Bispecific transduction enhancer

Abstract

To provide a bispecific transduction enhancer.SOLUTION: Compositions and methods for transducing immune cells invivo are provided in which multispecific antibodies (e.g., bispecific T cell engagers) are administered to render immune cells in a subject more susceptible to transduction by a vector, such as a lentiviral vector. The present disclosure provides a method of transducing an immune cell in a subject in need thereof, the method comprising: a) administering a multispecific antibody to render the immune cell in the subject more transducible; and b) administering a vector, optionally a viral vector.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 968,028, filed January 30, 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] Description of electronically submitted text files The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: Computer-readable format copy of the Sequence Listing (File name: UMOJ-004_01WO_SeqList_ST25.txt, Creation date: January 27, 2021, File size: 39.7 kilobytes).

[0003] Field The present disclosure relates generally to the in vivo transduction of immune cells to treat cancer and / or hematological malignancies. [Background technology]

[0004] background Cell therapy generally uses ex vivo transduction of immune cells to generate a population of therapeutic cells that are introduced into the patient. For example, T cells obtained from autologous or allogeneic sources can be transduced ex vivo with vectors encoding chimeric antigen receptors. The resulting CAR T-cells are then infused into the patient. Summary of the Invention [Problem to be solved by the invention]

[0005] Instead, it is desirable to generate therapeutic cells in vivo by delivering vectors to patients. Current methodologies for in vivo transduction of immune cells suffer from low efficiency. The present disclosure provides compositions and methods for in vivo transduction of immune cells to treat cancer and / or hematological malignancies. [Means for solving the problem]

[0006] overview The present disclosure provides methods of transducing immune cells in a subject in need thereof, the method comprising: a) administering a multispecific antibody to render immune cells in the subject more transducible; and b) administering a vector, optionally a viral vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the method transduces immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the vector is a lentiviral vector.

[0007] In some embodiments, the multispecific antibody comprises a T cell antigen-specific binding domain. In some embodiments, the T cell antigen is CD3, CD4, CD8, or TCR. In some embodiments, the multispecific antibody comprises a second antigen-specific binding domain. In some embodiments, the second antigen is CD19. In some embodiments, the second antigen is CD19, EpCAM, Her2 / neu, EGFR, CD66e, CD33, EphA2, or MCSP. In some embodiments, the second antigen is CD19, EpCAM, CD20, CD123, BCMA, B7-H3, CDE, or PSMA. In some embodiments, the second antigen is a myeloid cell antigen or a dendritic cell antigen. In some embodiments, the second antigen is CD33, DC-SIGN, CD11b, CD11c, or CD18. In some embodiments, the multispecific antibody is a bispecific antibody. In some embodiments, the bispecific antibody is a bispecific T cell engager (BiTE). In some embodiments, the BiTE is a CD19xCD3 BiTE. In some embodiments, the CD19xCD3 BiTE is blinatumomab.

[0008] In some embodiments, the multispecific antibody activates immune cells. In some embodiments, the multispecific antibody increases immune cell activation compared to administration of a vehicle control. In some embodiments, the multispecific antibody increases the number of immune cells in a subject's lymph nodes. In some embodiments, the multispecific antibody increases immune cell transduction compared to administration of a viral vector alone. In some embodiments, the multispecific antibody enhances in vivo transduction of immune cells by a viral vector. In some embodiments, the multispecific antibody reduces the effective concentration (EC50) of the viral vector. In some embodiments, the method achieves the same level of immune cell transduction without administering the multispecific antibody as a method comprising administering a higher concentration of viral vector.

[0009] In some embodiments, the vector is a viral vector comprising a polynucleotide encoding a T cell receptor or a chimeric antigen receptor. In some embodiments, the chimeric antigen receptor is an anti-CD19 chimeric antigen receptor. In some embodiments, the vector is a viral vector comprising a polynucleotide encoding a cytokine receptor. In some embodiments, the cytokine receptor is a drug-inducible cytokine receptor. In some embodiments, the vector further comprises one or more transgenes. In some embodiments, the viral vector comprises a transgene encoding a TGFβ dominant-negative receptor. In some embodiments, the lentiviral vector comprises one or more cell surface receptors that bind to a ligand on the target host cell, a heterologous viral envelope glycoprotein, a fusion glycoprotein, a T cell activation or costimulatory molecule, a ligand for CD19 or a functional fragment thereof, a cytokine or cytokine-based transduction enhancer, and / or a transmembrane protein comprising a mitogenic domain and / or a cytokine-based domain exposed on the surface of the lentiviral vector and / or conjugated to the surface. In some embodiments, the one or more T cell activation or costimulatory molecules comprise one or more T cell ligands. In some embodiments, the lentiviral vector is shunted by a coccivirus envelope protein. In some embodiments, the lentiviral vector is pseudotyped with a Nipah virus envelope protein. In some embodiments, the Nipah virus envelope protein is engineered to bind to EpCAM, CD4, or CD8.

[0010] In some embodiments, step a) and / or step b) of the method of transducing immune cells comprises subcutaneous administration. In some embodiments, step a) and / or step b) comprises intralymphatic administration. In some embodiments, step a) or step b) comprises intravenous administration. In some embodiments, both step a) and step b) comprise intravenous administration. In some embodiments, the multispecific antibody is administered at a dose of about 0.001 mg / kg to about 1 mg / kg.

[0011] In some embodiments, the multispecific antibody specifically binds to CD3 and CD19, and the vector is a lentiviral vector pseudotyped with a coccal virus envelope protein, and the vector comprises a polynucleotide encoding an anti-CD19 chimeric antigen receptor and a transgene encoding a TGFβ dominant-negative receptor.

[0012] The present disclosure provides methods for transducing immune cells in a subject in need thereof, the method comprising: a) administering a polynucleotide encoding a multispecific antibody to activate the immune cells in the subject; and b) administering a vector, optionally a viral vector. In some embodiments, the method transduces the immune cells. In some embodiments, the polynucleotide encoding the multispecific antibody is RNA. In some embodiments, the immune cells are T cells. In some embodiments, the vector is a lentiviral vector. In some embodiments, the multispecific antibody comprises a T cell antigen-specific binding domain. In some embodiments, the T cell antigen is CD3, CD4, CD8, or TCR. In some embodiments, the multispecific antibody comprises a second antigen-specific binding domain. In some embodiments, the second antigen is CD19. In some embodiments, the second antigen is CD19, EpCAM, Her2 / neu, EGFR, CD66e, CD33, EphA2, MCSP, CD22, CD79a, CD79b, or sIgM. In some embodiments, the second antigen is CD19, EpCAM, CD20, CD123, BCMA, B7-H3, CDE, or PSMA. In some embodiments, the second antigen is a lymph node antigen. In some embodiments, the multispecific antibody is a trispecific antibody. In some embodiments, the multispecific antibody is a bispecific antibody. In some embodiments, the bispecific antibody is a bispecific T cell engager (BiTE). In some embodiments, the BiTE is a CD19xCD3 BiTE. In some embodiments, the CD19xCD3 BiTE is blinatumomab. In some embodiments, the multispecific antibody activates immune cells. In some embodiments, the multispecific antibody increases immune cell activation compared to administration of a vehicle control. In some embodiments, the multispecific antibody increases the number of immune cells in the subject's lymph nodes. In some embodiments, the multispecific antibody increases immune cell transduction compared to administration of a viral vector alone. In some embodiments, the multispecific antibody enhances in vivo transduction of immune cells by a viral vector.In some embodiments, the multispecific antibody reduces the effective concentration (EC50) of the viral vector. In some embodiments, the method achieves the same level of immune cell transduction as a method comprising administering a higher concentration of the viral vector without administering the multispecific antibody. In some embodiments, step a) and / or step b) comprise subcutaneous administration. In some embodiments, step a) and / or step b) comprise intralymphatic administration. In some embodiments, step a) and / or step b) comprise intravenous administration. In some embodiments, the viral vector comprises a polynucleotide encoding a T cell receptor or a chimeric antigen receptor. In some embodiments, the chimeric antigen receptor is an anti-CD19 chimeric antigen receptor. In some embodiments, the viral vector comprises a polynucleotide encoding a cytokine receptor. In some embodiments, the cytokine receptor is a drug-inducible cytokine receptor. In some embodiments, the lentiviral vector comprises one or more cell surface receptors that bind to a ligand on the target host cell, a heterologous viral envelope glycoprotein, a fusion glycoprotein, a T cell activation or costimulatory molecule, a ligand for CD19 or a functional fragment thereof, a cytokine or cytokine-based transduction enhancer, and / or a transmembrane protein comprising a mitogenic domain and / or a cytokine-based domain exposed on the surface of the lentiviral vector and / or conjugated to the surface of the lentiviral vector. In some embodiments, the one or more T cell activation or costimulatory molecules comprise one or more T cell ligands. In some embodiments, the vector further comprises one or more transgenes. In some embodiments, the viral vector comprises a transgene encoding a TGFβ dominant-negative receptor. In some embodiments, the lentiviral vector is pseudotyped with a Cocalvirus envelope protein. In some embodiments, the lentiviral vector is pseudotyped with a Nipah virus envelope protein. In some embodiments, the Nipah envelope protein is engineered to bind to EpCAM, CD4, or CD8.In some embodiments, the multispecific antibody specifically binds to CD3 and CD19, the vector is a lentiviral vector pseudotyped with a coccivirus envelope protein, and the vector comprises a polynucleotide encoding an anti-CD19 chimeric antigen receptor and a transgene encoding a TGFβ dominant-negative receptor.

[0013] The present disclosure provides a method for the production of a multispecific antibody comprising administering to a subject in need thereof a multispecific antibody and a vector, optionally a viral vector. Combination therapies are provided for use in transducing immune cells in vivo.

[0014] The present disclosure provides a pharmaceutical composition comprising a multispecific antibody and a vector, optionally a viral vector.

[0015] The present disclosure provides a kit comprising 1) a multispecific antibody and 2) a vector, optionally a viral vector. The present disclosure also provides a kit comprising 1) a polynucleotide encoding the multispecific antibody and 2) a vector, optionally a viral vector. In some embodiments, the kit of the present disclosure is for use in a) transducing immune cells in a subject in need thereof; and / or b) treating a disease or disorder in a subject in need thereof.

[0016] The present disclosure provides methods of treating a disease or disorder in a subject in need thereof, the method comprising: a) administering a multispecific antibody to activate immune cells in the subject; and b) administering a vector, optionally a viral vector, before, after, or in conjunction with step a). In some embodiments, the method transduces immune cells. In some embodiments, the disease or disorder is cancer. In some embodiments, the disease or disorder is a hematological malignancy. In some embodiments, the hematological malignancy is B-cell lymphoma. In some embodiments, the method treats the disease or disorder more quickly than administration of the multispecific antibody alone and / or the vector alone. In some embodiments, the method provides a superior treatment outcome for the disease or disorder than administration of the multispecific antibody alone and / or the vector alone. In some embodiments, the multispecific antibody specifically binds to CD3 and CD19, the vector is a lentiviral vector pseudotyped with a coccivirus envelope protein, and the vector comprises a polynucleotide encoding an anti-CD19 chimeric antigen receptor and a transgene encoding a TGFβ dominant-negative receptor. In some embodiments, the method results in more rapid depletion of malignant B cells in the subject than administration of the multispecific antibody and / or vector alone, hi some embodiments, the method results in a lower number of residual malignant B cells and / or a lower rate of B-cell lymphoma relapse in the subject than administration of the multispecific antibody and / or vector alone. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 shows an embodiment in which blinatumomab is co-administered with a viral vector.

[0018] [Figure 2A] FIG. 2A shows flow cytometry to measure CD25+ T cells in experiments performed on primary T cells cultured at a 50:50 ratio with B cells.

[0019] [Figure 2B] FIG. 2B shows flow cytometry to measure CD25+ T cells in experiments performed on cultured primary T cells.

[0020] [Figure 3A] Figure 3A shows flow cytometry to measure T cells expressing anti-CD19 chimeric antigen receptors in experiments performed on primary T cells cultured at a 50:50 ratio with B cells.

[0021] [Figure 3B] Figure 3B shows flow cytometry to measure T cells expressing anti-CD19 chimeric antigen receptors in experiments performed on cultured primary T cells.

[0022] [Figure 4] FIG. 4 shows a graph of the ratio of B cells to T cells after cells were cultured in the presence of blinatumomab.

[0023] [Figure 5A] Figure 5A shows flow panel validation and gating strategy for anti-CD19CAR-TGFβ T cells generated and maintained in culture.

[0024] [Figure 5B] Figure 5B shows flow panel validation and gating strategy for anti-CD19CAR-TGFβ T cells generated in CD34 humanized mice.

[0025] [Figure 6] FIG. 6 shows bar graphs showing the transient activation of CD4+ T cells (left) and CD8+ T cells (right) upon administration of blinatumomab.

[0026] [Figure 7] FIG. 7 is a chart showing B cell counts over time with lentivirus and / or blinatumomab administration.

[0027] [Figure 8] Figure 8 shows representative flow cytometry plots of blood samples taken from mice on study day 33. Plots were gated on live CD3+ cell singlets. Intracellular anti-2A peptide staining was included in the panel as an alternative method for detecting CAR.

[0028] [Figure 9] FIG. 9 shows representative flow cytometry plots gated on viable cell singlets from spleen and bone marrow samples collected on study day 52 from the indicated study groups. DETAILED DESCRIPTION OF THE INVENTION

[0029] Detailed Description The present disclosure provides compositions and methods relating to the use of multispecific antibodies to facilitate the generation of genetically engineered target cell vectors. In some embodiments, the use of multispecific antibodies improves the transduction efficiency of vectors in vivo. In some embodiments, the transduction of target cells in a subject can be enhanced by administering one or more multispecific antibodies to the subject before, in conjunction with, or after administering a vector to the subject.

[0030] Without wishing to be bound by any particular theory, it is contemplated that the multispecific antibodies of the present disclosure may exert their effect through one or more mechanisms of action, including, but not limited to, the following: (i) stimulating target cells (e.g., immune cells) to enter a more activated and / or proliferative state, which can increase vector transduction efficiency; viral vector entry and payload delivery are typically more efficient when target cells are in an activated / proliferative state; (ii) causing immune cells to exit the G0 phase of the cell cycle; (iii) causing immune cells to replicate at least once; (iv) increasing the metabolic fitness of immune cells; and (v) attracting increased numbers of immune cells to physiologically relevant sites (e.g., lymph nodes).

[0031] As a result, using the methods and compositions described herein, significantly more cells can be transduced, and / or the same number of cells can be transduced with a lower effective concentration of vector. The compositions and methods of the present disclosure can facilitate the direct administration of vectors to subjects in need of treatment. Furthermore, by reducing the concentration at which vectors are effective in vivo, the compositions and methods of the present disclosure can limit side effects due to vector toxicity or off-target transduction. Thus, the present disclosure provides safer and more efficient gene therapy.

[0032] multispecific antibody The term multispecific antibody refers to an antibody molecule having two or more antigen-binding domains, e.g., two (bispecific), three (trispecific), or four (tetraspecific) binding domains. In some embodiments, a multispecific antibody is a bispecific antibody. In some embodiments, a multispecific antibody is a trispecific antibody. In some embodiments, Multispecific antibodies are constructs with more than three (eg, four, five, etc.) specificities.

[0033] The multispecific antibody molecules of the present disclosure can be constructed from various antibody fragments known in the art. For example, diabodies are composed of a non-covalent dimer of ScFv fragments. A bispecific antibody molecule is a bispecific antibody molecule composed of two Fab fragments linked by a hinge region, whereas F(ab')2 is a bispecific antibody molecule composed of two Fab fragments linked by a hinge region. Thus, those skilled in the art will recognize that different antibody fragments can be arranged in various combinations to generate bispecific or multispecific antibody molecules.

[0034] Various multispecific and / or bispecific formats include recombinant IgG-like dual targeting molecules, in which the two sides of the molecule each contain an Fab fragment or part of an Fab fragment of at least two different antibodies; IgG fusion molecules in which a single chain Fv molecule or stabilized diabody is attached to a heavy chain constant domain, Fc region or portion thereof; Fc fusion molecules in which different Fab fragments are attached together; ScFv and diabody-based antibodies and heavy chain antibodies (e.g., domain antibodies, nanobodies) in which different single chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are attached to each other or to another protein or carrier molecule, or multispecific antibodies generated by arm swapping. Exemplary multispecific and / or bispecific formats include dual targeting (DT)-Ig (GSK / Domantis), two-in-one antibodies (Genentech) and mAb2 (F-Star), dual variable domain (DVD)-Ig (Abbott), Ts2Ab (MedImmune / AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec) and TvAb (Roche), ScFv / Fc fusions (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS) and dual affinity retargeting technology (Fc-DART) (MacroGenics), F(ab)2 (Medarex / AMGEN), dual acting or bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), bivalent bispecific (Bi Dual-targeting molecules include Fab-Fv (UCB-Celltech), bispecific T cell engagers (BITE) (Micromet), tandem diabodies (Tandab) (Affimed), dual affinity retargeting technology (DART) (MacroGenics), single-chain diabodies (Academic), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack) and COMBODY (Epigen Biotech), dual-targeting nanobodies (Ablynx), and dual-targeting heavy chain-only domain antibodies. Various formats of bispecific antibodies are described, for example, in Chames and Baty (2009) Curr Opin Drug Disc Dev 12: 276 and Nunez-Prado et al., (2015) Drug Discovery Today 20(5):588-594. It has been done.

[0035] Examples of trispecific or tetraspecific antibody formats include, but are not limited to, Fab3, triabody, tetrabody, tribody, DVD-Ig, IgG-scFv, ScFv2-Fc, tandAbs, and DNL-Fab3.

[0036] In some embodiments, a multispecific antibody (e.g., a bispecific antibody) of the present disclosure comprises specific antigen-binding domains for a combination of two antigens selected from Table 1 below (each "x" mark indicates a combination):

[0037] [Table 1]

[0038] bispecific antibody As used herein, a bispecific antibody molecule refers to a molecule having two antigen-binding domains capable of binding to different antigens. Examples of bispecific antibody formats include, but are not limited to, bispecific T cell engagers (BiTEs), F(ab')2, F(ab')-ScFv2, di-scFv, diabodies, minibodies, scFv-Fc, DARTs, TandAbs, ScDiabodies, and ScDiabodies. Abody-CH3, diabody-CH3, triplebody, miniantibody, minibody, TriBi minibody, ScFv-CH3 KIH (knobs in holes), Fab-ScFv, SCFv-CH-CL-scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, intrabody, dock and lock antibody, ImmTAC, HSA Examples of such antibodies include ScFv-HAS, Sc diabody, Sc diabody-HAS, humabody, and tandem ScFv-toxic (see, for example, Christoph Spiess et al, Molecular Immunology 67 (2015) pages 95-106).

[0039] At least two binding domains and a variable domain of a multispecific antibody (VH / VL) can comprise a peptide linker (spacer peptide). The term "peptide linker," according to the present disclosure, includes an amino acid sequence that connects the amino acid sequences of one (variable and / or binding) domain and another (variable and / or binding) domain of the antibody construct of the present disclosure. Peptide linkers can also be used to fuse a third domain to another domain of the antibody construct of the present disclosure. The essential technical feature of such peptide linkers is that they do not contain any polymerization activity. Suitable peptide linkers include those described in U.S. Pat. Nos. 4,751,180 and 4,935,233 or WO 88 / 09344. Peptide linkers can also be used to connect other domains, modules, or regions (such as half-life extending domains) to the antibody construct of the present disclosure. Exemplary bispecific single chain antibody constructs are described in WO99 / 54440, Mack, J. Immunol. (1997), 158, 3965-3970, Mack, PNAS, (1995), 92, 7021-7025, Kufer, Cancer Immunol. Immunother., (1997), 45, 193-197, Loeffler, Blood, (2000), 95,6, 2098-2103, Bruehl, Immunol., (2001), 166, 2420-2426, Kipriyanov, J. Mol.Biol., (1999), 293, 41-56.

[0040] Bivalent (also called divalent) or bispecific single-chain variable fragments (bi-scFv or di-scFv with format (scFv)2) can be engineered by linking two scFv molecules (e.g., using a linker as described above). If such two scFv molecules have the same binding specificity, the resulting (scFv)2 molecule is preferably called bivalent (i.e., it has a valency of 2 for the same target epitope). If the two scFv molecules have different binding specificities, the resulting (scFv)2 molecule is preferably called bispecific. Linking can be achieved by producing a single peptide chain with two VH and two VL regions, resulting in a tandem scFv (see, for example, Kufer P. et al., (2004) Trends in Biotechnology 22(5):238-244). Another possibility is to create scFv molecules with a linker peptide (e.g., about 5 amino acids) that is too short for the two variable regions to fold together, thereby causing the scFv to dimerize. This type of entity is known as a diabody (see, e.g., Hollinger, Philipp et al., (July 1993) Proceedings of the National Academy of Sciences of the United States of America 90(14):6444-8).

[0041] Consistent with the present disclosure, either the first domain, the second domain, or the first and second domains can comprise a single domain antibody, each comprising a variable domain or at least the CDRs of a single domain antibody. A single domain antibody comprises only one (monomeric) antibody variable domain that can selectively bind to a specific antigen independently of other V regions or domains. The first single domain antibodies were engineered from heavy chain antibodies found in camelids, which are called VHH fragments. Cartilaginous fish also possess heavy chain antibodies (IgNAR), from which single domain antibodies called VNAR fragments can be derived. An alternative approach is to split the dimeric variable domains of typical immunoglobulins, such as those from humans or rodents, into monomers, thus obtaining VH or VL as single domain Abs. While the majority of research on single domain antibodies is currently based on heavy chain variable domains, nanobodies derived from light chains have also been shown to specifically bind to target epitopes. Examples of single domain antibodies are called sdAbs, nanobodies, or single variable domain antibodies.

[0042] Whether an antibody construct competes with another given antibody construct for binding can be measured in a competitive assay, such as a competitive ELISA or a cell-based competitive assay. Avidin-conjugated microparticles (beads) can also be used. Similar to an avidin-coated ELISA plate, when reacted with biotinylated proteins, each of these beads can be used as a substrate on which an assay can be performed. The antigen is coated onto the beads, which are then pre-coated with a first antibody. A second antibody is added, and any additional binding is determined. Possible means for reading out include flow cytometry.

[0043] Bispecific T cell engager "BiTE" generally refers to a single polypeptide chain molecule having two antigen-binding domains, one of which binds to an immune effector cell antigen (e.g., CD3) and the second of which binds to an antigen present on the surface of a target cell.

[0044] In some embodiments, one of the antigen-binding domains is specific for an immune cell, such as a T cell antigen, such as the CD3 receptor expressed on the surface of T cells. In some embodiments, the second antigen-binding domain binds to tumor cells via a tumor-specific molecule. Thus, BiTEs can form a link between T cells and tumor cells due to their specificity for antigens on T cells and tumor cells. This results in T cell activation, triggering the T cell to exert its cytotoxic effect on tumor cells independently of MHC I or costimulatory molecules. Examples of BITE-based therapies currently approved or undergoing clinical trials include, for example, blinatumomab (Blyncyto®), which targets CD19 and is intended for the treatment of non-Hodgkin's lymphoma and acute lymphoblastic leukemia, and solitomab, which targets EpCAM and is intended for the treatment of gastrointestinal and lung cancers.

[0045] In some embodiments, the bispecific antibodies described in this disclosure are BiTEs specific for at least one surface antigen on a T cell of interest, including, but not limited to, CD3, CD2, VLA-1, CD8, CD4, CCR6, CXCR5, CD25, CD31, CD45RO, CD197, CD127, CD38, CD27, CD196, CD277, and CXCR3, particularly CD2, CD3, CD31, and CD277.

[0046] BiTE molecules have been constructed against a variety of target antigens, including CD19, EpCAM, Her2 / neu, EGFR, CD66e (or CEA, CEACAM5), CD33, EphA2, MCSP (or HMW-MAA), CD22, CD79a, CD79b, and sIgM. BiTE molecules are typically produced as recombinant, glycosylated proteins secreted by higher eukaryotic cell lines.

[0047] In some embodiments, BiTEs of the present disclosure are composed of a non-target cell antigen-binding fragment and a target immune cell antigen-binding fragment linked together by a linker. Immune cells include, for example, natural killer (NK) cells, T cells, including cytotoxic T cells, or B cells, although myeloid lineage cells, such as monocytes or macrophages, dendritic cells, and neutrophilic granulocytes, can also be considered immune cells. Thus, in various embodiments, the immune cells are NK cells, T cells, B cells, monocytes, macrophages, dendritic cells, or neutrophilic granulocytes. As relevant herein, immune cells can be any target cell whose in vivo transduction is desired, and BiTEs have the effect of increasing the transduction efficiency of the virus on the immune cell. To avoid nonspecific interactions, bispecific antibodies can be selected that recognize antigens on immune effector cells that are at least overexpressed by the immune effector cells relative to other cells in the body. Such antigens can include, but are not limited to, CD3, CD16, CD25, CD28, CD64, CD89, NKG2D, and NKp46. In some embodiments, the immune effector cell antigen is a T cell antigen. In some embodiments, the immune effector cell antigen is CD3. Thus, in some embodiments, the BiTEs of the present disclosure are comprised of an antigen-binding fragment and an anti-CD3 antigen-binding fragment linked together by a linker.

[0048] First antigen of the multispecific antibody In some embodiments, the first antigen-binding domain of the multispecific antibody binds to an immune cell antigen. In some embodiments, the immune cell antigen is a T cell antigen. In some embodiments, the T cell antigen is selected from the group consisting of CD3, CD4, CD8, and TCR.

[0049] In some embodiments, the first antigen of the multispecific antibody is CD3. The CD3 receptor complex is a protein complex composed of four chains. In mammals, this complex contains one CD3γ (gamma) chain, one CD3δ (delta) chain, and two CD3ε (epsilon) chains. These chains associate with the T cell receptor (TCR) and the so-called ζ (zeta) chain to form the T cell receptor-CD3 complex, which generates activation signals in T lymphocytes. The CD3γ (gamma), CD3δ (delta), and CD3ε (epsilon) chains are highly related cell surface proteins of the immunoglobulin superfamily that contain a single extracellular immunoglobulin domain. The intracellular tail of the CD3 molecule contains a single conserved motif known as an immunoreceptor tyrosine-based activation motif, or ITAM for short, which is essential for the signal transduction ability of the TCR. The CD3 epsilon molecule is a polypeptide encoded by the CD3E gene located on chromosome 11 in humans. In some embodiments, the first antigen is CD3 epsilon. In some embodiments, the antigenic epitope comprises amino acid residues 1-27 of the extracellular domain of human CD3 epsilon.

