSF17 and PSMA, TNFRSF17 and CD23, TNFRSF17 and WT1, TNFRSF17 and CD44, TNFRSF17 and CD44v6, TNFRSF17 and CD174, TNFRSF17 and SLAMF7, TNFRSF17 and L1 CAM, TNFRSF17 and FLT3, TNFRSF17 and Sigle-6, TNFRSF17 and GD2, TNFRSF17 and PSCA, TNFRSF17 and NY-ESO-1, TNFRSF17 and GPNMB, TNFRSF17 and CD276, TNFRSF17 and CSPG4, TNFRSF17 and MAGEA3, TNFRSF17 and MAGEA4, TNFRSF17 and CD133, TNFRSF17 and TEM1, IL3RA and SDC1, IL3RA and CD19, IL3RA and CD20, IL3RA and CD22, IL3RA and BCMA, IL3RA and MS4A1, IL3RA and CD7, IL3RA and CD123, IL3RA and CD135, IL3RA and CD38, IL3RA and CD138, IL3RA and CD269, IL3RA and TNFRSF8, IL3RA and CD33, IL3RA and CD5, IL3RA and NCAM1, IL3RA and CD70, IL3RA and ULBP1, IL3RA and ULBP2, IL3RA and IL1RAP, IL3RA and CEACAM5, IL3RA and MET, IL3RA and EGFR, IL3RA and EGFRvIII, IL3RA and EPCAM, IL3RA and EPHA2, IL3RA and ERBB2, IL3RA and GPC3, IL3RA and MSLN, IL3RA and Muc1, IL3RA and PDCD1, IL3RA and CD274, IL3RA and KDR, IL3RA and IL13RA2, IL3RA and FOLH1, IL3RA and FAP, IL3RA and CA9, IL3RA and FOLR1, IL3RA and L1CAM, IL3RA and ROR1, IL3RA and ROR2, IL3RA and GPRC5D, IL3RA and PSMA, IL3RA and CD23, IL3RA and WT1, IL3RA and CD44, IL3RA and CD44v6, IL3RA and CD174, IL3RA and SLAMF7, IL3RA and L1 CAM, IL3RA and FLT3, IL3RA and Sigle-6, IL3RA and GD2, IL3RA and PSCA, IL3RA and NY-ESO-1, IL3RA and GPNMB, IL3RA and CD276, IL3RA and CSPG4, IL3RA and MAGEA3, IL3RA and MAGEA4, IL3RA and CD133, IL3RA and TEM1, SDC1 and CD19, SDC1 and CD20, SDC1 and CD22, SDC1 and BCMA, SDC1 and MS4A1, SDC1 and CD7, SDC1 and CD123, SDC1 and CD135, SDC1 and CD38, SDC1 and CD138, SDC1 and CD269, SDC1 and TNFRSF8, SDC1 and CD33, SDC1 and CD5, SDC1 and NCAM1, SDC1 and CD70, SDC1 and ULBP1, SDC1 and ULBP2, SDC1 and IL1RAP, SDC1 and CEACAM5, SDC1 and MET, SDC1 and EGFR, SDC1 and EGFRvIII, SDC1 and EPCAM, SDC1 and EPHA2, SDC1 and ERBB2, SDC1 and GPC3, SDC1 and MSLN, SDC1 and Muc1, SDC1 and PDCD1, SDC1 and CD274, SDC1 and KDR, SDC1 and IL13RA2, SDC1 and FOLH1, SDC1 and FAP, SDC1 and CA9, SDC1 and FOLR1, SDC1 and L1CAM, SDC1 and ROR1, SDC1 and ROR2, SDC1 and GPRC5D, SDC1 and PSMA, SDC1 and CD23, SDC1 and WT1, SDC1 and CD44, SDC1 and CD44v6, SDC1 and CD174, SDC1 and SLAMF7, SDC1 and L1 CAM, SDC1 and FLT3, DB1 / 154850959.6 78Attorney Ref. No.: 123690-5005-WO SDC1 and Sigle-6, SDC1 and GD2, SDC1 and PSCA, SDC1 and NY-ESO-1, SDC1 and GPNMB, SDC1 and CD276, SDC1 and CSPG4, SDC1 and MAGEA3, SDC1 and MAGEA4, SDC1 and CD133, SDC1 and TEM1, CD19 and CD20, CD19 and CD22, CD19 and BCMA, CD19 and MS4A1, CD19 and CD7, CD19 and CD123, CD19 and CD135, CD19 and CD38, CD19 and CD138, CD19 and CD269, CD19 and TNFRSF8, CD19 and CD33, CD19 and CD5, CD19 and NCAM1, CD19 and CD70, CD19 and ULBP1, CD19 and ULBP2, CD19 and IL1RAP, CD19 and CEACAM5, CD19 and MET, CD19 and EGFR, CD19 and EGFRvIII, CD19 and EPCAM, CD19 and EPHA2, CD19 and ERBB2, CD19 and GPC3, CD19 and MSLN, CD19 and Muc1, CD19 and PDCD1, CD19 and CD274, CD19 and KDR, CD19 and IL13RA2, CD19 and FOLH1, CD19 and FAP, CD19 and CA9, CD19 and FOLR1, CD19 and L1CAM, CD19 and ROR1, CD19 and ROR2, CD19 and GPRC5D, CD19 and PSMA, CD19 and CD23, CD19 and WT1, CD19 and CD44, CD19 and CD44v6, CD19 and CD174, CD19 and SLAMF7, CD19 and L1 CAM, CD19 and FLT3, CD19 and Sigle-6, CD19 and GD2, CD19 and PSCA, CD19 and NY-ESO-1, CD19 and GPNMB, CD19 and CD276, CD19 and CSPG4, CD19 and MAGEA3, CD19 and MAGEA4, CD19 and CD133, CD19 and TEM1, CD20 and CD22, CD20 and BCMA, CD20 and MS4A1, CD20 and CD7, CD20 and CD123, CD20 and CD135, CD20 and CD38, CD20 and CD138, CD20 and CD269, CD20 and TNFRSF8, CD20 and CD33, CD20 and CD5, CD20 and NCAM1, CD20 and CD70, CD20 and ULBP1, CD20 and ULBP2, CD20 and IL1RAP, CD20 and CEACAM5, CD20 and MET, CD20 and EGFR, CD20 and EGFRvIII, CD20 and EPCAM, CD20 and EPHA2, CD20 and ERBB2, CD20 and GPC3, CD20 and MSLN, CD20 and Muc1, CD20 and PDCD1, CD20 and CD274, CD20 and KDR, CD20 and IL13RA2, CD20 and FOLH1, CD20 and FAP, CD20 and CA9, CD20 and FOLR1, CD20 and L1CAM, CD20 and ROR1, CD20 and ROR2, CD20 and GPRC5D, CD20 and PSMA, CD20 and CD23, CD20 and WT1, CD20 and CD44, CD20 and CD44v6, CD20 and CD174, CD20 and SLAMF7, CD20 and L1 CAM, CD20 and FLT3, CD20 and Sigle-6, CD20 and GD2, CD20 and PSCA, CD20 and NY-ESO-1, CD20 and GPNMB, CD20 and CD276, CD20 and CSPG4, CD20 and MAGEA3, CD20 and MAGEA4, CD20 and CD133, CD20 and TEM1, CD22 and BCMA, CD22 and MS4A1, CD22 and CD7, CD22 and CD123, CD22 and CD135, CD22 and CD38, CD22 and CD138, CD22 and CD269, CD22 and TNFRSF8, CD22 and CD33, CD22 and CD5, CD22 and NCAM1, CD22 and CD70, CD22 and ULBP1, CD22 and ULBP2, CD22 and IL1RAP, CD22 and CEACAM5, CD22 and MET, CD22 and EGFR, CD22 and EGFRvIII, CD22 and EPCAM, DB1 / 154850959.6 79Attorney Ref. No.: 123690-5005-WO CD22 and EPHA2, CD22 and ERBB2, CD22 and GPC3, CD22 and MSLN, CD22 and Muc1, CD22 and PDCD1, CD22 and CD274, CD22 and KDR, CD22 and IL13RA2, CD22 and FOLH1, CD22 and FAP, CD22 and CA9, CD22 and FOLR1, CD22 and L1CAM, CD22 and ROR1, CD22 and ROR2, CD22 and GPRC5D, CD22 and PSMA, CD22 and CD23, CD22 and WT1, CD22 and CD44, CD22 and CD44v6, CD22 and CD174, CD22 and SLAMF7, CD22 and L1 CAM, CD22 and FLT3, CD22 and Sigle-6, CD22 and GD2, CD22 and PSCA, CD22 and NY-ESO-1, CD22 and GPNMB, CD22 and CD276, CD22 and CSPG4, CD22 and MAGEA3, CD22 and MAGEA4, CD22 and CD133, CD22 and TEM1, BCMA and MS4A1, BCMA and CD7, BCMA and CD123, BCMA and CD135, BCMA and CD38, BCMA and CD138, BCMA and CD269, BCMA and TNFRSF8, BCMA and CD33, BCMA and CD5, BCMA and NCAM1, BCMA and CD70, BCMA and ULBP1, BCMA and ULBP2, BCMA and IL1RAP, BCMA and CEACAM5, BCMA and MET, BCMA and EGFR, BCMA and EGFRvIII, BCMA and EPCAM, BCMA and EPHA2, BCMA and ERBB2, BCMA and GPC3, BCMA and MSLN, BCMA and Muc1, BCMA and PDCD1, BCMA and CD274, BCMA and KDR, BCMA and IL13RA2, BCMA and FOLH1, BCMA and FAP, BCMA and CA9, BCMA and FOLR1, BCMA and L1CAM, BCMA and ROR1, BCMA and ROR2, BCMA and GPRC5D, BCMA and PSMA, BCMA and CD23, BCMA and WT1, BCMA and CD44, BCMA and CD44v6, BCMA and CD174, BCMA and SLAMF7, BCMA and L1 CAM, BCMA and FLT3, BCMA and Sigle-6, BCMA and GD2, BCMA and PSCA, BCMA and NY-ESO-1, BCMA and GPNMB, BCMA and CD276, BCMA and CSPG4, BCMA and MAGEA3, BCMA and MAGEA4, BCMA and CD133, BCMA and TEM1, MS4A1 and CD7, MS4A1 and CD123, MS4A1 and CD135, MS4A1 and CD38, MS4A1 and CD138, MS4A1 and CD269, MS4A1 and TNFRSF8, MS4A1 and CD33, MS4A1 and CD5, MS4A1 and NCAM1, MS4A1 and CD70, MS4A1 and ULBP1, MS4A1 and ULBP2, MS4A1 and IL1RAP, MS4A1 and CEACAM5, MS4A1 and MET, MS4A1 and EGFR, MS4A1 and EGFRvIII, MS4A1 and EPCAM, MS4A1 and EPHA2, MS4A1 and ERBB2, MS4A1 and GPC3, MS4A1 and MSLN, MS4A1 and Muc1, MS4A1 and PDCD1, MS4A1 and CD274, MS4A1 and KDR, MS4A1 and IL13RA2, MS4A1 and FOLH1, MS4A1 and FAP, MS4A1 and CA9, MS4A1 and FOLR1, MS4A1 and L1CAM, MS4A1 and ROR1, MS4A1 and ROR2, MS4A1 and GPRC5D, MS4A1 and PSMA, MS4A1 and CD23, MS4A1 and WT1, MS4A1 and CD44, MS4A1 and CD44v6, MS4A1 and CD174, MS4A1 and SLAMF7, MS4A1 and L1 CAM, MS4A1 and FLT3, MS4A1 and Sigle-6, MS4A1 and GD2, MS4A1 and PSCA, DB1 / 154850959.6 80Attorney Ref. No.: 123690-5005-WO MS4A1 and NY-ESO-1, MS4A1 and GPNMB, MS4A1 and CD276, MS4A1 and CSPG4, MS4A1 and MAGEA3, MS4A1 and MAGEA4, MS4A1 and CD133, MS4A1 and TEM1, CD7 and CD123, CD7 and CD135, CD7 and CD38, CD7 and CD138, CD7 and CD269, CD7 and TNFRSF8, CD7 and CD33, CD7 and CD5, CD7 and NCAM1, CD7 and CD70, CD7 and ULBP1, CD7 and ULBP2, CD7 and IL1RAP, CD7 and CEACAM5, CD7 and MET, CD7 and EGFR, CD7 and EGFRvIII, CD7 and EPCAM, CD7 and EPHA2, CD7 and ERBB2, CD7 and GPC3, CD7 and MSLN, CD7 and Muc1, CD7 and PDCD1, CD7 and CD274, CD7 and KDR, CD7 and IL13RA2, CD7 and FOLH1, CD7 and FAP, CD7 and CA9, CD7 and FOLR1, CD7 and L1CAM, CD7 and ROR1, CD7 and ROR2, CD7 and GPRC5D, CD7 and PSMA, CD7 and CD23, CD7 and WT1, CD7 and CD44, CD7 and CD44v6, CD7 and CD174, CD7 and SLAMF7, CD7 and L1 CAM, CD7 and FLT3, CD7 and Sigle-6, CD7 and GD2, CD7 and PSCA, CD7 and NY-ESO-1, CD7 and GPNMB, CD7 and CD276, CD7 and CSPG4, CD7 and MAGEA3, CD7 and MAGEA4, CD7 and CD133, CD7 and TEM1, CD123 and CD135, CD123 and CD38, CD123 and CD138, CD123 and CD269, CD123 and TNFRSF8, CD123 and CD33, CD123 and CD5, CD123 and NCAM1, CD123 and CD70, CD123 and ULBP1, CD123 and ULBP2, CD123 and IL1RAP, CD123 and CEACAM5, CD123 and MET, CD123 and EGFR, CD123 and EGFRvIII, CD123 and EPCAM, CD123 and EPHA2, CD123 and ERBB2, CD123 and GPC3, CD123 and MSLN, CD123 and Muc1, CD123 and PDCD1, CD123 and CD274, CD123 and KDR, CD123 and IL13RA2, CD123 and FOLH1, CD123 and FAP, CD123 and CA9, CD123 and FOLR1, CD123 and L1CAM, CD123 and ROR1, CD123 and ROR2, CD123 and GPRC5D, CD123 and PSMA, CD123 and CD23, CD123 and WT1, CD123 and CD44, CD123 and CD44v6, CD123 and CD174, CD123 and SLAMF7, CD123 and L1 CAM, CD123 and FLT3, CD123 and Sigle-6, CD123 and GD2, CD123 and PSCA, CD123 and NY-ESO-1, CD123 and GPNMB, CD123 and CD276, CD123 and CSPG4, CD123 and MAGEA3, CD123 and MAGEA4, CD123 and CD133, CD123 and TEM1, CD135 and CD38, CD135 and CD138, CD135 and CD269, CD135 and TNFRSF8, CD135 and CD33, CD135 and CD5, CD135 and NCAM1, CD135 and CD70, CD135 and ULBP1, CD135 and ULBP2, CD135 and IL1RAP, CD135 and CEACAM5, CD135 and MET, CD135 and EGFR, CD135 and EGFRvIII, CD135 and EPCAM, CD135 and EPHA2, CD135 and ERBB2, CD135 and GPC3, CD135 and MSLN, CD135 and Muc1, CD135 and PDCD1, CD135 and CD274, CD135 and KDR, CD135 and IL13RA2, CD135 and FOLH1, CD135 and FAP, CD135 and CA9, CD135 and FOLR1, CD135 DB1 / 154850959.6 81Attorney Ref. No.: 123690-5005-WO and L1CAM, CD135 and ROR1, CD135 and ROR2, CD135 and GPRC5D, CD135 and PSMA, CD135 and CD23, CD135 and WT1, CD135 and CD44, CD135 and CD44v6, CD135 and CD174, CD135 and SLAMF7, CD135 and L1 CAM, CD135 and FLT3, CD135 and Sigle-6, CD135 and GD2, CD135 and PSCA, CD135 and NY-ESO-1, CD135 and GPNMB, CD135 and CD276, CD135 and CSPG4, CD135 and MAGEA3, CD135 and MAGEA4, CD135 and CD133, CD135 and TEM1, CD38 and CD138, CD38 and CD269, CD38 and TNFRSF8, CD38 and CD33, CD38 and CD5, CD38 and NCAM1, CD38 and CD70, CD38 and ULBP1, CD38 and ULBP2, CD38 and IL1RAP, CD38 and CEACAM5, CD38 and MET, CD38 and EGFR, CD38 and EGFRvIII, CD38 and EPCAM, CD38 and EPHA2, CD38 and ERBB2, CD38 and GPC3, CD38 and MSLN, CD38 and Muc1, CD38 and PDCD1, CD38 and CD274, CD38 and KDR, CD38 and IL13RA2, CD38 and FOLH1, CD38 and FAP, CD38 and CA9, CD38 and FOLR1, CD38 and L1CAM, CD38 and ROR1, CD38 and ROR2, CD38 and GPRC5D, CD38 and PSMA, CD38 and CD23, CD38 and WT1, CD38 and CD44, CD38 and CD44v6, CD38 and CD174, CD38 and SLAMF7, CD38 and L1 CAM, CD38 and FLT3, CD38 and Sigle-6, CD38 and GD2, CD38 and PSCA, CD38 and NY-ESO-1, CD38 and GPNMB, CD38 and CD276, CD38 and CSPG4, CD38 and MAGEA3, CD38 and MAGEA4, CD38 and CD133, CD38 and TEM1, CD138 and CD269, CD138 and TNFRSF8, CD138 and CD33, CD138 and CD5, CD138 and NCAM1, CD138 and CD70, CD138 and ULBP1, CD138 and ULBP2, CD138 and IL1RAP, CD138 and CEACAM5, CD138 and MET, CD138 and EGFR, CD138 and EGFRvIII, CD138 and EPCAM, CD138 and EPHA2, CD138 and ERBB2, CD138 and GPC3, CD138 and MSLN, CD138 and Muc1, CD138 and PDCD1, CD138 and CD274, CD138 and KDR, CD138 and IL13RA2, CD138 and FOLH1, CD138 and FAP, CD138 and CA9, CD138 and