Chimeric TCR constructs targeting BCMA and CD19 or CD20 and CD19, and uses of the same

Abstract

The present disclosure provides, in part, polypeptide dimers and polypeptides for improving cellular immunotherapy. Disclosed embodiments include bispecific polypeptide dimers (referred-to herein as chTCRs or chTCRs) that bind CD19 and BCMA, or that bind CD20 and CD19. In some embodiments, host cells expressing a bispecific chTCR that binds CD19 and BCMA are useful to treat systemic lupus erythematosus (SLE). Also provided are polynucleotides encoding the chTCRs, and vectors that comprise the polynucleotides. Also provided are host cells, such as T cells, that encode or express the chTCRs. Also provided are compositions and methods for using the same the same.

Description

[0001] CHIMERIC TCR CONSTRUCTS TARGETING BCMA AND CD19 OR CD20 AND CD19, AND USES OF THE SAME

[0002] STATEMENT OF GOVERNMENT INTEREST

[0003] This invention was made with government support under CAI 14536 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (360056-517W0-SL.xml; Size: 152,150 bytes; and Date of Creation: November 6, 2025) is herein incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] Genetically engineered T cells expressing transgenic T Cell Receptors (TCRs) or Chimeric Antigen Receptors (CARs) have proven to be relevant options to treat certain diseases. CAR T therapies have notably been successful for treating patients with refractory hematologic malignancies. CARs include an extracellular domain comprising an antibody single-chain variable fragment (scFv) or other binding domain that recognizes antigen, such as antigen expressed on the surface of target cells. CARs also include an intracellular domain comprising one or more signaling domains to activate the T cell and typically include the CD3^ cytoplasmic domain, often paired with a co-stimulatory signaling from, for example, CD28 or 4-1BB.

[0008] Despite clinical success, some limitations of CAR T cells have been observed. Namely, some CAR T therapies have been observed to induce toxicities that can be severe and potentially life-threatening such as Cytokine Release Syndrome (CRS). Antigen-independent CAR T cell activation, which has been referred-to as tonic signaling, is another observed limitation of some CARs as it fosters higher cytokine production, contributing to toxicities and driving early T cell exhaustion thus dampening long term functions and antitumor activity. Another observed limitation of CAR T cells to-date is a weak ability to sense target cells expressing low levels of antigen, which is thought to play a role in limiting clinical success by allowing escape of tumor cells with low levels of antigen. These limitations may originate from CAR-intrinsic signaling properties.

[0009] TCRs also have limitations. TCRs are MHC restricted - they recognize their antigen (peptides from intracellular proteins) presented on a given MHC molecule, thus limiting the number of patients eligible for each therapy to those that express both the antigen and the relevant MHC molecule. The antigen presentation pathway for T cell recognition involves multiple partners (MHC, TAP1 / 2, P2m) and can be altered in diseased cells (downregulation or mutations). Additionally, peripheral T cells are selected through thymic selection, a process where self-reactive, low affinity and high affinity TCRs are sequentially depleted. As a result, discovering high affinity TCRs (which have been described to provide preferred functions) specific for tumor antigens remains challenging.

[0010] BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Certain of the drawings herein show amino acid sequences. Unless specifically indictaed otherwise, an asterisk (*) indicates the end of the amino acid sequence expression product; i.e., a stop codon present in the encoding polynucleotide sequence.

[0012] Figures 1A-2A show amino acid sequences of elements of certain chimeric TCR (“chTCR”) (also referred to as “ChTCR” or “TCR / CAR”) expression constructs in accordance with the present disclosure.

[0013] Figures 2B-3H show amino acid sequences of certain chTCR expression constructs in accordance with the present disclosure.

[0014] Figure 4 relates to studies with a bispecific chTCR (“Bi-ChTCR”) targeting CD 19 and CD22, showing that bispecific chTCRs can be designed and have activity against antigenic cells. Top-left: Schematic of a bispecific chTCR construct. The CD3 proteins (labeled “a”, “8”, “y”, and “ ”) are shown in association with the chTCR at a cell membrane but are not part of the chTCR. Top-center: Knock out of endogenous TCRa and TCRP chains in T cells using base editing. Top-right: Expression of the bispecific chTCR, versus that of the indicated CAR targeting the same antigen(s). Bottom-left: Schematic of an experiment in which mice received a mixture of Nalm-6 GFP-FfLuc cells with different expression levels of CD19 and CD22. Bottom-center: Representative bioluminescence images of tumor burden in murine subjects treated with T cells expressing the indicated receptor. Bottom-right: Survival analysis of murine subjects treated with T cells expressing the indicated receptor.

[0015] Figure 5 Top: Schematic of a bispecific (anti-CD19 x anti-BCMA) chTCR expression construct of the present disclosure. Optionally, a furin cleavage sequence (R-A-K-R) immediately precedes each of the GSG-T2A and GSG-P2A sequences. Bottom-left: Schematic of a bispecific chTCR (in association with endogenous CD3 subunits) of the present disclosure, expressed on a cell surface along with a representative introduced cell surface marker (“EGFRtOPT”) of the present disclosure. In some embodiments, the anti-CD19 scFv (optionally, VL-linker-VH orientation) of the chTCR is in fusion with TRAC and the anti-BCMA scFv (optionally, VH-linker-VL orientation) is in fusion with TRBC. Bottom-right: Expression of the bispecific chTCR shown at top in primary T cells.