[0050] In some embodiments, the first antigen of the multispecific antibody is CD4 (cluster of differentiation 4). CD4 is a transmembrane glycoprotein of the immunoglobulin superfamily expressed on developing thymocytes, major histocompatibility class II (class II MHC)-restricted mature T lymphocytes, and, in humans, on cells of the macrophage / monocyte lineage. On lymphoid cells, CD4 plays a crucial role during thymocyte ontogeny and in the function of mature T cells. CD4 binds to a non-polymorphic region of class II MHC, which acts as a coreceptor for the T cell antigen receptor (TCR). This increases avidity between thymocytes and antigen-presenting cells and directly contributes to signal transduction through its association with the Src-like protein tyrosine kinase p56lck. CD4 is also a coreceptor for human and simian immunodeficiency viruses (HIV-1, HIV-2, and SIV). Specifically, CD4 is the receptor for the human immunodeficiency virus (HIV)-gp120 glycoprotein. Clinically, CD4 antibodies can be used to achieve immunological tolerance to grafts and transplants; to treat autoimmune diseases and immune deficiency-related disorders such as lupus, diabetes, rheumatoid arthritis, etc.; to treat CD4-expressing leukemias and lymphomas; and to treat HIV infection.

[0051] In some embodiments, the first antigen of the multispecific antibody is CD8 (cluster of differentiation 8). CD8 is a cell surface glycoprotein predominantly expressed on cytotoxic T lymphocytes, but also on dendritic cells, natural killer cells, natural killer T cells, and a subset of gamma delta T cells. This glycoprotein consists of two isoforms, α and β, which are encoded by different genes and expressed as αα homodimers or αβ heterodimers, the latter being predominant. The CD8 coreceptor stabilizes T cell receptor-MHC-I interactions and initiates intracellular signaling via lymphocyte-specific protein tyrosine kinase (Lck) phosphorylation of CD3-associated immunoreceptor tyrosine-based activation motifs (ITAMs) for activation. The amino acid sequence of full-length human CD8α has been provided in UniProt under accession number P01732. The amino acid sequence of full-length human CD8β has been provided in UniProt under accession number P10966. The term "CD8" includes full-length CD8α or CD8β, recombinant CD8, fragments thereof, and fusions thereof. The term also encompasses CD8α or CD8β or fragments thereof linked to, for example, a histidine tag, a mouse or human Fc, or a signal sequence.

[0052] In some embodiments, the first antigen of the multispecific antibody is CTLA-4 (cytotoxic T-lymphocyte-associated protein 4). CTLA-4, also known as CD152, is a single-pass type I membrane protein that forms disulfide-linked homodimers. Alternative splice variants encoding different isoforms have been characterized, including soluble isoforms that function as monomers. CTLA-4 surface expression is tightly regulated by restricted transport to the cell surface and rapid internalization. The extracellular region of CTLA-4 contains a single extracellular Ig(V) domain, followed by a transmembrane (TM) region and a small intracellular cytoplasmic tail (approximately 37 amino acids). The intracellular tail contains two tyrosine-based motifs that interact with several intracellular proteins, including the lipid kinase phosphatidylinositol 3-kinase (PI3K), the phosphatases SHP-2 and PP2A, and the clathrin adaptor proteins AP-1 and AP-2. CTLA4 is homologous to CD28 and, like CD28, binds to the ligands CD80 (B7-1) and CD86 (B7-2). However, unlike CD28, the binding of CTLA4 to B7 does not produce a stimulatory signal, but prevents the costimulatory signal normally provided by CD28. When naive T effector cells are activated through their T cell receptor (TCR), CTLA-4 is recruited to the cell surface and competes with CD28 (constitutively expressed on T cells) for CD80 / CD86, thereby blocking further signal transduction via the TCR and thus downregulating any further T cell response via TCR signaling. Thus, CTLA-4 acts as a negative regulator of T effector cell activation, reducing effector function and determining the efficacy and duration of T cell responses. In addition, CTLA-4 may play a role in enhancing the negative effects of regulatory T cells in the immune response to cancer. CTLA-4 has a much higher affinity for members of the B7 family than for CD28, and therefore its expression on T cells defines a dominant-negative regulator of T cells. Blockade of CTLA-4 has been reported to enhance T cell responses.

[0053] In some embodiments, the first antigen of the multispecific antibody is a T cell receptor (TCR). The TCR is a complex of membrane proteins involved in the activation of T cells in response to antigen presentation. The TCR is responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. The TCR is composed of a heterodimer of alpha (α) and beta (β) chains, although in some cells, the TCR consists of gamma and delta (γ / δ) chains. TCRs can exist in alpha / beta and gamma / delta forms, which are structurally similar but have distinct anatomical locations and functions. In some embodiments, the TCR can be engineered on any cell that contains a TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and gamma delta T cells. Engagement of the TCR with antigen and MHC leads to activation of the T lymphocyte through a series of biochemical events mediated by associated enzymes, coreceptors, and specialized accessory molecules. Each TCR chain is a member of the immunoglobulin superfamily and contains an N-terminal immunoglobulin (Ig)-variable (V) domain, an Ig-constant (C) domain, a transmembrane / transmembrane region, and a short C-terminal cytoplasmic tail. The variable domains of both the α- and β-TCR chains contain three hypervariable or complementarity-determining regions (CDRs). The constant domain of the TCR consists of a short connective sequence in which cysteine ​​residues form disulfide bonds, creating a link between the two chains. This structure allows the TCR to associate with other molecules, such as CD3, which in mammals possesses three distinct chains (γ, δ, and ε) and a ζ-chain. These accessory molecules have negatively charged transmembrane regions and are essential for signal transmission from the TCR into the cell. The CD3 and ζ-chains, together with the TCR, form what is known as the T cell receptor complex. Rhe signaling from the T cell complex is enhanced by simultaneous engagement of MHC molecules by specific coreceptors. On helper T cells, this co-receptor is CD4 (specific for class II MHC); whereas on cytotoxic T cells, this co-receptor is CD8 (specific for class I MHC).Co-receptors not only ensure the specificity of the TCR for the antigen, but also allow for long-term association between the antigen-presenting cell and the T cell, and also recruit essential molecules (e.g., LCK) inside the cell that are involved in signal transduction of activated T lymphocytes. Thus, the term "T cell receptor" is used in its conventional sense to mean a molecule that can recognize peptides when presented by MHC molecules.

[0054] Second antigen of multispecific antibody In some embodiments, the second antigen-binding domain of the multispecific antibody binds to an antigen present on the surface of a cell (e.g., a tumor cell) targeted by an immune effector cell. In some embodiments, the second antigen is selected from the group consisting of CD19, EpCAM, CD20, CD123, BCMA, B7-H3, and PSMA. In some embodiments, the second antigen is selected from the group consisting of CD19, EpCAM, Her2 / neu, EGFR, CD66e, CD33, EphA2, MCSP, CD22, CD79a, CD79b, and sIgM. In some embodiments, the second antigen is CD19.

[0055] In some embodiments, the second antigen of the multispecific antibody is EpCAM (epithelial cell adhesion molecule). EpCAM, also named CD326 or "tumor-associated calcium signal transducer 1," is a 40 kDa type I transmembrane molecule consisting of two epidermal growth factor-like extracellular domains, a cysteine-poor region, a transmembrane domain, and a short cytoplasmic tail. EpCAM refers to a glycoprotein. Human EpCAM is encoded by the GA733-2 gene on the long arm of chromosome 4 and is involved in cell-cell adhesion. EpCAM is expressed on most epithelial tissues. The sequence of EpCAM is well known in the art. Human EpCAM is a human cell surface glycoprotein antigen associated with cancers of various origins, including colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, lung cancer, cervical cancer, prostate cancer, and breast cancer. Malignant cell proliferation is often consistently associated with EpCAM expression at certain stages of tumor development, and high levels of EpCAM expression are negatively correlated with cell differentiation. High levels of EpCAM expression have been shown to correlate with poor survival among breast cancer patients.

[0056] In some embodiments, the second antigen of the multispecific antibody is CD19 (cluster of differentiation 19). CD19 is an antigenic determinant detectable on leukemia precursor cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD19 can be found under UniProt / Swiss-Prot accession number P15391, and the nucleotide sequence encoding human CD19 can be found under accession number NM_001178098. CD19 is expressed in most B-lineage cancers, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. It is also an early marker of B-cell precursor cells. In some embodiments, the CD19 protein is expressed on cancer cells. In some embodiments, "CD19" includes proteins that include mutations of the full-length wild-type protein, for example, point mutations, fragments, insertions, deletions, and splice variants.

[0057] In some embodiments, the second antigen of the multispecific antibody is CD20. CD20 is also known as B-lymphocyte CD20 antigen, MS4A1, B-lymphocyte surface antigen B1, Bp35, or leukocyte surface antigen Leu-16. The term CD20 includes human CD20 (AH003353; GenBank accession numbers M27395, J03574). The major form of human CD20 comprises a 297-amino acid protein described by GenBank protein ID 23110989. Examples of CD20 sequences include, but are not limited to, NCBI reference numbers NP_068769.2 and NP_690605.1. CD20 expression is found in lymphomas (e.g., B-cell non-Hodgkin's lymphoma (NHL)) and lymphocytic leukemias. Such lymphomas and lymphocytic leukemias include, for example, a) follicular lymphoma, b) small non-cleaved cell lymphoma / Burkitt lymphoma (including endemic Burkitt lymphoma, sporadic Burkitt lymphoma, and non-Burkitt lymphoma), c) marginal zone lymphoma (including extranodal marginal zone B-cell lymphoma (mucosa-associated lymphoid tissue lymphoma, MALT), nodal marginal zone B-cell lymphoma, and splenic marginal zone lymphoma), d) mantle cell lymphoma (MCL), e) large cell lymphoma (B-cell diffuse large cell lymphoma, In some embodiments, the CD20-expressing cancer is a B-cell non-Hodgkin's disease.In some embodiments, the CD20-expressing cancer is mantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell diffuse large cell lymphoma (DLCL), Burkitt's lymphoma, hairy cell leukemia, follicular lymphoma, multiple myeloma, marginal zone lymphoma, post-transplant lymphoproliferative disorder (PTLD), HIV-associated lymphoma, Waldenstrom's macroglobulinemia, or primary CNS lymphoma. In some embodiments, "CD20" includes proteins containing mutations of the full-length wild-type protein, e.g., point mutations, fragments, insertions, deletions, and splice variants.

[0058] In some embodiments, the second antigen of the multispecific antibody is CD123. CD123 is also known as cluster of differentiation 123, interleukin-3 receptor alpha chain, and IL3RA. CD123 is a type I transmembrane glycoprotein with an extracellular domain containing a predicted Ig-like domain and two FnIII domains. The term "CD123" can refer to any isoform of CD123. CD123 is preferentially expressed on certain types of pluripotent stem cells and cancer cells, such as leukemia cancer cells (e.g., acute myeloid leukemia cells). In some embodiments, "CD123" includes proteins containing mutations of the full-length wild-type protein, such as point mutations, fragments, insertions, deletions, and splice variants.

[0059] In some embodiments, the second antigen of the multispecific antibody is BCMA. The term "BCMA" refers to a B-cell maturation antigen. BCMA (also known as TNFRSF17, BCM, or CD269) is a member of the tumor necrosis receptor (TNFR) family and is predominantly expressed on terminally differentiated B cells, such as memory B cells and plasma cells. Its ligands are called B-cell-activating factor of the TNF family (BAFF) and proliferation-inducing ligand (APRIL). BCMA is involved in mediating the survival of plasma cells to maintain long-term humoral immunity. The BCMA gene is encoded on chromosome 16 and produces a 994-nucleotide primary mRNA transcript (NCBI accession NM_001192.2) that encodes a 184-amino acid protein (NP_001183.2). Additional transcript variants have been described with unknown significance (SmirnovaA S et al. Mol Immunol., 2008, 45(4):1179-1183). A second isoform, also known as TV4, has been identified (U niprot identifier Q02223-2). As used herein, "BCMA" includes proteins that contain mutations of full-length wild-type BCMA, e.g., point mutations, fragments, insertions, deletions, and splice variants. In some embodiments, BCMA is expressed on the cell surface of malignant B cells in a patient. In some embodiments, "BCMA" includes proteins that contain mutations of the full-length wild-type protein, e.g., point mutations, fragments, insertions, deletions, and splice variants.

[0060] In some embodiments, the second antigen of the multispecific antibody is PSMA. PSMA is also known as prostate-specific membrane antigen or folate hydrolase 1 (FOLH1). The amino acid sequence of human PSMA can be found under UniProt / Swiss-Prot accession number Q04609, and the NCBI reference sequence ID number for the amino acid sequence of human PSMA is NP_004467.1. PSMA is an integral, non-shed membrane glycoprotein that is highly expressed in prostate epithelial cells and is a cell surface marker for prostate cancer. In some embodiments, "PSMA" includes proteins that contain mutations of the full-length wild-type protein, such as point mutations, fragments, insertions, deletions, and splice variants.

[0061] In some embodiments, the second antigen of the multispecific antibody is HER2 / Neu. HER2 / neu is a 185 kDa receptor protein originally identified as the product of the ERBB2 transforming gene from neuroblastoma cells in chemically treated rats. HER2 / neu has been extensively investigated due to its role in several human cancers and mammalian development. The sequence of human HER2 / neu is available in GenBank under accession number X03363. HER2 / neu contains four domains: a ligand-binding extracellular domain; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxyl-terminal signaling domain with several tyrosine residues that can be phosphorylated. The sequence of the HER2 / neu extracellular domain (ECD) is available in Protein DataBank Record 1S78 (2004). HER2 / neu functions as a growth factor receptor and is often expressed by breast, ovarian, or lung cancer cells. HER2 / neu is overexpressed in 25-30% of human breast and ovarian cancers, and its overexpression is associated with aggressive clinical progression and poor prognosis in affected individuals. In some embodiments, "Her2 / neu" is a "Neu" includes proteins containing mutations of the full-length wild-type protein, such as point mutations, fragments, insertions, deletions and splice variants.

[0062] In some embodiments, the second antigen of the multispecific antibody is EGFR. EGFR, also known as human epidermal growth factor receptor, HER-1, or ErbB1, is a 170 kDa transmembrane receptor encoded by the c-erbB proto-oncogene and exhibits intrinsic tyrosine kinase activity. SwissProt database entry P00533 provides the sequence of EGFR. EGFR isoforms and variants (e.g., alternative RNA transcripts, truncated versions, polymorphisms, etc.) also exist, including but not limited to those identified by SwissProt database entry numbers P00533-1, P00533-2, P00533-3, and P00533-4. EGFR is known to bind to ligands including epidermal growth factor (EGF), transforming growth factor-alpha (TGf-amphiregulin, heparin-binding EGF (hb-EGF), betacellulin, and epiregulin. EGFR regulates numerous cellular processes through tyrosine kinase-mediated signaling pathways, including, but not limited to, activation of signaling pathways that control cell proliferation, differentiation, cell survival, apoptosis, angiogenesis, mitogenesis, and metastasis. In some embodiments, "EGFR" includes proteins containing mutations of the full-length wild-type protein, e.g., point mutations, fragments, insertions, deletions, and splice variants.

[0063] In some embodiments, the second antigen of the multispecific antibody is CD33 (cluster of differentiation 33). CD33 is an antigenic determinant detectable on normal precursor cells as well as on leukemia cells of the myeloid lineage. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD33 can be found under UniProt / Swiss-Prot accession number P20138, and the nucleotide sequence encoding human CD33 can be found under accession number NM_001772.3. In some embodiments, the CD33 protein is expressed on cancer cells. Certain hematological malignancies are characterized by the expression of CD33 on the surface of malignant cells. CD33-positive hematologic malignancies include, but are not limited to, acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia, thrombolytic leukemia, myelodysplastic syndrome, myeloproliferative disorder, refractory anemia, preleukemia syndrome, lymphoid leukemia, or undifferentiated leukemia. In some embodiments, "CD33" includes proteins containing mutations of the full-length wild-type protein, e.g., point mutations, fragments, insertions, deletions, and splice variants.

[0064] In some embodiments, the second antigen of the multispecific antibody is EphA2 (ephrin receptor A2). EphA2 is found to be overexpressed, mutated, or amplified in various cancers. The nucleotide sequence and / or amino acid sequence of the EphA2 polypeptide can be found in the literature or public databases, or the nucleotide sequence and / or amino acid sequence can be determined using cloning and sequencing techniques known to those skilled in the art. For example, the nucleotide sequence of human EphA2 can be found in the GenBank database (see, e.g., accession numbers BC037166, M59371, and M36395). The amino acid sequence of human EphA2 can be found in the GenBank database (see, e.g., accession numbers AAH37166 and AAA53375). In some embodiments, "EphA2" includes proteins containing mutations of the full-length wild-type protein, such as point mutations, fragments, insertions, deletions, and splice variants.

[0065] In some embodiments, the second antigen of the multispecific antibody is MCSP (melanoma chondroitin sulfate proteoglycan). MCSP is also known as chondroitin sulfate proteoglycan 4 (CSPG4), chondroitin sulfate proteoglycan NG2, high molecular weight-melanoma-associated antigen (HMW-MAA), and melanoma chondroitin sulfate proteoglycan. An exemplary amino acid sequence of human MCSP is set forth in Genbank accession number NP_001888. It is an early cell surface melanoma progression marker implicated in stimulating tumor cell proliferation, migration, and invasion. In some embodiments, "MCSP" includes proteins containing mutations of the full-length wild-type protein, such as point mutations, fragments, insertions, deletions, and splice variants.

[0066] In some embodiments, the second antigen of the multispecific antibody is CD66e, also known as carcinoembryonic antigen (CEA), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), or CD66. As a member of the immunoglobulin (Ig) family, it is one of six Ig It is a glycosylphosphatidylinositol (GPI) cell surface-anchored glycoprotein possessing a C2-type domain. Expression of CD66e can be found in many tumors of epithelial origin. The nucleotide and amino acid sequences encoding CEA are known in the art and can be easily retrieved by known methods. The amino acid sequence of human CEA is depicted in GenBank accession number NM_004363. In some embodiments, "CD66e" includes proteins containing mutations of the full-length wild-type protein, such as point mutations, fragments, insertions, deletions, and splice variants.

[0067] In some embodiments, the second antigen of the multispecific antibody is B7-H3 (also known as CD276), a member of the B7 family of immune cell-modulating molecules with a single transmembrane domain. In humans, the B7-H3 protein is expressed in two forms: variant 1 contains two V-like or C-like Ig domains, and variant 2 contains one V-like or C-like Ig domain. The C-terminal intracellular domain of B7-H3 contains 45 amino acids. It is expressed on the surface of a wide variety of tumor cells and tumor vasculature, including neuroblastoma, melanoma, renal cell carcinoma, prostate cancer, colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, ovarian cancer, and small cell lung cancer. B7-H3 expression correlates with poor prognosis in ovarian cancer, RCC, NSCLC, pancreatic cancer, prostate cancer, and colon cancer due to its potential role in inhibiting cytotoxic lymphocyte activity. In some embodiments, "B7-H3" includes proteins that include mutations of the full-length wild-type protein, for example, point mutations, fragments, insertions, deletions, and splice variants.

[0068] In some embodiments, the second antigen of the multispecific antibody is CD22. CD22, also known as SIGLEC-2 (UniProt P20273), is a cell surface receptor expressed on mature B cells. CD22 contains multiple Ig domains and is a member of the immunoglobulin superfamily. The extracellular domain of CD22 interacts with sialic acid moieties, including those present on the CD45 cell surface protein. CD22 is thought to function as an inhibitory receptor for B cell receptor signaling. CD22 is expressed on the surface of normal mature B lymphocytes as well as many types of malignant B cells, including, but not limited to, acute lymphocytic leukemia (B-ALL), chronic B lymphocytic leukemia (B-CLL), B lymphoma cells, such as Burkitt's lymphoma, AIDS-related lymphoma, and follicular lymphoma, and hairy cell leukemia. The restricted B cell expression of CD22, along with CD20 and CD19, makes it a target for therapeutic treatment of B cell malignancies. An example of a CD22-specific antibody is epratuzumab. In some embodiments, "CD22" includes proteins that include mutations of the full-length wild-type protein, such as point mutations, fragments, insertions, deletions, and splice variants.

[0069] In some embodiments, the second antigen of the multispecific antibody is CD79a. CD79a is an antigenic determinant known to be detectable in some malignant blood cancer cells, such as leukemia cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD79a can be found under accession number NP_001774.1 (isoform 1 precursor) or NP_067612.1 (isoform 2 precursor), and the mRNA sequence encoding them can be found under accession number NM_001783.3 (transcript variant 1) or NM_021601.3 (transcript variant 2). In some embodiments, the CD79a protein is expressed on cancer cells. In some embodiments, the multispecific antibody binds to an antigen within the extracellular domain of the CD79a protein. In some embodiments, "CD79a" includes proteins that include mutations of the full-length wild-type protein, for example, point mutations, fragments, insertions, deletions, and splice variants.

[0070] In some embodiments, the second antigen of the multispecific antibody is CD79b. CD79b is an antigenic determinant known to be detectable on some malignant blood cancer cells, such as leukemia cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD79b can be found under accession numbers NP_000617.1 (isoform 1 precursor), NP_067613.1 (isoform 2 precursor), or NP_001035022.1 (isoform 3 precursor), and the mRNA sequences encoding them can be found under accession numbers NM_000626.2 (transcript variant 1), NM_021602.2 (transcript variant 2), or NM_001039933.1 (transcript variant 3). In some embodiments, CD79b protein is expressed on cancer cells. In some embodiments, the multispecific antibody binds to an antigen within the extracellular domain of the CD79b protein. In some embodiments, "CD79b" includes proteins that include mutations of the full-length wild-type protein, such as point mutations, fragments, insertions, deletions, and splice variants.

[0071] In some embodiments, the second antigen of the multispecific antibody is sIgM. sIgM (surface immunoglobulin M) is typically expressed on B cells. In some embodiments, sIgM is expressed on cancer cells. In some embodiments, the multispecific antibody binds to an antigen within the extracellular domain of sIgM. In some embodiments, "sIgM" includes proteins containing mutations of the full-length wild-type protein, such as point mutations, fragments, insertions, deletions, and splice variants.

[0072] In some embodiments, the second antigen of the multispecific antibody is DC-SIGN. DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-binding nonintegrin) is also known as CD209 (cluster of differentiation 209), a protein encoded by the CD209 gene in humans. DC-SIGN is a C-type lectin receptor present on the surface of both macrophages and dendritic cells. DC-SIGN on macrophages recognizes and binds with high affinity to high-mannose N-glycans, a class of pathogen-associated molecular patterns (PAMPs) commonly found on viruses, bacteria, and fungi. This binding interaction activates phagocytosis. In myeloid cells and dendritic cells, DC-SIGN mediates dendritic cell rolling interactions with the blood endothelium and CD4+ T cell aggregation. Upon activation, it mediates recognition of pathogen haptens. In some embodiments, "DC-SIGN" includes proteins that contain mutations of the full-length wild-type protein, such as point mutations, fragments, insertions, deletions, and splice variants.

[0073] In some embodiments, the second antigen of the multispecific antibody is CD11b. CD11b (ITGAM; integrin αM) can form a heterodimer with CD18. It functions as a receptor for complement (C3bi), fibrinogen, or coagulation factor X. In humans, CD11b is strongly expressed on myeloid cells and weakly expressed on microglia in the brain, along with NK cells and some activated lymphocytes. In some embodiments, CD11b is also expressed on cancer cells, and targeting it results in anti-cancer effects. Exemplary related sequences of CD11b can be found in accession numbers NP_001139280.1, NP_000623.2, XP_011544153.1, XP_011544152.1, XP_006721108.1, AAH99660.1, and AH004143.2. In some embodiments, "CD11b" includes proteins that include mutations of the full-length wild-type protein, for example, point mutations, fragments, insertions, deletions, and splice variants.

[0074] In some embodiments, the second antigen of the multispecific antibody is CD11c. CD11c is also known as CD11C, CD11c antigen, integrin alpha X, complement component 3 receptor 4 subunit, ITGAX, LeuM5, integrin alpha X precursor, leukocyte adhesion glycoprotein p150, p95 alpha chain, and leukocyte adhesion receptor p150 subunit. The full-length CD11c protein has the amino acid sequence set forth in GenBank accession number NP_000878 and is encoded by the full-length nucleotide sequence set forth in GenBank accession number NM_000887. In some embodiments, "CD11c" includes proteins containing mutations of the full-length wild-type protein, such as point mutations, fragments, insertions, deletions, and splice variants.

[0075] In some embodiments, the second antigen of the multispecific antibody is CD18. CD18 is also known as integrin beta chain-2 or integrin β2, which in humans is encoded by the ITGB2 gene. CD18 can form a heterodimer with CD11b. Exemplary sequences of CD18 can be found in GenBank Accession No. NP_000202 (amino acid sequence) and GenBank Accession No. NM_000211 (nucleic acid). In some embodiments, "CD18" includes proteins containing mutations of the full-length wild-type protein, such as point mutations, fragments, insertions, deletions, and splice variants.

[0076] antibody subtype In some embodiments, the multispecific antibodies described herein are of the IgG1, IgG2, IgG3, or IgG4 isotype.

[0077] In some embodiments, the multispecific antibody is an IgG1 isotype. In some embodiments, the multispecific antibody is an IgG2 isotype. In some embodiments, the multispecific antibody is an IgG3 isotype. In some embodiments, the multispecific antibody is an IgG4 isotype.

[0078] In some embodiments, the multispecific antibody comprises one or more Fc substitutions that reduce binding of the multispecific antibody to Fcγ receptors (FcγRs).

[0079] In some embodiments, the one or more Fc substitutions are F234A / L235A in IgG4, L234A / L235A in IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S in IgG2, F234A / L235A in IgG4, S228P / F234A / L235A in IgG4, N297A in all Ig isotypes, V234A / G237A in IgG2, K214T / E233P / L234V / L235A / G236 deletion / A327G / P331A / D365 in IgG1. and S228P / F234A / L235A / G237A / P238S in IgG4, and S228P / F234A / L235A / G236 deletion / G237A / P238S in IgG4, wherein residue numbering is according to the EU index.

[0080] In some embodiments, the multispecific antibody further comprises a S228P substitution.

[0081] In some embodiments, the multispecific antibody comprises one or more asymmetric substitutions in the first CH3 domain, or in the second CH3 domain, or in both the first and second CH3 domains.

[0082] In some embodiments, the one or more asymmetric substitutions are selected from the group consisting of F450L / K409R, wild type / F409L_R409K, T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366SL368AY407V, L351YF405AY407V / T39 4W, T366I_K392MT394W / F405AY407V, T366LK392MT394W / F405AY407V, L351YY407A / T366AK409F, L351YY407A / T366VK409F, Y407A / T366AK409F and T350V_L351Y_F405AY407V / T350V_T366L_K392L_T394W.

[0083] Methods for generating antibodies Antibodies used in the methods of the disclosure that bind to specific antigens can be selected de novo, for example, from phage display libraries, where phage have been engineered to express human immunoglobulins or portions thereof, such as Fabs, single-chain antibodies (scFvs), or unpaired or paired antibody variable regions (Knappiket et al., J Mol Biol 296:57-86, 2000; Krebs et al., J Immunol Meth254:67-84, 2001; Vaughan et al., Nature Biotechnology 14:309-14, 1996; Sheets et al., PITAS (USA)95:6157-62, 1998; Hoogenboom and Winter, J Mol Biol 227:381,1991; Marks et al., J Mol Biol 222:581, 1991). Shi et al (2010) J. Mol. Biol. 397:385-96 and International Patent Publication No. W Phage display libraries expressing antibody heavy and light chain variable regions as fusion proteins with bacteriophage pIX coat protein, as described in O2009 / 085462. The antibody library can be screened for binding to a desired antigen, such as BCMA, CD3, CD38, CD123, CD19, CD33, PSMA, or the TMEFF2 extracellular domain. Positive clones obtained can be further characterized, and Fabs can be isolated from clonal lysates and subsequently cloned as full-length antibodies. Such phage display methods for isolating human antibodies are established in the art. See, for example: U.S. Patent Nos. 5,223,409; 5,403,484; 5,571,698; 5,427,908; 5,580,717; 5,969,108; 6,172,197; 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915; and 6,593,081.