FOLR1, CD138 and L1CAM, CD138 and ROR1, CD138 and ROR2, CD138 and GPRC5D, CD138 and PSMA, CD138 and CD23, CD138 and WT1, CD138 and CD44, CD138 and CD44v6, CD138 and CD174, CD138 and SLAMF7, CD138 and L1 CAM, CD138 and FLT3, CD138 and Sigle-6, CD138 and GD2, CD138 and PSCA, CD138 and NY-ESO-1, CD138 and GPNMB, CD138 and CD276, CD138 and CSPG4, CD138 and MAGEA3, CD138 and MAGEA4, CD138 and CD133, CD138 and TEM1, CD269 and TNFRSF8, CD269 and CD33, CD269 and CD5, CD269 and NCAM1, CD269 and CD70, CD269 and ULBP1, CD269 and ULBP2, CD269 and IL1RAP, CD269 and CEACAM5, CD269 and MET, CD269 and EGFR, CD269 and EGFRvIII, CD269 and EPCAM, CD269 and EPHA2, CD269 and ERBB2, CD269 and GPC3, CD269 and MSLN, DB1 / 154850959.6 82Attorney Ref. No.: 123690-5005-WO CD269 and Muc1, CD269 and PDCD1, CD269 and CD274, CD269 and KDR, CD269 and IL13RA2, CD269 and FOLH1, CD269 and FAP, CD269 and CA9, CD269 and FOLR1, CD269 and L1CAM, CD269 and ROR1, CD269 and ROR2, CD269 and GPRC5D, CD269 and PSMA, CD269 and CD23, CD269 and WT1, CD269 and CD44, CD269 and CD44v6, CD269 and CD174, CD269 and SLAMF7, CD269 and L1 CAM, CD269 and FLT3, CD269 and Sigle-6, CD269 and GD2, CD269 and PSCA, CD269 and NY-ESO-1, CD269 and GPNMB, CD269 and CD276, CD269 and CSPG4, CD269 and MAGEA3, CD269 and MAGEA4, CD269 and CD133, CD269 and TEM1, TNFRSF8 and CD33, TNFRSF8 and CD5, TNFRSF8 and NCAM1, TNFRSF8 and CD70, TNFRSF8 and ULBP1, TNFRSF8 and ULBP2, TNFRSF8 and IL1RAP, TNFRSF8 and CEACAM5, TNFRSF8 and MET, TNFRSF8 and EGFR, TNFRSF8 and EGFRvIII, TNFRSF8 and EPCAM, TNFRSF8 and EPHA2, TNFRSF8 and ERBB2, TNFRSF8 and GPC3, TNFRSF8 and MSLN, TNFRSF8 and Muc1, TNFRSF8 and PDCD1, TNFRSF8 and CD274, TNFRSF8 and KDR, TNFRSF8 and IL13RA2, TNFRSF8 and FOLH1, TNFRSF8 and FAP, TNFRSF8 and CA9, TNFRSF8 and FOLR1, TNFRSF8 and L1CAM, TNFRSF8 and ROR1, TNFRSF8 and ROR2, TNFRSF8 and GPRC5D, TNFRSF8 and PSMA, TNFRSF8 and CD23, TNFRSF8 and WT1, TNFRSF8 and CD44, TNFRSF8 and CD44v6, TNFRSF8 and CD174, TNFRSF8 and SLAMF7, TNFRSF8 and L1 CAM, TNFRSF8 and FLT3, TNFRSF8 and Sigle-6, TNFRSF8 and GD2, TNFRSF8 and PSCA, TNFRSF8 and NY-ESO-1, TNFRSF8 and GPNMB, TNFRSF8 and CD276, TNFRSF8 and CSPG4, TNFRSF8 and MAGEA3, TNFRSF8 and MAGEA4, TNFRSF8 and CD133, TNFRSF8 and TEM1, CD33 and CD5, CD33 and NCAM1, CD33 and CD70, CD33 and ULBP1, CD33 and ULBP2, CD33 and IL1RAP, CD33 and CEACAM5, CD33 and MET, CD33 and EGFR, CD33 and EGFRvIII, CD33 and EPCAM, CD33 and EPHA2, CD33 and ERBB2, CD33 and GPC3, CD33 and MSLN, CD33 and Muc1, CD33 and PDCD1, CD33 and CD274, CD33 and KDR, CD33 and IL13RA2, CD33 and FOLH1, CD33 and FAP, CD33 and CA9, CD33 and FOLR1, CD33 and L1CAM, CD33 and ROR1, CD33 and ROR2, CD33 and GPRC5D, CD33 and PSMA, CD33 and CD23, CD33 and WT1, CD33 and CD44, CD33 and CD44v6, CD33 and CD174, CD33 and SLAMF7, CD33 and L1 CAM, CD33 and FLT3, CD33 and Sigle-6, CD33 and GD2, CD33 and PSCA, CD33 and NY-ESO-1, CD33 and GPNMB, CD33 and CD276, CD33 and CSPG4, CD33 and MAGEA3, CD33 and MAGEA4, CD33 and CD133, CD33 and TEM1, CD5 and NCAM1, CD5 and CD70, CD5 and ULBP1, CD5 and ULBP2, CD5 and IL1RAP, CD5 and CEACAM5, CD5 and MET, DB1 / 154850959.6 83Attorney Ref. No.: 123690-5005-WO CD5 and EGFR, CD5 and EGFRvIII, CD5 and EPCAM, CD5 and EPHA2, CD5 and ERBB2, CD5 and GPC3, CD5 and MSLN, CD5 and Muc1, CD5 and PDCD1, CD5 and CD274, CD5 and KDR, CD5 and IL13RA2, CD5 and FOLH1, CD5 and FAP, CD5 and CA9, CD5 and FOLR1, CD5 and L1CAM, CD5 and ROR1, CD5 and ROR2, CD5 and GPRC5D, CD5 and PSMA, CD5 and CD23, CD5 and WT1, CD5 and CD44, CD5 and CD44v6, CD5 and CD174, CD5 and SLAMF7, CD5 and L1 CAM, CD5 and FLT3, CD5 and Sigle-6, CD5 and GD2, CD5 and PSCA, CD5 and NY-ESO-1, CD5 and GPNMB, CD5 and CD276, CD5 and CSPG4, CD5 and MAGEA3, CD5 and MAGEA4, CD5 and CD133, CD5 and TEM1, NCAM1 and CD70, NCAM1 and ULBP1, NCAM1 and ULBP2, NCAM1 and IL1RAP, NCAM1 and CEACAM5, NCAM1 and MET, NCAM1 and EGFR, NCAM1 and EGFRvIII, NCAM1 and EPCAM, NCAM1 and EPHA2, NCAM1 and ERBB2, NCAM1 and GPC3, NCAM1 and MSLN, NCAM1 and Muc1, NCAM1 and PDCD1, NCAM1 and CD274, NCAM1 and KDR, NCAM1 and IL13RA2, NCAM1 and FOLH1, NCAM1 and FAP, NCAM1 and CA9, NCAM1 and FOLR1, NCAM1 and L1CAM, NCAM1 and ROR1, NCAM1 and ROR2, NCAM1 and GPRC5D, NCAM1 and PSMA, NCAM1 and CD23, NCAM1 and WT1, NCAM1 and CD44, NCAM1 and CD44v6, NCAM1 and CD174, NCAM1 and SLAMF7, NCAM1 and L1 CAM, NCAM1 and FLT3, NCAM1 and Sigle-6, NCAM1 and GD2, NCAM1 and PSCA, NCAM1 and NY-ESO-1, NCAM1 and GPNMB, NCAM1 and CD276, NCAM1 and CSPG4, NCAM1 and MAGEA3, NCAM1 and MAGEA4, NCAM1 and CD133, NCAM1 and TEM1, CD70 and ULBP1, CD70 and ULBP2, CD70 and IL1RAP, CD70 and CEACAM5, CD70 and MET, CD70 and EGFR, CD70 and EGFRvIII, CD70 and EPCAM, CD70 and EPHA2, CD70 and ERBB2, CD70 and GPC3, CD70 and MSLN, CD70 and Muc1, CD70 and PDCD1, CD70 and CD274, CD70 and KDR, CD70 and IL13RA2, CD70 and FOLH1, CD70 and FAP, CD70 and CA9, CD70 and FOLR1, CD70 and L1CAM, CD70 and ROR1, CD70 and ROR2, CD70 and GPRC5D, CD70 and PSMA, CD70 and CD23, CD70 and WT1, CD70 and CD44, CD70 and CD44v6, CD70 and CD174, CD70 and SLAMF7, CD70 and L1 CAM, CD70 and FLT3, CD70 and Sigle-6, CD70 and GD2, CD70 and PSCA, CD70 and NY-ESO-1, CD70 and GPNMB, CD70 and CD276, CD70 and CSPG4, CD70 and MAGEA3, CD70 and MAGEA4, CD70 and CD133, CD70 and TEM1, ULBP1 and ULBP2, ULBP1 and IL1RAP, ULBP1 and CEACAM5, ULBP1 and MET, ULBP1 and EGFR, ULBP1 and EGFRvIII, ULBP1 and EPCAM, ULBP1 and EPHA2, ULBP1 and ERBB2, ULBP1 and GPC3, ULBP1 and MSLN, ULBP1 and Muc1, ULBP1 and PDCD1, DB1 / 154850959.6 84Attorney Ref. No.: 123690-5005-WO ULBP1 and CD274, ULBP1 and KDR, ULBP1 and IL13RA2, ULBP1 and FOLH1, ULBP1 and FAP, ULBP1 and CA9, ULBP1 and FOLR1, ULBP1 and L1CAM, ULBP1 and ROR1, ULBP1 and ROR2, ULBP1 and GPRC5D, ULBP1 and PSMA, ULBP1 and CD23, ULBP1 and WT1, ULBP1 and CD44, ULBP1 and CD44v6, ULBP1 and CD174, ULBP1 and SLAMF7, ULBP1 and L1 CAM, ULBP1 and FLT3, ULBP1 and Sigle-6, ULBP1 and GD2, ULBP1 and PSCA, ULBP1 and NY-ESO-1, ULBP1 and GPNMB, ULBP1 and CD276, ULBP1 and CSPG4, ULBP1 and MAGEA3, ULBP1 and MAGEA4, ULBP1 and CD133, ULBP1 and TEM1, ULBP2 and IL1RAP, ULBP2 and CEACAM5, ULBP2 and MET, ULBP2 and EGFR, ULBP2 and EGFRvIII, ULBP2 and EPCAM, ULBP2 and EPHA2, ULBP2 and ERBB2, ULBP2 and GPC3, ULBP2 and MSLN, ULBP2 and Muc1, ULBP2 and PDCD1, ULBP2 and CD274, ULBP2 and KDR, ULBP2 and IL13RA2, ULBP2 and FOLH1, ULBP2 and FAP, ULBP2 and CA9, ULBP2 and FOLR1, ULBP2 and L1CAM, ULBP2 and ROR1, ULBP2 and ROR2, ULBP2 and GPRC5D, ULBP2 and PSMA, ULBP2 and CD23, ULBP2 and WT1, ULBP2 and CD44, ULBP2 and CD44v6, ULBP2 and CD174, ULBP2 and SLAMF7, ULBP2 and L1 CAM, ULBP2 and FLT3, ULBP2 and Sigle-6, ULBP2 and GD2, ULBP2 and PSCA, ULBP2 and NY-ESO-1, ULBP2 and GPNMB, ULBP2 and CD276, ULBP2 and CSPG4, ULBP2 and MAGEA3, ULBP2 and MAGEA4, ULBP2 and CD133, ULBP2 and TEM1, IL1RAP and CEACAM5, IL1RAP and MET, IL1RAP and EGFR, IL1RAP and EGFRvIII, IL1RAP and EPCAM, IL1RAP and EPHA2, IL1RAP and ERBB2, IL1RAP and GPC3, IL1RAP and MSLN, IL1RAP and Muc1, IL1RAP and PDCD1, IL1RAP and CD274, IL1RAP and KDR, IL1RAP and IL13RA2, IL1RAP and FOLH1, IL1RAP and FAP, IL1RAP and CA9, IL1RAP and FOLR1, IL1RAP and L1CAM, IL1RAP and ROR1, IL1RAP and ROR2, IL1RAP and GPRC5D, IL1RAP and PSMA, IL1RAP and CD23, IL1RAP and WT1, IL1RAP and CD44, IL1RAP and CD44v6, IL1RAP and CD174, IL1RAP and SLAMF7, IL1RAP and L1 CAM, IL1RAP and FLT3, IL1RAP and Sigle-6, IL1RAP and GD2, IL1RAP and PSCA, IL1RAP and NY-ESO-1, IL1RAP and GPNMB, IL1RAP and CD276, IL1RAP and CSPG4, IL1RAP and MAGEA3, IL1RAP and MAGEA4, IL1RAP and CD133, IL1RAP and TEM1, CEACAM5 and MET, CEACAM5 and EGFR, CEACAM5 and EGFRvIII, CEACAM5 and EPCAM, CEACAM5 and EPHA2, CEACAM5 and ERBB2, CEACAM5 and GPC3, CEACAM5 and MSLN, CEACAM5 and Muc1, CEACAM5 and PDCD1, CEACAM5 and CD274, CEACAM5 and KDR, CEACAM5 and IL13RA2, CEACAM5 and FOLH1, CEACAM5 and FAP, CEACAM5 and CA9, CEACAM5 and FOLR1, DB1 / 154850959.6 85Attorney Ref. No.: 123690-5005-WO CEACAM5 and L1CAM, CEACAM5 and ROR1, CEACAM5 and ROR2, CEACAM5 and GPRC5D, CEACAM5 and PSMA, CEACAM5 and CD23, CEACAM5 and WT1, CEACAM5 and CD44, CEACAM5 and CD44v6, CEACAM5 and CD174, CEACAM5 and SLAMF7, CEACAM5 and L1 CAM, CEACAM5 and FLT3, CEACAM5 and Sigle-6, CEACAM5 and GD2, CEACAM5 and PSCA, CEACAM5 and NY-ESO-1, CEACAM5 and GPNMB, CEACAM5 and CD276, CEACAM5 and CSPG4, CEACAM5 and MAGEA3, CEACAM5 and MAGEA4, CEACAM5 and CD133, CEACAM5 and TEM1, MET and EGFR, MET and EGFRvIII, MET and EPCAM, MET and EPHA2, MET and ERBB2, MET and GPC3, MET and MSLN, MET and Muc1, MET and PDCD1, MET and CD274, MET and KDR, MET and IL13RA2, MET and FOLH1, MET and FAP, MET and CA9, MET and FOLR1, MET and L1CAM, MET and ROR1, MET and ROR2, MET and GPRC5D, MET and PSMA, MET and CD23, MET and WT1, MET and CD44, MET and CD44v6, MET and CD174, MET and SLAMF7, MET and L1 CAM, MET and FLT3, MET and Sigle-6, MET and GD2, MET and PSCA, MET and NY-ESO-1, MET and GPNMB, MET and CD276, MET and CSPG4, MET and MAGEA3, MET and MAGEA4, MET and CD133, MET and TEM1, EGFR and EGFRvIII, EGFR and EPCAM, EGFR and EPHA2, EGFR and ERBB2, EGFR and GPC3, EGFR and MSLN, EGFR and Muc1, EGFR and PDCD1, EGFR and CD274, EGFR and KDR, EGFR and IL13RA2, EGFR and FOLH1, EGFR and FAP, EGFR and CA9, EGFR and FOLR1, EGFR and L1CAM, EGFR and ROR1, EGFR and ROR2, EGFR and GPRC5D, EGFR and PSMA, EGFR and CD23, EGFR and WT1, EGFR and CD44, EGFR and CD44v6, EGFR and CD174, EGFR and SLAMF7, EGFR and L1 CAM, EGFR and FLT3, EGFR and Sigle-6, EGFR and GD2, EGFR and PSCA, EGFR and NY-ESO-1, EGFR and GPNMB, EGFR and CD276, EGFR and CSPG4, EGFR and MAGEA3, EGFR and MAGEA4, EGFR and CD133, EGFR and TEM1, EGFRvIII and EPCAM, EGFRvIII and EPHA2, EGFRvIII and ERBB2, EGFRvIII and GPC3, EGFRvIII and MSLN, EGFRvIII and Muc1, EGFRvIII and PDCD1, EGFRvIII and CD274, EGFRvIII and KDR, EGFRvIII and IL13RA2, EGFRvIII and FOLH1, EGFRvIII and FAP, EGFRvIII and CA9, EGFRvIII and FOLR1, EGFRvIII and L1CAM, EGFRvIII and ROR1, EGFRvIII and ROR2, EGFRvIII and GPRC5D, EGFRvIII and PSMA, EGFRvIII and CD23, EGFRvIII and WT1, EGFRvIII and CD44, EGFRvIII and CD44v6, EGFRvIII and CD174, EGFRvIII and SLAMF7, EGFRvIII and L1 CAM, EGFRvIII and FLT3, EGFRvIII and Sigle-6, EGFRvIII and GD2, EGFRvIII and PSCA, EGFRvIII and NY-ESO-1, EGFRvIII and GPNMB, DB1 / 154850959.6 86Attorney Ref. No.: 123690-5005-WO EGFRvIII and CD276, EGFRvIII and CSPG4, EGFRvIII and MAGEA3, EGFRvIII and MAGEA4, EGFRvIII and CD133, EGFRvIII and TEM1, EPCAM and EPHA2, EPCAM and ERBB2, EPCAM and GPC3, EPCAM and MSLN, EPCAM and Muc1, EPCAM and PDCD1, EPCAM and CD274, EPCAM and KDR, EPCAM and IL13RA2, EPCAM and FOLH1, EPCAM and FAP, EPCAM and CA9, EPCAM and FOLR1, EPCAM and L1CAM, EPCAM and ROR1, EPCAM and ROR2, EPCAM and GPRC5D, EPCAM and PSMA, EPCAM and CD23, EPCAM and WT1, EPCAM and CD44, EPCAM and CD44v6, EPCAM and CD174, EPCAM and SLAMF7, EPCAM and L1 CAM, EPCAM and FLT3, EPCAM and Sigle-6, EPCAM and GD2, EPCAM and PSCA, EPCAM and NY-ESO-1, EPCAM and GPNMB, EPCAM and CD276, EPCAM and CSPG4, EPCAM and MAGEA3, EPCAM and MAGEA4, EPCAM and CD133, EPCAM and TEM1, EPHA2 and ERBB2, EPHA2 and GPC3, EPHA2 and MSLN, EPHA2 and Muc1, EPHA2 and PDCD1, EPHA2 and CD274, EPHA2 and KDR, EPHA2 and IL13RA2, EPHA2 and FOLH1, EPHA2 and FAP, EPHA2 