[0016] Figure 6 shows a schematic of an in vivo study using bispecific (anti-CD19 x anti-CD20 chTCR T cells) to treat a transgenic mouse model of induced systemic lupus erythematosus (SLE).

[0017] Figure 7 shows flow cytometry data showing binding to recombinant CD 19 (percentage of cells that are receptor-positive) by T cells transduced to express: (left) a bi-chTCR targeting CD20 (Leul6-derived scFv in VL-linker-VH orientation) and CD 19 (FMC63 -derived scFv); (middle) a bi-chTCR targeting CD20 (Leul6-derived scFv in VH-linker-VL orientation) and CD19 (FMC63 -derived scFv); and (right) a bispecific CAR targeting CD20 (Leul6-derived scFv in VL-linker-VH orientation) and CD19 (FMC63 -derived scFv).

[0018] Figure 8 shows: (left) percent of T cells that are receptor-positive; (middle) geometric mean of binding to recombinant CD 19; and (right) geometric mean of TRAC / TRBC expression by T cells transduced to express the indicated construct (same constructs as in Figure 7). These data show that Bi-ChTCRs have improved binding to recombinant CD 19 over a bispecific CAR comprising the same scFv variable domains.

[0019] Figure 9 shows: (top) response (proliferation, measured as the percentage of Cell Trace Violet (“CTV”)-low cells) of T cells transduced to express the indicated construct (same constructs as in Figures 7 and 8) against Nalm6 cells positive for expression of CD20 and expressing the indicated level of CD 19; and (bottom) IL-2 production by the T cells against the Nalm6 cells expressing wild-type or low levels of CD 19, or with CD 19 knockout. For each test condition in the bottom panel (IL-2 production), the three bars correspond, from left to right: Bi- ChTCR with both scFvs in VL-linker-VH orientation; Bi-ChTCR with both scFvs in VH-linker- VL orientation; bispecific CAR. These data show that the Bi-ChTCRs have superior response to CD 19 High and Low cell lines.

[0020] Figures 10 and 11 relate to an in vivo experiment examining antitumor efficacy of an anti-BCMA x anti-CD19 bispecific chTCR of the present disclosure. Figure 10 shows characterization of BCMA expression by Nalm6 cells prior to injection into NSG mice. Cell lines expressing low levels of antigen are used to test sensitivity of the Bi-ChTCR to BCMA. Figure 11 shows bioluminescence imaging (BLI) data for NSG mice injected with 0.5M Nalm-6 BCMA-low cells per mouse (Day 0), followed by treatment with approx. 2M CD8+ / 2M CD4+ (i.e., approx. 4 million cells at a 1 :1 CD8+:CD4+ ratio) receptor-positive T cells on Day 3, followed by imaging on Day 9 and Day 16. These data show that CD11D5.3-based Bi-ChTCR shows early tumor control in a BCMA antigen-low mouse model.

[0021] Figure 12 shows (upper left) a cartoon representation of a double-stranded DNA expression vector. An EFla promoter is disposed on a first DNA strand and is operably linked to a polynucleotide encoding a chTCR. A MNDU3 (shown as “MND” in these Figures) promoter is disposed on an opposite DNA strand and is operably linked to a polynucleotide encoding a CD3 polypeptide or CD3 fusion polypeptide. At bottom right is a cartoon showing the encoded polypeptide expression products and promoters in further detail.

[0022] Figure 13 shows (upper left) a cartoon representation of a double-stranded DNA expression vector similar to that shown in Figure 12, except that the MNDU3 promoter is operably linked to a polynucleotide encoding an EGFR tag (e.g., a truncated EGFR tag as provided herein), and (bottom right) a cartoon showing the encoded polypeptide expression products and promoters in further detail.

[0023] Figure 14 shows a cartoon representation of a double-stranded DNA expression vector. An EFla promoter is operably linked to a polynucleotide encoding a chTCR in “full scFv” format and a second promoter (selected from hPGK, CMV, MNDU3, and RPBSA), disposed on an opposite strand to the first promoter, is operably linked to a polynucleotide encoding a truncated EGFR tag (“tEGFR”).

[0024] Figure 15 shows, at left, expression of an anti-CD19 chTCR (“full” scFv format, comprising a scFv comprising the VL and VH of FMC63 disposed on TRAC) in primary CD8+ human T cells when encoded by various lentiviral expression vectors. Each point represents data from cells isolated from a different healthy T cell donor. The expression vectors are:

[0025] “Full ChTCR WT (no tag)” encodes the full chTCR under control of the EFla promoter but does not encode the tEGFR. The full chTCR expression product comprises the TRAC- containing polypeptide separate from the TRBC-containing polypeptide by a RAKR sequence and a GSG-P2A self-cleaving peptide (i.e., a P2A sequence preceded immediately by a short Gly-Ser-Gly linker).