[0084] Multispecific antibodies (e.g., bispecific antibodies) can be generated in vitro in a cell-free environment by introducing asymmetric mutations into the CH3 regions of two monospecific homodimeric antibodies and forming bispecific heterodimeric antibodies from the two parent monospecific homodimeric antibodies under reducing conditions that allow disulfide bond isomerization, according to the method described in International Patent Publication No. WO 2011 / 131746. In this method, two monospecific bivalent antibodies are engineered to have specific substitutions in the CH3 domains that promote heterodimer stability; these antibodies are incubated together under reducing conditions sufficient to allow cysteines in the hinge regions to undergo disulfide bond isomerization; this generates bispecific antibodies by Fab arm exchange. Incubation conditions can be optimally restored to non-reducing conditions. Exemplary reducing agents that can be used include 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and beta-mercaptoethanol. In some embodiments, the reducing agent is selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. For example, incubation at a pH of 5-8, e.g., at a pH of 7.0, or at a pH of 7.4, in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol, at a temperature of at least 20°C for at least 90 minutes can be used.

[0085] Exemplary CH3 mutations that can be used in the first and second heavy chains of a bispecific antibody are K409R and / or F405L.

[0086] Additional CH3 mutations that can be used include techniques such as Duobody® mutations (Genmab), knob-in-hole mutations (Genentech), electrostatically matched mutations (Chugai Pharmaceutical Co., Ltd., Amgen, NovoNordisk, Oncomed), strand-exchange engineered domain bodies (SEEDbodies) (EMD Serono) and other asymmetric mutations (e.g., Zymeworks).

[0087] Duobody® mutations (Genmab) are disclosed, for example, in U.S. Pat. No. 9,150,663 and US2014 / 0303356 and include the mutations F405L / K409R, wild type / F405L_R409K, T350I_K370TF405L / K409R, K370W / K409R, D399AFGHILMNRSTVWY / K409R, T366ADEFGHILMQVY / K409R, L368ADEGHNRSTVQ / K409AGRH, D399FHKRQ / K409AGRH, F405IKLSTVW / K409AGRH and Y407LWQ / K409AGRH.

[0088] Knob-in-hole mutations, for example, are disclosed in WO1996 / 027011 and involve mutations at the interface of the CH3 domains, where an amino acid with a small side chain (hole) is introduced into the first CH3 domain and an amino acid with a large side chain (knob) is introduced into the second CH3 domain, resulting in preferential interaction between the first and second CH3 domains. Exemplary CH3 domain mutations that form knobs and holes are T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V.

[0089] As described in US2010 / 0015133, US2009 / 0182127, US2010 / 028637 or US2011 / 0123532, heavy chain heterodimer formation can be promoted using electrostatic interactions by substituting positively charged residues in the first CH3 region and negatively charged residues in the second CH3 region.

[0090] Other asymmetric mutations that can be used to promote heavy chain heterodimerization are L351YF405AY407V / T394W, T366IK392MT394W / F405AY407V, T366LK392MT394W / F405AY407V, L351YY407A / T366AK409F, L351YY407A / T366VK409F, Y407A / T366AK409F, or T350V_L351YF405AY407V / T350V_T366LK392LT394W, as described in US2012 / 0149876 or US2013 / 0195849.

[0091] As described in US20070287170, SEEDbody mutations involve substituting selected IgG residues with IgA residues to promote heavy chain heterodimerization.

[0092] Other exemplary mutations that can be used are R409D_K370E / D399K_E357K, S354C_T366W / Y349C_T366S_L368A_Y407V, Y349C_T366W / S354C_T366S_L368A_Y407V, ... Y349C_T366W / S354C_T366S_L368A_Y407V, Y349C_T366W / S354C_T366S_L368A_Y407V, Y349C_T366W / S354C_T366S_L368A_Y407V, Y349C_T366 368A_Y407V, T366K / L351D, L351K / Y349E, L351K / Y349D, L351K / L368E, L351YY407A / T366AK409F, L351YY407A / T366VK409F, K392D / D399K, K392D / E356K, K253ED282KK322D / D239KE240KK292D, K392D_K409D / D356K_D399K.

[0093] Additional bispecific or multispecific structures that can be used as T cell redirecting therapeutics include dual variable domain immunoglobulins (DVDs) (International Patent Publication No. WO2009 / 134776; DVDs are full-length antibodies comprising a heavy chain with the structure VH1-linker-VH2-CH and a light chain with the structure VL1-linker-VL2-CL; the linker is optionally present), structures containing various dimerization domains to connect two antibody arms with different specificities, such as leucine zippers or collagen dimerization domains (International Patent Publication No. WO2012 / 022811; U.S. Patent Nos. 5,932,448; 6,833,441), heavy chain-only antibodies such as two or more domain antibodies (dAbs) conjugated together, diabodies, camelid antibodies and engineered camelid antibodies, dual targeting (DT)-Ig (GSK / Domantis), two-in-one antibodies (Genentech), cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star) and CovX-body (CovX / Pfizer), IgG-like bispecific (InnClone / Eli Lilly), Ts2Ab (MedImmune / AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec) and TvAb (Roche), ScFv / Fc fusions (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), dual affinity retargeting technology (Fc-DART) (MacroGenics) and dual (ScFv)2-Fab (National Research Center for Antibody Medicine-China), dual-acting or bis-Fab (Genentech), dock-and-lock (DNL) (ImmunoMedics), bivalent bispecific (Biotecnol) and Fab-Fv (UCB-Celltech). ScFv-based antibodies, diabody-based antibodies and domain antibodies include, but are not limited to, bispecific T cell engagers (BiTEs) (Micromet), tandem diabodies (Tandab) (Affimed), dual affinity retargeting technology (DART) (MacroGenics), single chain diabodies (Academic), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack) and COMBODYs (Epigen Biotech), dual targeting nanobodies (Ablynx), dual targeting heavy chain-only domain antibodies.

[0094] Fc engineering of antibodies The Fc region of a T cell redirecting therapeutic, such as a bispecific or multispecific antibody, can comprise at least one substitution in the Fc region that reduces binding of the T cell redirecting therapeutic to activating Fcγ receptors (FcγRs) and / or reduces an Fc effector function, such as C1q binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or phagocytosis (ADCP).

[0095] Fc positions that can be substituted to reduce Fc binding to activating FcγRs and subsequently reduce effector function include substitutions L234A / L235A in IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S in IgG2, F234A / L235A in IgG4, S228P / F234A / L235A in IgG4, N297A in all Ig isotypes, V234A / G237A in IgG2, and K214T / E233P / L234V / L235A / G in IgG1. substitutions H268Q / V309L / A330S / P331S in IgG2; substitutions S267E / L328F in IgG1; substitutions L234F / L235E / D265A in IgG1; substitutions L234A / L235A / G237A / P238S / H268A / A330S / P331S in IgG1; substitutions S228P / F234A / L235A / G237A / P238S in IgG4; and substitutions S228P / F234A / L235A / G236 deletion / G237A / P238S in IgG4.

[0096] An Fc substitution that can be used to reduce CDC is the K322A substitution.

[0097] The well-known S228P substitution can also be made in IgG4 antibodies to enhance IgG4 stability.

[0098] An exemplary wild-type IgG1 comprises the amino acid sequence of SEQ ID NO: 31, as follows: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 31)

[0099] An exemplary wild-type IgG4 comprises the amino acid sequence of SEQ ID NO: 32, as follows: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 32)

[0100] In vivo delivery of polynucleotides encoding multispecific antibodies In some embodiments, instead of a multispecific antibody, a polynucleotide encoding the multispecific antibody is administered to a subject, which polynucleotide enables the production of the multispecific antibody in vivo. In some embodiments, administration of such a polynucleotide produces an effect in vivo similar to that of direct administration of the multispecific antibody. In some embodiments, administration of such a polynucleotide improves the in vivo transduction efficiency of the vector. In some embodiments, the polynucleotide is mRNA.

[0101] In some embodiments, in vivo delivery of such polynucleotides results in multispecific antibody expression over time (e.g., starting within hours and sustained for several days). In some embodiments, in vivo delivery of such polypeptides results in desirable pharmacokinetic, pharmacodynamic, and / or safety profiles of the encoded multispecific antibodies. In some embodiments, polynucleotides can be optimized by one or more means to prevent immune activation, increase stability, reduce any tendency to aggregate over time, and / or avoid impurities. Such optimization can include the use of modified nucleosides, modified and / or specific 5'UTR, 3'UTR, and / or poly(A) tail modifications for improved intracellular stability and translation efficiency (see, e.g., Stadle et al., 2017, Nat. Med.). Such modifications is known in the art.

[0102] Strategies for in vivo delivery of polynucleotides (e.g., mRNA) are known in the art. For a summary of strategies, see MolTher. 2019 Apr 10; 27(4): 710-728, the entire contents of which are incorporated herein by reference.

[0103] In some embodiments, polynucleotides encoding multispecific antibodies are co-formulated into lipid nanoparticles (LNPs). In some embodiments, the LNP formulation is composed of (1) an ionizable or cationic lipid or polymer material with a tertiary or quaternary amine to encapsulate the polyanionic mRNA; (2) a zwitterionic lipid (e.g., 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine [DOPE]) that resembles lipids in cell membranes; (3) cholesterol to stabilize the lipid bilayer of the LNP; and (4) polyethylene glycol (PEG)-lipids to provide a hydration layer to the nanoparticles, improving colloidal stability and reducing protein absorption.

[0104] In some embodiments, polynucleotides encoding multispecific antibodies are delivered in formulations with cationic or ionizable lipids and lipid-like agents. In some embodiments, the cationic lipids are alkylated quarternary ammonoids. The cationic lipid has a nium group and retains its cationic nature in a pH-independent manner. In some embodiments, the lipid is ionizable (e.g., as the pH decreases, it acquires a positive charge by protonation of the free amine). In some embodiments, the cationic lipid comprises N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). In some embodiments, the ionizable lipid comprises Dlin-MC3-DMA (MC3).

[0105] In some embodiments, the polynucleotide encoding the multispecific antibody is delivered in a formulation with a polymeric material. In some embodiments, the polymeric material comprises a low molecular weight polyethyleneimine (PEI) modified with a lipid chain. In some embodiments, the polymeric material comprises a poly(glycoamidoamine) polymer modified with a lipid chain. In some embodiments, the polymeric material comprises a poly(β-amino)ester (PBAE).

[0106] In some embodiments, polynucleotides encoding multispecific antibodies are expressed by formulation with dendrimers (e.g., polyamidoamine (PAMAM) or polypropylenimine-based dendrimers) or cell-penetrating peptides (CPPs). In some embodiments, the polynucleotide is covalently linked to the CPP.

[0107] Examples of multispecific antibodies In some embodiments, the multispecific antibody is a BCMAxCD3 bispecific antibody, a GPRC5DxCD3 bispecific antibody, a CD33xCD3 bispecific antibody, a CD19xCD3 bispecific antibody, a CD123xCD3 bispecific antibody, a PSMAxCD3 bispecific antibody, or a TMEFF2xCD3 bispecific antibody.

[0108] In some embodiments, the multispecific antibody is a BCMAxCD3 bispecific antibody. In some embodiments, the multispecific antibody is a GPRC5DxCD3 bispecific antibody. In some embodiments, the multispecific antibody is a CD33xCD3 bispecific antibody. In some embodiments, the multispecific antibody is a CD19xCD3 bispecific antibody. In some embodiments, the multispecific antibody is a CD123xCD3 bispecific antibody. In some embodiments, the multispecific antibody is a PSMAxCD3 bispecific antibody. In some embodiments, the multispecific antibody is a TMEFF2xCD3 bispecific antibody.

[0109] In some embodiments, the multispecific antibody binds to CD3 epsilon (CDR), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C.

[0110] In some embodiments, the multispecific antibody that binds to CD19 is selected from the group consisting of blinatumomab, axicabtagene ciloreucel, tisagenlecleucel-t, inebilizumab, lisocabtagene maraleucel, XmAb-5574, CIK-CAR.CD19, ICTCAR-011, IM-19, JCAR-014, loncastuximabtesirine, MB-CART2019.1, OXS-155 and comprising the CD19-binding domain of 0, PBCAR-0191, PCAR-019, PCAR-119, Sen1-001, TI-1007, XmAb-5871, PTG-01, PZ01, Sen1_1904A, Sen11904B, UCART-19, CSG-CD19, DI-B4, ET-190, GC-007F or GC-022.

[0111] In some embodiments, multispecific antibodies that bind to CD19 comprise blinatumomab, axicabtagene ciloreucel, tisagenlecleucel-t, inebilizumab, lisocabtagene maraleucel, XmAb-5574, CIK-CAR.CD19, ICTCAR-011, IM-19, JCAR-014, loncastuximab tesirin, MB-CART2019.1, OXS-1550, PBCAR-0191, PCAR-019, PCAR-119, Sen1-001, TI-1007, XmAb-5871, PTG-01, PZ01, Sen1_1904A, Sen1_1904B, UCART-19, CSG-CD19, DI-B4, ET-190, GC-007F, or GC-022.

[0112] In some embodiments, the multispecific antibody used in the present disclosure is blinatumomab. Blinatumomab is a CD19 / CD3-bispecific antibody construct of the bispecific T-cell engager (BiTE) class and comprises the amino acid sequence of SEQ ID NO: 34: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGS QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGG GGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGG SGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKHHHHHH (SEQ ID NO: 34).

[0113] The CD19 binding region of blinatumomab contains the following CDRs: CDRL1: QSVDYDGDSY (SEQ ID NO: 35) CDRL2: DAS (SEQ ID NO: 36) CDRL3: QQSTEDPWT (SEQ ID NO: 37) CDRH1: GYAFSSYW (SEQ ID NO: 38) CDRH2: IWPGDGDT (SEQ ID NO: 39) CDRH3: ARRETTTVGRYYYAMDY (SEQ ID NO: 40)

[0114] The CD3 binding region of blinatumomab contains the following CDRs: CDRH1: GYTFTRYT (SEQ ID NO: 41) CDRH2: INPSRGYT (SEQ ID NO: 42) CDRH3: ARYYDDHYCLDY (SEQ ID NO: 43) CDRL1: SSVSY (SEQ ID NO: 44) CDRL2: DTS (SEQ ID NO: 45) CDRL3: QQWSSNP (SEQ ID NO: 46)

[0115] In some embodiments, the multispecific antibody is a CD19xCD3 bispecific antibody comprising a CD19 binding region comprising the CDRs according to SEQ ID NOs: 35 to 40 and / or a CD3 binding region comprising the CDRs according to SEQ ID NOs: 41 to 46. In some embodiments, the CD19xCD3 bispecific antibody is a CD19xCD3 BiTE.

[0116] method Various embodiments of the present disclosure provide methods of using multispecific antibodies as enhancers of vector (e.g., non-viral or viral vector) transduction. In some embodiments, the multispecific antibodies facilitate cell transduction of vectors, such as vectors designed for gene therapy and / or treatment of cancer or hematologic malignancies in vivo. In some embodiments, both the multispecific antibody and the vector are administered in vivo. In various embodiments, the multispecific antibody can be administered before, in conjunction with, or after administration of the viral vector.

[0117] In some embodiments, the multispecific antibodies and vectors are administered to a subject to treat and / or prevent a disease, disorder, or condition, hi some embodiments, the multispecific antibodies and vectors are administered to a subject for research and / or drug development purposes.

[0118] effect In some embodiments, administration of the multispecific antibody in a subject results in activation of immune cells, hi some embodiments, the activation of immune cells is mediated by binding of the multispecific antibody to both immune cells and cells expressing a specific antigen.

[0119] In some embodiments, immune cell activation is measured by the level of one or more cell markers. In some embodiments, immune cell activation is measured by the percentage of immune cells that are positive for one or more cell markers. In some embodiments, the immune cells are T cells (T lymphocytes) or NK cells. In some embodiments, the immune cells are CD4+ T cells or CD8+ T cells. In some embodiments, the one or more cell markers are selected from the group consisting of CD71, CD25, CD69, Ki67, and any combination thereof.

[0120] In some embodiments, immune cell activation is measured by the percentage of immune cells that are CD71 positive. In some embodiments, administration of the multispecific antibody increases the percentage of CD71+ immune cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments, immune cell activation is measured by the level of CD71 expressed on the surface of immune cells. In some embodiments, administration of the multispecific antibody increases the level of CD71 expressed on the surface of immune cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7-fold, or at least 10-fold.

[0121] In some embodiments, immune cell activation is measured by the percentage of immune cells that are CD25 positive. In some embodiments, administration of the multispecific antibody increases the percentage of CD25+ immune cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments, immune cell activation is measured by the level of CD25 expressed on the surface of immune cells. In some embodiments, administration of the multispecific antibody increases the level of CD25 expressed on the surface of immune cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7-fold, or at least 10-fold.

[0122] In some embodiments, immune cell activation is measured by the percentage of immune cells that are CD69 positive. In some embodiments, administration of the multispecific antibody increases the percentage of CD69+ immune cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments, immune cell activation is measured by the level of CD69 expressed on the surface of immune cells. In some embodiments, administration of the multispecific antibody increases the level of CD69 expressed on the surface of immune cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7-fold, or at least 10-fold.

[0123] In some embodiments, immune cell activation is measured by the percentage of immune cells that are Ki67 positive. In some embodiments, administration of a multispecific antibody increases the percentage of Ki67+ immune cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments, immune cell activation is measured by the level of Ki67 expressed on the surface of immune cells. In some embodiments, administration of a multispecific antibody increases the level of Ki67 expressed on the surface of immune cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7-fold, or at least 10-fold.

[0124] In some embodiments, administration of the multispecific antibody in a subject results in an increase in immune cells that are susceptible and / or readily receptive to transduction with the vector.

[0125] In some embodiments, administration of the multispecific antibody in a subject results in active proliferation of immune cells, which in some embodiments increases the number of and / or susceptibility to transduction by the vector.

[0126] In some embodiments, administration of the multispecific antibody in a subject results in a decrease in the number of G0 phase immune cells (e.g., T cells) and / or an increase in the number of non-G0 phase immune cells (e.g., T cells).

[0127] In some embodiments, administration of the multispecific antibody in a subject increases the number and / or percentage of immune cells that are metabolically adapted to vector transduction.

[0128] In some embodiments, administration of the multispecific antibody in a subject results in the accumulation of immune cells in lymph nodes. In some embodiments, administration of the multispecific antibody in a subject results in the accumulation of immune cells at the tumor site.

[0129] In some embodiments, administration of the multispecific antibody in a subject facilitates entry of a vector (e.g., a viral particle) into a target immune cell. In some embodiments, administration of the multispecific antibody in a subject enhances the infectious titer of the vector particle. In some embodiments, administration of the multispecific antibody in a subject increases cellular uptake of the vector particle by immune cells.

[0130] In some embodiments, the multispecific antibody is a bispecific antibody. In some embodiments, the vector is a lentiviral vector. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell herein is a subset of immune cells in vivo that can be recognized by at least one antigen-specific binding domain of the multispecific antibody. In some embodiments, the immune cell is present in a lymph node.

[0131] Dosage schedule In some embodiments of the methods described herein, transduction (e.g., retroviral transduction, e.g., lentiviral transduction) of T lymphocytes (e.g., primary human T lymphocytes) may be enhanced by administering a multispecific antibody to the subject prior to, in conjunction with, or after administration of the vector to the subject for any of the time periods disclosed herein, or any combination thereof.

[0132] In some embodiments, the multispecific antibody is administered before the vector is administered. In some embodiments, the multispecific antibody is administered about 0.5 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 9 hours, about 12 hours, about 16 hours, about 20 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days or more before the vector is administered. In some embodiments, when the multispecific antibody and / or vector are administered repeatedly, the time intervals listed herein are calculated based on the interval between the last administration of the multispecific antibody and the first administration of the vector.

[0133] In some embodiments, the multispecific antibody is administered after the vector is administered. In some embodiments, the multispecific antibody is administered about 0.5 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 9 hours, about 12 hours, or about 16 hours after the vector is administered. In some embodiments, when the multispecific antibody and / or vector are administered repeatedly, the time intervals listed herein are calculated based on the interval between the last administration of the vector and the first administration of the multispecific antibody.

[0134] In some embodiments, the multispecific antibody is administered in conjunction with a vector. As used herein, the term "in conjunction with" does not limit the administration of therapeutic agents at exactly the same time, but rather means that the multispecific antibody and vector are administered to a subject and / or cells sequentially and within a time interval so that they can act together on target cells. For example, each agent can be administered sufficiently close in time to produce the desired therapeutic or prophylactic effect, for example, within about 10 minutes, about 20 minutes, about 30 minutes, about 60 minutes, about 2 hours, about 3 hours, about 6 hours, about 12 hours, or about 24 hours. Each agent can be administered to a subject separately in any appropriate form and by any suitable route. Each agent in a concurrent administration can be administered in the same pharmaceutical agent (simultaneously), in separate pharmaceutical agents administered one after the other in any order, or sequentially in any order.

[0135] In some embodiments, when the multispecific antibody and / or vector are administered repeatedly, at least one administration of the multispecific antibody is administered in conjunction with at least one administration of the vector. In some embodiments, the first or only administration of the vector is administered in conjunction with the last or only administration of the multispecific antibody. In some embodiments, the first or only administration of the multispecific antibody is administered in conjunction with the last or only administration of the vector. In some embodiments, each administration of the multispecific antibody is administered in conjunction with an administration of the vector. In some embodiments, each administration of the vector is administered in conjunction with an administration of the multispecific antibody.

[0136] The present disclosure further contemplates that one or more additional agents that improve the transduction efficiency of the vector can be used in combination with the multispecific antibodies and vectors described herein, and the one or more additional agents can be administered before, in conjunction with, or after administration of the multispecific antibody and / or vector to a subject.

[0137] Dosage Vector dosage The vectors can be used to infect cells in vivo at any effective dosage, hi some embodiments, the vectors are administered to a subject in vivo by direct injection into a cell, tissue, organ, or subject in need of treatment.

[0138] Viral vectors can also be delivered according to viral titer (TU / mL). The amount of lentivirus directly injected is determined by the total TU and can vary based on both the volume that can be feasibly injected at the site and the type of tissue being injected. In some embodiments, approximately 1 x 10 per injection. 5 ~1×10 6 , about 1×10 5 ~1×10 7 , 1×10 5 ~1×10 7 , about 1×10 6 ~1×10 9 , about 1×10 7 ~1×10 10 , about 1×107 ~1×10 11 Or about 1 x 10 9 ~1×10 11 or higher delivered virus titers can be used. In some embodiments, titers of about 1 x 10 per injection are used. 6 ~1×10 7 , about 1×10 6 ~1×10 8 , 1×10 6 ~1×10 9 , about 1×10 7 ~1×10 10 , about 1×10 8 ~1×10 11 , about 1×10 8 ~1×10 12 Or about 1 x 10 10 ~1×10 12 For example, brain injection sites may only allow very small volumes of virus to be injected, so high titer preps are preferred, approximately 1 x 10 per injection. 6 ~1×10 7 , about 1×10 6 ~1×10 8 , 1×10 6 ~1×10 9 , about 1×10 7 ~1×10 10 , about 1×10 8 ~1×10 11 , about 1×10 8 ~1×10 12 Or about 1 x 10 10 ~1×10 12 or more TU can be used. However, systemic delivery can accommodate much higher TU, approximately 1 x 10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 , about 1×10 14 or about 1 x 10 15 can deliver a load of

[0139] In some embodiments, the vector is administered at a dose of about 1 x 10 per kilogram (vg) of total subject body weight. 12 ~5×10 14 In some embodiments, the vector is administered at a dose of between about 1 x 10 vector genomes (vg) and about 1 x 10 vector (vg / kg). 13 ~5×10 14 In some embodiments, the vector is administered at a dose of between about 5×10 13 ~3×10 14 In some embodiments, the vector is administered at a dose of between about 5×10 13 ~1×10 14 In some embodiments, the vector is administered at a dose of between about 1 x 10 vg / kg. 12 Less than 3 × 10 vg / kg 12 Less than 5 × 10 vg / kg 12 Less than 7 × 10 vg / kg 12 Less than 1 × 10 vg / kg 13 Less than 3 × 10 vg / kg 13 Less than 5 × 10 vg / kg 13 Less than 7 × 10 vg / kg 13 Less than 1 × 10 vg / kg 14 Less than 3 × 10 vg / kg 14 Less than 5 × 10 vg / kg 14 Less than 7 × 10 vg / kg 14 Less than 1 × 10 vg / kg 15 Less than 3 × 10 vg / kg 15 Less than 5 × 10 vg / kg 15 vg / kg or less than 7 × 10 15 It is administered at a dose of less than vg / kg.

[0140] In some embodiments, the vector is administered at a dose of about 1 x 10 per kilogram of total subject body weight (vp). 12 ~5×10 14 In some embodiments, the vector is administered at a dose of between about 1 x 10 vector particles (vp) and about 1 x 10 vector (vp / kg). 13 ~5×10 14 In some embodiments, the vector is administered at a dose of between about 5×10 vp / kg.13 ~3×10 14 In some embodiments, the vector is administered at a dose of between about 5×10 vp / kg. 13 ~1×10 14 In some embodiments, the vector is administered at a dose of between about 1 x 10 vp / kg. 12 Less than 3 × 10 vp / kg 12 Less than 5 × 10 vp / kg 12 Less than 7 × 10 vp / kg 12 Less than 1 × 10 vp / kg 13 Less than 3 × 10 vp / kg 13 Less than 5 × 10 vp / kg 13 Less than 7 × 10 vp / kg 13 Less than 1 × 10 vp / kg 14 Less than 3 × 10 vp / kg 14 Less than 5 × 10 vp / kg 14 Less than 7 × 10 vp / kg 14 Less than 1 × 10 vp / kg 15 Less than 3 × 10 vp / kg 15 Less than 5 × 10 vp / kg 15 vp / kg or less than 7 × 10 15 It is administered at a dose of less than vp / kg.