and CA9, EPHA2 and FOLR1, EPHA2 and L1CAM, EPHA2 and ROR1, EPHA2 and ROR2, EPHA2 and GPRC5D, EPHA2 and PSMA, EPHA2 and CD23, EPHA2 and WT1, EPHA2 and CD44, EPHA2 and CD44v6, EPHA2 and CD174, EPHA2 and SLAMF7, EPHA2 and L1 CAM, EPHA2 and FLT3, EPHA2 and Sigle-6, EPHA2 and GD2, EPHA2 and PSCA, EPHA2 and NY-ESO-1, EPHA2 and GPNMB, EPHA2 and CD276, EPHA2 and CSPG4, EPHA2 and MAGEA3, EPHA2 and MAGEA4, EPHA2 and CD133, EPHA2 and TEM1, ERBB2 and GPC3, ERBB2 and MSLN, ERBB2 and Muc1, ERBB2 and PDCD1, ERBB2 and CD274, ERBB2 and KDR, ERBB2 and IL13RA2, ERBB2 and FOLH1, ERBB2 and FAP, ERBB2 and CA9, ERBB2 and FOLR1, ERBB2 and L1CAM, ERBB2 and ROR1, ERBB2 and ROR2, ERBB2 and GPRC5D, ERBB2 and PSMA, ERBB2 and CD23, ERBB2 and WT1, ERBB2 and CD44, ERBB2 and CD44v6, ERBB2 and CD174, ERBB2 and SLAMF7, ERBB2 and L1 CAM, ERBB2 and FLT3, ERBB2 and Sigle-6, ERBB2 and GD2, ERBB2 and PSCA, ERBB2 and NY-ESO-1, ERBB2 and GPNMB, ERBB2 and CD276, ERBB2 and CSPG4, ERBB2 and MAGEA3, ERBB2 and MAGEA4, ERBB2 and CD133, ERBB2 and TEM1, GPC3 and MSLN, GPC3 and Muc1, GPC3 and PDCD1, GPC3 and CD274, GPC3 and KDR, GPC3 and IL13RA2, GPC3 and FOLH1, GPC3 and FAP, GPC3 and CA9, GPC3 and FOLR1, GPC3 and L1CAM, GPC3 and ROR1, GPC3 and ROR2, GPC3 and GPRC5D, GPC3 and PSMA, GPC3 and CD23, GPC3 and WT1, GPC3 and CD44, GPC3 and CD44v6, GPC3 and CD174, GPC3 and SLAMF7, GPC3 and L1 CAM, GPC3 and FLT3, GPC3 and Sigle-6, GPC3 DB1 / 154850959.6 87Attorney Ref. No.: 123690-5005-WO and GD2, GPC3 and PSCA, GPC3 and NY-ESO-1, GPC3 and GPNMB, GPC3 and CD276, GPC3 and CSPG4, GPC3 and MAGEA3, GPC3 and MAGEA4, GPC3 and CD133, GPC3 and TEM1, MSLN and Muc1, MSLN and PDCD1, MSLN and CD274, MSLN and KDR, MSLN and IL13RA2, MSLN and FOLH1, MSLN and FAP, MSLN and CA9, MSLN and FOLR1, MSLN and L1CAM, MSLN and ROR1, MSLN and ROR2, MSLN and GPRC5D, MSLN and PSMA, MSLN and CD23, MSLN and WT1, MSLN and CD44, MSLN and CD44v6, MSLN and CD174, MSLN and SLAMF7, MSLN and L1 CAM, MSLN and FLT3, MSLN and Sigle-6, MSLN and GD2, MSLN and PSCA, MSLN and NY-ESO-1, MSLN and GPNMB, MSLN and CD276, MSLN and CSPG4, MSLN and MAGEA3, MSLN and MAGEA4, MSLN and CD133, MSLN and TEM1, Muc1 and PDCD1, Muc1 and CD274, Muc1 and KDR, Muc1 and IL13RA2, Muc1 and FOLH1, Muc1 and FAP, Muc1 and CA9, Muc1 and FOLR1, Muc1 and L1CAM, Muc1 and ROR1, Muc1 and ROR2, Muc1 and GPRC5D, Muc1 and PSMA, Muc1 and CD23, Muc1 and WT1, Muc1 and CD44, Muc1 and CD44v6, Muc1 and CD174, Muc1 and SLAMF7, Muc1 and L1 CAM, Muc1 and FLT3, Muc1 and Sigle-6, Muc1 and GD2, Muc1 and PSCA, Muc1 and NY- ESO-1, Muc1 and GPNMB, Muc1 and CD276, Muc1 and CSPG4, Muc1 and MAGEA3, Muc1 and MAGEA4, Muc1 and CD133, Muc1 and TEM1, PDCD1 and CD274, PDCD1 and KDR, PDCD1 and IL13RA2, PDCD1 and FOLH1, PDCD1 and FAP, PDCD1 and CA9, PDCD1 and FOLR1, PDCD1 and L1CAM, PDCD1 and ROR1, PDCD1 and ROR2, PDCD1 and GPRC5D, PDCD1 and PSMA, PDCD1 and CD23, PDCD1 and WT1, PDCD1 and CD44, PDCD1 and CD44v6, PDCD1 and CD174, PDCD1 and SLAMF7, PDCD1 and L1 CAM, PDCD1 and FLT3, PDCD1 and Sigle-6, PDCD1 and GD2, PDCD1 and PSCA, PDCD1 and NY-ESO-1, PDCD1 and GPNMB, PDCD1 and CD276, PDCD1 and CSPG4, PDCD1 and MAGEA3, PDCD1 and MAGEA4, PDCD1 and CD133, PDCD1 and TEM1, CD274 and KDR, CD274 and IL13RA2, CD274 and FOLH1, CD274 and FAP, CD274 and CA9, CD274 and FOLR1, CD274 and L1CAM, CD274 and ROR1, CD274 and ROR2, CD274 and GPRC5D, CD274 and PSMA, CD274 and CD23, CD274 and WT1, CD274 and CD44, CD274 and CD44v6, CD274 and CD174, CD274 and SLAMF7, CD274 and L1 CAM, CD274 and FLT3, CD274 and Sigle-6, CD274 and GD2, CD274 and PSCA, CD274 and NY-ESO-1, CD274 and GPNMB, CD274 and CD276, CD274 and CSPG4, CD274 and MAGEA3, CD274 and MAGEA4, CD274 and CD133, CD274 and TEM1, KDR and IL13RA2, KDR and FOLH1, KDR and FAP, KDR and CA9, KDR and FOLR1, KDR and L1CAM, KDR and ROR1, KDR and ROR2, KDR and GPRC5D, KDR DB1 / 154850959.6 88Attorney Ref. No.: 123690-5005-WO and PSMA, KDR and CD23, KDR and WT1, KDR and CD44, KDR and CD44v6, KDR and CD174, KDR and SLAMF7, KDR and L1 CAM, KDR and FLT3, KDR and Sigle-6, KDR and GD2, KDR and PSCA, KDR and NY-ESO-1, KDR and GPNMB, KDR and CD276, KDR and CSPG4, KDR and MAGEA3, KDR and MAGEA4, KDR and CD133, KDR and TEM1, IL13RA2 and FOLH1, IL13RA2 and FAP, IL13RA2 and CA9, IL13RA2 and FOLR1, IL13RA2 and L1CAM, IL13RA2 and ROR1, IL13RA2 and ROR2, IL13RA2 and GPRC5D, IL13RA2 and PSMA, IL13RA2 and CD23, IL13RA2 and WT1, IL13RA2 and CD44, IL13RA2 and CD44v6, IL13RA2 and CD174, IL13RA2 and SLAMF7, IL13RA2 and L1 CAM, IL13RA2 and FLT3, IL13RA2 and Sigle-6, IL13RA2 and GD2, IL13RA2 and PSCA, IL13RA2 and NY-ESO-1, IL13RA2 and GPNMB, IL13RA2 and CD276, IL13RA2 and CSPG4, IL13RA2 and MAGEA3, IL13RA2 and MAGEA4, IL13RA2 and CD133, IL13RA2 and TEM1, FOLH1 and FAP, FOLH1 and CA9, FOLH1 and FOLR1, FOLH1 and L1CAM, FOLH1 and ROR1, FOLH1 and ROR2, FOLH1 and GPRC5D, FOLH1 and PSMA, FOLH1 and CD23, FOLH1 and WT1, FOLH1 and CD44, FOLH1 and CD44v6, FOLH1 and CD174, FOLH1 and SLAMF7, FOLH1 and L1 CAM, FOLH1 and FLT3, FOLH1 and Sigle-6, FOLH1 and GD2, FOLH1 and PSCA, FOLH1 and NY- ESO-1, FOLH1 and GPNMB, FOLH1 and CD276, FOLH1 and CSPG4, FOLH1 and MAGEA3, FOLH1 and MAGEA4, FOLH1 and CD133, FOLH1 and TEM1, FAP and CA9, FAP and FOLR1, FAP and L1CAM, FAP and ROR1, FAP and ROR2, FAP and GPRC5D, FAP and PSMA, FAP and CD23, FAP and WT1, FAP and CD44, FAP and CD44v6, FAP and CD174, FAP and SLAMF7, FAP and L1 CAM, FAP and FLT3, FAP and Sigle-6, FAP and GD2, FAP and PSCA, FAP and NY-ESO-1, FAP and GPNMB, FAP and CD276, FAP and CSPG4, FAP and MAGEA3, FAP and MAGEA4, FAP and CD133, FAP and TEM1, CA9 and FOLR1, CA9 and L1CAM, CA9 and ROR1, CA9 and ROR2, CA9 and GPRC5D, CA9 and PSMA, CA9 and CD23, CA9 and WT1, CA9 and CD44, CA9 and CD44v6, CA9 and CD174, CA9 and SLAMF7, CA9 and L1 CAM, CA9 and FLT3, CA9 and Sigle-6, CA9 and GD2, CA9 and PSCA, CA9 and NY-ESO-1, CA9 and GPNMB, CA9 and CD276, CA9 and CSPG4, CA9 and MAGEA3, CA9 and MAGEA4, CA9 and CD133, CA9 and TEM1, FOLR1 and L1CAM, FOLR1 and ROR1, FOLR1 and ROR2, FOLR1 and GPRC5D, FOLR1 and PSMA, FOLR1 and CD23, FOLR1 and WT1, FOLR1 and CD44, FOLR1 and CD44v6, FOLR1 and CD174, FOLR1 and SLAMF7, FOLR1 and L1 CAM, FOLR1 and FLT3, FOLR1 and Sigle-6, FOLR1 and GD2, FOLR1 and PSCA, FOLR1 and NY-ESO-1, FOLR1 and GPNMB, FOLR1 and CD276, FOLR1 and CSPG4, DB1 / 154850959.6 89Attorney Ref. No.: 123690-5005-WO FOLR1 and MAGEA3, FOLR1 and MAGEA4, FOLR1 and CD133, FOLR1 and TEM1, L1CAM and ROR1, L1CAM and ROR2, L1CAM and GPRC5D, L1CAM and PSMA, L1CAM and CD23, L1CAM and WT1, L1CAM and CD44, L1CAM and CD44v6, L1CAM and CD174, L1CAM and SLAMF7, L1CAM and L1 CAM, L1CAM and FLT3, L1CAM and Sigle-6, L1CAM and GD2, L1CAM and PSCA, L1CAM and NY-ESO-1, L1CAM and GPNMB, L1CAM and CD276, L1CAM and CSPG4, L1CAM and MAGEA3, L1CAM and MAGEA4, L1CAM and CD133, L1CAM and TEM1, ROR1 and ROR2, ROR1 and GPRC5D, ROR1 and PSMA, ROR1 and CD23, ROR1 and WT1, ROR1 and CD44, ROR1 and CD44v6, ROR1 and CD174, ROR1 and SLAMF7, ROR1 and L1 CAM, ROR1 and FLT3, ROR1 and Sigle-6, ROR1 and GD2, ROR1 and PSCA, ROR1 and NY-ESO-1, ROR1 and GPNMB, ROR1 and CD276, ROR1 and CSPG4, ROR1 and MAGEA3, ROR1 and MAGEA4, ROR1 and CD133, ROR1 and TEM1, ROR2 and GPRC5D, ROR2 and PSMA, ROR2 and CD23, ROR2 and WT1, ROR2 and CD44, ROR2 and CD44v6, ROR2 and CD174, ROR2 and SLAMF7, ROR2 and L1 CAM, ROR2 and FLT3, ROR2 and Sigle-6, ROR2 and GD2, ROR2 and PSCA, ROR2 and NY-ESO-1, ROR2 and GPNMB, ROR2 and CD276, ROR2 and CSPG4, ROR2 and MAGEA3, ROR2 and MAGEA4, ROR2 and CD133, ROR2 and TEM1, GPRC5D and PSMA, GPRC5D and CD23, GPRC5D and WT1, GPRC5D and CD44, GPRC5D and CD44v6, GPRC5D and CD174, GPRC5D and SLAMF7, GPRC5D and L1 CAM, GPRC5D and FLT3, GPRC5D and Sigle-6, GPRC5D and GD2, GPRC5D and PSCA, GPRC5D and NY-ESO-1, GPRC5D and GPNMB, GPRC5D and CD276, GPRC5D and CSPG4, GPRC5D and MAGEA3, GPRC5D and MAGEA4, GPRC5D and CD133, GPRC5D and TEM1, PSMA and CD23, PSMA and WT1, PSMA and CD44, PSMA and CD44v6, PSMA and CD174, PSMA and SLAMF7, PSMA and L1 CAM, PSMA and FLT3, PSMA and Sigle-6, PSMA and GD2, PSMA and PSCA, PSMA and NY-ESO-1, PSMA and GPNMB, PSMA and CD276, PSMA and CSPG4, PSMA and MAGEA3, PSMA and MAGEA4, PSMA and CD133, PSMA and TEM1, CD23 and WT1, CD23 and CD44, CD23 and CD44v6, CD23 and CD174, CD23 and SLAMF7, CD23 and L1 CAM, CD23 and FLT3, CD23 and Sigle-6, CD23 and GD2, CD23 and PSCA, CD23 and NY- ESO-1, CD23 and GPNMB, CD23 and CD276, CD23 and CSPG4, CD23 and MAGEA3, CD23 and MAGEA4, CD23 and CD133, CD23 and TEM1, WT1 and CD44, WT1 and CD44v6, WT1 and CD174, WT1 and SLAMF7, WT1 and L1 CAM, WT1 and FLT3, WT1 and Sigle-6, WT1 and GD2, WT1 and PSCA, WT1 and NY-ESO-1, WT1 and GPNMB, WT1 and CD276, WT1 DB1 / 154850959.6 90Attorney Ref. No.: 123690-5005-WO and CSPG4, WT1 and MAGEA3, WT1 and MAGEA4, WT1 and CD133, WT1 and TEM1, CD44 and CD44v6, CD44 and CD174, CD44 and SLAMF7, CD44 and L1 CAM, CD44 and FLT3, CD44 and Sigle-6, CD44 and GD2, CD44 and PSCA, CD44 and NY-ESO-1, CD44 and GPNMB, CD44 and CD276, CD44 and CSPG4, CD44 and MAGEA3, CD44 and MAGEA4, CD44 and CD133, CD44 and TEM1, CD44v6 and CD174, CD44v6 and SLAMF7, CD44v6 and L1 CAM, CD44v6 and FLT3, CD44v6 and Sigle-6, CD44v6 and GD2, CD44v6 and PSCA, CD44v6 and NY-ESO-1, CD44v6 and GPNMB, CD44v6 and CD276, CD44v6 and CSPG4, CD44v6 and MAGEA3, CD44v6 and MAGEA4, CD44v6 and CD133, CD44v6 and TEM1, CD174 and SLAMF7, CD174 and L1 CAM, CD174 and FLT3, CD174 and Sigle-6, CD174 and GD2, CD174 and PSCA, CD174 and NY-ESO-1, CD174 and GPNMB, CD174 and CD276, CD174 and CSPG4, CD174 and MAGEA3, CD174 and MAGEA4, CD174 and CD133, CD174 and TEM1, SLAMF7 and L1 CAM, SLAMF7 and FLT3, SLAMF7 and Sigle-6, SLAMF7 and GD2, SLAMF7 and PSCA, SLAMF7 and NY-ESO-1, SLAMF7 and GPNMB, SLAMF7 and CD276, SLAMF7 and CSPG4, SLAMF7 and MAGEA3, SLAMF7 and MAGEA4, SLAMF7 and CD133, SLAMF7 and TEM1, L1 CAM and FLT3, L1 CAM and Sigle-6, L1 CAM and GD2, L1 CAM and PSCA, L1 CAM and NY-ESO-1, L1 CAM and GPNMB, L1 CAM and CD276, L1 CAM and CSPG4, L1 CAM and MAGEA3, L1 CAM and MAGEA4, L1 CAM and CD133, L1 CAM and TEM1, FLT3 and Sigle-6, FLT3 and GD2, FLT3 and PSCA, FLT3 and NY-ESO-1, FLT3 and GPNMB, FLT3 and CD276, FLT3 and CSPG4, FLT3 and MAGEA3, FLT3 and MAGEA4, FLT3 and CD133, FLT3 and TEM1, Sigle-6 and GD2, Sigle-6 and PSCA, Sigle-6 and NY-ESO-1, Sigle-6 and GPNMB, Sigle-6 and CD276, Sigle-6 and CSPG4, Sigle-6 and MAGEA3, Sigle-6 and MAGEA4, Sigle-6 and CD133, Sigle-6 and TEM1, GD2 and PSCA, GD2 and NY-ESO-1, GD2 and GPNMB, GD2 and CD276, GD2 and CSPG4, GD2 and MAGEA3, GD2 and MAGEA4, GD2 and CD133, GD2 and TEM1, PSCA and NY-ESO-1, PSCA and GPNMB, PSCA and CD276, PSCA and CSPG4, PSCA and MAGEA3, PSCA and MAGEA4, PSCA and CD133, PSCA and TEM1, NY-ESO-1 and GPNMB, NY-ESO-1 and CD276, NY-ESO-1 and CSPG4, NY-ESO-1 and MAGEA3, NY-ESO-1 and MAGEA4, NY- ESO-1 and CD133, NY-ESO-1 and TEM1, GPNMB and CD276, GPNMB and CSPG4, GPNMB and MAGEA3, GPNMB and MAGEA4, GPNMB and CD133, GPNMB and TEM1, CD276 and CSPG4, CD276 and MAGEA3, CD276 and MAGEA4, CD276 and CD133, CD276 and TEM1, CSPG4 and MAGEA3, CSPG4 and MAGEA4, CSPG4 and CD133, CSPG4 and DB1 / 154850959.6 91Attorney Ref. No.: 123690-5005-WO TEM1, MAGEA3 and MAGEA4, MAGEA3 and CD133, MAGEA3 and TEM1, MAGEA4 and CD133, MAGEA4 and TEM1, and CD133 and TEM1.