[0026] “Full ChTCR P2A Middle tEGFR” encodes the following under control of the EFla promoter: TRBC_RAKR GSG_P2A tEGFR _RAKR _GSG_P2A FMC63 scFv_TRAC

[0027] “Full ChTCR P2A End tEGFR” encodes the following under control of the EFla promoter: TRBC RAKR _GSG_P2A FMC63 scFv_TRAC_RAKR GSG_P2A tEGFR “Full ChTCR hPGK tEGFR”, “Full ChTCR CMV tEGFR”, “Full ChTCR MND tEGFR”, and “Full ChTCR RPBSA tEGFR”, vectors encode (TRBC_RAKR GSG_P2A FMC63 scFv_TRAC) under control of the EFla promoter, and the tEGFR under conrol of a hPGK, CMV, MNDU3, or RPBSA promoter, respectively.

[0028] The bar graph at the upper right of Figure 15 shows transduction efficiency (percentage of T cells positive for binding recombinant CD 19) of the indicated expression constructs in primary CD8+ human T cells.

[0029] Figures 16A-17 provide additional data for primary CD8+ human T cells transduced with the expression vectors shown in Figure 15: (16A) amount of CD 19 binding, a measure of how many anti-CD19 chTCR molecules are being expressed; (16B) expression of TRAC / TRBC; (17) expression of tEGFR.

[0030] Figure 18 shows a cartoon representation of a double-standed DNA expression vector. An EFla promoter is disposed on a first DNA strand and is operably linked to a polynucleotide encoding a chTCR in full-scFv format (anti -CD 19 scFv comprising VL and VH of FMC63 disposed on TRAC). A MNDU3 or EFla promoter is disposed on an opposite DNA strand and is operably linked to a polynucleotide encoding a wild-type human CD3(^ polypeptide.

[0031] Figure 19 shows (left) flow cytometry data for binding recombinant CD 19 and expression of TRAC / TRBC and (right) % CD19-binding, of primary CD8+ human T cells transduced with the indicated expression vector. Certain expression vectors used two promoters: EFla and EF la, or EF 1 a and MNDU3.

[0032] “WT Full ChTCR” encodes the following under control of an EFla promoter: TRBC RAKR _GSG_P2A FMC63 scFv TRAC RAKR GSG P2A ( 1)32

[0033] “Full ChTCR P2A Middle WT CD3z” encodes the following under control of an EFla promoter: TRBC RAKR _GSG_P2A ( 1)32 RAKR _GSG_P2A FMC63 scFv_TRAC

[0034] “Full ChTCR P2A EFla WT CD3z” encodes: under control of a first EFla promoter, TRBC_RAKR GSG_P2A FMC63 scFv_TRAC, and under control of a second EFla promoter on the opposite strand, CD3 .

[0035] “Full ChTCR P2A MND WT CD3z” encodes: under control of an EFla promoter, TRBC_RAKR_GSG_P2A FMC63 scFv_TRAC, and under control of a MNDU3 promoter on the opposite strand, CD3 .

[0036] Two batches of transduced primary CD8+ human T cells were used to generate the bar graphs at the right of the figure (one batch per graph). Figure 20 shows median fluorescence intensity (MFI) readouts indicating CD19-binding, TRAC / TRBC expression, and CD3(^ expression in primary CD8+ human T cells transduced with the indicated construct (as in Figure 63).

[0037] Figure 21 shows flow cytometry data showing (left) expression of tEGFR in primary CD8+ human T cells transduced with a DNA expression construct encoding: a bispecific chTCR (anti-BCMA x anti-CD19, with scFv comprising VH and VL from Cl 1D5.3 fused to TRBC and scFv comprising VL and VH from FMC63 fused to TRAC) under control of an EFla promoter; and tEGFRopt under control of a MNDU3 promoter, and (right) BCMA-binding and TRAC / TRBC expression in tEGFR+ T cells.

[0038] DETAILED DESCRIPTION

[0039] The present disclosure provides polypeptide dimers (referred-to herein as a chimeric TCRs, chTCRs, ChTCRs or TCR / CARs) that bind BCMA and CD 19 or CD20 and CD 19. The polypeptide dimers can confer to a host cell (e.g. a T cell) target-specificity of a CAR while leveraging natural T cell signaling properties. chTCRs can be efficiently expressed at the surface of, for example, host T cells and confer to host T cells a diversified and highly sensitive signaling complex that acts with native T cell signaling properties. In some embodiments, a T cell expressing a chTCR has one or more of the following properties as compared to a T cell expressing a CAR that binds the same target(s): increased sensitivity to antigen; increased production of one or more cytokine (e.g., IFN-y, IL-2); increased killing against cells expressing the target(s); increased proliferation when in the presence of the target(s); reduced basal or nonspecific activation; increased survival of a model mammal comprising cells expressing the target(s); increased antitumor activity against tumor cells expressing the target(s); lower target EC50 for production of IL-2 and / or IFN-y; higher avidity (e.g., a greater amount of chTCR- expressing cells bound to target); and association of the chTCR with one or more CD3 proteins.

[0040] Also provided are methods of using any of the presently disclosed chTCRs, CCRs, fusion polypeptides, polynucleotides, vectors, host cells, and compositions to treat a disease or disorder characterized by expression of (human) CD 19 and / or (human) BCMA, preferably both CD 19 and BCMA, or a disease or disorder characterized by expression of (human) CD 19 and / or (human) CD20, preferably both CD 19 and CD20.