[0141] Antibody dosage and timing The dose of multispecific antibody (e.g., bispecific antibody) given to a subject with a disease or disorder (e.g., cancer, such as a hematological malignancy) is sufficient to improve vector transduction efficiency as described herein (an "effective amount"). A dose of multispecific antibody sufficient to induce cytotoxicity or other secondary therapeutic effect in cells can be used, but more preferably, a reduced dose is used, especially when non-target cells (e.g., B cells for a CD3xCD19 bispecific) are non-malignant cells or cells whose generation after transduction is desirable for the therapeutic effect of the target immune cells (e.g., T cells). In some embodiments, the selected dose is 10, 100, 1000, 5000, or 10,000 times lower than the dose of multispecific antibody used in monotherapy. In some embodiments, the dose comprises about 5 μg to about 10 mg / kg, e.g., about 0.005 mg to about 3 mg / kg or about 0.5 mg to about 2.5 mg / kg, or about 0.4 mg / kg, about 0.8 mg / kg, about 1.6 mg / kg, or about 2.4 mg / kg of antibody. Suitable doses include, for example, about 0.01, 0.02, 0.05, 0.07, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0 mg / kg. In some embodiments, the dose comprises from about 0.05 μg to about 1 mg / kg, e.g., from about 0.5 μg to about 0.30 mg / kg or from about 0.005 mg to about 0.25 mg / kg, or about 0.04 mg / kg, about 0.08 mg / kg, about 0.16 mg / kg, or about 0.24 mg / kg of antibody. Suitable doses include, for example, about 0.001, 0.002, 0.005, 0.007, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.01, 0.015, 0.016, 0.017, 0.018, 0.019, 0.02, 0.021, 0.022, 0.023, 0.024, 0.025, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.1 mg / kg.

[0142] A fixed unit dose of the multispecific antibody can also be given, for example, about 1, 2, 5, 10, 20, 50, 100, 200, 500, or 1000 mg, or the dose can be measured based on the surface area of ​​the patient, for example, about 500, 400, 300, 250, 200, 100, 50, 20, 10, 5, 2, or 1 mg / m 2 In some embodiments, the fixed unit dose of the multispecific antibody is, for example, about 0.1, 0.2, 0.5, 0.1, 0.2, 0.5, 0.1, 0.2, 0.5, or 1 mg, or the dose can be based on the surface area of ​​the patient, for example, about 50, 40, 30, 25, 20, 10, 5, 2, 1, 0.5, 0.2, or 0.1 mg / m 2 In some embodiments, the fixed unit dose of the multispecific antibody can be, for example, about 0.01, 0.02, 0.05, 0.01, 0.02, 0.05, 0.01, 0.02, 0.05, or 0.1 mg, or the dose can be based on a patient's surface area, for example, about 5, 4, 3, 2.5, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.02, or 0.01 mg / m 2 can be based on

[0143] The multispecific antibody can be administered before, during, or after administration of the vector (e.g., a viral vector). In some embodiments, the multispecific antibody is administered 1 week, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day before the vector. In some embodiments, the multispecific antibody is administered 1 to 4 hours, 4 to 8 hours, or about 1 hour, about 2 hours, about 3 hours, or about 4 hours before administration of the vector. In some embodiments, the multispecific antibody is administered concomitantly with administration of the vector. In some embodiments, the multispecific antibody is administered after the vector (e.g., 1 to 4 hours, 1 to 8 hours, or 1 day after the vector).

[0144] While multispecific antibodies in current treatments are generally administered repeatedly (i.e., on a weekly, bi-weekly, or monthly schedule), the methods of the present disclosure Thus, the multispecific antibody can be administered only once, twice, or three times. In certain embodiments, the administration of the multispecific antibody is performed exactly once. Similarly, a single injection of the multispecific antibody is administered before or in conjunction with administration of the vector. If repeated administration of the vector is desired, one can choose to repeat the administration protocol for the multispecific antibody each time treatment with the vector is performed.

[0145] Route of administration In some embodiments, the vector is administered parenterally, intravenously, intramuscularly, subcutaneously, The vector is administered by a route selected from the group consisting of intratumoral and intralymphatic. In some embodiments, the vector is administered multiple times. In some embodiments, the vector is administered by intralymphatic injection of the vector. In some embodiments, the vector is administered by injection of the vector at the tumor site (i.e., intratumoral). In some embodiments, the vector is administered subcutaneously. In some embodiments, the vector is administered systemically. In some embodiments, the vector is administered intravenously. In some embodiments, the vector is administered intra-arterially. In some embodiments, the vector is a lentiviral vector.

[0146] In some embodiments, the multispecific antibody is administered by a route selected from the group consisting of parenteral, intravenous, intramuscular, subcutaneous, intratumoral, and intralymphatic. In some embodiments, the antibody is administered multiple times. In some embodiments, the antibody is administered by intralymphatic injection of the antibody. In some embodiments, the antibody is administered by injection of the antibody into the tumor site (i.e., intratumoral). In some embodiments, the antibody is administered subcutaneously. In some embodiments, the antibody is administered systemically. In some embodiments, the antibody is administered intravenously. In some embodiments, the antibody is administered intra-arterially. In some embodiments, the antibody is a bispecific antibody.

[0147] The multispecific antibody and the do not have to share the same mode of administration, e.g. One agent (eg, an antibody) can be administered systematically, while the second agent (eg, a vector) can be administered intralymphatically.

[0148] Transduction efficiency In some embodiments, compositions and methods of the disclosure that employ multispecific antibodies can increase the transduction efficiency of a viral vector by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more compared to the transduction efficiency of a viral vector that does not employ such multispecific antibodies.

[0149] In some embodiments, compositions and methods of the disclosure that employ multispecific antibodies can increase the transduction efficiency of a viral vector by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 5-fold, at least about 7-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 70-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 500-fold, at least about 700-fold, at least about 1000-fold or more compared to the transduction efficiency of a viral vector that does not employ such multispecific antibodies.

[0150] Combinatorial Therapy The present disclosure further contemplates that one or more additional agents that improve the transduction efficiency of the vector can be used in combination with the multispecific antibodies and vectors described herein.

[0151] In some embodiments, the method further comprises administering to the subject one or more anti-cancer treatments.

[0152] In some embodiments, the one or more anti-cancer therapies are selected from the group consisting of autologous stem cell transplant (ASCT), radiation, surgery, chemotherapeutic agents, immunomodulatory agents, and targeted cancer therapies.

[0153] In some embodiments, the one or more anticancer therapies are lenalidomide, thalidomide, pomalidomide, bortezomib, carfilzomib, elotuzumab, ixazomib, melphalan, dexamethasone, vincristine, cyclophosphamide, hydroxydaunorubicin, prednisone, rituximab, imatinib, dasatinib, nilotinib, bosutinib, ponatinib, bafetinib, saracatinib, or rifametinib. nib), tozasertib or danusertib, cytarabine, daunorubicin, idarubicin, mitoxantrone, hydroxyurea, decitabine, cladribine, fludarabine, topotecan, etoposide, 6-thioguanine, corticosteroids, methotrexate, 6-mercaptopurine, azacitidine, arsenic trioxide and all-trans retinoic acid, or any combination thereof.

[0154] vector Vector can be virus or non-virus vector.Exemplary non-virus vector includes, for example, naked DNA, cationic liposome complex, cationic polymer complex, cationic liposome-polymer complex and exosome.Example of virus vector includes but is not limited to, adenovirus vector, retrovirus vector, lentivirus vector, herpesvirus vector and adeno-associated virus (AAV) vector.

[0155] In some embodiments, the vector comprises a polynucleotide. In some embodiments, the polynucleotide encodes at least one therapeutic polypeptide. The term "therapeutic polypeptide" refers to a polypeptide that is being developed for therapeutic use or has been developed for therapeutic use. In some embodiments, the therapeutic polypeptide is expressed in target cells (e.g., host T cells) for therapeutic use. In some embodiments, the therapeutic polypeptide comprises a T cell receptor, a chimeric antigen receptor, or a cytokine receptor.

[0156] In some embodiments, the vectors described herein are retroviral vectors. In some embodiments, the vectors are lentiviral vectors. In some embodiments, the vectors are adeno-associated viral vectors.

[0157] The term viral vector can refer to either a vector or viral particle capable of transferring a nucleic acid into a cell, or the transferred nucleic acid itself. A viral vector contains structural and / or functional genetic elements that are primarily derived from a virus. The term "retroviral vector" refers to a viral vector or a portion thereof that contains structural and functional genetic elements that are primarily derived from a retrovirus. The term "lentiviral vector" refers to a viral vector or a portion thereof that contains structural and functional genetic elements, including LTRs, that are primarily derived from a lentivirus. The term "hybrid" refers to a vector, LTR, or other nucleic acid that contains both retroviral sequences, e.g., lentiviral sequences, and non-lentiviral sequences. In some embodiments, a hybrid vector refers to a vector or transfer plasmid that contains retroviral sequences, e.g., lentiviral sequences, for reverse transcription, replication, integration, and / or packaging.

[0158] Vector-type retroviral vector Retroviruses include lentiviruses, gamma-retroviruses and Retroviruses include alpha-retroviruses, each of which can be used to deliver polynucleotides to cells using methods known in the art. Lentiviruses are complex retroviruses that contain the common retroviral genes gag, pol, and env, as well as other genes with regulatory or structural functions. Their greater complexity allows the virus to modulate its life cycle, such as during the course of latent infection. Exemplary lentiviruses include, but are not limited to, HIV (human immunodeficiency viruses, including HIV types 1 and 2); visna-maedi virus (VMV); caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In some embodiments, the backbone is an HIV-based vector backbone (i.e., HIV cis-acting sequence elements). Retroviral vectors have been generated by multiple attenuation of HIV virulence genes, for example, genes env, vif, vpr, vpu and nef have been deleted, making the vector biologically safe.

[0159] Exemplary lentiviral vectors include those described in Naldini et al. (1996) Science 272:263-7; Zufferey et al. (1998) J. Virol. 72:9873-9880; Dull et al. (1998) J. Virol. 72:8463-8471; U.S. Patent No. 6,013,516; and U.S. Patent No. 5,994,136, each of which is incorporated herein by reference in its entirety. Generally, these vectors are configured to carry the necessary sequences for selection of cells containing the vector, for incorporating exogenous nucleic acid into lentiviral particles, and for transfer of nucleic acid into target cells.

[0160] The commonly used lentiviral vector system is the so-called third-generation system. Third-generation lentiviral vector systems contain four types of plasmids. The "transfer plasmid" encodes the polynucleotide sequence delivered to target cells by the lentiviral vector system. Transfer plasmids generally contain one or more transgene sequences of interest flanked by long terminal repeat (LTR) sequences, which facilitate integration of the transfer plasmid sequence into the host genome. For safety reasons, transfer plasmids are generally designed to eliminate the replication capacity of the resulting vector. For example, transfer plasmids lack the genetic elements necessary for the production of infectious particles in host cells. In addition, transfer plasmids can be designed with a deletion of the 3' LTR, rendering the virus "self-inactivating" (SIN). See Dullet et al. (1998) J. Virol. 72:8463-71; Miyoshi et al. (1998) J. Virol. 72:8150-57. Viral particles can also contain a 3' untranslated region (UTR) and a 5' UTR. The UTR contains retroviral regulatory elements that support packaging, reverse transcription, and integration of the proviral genome into the cell after contact of the cell with the retroviral particle.

[0161] Third-generation systems also generally contain two "packaging plasmids" and one "envelope plasmid." The "envelope plasmid" generally encodes the Env gene operably linked to a promoter. In an exemplary third-generation system, the Env gene is VSV-G and the promoter is a CMV promoter. As an additional safety feature, third-generation systems use two packaging plasmids, one encoding gag and pol, and the other encoding rev—an improvement over the single packaging plasmid of the so-called second-generation system. While safer, third-generation systems can be more cumbersome to use and result in lower virus titers due to the addition of yet another plasmid. Exemplary packing plasmids are pMD2.G, Including, but not limited to, pRSV-rev, pMDLG-pRRE and pRRL-GOI.

[0162] Many retroviral vector systems rely on the use of a "packaging cell line." Generally, a packaging cell line is a cell line that, when a transfer plasmid, packaging plasmid(s), and envelope plasmid are introduced into the cell, allows the cell to produce infectious retroviral particles. Various methods of introducing plasmids into cells can be used, including transfection or electroporation. In some cases, the packaging cell line is adapted for high-efficiency packaging of the retroviral vector system into retroviral particles.

[0163] As used herein, the term "retroviral vector" or "lentiviral vector" refers to a viral particle that contains a polynucleotide encoding a heterologous protein (e.g., a chimeric antigen receptor), one or more capsid proteins, and other proteins necessary for transduction of the polynucleotide into a target cell. Retroviral and lentiviral particles generally contain an RNA genome (derived from a transfer plasmid), a lipid bilayer envelope in which the Env protein is embedded, and other accessory proteins, including integrase, protease, and matrix protein.

[0164] The ex vivo efficiency of retroviral or lentiviral vector systems can be evaluated in various ways known in the art, including measuring vector copy number (VCN) or vector genome (vg), such as by quantitative polymerase chain reaction (qPCR), or viral titer expressed in infectious units per milliliter (IU / mL).For example, titer can be evaluated using a functional assay performed in cultured tumor cell line HT1080, as described in Humbert et al. Development of Third-generation Cocal Envelope Producer Cell Lines for Robust Retroviral Gene Transfer into Hematopoietic Stem Cells and T-cells.Molecular Therapy 24:1237-1246 (2016).When titer is evaluated in a constantly dividing cultured cell line, no stimulation is required, and therefore the measured titer is not affected by surface manipulation of retroviral particles. Other methods for assessing the efficiency of retroviral vector systems are provided in Gaerertsetal. Comparison of retroviral vector titration methods. BMC Biotechnol. 6:34 (2006).

[0165] In some embodiments, the retroviral and / or lentiviral particles of the present disclosure comprise a polynucleotide comprising a sequence encoding a receptor that specifically binds to a hapten. In some embodiments, the sequence encoding the receptor that specifically binds to a hapten is operably linked to a promoter. Exemplary promoters include, but are not limited to, a cytomegalovirus (CMV) promoter, a CAG promoter, an SV40 promoter, an SV40 / CD43 promoter, and an MND promoter.

[0166] In some embodiments, the retroviral particle comprises a transduction enhancer. In some embodiments, the retroviral particle comprises a polynucleotide comprising a sequence encoding a T cell activator protein. In some embodiments, the retroviral particle comprises a polynucleotide comprising a sequence encoding a hapten-binding receptor. In some embodiments, the retroviral particle comprises a tagging protein.

[0167] In some embodiments, each retroviral particle comprises a polynucleotide comprising, in 5' to 3' order: (i) a 5' long terminal repeat (LTR) or untranslated region (UTR), (ii) a promoter, (iii) a sequence encoding a receptor that specifically binds to a hapten, and (iv) a 3' LTR or UTR.

[0168] In some embodiments, the retroviral particle contains a cell surface receptor that binds to a ligand on the target host cell, enabling host cell transduction. The viral vector can contain a heterologous viral envelope glycoprotein, resulting in a pseudotyped viral vector. For example, the viral envelope glycoprotein can be derived from RD114 or one of its variants, VSV-G, gibbon ape leukemia virus (GALV), or an amphotropic envelope, measles envelope, or baboon retrovirus envelope glycoprotein. In some embodiments, the cell surface receptor is the VSV G protein from the Kokal strain or a functional variant thereof. In some embodiments, the viral fusion glycoprotein comprises the amino acid sequence of SEQ ID NO: 33 (Kokal G protein). In some embodiments, the viral fusion glycoprotein comprises an amino acid sequence at least 95% identical to SEQ ID NO: 33 (Kokal G protein). In some embodiments, the viral fusion glycoprotein comprises an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 33 (Cocal G protein), as follows: NFLLLTFIVLPLCSHAKFSIVFPQSQKGNWKNVPSSYHYCPSSSDQNWHNDLLGITMKVKMPKTHKAIQADGWMCHAAKWITTCDFRWYGPKYITHSIHSIQPTSEQCKESIKQTKQGTWMSPGFPP QNCGYATVTDSVAVVVQATPHHVLVDEYTGEWIDSQFPNGKCETEECETVHNSTVWYSDYKVTGLCDATLVDTEITFFSEDGKKESIGKPNTGYRSNYFAYEKGDKVCKMNYCKHAGVRLPSGVWFEF VDQDVYAAAKLPECPVGATISAPTQTSVDVSLILDVERILDYSLCQETWSKIRSKQPVSPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRIDIDNPIISKMVGKISGSQTERELWTEWFPYEGVE IGPNGILKTPTGYKFPLFMIGHGMLDSDLHKTSQAEVFEHPHLAEAPKQLPEEETLFFGDTGISKNPVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK (SEQ ID NO: 33)

[0169] Various fusion glycoproteins can be used to pseudotype lentiviral vectors. The most commonly used example is the envelope glycoprotein from vesicular stomatitis virus (VSVG), but many other viral proteins have also been used to pseudotype lentiviral vectors. See Joglekaret et al. Human Gene Therapy Methods 28:291-301 (2017). The present disclosure contemplates the substitution of various fusion glycoproteins. Notably, some fusion glycoproteins result in higher vector efficiency.

[0170] In some embodiments, pseudotyping of the fusion glycoprotein or functional variant thereof facilitates targeted transduction of specific cell types, including, but not limited to, T cells or NK-cells. In some embodiments, the fusion glycoprotein or functional variant thereof is selected from the group consisting of human immunodeficiency virus (HIV) gp160, murine leukemia virus (MLV) gp70, gibbon ape leukemia virus (GALV) gp70, feline leukemia virus (RD114) gp70, amphotropic retrovirus (Ampho) gp70, 10A1 MLV (10A1) gp70, ecotropic retrovirus (Eco) gp70, baboon ape (ape) leukemia virus (BaEV) gp70, measles virus (MV) H gp70, and the like. and F, Nipah virus (NiV) H and F, rabies virus (RabV) G, Mokola virus (MOKV) G, Ebola Zaire virus (EboZ) G, lymphocytic choriomeningitis virus (LCMV) GP1 and GP2, baculovirus GP64, chikungunya virus (CHIKV) E1 and E2, Ross River virus (RRV) E1 and E2, Semliki Forest virus (SFV) E1 and E2, Sindbis virus (SV) E1 and E2, Venezuelan equine encephalitis virus (VEEV) E1 and E2, Western equine encephalitis virus (WEEV) E1 and E2, influenza A, B, C, or D The full-length polypeptide(s), functional fragment(s), homolog(s) or functional variant(s) of HA, fowl plague virus (FPV) HA, vesicular stomatitis virus VSV-G or Chandipura virus and Piry virus CNV-G and PRV-G.

[0171] In some embodiments, the fusion glycoprotein or functional variant thereof is selected from the group consisting of vesicular stomatitis Alagoas virus (VSAV), Carajas vegiclovirus (CJSV), Chandipura vegiclovirus (CHPV), Kocal vegiclovirus (COCV), vesicular stomatitis Indiana virus (VSIV), Isfa virus (ISF), and the like. Isfahan Vesiculovirus (ISFV), Maraba Vesiculovirus ( MARAV), Vesicular stomatitis New Jersey virus (VSNJV), Bas-Congo virus (BASV) G protein full-length polypeptide, functional In some embodiments, the fusion glycoprotein or functional variant thereof is a cocalvirus G protein.

[0172] In some embodiments, the fusion glycoprotein or functional variant thereof is a full-length polypeptide, functional fragment, homolog, or functional variant of the G protein of Vesicular Stomatitis Alagoas virus (VSAV), Carajas vegiculovirus (CJSV), Chandipura vegiculovirus (CHPV), Kocar vegiculovirus (COCV), Vesicular Stomatitis Indiana virus (VSIV), Isfahan vegiculovirus (ISFV), Maraba vegiculovirus (MARAV), Vesicular Stomatitis New Jersey virus (VSNJV), or Bascongovirus (BASV). In some embodiments, the fusion glycoprotein or functional variant thereof is a Kocarvirus G protein.

[0173] In some embodiments, the vector is a Nipah virus (NiV) envelope-pseudotyped lentiviral particle ("Nipah envelope-pseudotyped vector"). In some embodiments, the Nipah envelope-pseudotyped vector is pseudotyped using the Nipah virus envelope glycoproteins NiV-F and NiV-G. In some embodiments, the NiV-F and / or NiV-G glycoproteins in such Nipah envelope-pseudotyped vectors are modified variants. In some embodiments, the NiV-F and / or NiV-G glycoproteins in such Nipah envelope-pseudotyped vectors are modified to include an antigen-binding domain. In some embodiments, the antigen is EpCAM, CD4, or CD8. In some embodiments, the Nipah envelope-pseudotyped vector can efficiently transduce cells expressing EpCAM, CD4, or CD8. See U.S. Patent No. 9,486,539 and Bender et al. PLoS Pathog. 2016 Jun; 12(6): e1005641.

[0174] In some embodiments, the retroviral vector is surface-engineered. Exemplary methods for surface-engineering retroviral vectors are provided, for example, in WO2019 / 200056, PCT / US2019 / 062675 and US62 / 916,110, each of which is incorporated herein by reference in its entirety.

[0175] The present disclosure provides various non-viral proteins capable of viral surface display. In some embodiments, the non-viral protein is a costimulatory molecule. Traditionally, in vitro lentiviral transduction requires the addition of an exogenous activator, such as "stimbeads," e.g., Dynabeads™ human T-activator CD3 / CD28. In some embodiments, the retroviral (e.g., lentiviral) vectors of the present disclosure incorporate one or more copies of a non-viral protein, such as a T cell activation or costimulatory molecule(s). Incorporation of T cell activation or costimulatory molecule(s) in the vector can provide a vector that can activate and efficiently transduce T cells in the absence or presence of a lower amount of an exogenous activator, i.e., without stimbeads or equivalent agents. This allows the vector to further enhance in vivo transduction of T cells using multispecific antibodies according to the methods disclosed herein.

[0176] In some embodiments, the T cell activating or costimulatory molecule can be selected from the group consisting of an anti-CD3 antibody, CD28 ligand (CD28L), and 41bb ligand (41BBL or CD137L). Various T cell activating or costimulatory molecules are known in the art and include, without limitation, agents that specifically bind to any of the T cell expressed proteins, CD3, CD28, CD134, also known as OX40, or 41bb, also known as 4-1BB or CD137 or TNFRSF9. For example, an agent that specifically binds to CD3 can be an anti-CD3 antibody (e.g., OKT3, CRIS-7, or I2C) or an antigen-binding fragment of an anti-CD3 antibody.

[0177] In some embodiments, the agent that specifically binds to CD3 is a single-chain Fv fragment (scFv) of an anti-CD3 antibody. In some embodiments, the T cell activating or costimulatory molecule is selected from the group consisting of an anti-CD3 antibody, a ligand of CD28 (e.g., CD28L), and a 41bb ligand (41BBL or CD137L). CD86, also known as B7-2, is a ligand for both CD28 and CTLA-4. In some embodiments, the ligand of CD28 is CD86. CD80 is an additional ligand for CD28. In some embodiments, the ligand of CD28 is CD80. In some embodiments, the ligand of CD28 is an anti-CD28 antibody or anti-CD28 scFv attached to a transmembrane domain for display on the surface of the vector. Vectors containing one or more T cell activating or costimulatory molecule(s) can be made by engineering packaging cell lines by the methods provided in WO2016 / 139463; or by expression of the T cell activating or costimulatory molecule(s) from a polycistronic helper vector as described in PCT / US2019 / 062675.

[0178] In some embodiments, the vector comprises a ligand for CD19, or a functional fragment thereof, linked to its native or heterologous transmembrane domain. In some embodiments, CD19 acts as a ligand for blinatumomab, thereby providing an adapter for binding the particle to T cells via the anti-CD3 portion of blinatumomab. In some embodiments, another type of particle surface ligand can function to bind appropriately surface-engineered lentiviral particles to T cells using a multispecific antibody comprising a binding moiety for the particle surface ligand. In some embodiments, the multispecific antibody is a bispecific antibody, e.g., a bispecific T cell engager (BiTE).

[0179] The non-viral protein may be a cytokine. In some embodiments, the cytokine may be selected from the group consisting of IL-15, IL-7, and IL-2. If the non-viral protein used is a soluble protein (such as an scFv or cytokine), it can be tethered to the surface of the lentiviral particle by fusion to a transmembrane domain, such as the transmembrane domain of CD8. Alternatively, it can be indirectly tethered to the lentiviral particle by using a transmembrane protein engineered to bind to the soluble protein. The further inclusion of one or more cytoplasmic residues can increase the stability of the fusion protein.

[0180] In some embodiments, the surface-engineered vector comprises a transmembrane protein comprising a mitogenic domain and / or a cytokine-based domain. In certain embodiments, the mitogenic domain binds to a T cell surface antigen such as CD3, CD28, CD134, and CD137. In some embodiments, the mitogenic domain binds to the CD3 epsilon chain.

[0181] CD28 is a protein expressed on T cells that provides a costimulatory signal required for T cell activation and survival. T cell stimulation via CD28 in addition to the T cell receptor (TCR) can provide a powerful signal for the production of various interleukins (especially IL-6).

[0182] CD134, also known as OX40, is a member of the TNFR superfamily of receptors that, unlike CD28, is not constitutively expressed on resting naive T cells. OX40 is a secondary costimulatory molecule expressed 24–72 hours after activation; its ligand, OX40L, is also not expressed on resting antigen-presenting cells but is expressed after antigen-presenting cell activation. OX40 expression is dependent on full T cell activation; in the absence of CD28, OX40 expression is delayed and reduced to 4-fold lower levels.

[0183] CD137, also known as 4-1BB, is a member of the tumor necrosis factor (TNF) receptor family. CD137 can be expressed by activated T cells, but it is not expressed by CD8 On T cells, CD137 expression is seen to a greater extent than on CD4 T cells. In addition, CD137 expression is found on dendritic cells, follicular dendritic cells, natural killer cells, granulocytes, and cells of the vascular wall at sites of inflammation. The best-characterized activity of CD137 is its costimulatory activity for activated T cells. Crosslinking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity.

[0184] The mitogenic domain can comprise all or part of an antibody or other molecule that specifically binds to a T cell surface antigen. The antibody can activate TCR or CD28. The antibody can bind to TCR, CD3, or CD28. Examples of such antibodies include OKT3, 15E8, and TGN1412. Other suitable antibodies include: Anti-CD28:CD28.2, 10F3 Anti-CD3 / TCR: UCHT1, YTH12.5, TR66 Includes:

[0185] The mitogenic domain can include a binding domain from OKT3, 15E8, TGN1412, CD28.2, 10F3, UCHT1, YTH12.5, or TR66.

[0186] The mitogenic domain can comprise all or part of a costimulatory molecule, such as OX40L and 41BBL. For example, the mitogenic domain can comprise a binding domain from OX40L or 41BBL.