[0312] In some embodiments, the CAR or TCR described herein is bispecific and has affinity for CD19 and CD22; CD19 and CD20; CD20 and CD22; BCMA and CD19; BCMA and CD19; or BCMA and CD22.

[0313] In some embodiments, the CAR or TCR described herein is tri-specific and has affinity for CD19, CD20, and CD22; CD19, CD20, and BCMA; or CD19, CD22, and BCMA; CD20, CD22, and BCMA.

[0314] In some embodiments, the DNA encoding the CAR of the present disclosure is operably linked to a promoter such as a synthetic promoter, a constitutive promoter, or an inducible promoter. Useful constitutive promoters include an ubiquitin C promoter, an elongation factor- 1 alpha promoter (EFla promoter), a CMV promoter, and any other constitutive promoter known to those skilled in the art. Useful inducible promoters are described, e.g., in Ede et al, ACS Synth Biol, 2016, 5(5):395-404, and can include cell-type specific promoters and inducible switch promoters. Illustrative non-limiting constitutive promoters are described in PLoS One, 2010, 5(8):el24l3. In some embodiments, the promoter is the EFla promoter. V. Extracorporeal transfection

[0315] As used herein “extracorporeal” is used in reference to cells, such as peripheral blood or bone marrow cells, harvested or extracted from the body and the modification of those cells prior to their intended return (reinfusion). Modification of cells generally relates to cell separation and washing procedures and exposure to transfection agents (e.g., LNPs, tLNPs), over a time interval of several hours. Such procedures are typically conducted within a single institution.

[0316] Extracorporeal typically involves minimal manipulation or modification of subject cells, including limited periods of cell culture and expansion. In some embodiments, unlike conventional CAR-T / TCR manufacturing processes, the extracorporeal method described herein does not require treatment of target immune effector cells with an activation agent ex vivo, such as cytokines including but not limited to IL-2, IL-7, IL-12, IL-15, IL-18, IL-21; IL-23, any combination thereof; CTS™; OKT-3; anti-CD3 / CD28 agents (e.g., Dynabeads™); or MACS® GMP T Cell TransAct™. It also includes the avoidance of refrigerated or cryogenic storage or shipment over extended periods spanning multiple days or longer. DB1 / 154850959.6 92Attorney Ref. No.: 123690-5005-WO

[0317] As used herein “transfection” or “transfecting” refers to the introduction of nucleic acids into cells by non-viral methods. Transfection can be mediated by calcium phosphate, cationic polymers, magnetic beads, electroporation, and lipid-based reagents. In preferred embodiments disclosed herein the transfection is mediated by nanoparticles (NP) including targeted lipid nanoparticles LNPs (tLNPs).

[0318] In some embodiments, the present disclosure provides herein a method for extracorporeal transfection of an immune effector cell collected from a subject in need thereof. The immune effector cell is present in the blood drawn from the subject, and the extracorporeal transfection is achieved by contacting blood, or a cell-containing fraction thereof, from the subject with a nanoparticle ex vivo. In some embodiments, the nanoparticle contains a surface- exposed immune effector binding moiety; and a deoxyribonucleic acid encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR).

[0319] In some embodiments, the contacting of the blood, or a cell-containing fraction thereof, with a nanoparticle comprises drawing blood from the subject and admixing a solution comprising the nanoparticle with the drawn blood that contains the immune effector cells.

[0320] In some embodiments, the blood is drawn into a closed system and the admixing comprises adding the solution into the closed system, for example, a closed vein-to-vein system including but not limited to apheresis system. In some embodiments of the manufacturing processes described herein, immune effector cells are collected using standard apheresis equipment, such as Cobe® Spectra, Spectra Optia®, Fenwal™ Amicus® or equivalent. In some embodiments, cells from the circulating blood of a subject are obtained by leukopheresis, erythrocytapheresis, thrombapheresis, thrombocytapheresis, plasmapheresis, or plateletpheresis. In some embodiments, the leukopheresis process yielded approximately 100-500 mL of apheresis product from a patient (i.e., a subject). The apheresis product is subjected to the transfection on-site, namely, transfection at the point-of-care, such as bedside.

[0321] As a non-limiting example, an apheresis device can include one or more connections configured to move whole blood and / or blood components to and from blood component separation devices. Blood component separation devices may be housed within the apheresis devices and often includes centrifuges. The apheresis device may be used for whole blood separation processes, where whole blood is withdrawn form a donor or a subject or a patient or DB1 / 154850959.6 93Attorney Ref. No.: 123690-5005-WO source and provided to a blood component separation device where the whole blood is separated into various compounds, components, or elements some of which are collected and a remainder is returned to the donor or the subject or the patient or the source.

[0322] Exemplary description of apheresis and equivalent systems and their use can be found in U.S. Patent Publication No.20250041501, U.S. Patent No.8,123,713; 7,780,618; 10,704,023; 9,435,736, 8,057,376, each of which is hereby incorporated by reference in its entireties.

[0323] In some embodiments, during blood drawing, the blood is collected into an apheresis bag and admixed by adding a solution containing the nanoparticle of the present disclosure to the apheresis bag.

[0324] In some embodiments, the drawn blood is processed and enriched during apheresis. In some embodiments, the apheresis product is a “leukopheresis” product. As used herein, the term “leukopheresis” refers to the bulk mononuclear cells present in the blood, namely the separation and collection of leukocytes, white blood cells (WBCs), from plasma and red blood cells.

[0325] In some embodiments, the enriched apheresis product comprises about 5% to about 25% of the total peripheral blood mononuclear cell component. In some embodiments, the enriched apheresis product is a population of lymphoid cells or a lymphoid cell. In this embodiment, the lymphoid cell is selected from the group consisting of a T cell, a B cell, a natural killer (NK) cell, a CD8+ T cell, a CD4+ T cell, a cytotoxic T lymphocyte, a regulatory T cell, and any combination thereof.

[0326] In some embodiments, the enriched apheresis product has a predetermined volume and / or a predetermined hematocrit regardless of the number of PBMC collection cycles executed by the Apheresis system and / or the number of pre-products used to produce the enriched apheresis product.

[0327] In some embodiments, the predetermined volume is about 120 ml to about 400 mL. In some embodiments, the predetermined volume is about 120 ml, about 150 ml, about 175 ml, about 180 ml, about 200 ml, about 225 ml, about 250 ml, about 275 ml, about 300 ml, about 325 ml, about 350 ml, about 375 ml, or about 400 ml or less. In some embodiments, the apheresis is configured with a specific target yield of PBMCs to be collected and treated. The specific target yield of PBMCs to be collected and treated may be assessed by the apheresis system and / or by DB1 / 154850959.6 94Attorney Ref. No.: 123690-5005-WO entering the subject's PBMC pre-count. Based on the target PBMC yield and the number of PBMCs collected during each mononuclear collection cycle, the controller of the apheresis system may determine the number of collection cycles to execute. As an example, in some embodiments, if the target PBMC yield is about 5×109PBMCs, the apheresis system will collect about 1×109PBMCs per mononuclear collection cycle, then the controller will determine that it is appropriate to execute the mononuclear collection cycle five times.

[0328] In some embodiments, the target PBMC yield is at least about 0.1×107, at least about 0.2×107, at least about 0.3×107, at least about 0.4×107, at least about 0.5×107, at least about 0.6×107, at least about 0.7×107, at least about 0.8×107, at least about 0.9×107, at least about 1×107, at least about 2×107, at least about 4×107, at least about 6×107, at least about 8×107, at least about 9×107, at least about 1×108, at least about 2×108, at least about 3×108, at least about 4×108, or at least about 5×108cells / mL. In some embodiments, the target PBMC yield is from about 0.5×106cells / mL to about 4×106cells / mL. In some embodiments, target PBMC yield is from about 0.5×106cells / mL to about 1×108cells / mL. In some embodiments, target PBMC yield is from about 4.0×106cells / mL to about 1×108cells / mL.

[0329] In some embodiments, the predetermined hematocrit is about 0% to about 10%. In another embodiment, the predetermined hematocrit is about 2%. In some embodiments, the predetermined volume is approximately 200 mL and the predetermined hematocrit is approximately 2%. The predetermined volume and / or the predetermined hematocrit may vary without departing from the scope of the present disclosure.

[0330] In some embodiments, an anticoagulant is added to the solution comprising the nanoparticle or the drawn blood prior to, during, or after the admixing. In some embodiments, an anticoagulant is added to the solution comprising the nanoparticle or the drawn blood. In some embodiments, an anticoagulant is added to the drawn blood. In some embodiments, such anticoagulant is added through an anticoagulant pump to draw anticoagulant from an anticoagulant bag and mix the anticoagulant with the content in the apheresis system.

[0331] In some embodiments, the anticoagulant is sodium citrate. Sodium citrate is widely accepted because healthy donors are able to handle it better. Heparin can also be used, for example, for sicker populations. Using heparin will maintain a static state of calcium ion activity. Citrate based anticoagulants bind with calcium ions. Decrease in calcium ions may cause DB1 / 154850959.6 95Attorney Ref. No.: 123690-5005-WO increase in cardiac adverse events such as arrhythmias. For this reason, heparin has been safely used in cardiac applications for many years. Heparin is standard for the care for patients undergoing vascular procedures. In another embodiment, the anticoagulant can include ethylenediaminetetraacetate (EDTA), and / or fluoride.

[0332] In some embodiments, the apheresis of blood drawn from a subject, and the admixing of the blood with a nanoparticle composition / solution described herein, are performed sequentially, or concurrently.

[0333] In some embodiments, apheresis of blood drawn from a subject separates the blood components, thereby generating a first blood fraction containing immune effector cells and a second blood fraction free of immune effector cells. The blood fraction contains immune effector cells, including peripheral blood mononuclear cells (PBMCs), is used for transfection. In some embodiments, the separation is followed by admixing a solution comprising the nanoparticle with the first blood fraction containing the immune effector cells. In some embodiments, the apheresis is leukopheresis. In some embodiments, the first blood fraction is fractionated into an apheresis bag and the admixing comprises adding the solution into the pheresis bag.

[0334] In another embodiments, the contacting and transfection of the immune effector cells is achieved by admixing a solution comprising the nanoparticle with whole blood drawn from the subject, then followed by apheresis of the admixed whole blood, thereby generating a first blood fraction comprising immune effector cells and a second blood fraction free of immune effector cells. The first blood fraction comprises immune effector cells transfected with the nanoparticle described herein. In some embodiments, the apheresis is leukopheresis. In some embodiments, the first blood fraction is fractionated into an apheresis bag and the admixing comprises adding the solution into the pheresis bag.

[0335] In some embodiments, following the contacting and transfection, the immune effector cells in the first blood fraction are separated from free nanoparticles, including isolated / unbound nanoparticles, and / or other unwanted components from the apheresis. In some embodiments, the cells in the first blood fraction collected by apheresis are washed to remove the free / unbound nanoparticles, the plasma fraction, and other undesirable components, and to place the cells in an appropriate buffer or media, such as phosphate buffered saline (PBS) or wash solution lacks DB1 / 154850959.6 96Attorney Ref. No.: 123690-5005-WO calcium and may lack magnesium or may lack many if not all divalent cations, subsequent modifications, or re-infusion back to the subject.

[0336] As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow- through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer’s instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca2+-free, Mg2+ free PBS, PlasmaLyte A, or another saline solution with or without a buffer.

[0337] In some embodiments, the immune effector cells in the first blood fraction are separated by plasmapheresis, filtering with a spinning membrane filter, or centrifugation, or a combination of any of these procedures. In some embodiments, the immune effector cells in the first blood fraction are separated by plasmapheresis. In some embodiments, the immune effector cells in the first blood fraction are separated by a spinning membrane filter. In some embodiments, the immune effector cells in the first blood fraction are separated by centrifugation.

[0338] In some embodiments, the immune effector cells in the first blood fraction are further separated by plasmapheresis, a process that selectively removes plasma while retaining cellular components, thereby allowing for the enrichment of immune effector cells. Plasmapheresis is advantageous in maintaining cell integrity and reducing unwanted plasma proteins that may interfere with subsequent processing. In some embodiments, the immune effector cells in the first blood fraction are separated by a spinning membrane filter, which utilizes a semi-permeable membrane and centrifugal force to selectively filter out smaller plasma components while retaining immune effector cells. This technique can provide a high-purity fraction of immune cells while minimizing shear stress that could impact cell functionality. In some embodiments, the immune effector cells in the first blood fraction are separated by centrifugation, wherein the differential densities of blood components are exploited to isolate the immune effector cells. Centrifugation conditions, such as speed, duration, and gradient media, may be optimized to maximize recovery while minimizing cell damage. Density gradient centrifugation, for example, may be employed to enhance the separation of lymphocytes, monocytes, and other immune effector cells from red blood cells and platelets. DB1 / 154850959.6 97Attorney Ref. No.: 123690-5005-WO

[0339] In some embodiments, a combination of plasmapheresis, spinning membrane filtration, and centrifugation may be used sequentially or in parallel to achieve optimal separation of immune effector cells. This multi-step approach may be beneficial in cases where high-purity cell fractions are required for therapeutic applications, such as cell infusion or genetic modification.

[0340] In some embodiments, the transfection of the immune effector cells collected comprises admixing the cells, the drawn blood comprising the immune effector cells, or a fraction of the blood comprising such cells, with the nanoparticles of the present disclosure. In some embodiments, the admixing may be performed in the apheresis system during the apheresis. In some embodiments, the admixing may be performed prior to the apheresis. In other embodiments, the admixing may be performed subsequent to the apheresis. In yet other embodiments, the admixing may be performed in a closed system, such as a closed vein-to-vein system.

[0341] In some embodiments, the admixing is performed at a cell density from about 250,000 cells per mL to about 1 million cells per mL. In some embodiments, the cell density is from about 250,000 cells per mL to about 1 million cells per mL. In some embodiments, the cell density is from about 250,000 cells per mL to about 500,000 cells per mL. In some embodiments, the cell density is from about 500,000 cells per mL to about 750,000 cells per mL. In some embodiments, the cell density is from about 750,000 cells per mL to about 1 million cells per mL. In some embodiments, the admixing is performed at a cell density of from about 1 million cells per mL to about 20 million cells per mL. In some embodiments, the admixing is performed at a cell density of at least 5 million cells per mL. In some embodiments, the admixing is performed at a cell density of at least 10 million cells per mL. In some embodiments, the admixing is performed at a cell density of at least 15 million cells per mL. In some embodiments, the admixing is performed at a cell density of at least 20 million cells per mL.

[0342] In some embodiments, the cell density for admixing is within a specific range selected from the group consisting of: between 250,000 cells per mL and 500,000 cells per mL; between 250,000 cells per mL and 750,000 cells per mL; between 250,000 cells per mL and 1 million cells per mL; between 250,000 cells per mL and 5 million cells per mL; between 250,000 cells per mL and 10 million cells per mL; between 250,000 cells per mL and 15 million cells per mL; DB1 / 154850959.6 98Attorney Ref. No.: 123690-5005-WO between 250,000 cells per mL and 20 million cells per mL; between 500,000 cells per mL and 750,000 cells per mL; between 500,000 cells per mL and 1 million cells per mL; between 500,000 cells per mL and 5 million cells per mL; between 500,000 cells per mL and 10 million cells per mL; between 500,000 cells per mL and 15 million cells per mL; between 500,000 cells per mL and 20 million cells per mL; between 750,000 cells per mL and 1 million cells per mL; between 750,000 cells per mL and 5 million cells per mL; between 750,000 cells per mL and 10 million cells per mL; between 750,000 cells per mL and 15 million cells per mL; between 750,000 cells per mL and 20 million cells per mL; between 1 million cells per mL and 5 million cells per mL; between 1 million cells per mL and 10 million cells per mL; between 1 million cells per mL and 15 million cells per mL; between 1 million cells per mL and 20 million cells per mL; between 5 million cells per mL and 10 million cells per mL; between 5 million cells per mL and 15 million cells per mL; between 5 million cells per mL and 20 million cells per mL; between 10 million cells per mL and 15 million cells per mL; between 10 million cells per mL and 20 million cells per mL; and between 15 million cells per mL and 20 million.

[0343] “DNA copy number” is to refer to the minicircle DNA, or the like, which is to encode for the CAR (or TCR or the like) in accordance with the disclosure. It is understood that copy number is to refer to the DNA, that is to be incorporated in a tLNP (or, likewise, nanoparticle or LNP as used herein), and the DNA copy number is calculated based on the molecular weight of the DNA. Of tLNPs as prepared in accordance with the disclosure, the molecular weight of the DNA incorporated therein is known, and the amount of DNA included in a tLNP preparation can easily be determined (e.g. using qPCR or the like), and thus, the ratio of DNA copy number per cell when can easily be selected or determined, when admixing a tLNP preparation and cells contained in a certain volume e.g. whole blood, having a certain cell concentration (cell / mL).