[0041] Also provided are methods of using any of the presently disclosed chTCRs, CCRs, fusion polypeptides, polynucleotides, vectors, host cells, and compositions in the preparation of a medicament to treat a disease or disorder characterized by expression of CD19 and / or BCMA, preferably both CD 19 and BCMA, or a disease or disorder characterized by expression of CD 19 and / or CD20, preferably both CD 19 and CD20.

[0042] In some embodiments, host cells (e.g., T cells) expressing a chTCR that binds CD 19 and BCMA are useful to treat an autoimmune disease, such as systemic lupus erythematosus (SLE). In some embodiments, a subject receiving therapy as provided herein does not receive lymphodepleting therapy (e.g., lymphodepleting chemotherapy) or has not received lymphodepleting therapy (e.g., lymphodepleting chemotherapy), prior to the administering of a composition (e.g., a T cell composition) as provided herein.

[0043] Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide additional definitions of certain terms to be used herein. Still more definitions are set forth throughout this disclosure.

[0044] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, is to be understood to include any integer within the recited range, unless otherwise indicated. As used herein, the term "about" means ± 20% of the indicated range, value, or structure, unless otherwise indicated. "About" includes ±15%, ±10%, and ±5%. It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the enumerated components. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination of the alternatives. As used herein, the terms "include," "have," and "comprise" are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.

[0045] "Optional" or "optionally" means that the subsequently described element, component, event, or circumstance may or may not occur, and that the description includes instances in which the element, component, event, or circumstance occurs and instances in which they do not.

[0046] In addition, it should be understood that the individual constructs, or groups of constructs, derived from the various combinations of the structures and subunits described herein, are disclosed by the present application to the same extent as if each construct or group of constructs was set forth individually. Thus, selection of particular structures or particular subunits is within the scope of the present disclosure. The term "consisting essentially of' is not equivalent to "comprising" and refers to the specified materials or steps of a claim, or to those that do not materially affect the basic characteristics of a claimed subject matter. For example, a protein domain, region, or module (e.g., a protein domain, linker, signal peptide) or a protein (which may have one or more domains, regions, or modules) "consists essentially of a particular amino acid sequence when the amino acid sequence of a domain, region, module, or protein includes extensions, deletions, mutations, or a combination thereof (e.g., amino acids at the amino- or carboxy -terminus or between domains) that, in combination, contribute to at most 20% (e.g, at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of a domain, region, module, or protein and do not substantially affect (i.e., do not reduce the activity by more than 50%, such as no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) the activity of the domain(s), region(s), module(s), or protein (e.g., the target binding affinity of a binding protein).

[0047] As used herein, "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y- carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

[0048] As used herein, "mutation" refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively. A mutation can result in several different types of change in sequence, including substitution, insertion or deletion of nucleotide(s) or amino acid(s).

[0049] A "conservative substitution" refers to amino acid substitutions that do not significantly affect or alter binding characteristics of a particular protein. Generally, conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include a substitution found in one of the following groups: Group 1 : Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3: Asparagine (Asn or N), Glutamine (Gin or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (He or I), Leucine (Leu or L), Methionine (Met or M), Valine (Vai or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp or W). Additionally or alternatively, amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Vai, Leu, and He. Other conservative substitutions groups include: sulfur- containing: Met and Cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gin; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gin; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, He, Vai, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company. Variant proteins, peptides, polypeptides, and amino acid sequences of the present disclosure can, in certain embodiments, comprise one or more conservative substitutions relative to a reference amino acid sequence.

[0050] As used herein, "protein" or "polypeptide" refers to a polymer of amino acid residues. Proteins apply to naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid and non-naturally occurring amino acid polymers.

[0051] As used herein, "fusion protein" or "fusion polypeptide" refers to a protein that, in a single chain, has at least two distinct domains and / or motifs, wherein the domains or motifs are not naturally found together (e.g., in the given arrangement, order, or number, or at all) in a protein. In certain embodiments, a fusion protein comprises at least two distinct domains and / or motifs that are not found together in a single naturally occurring peptide or polypeptide. In certain embodiments, a fusion protein comprises amino acid sequences from two or more distinct polypeptides. TCR / CARs include one or more fusion polypeptides. CCRs comprise a fusion polypeptide. A polynucleotide encoding a fusion protein may be constructed using PCR, recombinantly engineered, or the like, or such fusion proteins can be synthesized. A fusion protein may further contain other components, such as a tag, a linker, or a transduction marker. In certain embodiments, a fusion protein expressed or produced by a host cell (e.g., a T cell) locates to the cell surface, where the fusion protein can be anchored to the cell membrane. "Nucleic acid molecule" or "polynucleotide" refers to a polymeric compound including covalently linked nucleotides, which can be made up of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine ring). Purine bases include adenine, guanine, hypoxanthine, and xanthine, and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid molecules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA, either of which may be single or double-stranded. If single-stranded, the nucleic acid molecule may be the coding strand or non-coding (anti-sense strand). A nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence. Some versions of the nucleotide sequences may also include intron(s) to the extent that the intron(s) would be removed through co- or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence as the result of the redundancy or degeneracy of the genetic code, or by splicing.

[0052] In some embodiments, a polynucleotide or expression construct of the present disclosure comprises DNA. In some embodiments, a polynucleotide or expression construct of the present disclosure comprises RNA. In some embodiments, a polynucleotide or expression construct of the present disclosure comprises mRNA. In some embodiments, a polynucleotide or expression construct of the present disclsoure comprises circRNA, saRNA, or both.