[0187] In some embodiments, the vector comprises an anti-CD3ε antibody or an antigen-binding fragment thereof linked to a transmembrane domain. An exemplary anti-CD3ε antibody is OKT3. OKT3, also known as muromonab-CD3, is a monoclonal antibody targeted to the CD3ε chain. It is clinically used to reduce acute rejection in patients with organ transplants. It was the first monoclonal antibody approved for clinical use in humans. The CDRs of OKT3 are as follows: CDRH1: GYTFTRY (SEQ ID NO: 1) CDRH2:NPSRGY (SEQ ID NO: 2) CDRH3: YYDDHYCLDY (SEQ ID NO: 3) CDRL1: SASSSVSYMN (SEQ ID NO: 4) CDRL2: DTSKLAS (SEQ ID NO: 5) CDRL3: QQWSSNPFT (SEQ ID NO: 6)

[0188] 15E8 is a mouse monoclonal antibody against human CD28. Its CDRs are as follows: CDRH1:GFSLTSY (SEQ ID NO: 7) CDRH2:WAGGS (SEQ ID NO:8) CDRH3: DKRAPGKLYYGYPDY (SEQ ID NO: 9) CDRL1: RASESVEYYVTSLMQ (SEQ ID NO: 10) CDRL2: AASNVES (SEQ ID NO: 11) CDRL3: QQTRKVPST (SEQ ID NO: 12)

[0189] In some embodiments, the vector comprises an anti-CD28 antibody or antigen-binding fragment thereof linked to a transmembrane domain. TGN1412 (also known as CD28-SuperMAB) is a humanized monoclonal antibody that not only binds to the CD28 receptor but is also a strong agonist of it. Its CDRs are as follows: CDRH1: GYTFSY (SEQ ID NO: 13) CDRH2: YPGNVN (SEQ ID NO: 14) CDRH3: SHYGLDWNFDV (SEQ ID NO: 15) CDRL1: HASQNIYVLN (SEQ ID NO: 16) CDRL2: KASNLHT (SEQ ID NO: 17) CDRL3: QQGQTYPYT (SEQ ID NO: 18)

[0190] In some embodiments, the vector comprises a CD134 ligand or a functional fragment thereof linked to the transmembrane domain. OX40L is a native ligand of CD134, which is expressed on cells such as DC2 (a subtype of dendritic cells) and enables the amplification of Th2 cell differentiation. OX40L is also named CD252 (cluster of differentiation 252).

[0191] The sequence of OX40L is: MERVQPLEENVGNAARPRFERNKLLLVASVIQGLGLLLCFTYICLHFSALQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL (SEQ ID NO: 19)

[0192] In some embodiments, the vector comprises a ligand of 4-1BB or a functional fragment thereof linked to its native or heterologous transmembrane domain. 4-1BBL is a cytokine belonging to the tumor necrosis factor (TNF) ligand family. This transmembrane cytokine is a bidirectional signal transducer that acts as a ligand for 4-1BB, a costimulatory receptor molecule in T lymphocytes. 4-1BBL has been shown to reactivate anergic T lymphocytes in addition to promoting T lymphocyte proliferation.

[0193] The sequence of 41BBL is: MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE (SEQ ID NO: 20)

[0194] Transduction enhancer spacer domain The mitogenic transduction enhancer and / or cytokine-based transduction enhancer can include a "spacer sequence" to connect the antigen-binding domain with the transmembrane domain. The flexible spacer allows the antigen-binding domains to orient in different directions, facilitating binding. As used herein, the term "linked to" refers to chemical linkage, direct C- to N-terminal fusion of two proteins; chemical linkage to a non-peptide spacer; chemical linkage to a polypeptide spacer; and C- to N-terminal fusion of two proteins to a polypeptide spacer, e.g., a spacer sequence, via a peptide bond.

[0195] The spacer sequence can comprise, for example, an lgG1 Fc region, an lgG1 hinge, or a human CD8 stalk or a mouse CD8 stalk. The human IgG1 spacer may comprise an alternative linker sequence with similar length and / or domain spacing characteristics to the IgG1 Fc region, IgG1 hinge, or CD8 stalk. The human IgG1 spacer may be modified to remove the Fc binding motif. In some embodiments, the spacer sequence may be derived from a human protein.

[0196] Examples of amino acid sequences for these spacers are shown below. Human IgG1 hinge-CH2CH3: AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD(array Number 21) Human CD8 stalk: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI (SEQ ID NO: 22) Human lgG1 hinge: AEPKSPDKTHTCPPCPKDPK (SEQ ID NO: 23) CD2 ectodomain: KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD (SEQ ID NO: 24) CD34 ectodomain: SLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKT (SEQ ID NO: 25)

[0197] The transmembrane domain is a membrane-spanning sequence of a mitogenic transduction enhancer and / or a cytokine-based transduction enhancer. The transmembrane domain can comprise a hydrophobic alpha helix. The transmembrane domain can be derived from CD28. In some embodiments, the transmembrane domain is derived from a human protein.

[0198] An alternative to the transmembrane domain is a membrane targeting domain, such as a GPI anchor. GPI anchoring is a post-translational modification that occurs in the endoplasmic reticulum. Pre-assembled GPI anchor precursors are transferred to proteins with C-terminal GPI signal sequences. During processing, the GPI anchor replaces the GPI signal sequence and is linked to the target protein by an amide bond. The GPI anchor targets the mature protein to the membrane. In some embodiments, the tagged protein comprises a GPI signal sequence.

[0199] The viral vector of the present disclosure can include a cytokine-based transduction enhancer in the viral envelope. In some embodiments, the cytokine-based transduction enhancer is derived from the host cell during viral vector production. In some embodiments, the cytokine-based transduction enhancer is produced by the host cell and expressed on the cell surface. When the nascent viral vector buds from the host cell membrane, the cytokine-based transduction enhancer can be incorporated into the viral envelope as part of the packaging cell-derived lipid bilayer.

[0200] A cytokine-based transduction enhancer can include a cytokine domain and a transmembrane domain. It can have the structure CS-TM, where C is the cytokine domain, S is an optional spacer domain (e.g., a spacer sequence), and TM is the transmembrane domain. The spacer domain and transmembrane domain are as defined above.

[0201] The cytokine domain can include a T cell activating cytokine, such as those derived from IL2, IL7, and IL15, or functional fragments thereof. As used herein, a "functional fragment" of a cytokine is a fragment of a polypeptide that retains the ability to bind to its particular receptor and activate T cells.

[0202] IL2 is a factor secreted by T cells to regulate the growth and differentiation of T cells and certain B cells. IL2 is a lymphokine that induces proliferation of responding T cells. It is secreted as a single glycosylated polypeptide, and cleavage of a signal sequence is required for its activity. Solution NMR suggests that the structure of IL2 comprises a bundle of four helices (designated A-D) flanked by two shorter helices and several poorly defined loops. Residues in helix A and in the loop region between helices A and B are important for receptor binding. The sequence of IL2 is: MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(Sequence Number 26)

[0203] IL7 is a cytokine that functions as a growth factor for early lymphoid cells of both the B and T cell lineages. The sequence of IL7 is: MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO: 27)

[0204] IL-15 is a cytokine with structural similarity to IL-2. Like IL-2, IL-15 binds to and signals through a complex composed of the IL-2 / IL-15 receptor beta chain and common gamma chain. IL-15 is secreted by mononuclear phagocytes and some other cells after infection with a virus(ies). This cytokine induces cell proliferation of natural killer cells; cells of the innate immune system whose primary role is to kill virus-infected cells. The sequence of IL-15 is: MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 28)

[0205] The cytokine-based transduction enhancer can comprise one of the following sequences, or a functional fragment or variant thereof: Membrane-IL7: MAHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPT KSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGGGSPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV (SEQ ID NO: 29) Membrane-IL15: MGLVRRGARAGPRMPRGWTALCLLSLLPSGFMAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV (SEQ ID NO: 30)

[0206] The cytokine-based transduction enhancer may comprise a variant of the sequence set forth as SEQ ID NO: 29 or 30 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence is a cytokine-based transduction enhancer that has the required property, i.e., the ability to activate T cells when present in the envelope protein of a retroviral or lentiviral vector.

[0207] The present disclosure further provides various retroviral vectors, including, but not limited to, gamma-retroviral vectors, alpha-retroviral vectors, and lentiviral vectors.

[0208] AAV In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. AAV is a 4.7 kb single-stranded DNA virus. Because wild-type AAV is non-pathogenic and has no etiological association with any known disease, recombinant vectors based on AAV are associated with excellent clinical safety. In addition, AAV offers the ability for highly efficient gene delivery and sustained transgene expression in multiple tissues. "AAV vector" refers to a vector derived from an adeno-associated virus serotype, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAVrh.74, etc. An AAV vector can be deleted in whole or in part of one or more of the AAV wild-type genes, for example, the rep and / or cap genes, but retain the sequence of functional flanking inverted terminal repeats (ITRs). Functional ITR sequences are necessary for the rescue, replication, and packaging of AAV virions. Thus, an AAV vector is defined herein as comprising at least a sequence (e.g., a functional ITR) required in cis for viral replication and packaging. The ITRs do not need to be wild-type nucleotide sequences and may be altered, for example, by nucleotide insertion, deletion, or substitution, as long as the sequence provides functional rescue, replication, and packaging. AAV vectors may also include other modifications, including, but not limited to, one or more modified capsid proteins (e.g., VP1, VP2, and / or VP3). For example, capsid proteins may be modified to alter tropism and / or reduce immunogenicity.

[0209] Because wild-type AAV is non-pathogenic and has no etiological association with any known disease, recombinant vectors based on AAV are associated with excellent clinical safety. In addition, AAV provides the ability for highly efficient gene delivery and sustained transgene expression in multiple tissues. Various serotypes of AAV are known, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAVrh.74, etc. AAV vectors can be completely or partially deleted from one or more of the AAV wild-type genes, such as the rep and / or cap genes, but retain the sequence of functional adjacent inverted terminal repeats (ITRs). The serotype of a recombinant AAV vector is determined by its capsid. International Patent Publication No. WO2003042397A2 discloses various capsid sequences, including those of AAV1, AAV2, AAV3, AAV8, AAV9, and rh10. International Patent Publication No. WO2013078316A1 discloses the polypeptide sequence of VP1 from AAVrh74. Many different naturally occurring or genetically modified AAV capsid sequences are known in the art.

[0210] AAV vectors useful in practicing the present disclosure can be packaged into AAV virions (virus particles) using a variety of systems, including adenovirus-based systems and helper-free systems. Standard methods in AAV biology are described in Kwon and Schaffer. Pharm Res. (2008) 25(3):489-99; Wu et al. Mol. Ther. (2006) 14(3):316-27., Burger et al. Mol. Ther. (2004) 10(2):302-17; Grimmet al. Curr Gene Ther. (2003) 3(4):281-304; Deyle DR, Russell DW. Curr Opin Mol Ther. (2009) 11(4):442-447; McCarty et al. Gene Ther. (2001) 8(16):1248-54; and Duanet et al. Mol Ther. (2001) 4(4):383-91. Helper-free systems included those described in US 6,004,797; US 7,588,772; and US 7,094,604.

[0211] Gene delivery viral vectors useful in the practice of the present disclosure can be constructed using methodologies known in the art of molecular biology.Typically, viral vectors carrying transgenes are assembled from polynucleotides encoding transgenes, suitable regulatory elements, and elements necessary for the production of viral proteins that mediate cell transduction.Such recombinant viruses can be produced by techniques known in the art, for example, by transfecting packaging cells or by transient transfection with helper plasmids or viruses.Examples of viral packaging cells include, but are not limited to, HeLa cells, SF9 cells (optionally with baculovirus helper vectors), 293 cells, etc.As described in US20170218395A1, AAV vectors can be produced using a system based on herpesvirus. Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in WO95 / 14785, WO96 / 22378, U.S. Pat. No. 5,882,877, U.S. Pat. No. 6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056 and WO94 / 19478, the entire contents of each of which are incorporated herein by reference.

[0212] Illustrative examples of viral vectors that can be used in the compositions and methods of the present disclosure are disclosed in WO2016 / 139463; WO2017 / 165245; WO2018111834, each of which is incorporated herein in its entirety.

[0213] Non-viral vectors In some embodiments, the compositions and methods of the present disclosure can be used with non-viral vectors. Exemplary non-viral vectors are provided, for example, in Smith et al. Nat Nanotechnol. 12 (8): 813-820 (2017). In some embodiments, the non-viral vector is a type of nanoparticle. In some embodiments, the nanoparticle is polymer-based. In some embodiments, the non-viral vector is liposome-based. In some embodiments, the nanoparticle is provided with an immune cell targeting molecule. In some embodiments, the nanoparticle is loaded with a polynucleotide molecule encoding one or more expression cassettes.

[0214] Chimeric Antigen Receptor In some embodiments, the vectors described herein are used to transduce cells (e.g., T lymphocytes) with nucleic acid sequences (polynucleotides) encoding one or more chimeric antigen receptors (CARs). In some embodiments, transduction of the vector results in expression of one or more CARs in the transduced cells.

[0215] CAR is an artificial membrane-bound protein that directs T lymphocytes to antigens and stimulates them to kill cells displaying the antigen. See, for example, Eshhar, U.S. Patent No. 7,741,465. Generally, CAR is a genetically engineered receptor that includes an extracellular domain that binds to an antigen, for example, an antigen on a cell, an optional linker, a transmembrane domain, and an intracellular (cytoplasmic) domain that includes a costimulatory domain and / or a signal transduction domain that transmits activation signals to immune cells. Using CAR, a single receptor can be programmed to both recognize specific antigens and, when bound to the antigen, activate immune cells to attack and destroy cells bearing the antigen. When these antigens are present on tumor cells, immune cells expressing CAR can target and kill tumor cells. When a CAR is expressed, for example, on the surface of a T lymphocyte, and the extracellular domain of the CAR binds to an antigen, all other conditions are met, and the intracellular signaling domain transmits a signal to the T lymphocyte to activate and / or proliferate the T lymphocyte, and, if the antigen is present on the cell surface, to kill the cell expressing the antigen. Because T lymphocytes require two signals, a primary activation signal and a costimulatory signal, for maximum activation, the CAR may contain a stimulatory domain and a costimulatory domain, and thus, when an antigen binds to the extracellular domain, transmission of both the primary activation signal and the costimulatory signal occurs.

[0216] CAR intracellular domain In some embodiments, the intracellular domain of a CAR is or includes an intracellular domain or motif of a protein that is expressed on the surface of T lymphocytes and induces activation and / or proliferation of the T lymphocytes. Such a domain or motif can transmit the primary antigen binding signal required for T lymphocyte activation in response to antigen binding to the extracellular portion of the CAR. Typically, this domain or motif includes or is an ITAM (immunoreceptor tyrosine-based activation motif). ITAM-containing polypeptides suitable for CARs include, for example, the zeta CD3 chain (CD3ζ) or an ITAM-containing portion thereof. In some embodiments, the intracellular domain is a CD3ζ intracellular signaling domain. In some embodiments, the intracellular domain is derived from a lymphocyte receptor chain, a TCR / CD3 complex protein, an Fc receptor subunit, or an IL-2 receptor subunit. In some embodiments, the intracellular signaling domain of a CAR can be derived from, for example, 003ζ, CD3ε, CD22, CD79a, CD66d, or CD39 signaling domain. An "intracellular signaling domain" refers to the portion of a CAR polypeptide that is involved in conveying the message of effective CAR binding to a target antigen inside an immune effector cell to elicit effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including release of cytotoxic factors against a target cell to which the CAR is bound, or other cellular responses elicited after binding of an antigen to the extracellular CAR domain.

[0217] In some embodiments, the CAR further comprises one or more costimulatory domains or motifs, for example, as part of the intracellular domain of the polypeptide. Costimulatory molecules are well-known cell surface molecules that provide a second signal, other than antigen receptors or Fc receptors, necessary for the efficient activation and function of T lymphocytes upon antigen binding. The one or more costimulatory domains or motifs can be or include, for example, one or more of the following: a costimulatory CD27 polypeptide sequence, a costimulatory CD28 polypeptide sequence, a costimulatory OX40 (CD134) polypeptide sequence, a costimulatory 4-1BB (CD137) polypeptide sequence, or a costimulatory-inducible T cell costimulatory (ICOS) polypeptide sequence, or other costimulatory domains or motifs, or any combination thereof. In some embodiments, the one or more costimulatory domains are selected from the group consisting of the intracellular domains of 4-1BB, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70.

[0218] In some embodiments, the intracellular domain can be further modified to encode a detectable protein, for example, a fluorescent protein (e.g., green fluorescent protein) or any known variant thereof.

[0219] CAR transmembrane region The transmembrane region can be any transmembrane region that can be incorporated into a functional CAR, for example, a transmembrane region from a CD4 or CD8 molecule.

[0220] In some embodiments, the transmembrane domain of the CAR is selected from the group consisting of CD8, the transmembrane domain of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1 BB (CD137), 4-1 BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITG AX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT It can be derived from AM, 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 / or NKG2C.

[0221] CAR linker region The optional linker of a CAR placed between the extracellular domain and the transmembrane domain can be a polypeptide of approximately 2 to 100 amino acids in length. The linker can contain or consist of flexible residues such as glycine and serine, thus allowing adjacent protein domains to move freely relative to each other. For example, if it is desirable to ensure that two adjacent domains do not sterically interfere with each other, a longer linker can be used. The linker can be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (e.g., T2A), 2A-like linkers, or functional equivalents and combinations thereof. In some embodiments, the linker is derived from the hinge region or a portion of the hinge region of any immunoglobulin.

[0222] CAR extracellular domain In some embodiments, the nucleic acid transduced into a cell using the methods described herein comprises a sequence encoding a polypeptide, wherein the extracellular domain of the polypeptide binds to an antigen of interest. In some embodiments, the extracellular domain comprises a receptor or portion of a receptor that binds to the antigen. In some embodiments, the extracellular domain comprises or is an antibody or antigen-binding portion thereof. In some embodiments, the extracellular domain comprises or is a single-chain Fv domain. The single-chain Fv domain can, for example, comprise a VL and a VH linked by a flexible linker, wherein the VL and VH are derived from an antibody that binds to the antigen.

[0223] In some embodiments, the extracellular domain of the CAR can contain any polypeptide that binds to a desired antigen (e.g., prostate neoantigen). The extracellular domain can comprise an scFv, a portion of an antibody, or an alternative scaffold. CARs can also be engineered to bind to two or more desired antigens, which can be arranged in tandem and separated by a linker sequence. For example, one or more domain antibodies, scFvs, llama VHH antibodies, or other VH-only antibody fragments can be assembled in tandem by a linker to provide bispecific or multispecificity to the CAR.

[0224] The antigen to which the extracellular domain of the polypeptide binds can be any antigen of interest, for example, an antigen on a tumor cell. The tumor cell can be, for example, a cell in a solid tumor or a cell of a blood cancer. The antigen can be any antigen expressed on cells of any tumor or cancer type, such as lymphoma, lung cancer, breast cancer, prostate cancer, adrenocortical carcinoma, thyroid cancer, nasopharyngeal carcinoma, melanoma (e.g., malignant melanoma), skin cancer, colorectal cancer, desmoid tumor, desmoplastic small round cell tumor, endocrine tumor, Ewing's sarcoma, peripheral primitive neuroectodermal tumor, solid germ cell tumor, hepatoblastoma, neuroblastoma, non-rhabdomyosarcoma soft tissue sarcoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, Wilms' tumor, glioblastoma, myxoma, fibroma, lipoma, etc. In some embodiments, the lymphoma is selected from the group consisting of chronic lymphocytic leukemia (small lymphocytic lymphoma), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenstrom's macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma, In some embodiments, the cancer may be chronic lymphocytic leukemia (CLL), T-lymphocytic prolymphocytic leukemia, T-lymphocytic large granular lymphocytic leukemia, aggressive NK-cell leukemia, adult T-lymphocytic leukemia / lymphoma, extranodal NK / T-lymphocytic lymphoma, nasal type, enteropathy-type T-lymphocytic lymphoma, hepatosplenic T-lymphocytic lymphoma, blastic NK-cell lymphoma, mycosis fungoides, Sézary syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T-lymphocytic lymphoma, peripheral T-lymphocytic lymphoma (unspecified), anaplastic large cell lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma. In some embodiments, the cancer is chronic lymphocytic leukemia (CLL), the B cells of the CLL have a normal karyotype. In some embodiments where the cancer is chronic lymphocytic leukemia (CLL), the B cells of the CLL have a 17p deletion, an 11q deletion, a 12q trisomy, a 13q deletion, or a p53 deletion.

[0225] In some embodiments, the antigen is a tumor-associated antigen (TAA) or tumor-specific antigen (TSA). In some embodiments, the tumor-associated antigen or tumor-specific antigen includes, but is not limited to, B-cell maturation antigen (BCMA), B-cell activating factor (BAFF), Her2, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), EGFRvIII, cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD19, CD20, CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GRF), and the like. protein) (GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin , thyroglobulin, thyroid transcription factor-1, vascular endothelial growth factor receptor (VEGFR), dimeric form of pyruvate kinase isoenzyme type M2 (tumor M2-PK), abnormal ras protein, or abnormal p53 protein.

[0226] In some embodiments, the TAA or TSA is a cancer / testis (CT) antigen, such as BAGE, CAGE, CTAGE, FATE, GAGE, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-ESO-1, NY-SAR-35, OY-TES-1, SPANXB1, SPA17, SSX, SYCP1, or TPTE.

[0227] In some embodiments, the TAA or TSA is a carbohydrate or ganglioside, such as fuc-GM1, GM2 (carcinoembryonic antigen-immunogenic-1; OFA-I-1); GD2 (OFA-I-2), GM3, GD3, etc.

[0228] In some embodiments, the TAA or TSA is selected from the group consisting of alpha-actinin-4, Bage-1, BCR-ABL, Bcr-Abl fusion protein, beta-catenin, CA125, CA15-3 (CA27.29\BCAA), CA195, CA242, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, CEA, coa-1, dek-can fusion protein, EBNA, EF2, Epstein-Barr virus antigen, ETV6-AML1 fusion protein, and the like. quality, HLA-A2, HLA-All, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Gage3, 4, 5, 6, and 7, GnTV, Herv-K-mel, Lage-1, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, TRP2-Int2, gp100(Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, RAGE, GAGE-1, GAGE-2, p15(58), RAGE, SCP-1, Hom / Mel-40, PRAME, p53, H-Ras, HER-2 / neu, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA19-9, CA72-4, and CAM 17.1, NuMa, K-ras, 13-catenin, Mum-1, p16, TAGE, PSMA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, CD68\KP1, CO-029 , FGF-5, G250, Ga733(EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG7 2, TLP, TPS, CD19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Sp17), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T-cell receptor gamma alternative reading frame protein), Trp-p8, STEAP1 (prostatic six-transmembrane epithelial antigen 1), aberrant Ras protein, or aberrant p53 protein. In some embodiments, the tumor-associated or tumor-specific antigen is integrin αvβ3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), or Ral-B. Other tumor-associated and tumor-specific antigens are known to those skilled in the art.

[0229] Antibodies and scFvs that bind to TSAs and TAAs include antibodies and scFVs known in the art, as well as the nucleotide sequences encoding them.

[0230] In some embodiments, the antigen is not considered to be a TSA or TAA, but is nevertheless an antigen associated with tumor cells or tumor-induced damage.In some embodiments, for example, the antigen is, for example, a growth factor, cytokine, or interleukin, such as an angiogenesis- or vasculogenesis-related growth factor, cytokine, or interleukin.Such growth factors, cytokines, or interleukins can include, for example, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), or interleukin-8 (IL-8).Tumors can also create a local hypoxic environment in the tumor.Therefore, in some embodiments, the antigen is a hypoxia-related factor, for example, HIF-1α, HIF-1β, HIF-2a, HIF-2β, HIF-3α, or HIF-3β. Tumors can also cause localized damage to normal tissues, resulting in the release of molecules known as damage-associated molecular pattern molecules (DAMPs; also known as alarmins). Thus, in some embodiments, the antigen is a DAMP, such as heat shock protein, chromatin-associated protein high mobility group box 1 (HMGB1), S100A8 (MRP8, calgranulin A), S100A9 (MRP14, calgranulin B), serum amyloid A (SAA), or can be deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate.

[0231] In some embodiments of the polypeptides described herein, the extracellular domain is joined to the transmembrane domain directly or by a linker, spacer, or hinge polypeptide sequence, eg, a sequence derived from CD28 or a sequence derived from CTLA4.

[0232] In some embodiments, the extracellular domain that binds to a desired antigen may be derived from an antibody or antigen-binding fragment thereof generated using the techniques described herein.

[0233] CAR example Non-limiting examples of chimeric antigen receptors that can be used in conjunction with the compositions and methods of the present disclosure are disclosed in WO2019 / 200056; PCT / US2019 / 062675; US62 / 916,110; WO2015 / 017214; WO / 2018 / 148224; WO2019156795, each of which is incorporated herein in its entirety.

[0234] Gene editing Numerous gene editing methods are known in the art, and additional methods are constantly being developed. The methods and compositions of the present disclosure can deliver various genetic payloads, including polynucleotides intended for insertion into the genome of target cells and / or gene editing systems (CRISPR-Cas, meganucleases, homing endonucleases, zinc finger enzymes, etc.). In several embodiments, polynucleotides (e.g., transgenes), enzymes, and / or guide RNAs are delivered by one, two, three, or more vectors of the same type (e.g., lentivirus, AAV, etc.) or different types (e.g., including a combination of a non-viral vector and a viral vector or a combination of different types of viral vectors). The methods and systems of the present disclosure can be used to generate point mutation(s), insertions, deletions, etc. Random mutagenesis and multilocus gene editing are also within the scope of the present disclosure.

[0235] target immune cells Non-limiting examples of cells that can be targeted by the vectors described herein include T lymphocytes, dendritic cells (DCs), Treg cells, B cells, natural killer cells, and macrophages.

[0236] T cells T cells ("T lymphocytes") are a type of lymphocyte (itself a type of white blood cell) that plays a central role in cell-mediated immunity. Several T cell subsets exist, each with distinct functions. T cells can be distinguished from other lymphocytes, such as B cells and NK cells, by the presence of T cell receptors (TCRs) on their cell surface. The TCR is responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules and is composed of two distinct protein chains. In 95% of T cells, the TCR consists of an alpha (α) chain and a beta (β) chain. When the TCR engages an antigen peptide and MHC (peptide / MHC complex), a series of biochemical events mediated by associated enzymes, coreceptors, specialized adapter molecules, and activated or released transcription factors activates the T lymphocyte.

[0237] In some embodiments, the cells used in the methods provided herein are primary T lymphocytes (e.g., primary human T lymphocytes). The primary T lymphocytes used in the methods provided herein can be naive T lymphocytes or MHC-restricted T lymphocytes. In some embodiments, the T lymphocytes are CD4 + In another embodiment, the T lymphocytes are CD8 + In some embodiments, the primary T lymphocytes are tumor-infiltrating lymphocytes (TILs). In some embodiments, the primary T lymphocytes are isolated from a tumor biopsy or expanded from T lymphocytes isolated from a tumor biopsy. In some embodiments, the primary T lymphocytes are isolated from peripheral blood, umbilical cord blood, or lymph, or expanded from T lymphocytes isolated from peripheral blood, umbilical cord blood, or lymph. In some embodiments, the T lymphocytes are allogeneic to a particular individual, e.g., the recipient of the T lymphocytes. In certain other embodiments, the T lymphocytes are not allogeneic to a particular individual, e.g., the recipient of the T lymphocytes. In some embodiments, the T lymphocytes are autologous to a particular individual, e.g., the recipient of the T lymphocytes.