[0344] In some embodiments, the contacting is performed at a ratio of DNA copy number to cells of at least 6×102DNA copies per cell, at least 8×102DNA copies per cell, at least 6×103DNA copies per cell, at least 8×103DNA copies per cell, at least 6×104DNA copies per cell, at least 8×104DNA copies per cell, at least 1×105DNA copies per cell, at least 2×105DNA copies per cell, at least 4×105DNA copies per cell, at least 6×105DNA copies per cell, at least 8×105DNA copies per cell, at least 1×106DNA copies per cell, 3×106DNA copies per cell, at least 6×106DNA copies per cell, at least 8×106DNA copies per cell, at least 1×107DNA copies per cell, at least 3×107DNA copies per cell, or at least 6×107DNA copies per cell. DB1 / 154850959.6 99Attorney Ref. No.: 123690-5005-WO

[0345] In some embodiments, the contacting is performed at a ratio of DNA copy number to cells of from 6×102to 6×107DNA copies per cell. In some embodiments, the contacting is performed at a ratio of DNA copy number to cells of from 6×102to 6×103DNA copies per cell. In some embodiments, the contacting is performed at a ratio of DNA copy number to cells of from 6×103to 6×104DNA copies per cell. In some embodiments, the contacting is performed at a ratio of DNA copy number to cells of from 6×104to 6×105DNA copies per cell. In some embodiments, the contacting is performed at a ratio of DNA copy number to cells of from 6×105to 6×106DNA copies per cell. In some embodiments, the contacting is performed at a ratio of DNA copy number to cells of from 6×106to 6×107DNA copies per cell.

[0346] In some embodiments, the contacting is performed at a ratio of DNA copy number to cells of 8×10² DNA copies per cell to 8×10³ DNA copies per cell; 8×10² DNA copies per cell to 8×10⁴ DNA copies per cell; 8×10² DNA copies per cell to 2×10⁵ DNA copies per cell; 8×10² DNA copies per cell to 4×10⁵ DNA copies per cell; 8×10² DNA copies per cell to 8×10⁵ DNA copies per cell; 8×10² DNA copies per cell to 1×10⁶ DNA copies per cell; 8×10² DNA copies per cell to 3×10⁶ DNA copies per cell; 8×10² DNA copies per cell to 6×10⁶ DNA copies per cell; 8×10³ DNA copies per cell to 8×10⁴ DNA copies per cell; 8×10³ DNA copies per cell to 2×10⁵ DNA copies per cell; 8×10³ DNA copies per cell to 4×10⁵ DNA copies per cell; 8×10³ DNA copies per cell to 8×10⁵ DNA copies per cell; 8×10³ DNA copies per cell to 1×10⁶ DNA copies per cell; 8×10³ DNA copies per cell to 3×10⁶ DNA copies per cell; 8×10³ DNA copies per cell to 6×10⁶ DNA copies per cell; 8×10⁴ DNA copies per cell to 2×10⁵ DNA copies per cell; 8×10⁴ DNA copies per cell to 4×10⁵ DNA copies per cell; 8×10⁴ DNA copies per cell to 8×10⁵ DNA copies per cell; 8×10⁴ DNA copies per cell to 1×10⁶ DNA copies per cell; 8×10⁴ DNA copies per cell to 3×10⁶ DNA copies per cell; 8×10⁴ DNA copies per cell to 6×10⁶ DNA copies per cell; 2×10⁵ DNA copies per cell to 4×10⁵ DNA copies per cell; 2×10⁵ DNA copies per cell to 8×10⁵ DNA copies per cell; 2×10⁵ DNA copies per cell to 1×10⁶ DNA copies per cell; 2×10⁵ DNA copies per cell to 3×10⁶ DNA copies per cell; 2×10⁵ DNA copies per cell to 6×10⁶ DNA copies per cell; 4×10⁵ DNA copies per cell to 8×10⁵ DNA copies per cell; 4×10⁵ DNA copies per cell to 1×10⁶ DNA copies per cell; 4×10⁵ DNA copies per cell to 3×10⁶ DNA copies per cell; 4×10⁵ DNA copies per cell to 6×10⁶ DNA copies per cell; 8×10⁵ DNA copies per cell to 1×10⁶ DNA copies per cell; 8×10⁵ DNA copies per cell to 3×10⁶ DNA copies per cell; 8×10⁵ DNA copies per cell to 6×10⁶ DNA copies per cell; 1×10⁶ DNA copies per cell to 3×10⁶ DNA copies per cell; DB1 / 154850959.6 100Attorney Ref. No.: 123690-5005-WO 1×10⁶ DNA copies per cell to 6×10⁶ DNA copies per cell; or 3×10⁶ DNA copies per cell to 6×10⁶ DNA copies per cell.

[0347] In some embodiments, the contacting is performed at a nanoparticle dose of at least 2×10-6ng per cell, at least 5×10-6ng per cell, at least 2×10-5ng per cell, at least 5×10-5ng per cell, at least 2×10-4ng per cell, at least 5×10-4ng per cell, at least 0.00125 ng per cell, at least 0.00125 ng per cell, at least 0.0025 ng per cell, at least 0.005 ng per cell, at least 0.01 ng per cell, at least 0.02 ng per cell, or at least 0.04 ng per cell.

[0348] In some embodiments, the contacting is performed at a nanoparticle dose within a range of 2×10⁻⁶ ng per cell to 5×10⁻⁶ ng per cell; 2×10⁻⁶ ng per cell to 2×10⁻⁵ ng per cell; 2×10⁻⁶ ng per cell to 5×10⁻⁵ ng per cell; 2×10⁻⁶ ng per cell to 2×10⁻⁴ ng per cell; 2×10⁻⁶ ng per cell to 5×10⁻⁴ ng per cell; 2×10⁻⁶ ng per cell to 0.00125 ng per cell; 2×10⁻⁶ ng per cell to 0.0025 ng per cell; 2×10⁻⁶ ng per cell to 0.005 ng per cell; 2×10⁻⁶ ng per cell to 0.01 ng per cell; 2×10⁻⁶ ng per cell to 0.02 ng per cell; 2×10⁻⁶ ng per cell to 0.04 ng per cell; 5×10⁻⁶ ng per cell to 2×10⁻⁵ ng per cell; 5×10⁻⁶ ng per cell to 5×10⁻⁵ ng per cell; 5×10⁻⁶ ng per cell to 2×10⁻⁴ ng per cell; 5×10⁻⁶ ng per cell to 5×10⁻⁴ ng per cell; 5×10⁻⁶ ng per cell to 0.00125 ng per cell; 5×10⁻⁶ ng per cell to 0.0025 ng per cell; 5×10⁻⁶ ng per cell to 0.005 ng per cell; 5×10⁻⁶ ng per cell to 0.01 ng per cell; 5×10⁻⁶ ng per cell to 0.02 ng per cell; 5×10⁻⁶ ng per cell to 0.04 ng per cell; 2×10⁻⁵ ng per cell to 5×10⁻⁵ ng per cell; 2×10⁻⁵ ng per cell to 2×10⁻⁴ ng per cell; 2×10⁻⁵ ng per cell to 5×10⁻⁴ ng per cell; 2×10⁻⁵ ng per cell to 0.00125 ng per cell; 2×10⁻⁵ ng per cell to 0.0025 ng per cell; 2×10⁻⁵ ng per cell to 0.005 ng per cell; 2×10⁻⁵ ng per cell to 0.01 ng per cell; 2×10⁻⁵ ng per cell to 0.02 ng per cell; or 2×10⁻⁵ ng per cell to 0.04 ng per cell.

[0349] In some embodiments, the contacting comprises admixing a solution comprising the nanoparticle with the drawn blood at a weight-to-volume ratio of nanoparticle dose to drawn blood of at least 0.1 ng / mL, at least 1 ng / mL, at least 5 ng / mL, at least 10 ng / mL, at least 25 ng / mL, at least 50 ng / mL, at least 100 ng / mL, at least 200 ng / mL, at least 400 ng / mL, at least 800 ng / mL, at least 1000 ng / mL, at least 1200 ng / mL, at least 1400 ng / mL, at least 1600 ng / mL, at least 1800 ng / mL, or at least 2000 ng / mL.

[0350] In some embodiments, the contacting comprises admixing a solution comprising the nanoparticle with the drawn blood at a weight-to-volume ratio of nanoparticle dose to drawn blood within a range of 0.1 ng / mL to 1 ng / mL; 0.1 ng / mL to 5 ng / mL; 0.1 ng / mL to 10 ng / mL; DB1 / 154850959.6 101Attorney Ref. No.: 123690-5005-WO 0.1 ng / mL to 25 ng / mL; 0.1 ng / mL to 50 ng / mL; 0.1 ng / mL to 100 ng / mL; 0.1 ng / mL to 200 ng / mL; 0.1 ng / mL to 400 ng / mL; 0.1 ng / mL to 800 ng / mL; 0.1 ng / mL to 1000 ng / mL; 0.1 ng / mL to 1200 ng / mL; 0.1 ng / mL to 1400 ng / mL; 0.1 ng / mL to 1600 ng / mL; 0.1 ng / mL to 1800 ng / mL; 0.1 ng / mL to 2000 ng / mL; 1 ng / mL to 5 ng / mL; 1 ng / mL to 10 ng / mL; 1 ng / mL to 25 ng / mL; 1 ng / mL to 50 ng / mL; 1 ng / mL to 100 ng / mL; 1 ng / mL to 200 ng / mL; 1 ng / mL to 400 ng / mL; 1 ng / mL to 800 ng / mL; 1 ng / mL to 1000 ng / mL; 1 ng / mL to 1200 ng / mL; 1 ng / mL to 1400 ng / mL; 1 ng / mL to 1600 ng / mL; 1 ng / mL to 1800 ng / mL; 1 ng / mL to 2000 ng / mL; 5 ng / mL to 10 ng / mL; 5 ng / mL to 25 ng / mL; 5 ng / mL to 50 ng / mL; 5 ng / mL to 100 ng / mL; 5 ng / mL to 200 ng / mL; 5 ng / mL to 400 ng / mL; 5 ng / mL to 800 ng / mL; 5 ng / mL to 1000 ng / mL; 5 ng / mL to 1200 ng / mL; 5 ng / mL to 1400 ng / mL; 5 ng / mL to 1600 ng / mL; 5 ng / mL to 1800 ng / mL; or 5 ng / mL to 2000 ng / mL.

[0351] In some embodiments, the contacting is performed at a defined ratio of DNA copy number to cells. It is understood that the DNA copy number is to refer to the total number of DNA molecules, such as the total number of double stranded DNA as comprised in a nanoparticle in accordance with the disclosure, such as in an LNP or tLNP. For example in the context of minicircle DNA comprised in a tLNP, copy number refers to the number of double stranded minicircle DNA molecules. The copy number of a nanoparticle can easily be calculated, either based on the amount of DNA that is incorporated in a preparation, or by determining the DNA concentration (e.g. via qPCR) and thus DNA copy number concentration and / or total DNA copy number comprised in an tLNP dose (i.e. amount of DNA comprised in a defined volume of tLNP prepared). Hence, the ratio of DNA copy number per cell can easily be selected or determined, when admixing a certain amount or defined dose of a tLNP preparation and cells contained in a certain volume e.g. whole blood, having a certain cell concentration (cell / mL). Preferably, the total dose of nucleic acid comprises DNA to mRNA in a weight ratio of about 1:1. Total nucleic acid may thus preferably comprise mcDNA and an mRNA encoding transposase, or the like, and as described herein and as shown in the examples, in a 1:1 ratio by weight of about 1:1. More preferably, the ratio is 1:1.

[0352] Hence, in some embodiments, the contacting is performed at a ratio of DNA copy number to cells of at least 6×102DNA copies per cell, at least 8×102DNA copies per cell, at least 6×103DNA copies per cell, at least 8×103DNA copies per cell, at least 6×104DNA copies per cell, at least 8×104DNA copies per cell, at least 1×105DNA copies per cell, at least 2×105DB1 / 154850959.6 102Attorney Ref. No.: 123690-5005-WO DNA copies per cell, at least 4×105DNA copies per cell, at least 6×105DNA copies per cell, at least 8×105DNA copies per cell, at least 1×106DNA copies per cell, 3×106DNA copies per cell, at least 6×106DNA copies per cell, at least 8×106DNA copies per cell, or at least 1×107DNA copies per cell.

[0353] In some embodiments, the contacting is performed at a ratio of DNA copy number to cells of 8×10² DNA copies per cell to 8×10³ DNA copies per cell; 8×10² DNA copies per cell to 8×10⁴ DNA copies per cell; 8×10² DNA copies per cell to 2×10⁵ DNA copies per cell; 8×10² DNA copies per cell to 4×10⁵ DNA copies per cell; 8×10² DNA copies per cell to 8×10⁵ DNA copies per cell; 8×10² DNA copies per cell to 1×10⁶ DNA copies per cell; 8×10² DNA copies per cell to 3×10⁶ DNA copies per cell; 8×10² DNA copies per cell to 6×10⁶ DNA copies per cell; 8×10³ DNA copies per cell to 8×10⁴ DNA copies per cell; 8×10³ DNA copies per cell to 2×10⁵ DNA copies per cell; 8×10³ DNA copies per cell to 4×10⁵ DNA copies per cell; 8×10³ DNA copies per cell to 8×10⁵ DNA copies per cell; 8×10³ DNA copies per cell to 1×10⁶ DNA copies per cell; 8×10³ DNA copies per cell to 3×10⁶ DNA copies per cell; 8×10³ DNA copies per cell to 6×10⁶ DNA copies per cell; 8×10⁴ DNA copies per cell to 2×10⁵ DNA copies per cell; 8×10⁴ DNA copies per cell to 4×10⁵ DNA copies per cell; 8×10⁴ DNA copies per cell to 8×10⁵ DNA copies per cell; 8×10⁴ DNA copies per cell to 1×10⁶ DNA copies per cell; 8×10⁴ DNA copies per cell to 3×10⁶ DNA copies per cell; 8×10⁴ DNA copies per cell to 6×10⁶ DNA copies per cell; 2×10⁵ DNA copies per cell to 4×10⁵ DNA copies per cell; 2×10⁵ DNA copies per cell to 8×10⁵ DNA copies per cell; 2×10⁵ DNA copies per cell to 1×10⁶ DNA copies per cell; 2×10⁵ DNA copies per cell to 3×10⁶ DNA copies per cell; 2×10⁵ DNA copies per cell to 6×10⁶ DNA copies per cell; 4×10⁵ DNA copies per cell to 8×10⁵ DNA copies per cell; 4×10⁵ DNA copies per cell to 1×10⁶ DNA copies per cell; 4×10⁵ DNA copies per cell to 3×10⁶ DNA copies per cell; 4×10⁵ DNA copies per cell to 6×10⁶ DNA copies per cell; 8×10⁵ DNA copies per cell to 1×10⁶ DNA copies per cell; 8×10⁵ DNA copies per cell to 3×10⁶ DNA copies per cell; 8×10⁵ DNA copies per cell to 6×10⁶ DNA copies per cell; 1×10⁶ DNA copies per cell to 3×10⁶ DNA copies per cell; 1×10⁶ DNA copies per cell to 6×10⁶ DNA copies per cell; or 3×10⁶ DNA copies per cell to 6×10⁶ DNA copies per cell.

[0354] In some embodiments, the contacting is performed at a defined nanoparticle dose of total nucleic acid (in weight) to cells (number). Nanoparticles, in accordance with the invention DB1 / 154850959.6 103Attorney Ref. No.: 123690-5005-WO are to comprise nucleic acid, e.g. an mRNA and DNA, such as a combination of an mRNA encoding a transposase and an mcDNA with compatible ITRs. The total amount of nucleic acid that is comprised in a nanoparticle can easily be calculated, either based on the amount of nucleic acid (such as DNA and RNA) that is incorporated in a preparation, or by determining the nucleic acid concentration (e.g. via qPCR) and thus nucleic acid copy number concentration, while knowing the length of the nucleic acids (e.g. of mRNA and e.g. minicircle DNA), or any other suitable means (e.g. utilizing spectrophotometry and / or suitable dyes). Hence, the nanoparticle dose (i.e. ng of nucleic acid per cell) can easily be selected or determined, when admixing a certain amount or defined dose of a tLNP preparation and cells contained in a certain volume e.g. whole blood, having a certain cell concentration (cell / mL).

[0355] In some embodiments, the contacting is performed at a nanoparticle dose of at least 2×10-6ng of nucleic acid per cell, at least 5×10-6ng of nucleic acid per cell, at least 2×10-5ng per cell, at least 5×10-5ng nucleic acid per cell, at least 2×10-4ng nucleic acid per cell, at least 5×10-4ng nucleic acid per cell, at least 0.00125 ng nucleic acid per cell, at least 0.00125 ng nucleic acid per cell, at least 0.0025 ng nucleic acid per cell, at least 0.005 ng nucleic acid per cell, at least 0.01 ng nucleic acid per cell, at least 0.02 ng nucleic acid per cell, or at least 0.04 ng nucleic acid per cell.