[0053] A polynucleotide or expression construct can, for example, be comprised, contained, and / or delivered to a subject or a host cell in a carrier, wherein the carrier comprises a lipid, a lipid-derived delivery vehicle, such as a liposome, a solid lipid nanoparticle, an oily suspension, a submicron lipid emulsion, a lipid microbubble, an inverse lipid micelle, a cochlear liposome, a lipid microtubule, a lipid microcylinder, lipid nanoparticle (LNP), a lipopolyplex (LPP), a cationic polypeptide, a polymeric nanoparticle, or a nanoscale platform, such as a nanoemulsion.

[0054] Variants of nucleic acid molecules of this disclosure are also contemplated. Variant nucleic acid molecules are at least 70%, 75%, 80%, 85%, 90%, and are preferably 95%, 96%, 97%, 98%, 99%, or 99.9% identical a nucleic acid molecule of a defined or reference polynucleotide as described herein, or that hybridize to a polynucleotide under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68°C or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42°C. Nucleic acid molecule variants retain the capacity to encode a fusion protein or a binding domain thereof having a functionality described herein, such as specifically binding a target molecule. "Percent sequence identity" refers to a relationship between two or more sequences, as determined by comparing the sequences. Preferred methods to determine sequence identity are designed to give the best match between the sequences being compared. For example, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment). Further, non-homologous sequences may be disregarded for comparison purposes. The percent sequence identity referenced herein is calculated over the length of the reference sequence, unless indicated otherwise. Methods to determine sequence identity and similarity can be found in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using a BLAST program (e.g., BLAST 2.0, BLASTP, BLASTN, or BLASTX). The mathematical algorithm used in the BLAST programs can be found in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997. Within the context of this disclosure, it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. "Default values" mean any set of values or parameters which originally load with the software when first initialized.

[0055] The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated. Such nucleic acid could be part of a vector and / or such nucleic acid or polypeptide could be part of a composition (e.g., a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide. In some embodiments, a composition of the present disclosure can be "isolated" in the sense that it is physically separated from and not comprised within a subject to whom the composition can be, was, or is to be administered.

[0056] The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region ("leader and trailer") as well as intervening sequences (introns) between individual coding segments (exons).

[0057] A "functional variant" refers to a polypeptide or polynucleotide that is structurally similar or substantially structurally similar to a parent or reference compound of this disclosure, but differs, in some contexts slightly, in composition (e.g., one base, atom or functional group is different, added, or removed), such that the polypeptide or encoded polypeptide is capable of performing at least one function of the encoded parent polypeptide with at least 50% efficiency, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent polypeptide. In other words, a functional variant of a polypeptide or encoded polypeptide of this disclosure has "similar binding," "similar affinity" or "similar activity" when the functional variant displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide, such as an assay for measuring binding affinity (e.g., Biacore® or tetramer staining measuring an association (Ka) or a dissociation (KD) constant) or avidity; or an assay measuring TCR signaling or an activity stimulated thereby (e.g. as exemplified herein, such as measuring IFN-g production, IL-2 production, intracellular calcium flux, cellular avidity as determined by the percentage of cells in a sample that bind to antigen, proliferation, specific cytotoxicity against a target cell, NF AT expression, NFkB expression, AP-1 expression, Nur77 expression) optionally in the presence of PVR or PVR-expressing cells.))

[0058] As used herein, a "functional portion" or "functional fragment" refers to a polypeptide or polynucleotide that comprises only a domain, portion or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% activity associated with the domain, portion or fragment of the parent or reference compound, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent polypeptide, or provides a biological benefit (e.g., T cell signaling and / or activity following binding to antigen). A "functional portion" or "functional fragment" of a polypeptide or encoded polypeptide of this disclosure has "similar binding" or "similar activity" when the functional portion or fragment displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide (preferably no more than 20% or 10%, or no more than a log difference as compared to the parent or reference with regard to affinity), such as an assay for measuring binding affinity or measuring effector function (e.g., cytokine release).

[0059] As used herein, "heterologous" or "non-endogenous" or "exogenous" refers to any gene, protein, compound, nucleic acid molecule, or activity that is not native to a host cell or a subject, or any gene, protein, compound, nucleic acid molecule, or activity native to a host cell or a subject that has been altered. Heterologous, non-endogenous, or exogenous includes genes, proteins, compounds, or nucleic acid molecules that have been mutated or otherwise altered such that the structure, activity, or both is different as between the native and altered genes, proteins, compounds, or nucleic acid molecules. In certain embodiments, heterologous, non-endogenous, or exogenous genes, proteins, or nucleic acid molecules (e.g., receptors, ligands, etc.) may not be endogenous to a host cell or a subject, but instead nucleic acids encoding such genes, proteins, or nucleic acid molecules may have been added to a host cell by conjugation, transformation, transfection, electroporation, or the like, wherein the added nucleic acid molecule may integrate into a host cell genome or can exist as extra-chromosomal genetic material (e.g., as a plasmid or other self-replicating vector). It will be appreciated that in the case of a host cell that comprises a heterologous polynucleotide, the polynucleotide is "heterologous" to progeny of the host cell, whether or not the progeny were themselves manipulated to, for example, introduce the polynucleotide.