[0238] In some embodiments, the primary T lymphocytes used in the methods described herein are isolated from tumors, for example, tumor-infiltrating lymphocytes.In some embodiments, these T lymphocytes are specific for tumor-specific antigens (TSA) or tumor-associated antigens (TAA).In some embodiments, primary T lymphocytes are obtained from individuals, expanded as needed, and then transduced with nucleic acids encoding one or more chimeric antigen receptors (CARs) using the methods described herein, and then expanded as needed.T lymphocytes can be expanded, for example, by contacting T lymphocytes in culture with antibodies against CD3 and / or CD28, for example, antibodies attached to the surface of beads or cell culture plates; see, for example, U.S. Patent Nos. 5,948,893; 6,534,055; 6,352,694; 6,692,964; 6,887,466; and 6,905,681. In some embodiments, the antibodies are anti-CD3 and / or anti-CD28, and the antibodies are not bound to a solid surface (e.g., the antibody and T lymphocytes are contacted in solution). In some embodiments, either the anti-CD3 antibody or the anti-CD28 antibody is bound to a solid surface (e.g., beads, tissue culture dish plastic), and the other antibody is not bound to a solid surface (e.g., in solution).

[0239] NK cells Natural killer (NK) cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. NK cells typically comprise approximately 10–15% of the mononuclear cell fraction in normal peripheral blood. In humans, NK cells do not express the T cell antigen receptor (TCR), CD3, or surface immunoglobulin (Ig) B cell receptor, but typically express the surface markers CD16 (FcγRIII) and CD56. NK cells are cytotoxic; small granules in their cytoplasm contain specialized proteins, such as perforin and proteases known as granzymes. When perforin is released in close proximity to a cell destined for death, it forms pores in the target cell's plasma membrane, through which granzymes and related molecules can enter and induce apoptosis. One granzyme, granzyme B (also known as granzyme 2 and cytotoxic T lymphocyte-associated serine esterase 1), is a serine protease crucial for the rapid induction of target cell apoptosis in cell-mediated immune responses.

[0240] NK cells are activated in response to interferon or macrophage-derived cytokines. Activated NK cells are called lymphokine-activated killer (LAK) cells. NK cells have two types of surface receptors, called "activating receptors" and "inhibitory receptors," that control the cytotoxic activity of the cells.

[0241] Among other activities, NK cells play a role in host rejection of tumors. Many cancer cells have reduced or no class I MHC expression and can be targeted by NK cells. Natural killer cells can be activated by cells that lack or display reduced levels of major histocompatibility complex (MHC) proteins. In addition to participating in direct cytotoxic killing, NK cells also play a role in cytokine production, which may be important for controlling cancer and infectious diseases. Activated and expanded NK and LAK cells have been used for both ex vivo and in vivo treatment of patients with advanced cancers, with some success against bone marrow-related diseases such as leukemia, breast cancer, and certain types of lymphoma.

[0242] Pharmaceutical Compositions and Formulations The formulations and compositions of the disclosure may include any number of multispecific antibody and / or vector combinations, and optionally one or more additional pharmaceutical agents (polypeptides, polynucleotides, compounds, etc.) formulated as pharmaceutically or physiologically acceptable compositions for administration to cells, tissues, organs, or animals, alone or in combination with one or more other therapeutic modalities. In some embodiments, the one or more additional pharmaceutical agents further increase the transduction efficiency of the vector.

[0243] In some embodiments, the present disclosure provides compositions comprising a therapeutically effective amount of a multispecific antibody (e.g., a bispecific antibody) described herein formulated together with one or more pharmaceutically acceptable carriers (excipients) and / or diluents. In some embodiments, the composition further comprises other agents, such as cytokines, growth factors, hormones, small molecules, or various pharmaceutically active agents.

[0244] In some embodiments, antibody compositions and formulations used in accordance with the present disclosure can be prepared by mixing antibodies having the desired degree of purity with pharmaceutically acceptable carriers, excipients, or stabilizers as needed for storage in the form of lyophilized formulations or aqueous solutions (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used. In some embodiments, one or more pharmaceutically acceptable surfactants (surfactants), buffers, isotonicity agents, salts, amino acids, sugars, stabilizers, and / or antioxidants are used in the formulations.

[0245] Suitable pharmaceutically acceptable surfactants include, but are not limited to, polyethylene-sorbitan-fatty acid esters, polyethylene-polypropylene glycol, polyoxyethylene-stearate, and sodium dodecyl sulfate. Suitable buffers include, but are not limited to, histidine buffers, citrate buffers, succinate buffers, acetate buffers, and phosphate buffers.

[0246] An isotonic agent is used to produce an isotonic formulation. An isotonic formulation is a liquid or a liquid reconstituted from a solid form, such as a lyophilized form, that has the same tonicity as some other solution to which it is compared, such as physiological saline solution or serum. Suitable isotonic agents include, but are not limited to, salts, including but not limited to sodium chloride (NaCl) or potassium chloride, sugars, including but not limited to glucose, sucrose, trehalose, or any component from the group of amino acids, sugars, salts, and combinations thereof. In some embodiments, the isotonic agent is generally used in a total amount of about 5 mM to about 350 mM.

[0247] Non-limiting examples of salts include salts of any combination of cations sodium, potassium, calcium, or magnesium with anions chloride, phosphate, citrate, succinate, sulfate, or mixtures thereof. Non-limiting examples of amino acids include arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, and proline. Non-limiting examples of sugars according to the present disclosure include trehalose, sucrose, mannitol, sorbitol, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine (also referred to as "meglumine"), galactosamine, and neuraminic acid, and combinations thereof. Non-limiting examples of stabilizers include the amino acids and sugars described above, as well as commercially available cyclodextrins and dextrans of any type and molecular weight known in the art. Non-limiting examples of antioxidants include excipients such as methionine, benzyl alcohol, or any other excipient used to minimize oxidation.

[0248] In some embodiments, the present disclosure provides compositions comprising a therapeutically effective amount of a vector described herein formulated together with one or more pharmaceutically acceptable carriers (excipients) and / or diluents (e.g., pharmaceutically acceptable cell culture media). In some embodiments, the compositions further comprise other agents, such as cytokines, growth factors, hormones, small molecules, or various pharmaceutically active agents.

[0249] The phrase " pharmaceutically acceptable " refers to molecular entities and compositions that do not cause allergic reactions or similar adverse reactions when administered to humans.The preparation of aqueous compositions containing proteins as active ingredients is well understood in the art.Typically, such compositions are prepared as injections, either as liquid solutions or suspensions; solid forms suitable for dissolving or suspending in liquid before injection can also be prepared.Preparations can also be emulsified.

[0250] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutical active substances is well known in the art. Except as a conventional media or agent is incompatible with the active ingredient, the use of any conventional media or agent in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0251] As used herein, "pharmaceutically acceptable carrier" encompasses any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, including pharmaceutically acceptable cell culture media. In some embodiments, compositions containing a carrier are suitable for parenteral administration, e.g., intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except where a conventional media or agent is incompatible with the transduced cells, use of any conventional media or agent in the pharmaceutical compositions of the present disclosure is contemplated.

[0252] The composition may further comprise one or more polypeptides, polynucleotides, vectors containing the same, or compounds that increase the transduction efficiency of the vector, formulated in a pharmaceutically or physiologically acceptable solution for administration to cells or animals alone or in combination with one or more other therapeutic modalities. It will also be understood that, if desired, the compositions of the present disclosure can be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, or various pharmaceutically active agents. There is virtually no limit to other components that can also be included in the composition, provided that the additional agents do not adversely affect the ability of the composition to deliver its intended therapy.

[0253] The present disclosure also provides pharmaceutical compositions comprising an expression cassette or vector (e.g., a therapeutic vector) disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical composition comprises a lentiviral vector comprising an expression cassette disclosed herein, where, for example, the expression cassette comprises one or more polynucleotide sequences encoding one or more chimeric antigen receptors (CARs) and variants thereof.

[0254] Pharmaceutical compositions containing the expression cassette or vector may be in any form suitable for the selected mode of administration, e.g., intraventricular, intramyocardial, intracoronary, intravenous, intraarterial, intrarenal, intraurethral, ​​epidural, intrathecal, or intramuscular administration. Vectors can be administered to animals and humans as the sole active agent or in combination with other active agents, in unit dosage forms, or in admixture with conventional pharmaceutical supports. In some embodiments, the pharmaceutical composition comprises cells transduced ex vivo with any of the vectors according to the present disclosure.

[0255] In some embodiments, the vector (e.g., lentiviral vector) or pharmaceutical composition comprising the vector is effective when administered systemically. For example, the viral vector of the present disclosure is effective in some cases when administered intravenously to a subject (e.g., a primate, such as a non-human primate or a human). In some embodiments, the viral vector of the present disclosure, when administered systemically, can induce the expression of CAR in various immune cells (e.g., in T cells, dendritic cells, NK cells).

[0256] In various embodiments, the pharmaceutical composition contains pharmaceutically acceptable vehicles (e.g., carriers, diluents, and excipients) for injectable formulations. Exemplary excipients include poloxamers. Formulation buffers for viral vectors generally contain salts to prevent aggregation and other excipients (e.g., poloxamers) to reduce vector adhesion. These may be in isotonic sterile saline solution (such as monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or a mixture of such salts), or may be dried, particularly freeze-dried, compositions that can be optionally reconstituted into an injectable solution upon addition of sterile water or saline. In some embodiments, the formulation is stable for storage and use when frozen (e.g., below 0°C, about -60°C, or about -72°C).

[0257] The formulation of pharmaceutical compositions, pharmaceutically acceptable excipients and carrier solutions of the present disclosure will be well known to those of skill in the art, as will the development of appropriate administration and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including, for example, oral, parenteral, intravenous, intranasal, and intramuscular administration and formulations.

[0258] In certain circumstances, it is desirable to deliver the compositions disclosed herein parenterally, intravenously, intramuscularly, or intraperitoneally.For example, United States Patent No. 5,543,158; United States Patent No. 5,641,515 and United States Patent No. 5,399,363 (each of which is specifically incorporated herein in its entirety by reference).The solution of active compound as free base or pharmacologically acceptable salt can be prepared in water, suitably mixed with surfactant such as hydroxypropylcellulose.Dispersion can also be prepared in glycerol, liquid polyethylene glycol, and their mixture, and in oil.Under normal conditions of storage and use, these preparations contain preservatives to prevent microbial growth.

[0259] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases, the form should be sterile and fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In some embodiments, isotonic agents, for example, sugars or sodium chloride, are added. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0260] For parenteral administration as an aqueous solution, for example, the solution should be appropriately buffered if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous medium that can be used will be known to those skilled in the art in light of this disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid, or injected into the proposed infusion site (see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins, 2005). Depending on the condition of the subject to be treated, Some variation in dosage is inevitable. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as appropriate per FDA Office of Biologics standards.

[0261] In some embodiments, the present disclosure provides formulations or compositions suitable for viral vector-based delivery (i.e., viral-mediated transduction), including but not limited to retroviral (e.g., lentiviral) vectors.

[0262] disease The present disclosure also provides a method for enhancing the effectiveness of immunotherapy that can be used to treat a disease, disorder, or condition. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is a hematological malignancy or a solid tumor. In some embodiments, the subject is relapsed from treatment with a previous anti-cancer therapeutic agent or is refractory to treatment with a previous anti-cancer therapeutic agent.

[0263] Hematologic malignancies In some embodiments, the cancer is a hematological malignancy.

[0264] In some embodiments, the hematological malignancy is lymphoma, B-cell malignancy, Hodgkin's lymphoma, non-Hodgkin's lymphoma, DLBLC, FL, MCL, marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), CLL, ALL, AML, Waldenstrom's macroglobulinemia, or T-cell lymphoma.

[0265] In some embodiments, the solid tumor is lung cancer, liver cancer, cervical cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma, glioblastoma, prostate cancer, esophageal cancer, or gastric cancer. WO2019057124A1 discloses cancers suitable for treatment with T cell redirecting therapeutics that bind to CD19.

[0266] In some embodiments, the hematological malignancy is selected from the group consisting of multiple myeloma, smoldering multiple myeloma, monoclonal gammopathy of undetermined significance (MGUS), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, plasma cell leukemia, light chain amyloidosis (AL), precursor B-cell lymphoblastic leukemia, pre- B-cell lymphoblastic leukemia, acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic lymphocytic leukemia (CLL), B-cell malignancies, chronic myeloid leukemia (CML), hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm, Hodgkin's lymphoma, non-Hodgkin's lymphoma, marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), plasma cell leukemia, anaplastic large cell lymphoma (ALCL), leukemia or lymphoma.

[0267] In some embodiments, the hematological malignancy is multiple myeloma.

[0268] In some embodiments, the multiple myeloma is newly diagnosed multiple myeloma.

[0269] In some embodiments, the multiple myeloma is relapsed or refractory multiple myeloma.

[0270] In some embodiments, multiple myeloma is high-risk multiple myeloma.It is known that the subject with high-risk multiple myeloma relapses early and has poor prognosis and outcome.If subject has one or more of the following cytogenetic abnormalities, this subject can be classified as having high-risk multiple myeloma: t(4;14)(p16;q32), t(14;16)(q32;q23), del17p, 1qAmp, t(4;14)(p16;q32) and t(14;16)(q32;q23), t(4;14)(p16;q32) and del17p, t(14;16)(q32;q23) and del17p, or t(4;14)(p16;q32), t(14;16)(q32;q23) and del17p.

[0271] In some embodiments, the subject with high-risk multiple myeloma has one or more chromosomal abnormalities including t(4;14)(p16;q32), t(14;16)(q32;q23), del17p, 1qAmp, t(4;14)(p16;q32) and t(14;16)(q32;q23), t(4;14)(p16;q32) and del17p, t(14;16)(q32;q23) and del17p; or t(4;14)(p16;q32), t(14;16)(q32;q23) and del17p, or any combination thereof.

[0272] Various qualitative and / or quantitative methods can be used to determine disease recurrence or refractoryness. Possible associated symptoms include, for example, a decline or plateau in the patient's health or the re-establishment or worsening of various symptoms associated with solid tumors, and / or the spread of cancerous cells from one location in the body to other organs, tissues, or cells.

[0273] Cellular genetic abnormalities can be detected, for example, by fluorescence in situ hybridization (FISH). Regarding chromosomal translocation, oncogenes are translocated to the IgH region on chromosome 14q32, resulting in the dysregulation of these genes. t(4;14)(p16;q32) involves the translocation of fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET domain-containing protein (MMSET) (also known as WHSC1 / NSD2), and t(14;16)(q32;q23) involves the translocation of MAF transcription factor C-MAF. 17p deletion (del17p) involves the loss of the p53 locus.

[0274] In some embodiments, the multiple myeloma is relapsed or refractory to treatment with an anti-CD38 antibody, lenalinomide, bortezomib, pomalidomide, carfilzomib, elotuzumab, ixazomib, melphalan, or thalidomide, or any combination thereof.

[0275] In some embodiments, the hematological malignancy is AML.

[0276] In some embodiments, the AML is AML with at least one genetic abnormality, AML with multilineage dysplasia, therapy-related AML, undifferentiated AML, AML with minimal maturation, AML with maturation, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, acute megakaryoblastic leukemia, acute basophilic leukemia, acute panmyelosis with fibrosis, or myeloid sarcoma.

[0277] In some embodiments, the AML is AML with at least one genetic abnormality. In some embodiments, the AML is AML with multilineage dysplasia. In some embodiments, the AML is therapy-related AML. In some embodiments, the AML is undifferentiated AML. In some embodiments, the AML is AML with minimal maturation. In some embodiments, the AML is AML with maturation. In some embodiments, the AML is acute myelomonocytic leukemia. In some embodiments, the AML is acute monocytic leukemia. In some embodiments, the AML is acute erythroleukemia. In some embodiments, the AML is acute megakaryoblastic leukemia. In some embodiments, the AML is acute basophilic leukemia. In some embodiments, the AML is acute panmyelosis with fibrosis. In some embodiments, the AML is myeloid sarcoma.

[0278] In some embodiments, the at least one genetic abnormality is a translocation between chromosomes 8 and 21, a translocation or inversion in chromosome 16, a translocation between chromosomes 15 and 17, an alteration in chromosome 11, or a mutation in fins-related tyrosine kinase 3 (FLT3), nucleophosmin (NPM1), isocitrate dehydrogenase 1 (IDH1), isocitrate dehydrogenase 2 (IDH2), DNA (cytosine-5)-methyltransferase 3 (DNMT3A), CCAAT / enhancer-binding protein alpha (CEBPA), U2 small nuclear RNA cofactor 1 (U2AF1), enhancer of zeste2 polycomb repressive complex 2 subunit (EZH2), structural maintenance of chromosome 1A (SMC1A), or structural maintenance of chromosome 3 (SMC3).

[0279] In some embodiments, the at least one genetic abnormality is a translocation between chromosomes 8 and 21. In some embodiments, the at least one genetic abnormality is a translocation or inversion in chromosome 16. In some embodiments, the at least one genetic abnormality is a translocation between chromosomes 15 and 17. In some embodiments, the at least one genetic abnormality is an alteration in chromosome 11. In some embodiments, the at least one genetic abnormality is a mutation in fins-related tyrosine kinase 3 (FLT3). In some embodiments, the at least one genetic abnormality is a mutation in nucleophosmin (NPM1). In some embodiments, the at least one genetic abnormality is a mutation in isocitrate dehydrogenase 1 (IDH1). In some embodiments, the at least one genetic abnormality is a mutation in isocitrate dehydrogenase 2 (IDH2). In some embodiments, the at least one genetic abnormality is a mutation in DNA (cytosine-5)-methyltransferase 3 (DNMT3A). In some embodiments, the at least one genetic abnormality is a mutation in CCAAT / enhancer-binding protein alpha (CEBPA). In some embodiments, the at least one genetic abnormality is a mutation in U2 small nuclear RNA cofactor 1 (U2AF1). In some embodiments, the at least one genetic abnormality is a mutation in enhancer of zeste2 polycomb repressive complex 2 subunit (EZH2). In some embodiments, the at least one genetic abnormality is a mutation in structural maintenance of chromosome 1A (SMC1A). In some embodiments, the at least one genetic abnormality is a mutation in structural maintenance of chromosome 3 (SMC3).

[0280] In some embodiments, the at least one genetic abnormality is a translocation t(8;21)(q22;q22), an inversion inv(16)(p13;q22), a translocation t(16;16)(p13;q22), a translocation t(15;17)(q22;q12), a mutation FLT3-ITD, a mutation R132H or R100Q / R104V / F108L / R119Q / I130V in IDH1, or a mutation R140Q or R172 in IDH2.

[0281] In some embodiments, the at least one genetic abnormality is a translocation t(8;21)(q22;q22). In some embodiments, the at least one genetic abnormality is an inversion inv(16)(p13;q22). In some embodiments, the at least one genetic abnormality is a translocation t(16;16)(p13;q22). In some embodiments, the at least one genetic abnormality is a translocation t(15;17)(q22;q12). In some embodiments, the at least one genetic abnormality is a mutation FLT3-ITD. In some embodiments, the at least one genetic abnormality is a mutation R132H in IDH1. In some embodiments, the at least one genetic abnormality is a mutation R100Q / R104V / F108L / R119Q / I130V in IDH1. In some embodiments, the at least one genetic abnormality is a mutation R140Q in IDH2. In some embodiments, the at least one genetic abnormality is a mutation R172 in IDH2.

[0282] In some embodiments, the hematological malignancy is ALL.

[0283] In some embodiments, the ALL is B-lineage ALL, T-lineage ALL, adult ALL, or childhood ALL.

[0284] In some embodiments, the ALL is B-lineage ALL. In some embodiments, the ALL is T-lineage ALL. In some embodiments, the ALL is adult ALL. In some embodiments, the ALL is childhood ALL.

[0285] In some embodiments, the subject with ALL has a Philadelphia chromosome or is resistant or has acquired resistance to treatment with a BCR-ABL kinase inhibitor.

[0286] In some embodiments, the subject with ALL has a Philadelphia chromosome. In some embodiments, the subject with ALL is resistant or has acquired resistance to treatment with a BCR-ABL kinase inhibitor.

[0287] Ph chromosome is present in approximately 20% of adults with ALL and a small proportion of children with ALL, and is associated with poor prognosis.At relapse, patients with Ph+ positive ALL may be receiving tyrosine kinase inhibitor (TKI) regimen, and thus may develop resistance to TKI.Therefore, the methods described herein can be administered to subjects who have developed resistance to selective or partially selective BCR-ABL inhibitors.Exemplary BCR-ABL inhibitors are, for example, imatinib, dasatinib, nilotinib, bosutinib, ponatinib, bafetinib, saracatinib, tozasertib or danusertib.

[0288] Other chromosomal rearrangements identified in patients with B-lineage ALL are t(v;11q23) (MLL rearrangement), t(1;19)(q23;p13.3);TCF3-PBX1 (E2A-PBX1), t(12;21)(p13;q22);ETV6-RUNX1 (TEL-AML1), and t(5;14)(q31;q32);IL3-IGH.

[0289] In some embodiments, the subject has ALL with t(v;11q23) (MLL rearrangement), t(1;19)(q23;p13.3);TCF3-PBX1 (E2A-PBX1), t(12;21)(p13;q22);ETV6-RUNX1 (TEL-AML1), or t(5;14)(q31;q32);IL3-IGH chromosomal rearrangement.

[0290] Chromosomal rearrangements can be identified using well-known methods, such as fluorescent in situ hybridization, karyotyping, pulsed-field gel electrophoresis, or sequencing.

[0291] In some embodiments, the hematological malignancy is smoldering multiple myeloma, MGUS, ALL, DLBLC, BL, FL, MCL, Waldenstrom's macroglobulinemia, plasma cell leukemia, AL, precursor B-cell lymphoblastic leukemia, precursor B-cell lymphoblastic leukemia, myelodysplastic syndrome (MDS), CLL, B-cell malignancy, CML, HCL, blastic plasmacytoid dendritic cell neoplasm, Hodgkin's lymphoma, non-Hodgkin's lymphoma, MZL, MALT, plasma cell leukemia, ALCL, leukemia, or lymphoma.

[0292] solid tumors In some embodiments, the cancer is a solid tumor.

[0293] In some embodiments, the solid tumor is prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), liver cancer, cervical cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, melanoma, esophageal cancer, gastric cancer, stomach cancer, renal cancer, bladder cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma, head and neck cancer, glioma, glioblastoma, colorectal cancer, thyroid cancer, epithelial carcinoma, or adenocarcinoma.

[0294] In some embodiments, the prostate cancer is recurrent prostate cancer. In some embodiments, the prostate cancer is refractory prostate cancer. In some embodiments, the prostate cancer is malignant prostate cancer. In some embodiments, the prostate cancer is castration-resistant prostate cancer.

[0295] definition The term "identical" or percent "identity," in reference to two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are identical or have a specified percentage of the same amino acid residues or nucleotides (i.e., share at least about 80% identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity) with a reference sequence over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are therefore said to be "substantially identical." This definition also refers to the complement of a test sequence. In some embodiments, identity exists over a region that is at least about 25 amino acids or nucleotides in length, for example, over a region that is 50, 100, 200, 300, 400 amino acids or nucleotides in length, or over the entire length of the reference sequence.

[0296] For sequence comparison, typically, one sequence acts as the reference sequence to which test sequence is compared.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, and if necessary, partial sequence coordinates are designated, and sequence algorithm program parameters are also designated.Default program parameters can be used, or alternative parameters can be designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence and reference sequence based on program parameters.In some embodiments, BLAST and BLAST 2.0 algorithm and default parameters are used.

[0297] "Comparison window," as used herein, refers to any one of the number of contiguous positions selected from the group consisting of 20 to 600, usually about 50 to about 200, more usually about 100 to about 150, within which a sequence can be compared to a reference sequence of the same number of contiguous positions after optimally aligning the two sequences. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), or by the homology algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970). Sequence identity can be determined by sequence alignment algorithms, by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., eds., Current Protocols in Molecular Biology (1995 supplement)). Percent sequence identity and sequence similarity Examples of suitable algorithms for determining similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., J. Mol. Biol. 215: 403-410, respectively. (1990) and Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1977). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the world wide web at ncbi.nlm.nih.gov / ).

[0298] As described below, an indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the antibody generated against the polypeptide encoded by the second nucleic acid.Therefore, a polypeptide is typically substantially identical to a second polypeptide, for example, when the two peptides differ only by conservative substitutions.Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize with each other under stringent conditions.Another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify these sequences.

[0299] As used herein, "administering" refers to local and systemic administration, including, for example, enteral, parenteral, pulmonary, and topical / transdermal administration. Routes of administration of pharmaceutical ingredients (e.g., vectors) used in the methods described herein include, for example, oral (per os (PO)) administration, nasal or inhalation administration, administration as a suppository, topical contact, transdermal delivery (e.g., via a transdermal patch), intrathecal (IT) administration, intravenous ("iv") administration, intraperitoneal ("ip") administration, intramuscular ("im") administration, intralesional administration, or subcutaneous ("sc") administration to a subject, or implantation of a slow-release device, e.g., a mini-osmotic pump, a depot formulation, or the like. Administration can be by any route, including parenteral and transmucosal (e.g., oral, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intrarenal, intraurethral, ​​intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, iontophoretic, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like.

[0300] The terms "systemic administration" and "systemically administered" refer to a method of administering a pharmaceutical ingredient or composition to a mammal such that the pharmaceutical ingredient or composition is delivered by the circulatory system to sites within the body, including sites targeted for pharmaceutical action. Systemic administration includes, but is not limited to, oral, intranasal, rectal, and parenteral (e.g., other than through the digestive tract, such as intramuscular, intravenous, intraarterial, transdermal, and subcutaneous) administration.

[0301] The term "co-administering" or "concurrent administration," when used with respect to, for example, a pharmaceutical component (e.g., a vector) and / or analog thereof and another active agent (e.g., a multispecific antibody), refers to administering the pharmaceutical component and / or analog and the active agent such that they both achieve their physiological effect simultaneously. However, the two agents need not be administered together. In some embodiments, administration of one agent may precede administration of the other. A simultaneous physiological effect does not necessarily require that both agents be present in the circulation at the same time. However, in some embodiments, co-administration typically results in both agents being present in the body (e.g., in plasma) at a significant fraction (e.g., 20% or more, e.g., 30% or 40% or more, e.g., 50% or 60% or more, e.g., 70% or 80% or 90% or more) of their peak serum concentration for any given dose.

[0302] The term "effective amount" or "pharmaceutically effective amount" refers to the amount and / or dosage and / or dosage regime of one or more pharmaceutical components (e.g., vectors) necessary to bring about a desired result.

[0303] The phrase "cause to be administered" refers to an action taken by a medical professional (e.g., physician) or person managing a subject's medical care to administer and / or permit the administration of the agent(s) / compound(s) in question to a subject. Causing administration may involve diagnosing and / or determining an appropriate therapeutic or prophylactic regimen and / or prescribing a particular agent(s) / compound to a subject. Such prescribing may include, for example, drafting a prescription form, annotating a medical record, etc.

[0304] As used herein, the terms "treating" and "treatment" refer to delaying the onset of, arresting or reversing the progression of, reducing the severity of, or alleviating or preventing either the disease or condition to which the term applies, or one or more symptoms of such disease or condition. The terms "treating" and "treatment" also encompass preventing, alleviating, ameliorating, reducing, inhibiting, eliminating, and / or reversing one or more symptoms of a disease or condition.