[0356] In some embodiments, the contacting is performed at a nanoparticle dose within a range of 2×10⁻⁶ ng nucleic acid per cell to 5×10⁻⁶ ng nucleic acid per cell; 2×10⁻⁶ ng nucleic acid per cell to 2×10⁻⁵ ng nucleic acid per cell; 2×10⁻⁶ ng nucleic acid per cell to 5×10⁻⁵ ng nucleic acid per cell; 2×10⁻⁶ ng nucleic acid per cell to 2×10⁻⁴ ng nucleic acid per cell; 2×10⁻⁶ ng nucleic acid per cell to 5×10⁻⁴ ng nucleic acid per cell; 2×10⁻⁶ ng nucleic acid per cell to 0.00125 ng nucleic acid per cell; 2×10⁻⁶ ng nucleic acid per cell to 0.0025 ng nucleic acid per cell; 2×10⁻⁶ ng nucleic acid per cell to 0.005 ng nucleic acid per cell; 2×10⁻⁶ ng nucleic acid per cell to 0.01 ng nucleic acid per cell; 2×10⁻⁶ ng nucleic acid per cell to 0.02 ng nucleic acid per cell; 2×10⁻⁶ ng nucleic acid per cell to 0.04 ng nucleic acid per cell; 5×10⁻⁶ ng nucleic acid per cell to 2×10⁻⁵ ng nucleic acid per cell; 5×10⁻⁶ ng nucleic acid per cell to 5×10⁻⁵ ng nucleic acid per cell; 5×10⁻⁶ ng nucleic acid per cell to 2×10⁻⁴ ng nucleic acid per cell; 5×10⁻⁶ ng nucleic acid per cell to 5×10⁻⁴ ng nucleic acid per cell; 5×10⁻⁶ ng nucleic acid per cell to 0.00125 ng nucleic acid per cell; 5×10⁻⁶ ng nucleic acid per cell to 0.0025 ng nucleic acid per cell; 5×10⁻⁶ ng nucleic acid per cell to DB1 / 154850959.6 104Attorney Ref. No.: 123690-5005-WO 0.005 ng nucleic acid per cell; 5×10⁻⁶ ng nucleic acid per cell to 0.01 ng nucleic acid per cell; 5×10⁻⁶ ng nucleic acid per cell to 0.02 ng nucleic acid per cell; 5×10⁻⁶ ng nucleic acid per cell to 0.04 ng nucleic acid per cell; 2×10⁻⁵ ng nucleic acid per cell to 5×10⁻⁵ ng nucleic acid per cell; 2×10⁻⁵ ng nucleic acid per cell to 2×10⁻⁴ ng nucleic acid per cell; 2×10⁻⁵ ng nucleic acid per cell to 5×10⁻⁴ ng nucleic acid per cell; 2×10⁻⁵ ng nucleic acid per cell to 0.00125 ng nucleic acid per cell; 2×10⁻⁵ ng nucleic acid per cell to 0.0025 ng nucleic acid per cell; 2×10⁻⁵ ng nucleic acid per cell to 0.005 ng nucleic acid per cell; 2×10⁻⁵ ng nucleic acid per cell to 0.01 ng nucleic acid per cell; 2×10⁻⁵ ng nucleic acid per cell to 0.02 ng nucleic acid per cell; or 2×10⁻⁵ ng nucleic acid per cell to 0.04 ng nucleic acid per cell.

[0357] In some embodiments, the contacting comprises admixing a solution comprising the nanoparticle with the drawn blood at a weight-to-volume ratio of nanoparticle dose to drawn blood of at least 0.1 ng nucleic acid / mL, at least 1 ng nucleic acid / mL, at least 5 ng nucleic acid / mL, at least 10 ng nucleic acid / mL, at least 25 ng nucleic acid / mL, at least 50 ng nucleic acid / mL, at least 100 ng nucleic acid / mL, at least 200 ng nucleic acid / mL, at least 400 ng nucleic acid / mL, at least 800 ng nucleic acid / mL, at least 1000 ng nucleic acid / mL, at least 1200 ng nucleic acid / mL, at least 1400 ng nucleic acid / mL, at least 1600 ng nucleic acid / mL, at least 1800 ng nucleic acid / mL, or at least 2000 ng nucleic acid / mL.

[0358] In some embodiments, the contacting comprises admixing a solution comprising the nanoparticle with the drawn blood at a weight-to-volume ratio of nanoparticle dose to drawn blood within a range of 0.1 ng nucleic acid / mL to 1 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 5 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 10 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 25 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 50 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 100 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 200 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 400 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 800 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 1000 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 1200 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 1400 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 1600 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 1800 ng nucleic acid / mL; 0.1 ng nucleic acid / mL to 2000 ng nucleic acid / mL; 1 ng nucleic acid / mL to 5 ng nucleic acid / mL; 1 ng nucleic acid / mL to 10 ng nucleic acid / mL; 1 ng nucleic acid / mL to 25 ng nucleic acid / mL; 1 ng nucleic acid / mL to 50 ng nucleic acid / mL; 1 ng nucleic acid / mL to 100 ng nucleic acid / mL; 1 ng nucleic DB1 / 154850959.6 105Attorney Ref. No.: 123690-5005-WO acid / mL to 200 ng nucleic acid / mL; 1 ng nucleic acid / mL to 400 ng nucleic acid / mL; 1 ng nucleic acid / mL to 800 ng nucleic acid / mL; 1 ng nucleic acid / mL to 1000 ng nucleic acid / mL; 1 ng nucleic acid / mL to 1200 ng nucleic acid / mL; 1 ng nucleic acid / mL to 1400 ng nucleic acid / mL; 1 ng nucleic acid / mL to 1600 ng nucleic acid / mL; 1 ng nucleic acid / mL to 1800 ng nucleic acid / mL; 1 ng nucleic acid / mL to 2000 ng nucleic acid / mL; 5 ng nucleic acid / mL to 10 ng nucleic acid / mL; 5 ng nucleic acid / mL to 25 ng nucleic acid / mL; 5 ng nucleic acid / mL to 50 ng nucleic acid / mL; 5 ng nucleic acid / mL to 100 ng nucleic acid / mL; 5 ng nucleic acid / mL to 200 ng nucleic acid / mL; 5 ng nucleic acid / mL to 400 ng nucleic acid / mL; 5 ng nucleic acid / mL to 800 ng nucleic acid / mL; 5 ng nucleic acid / mL to 1000 ng nucleic acid / mL; 5 ng nucleic acid / mL to 1200 ng nucleic acid / mL; 5 ng nucleic acid / mL to 1400 ng nucleic acid / mL; 5 ng nucleic acid / mL to 1600 ng nucleic acid / mL; 5 ng nucleic acid / mL to 1800 ng nucleic acid / mL; or 5 ng nucleic acid / mL to 2000 ng nucleic acid / mL.

[0359] Table 3 below is a list of exemplary lipid nanoparticle (LNP) doses calculated in weight- to-cell ratio (ng / cell) or DNA copy number (DNA copy number per cell). Herein the mcDNA has a size of about 3 kB. In these exemplary nanoparticles, RNA and mcDNA is packaged in a 1:1 ratio by weight. The tLNP dose listed in the table is the total weight of nucleic acid (i.e. RNA and mcDNA) as contained in lipid nanoparticles which is contacted with a defined amount of cells, i.e.20.000 cells. From the dose contacted with this number of cells, the dose nucleic acid per cell can easily be calculated. For the number of DNA copies per cell, if one assumes that the total dose of nucleic acid corresponds with 100% mcDNA, this means 1 ng nucleic acid corresponds with 3.26 x 108copies of mcDNA. As in this scenario, the ratio of mcDNA to RNA is 1:1 by weight, this means the actual copy number in the dose needs to be needs to be divided by 2, to get the actual copy number. For the 1 ng, this means a DNA copy number of 1.63 x 108. When brought into contact with 20,000 cells, this translates to 8,150 DNA copies per cell.

[0360] Table 3. lipid nanoparticle (LNP) doses and DNA copy numbers. tLNP dose ng nucleic tLNP dose (ng DNA copy DNA copies per acid / cell nucleic acid) number per dose cell 00.000005 0.1 1.63E+07 8.15E+020.00005 1 1.63E+08 8.15E+03DB1 / 154850959.6 106Attorney Ref. No.: 123690-5005-WO 0.0005 10 1.63E+09 8.15E+040.00125 25 4.07E+09 2.04E+050.0025 50 8.15E+09 4.07E+050.005 100 1.63E+10 8.15E+050.01 200 3.26E+10 1.63E+060.02 400 6.52E+10 3.26E+060.04 800 1.30E+11 6.52E+06

[0361] In some embodiments, the admixing immediately follows drawing the blood from the subject, ensuring there is minimal delay between blood collection and processing. Immediate admixing may help preserve cell viability and functionality by preventing prolonged exposure of immune effector cells to potentially suboptimal conditions.

[0362] In some embodiments, the admixing occurs within twenty-four hours of drawing the blood from the subject, twelve hours of drawing the blood from the subject, within four hours of drawing the blood from the subject, within an hour of drawing the blood from the subject. In some embodiments, the admixing occurs within a specific range of time intervals selected from the group consisting of twenty-four hours and twelve hours, twenty-four hours and four hours, twenty-four hours and one hour, twelve hours and four hours, twelve hours and one hour, and four hours and one hour of drawing the blood from the subject.

[0363] In other embodiments, the admixing is performed within 1 hour, forth-five minutes, thirty minutes, fifteen minutes, ten minutes, five minutes, or one minute of drawing the blood from the subject. In some embodiments, the admixing occurs within a specific range of time intervals selected from the group consisting of one hour and forty-five minutes, one hour and thirty minutes, one hour and fifteen minutes, one hour and ten minutes, one hour and five minutes, one hour and one minute, forty-five minutes and thirty minutes, forty-five minutes and fifteen minutes, forty-five minutes and ten minutes, forty-five minutes and five minutes, forty- five minutes and one minute, thirty minutes and fifteen minutes, thirty minutes and ten minutes, thirty minutes and five minutes, thirty minutes and one minute, fifteen minutes and ten minutes, fifteen minutes and five minutes, fifteen minutes and one minute, ten minutes and five minutes, ten minutes and one minute, or five minutes and one minute after drawing the blood from the subject. DB1 / 154850959.6 107Attorney Ref. No.: 123690-5005-WO

[0364] In yet other embodiments, the admixing is performed concurrently with the drawing of the blood from the subject. In some embodiment, the concurrent admixing is achieved by a real- time or inline mixing approach, for example by using specialized blood collection devices or systems that introduce the reagents immediately as the blood is drawn from a subject. This method could help further minimize processing time and reduce handling procedure steps, thereby ensuring rapid and efficient preparation of the treated blood fraction.

[0365] After the transfection, the immune effector cells, or the drawn blood or a fraction thereof containing the immune effector cells, are returned to the subject by infusion.

[0366] In some embodiments, the immune effector cells, or the drawn blood or a fraction thereof containing the immune effector cells, are infused to the subject within thirty-six hours, twenty-four hours, twelve hours, six hours, four hours, two hours, or one hour of drawing the blood from the subject. In some embodiments, the infusion occurs within a specific range of time interval selected from the group consisting of between thirty-six hours and twenty-four hours, between thirty-six hours and twelve hours, between thirty-six hours and six hours, between thirty-six hours and four hours, between thirty-six hours and two hours, between thirty-six hours and one hour, between twenty-four hours and twelve hours, between twenty-four hours and six hours, between twenty-four hours and four hours, between twenty-four hours and two hours, between twenty-four hours and one hour, between twelve hours and six hours, between twelve hours and four hours, between twelve hours and two hours, between twelve hours and one hour, between six hours and four hours, between six hours and two hours, between six hours and one hour, between four hours and two hours, between four hours and one hour, and between two hours and one hour, of drawing the blood from the subject. EXAMPLES Example 1 – Methods for extracorporeal transfection of an immune effector cell

[0367] To show the potential of extracorporeal transfection for CAR-T manufacturing using tLNPs, an in vitro test was performed to assess the capability of tLNPs to transfect and activate T cells in whole blood in a short time period. For this purpose, LNPs encapsulating minicircle DNA encoding GFP (used as reporter) were formulated, after which they were post-inserted with PEG lipids conjugated to anti-CD7 VHH and an anti CD3scFv. This post-insertion ensures the DB1 / 154850959.6 108Attorney Ref. No.: 123690-5005-WO tLNPs are specifically targeted to T- and NK cells and can induce T cell activation by engaging CD3 on the surface of T cells.

[0368] These targeted and activating tLNPs were incubated with healthy donor blood for 10 minutes, 2 hours and 6 hours. After the indicated incubation times, PBMC were isolated from the blood using Ficoll Paque density centrifugation. The PBMCs were subjected to rigorous washing after the initial isolation, to ensure removal of the excess tLNPs, that were not bound to- or taken up by cells. Subsequently, PBMCs were cultured for 4 days, to allow GFP expression driven by DNA (Figure 2). Transfection efficiency of T cells was determined by measuring GFP signal. T- cell transfection was achieved in all tested conditions, a tLNP incubation period as short as 10 minutes lead to a transfection efficiency of 5%, which increased up to almost 20% when the incubation was prolonged to 6 hours (Figure 2, T cells chart). B cell transfection was not seen at any of the time points (Figure 2, B cells chart).

[0369] 1. Material and Methods

[0370] 1.1 tLNP production

[0371] Lipid nanoparticles (LNPs) were synthesized using standard procedures employing a microfluidic device to combine an aqueous phase containing nucleic acids with an ethanol phase containing lipid and cholesterol components. The aqueous phase comprised nucleic acids such as, minicircle DNA encoding for GFP or CD19 CAR, and SB100X mRNA in a citrate buffer, while the ethanol phase contained specific types of phospholipids, ionizable lipids, cholesterol, and PEGylated lipids at predetermined molar ratios. The aqueous and ethanol phases were subsequently mixed in the microfluidic device at a specific ratio. The resulting LNPs were then targeted with DSPE-PEG-VHH-CD7 and CD3 scFv using a post-insertion method. The concentration of encapsulated nucleic acid and the encapsulation efficiency (EE%) of the targeted LNPs were determined using the Quant-iT Ribogreen Assay kit.

[0372] 1.2 Whole blood tLNP transfection

[0373] Healthy donor blood was collected using VACUETTE TUBE 2 ml 9NC Coagulation sodium citrate 3.2% (Greiner). The blood was plated 500 µl per well in a 24 well plate and tLNP encapsulating a 3000 base pair minicircle DNA (mcDNA) encoding a green fluorescent protein gene flanked by Sleeping Beauty ITRs and an mRNA encoding the SB100x transposase at a 1:1 DB1 / 154850959.6 109Attorney Ref. No.: 123690-5005-WO mass ratio was added at an amount of 6.5 µg total nucleic acids, corresponding to a dose of approximately 5.3x10^7 mcDNA per cell. After the indicated transfection time, PBMCs were isolated by ficoll-paque density gradient centrifugation (Gibco) using 15 ml Sepmate Tubes (Stemcell Technologies) according to manufacturer’s protocol. The isolated PBMCs were maintained for 4 days at 37 degrees 5% CO2 in RPMI 1640 (Thermo Scientific) medium containing 2.5% heath inactivated pooled human Serum (Sanquin), 1% Pen / Strep (Gibco), 2mM 2-mercaptoethanol (Gibco)and 50 iU / ml IL-2 (Mitenyi Biotec). After four days, GFP expression on T- and B cells was measured on FACS Canto, B cells were stained using CD19-PE (Antibodies- online), and T cells using CD3-APC ( BD biosciences), as illustrated in Figure 3.

[0374] See also Nawaz, W. et al. AAV-mediated in vivo CAR gene therapy for targeting human T-cell leukemia. Blood Cancer J 11, 119 (2021); Rogers, G.L. et al. Optimization of AAV6 transduction enhances site-specific genome editing of primary human lymphocytes. Mol Ther Methods Clin Dev 23, 198-209 (2021); Lam, N. et al. Anti-BCMA chimeric antigen receptors with fully human heavy-chain-only antigen recognition domains. Nat Commun 11, 283 (2020); Ghassemi, S. et al. Rapid manufacturing of non-activated potent CAR T cells. Nat Biomed Eng 6, 118-128 (2022), each of which is hereby incorporated by reference in its entirety for all purposes. Example 2

[0375] 2.1 – Specific targeting to T cells and NK cells with CD7-targeted lipid nanoparticles

[0376] To measure the targeting specificity of LNPs to T cells and NK cells in vitro, mRNA conjugated with fluorescent dye Cy5 was encapsulated in lipid nanoparticles (LNPs). The loaded LNPs were then either functionalized by integrating PEG lipids conjugated to anti-CD7 VHH (tLNPs) or left unfunctionalized (untargeted). The anti-CD7 VHH is surface exposed in the tLNPs. The functionalized (tLNPs) and unfunctionalized (untargeted) were added to aliquots of peripheral blood mononuclear cells (PBMCs) for 2 hours. Cell transfection following the incubation was determined by measuring the Cy5 signal of each cell type using flow cytometry. Figure 4 illustrates the specific targeting of CD7-targeted LNPs to T cells and / or NK cells. Specifically, in the PMBC population incubated with the untargeted LNPs, less than half of the cells showing Cy5 transfection were either T-cells or NK-cells, consistent with indiscriminate transfection of cell type. However, in the PMBC population incubated with the targeted LNPs DB1 / 154850959.6 110Attorney Ref. No.: 123690-5005-WO (tLNP), nearly all of the cells showing Cy5 transfection were either T-cells or NK-cells, demonstrating selective targeting and transfection of these cell types.

[0377] 2.2 – Efficient generation of proliferative CAR-T cells in whole blood within 6 hours of incubation time; proliferation over time can be induced by the addition of target cells (antigen-induced proliferation)

[0378] To attain stable DNA expression of a chimeric antigen receptor (CAR) in T cells, an LNP formulation co-delivering DNA and mRNA was prepared and targeted to T cells. Briefly, LNPs encapsulating a 3800 base pair minicircle DNA (mcDNA) encoding an anti-CD19 CAR flanked by Sleeping Beauty ITRs and an mRNA encoding the SB100x transposase were formulated. The mcDNA and mRNAs were encapsulated at a 1:1 mass ratio. These LNPs were then functionalized with an anti-CD7 variable heavy domain of heavy chain (VHH) and an anti- CD3 single-chain variable fragment (scFv) by inserting lipids conjugated to the anti-CD7 VHH and lipids conjugated to the anti-CD3 scFv into the LNPs using a previously described post- insertion method (Swart, L. E. et al. A robust post-insertion method for the preparation of targeted siRNA LNPs. Int J Pharm 620, 121741 (2022)).

[0379] The loaded LNPs were then added to 500 µl of whole human blood at amounts of 50 ng, 100 ng, 400 ng, and 800 ng, measured as total nucleic acid mass. These doses correspond to approximately 3.2x10^5, 6.4x10^5, 2.6x10^6, and 5.2x10^6 mcDNA per cell, respectively. The blood was then incubated while shaking for 6 hours at 37 degrees °C. After the 6-hour incubation period, PBMCs were isolated from the whole blood, washed to further remove any remaining free tLNPs, and resuspended in culture medium.