[0060] The term "homologous" or "homolog" refers to a gene, protein, compound, nucleic acid molecule, or activity found in or derived from a host cell, species, or strain. For example, a heterologous or exogenous polynucleotide or gene encoding a polypeptide may be homologous to a native polynucleotide or gene and encode a homologous polypeptide or activity, but the polynucleotide or polypeptide may have an altered structure, sequence, expression level, or any combination thereof. A non-endogenous polynucleotide or gene, as well as the encoded polypeptide or activity, may be from the same species, a different species, or a combination thereof.

[0061] As used herein, the term "endogenous" or "native" refers to a polynucleotide, gene, protein, compound, molecule, or activity that is normally present in a host cell or a subject.

[0062] The term "expression", as used herein, refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene. The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof. An expressed nucleic acid molecule is typically operably linked to an expression control sequence (e.g., a promoter).

[0063] The term "operably linked" refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). "Unlinked" means that the associated genetic elements are not closely associated with one another and the function of one does not affect the other.

[0064] As used herein, "expression vector" refers to a DNA construct containing a nucleic acid molecule that is operably linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. In the present specification, "plasmid," "expression plasmid," "virus" and "vector" are often used interchangeably.

[0065] The term "introduced" in the context of inserting a nucleic acid molecule into a cell, means "transfection", or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell wherein the nucleic acid molecule may be incorporated into the genome of a cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA). As used herein, the term "engineered," "recombinant" or "non-natural" refers to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or has been modified by introduction of an exogenous nucleic acid molecule, wherein such alterations or modifications are introduced by genetic engineering ( / .< ., human intervention). Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding proteins, fusion proteins or enzymes, or other nucleic acid molecule additions, deletions, substitutions or other functional disruption of a cell’s genetic material. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a polynucleotide, gene or operon.

[0066] As described herein, more than one heterologous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof. When two or more heterologous nucleic acid molecules are introduced into a host cell, it is understood that the two or more heterologous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into the host chromosome at a single site or multiple sites, or any combination thereof. The number of referenced heterologous nucleic acid molecules or protein activities refers to the number of encoding nucleic acid molecules or the number of protein activities, not the number of separate nucleic acid molecules introduced into a host cell.

[0067] The term "construct" refers to any polynucleotide that contains a recombinant nucleic acid molecule. A construct may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into a genome. A "vector" is a nucleic acid molecule that is capable of transporting another nucleic acid molecule. Vectors may be, for example, plasmids, cosmids, viruses, a RNA vector or a linear or circular DNA or RNA molecule that may include chromosomal, non-chromosomal, semi -synthetic or synthetic nucleic acid molecules. Vectors of the present disclosure also include transposon systems (e.g., Sleeping Beauty, see, e.g., Geurts et al., Mol. Ther. 5:108, 2003: Mates et al., Nat. Genet. 41.753, 2009). Exemplary vectors are those capable of autonomous replication (episomal vector), capable of delivering a polynucleotide to a cell genome (e.g., viral vector), or capable of expressing nucleic acid molecules to which they are linked (expression vectors).

[0068] As used herein, the term "host" refers to a cell (e.g., T cell) or microorganism targeted for genetic modification with a heterologous nucleic acid molecule to produce a polypeptide of interest (e.g, a fusion protein of the present disclosure). In certain embodiments, a host cell may optionally possess or be modified to include other genetic modifications that confer desired properties related or unrelated to, e.g., biosynthesis of the heterologous protein (e.g., inclusion of a detectable marker; deleted, altered or truncated endogenous host cell protein; expression of an antigen-binding protein).

[0069] As used herein, "enriched" or "depleted" with respect to amounts of cell types in a mixture refers to an increase in the number of the "enriched" type, a decrease in the number of the "depleted" cells, or both, in a mixture of cells resulting from one or more enriching or depleting processes or steps. Thus, depending upon the source of an original population of cells subjected to an enriching process, a mixture or composition may contain 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more (in number or count) of the "enriched" cells. Cells subjected to a depleting process can result in a mixture or composition containing 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% percent or less (in number or count) of the "depleted" cells. In certain embodiments, amounts of a certain cell type in a mixture will be enriched and amounts of a different cell type will be depleted, such as enriching for CD4+cells while depleting CD8+cells, or enriching for CD62L+cells while depleting CD62L cells, or combinations thereof.

[0070] "T cell receptor" (TCR) refers to a multi-protein complex (each component protein having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail; see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rdEd., Current Biology Publications, p. 4:33, 1997) capable of binding to an antigen peptide bound to a MHC receptor. A TCR can be found on the surface of a cell or in soluble form and generally is comprised of a heterodimer having a and P chains (also known as TCRa and TCRP, respectively), or y and 5 chains (also known as TCRy and TCR5, respectively). The extracellular portion of TCR chains (e.g., a-chain, P-chain) contain two immunoglobulin domains, a variable domain (e.g., a-chain variable domain or Va, P-chain variable domain or Vp; typically amino acids 1 to 116 based on Kabat numbering (Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5thed.) at the N-terminus, and one constant domain (e.g., a- chain constant domain or Ca, typically amino acids 117 to 259 based on Kabat, P-chain constant domain or Cp, typically amino acids 117 to 295 based on Kabat) adjacent to the cell membrane. Non-limiting examples of TCR constant domain amino acid sequences are provided herein. The variable domains contain complementary determining regions (CDRs) separated by framework regions (FRs) (see, e.g., lores et al., Proc. Nat'l Acad. Set. U.S.A. 57:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). The source of a TCR or TCR binding domain as used in the present disclosure may be from various animal species, such as a human, mouse, rat, rabbit, non-human primate, or other mammal. chTCRs of the present disclosure are structurally distinct from TCRs.