[0305] The term "mitigating" refers to the reduction or elimination of one or more symptoms of the lesion or disease, and / or the reduction or delay in the rate of onset or severity of one or more symptoms of the lesion or disease, and / or the prevention of the lesion or disease. In some embodiments, the reduction or elimination of one or more symptoms of the lesion or disease can include, for example, a measurable and sustained reduction in tumor volume.

[0306] As used herein, the phrase "consisting essentially of" refers to the genus or species of active pharmaceutical agent(s) listed for the method or composition, and may further include other agents that do not themselves have substantial activity for the listed indication or purpose.

[0307] The terms "subject," "individual," and "patient" refer interchangeably to a mammal, preferably a human or non-human primate, but also to domesticated mammals (e.g., canines or felines), laboratory mammals, and agricultural mammals. In various embodiments, the subject can be a human (e.g., an adult male, adult female, adolescent male, adolescent female, boy, girl).

[0308] The term "vector" as used herein refers to a macromolecular complex that can deliver an exogenous nucleic acid molecule into a cell independently of another agent. As used herein, naked nucleic acid molecules such as plasmids are excluded from the term vector because they cannot effectively transduce target cells independently of other factors (e.g., transfection reagents or electroporation). A vector can be a viral vector or a non-viral vector. Viral vectors include retroviral vectors and lentiviral vectors. Non-viral vectors are limited to liposomes, nanoparticles, and other encapsulation systems for delivering polynucleotides into cells.

[0309] As used herein, the term "expression cassette" refers to a DNA segment that, under appropriate circumstances, is capable of driving expression of a polynucleotide ("transgene") encoding a polypeptide (e.g., a chimeric antigen receptor) incorporated into the expression cassette. When introduced into a host cell, the expression cassette is capable of, among other things, directing the cellular machinery to transcribe the transgene into RNA, which is then typically further processed and ultimately translated into a polypeptide. The expression cassette can be included in a vector (e.g., a viral vector). Generally, the term expression cassette excludes polynucleotide sequences 5' to the 5' ITR and 3' to the 3' ITR.

[0310] The term "derived" refers to cells obtained from their biological source and in Used to indicate grown in vitro or otherwise manipulated (e.g., cultured in growth medium to expand the population and / or create a cell line).

[0311] The term "transduce" refers to the introduction of a nucleic acid into a cell or host organism by a vector (e.g., a lentiviral vector). Thus, the introduction of a transgene into a cell by a viral vector can be referred to as "transduction" of the cell. The transgene may or may not be integrated into the genomic nucleic acid of the transduced cell. If the introduced transgene is integrated into the nucleic acid (genomic DNA) of the recipient cell or organism, the transgene may be stably maintained in the cell. Alternatively, the introduced transgene may exist extrachromosomally or only transiently in the recipient cell or host organism. Thus, a "transduced cell" is a cell into which a transgene has been introduced by transduction. Thus, a "transduced" cell is a cell into which a polynucleotide has been introduced.

[0312] The term "transduction efficiency" is an expression of the proportion of cells that express or are transduced with a transgene when a cell culture is contacted with vector particles. In some embodiments, efficiency can be expressed as the number of cells that express the transgene when a given number of cells are contacted with a given number of vector particles. In some embodiments, "relative transduction efficiency" is the ratio of cells transduced by a given number of viral particles in one condition to the proportion of cells transduced by the same number of particles in another condition containing a similar number of cells of the same cell type. Relative transduction efficiency is most often used to compare the effect of a modulator of transduction efficiency on cells and / or animals treated with the modulator or on cells and / or animals that are not treated with the modulator.

[0313] All patents, patent publications, and other publications referenced or identified in this specification are individually and expressly incorporated by reference herein in their entirety for all purposes.

[0314] Further Numbered Embodiments - Section A One set of numbered embodiments of the present disclosure are provided as follows: Clause 1. A method of transducing immune cells in a subject in need thereof, comprising: a) administering a multispecific antibody to render immune cells in the subject more transducible; b) administering a vector, optionally a viral vector; and transducing immune cells.

[0315] Clause 2. The method of clause 1, wherein the immune cell is a T cell.

[0316] Clause 3. The method of clause 1 or 2, wherein the vector is a lentiviral vector.

[0317] Clause 4. The method of any one of clauses 1-3, wherein the multispecific antibody comprises a T cell antigen-specific binding domain.

[0318] Clause 5. The method of Clause 4, wherein the T cell antigen is CD3, CD4, CD8 or TCR.

[0319] Clause 6. The method of any one of clauses 1-5, wherein the multispecific antibody comprises a second antigen-specific binding domain.

[0320] Clause 7. The method of Clause 6, wherein the second antigen is CD19.

[0321] Clause 8. The method of Clause 6, wherein the second antigen is CD19, EpCAM, Her2 / neu, EGFR, CD66e, CD33, EphA2, or MCSP.

[0322] Clause 9. The method of Clause 6, wherein the second antigen is CD19, EpCAM, CD20, CD123, BCMA, B7-H3, CDE, or PSMA.

[0323] Clause 10. The method of Clause 6, wherein the second antigen is a myeloid cell antigen or a dendritic cell antigen.

[0324] Clause 11. The method of Clause 10, wherein the second antigen is CD33, DC-SIGN, CD11b, CD11c, or CD18.

[0325] Clause 12. The method of any one of clauses 1 to 11, wherein the multispecific antibody is a bispecific antibody.

[0326] Clause 13. The method of Clause 12, wherein the bispecific antibody is a bispecific T cell engager (BiTE).

[0327] Clause 14. The method of clause 13, wherein the BiTE is a CD19xCD3 BiTE.

[0328] Clause 15. The method of clause 14, wherein the CD19xCD3 BiTE is blinatumomab.

[0329] Clause 16. The method of any one of clauses 1 to 15, wherein the multispecific antibody activates immune cells.

[0330] Clause 17. The method of any one of clauses 1-16, wherein the multispecific antibody increases immune cell activation compared to administration of a vehicle control.

[0331] Clause 18. The method of any one of clauses 1-18, wherein the multispecific antibody increases the number of immune cells in the lymph nodes of the subject.

[0332] Clause 19. The method of any one of clauses 1-18, wherein the multispecific antibody increases transduction of immune cells compared to administration of the viral vector alone.

[0333] Clause 20. The method of any one of clauses 1-19, wherein the multispecific antibody enhances in vivo transduction of immune cells by the viral vector.

[0334] Clause 21. The method of any one of clauses 1 to 20, wherein the multispecific antibody reduces the effective concentration (EC50) of the viral vector.

[0335] Clause 22. The method of any one of clauses 1 to 21, wherein the method achieves the same level of immune cell transduction without administering a multispecific antibody as a method comprising administering a higher concentration of viral vector.

[0336] Clause 23. The method of any one of clauses 1 to 22, wherein step a) and / or step b) comprises subcutaneous administration.

[0337] Clause 24. The method of any one of clauses 1 to 23, wherein step a) and / or step b) comprises intralymphatic administration.

[0338] Clause 25. The method of any one of clauses 1 to 24, wherein the viral vector comprises a polynucleotide encoding a T cell receptor or a chimeric antigen receptor.

[0339] Clause 26. The method of Clause 25, wherein the chimeric antigen receptor is an anti-CD19 chimeric antigen receptor.

[0340] Clause 27. The method of any one of clauses 1-26, wherein the viral vector comprises a polynucleotide encoding a cytokine receptor.

[0341] Clause 28. The method of Clause 27, wherein the cytokine receptor is a drug-inducible cytokine receptor.

[0342] Clause 29. The method of any one of clauses 1-28, wherein the vector further comprises one or more transgenes.

[0343] Clause 30. The method of Clause 29, wherein the viral vector comprises a transgene encoding a TGFβ dominant-negative receptor.

[0344] Clause 31. The method of any one of clauses 3 to 30, wherein the lentiviral vector comprises one or more cell surface receptors that bind to a ligand on the target host cell, a heterologous viral envelope glycoprotein, a fusion glycoprotein, a T cell activation or costimulatory molecule, a ligand for CD19 or a functional fragment thereof, a cytokine or cytokine-based transduction enhancer, and / or a transmembrane protein comprising a mitogenic domain and / or a cytokine-based domain exposed on the surface of the lentiviral vector and / or conjugated to the surface thereof.

[0345] Clause 32. The method of Clause 31, wherein the one or more T cell activating or costimulatory molecules comprise one or more T cell ligands.

[0346] Clause 33. The method of any one of clauses 3 to 32, wherein the lentiviral vector is pseudotyped with a coccivirus envelope protein.

[0347] Clause 34. The method of any one of clauses 3 to 33, wherein the lentiviral vector is pseudotyped with a Nipah virus envelope protein.

[0348] Clause 35. The method of Clause 34, wherein the Nipah envelope protein is engineered to bind to EpCAM, CD4 or CD8.

[0349] Clause 36. The method of any one of clauses 1 to 35, wherein step a) or step b) comprises intravenous administration.

[0350] Clause 37. The method of Clause 36, wherein both step a) and step b) comprise intravenous administration.

[0351] Clause 38. The method of any one of clauses 1 to 37, wherein the multispecific antibody is administered at a dose of about 0.001 mg / kg to about 1 mg / kg.

[0352] Clause 39. The method of any one of clauses 1-38, wherein the multispecific antibody specifically binds to CD3 and CD19, the vector is a lentiviral vector pseudotyped with a coccivirus envelope protein, and the vector comprises a polynucleotide encoding an anti-CD19 chimeric antigen receptor and a transgene encoding a TGFβ dominant negative receptor.

[0353] Clause 40. A method of transducing immune cells in a subject in need thereof, comprising: a) administering a polynucleotide encoding a multispecific antibody to activate immune cells in a subject; b) administering a vector, optionally a viral vector; and transducing immune cells.

[0354] Clause 41. The method of Clause 40, wherein the polynucleotide encoding the multispecific antibody is RNA.

[0355] Clause 42. The method of clause 40 or 41, wherein the immune cell is a T cell.

[0356] Clause 43. The method of any one of clauses 40 to 42, wherein the vector is a lentiviral vector.

[0357] Clause 44. The method of any one of Clauses 40 to 43, wherein the multispecific antibody comprises a T cell antigen-specific binding domain.

[0358] Clause 45. The method of Clause 44, wherein the T cell antigen is CD3, CD4, CD8 or TCR.

[0359] Clause 46. The method of any one of Clauses 40 to 45, wherein the multispecific antibody comprises a second antigen-specific binding domain.

[0360] Clause 47. The method of Clause 46, wherein the second antigen is CD19.

[0361] Clause 48. The method of Clause 46, wherein the second antigen is CD19, EpCAM, Her2 / neu, EGFR, CD66e, CD33, EphA2, MCSP, CD22, CD79a, CD79b, or sIgM.

[0362] Clause 49. The method of Clause 46, wherein the second antigen is CD19, EpCAM, CD20, CD123, BCMA, B7-H3, CDE, or PSMA.

[0363] Clause 50. The method of Clause 46, wherein the second antigen is a lymph node antigen.

[0364] Clause 51. The method of any one of Clauses 40 to 50, wherein the multispecific antibody is a trispecific antibody.

[0365] Clause 52. The method of any one of Clauses 40 to 50, wherein the multispecific antibody is a bispecific antibody.

[0366] Clause 53. The method of Clause 52, wherein the bispecific antibody is a bispecific T cell engager (BiTE).

[0367] Clause 54. The method of clause 53, wherein the BiTE is a CD19xCD3 BiTE.

[0368] Clause 55. The method of clause 54, wherein the CD19xCD3 BiTE is blinatumomab.

[0369] Clause 56. The method of any one of Clauses 40 to 55, wherein the multispecific antibody activates immune cells.

[0370] Clause 57. The method of any one of clauses 40-56, wherein the multispecific antibody increases immune cell activation compared to administration of a vehicle control.

[0371] Clause 58. The method of any one of clauses 40 to 57, wherein the multispecific antibody increases the number of immune cells in the lymph nodes of the subject.

[0372] Clause 59. The method of any one of clauses 40-58, wherein the multispecific antibody increases transduction of immune cells compared to administration of the viral vector alone.

[0373] Clause 60. The method of any one of clauses 40-59, wherein the multispecific antibody enhances in vivo transduction of immune cells by the viral vector.

[0374] Clause 61. The method of any one of clauses 40 to 60, wherein the multispecific antibody reduces the effective concentration (EC50) of the viral vector.

[0375] Clause 62. The method of any one of clauses 40-61, wherein the method achieves the same level of immune cell transduction without administering a multispecific antibody as a method comprising administering a higher concentration of viral vector.

[0376] Clause 63. The method of any one of clauses 40 to 62, wherein step a) and / or step b) comprises subcutaneous administration.

[0377] Clause 64. The method of any one of clauses 40 to 63, wherein step a) and / or step b) comprises intralymphatic administration.

[0378] Clause 65. The method of any one of clauses 40 to 64, wherein step a) and / or step b) comprises intravenous administration.

[0379] Clause 66. The method of any one of clauses 40 to 65, wherein the viral vector comprises a polynucleotide encoding a T cell receptor or a chimeric antigen receptor.

[0380] Clause 67. The method of Clause 66, wherein the chimeric antigen receptor is an anti-CD19 chimeric antigen receptor.

[0381] Clause 68. The method of any one of Clauses 40 to 67, wherein the viral vector comprises a polynucleotide encoding a cytokine receptor.

[0382] Clause 69. The method of Clause 68, wherein the cytokine receptor is a drug-inducible cytokine receptor.

[0383] Clause 70. The method of any one of clauses 43 to 69, wherein the lentiviral vector comprises one or more cell surface receptors that bind to a ligand on the target host cell, a heterologous viral envelope glycoprotein, a fusion glycoprotein, a T cell activation or costimulatory molecule, a ligand for CD19 or a functional fragment thereof, a cytokine or cytokine-based transduction enhancer, and / or a transmembrane protein comprising a mitogenic domain and / or a cytokine-based domain exposed on the surface of the lentiviral vector and / or conjugated to the surface thereof.

[0384] Clause 71. The method of Clause 70, wherein the one or more T cell activating or costimulatory molecules comprise one or more T cell ligands.

[0385] Clause 72. The method of any one of clauses 43 to 70, wherein the vector further comprises one or more transgenes.

[0386] Clause 73. The method of Clause 72, wherein the viral vector comprises a transgene encoding a TGFβ dominant-negative receptor.

[0387] Clause 74. The method of any one of Clauses 43 to 73, wherein the lentiviral vector is pseudotyped with a coccivirus envelope protein.

[0388] Clause 75. The method of any one of clauses 43 to 74, wherein the lentiviral vector is pseudotyped with a Nipah virus envelope protein.

[0389] Clause 76. The method of Clause 75, wherein the Nipah envelope protein is engineered to bind to EpCAM, CD4 or CD8.

[0390] Clause 77. The method of any one of clauses 43-77, wherein the multispecific antibody specifically binds to CD3 and CD19, the vector is a lentiviral vector pseudotyped with a coccivirus envelope protein, and the vector comprises a polynucleotide encoding an anti-CD19 chimeric antigen receptor and a transgene encoding a TGFβ dominant-negative receptor.

[0391] Article 78. A combination therapy comprising a multispecific antibody and a vector, optionally a viral vector, for use in transducing immune cells in vivo.

[0392] Clause 79. A pharmaceutical composition comprising a multispecific antibody and a vector, optionally a viral vector.

[0393] Clause 80. A kit comprising: 1) a polyspecific antibody; and 2) a vector, optionally a viral vector.

[0394] Clause 81. A kit comprising: 1) a polynucleotide encoding a multispecific antibody; and 2) a vector, optionally a viral vector.

[0395] Article 82. a) Transduction of immune cells in a subject in need thereof; and / or b) Treating a disease or disorder in a subject in need thereof 82. A kit according to clause 80 or 81 for use in

[0396] Article 83. A method of treating a disease or disorder in a subject in need thereof, comprising: a) administering a multispecific antibody to activate immune cells in a subject; b) administering a vector, optionally a viral vector, before, after and in conjunction with step a); A method comprising:

[0397] Clause 84. The method of Clause 83, wherein the immune cells are transduced.

[0398] Clause 85. The method of clause 83 or 84, wherein the disease or disorder is cancer.

[0399] Clause 86. The method of clause 83 or 84, wherein the disease or disorder is a hematological malignancy.

[0400] Clause 87. The method of Clause 86, wherein the hematological malignancy is a B-cell lymphoma.

[0401] Clause 88. The method of any one of clauses 85-87, which treats the disease or disorder more quickly than administration of the multispecific antibody alone and / or the vector alone.

[0402] Clause 89. The method of any one of clauses 83-88, which results in a superior outcome for the treatment of the disease or disorder than administration of the multispecific antibody alone and / or the vector alone.

[0403] Clause 90. The method of any one of clauses 83 to 89, wherein the multispecific antibody specifically binds to CD3 and CD19, the vector is a lentiviral vector pseudotyped with a coccivirus envelope protein, and the vector comprises a polynucleotide encoding an anti-CD19 chimeric antigen receptor and a transgene encoding a TGFβ dominant negative receptor.

[0404] Clause 91. The method of clause 87 or 90, which results in more rapid depletion of malignant B cells in a subject than administration of the multispecific antibody alone and / or the vector alone.

[0405] Clause 92. The method of any one of clauses 87 or 90-91, which results in a lower number of residual malignant B cells and / or a lower B-cell lymphoma relapse rate in the subject than administration of the multispecific antibody alone and / or the vector alone.

[0406] Further Numbered Embodiments - Section B Another set of further numbered embodiments of the present disclosure are provided in the following clauses: Clause 1. A vector comprising a polynucleotide for use in a method of treatment, the method comprising: (a) administering a multispecific antibody to render immune cells in the subject more transducible; (b) administering a vector to a subject to transduce an immune cell, wherein the transduction comprises delivery of a polynucleotide to the cell; A vector comprising:

[0407] Clause 2. A multispecific antibody for use in a method of treatment, the method comprising: (a) administering a multispecific antibody to render immune cells in the subject more transducible; (b) administering to a subject a vector comprising the polynucleotide to transduce an immune cell, wherein the transduction comprises delivery of the polynucleotide to the cell; A multispecific antibody comprising:

[0408] Clause 3. A polynucleotide for use in a method of treatment, the method comprising: (a) administering a multispecific antibody to render immune cells in the subject more transducible; (b) administering to a subject a vector comprising the polynucleotide to transduce an immune cell, wherein the transduction comprises delivery of the polynucleotide to the cell; A polynucleotide comprising:

[0409] Clause 4. A vector for use according to clause 1, a multispecific antibody for use according to clause 2, or a polynucleotide for use according to clause 3, wherein the immune cell is a T cell.

[0410] Clause 5. A vector for use according to clause 1 or clause 4, a multispecific antibody for use according to clause 2 or clause 4, or a polynucleotide for use according to clause 3 or clause 4, wherein the multispecific antibody activates immune cells, and optionally the activation leads to increased CD71 expression.

[0411] Clause 6. A vector for use according to any one of clauses 1 and 4-5, a multispecific antibody for use according to any one of clauses 2 and 4-5, or a polynucleotide for use according to any one of clauses 3-5, wherein the multispecific antibody increases activation of immune cells compared to administration of a vehicle control, and optionally, the activation leads to increased CD71 expression.

[0412] Clause 7. The multispecific antibody (i) increasing the number of immune cells in the lymph nodes of a subject; and / or (ii) increasing the transduction of immune cells compared to administration of the vector alone; and / or (iii) enhance the in vivo transduction of immune cells by the vector; and / or (iv) reducing the effective concentration (EC50) of the vector; A vector for use according to any one of clauses 1 and 4 to 6, a multispecific antibody for use according to any one of clauses 2 and 4 to 6, or a polynucleotide for use according to any one of clauses 3 to 6.

[0413] Clause 8. A vector for use according to any one of clauses 1 and 4 to 7, a multispecific antibody for use according to any one of clauses 2 and 4 to 7, or a polynucleotide for use according to any one of clauses 3 to 7, wherein the method achieves the same level of immune cell transduction as a method comprising administering a higher concentration of the vector without administering the multispecific antibody.

[0414] Clause 9. Step a) and / or step b) (i) subcutaneous administration; or (ii) intralymphatic administration; or (iii) Intravenous administration Including, Optionally, both step a) and step b) comprise intravenous administration. A vector for use according to any one of clauses 1 and 4 to 8, a multispecific antibody for use according to any one of clauses 2 and 4 to 8, or a polynucleotide for use according to any one of clauses 3 to 8.

[0415] Clause 10. The vector for use according to any one of clauses 1 and 4 to 9, the multispecific antibody for use according to any one of clauses 2 and 4 to 9, or the polynucleotide for use according to any one of clauses 3 to 9, wherein the multispecific antibody is administered at a dose of from about 0.001 mg / kg to about 1 mg / kg.

[0416] Clause 11. A vector for use according to any one of clauses 1 and 4 to 10, a multispecific antibody for use according to any one of clauses 2 and 4 to 10, or a polynucleotide for use according to any one of clauses 3 to 10, wherein step b) is performed before, after and / or in conjunction with step a).

[0417] Clause 12. A vector for use according to any one of clauses 1 and 4 to 11, a multispecific antibody for use according to any one of clauses 2 and 4 to 11, or a polynucleotide for use according to any one of clauses 3 to 11, wherein the method is for treating cancer.

[0418] Clause 13. A vector for use according to any one of clauses 1 and 4-12, a multispecific antibody for use according to any one of clauses 2 and 4-12, or a polynucleotide for use according to any one of clauses 3-12, wherein the method is for treating a hematological malignancy, and optionally the hematological malignancy is a B-cell lymphoma.

[0419] Clause 14. The method is for treating a disease or disorder, the method treats the disease or disorder more quickly than administration of the multispecific antibody alone and / or the vector alone; or the method results in a superior outcome for the treatment of the disease or disorder than administration of the multispecific antibody alone and / or the vector alone; A vector for use according to any one of clauses 1 and 4 to 13, a multispecific antibody for use according to any one of clauses 2 and 4 to 13, or a polynucleotide for use according to any one of clauses 3 to 13.

[0420] Clause 15. A vector, multispecific antibody, or polynucleotide for use according to clause 13, wherein the method results in faster depletion of malignant B cells in the subject than administration of the multispecific antibody alone and / or the vector alone.

[0421] Clause 16. A vector, multispecific antibody, or polynucleotide for use according to clause 13 or clause 15, wherein the method results in a lower number of residual malignant B cells and / or a lower relapse rate of B-cell lymphoma in the subject than administration of the multispecific antibody alone and / or the vector alone.

[0422] Clause 17. A vector for use according to any one of clauses 1 and 4 to 16, a multispecific antibody for use according to any one of clauses 2 and 4 to 16, or a polynucleotide for use according to any one of clauses 3 to 16, wherein the multispecific antibody is administered as a polynucleotide encoding the multispecific antibody.

[0423] Clause 18. A pharmaceutical composition comprising a multispecific antibody and a vector.

[0424] Clause 19. A kit comprising: 1) a multispecific antibody; and 2) a vector.

[0425] Clause 20. A kit comprising: 1) a polynucleotide encoding a multispecific antibody; and 2) a vector.

[0426] Clause 21. A vector, multispecific antibody, or polynucleotide for use according to clause 17, or a kit according to clause 20, wherein the polynucleotide encoding the multispecific antibody is RNA.

[0427] Clause 22. A vector for use according to any one of clauses 1, 4 to 17 and 21, a multispecific antibody for use according to any one of clauses 2, 4 to 17 and 21, a polynucleotide for use according to any one of clauses 3 to 17 and 21, a pharmaceutical composition according to clause 18 or a kit according to any one of clauses 19 to 21, wherein the vector is a viral vector, optionally a lentiviral vector.

[0428] Clause 23. A vector for use according to any one of clauses 1, 4 to 17 and 21 to 22, a multispecific antibody for use according to any one of clauses 2, 4 to 17 and 21 to 22, a polynucleotide for use according to any one of clauses 3 to 17 and 21 to 22, a pharmaceutical composition according to clause 18 or clause 22 or a kit according to any one of clauses 19 to 22, wherein the multispecific antibody comprises a T cell antigen-specific binding domain, and optionally the T cell antigen is CD3, CD4, CD8 or TCR.

[0429] Article 24. the multispecific antibody comprises a second antigen-specific binding domain; as needed, (i) the second antigen is CD19, or (ii) the second antigen is CD19, EpCAM, Her2 / neu, EGFR, CD66e, CD33, EphA2, or MCSP; or (iii) the second antigen is CD19, EpCAM, Her2 / neu, EGFR, CD66e, CD33, EphA2, MCSP, CD22, CD79a, CD79b, or sIgM; (iv) the second antigen is CD19, EpCAM, CD20, CD123, BCMA, B7-H3, CDE, or PSMA; or (v) the second antigen is a lymph node antigen; or (vi) the second antigen is a myeloid cell or dendritic cell antigen, optionally CD33, DC-SIGN, CD11b, CD11c, or CD18; a vector for use according to any one of clauses 1, 4 to 17 and 21 to 23, a multispecific antibody for use according to any one of clauses 2, 4 to 17 and 21 to 23, a polynucleotide for use according to any one of clauses 3 to 17 and 21 to 23, a pharmaceutical composition according to any one of clauses 18 and 22 to 23, or a kit according to any one of clauses 19 to 23.

[0430] Clause 25. A vector for use according to any one of clauses 1, 4 to 17 and 21 to 24, a multispecific antibody for use according to any one of clauses 2, 4 to 17 and 21 to 24, a polynucleotide for use according to any one of clauses 3 to 17 and 21 to 24, a pharmaceutical composition according to any one of clauses 18 and 22 to 24, or a kit according to any one of clauses 19 to 24, wherein the multispecific antibody is a bispecific antibody or trispecific antibody, optionally the bispecific antibody is a bispecific T cell engager (BiTE), optionally the BiTE is a CD19xCD3 BiTE, optionally the CD19xCD3 BiTE is blinatumomab.

[0431] Clause 26. A vector for use according to any one of clauses 1, 4 to 17 and 21 to 25, a multispecific antibody for use according to any one of clauses 2, 4 to 17 and 21 to 25, a polynucleotide for use according to any one of clauses 3 to 17 and 21 to 25, a pharmaceutical composition according to any one of clauses 18 and 22 to 25 or a kit according to any one of clauses 19 to 25, wherein the polynucleotide delivered to the cell encodes at least one therapeutic polypeptide.

[0432] Clause 27. A vector, multispecific antibody, or polynucleotide for use according to clause 26, or a pharmaceutical composition or kit according to clause 26, wherein at least one therapeutic polypeptide comprises a T cell receptor or a chimeric antigen receptor, and optionally the T cell receptor or chimeric antigen receptor targets an antigen associated with cancer or hematological malignancy.

[0433] Clause 28. A vector, a multispecific antibody, or a polynucleotide for use according to clause 27, or a pharmaceutical composition or kit according to clause 27, wherein the chimeric antigen receptor is an anti-CD19 chimeric antigen receptor.