[0380] To evaluate transfection and integration of the CAR DNA into the genome of T-cells, the percentage of CD3+ cells that expressed the anti-CD19 CAR were determined over time, as CD3+ is a defining marker for T cells. Figure 5 shows these percentages as a function of time post-transfection. As indicated by these results, all four doses of tLNP treatment were effective in transfection and stable genomic integration. DB1 / 154850959.6 111Attorney Ref. No.: 123690-5005-WO

[0381] 2.3 – Efficient LNP-mediated T-cell transfection in whole blood leading to generation of proliferative CAR-T cells within 1 hour of incubation time

[0382] To study conditions for efficient LNP-mediated T-cell transfection to generate proliferative CAR-T cells in whole blood, minicircle CAR CD19 and SB100x mRNA were loaded into CD7 / CD3-targeted LNPs, as described in Example 2.2. Different amounts of the loaded LNPs (50 ng, 100 ng, 400 ng, and 800 ng) were added to 500 µl of whole blood and incubated (while shaking) for different durations (30 min, 1 hour, 2 hours, 4 hours, and 6 hours) at 37 degrees °C. After the respective incubation period, PBMCs were isolated from the whole blood, washed, and resuspended in culture medium. To evaluate T-cell proliferation upon presentation of CD19 (antigen-induced proliferation), each culture was split 10-days post incubation and NALM6 tumor cells were added to half of the cultures at a 1: 1 ratio of T cells in the cultures. NALM6 is a B cell precursor leukemia cell line expressing CD19. The NALM6 cell line is available, for example, under accession number ACC 128 from the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH.

[0383] The percentage of CD3+ cells expressing the anti-CD19 CAR in each culture was determined over time, as described in Example 2.2. Figure 6 shows these percentages as a function of time post-transfection. Significantly, proliferation of cells treated with all four doses for at least one hour were stimulated by exposure to the NALM6 cells, as indicated by the increase in the percentage of CD3+ cells that were also CAR CD19+.

[0384] 2.4 – Efficient LNP-mediated T-cell transfection in isolated PBMCs leading to generation of CAR-T cells within 4 hours of incubation time

[0385] To study conditions for efficient LNP-mediated T-cell transfection of peripheral blood mononuclear cells (PMBC) cells separated from whole blood to generate proliferative CAR-T cells, minicircle CAR CD19 and SB100x mRNA were loaded into CD7 / CD3-targeted LNPs, as described in Example 2.2. PBMCs isolated from whole blood were then incubated with the targeted LNPs (tLNPs) for 4 hours at a range of cell densities and tLNP concentrations. As indicated in Figure 7, incubation was performed using cell densities ranging from 300,000 cells / mL (mio / mL) to 20 mio / mL, at concentrations of tLNP from 0.01 ng / cell to 0.01 ng / cell, measured as total nucleic acids. These correspond to doses of approximately 1.3x10^5 to 1.3x10^6 mcDNA per cell. Incubation (transfection) was done in 100 µl total volume in LNP DB1 / 154850959.6 112Attorney Ref. No.: 123690-5005-WO freezing medium (TBS, 8% sucrose) directly or diluted in PBS. Incubation was done for 4 hours in an incubator (not shaking) at 37 °C. After the incubation, cells were washed and resuspended in culture medium.

[0386] Five days after incubation, the percentage of CD3+ cells expressing the anti-CD19 CAR in each culture was determined, as described in Example 2.2. Figure 7 shows these percentages as functions of cell density and tLNP dose. The data shown in Figure 7, suggests dose- dependent and cell culture density-dependent effects of the tLNP treatment.

[0387] 2.5 – Stable CAR expression induced by LNP-mediated T-cell transfection in isolated PBMCs within 10 minutes of incubation time

[0388] To study and compare conditions for stable CAR expression induced by LNP-mediated T-cell transfection in isolated PBMCs or whole blood, minicircle CAR CD19 and SB100x mRNA were loaded into CD7 / CD3-targeted LNPs, as described in Example 2.2. Whole blood transfection was performed by adding 16 µg of loaded LNPs, measured as total nucleic acid mass, into 25 ml of blood. The incubation was performed at room temperature in a 50 ml tube that was rolling. After 4 hours of incubation, PBMCs were isolated from the blood, washed, and kept in the culture. tLNP transfection of PBMC was performed using cells from the same healthy donor as the whole blood transfection. PBMCs were isolated and were transfected at a cell density of 30 million / ml and at a LNP dose of 0.003 ng LNP / cell, measured as total nucleic acid mass, in a total volume of 60 µl in LNP freezing medium (TBS, 8% sucrose). Incubation was performed for 10 minutes at room temperature (no shaking). After the incubation, the cells were washed in PBS and resuspended in the medium. Cells were then cultured for 4 weeks and the percentage of CD3+ cells expressing the anti-CD19 CAR in each culture was determined over time, as described in Example 2.2. Figure 8 shows these percentages as a function of time.

[0389] An advantage of the disclosure is the recognition that activation of resting immune effector cells, such as T-cells, reorganizes the nuclear membrane, facilitating importation of recombinant DNA transfected in the cell by the disclosed LNPs for stable integration into the genome. This can be achieved by stimulating a cell-surface receptor via a non-activating immune effector binding moiety, e.g., by functionalizing the LNP with an anti-CD3 immune effector binding moiety. Alternatively, this can be achieved by including an mRNA encoding a CAR / TCR in the LNP to promote early expression of the CAR / TCR, facilitating activation of the DB1 / 154850959.6 113Attorney Ref. No.: 123690-5005-WO T-cell. In some embodiments, a non-activating immune effector binding moiety, an mRNA encoding a CAR / TCR in the LNP, or both are utilized in the LNP to promote genomic integration, as well as stable expression of CAR / TCR in the immune effector cell after transfection. As a result, the LNP described herein facilitates the rapid preparation of CAR-T cells allowing the process to be performed bedside / same day and eliminating the need for lengthy CAR-T production processes.

[0390] Based on the data provided in the examples, the described LNP-mediated transfection approach demonstrates efficient generation of CAR-T cells in a rapid manner. Specifically, the data illustrate that incubation of peripheral blood mononuclear cells (PBMCs) or whole blood with tLNPs containing minicircle CAR DNA and SB100x mRNA resulted in stable CAR expression in T cells. These results support the theory that activation of resting T-cells reorganizes the nuclear membrane, facilitating the importation and genomic integration of recombinant DNA transfected via the disclosed LNPs. Furthermore, the rapid expression of CAR in CD3+ cells, observed within hours post-transfection, corroborates the role of functionalized tLNPs in promoting early activation and genomic integration. The ability to achieve stable CAR expression in a short timeframe enables bedside or same-day CAR-T preparation, eliminating the need for prolonged ex vivo expansion and conventional manufacturing steps. REFERENCES CITED AND ALTERNATIVE EMBODIMENTS

[0391] All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0392] The present invention can be implemented as a computer program product that includes a computer program mechanism embedded in a non-transitory computer-readable storage medium. For instance, the computer program product could contain instructions for operating the user interfaces disclosed herein and described with respect to the Figures. These program modules can be stored on a CD-ROM, DVD, magnetic disk storage product, USB key, or any other non-transitory computer readable data or program storage product. DB1 / 154850959.6 114Attorney Ref. No.: 123690-5005-WO

[0393] Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only. The embodiments were chosen and described in order to best explain the principles of the invention and its practical applications, to thereby enable others skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated. The invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. DB1 / 154850959.6 115

Claims

Attorney Ref. No.: 123690-5005-WO WHAT IS CLAIMED IS:

1. A method for making a transfected immune effector cell, the method comprising contactingblood, or a cell-containing fraction thereof, from a subject with a nanoparticle ex vivo, wherein the nanoparticle comprises: a surface-exposed immune effector binding moiety; and a deoxyribonucleic acid encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR).2 The method of claim 1, wherein the contacting comprises drawing blood from the subject andadmixing a solution comprising the nanoparticle with the drawn blood.3 The method of claim 2, wherein the blood is drawn into an apheresis bag and the admixingcomprises adding the solution into the apheresis bag.4 The method of claim 2 or 3, wherein an anticoagulant is added to the solution comprising thenanoparticle prior to, during, or after the admixing.5 The method of claim 4, wherein an anticoagulant is sodium citrate.6 The method of claim 2 or 3, further comprising, after the admixing, returning the blood to thesubject.7 The method of claim 1, wherein the contacting comprises apheresis of blood drawn from thesubject, thereby generating a first blood fraction comprising immune effector cells and a second blood fraction free of immune effector cells, followed by admixing a solution comprising the nanoparticle with the first blood fraction.8 The method of claim 1, wherein the contacting comprises admixing a solution comprising thenanoparticle with blood drawn from the subject, followed by apheresis of the blood, thereby generating a first blood fraction comprising immune effector cells and a second blood fraction free of immune effector cells. DB1 / 154850959.6 116Attorney Ref. No.: 123690-5005-WO9. The method of any one of claims 1 to 8, wherein the immune effector cells comprise peripheralblood mononuclear cells (PBMCs).

10. The method of claim 7 or 8, wherein the apheresis is leukopheresis.

11. The method of any one of claims 7 to 10, wherein the first blood fraction is fractionated into anapheresis bag and the admixing comprises adding the solution into the apheresis bag.

12. The method of any one of claims 7 to 11, further comprising, after the contacting, separatingcells in the first blood fraction from free nanoparticles.

13. The method of claim 12, wherein the separating comprises washing the cells.

14. The method of 12 or 13, wherein the separating comprises plasmapheresis, filtering with aspinning membrane filter, or centrifugation.

15. The method of any one of claims 7 to 14, further comprising, returning cells from the first bloodfraction to the subject.

16. The method of any one of claims 1 to 15, wherein the contacting occurs in a closed vein-to-veinsystem.

17. The method of any one of claims 1-16, wherein the contacting is performed at a cell density of atleast 250,000 cells per mL, at least 1 million cells per mL, at least 5 million cells per mL, or at least 10 million cells per mL.

18. The method of any one of claims 1-16, wherein the contacting is performed at a cell density of atleast 1 million cells per mL.

19. The method of any one of claims 1-16, wherein the contacting is performed at a cell density of atleast 5 million cells per mL. DB1 / 154850959.6 117Attorney Ref. No.: 123690-5005-WO20. The method of any one of claims 1-16, wherein the contacting is performed at a cell density offrom 1 million cells per mL to 20 million cells per mL.

21. The method of any one of claims 1-16, wherein the contacting is performed at a ratio of DNAcopy number to cells of at least 6×102DNA copies per cell, at least 8×102DNA copies per cell, at least 6×103DNA copies per cell, at least 8×103DNA copies per cell, at least 6×104DNA copies per cell, at least 8×104DNA copies per cell, at least 1×105DNA copies per cell, at least 2×105DNA copies per cell, at least 4×105DNA copies per cell, at least 6×105DNA copies per cell, at least 8×105DNA copies per cell, at least 1×106DNA copies per cell, 3×106DNA copies per cell, or at least 6×106DNA copies per cell.

22. The method of any one of claims 1-16, wherein the contacting is performed at a ratio of DNAcopy number to cells of from 6×102to 6×107DNA copies per cell.

23. The method of any one of claims 1-16, wherein the contacting is performed at a ratio of DNAcopy number to cells of from 6×103to 6×107DNA copies per cell.

24. The method of any one of claims 2-23, wherein the admixing occurs within twenty-four hours ofdrawing the blood from the subject, within four hours of drawing the blood from the subject, or within an hour of drawing the blood from the subject.

25. The method of any one of claims 2-23, wherein the admixing occurs within fifteen minutes ofdrawing the blood from the subject.

26. The method of any one of claims 2-23, wherein the admixing occurs within five minutes ofdrawing the blood from the subject.

27. The method of any one of claims 2-26, wherein the blood, or cell-containing fraction thereof, iscontacted with the nanoparticle for no more than six hours.

28. The method of any one of claims 2-26, wherein the blood, or cell-containing fraction thereof, iscontacted with the nanoparticle for no more than two hours. DB1 / 154850959.6 118Attorney Ref. No.: 123690-5005-WO29. The method of any one of claims 2-26, wherein the blood, or cell-containing fraction thereof, iscontacted with the nanoparticle for no more than one hour.

30. The method of any one of claims 2-26, wherein the blood, or cell-containing fraction thereof, iscontacted with the nanoparticle for no more than 30 minutes.

31. The method of any one of claims 2-26, wherein the blood, or cell-containing fraction thereof, iscontacted with the nanoparticle for no more than 15 minutes.

32. The method of any one of claims 2-31, wherein the blood or cells from the first blood fraction isreturned to the subject within twenty-four hours of drawing the blood from the subject, within twelve hours of drawing the blood from the subject, or within four hours of drawing the blood from the subject.

33. The method of any one of claims 2-31, wherein the blood or cells from the first blood fraction isreturned to the subject within two hours of drawing the blood from the subject.

34. The method of any one of claims 2-31, wherein the blood or cells from the first blood fraction isreturned to the subject within one hour of drawing the blood from the subject.

35. The method of any one of claims 1-34, wherein the nanoparticle is a lipid nanoparticle (LNP).

36. The method of any one of claims 1-35, wherein the immune effector cell is a T-cell.

37. The method of any one of claims 1-35, wherein the immune effector cell is an NK-cell38. The method of claim 36, wherein the surface-exposed immune effector binding moiety bindsCD2, CD4, CD3, CD5, CD7, CD8, CD25, CD127, CD152, CD11, or CD28.

39. The method of claim 38, wherein the CD7 binding moiety is an anti-CD7 antibody or antigenbinding domain thereof.

40. The method of claim 38 or 39, wherein the CD7 binding moiety is an anti-CD7 VHH.DB1 / 154850959.6 119Attorney Ref. No.: 123690-5005-WO41. The method of any one of claims 38-40, wherein the CD7 binding moiety comprises an aminoacid sequence selected from the group consisting of SEQ ID NOS: 142, 146, 150, 154, 158, 162, 166, 170, and 174.

42. The method of any one of claims 38-40, wherein the CD7 binding moiety comprises:CDRs1-3 as set forth in SEQ ID NOs: 143-145, respectively, CDRs1-3 as set forth in SEQ ID NOs: 147-149, respectively, CDRs1-3 as set forth in SEQ ID NOs: 151-153, respectively, CDRs1-3 as set forth in SEQ ID NOs: 155-157, respectively, CDRs1-3 as set forth in SEQ ID NOs: 159-161, respectively, CDRs1-3 as set forth in SEQ ID NOs: 163-165, respectively, CDRs1-3 as set forth in SEQ ID NOs: 167-169, respectively, CDRs1-3 as set forth in SEQ ID NOs: 171-173, respectively, or CDRs1-3 as set forth in SEQ ID NOs: 175-177, respectively.

43. The method of any one of claims 1-42, wherein the nanoparticle comprises a surface-exposedCD3 binding moiety.

44. The method of claim 43, wherein the CD3 binding moiety is an anti-CD3 antibody or antigenbinding domain thereof.

45. The method of claim 44, wherein the CD3 binding moiety comprises an anti-CD3 ScFv.

46. The method of claim 45, wherein the CD3 binding moiety comprisesa heavy chain variable region (VH) as set forth in SEQ ID NOS: 179, and a light chain variable region (VL) as set forth in SEQ ID NOS: 181; or a heavy chain variable region (VH) as set forth in SEQ ID NOS: 189, and a light chain variable region (VL) as set forth in SEQ ID NOS: 191.

47. The method of any one of claims 38-40, wherein the CD3 binding moiety comprises:VH-CDRs 1-3 as set forth in SEQ ID NOs: 182-184, respectively, and VL-CDRs 1-3 CDRs1 as set forth in SEQ ID NOs: 185-187, respectively; or DB1 / 154850959.6 120Attorney Ref. No.: 123690-5005-WO VH-CDRs 1-3 as set forth in SEQ ID NOs: 192-194, respectively, and VL-CDRs 1-3 CDRs1 as set forth in SEQ ID NOs: 195-197, respectively.

48. The method of any one of claims 1-47, wherein the deoxyribonucleic acid encoding the CAR orTCR is a plasmid, a minicircle DNA, a doggybone DNA, a miniplasmid DNA, Tiny plasmid, nanoplasmid or a close-ended DNA (ceDNA).

49. The method of any one of claims 1-47, wherein the deoxyribonucleic acid encoding the CAR orTCR is a minicircle DNA.

50. The method of any one of claims 1-47, wherein the deoxyribonucleic acid encoding the CAR orTCR is an open-ended DNA, or a single-stranded DNA (ssDNA).

51. The method of any one of claims 1-50, wherein:the deoxyribonucleic acid encoding the CAR or TCR comprises a transposable element comprising a gene sequence encoding the CAR or TCR flanked by inverted terminal repeats (ITRs); and the nanoparticle further comprises a nucleic acid encoding a transposase with specificity for the ITRs.

52. The method of claim 51, wherein the transposable element is selected from the group consistingof a Sleeping Beauty (SB) 10 transposable element, a Sleeping Beauty 11 transposable element, a Sleeping Beauty 100X transposable element, a PiggyBac transposable element, a PiggyBat transposable element, a Mu transposable element, a TcBuster transposable element, a Mos1 transposable element, a Tol1 transposable element, a Tol2 transposable element, a Frog Prince transposable element, a spinON transposable element, a HimarI transposable element, a Passport transposable element, a Minos transposable element, a hAT transposable element, an Hsmar1 transposable element, a Harbinger transposable element, a Harbinger3-DR transposable element, an Ac / Ds transposable element, and a PIF transposable element.

53. The method of claim 51, wherein the transposable element is a Sleeping Beauty (SB)transposable element. DB1 / 154850959.6 121Attorney Ref. No.: 123690-5005-WO54. The method of claim 51, wherein the transposable element is a piggyBac (PB) transposableelement.

55. The method of any one of claims 1-50, wherein the nanoparticle comprises one or more nucleicacids encoding a recombinase gene-editing system or a nuclease gene-editing system, wherein the recombinase gene-editing system or the nuclease gene-editing system has specificity for the deoxyribonucleic acid encoding the CAR or TCR.

56. The method of claim 55, wherein the recombinase gene-editing system is selected from thegroup consisting of a Cre system, an FLP system, a lamda integrase system, a phiC31 integrase system, a Bxb1 integrase system, a gamma-delta resolvase system, a Tn3 resolvase system, and a Gin invertase system.