[0071] "CD3" is a multi-protein complex of six chains (see, Abbas and Lichtman, 2003;

[0072] Janeway et al., p. 172 and 178, 1999). In mammals, the complex generally comprises a CD3y chain, a CD36 chain, two CD3s chains, and a homodimer of CD3(^ chains. The CD3y, CD36, and CD3s chains are related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3y, CD36, and CD3s chains are negatively charged, which is thought to allow these chains to associate with positively charged regions of T cell receptor chains. The intracellular tails of the CD3 complex proteins contain immunoreceptor tyrosine-based activation motifs or IT AMs, which are thought to be important for T cell signaling in response to antigen binding.

[0073] CD3, as well as the protein subunits, domains, and sequences therefrom, may be from various animal species, including human, mouse, rat, or other mammals. In preferred embodiments, CD3 proteins are human CD3 proteins.

[0074] In certain embodiments, a TCR is found on the surface of T cells (also referred to as T lymphocytes) and associates with the CD3 complex. In certain embodiments, a TCR complex comprises a TCR or a functional portion thereof; a dimer comprising two CD3(^ chains, or functional portions or variants thereof; a dimer comprising a CD36 chain and a CDe chain, or functional portions or variants thereof; and a dimer comprising a CD3y chain and a CDe chain, or functional portions or variants thereof, any one or more of which may be endogenous or heterologous to the T cell. chTCRs of the present disclosure associate with the CD3 complex.

[0075] "Major histocompatibility complex molecules" (MHC molecules) refer to glycoproteins that deliver peptide antigens to a cell surface. MHC class I molecules are heterodimers consisting of a membrane spanning a chain (with three a domains) and a non-covalently associated P2 microglobulin. MHC class II molecules are composed of two transmembrane glycoproteins, a and P, both of which span the membrane. Each chain has two domains. MHC class I molecules deliver peptides originating in the cytosol to the cell surface, where a peptide:MHC complex is recognized by CD8+T cells. MHC class II molecules deliver peptides originating in the vesicular system to the cell surface, where they are recognized by CD4+T cells. An MHC molecule may be from various animal species, including human, mouse, rat, cat, dog, goat, horse, or other mammals.

[0076] "CD4" refers to an immunoglobulin co-receptor glycoprotein that can assist the TCR in binding to antigen:MHC and communicating with antigen-presenting cells (see, Campbell & Reece, Biology 909 (Benjamin Cummings, Sixth Ed., 2002); UniProtKB P01730). CD4 is found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells, and includes four immunoglobulin domains (DI to D4) that are expressed at the cell surface. During antigen recognition, CD4 is recruited, along with the TCR complex, to bind to different regions of the MHCII molecule (CD4 binds MHCII P2, while the TCR complex binds antigen:MHCII al / pi).

[0077] As used herein, the term "CD8 co-receptor" or "CD8" means the cell surface glycoprotein CD8, either as an alpha-alpha homodimer or an alpha-beta heterodimer. The CD8 co-receptor can assist in the function of cytotoxic T cells (CD8+) and functions through signaling via its cytoplasmic tyrosine phosphorylation pathway (Gao and Jakobsen, Immunol. Today 27:630-636, 2000; Cole and Gao, Cell. Mol. Immunol. 7:81-88, 2004). In humans, there are five (5) different CD8 beta chains (see UniProtKB identifier Pl 0966) and a single CD8 alpha chain (see UniProtKB identifier P01732).

[0078] The term "variable region" or "variable domain" refers to the domain an antibody heavy or light chain (or, for TCRs, of a TCR of a TCR a-chain or P-chain (or y-chain and 8-chain for y6 TCRs)), that is involved in binding to antigen (i.e., contains amino acids and / or other structures that contact antigen and result in binding). The variable domains of cognate chains generally have similar structures, with each domain comprising four generally conserved framework regions (FRs) and three CDRs. In both TCRs and antibodies, framework regions separate CDRs and CDRs are situated between framework regions (i.e., in primary structure).

[0079] The terms "complementarity determining region," and "CDR," are synonymous with "hypervariable region" or "HVR," and refer to sequences of amino acids within TCR or antibody variable regions, which, in general, confer antigen specificity and / or binding affinity and are separated from one another in primary structure by framework sequence. In some cases, framework amino acids can also contribute to binding, e.g., may also contact the antigen or antigen-containing molecule. In general, there are three CDRs in each variable region (e.g, three CDRs in each of the antibody heavy chain and light chain variable regions). Variable domain sequences can be aligned to a numbering scheme (e.g, Kabat, EU, International Immunogenetics Information System (IMGT) and Aho), which can allow equivalent residue positions to be annotated and for different molecules to be compared using Antigen receptor Numbering And Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298- 300).