[0434] Clause 29. A vector, multispecific antibody, or polynucleotide for use according to clause 26, or a pharmaceutical composition or kit according to clause 26, wherein at least one therapeutic polypeptide comprises a cytokine receptor, and optionally the cytokine receptor is a drug-inducible cytokine receptor.

[0435] Clause 30. A vector for use according to any one of clauses 1, 4 to 17 and 21 to 29, a multispecific antibody for use according to any one of clauses 2, 4 to 17 and 21 to 29, a polynucleotide for use according to any one of clauses 3 to 17 and 21 to 29, a pharmaceutical composition according to any one of clauses 18 and 22 to 29 or a kit according to any one of clauses 19 to 29, wherein the vector further comprises one or more transgenes, optionally wherein the one or more transgenes comprise a transgene encoding a TGFβ dominant negative receptor.

[0436] Clause 31. A vector for use according to any one of clauses 1, 4 to 17 and 21 to 30, a multispecific antibody for use according to any one of clauses 2, 4 to 17 and 21 to 30, a polynucleotide for use according to any one of clauses 3 to 17 and 21 to 30, a pharmaceutical composition according to any one of clauses 18 and 22 to 30 or a kit according to any one of clauses 19 to 30, wherein the vector comprises one or more cell surface receptors that bind to a ligand on the target host cell, a heterologous viral envelope glycoprotein, a fusion glycoprotein, a T cell activating or costimulatory molecule, a ligand for CD19 or a functional fragment thereof, a cytokine or cytokine-based transduction enhancer, and / or a transmembrane protein comprising a mitogenic domain and / or a cytokine-based domain exposed on the surface of the vector and / or conjugated to its surface, and optionally the one or more T cell activating or costimulatory molecules comprise one or more T cell ligands.

[0437] Clause 32. A vector for use according to any one of clauses 1, 4 to 17 and 21 to 31, a multispecific antibody for use according to any one of clauses 2, 4 to 17 and 21 to 31, a polynucleotide for use according to any one of clauses 3 to 17 and 21 to 31, a pharmaceutical composition according to any one of clauses 18 and 22 to 31 or a kit according to any one of clauses 19 to 31, wherein the vector is a lentiviral vector, and the lentiviral vector is pseudotyped with a Cocalvirus envelope protein and / or a Nipah virus envelope protein, and optionally the Nipah envelope protein is engineered to bind to EpCAM, CD4 or CD8.

[0438] Clause 33. A vector for use according to any one of clauses 1, 4 to 17 and 21 to 32, a multispecific antibody for use according to any one of clauses 2, 4 to 17 and 21 to 32, a polynucleotide for use according to any one of clauses 3 to 17 and 21 to 32, a pharmaceutical composition according to any one of clauses 18 and 22 to 32 or a kit according to any one of clauses 19 to 32, wherein the multispecific antibody specifically binds to CD3 and CD19, and the vector is a lentiviral vector pseudotyped with coccivirus envelope protein, and the vector comprises a polynucleotide encoding an anti-CD19 chimeric antigen receptor and a transgene encoding a TGFβ dominant negative receptor.

[0439] Clause 34. A kit according to any one of clauses 19 to 33 for use in a method of treatment. [Example]

[0440] Example 1 Blinatumomab enhances lentiviral vector-mediated T cell transduction This example relates to the use of a CD19xCD3 bispecific antibody (blinatumomab) via subcutaneous, intralymphatic, or intratumoral injection to activate T cells in lymph nodes, thereby increasing transduction by surface-engineered lentiviral vectors.

[0441] Lentiviral vector VivoVec Lentiviral vectors (VivoVec) were generated using a modified third-generation packaging system. To generate lentiviral particles, an envelope plasmid encoding a 2A-linked polycistronic expression construct (CD86-2A-anti-CD3scFv-2A-CD137L-2A-COCVG) was cotransfected into 293 cells with a transfer plasmid encoding an anti-CD19 chimeric antigen receptor operably linked to the constitutive CMV or MND promoter, flanked by 5' and 3' long terminal repeats; and two packaging plasmids encoding the gag and pol genes, and the rev gene, respectively.

[0442] Expression of CD86, a transmembrane fusion anti-CD3 single-chain variable fragment, CD137L, and the cocalvirus G protein (COCVG) from an envelope plasmid results in a surface-engineered lentiviral vector that is specific for CD3-expressing cells (anti-CD3); stimulates T cells (CD86 and CD137L); and transduces T cells (pseudotyped with the cocalvirus G protein). The genome of the lentiviral vector is RNA transcribed by the Pol protein from the transfer plasmid. Gag mediates viral packaging of the G protein, Gag, and Rev proteins along with the RNA genome.

[0443] VSV particles Lentiviral vectors (VSV particles) were generated using a modified third-generation packaging system. To generate lentiviral particles, an envelope plasmid encoding the G protein of vesicular stomatitis virus (VSV) was cotransfected into 293 cells with a transfer plasmid encoding an anti-CD19 chimeric antigen receptor operably linked to the constitutive CMV or MND promoter, flanked by 5' and 3' long terminal repeats; and two packaging plasmids encoding the gag and pol genes, and the rev gene, respectively.

[0444] Expression of the VSV G protein from the envelope plasmid results in a lentiviral vector pseudotyped with the VSV G protein. The genome of the lentiviral vector is RNA transcribed from the transfer plasmid by the Pol protein. Gag mediates viral packaging of the G protein, Gag, and Rev proteins along with the RNA genome.

[0445] Multispecific (bispecific) antibodies Blinatumomab is a bispecific antibody of the "BiTE" class that has been clinically approved for the treatment of CD19-expressing hematologic malignancies, precursor B-cell acute lymphoblastic leukemia (ALL). Blinatumomab's therapeutic mechanism of action involves the creation of an "immune synapse" between T cells and tumor B cells, inducing the T cells to kill the tumor B cells. Here, the biochemical activity of blinatumomab is applied for administration to patients (with or without hematologic malignancies) for the purpose of engaging normal B cells with T cells in vivo for an alternative purpose: making the T cells susceptible to lentiviral transduction.

[0446] Demonstration of increased transduction in vitro Purified human primary T cells were thawed and resuspended in 3 mL of culture medium (RPMI-1640 + 10% fetal bovine serum). T cells were used alone or with B cells at a 50:50 ratio. Treatments tested were: vehicle control; blinatumomab alone; blinatumomab + lentiviral particles (VivoVec); lentiviral particles pseudotyped with control VSV; and blinatumomab + lentiviral particles pseudotyped with control VSV.

[0447] Three days after the initiation of culture, cells were harvested and analyzed by flow cytometry for expression of the T cell activation surface marker CD25 (Figures 2A and 2B) and for expression of the vector-delivered anti-CD19 chimeric antigen receptor (CCR) (Figures 3A and 3B). Blinatumomab ("Blina") increased the concentration of CD25+ activated T cells from approximately 7-17% to approximately 87-92% (Figure 2A). This effect was dependent on the presence of B cells in the culture; in their absence, no increase in activation was observed in blinatumomab-treated samples compared to untreated samples (Figure 2B). Blinatumomab (Blina) increased the percentage of T cells transduced by VivoVec vectors from approximately 12% to approximately 41%; blinatumomab (Blina) also increased transduction by VSV particles (VSVG) from approximately 4% to approximately 24% (Figure 3A). This effect was dependent on the presence of B cells in the culture; in the absence of B cells, VivoVec transduced approximately 10-11% of T cells, while VSV particles transduced approximately 4% of T cells (Figure 3B).

[0448] The ratio of B cells to T cells in the cultures was assessed for samples with a 50:50 B cell:T cell ratio. B cell expansion resulted in a final ratio of approximately 80:20. This ratio was similar for all treatments tested, demonstrating that blinatumomab can be used for T cell activation at concentrations that do not lead to rapid B cell killing (Figure 4).

[0449] Demonstration of increased transduction in vivo Subjects (mice, primates, or humans) are administered blinatumomab at a subclinical dose (in humans, approximately 1 mg to approximately 10 mg in a single injection on day 1) prior to or in conjunction with a lentiviral vector such as VivoVec. Lymph node biopsies are used to isolate T cells, which are assayed for expression of a transgene (e.g., an anti-CD19 chimeric antigen receptor). Subjects with B-cell malignancies are treated with a combination of VivoVec and blinatumomab. Reductions in disease progression, as measured by B-cell burden and tumor size, are observed.

[0450] Example 2 Blinatumomab-mediated in vivo transduction of T cells by lentiviral vectors In vivo transduction of T cells with a lentiviral vector encoding an anti-CD19 CAR in CD34-humanized NSG mice was evaluated using blinatumomab as a tool to activate T cells in vivo to facilitate transduction. The key questions addressed in this study were: 1) whether blinatumomab promotes CAR generation, 2) what dose of blinatumomab is required for CAR T cell generation, and 3) whether CAR+ cells can be detected and whether this correlates with B cell depletion.

[0451] Test Design Virus preparation and QC data Viral payload: Lentiviral particles pseudotyped with U4367EA110_5, pRRL-MND-human-Frb-CD19_CAR-TGFβdn-VTw.cocal, carrying an anti-CD19-CAR payload and also expressing Frb and TGFβ dominant-negative receptor (CD19CAR-TGFβ) payload, were produced at Fred Hutchinson following a protocol similar to that used in Example 1. Endotoxin activity was 2.1 EU / mL as measured by a Chromogenic Endotoxin Quant Kit (Cat# A39552S). Cultures were negative for mycoplasma as determined by a Lonza MycoAlert Mycoplasma Detection kit.

[0452] Animal Testing Protocol Animal studies were performed at Lumigenics LLC using CD34+ humanized mice (Jackson Laboratories). HuNSG mice 18-26 weeks after CD34+ HSC implantation were used for this study. Mice were allowed to acclimate for one week after arrival. Blood was collected on study day -2 to assess engraftment. Mice were assigned to study groups to ensure that human T cell characteristics were equivalent between groups.

[0453] Blood was collected weekly for the duration of the study, beginning on day -2, into EDTA-coated tubes. At least 70 μL was collected per blood draw. Samples were mixed by inversion and transported overnight in cryopacks to Umoja. Body weights were measured twice weekly for the length of the study. Animals that showed weight loss of more than 20% of their initial body weight were euthanized and recorded as "conditional deaths."

[0454] Mice in the blinatumomab treatment group were treated intravenously (IV) on study days 1, 2, and 4. Mice in the appropriate groups were treated with lentivirus by IV injection on study day 4.

[0455] At study completion on day 52, blood, small sections of spleen, and femurs were shipped in cryopacks to Umoja Biopharma for analysis by flow cytometry.

[0456] Study endpoints include: 1) CAR-T cell transduction and expansion by flow cytometry, 2) CAR-T cell phenotype by flow cytometry, 3) B-cell depletion, and 4) toxicity, survival.

[0457] Table 2 below outlines the trial timeline.

[0458] [Table 2]

[0459] result Flow cytometry panel validation: Our flow cytometry panel was validated against anti-CD19CAR-TGFβ T cells generated and maintained in culture (Figure 5A) and in humanized mice (Figure 5B). T cells were used as a positive control. In Figure 5A, ex vivo-produced CAR-TGFβ T cells were used to validate CAR T cell detection by detecting the TGFβ double-negative receptor. All populations were gated by debris exclusion / singlets / live cells / human CD45. CAR T cells were defined as CD3+ and FITC+. Non-CAR T cells were defined as CD3+ and FITC-. A non-CAR T cell population was used as a negative control to define positive staining for the TGFβ double-negative receptor. Figure 5B shows the gating scheme for identifying CD3+ T cells that are either CD4+ or CD8+ and express the CD25 or CD71 activation markers.

[0460] As shown in Figure 6, blinatumomab administration activated T cells, as measured by CD71 expression in both CD4 and CD8 T cells 5 days after injection ("+" indicates the "low blin" group, "++" indicates the "high blin" group; " ** "," *** " and " **** " is (P values ​​of <0.01, <0.001, and <0.0001 are shown, respectively.) CD25 was included in the flow panel but was not used as an in vivo activation marker because the observed CD25 expression was very low in all groups at all time points.

[0461] Circulating B cells were also measured in mice treated with 0.004 mg / kg blinatumomab (low blinatumomab) or 0.04 mg / kg blinatumomab (high blinatumomab) with or without CD19CAR-TGFβ cocal lentiviral treatment (Figure 7). The blinatumomab-only treatment group showed immediate B cell depletion at approximately day 5, but B cell numbers subsequently increased. On the other hand, CD19CAR-TGFβ cocal lentiviral treatment resulted in significant and sustained B cell depletion, regardless of blinatumomab administration. B cell depletion began at approximately day 12 in mice treated with lentivirus alone, while B cell depletion began at approximately day 5 in mice treated with lentivirus plus blinatumomab. All mice in the lentivirus-treated groups had few or no circulating B cells throughout study days 12 through 52 (Figure 7). The blinatumomab-only group served as a negative gating control, and no significant populations of CAR T cells were observed in either group throughout the study period (Figure 8).

[0462] On study day 52, complete B cell eradication was observed in the bone marrow and spleen of lentivirus-treated mice (Figure 9). We speculate that due to rapid B cell depletion, a transient CAR+ population existed at levels below our detection threshold.

[0463] In summary, intravenous administration of a lentivirus bearing a CD19CAR-TGFβ payload was sufficient to induce significant and sustained B cell depletion in CD34+ humanized mice. Blinatumomab administration accelerated B cell depletion. At the end of the study, no B cells were detected in the spleen or bone marrow of the lentivirus-treated group. In contrast, mice treated with blinatumomab but not lentivirus recovered circulating B cell populations after transient depletion at both dose levels, with readily detectable B cell populations in the bone marrow and spleen (Figure 9). These results are consistent with the predicted activity of in vivo anti-CD19 CAR T cell generation.

[0464] Example 3 Co-administration of blinatumomab and lentiviral vector This example relates to the co-administration of a CD19xCD3 bispecific antibody (blinatumomab) and a surface-engineered lentiviral vector containing a transgene encoding an anti-CD19 CAR.

[0465] CD34+ humanized mice are co-administered with blinatumomab and lentiviral vector. In corresponding control groups, mice receive either blinatumomab alone, lentiviral vector alone, or sham solution. Administration can be subcutaneous, intralymphatic, and / or intratumoral. Blood and tissue samples (e.g., liver, lung, spleen, bone marrow) are collected for analysis of the following factors: 1) CAR-T cell transduction and expansion as analyzed by flow cytometry, 2) CAR-T cell phenotype as analyzed by flow cytometry, 3) B cell count, and 4) toxicity and survival. Results of the co-administered groups are compared with the control group. In certain embodiments, for example, the following are provided: (Item 1) 1. A method for transducing immune cells in a subject in need thereof, comprising: a) administering a multispecific antibody to render immune cells in the subject more transducible; b) administering a vector, optionally a viral vector; wherein said immune cells are transduced. (Item 2) Item 10. The method of item 1, wherein the immune cells are T cells. (Item 3) Item 10. The method of item 1, wherein the vector is a lentiviral vector. (Item 4) 3. The method of claim 2, wherein the multispecific antibody comprises a T cell antigen-specific binding domain. (Item 5) 5. The method of claim 4, wherein the T cell antigen is CD3, CD4, CD8 or TCR. (Item 6) 5. The method of claim 4, wherein the multispecific antibody comprises a second antigen-specific binding domain. (Item 7) 7. The method of claim 6, wherein the second antigen is CD19. (Item 8) 7. The method of item 6, wherein the second antigen is CD19, EpCAM, Her2 / neu, EGFR, CD66e, CD33, EphA2, or MCSP. (Item 9) 7. The method of item 6, wherein the second antigen is CD19, EpCAM, CD20, CD123, BCMA, B7-H3, CDE, or PSMA. (Item 10) Item 11. The method according to Item 6, wherein the second antigen is a myeloid cell antigen or a dendritic cell antigen. 11. The method of item 10, wherein the second antigen is CD33, DC-SIGN, CD11b, CD11c, or CD18. (Item 12) 2. The method of claim 1, wherein the multispecific antibody is a bispecific antibody. (Item 13) 13. The method of claim 12, wherein the bispecific antibody is a bispecific T cell engager (BiTE). (Item 14) 14. The method of item 13, wherein the BiTE is a CD19xCD3 BiTE. (Item 15) Item 16. The method of item 14, wherein the CD19×CD3 BiTE is blinatumomab. 16. The method of any one of items 1 to 15, wherein the multispecific antibody activates the immune cells. (Item 17) 17. The method of claim 16, wherein the multispecific antibody increases activation of the immune cells compared to administration of a vehicle control. (Item 18) 16. The method of any one of items 1 to 15, wherein the multispecific antibody increases the number of immune cells in lymph nodes of the subject. (Item 19) 16. The method of any one of items 1 to 15, wherein the multispecific antibody increases transduction of the immune cells compared to administration of the viral vector alone. (Item 20) 16. The method of any one of items 1 to 15, wherein the multispecific antibody enhances in vivo transduction of the immune cell by the viral vector. (Item 21) The polyspecific antibody is capable of inhibiting the effective concentration (EC 50 21. The method according to item 20, wherein the amount of erythrocyte stimulating factor (E1) is reduced. (Item 22) 16. The method of any one of items 1 to 15, wherein the method achieves the same level of immune cell transduction without administering the multispecific antibody as a method comprising administering a higher concentration of the viral vector. (Item 23) Item 24. The method according to item 1, wherein step a) and / or step b) comprises subcutaneous administration. 2. The method of claim 1, wherein step a) and / or step b) comprises intralymphatic administration. (Item 25) 3. The method of claim 2, wherein the vector is a viral vector comprising a polynucleotide encoding a T cell receptor or a chimeric antigen receptor. (Item 26) 26. The method of claim 25, wherein the chimeric antigen receptor is an anti-CD19 chimeric antigen receptor. (Item 27) 3. The method of claim 2, wherein the vector is a viral vector comprising a polynucleotide encoding a cytokine receptor. (Item 28) 28. The method of claim 27, wherein the cytokine receptor is a drug-inducible cytokine receptor. (Item 29) 26. The method of claim 25, wherein the vector further comprises one or more transgenes. (Item 30) 30. The method of claim 29, wherein the viral vector comprises the transgene encoding a TGFβ dominant-negative receptor. (Item 31) 4. The method of claim 3, wherein the lentiviral vector comprises one or more cell surface receptors that bind to a ligand on a target host cell, a heterologous viral envelope glycoprotein, a fusion glycoprotein, a T cell activation or costimulatory molecule, a ligand for CD19 or a functional fragment thereof, a cytokine or cytokine-based transduction enhancer, and / or a transmembrane protein comprising a mitogenic domain and / or a cytokine-based domain exposed on the surface of the lentiviral vector and / or conjugated to the surface of the lentiviral vector. (Item 32) 32. The method of claim 31, wherein the one or more T cell activating or costimulatory molecules comprise one or more T cell ligands. (Item 33) 32. The method of claim 31, wherein the lentiviral vector is pseudotyped with a coccivirus envelope protein. (Item 34) 32. The method of claim 31, wherein the lentiviral vector is pseudotyped with a Nipah virus envelope protein. (Item 35) 35. The method of claim 34, wherein the Nipah envelope protein is engineered to bind to EpCAM, CD4, or CD8. (Item 36) 2. The method of claim 1, wherein step a) or step b) comprises intravenous administration. (Item 37) 37. The method of claim 36, wherein both step a) and step b) comprise intravenous administration. (Item 38) 38. The method of item 36 or 37, wherein the multispecific antibody is administered at a dose of about 0.001 mg / kg to about 1 mg / kg. (Item 39) 2. The method of claim 1, wherein the multispecific antibody specifically binds to CD3 and CD19, the vector is a lentiviral vector pseudotyped with a coccivirus envelope protein, and the vector comprises a polynucleotide encoding an anti-CD19 chimeric antigen receptor and a transgene encoding a TGFβ dominant-negative receptor. (Item 40) 1. A method for transducing immune cells in a subject in need thereof, comprising: a) administering a polynucleotide encoding a multispecific antibody to activate immune cells in the subject; b) administering a vector, optionally a viral vector; wherein said immune cells are transduced. (Item 41) 41. The method of claim 40, wherein the polynucleotide encoding the multispecific antibody is RNA. (Item 42) 41. The method of claim 40, wherein the immune cells are T cells. (Item 43) 41. The method of claim 40, wherein the vector is a lentiviral vector. (Item 44) Item 45. The method of Item 42, wherein the multispecific antibody comprises a T cell antigen-specific binding domain. 45. The method of claim 44, wherein the T cell antigen is CD3, CD4, CD8 or TCR. (Item 46) 47. The method of claim 44, wherein the multispecific antibody comprises a second antigen-specific binding domain. 47. The method of claim 46, wherein the second antigen is CD19. (Item 48) 47. The method of item 46, wherein the second antigen is CD19, EpCAM, Her2 / neu, EGFR, CD66e, CD33, EphA2, MCSP, CD22, CD79a, CD79b, or sIgM. (Item 49) 47. The method of item 46, wherein the second antigen is CD19, EpCAM, CD20, CD123, BCMA, B7-H3, CDE, or PSMA. (Item 50) 47. The method of claim 46, wherein the second antigen is a lymph node antigen. (Item 51) 41. The method of claim 40, wherein the multispecific antibody is a trispecific antibody. (Item 52) 41. The method of claim 40, wherein the multispecific antibody is a bispecific antibody. (Item 53) 53. The method of claim 52, wherein the bispecific antibody is a bispecific T cell engager (BiTE). (Item 54) 54. The method of item 53, wherein the BiTE is a CD19xCD3 BiTE. (Item 55) Item 56. The method of item 54, wherein the CD19xCD3 BiTE is blinatumomab. 56. The method of any one of items 40 to 55, wherein the multispecific antibody activates the immune cells. (Item 57) 56. The method of any one of items 40 to 55, wherein the multispecific antibody increases activation of the immune cells compared to administration of a vehicle control. (Item 58) 56. The method of any one of items 40 to 55, wherein the multispecific antibody increases the number of immune cells in lymph nodes of the subject. (Item 59) 56. The method of any one of items 40 to 55, wherein the multispecific antibody increases transduction of the immune cells compared to administration of the viral vector alone. (Item 60) 56. The method of any one of items 40 to 55, wherein the multispecific antibody enhances in vivo transduction of the immune cell by the viral vector. (Item 61) The polyspecific antibody is capable of inhibiting the effective concentration (EC 50 Item 61. The method according to Item 60, wherein the amount of erythrocyte stimulating factor (E1) is reduced. (Item 62) 61. The method of item 60, wherein the method achieves the same level of immune cell transduction without administering the multispecific antibody as a method comprising administering a higher concentration of the viral vector. (Item 63) 41. The method of claim 40, wherein step a) and / or step b) comprises subcutaneous administration. (Item 64) 41. The method of claim 40, wherein step a) and / or step b) comprises intralymphatic administration. (Item 65) 41. The method of claim 40, wherein step a) and / or step b) comprises intravenous administration. (Item 66) 41. The method of claim 40, wherein the viral vector comprises a polynucleotide encoding a T cell receptor or a chimeric antigen receptor. (Item 67) 67. The method of claim 66, wherein the chimeric antigen receptor is an anti-CD19 chimeric antigen receptor. (Item 68) 41. The method of claim 40, wherein the viral vector comprises a polynucleotide encoding a cytokine receptor. (Item 69) 69. The method of item 68, wherein the cytokine receptor is a drug-inducible cytokine receptor. (Item 70) 44. The method of claim 43, wherein the lentiviral vector comprises one or more cell surface receptors that bind to a ligand on a target host cell, a heterologous viral envelope glycoprotein, a fusion glycoprotein, a T cell activation or costimulatory molecule, a ligand for CD19 or a functional fragment thereof, a cytokine or cytokine-based transduction enhancer, and / or a transmembrane protein comprising a mitogenic domain and / or a cytokine-based domain exposed on the surface of the lentiviral vector and / or conjugated to the surface thereof. (Item 71) 71. The method of claim 70, wherein the one or more T cell activating or costimulatory molecules comprise one or more T cell ligands. (Item 72) 41. The method of claim 40, wherein the vector further comprises one or more transgenes. (Item 73) 73. The method of claim 72, wherein the viral vector comprises the transgene encoding a TGFβ dominant-negative receptor. (Item 74) 44. The method of claim 43, wherein the lentiviral vector is pseudotyped with a coccivirus envelope protein. (Item 75) 44. The method of claim 43, wherein the lentiviral vector is pseudotyped with a Nipah virus envelope protein. (Item 76) 76. The method of claim 75, wherein the Nipah envelope protein is engineered to bind to EpCAM, CD4, or CD8. (Item 77) 44. The method of claim 43, wherein the multispecific antibody specifically binds to CD3 and CD19, the vector is a lentiviral vector pseudotyped with a coccivirus envelope protein, and the vector comprises a polynucleotide encoding an anti-CD19 chimeric antigen receptor and a transgene encoding a TGFβ dominant-negative receptor. (Item 78) A combination therapy comprising a multispecific antibody and a vector, optionally a viral vector, for use in transducing immune cells in vivo. (Item 79) A pharmaceutical composition comprising a multispecific antibody and a vector, optionally a viral vector. (Item 80) A kit comprising: 1) a multispecific antibody; and 2) a vector, optionally a viral vector. (Item 81) A kit comprising: 1) a polynucleotide encoding a multispecific antibody; and 2) a vector, optionally a viral vector. (Item 82) a) transduction of immune cells in a subject in need thereof; and / or b) Treating a disease or disorder in a subject in need thereof 82. The kit according to item 80 or 81 for use in (Item 83) 1. A method of treating a disease or disorder in a subject in need thereof, comprising: a) administering a multispecific antibody to activate immune cells in the subject; b) administering a vector, optionally a viral vector, before, after and in conjunction with step a); A method comprising: (Item 84) 84. The method of item 83, wherein the immune cells are transduced. (Item 85) 85. The method of item 83 or 84, wherein the disease or disorder is cancer. (Item 86) 85. The method of item 83 or 84, wherein the disease or disorder is a hematological malignancy. (Item 87) 87. The method of item 86, wherein the hematological malignancy is a B-cell lymphoma. (Item 88) 86. The method of item 85, wherein the disease or disorder is treated more quickly than by administration of the multispecific antibody alone and / or the vector alone. (Item 89) 86. The method of item 85, wherein the method results in a superior treatment outcome for the disease or disorder than administration of the multispecific antibody alone and / or the vector alone. (Item 90) 86. The method of claim 85, wherein the multispecific antibody specifically binds to CD3 and CD19, the vector is a lentiviral vector pseudotyped with a coccivirus envelope protein, and the vector comprises a polynucleotide encoding an anti-CD19 chimeric antigen receptor and a transgene encoding a TGFβ dominant-negative receptor. (Item 91) 88. The method of item 87, wherein the method results in faster depletion of malignant B cells in the subject than administration of the multispecific antibody alone and / or the vector alone. (Item 92) 88. The method of item 87, wherein the method results in a lower number of residual malignant B cells and / or a lower relapse rate of the B-cell lymphoma in the subject than administration of the multispecific antibody alone and / or the vector alone.

Claims

[Claim 1] The invention described in the present specification.

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