57. The method of 55, wherein the nuclease gene-editing system is select from the group consistingof a CRISPR / Cas system, an ARCUS system, a TALEN system, a meganuclease system, and a zinc finger nuclease system.

58. The method of any one of claims 1-57, wherein the CAR or TCR has affinity for a cell-surfaceantigen selected from the group of genes consisting of TNFRSF17, IL3RA, SDC1, CD19, CD20, CD22, BCMA, MS4A1, CD7, CD123, CD135, CD38, CD138, CD269, TNFRSF8, CD33, CD38, CD5, NCAM1, CD70, ULBP1, ULBP2, IL1RAP, CEACAM5, MET, EGFR, EGFRvIII, EPCAM, EPHA2, ERBB2, GPC3, MSLN, Muc1, PDCD1, CD274, KDR, IL13RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, ROR2, GPRC5D, PSMA, CD23, WT1, CD44, CD44v6, CD174, SLAMF7, L1 CAM, FLT3, Sigle-6, GD2, PSCA, NY-ESO-1, GPNMB, CD276, CSPG4, MAGEA3, MAGEA4, CD133, TEM1, and combinations thereof.

59. The method of any one of claims 1-58, wherein the CAR or TCR has affinity for a single surfaceantigen.

60. The method of any one of claims 1-58, wherein the CAR or TCR has affinity for two surfaceantigens. DB1 / 154850959.6 122Attorney Ref. No.: 123690-5005-WO61. The method of any one of claims 1-58, wherein the CAR or TCR has affinity for three, or more,surface antigens.

62. The method of any one of claims 1-58, wherein the CAR or TCR has affinity for CD19, CD20,CD22, BCMA, or combinations thereof.

63. The method of claim 62, wherein the CAR or TCR has affinity for a pair of cell-surface antigensselected from the group consisting of: CD19 and CD22; CD19 and CD20; CD20 and CD22; BCMA and CD19; BCMA and CD19; and BCMA and CD22.

64. The method of any one of claims 1-63, wherein the CAR has affinity for CD19.

65. The method of claim 64, wherein the CAR has a polypeptide sequence of the CAR used inlisocabtagene maraleucel, tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, or obecabtagene autoleucel.

66. The method of any one of claims 1-65, wherein the CAR has affinity for BCMA.

67. The method of claim 66, wherein the CAR has a polypeptide sequence of the CAR used inidecabtagene vicleucel or ciltacabtagene autoleucel.

68. The method of any one of claims 1-54, wherein the CAR or TCR binds a T cell via interactionwith one or more of the following: T-cell γ chains, T-cell β chains, T-cell δ chains, T-cell constant chains, CCR7, CD3, CD4, CD5, CD7, CD8, CD11b, CD11c, CD16, CD19, CD20, CD21, CD22, CD25, CD28, CD34, CD35, CD40, CD45RA, CD45RO, CD52, CD56, CD62L, CD68, CD80, CD95, CD117, CD127, CD133, CD137 (4-1BB), CD163, F4 / 80, IL-4Rα, Sca-1, DB1 / 154850959.6 123Attorney Ref. No.: 123690-5005-WO CTLA-4, GITR, GARP, LAP, granzyme B, LFA-1, transferrin receptor, and combinations thereof.

69. The method of any one of claims 1-68, wherein the method comprises no treatment of theimmune effector cell with an activation agent ex vivo.

70. The method of claim 69, wherein the activation agent is a cytokine.

71. The method of claim 69, wherein the activation agent is CTS™ (Cell Therapy Systems).

72. The method of claim 69, wherein the activation agent is OKT-3.

73. The method of claim 69, wherein the activation agent is Dynabeads™ CD3 / CD28.

74. The method of claim 69, wherein the activation agent is MACS® GMP T Cell TransAct™.

75. The method of any one of claims 1-74, wherein the subject has a disease or disorder, and themethod further comprises treating the disease or disorder in the subject comprising administering a therapeutically effective amount of the transfected immune effector cells to the subject.

76. The method of claim 75, wherein the disease or disorder is a cancer, an autoimmune disease, oran infectious disease.

77. The method of claim 75, wherein the subject has a cancer selected from the group consisting oflymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, leukemia, liquid tumors, liver cancer, lung cancer, lymphoma, malignant mesothelioma, mastocytoma, melanoma, multiple myeloma, DB1 / 154850959.6 124Attorney Ref. No.: 123690-5005-WO nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, and urinary bladder cancer.

78. The method of claim 75, wherein the subject has an autoimmune disease selected from the groupconsisting of Arthritis, chronic obstructive pulmonary disease, ankylosing spondylitis, Crohn's disease (one of two types of idiopathic inflammatory bowel disease “IBD”), dermatomyositis, diabetes mellitus type 1, endometriosis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's disease, hidradenitis suppurativa, Kawasaki disease, IgA nephropathy, idiopathic thrombocytopenia purpura, interstitial cystitis, lupus erythematosus, mixed connective tissue disease, morphea, myasthenia gravis, narcolepsy, neuromyotonia, pemphigus vulgaris, pernicious anaemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, relapsing polychondritis, rheumatoid arthritis, schizophrenia, scleroderma, Sjogren's syndrome, stiff person syndrome, temporal arteritis (also known as “giant cell arteritis”), ulcerative colitis (one of two types of idiopathic inflammatory bowel disease “IBD”), vasculitis, vitiligo, and Wegener's granulomatosis.

79. The method of claim 75, wherein the subject has an infectious disease selected from the groupconsisting of Acquired Immunodeficiency Syndrome (AIDS), cute or chronic bacterial infection, acute or chronic parasitic infection, acute or chronic viral infection, acute or chronic fungal infection, meningitis, hepatitis (A, B or C, or other viral hepatitis), peritonitis, pneumonia, epiglottitis, malaria, dengue hemorrhagic fever, leishmaniasis, streptococcal myositis, mycobacterium tuberculosis, mycobacterium avium intracellulare, Pneumocystis carinii pneumonia, orchitis / epidydimitis, legionella, Lyme disease, influenza A, epstein-barr virus, viral encephalitis / aseptic meningitis, and pelvic inflammatory disease.

80. A nanoparticle for use in extracorporeal transfection of an immune effector cell, the transfectioncomprising contacting blood, or a cell-containing fraction thereof, from a subject with a nanoparticle ex vivo, wherein the nanoparticle comprises: a surface-exposed immune effector binding moiety; and a nucleic acid encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR). DB1 / 154850959.6 125Attorney Ref. No.: 123690-5005-WO81. A nanoparticle comprising:a surface-exposed CD7 binding moiety; a surface-exposed CD3 binding moiety; and a deoxyribonucleic acid encoding a polypeptide or functional RNA.

82. The nanoparticle of claim 81, wherein the nanoparticle is a lipid nanoparticle (LNP).

83. The nanoparticle of claim 81 or 82, wherein the CD7 binding moiety is an anti-CD7 antibody orantigen binding domain thereof.

84. The nanoparticle of claim 81 or 82, wherein the CD7 binding moiety is an anti-CD7 VHH.

85. The nanoparticle of any one of claims 81-84, wherein the CD7 binding moiety comprises anamino acid sequence selected from the group consisting of SEQ ID NOS: 142, 146, 150, 154, 158, 162, 166, 170, and 174.

86. The nanoparticle of any one of claims 81-84, wherein the CD7 binding moiety comprises:CDRs1-3 as set forth in SEQ ID NOs: 143-145, respectively, CDRs1-3 as set forth in SEQ ID NOs: 147-149, respectively, CDRs1-3 as set forth in SEQ ID NOs: 151-153, respectively, CDRs1-3 as set forth in SEQ ID NOs: 155-157, respectively, CDRs1-3 as set forth in SEQ ID NOs: 159-161, respectively, CDRs1-3 as set forth in SEQ ID NOs: 163-165, respectively, CDRs1-3 as set forth in SEQ ID NOs: 167-169, respectively, CDRs1-3 as set forth in SEQ ID NOs: 171-173, respectively, or CDRs1-3 as set forth in SEQ ID NOs: 175-177, respectively.

87. The nanoparticle of any one of claims 81-86, wherein the nanoparticle is an LNP and the CD7binding moiety is conjugated to a surface-exposed lipid of the LNP.

88. The nanoparticle of any one of claims 81-87, wherein the CD3 binding moiety is an anti-CD3antibody or antigen binding domain thereof. DB1 / 154850959.6 126Attorney Ref. No.: 123690-5005-WO89. The nanoparticle of claim 88, wherein the CD3 binding moiety comprises an anti-CD3 ScFv.

90. The nanoparticle of claim 88 or 89, wherein the CD3 binding moiety comprisesa heavy chain variable region (VH) as set forth in SEQ ID NOS: 179, and a light chain variable region (VL) as set forth in SEQ ID NOS: 181; or a heavy chain variable region (VH) as set forth in SEQ ID NOS: 189, and a light chain variable region (VL) as set forth in SEQ ID NOS: 191.

91. The nanoparticle of claim 88 or 89, wherein the CD3 binding moiety comprises:VH-CDRs 1-3 as set forth in SEQ ID NOs: 182-184, respectively, and VL-CDRs 1-3 CDRs1 as set forth in SEQ ID NOs: 185-187, respectively; or VH-CDRs 1-3 as set forth in SEQ ID NOs: 192-194, respectively, and VL-CDRs 1-3 CDRs1 as set forth in SEQ ID NOs: 195-197, respectively.

92. The nanoparticle of any one of claims 81-91, wherein nanoparticle is an LNP and the CD3binding moiety is conjugated to a surface-exposed lipid of the LNP.

93. The nanoparticle of any one of claims 81-92, wherein the deoxyribonucleic acid encodes achimeric antigen receptor (CAR) or T-cell receptor (TCR).

94. The nanoparticle of any one of claims 81-93, wherein the deoxyribonucleic acid is a plasmid, aminicircle DNA, a doggybone DNA, a miniplasmid DNA, a Tiny plasmid, a nanoplasmid or a close-ended DNA (ceDNA).

95. The nanoparticle of any one of claims 81-93, wherein the deoxyribonucleic acid is a minicircleDNA.

96. The nanoparticle of any one of claims 81-93, wherein the deoxyribonucleic acid is an open-ended DNA or a single-stranded DNA (ssDNA).

97. The nanoparticle of any one of claims 81-96, wherein:DB1 / 154850959.6 127Attorney Ref. No.: 123690-5005-WO the deoxyribonucleic acid comprises a transposable element comprising a gene sequence encoding the polypeptide or functional RNA flanked by inverted terminal repeats (ITRs); and the nanoparticle further comprises a nucleic acid encoding a transposase with specificity for the ITRs.

98. The nanoparticle of claim 97, wherein the transposable element is selected from the groupconsisting of a Sleeping Beauty (SB) 10 transposable element, a Sleeping Beauty 11 transposable element, a Sleeping Beauty 100X transposable element, a PiggyBac transposable element, a PiggyBat transposable element, a Mu transposable element, a TcBuster transposable element, a Mos1 transposable element, a Tol1 transposable element, a Tol2 transposable element, a Frog Prince transposable element, a spinON transposable element, a HimarI transposable element, a Passport transposable element, a Minos transposable element, a hAT transposable element, an Hsmar1 transposable element, a Harbinger transposable element, a Harbinger3-DR transposable element, an Ac / Ds transposable element, and a PIF transposable element.

99. The nanoparticle of claim 97, wherein the transposable element is a Sleeping Beauty (SB)transposable element.

100. The nanoparticle of claim 97, wherein the transposable element is a piggyBac (PB)transposable element.

101. The nanoparticle of any one of claims 81-100, wherein the nanoparticle comprises one ormore nucleic acids encoding a recombinase gene-editing system or a nuclease gene-editing system, wherein the recombinase gene-editing system or the nuclease gene-editing system has specificity for the deoxyribonucleic acid encoding the polypeptide or functional RNA.

102. The nanoparticle of claim 101, wherein the recombinase gene-editing system is selectedfrom the group consisting of a Cre system, an FLP system, a lamda integrase system, a phiC31 integrase system, a Bxb1 integrase system, a gamma-delta resolvase system, a Tn3 resolvase system, and a Gin invertase system. DB1 / 154850959.6 128Attorney Ref. No.: 123690-5005-WO103. The nanoparticle of 101, wherein the nuclease gene-editing system is select from the groupconsisting of a CRISPR / Cas system, an ARCUS system, a TALEN system, a meganuclease system, and a zinc finger nuclease system.

104. The nanoparticle of any one of claims 93-103, wherein the CAR or TCR has affinity for acell-surface antigen selected from the group of genes consisting of TNFRSF17, IL3RA, SDC1, CD19, CD20, CD22, BCMA, MS4A1, CD7, CD123, CD135, CD38, CD138, CD269, TNFRSF8, CD33, CD38, CD5, NCAM1, CD70, ULBP1, ULBP2, IL1RAP, CEACAM5, MET, EGFR, EGFRvIII, EPCAM, EPHA2, ERBB2, GPC3, MSLN, Muc1, PDCD1, CD274, KDR, IL13RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, ROR2, GPRC5D, PSMA, CD23, WT1, CD44, CD44v6, CD174, SLAMF7, L1 CAM, FLT3, Sigle-6, GD2, PSCA, NY-ESO-1, GPNMB, CD276, CSPG4, MAGEA3, MAGEA4, CD133, TEM1, and combinations thereof.

105. The nanoparticle of any one of claims 93-104, wherein the CAR or TCR has affinity for asingle surface antigen.

106. The nanoparticle of any one of claims 93-104, wherein the CAR or TCR has affinity fortwo surface antigens.

107. The nanoparticle of any one of claims 93-104, wherein the CAR or TCR has affinity forthree, or more, surface antigens.

108. The nanoparticle of any one of claims 93-107, wherein the CAR or TCR has affinity forCD19, CD20, CD22, BCMA, or a combination thereof.

109. nanoparticle of any one of claims 93-107, wherein the CAR or TCR has affinity for a pairof cell-surface antigens selected from the group consisting of: CD19 and CD22; CD19 and CD20; CD20 and CD22; BCMA and CD19; BCMA and CD19; and DB1 / 154850959.6 129Attorney Ref. No.: 123690-5005-WO BCMA and CD22.

110. The nanoparticle of any one of claims 93-109, wherein the CAR has affinity for CD19.

111. The nanoparticle of claim 110, wherein the CAR has a polypeptide sequence of the CARused in lisocabtagene maraleucel, tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, or obecabtagene autoleucel.

112. The nanoparticle of any one of claims 93-111, wherein the CAR has affinity for BCMA.

113. The nanoparticle of claim 112, wherein the CAR has a polypeptide sequence of the CARused in idecabtagene vicleucel or ciltacabtagene autoleucel.

114. The nanoparticle of any one of claims 93-103, wherein the CAR or TCR binds a T cell viainteraction with one or more of the following: T-cell γ chains, T-cell β chains, T-cell δ chains, T- cell constant chains, CCR7, CD3, CD4, CD5, CD7, CD8, CD11b, CD11c, CD16, CD19, CD20, CD21, CD22, CD25, CD28, CD34, CD35, CD40, CD45RA, CD45RO, CD52, CD56, CD62L, CD68, CD80, CD95, CD117, CD127, CD133, CD137 (4-1BB), CD163, F4 / 80, IL-4Rα, Sca-1, CTLA-4, GITR, GARP, LAP, granzyme B, LFA-1, transferrin receptor, and combinations thereof.

115. Use of a nanoparticle in the manufacture of a medicament for extracorporeal transfectionof an immune effector cell, the transfection comprising contacting blood, or a cell-containing fraction thereof, from a subject with a nanoparticle, wherein the nanoparticle comprises: a surface-exposed immune effector binding moiety; and a nucleic acid encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR).

116. The use of claim 115, further comprising one or more of the features recited in any ofclaims 1-79.

117. A nanoparticle comprising:a surface-exposed CD7 binding moiety; DB1 / 154850959.6 130Attorney Ref. No.: 123690-5005-WO a surface-exposed CD3 binding moiety; a deoxyribonucleic acid comprising a transposable element comprising a gene sequence encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR) flanked by inverted terminal repeats (ITRs); and an mRNA encoding a transposase with specificity for the ITRs.

118. A method for making a transfected T-cell, the method comprising contacting blood from asubject with a nanoparticle ex vivo, wherein the nanoparticle comprises: a surface-exposed CD7 binding moiety; a surface-exposed CD3 binding moiety; a deoxyribonucleic acid comprising a transposable element comprising a gene sequence encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR) flanked by inverted terminal repeats (ITRs); and an mRNA encoding a transposase with specificity for the ITRs.

119. A method for making a transfected T-cell, the method comprising isolating peripheralblood mononuclear cells (PBMCs) from blood from a subject and contacting the isolated PMBCs with a nanoparticle ex vivo, wherein the nanoparticle comprises: a surface-exposed CD7 binding moiety; a surface-exposed CD3 binding moiety; a deoxyribonucleic acid comprising a transposable element comprising a gene sequence encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR) flanked by inverted terminal repeats (ITRs); and an mRNA encoding a transposase with specificity for the ITRs.

120. A method for treating a subject in need thereof, the method comprising administering tothe subject a transfected immune effector cell prepared by contacting blood, or a cell-containing fraction thereof, from a subject with a nanoparticle ex vivo, wherein the nanoparticle comprises: a surface-exposed immune effector binding moiety; and a deoxyribonucleic acid encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR). DB1 / 154850959.6 131Attorney Ref. No.: 123690-5005-WO121. A method for treating a subject in need thereof, the method comprising administering tothe subject a transfected T-cell prepared by contacting blood, or a T-cell-containing fraction thereof, from a subject with a nanoparticle ex vivo, wherein the nanoparticle comprises: a surface-exposed CD7 binding moiety; a surface-exposed CD3 binding moiety; a deoxyribonucleic acid comprising a transposable element comprising a gene sequence encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR) flanked by inverted terminal repeats (ITRs); and an mRNA encoding a transposase with specificity for the ITRs and a deoxyribonucleic acid encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR). DB1 / 154850959.6 132

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