[0080] "Treat" or "treatment" or "ameliorate" refers to medical management of a disease, disorder, or condition of a human subject, unless otherwise indicated. A subject may also be a non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat. In general, an appropriate dose or treatment regimen comprising a host cell expressing a fusion protein of the present disclosure, and optionally an adjuvant, is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit. Therapeutic or prophylactic / preventive benefit includes improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease; stabilization of disease state; delay of disease progression; remission; survival; prolonged survival; or any combination thereof. In some embodiments, a benefit of a cellular immunotherapy of this disclosure can further include a reduction (e.g., in number or severity) or absence of a cytokine-related toxicity, such as a cytokine release syndrome.

[0081] A "therapeutically effective amount" or "effective amount" of a composition (fusion protein, host cell expressing a fusion protein, polynucleotide, vector, polypeptide dimer, or the like) of this disclosure, refers to an amount of the composition sufficient to result in a therapeutic effect, including improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease, stabilization of disease state; delay of disease progression; remission; survival; or prolonged survival in a statistically significant manner.

[0082] When referring to an individual active ingredient, administered alone, a therapeutically effective amount refers to the effects of that ingredient alone. When referring to a combination, a therapeutically effective amount refers to the combined amounts of active ingredients or combined adjunctive active ingredient with a cell expressing an active ingredient that results in a therapeutic effect, whether administered serially or simultaneously. A combination may also be a cell expressing more than one active ingredient.

[0083] The term "pharmaceutically acceptable excipient or carrier" or "physiologically acceptable excipient or carrier" refer to biologically compatible vehicles, e.g, physiological saline, which are described in greater detail herein, that are suitable for administration to a human or other non-human mammalian subject and generally recognized as safe or not causing a serious adverse event.

[0084] As used herein, "statistically significant" refers to a p-value of 0.050 or less when calculated using the Student’s t-test and indicates that it is unlikely that a particular event or result being measured has arisen by chance.

[0085] As used herein, the term "adoptive immune therapy" or "adoptive immunotherapy" refers to administration of naturally occurring or genetically engineered, disease-antigen-specific immune cells (e.g., T cells). Adoptive cellular immunotherapy may be autologous (immune cells are from the recipient), allogeneic (immune cells are from a donor of the same species) or syngeneic (immune cells are from a donor genetically identical to the recipient). chTCRs

[0086] Disclosed embodiments include hybrid receptor polypeptides (also referred-to herein as chTCRs) that bind BCMA and CD 19, or CD20 and CD 19, and confer to a host cell (e.g. a T cell) target-specificity of a CAR while leveraging natural T cell signaling properties. chTCRs can be efficiently expressed at the surface of host cells and confer to host cells a diversified and highly sensitive signaling complex that acts with native T cell signaling properties. In some embodiments, a chTCR comprises (1) a first polypeptide comprising a first TCR constant domain and (2) a second polypeptide comprising a second TCR constant domain, wherein the first TCR constant domain and the second TCR constant domain associate to form a dimer. The association can comprise one or more native interaction between cognate TCR constant domains (e.g. a native disulfide bond), one or more engineered interaction between the TCR constant domains (e.g. one or more disulfide bonds introduced by protein engineering), or both. The first and second polypeptides each comprise a scFv (one specific for BCMA and comprising the VH and VL amino acid sequences shown in Figures 1A, the other specific for CD 19 and comprising the VH and VL amino acid sequences shown in Figure IB). In some embodiments, a chTCR comprises the VH, VL, and linker amino acids shown in Figure 1 A, and the VH, VL, and linker amino acids shown in Figure IB.

[0087] In other embodiments, the first and second polypeptides each comprise a scFv (one specific for CD20 and the other specific for CD 19).

[0088] In some embodiments, the first TCR constant domain comprises a human TCR alphachain constant domain (Ca) or an engineered variant thereof and the second TCR constant domain comprises a human TCR beta-chain constant domain (CP) or an engineered variant thereof. In other embodiments, the first TCR constant domain comprises a human CP or an engineered variant thereof and the second TCR constant domain comprises a human Ca or an engineered variant thereof.

[0089] It will be understood that the terms “TRAC” and “TRBC”, when referring to a chTCR, may be used interchangeably with the terms TCR Ca and TCR CP, respectively, and include embodiments comprising the variant sequences and modifications described herein. A TRAC or TRBC may, but need not necessarily, comprise the native amino acid sequence encoded by a(n e.g. human) TRAC or TRBC, respectively. When describing an endogenous gene locus encoding a TCR alpha chain constant domain or beta chain constant domain, the term TRAC or TRBC, respectively, may be used.

[0090] In certain preferred embodiments, TCR constant domain sequences are human or comprise engineered variants of human sequences. TCR constant domains may be engineered to improve pairing, expression, stability, or any combination of these. See, e.g., Cohen et al., Cancer Res, 2007; Kuball et al., Blood 2007; and Haga-Friedman et al., Journal of Immunology 2009. Examples of engineering in TCR Ca and CP are illustrated in Figure 7; these can include mutation of a native amino acid to a cysteine so that a disulfide bond forms between the introduced cysteine of one TCR constant domain and a native cysteine of the other TCR constant domain. Such mutations can include T48C in Ca, T57C in CP, or both. Mutations to improve stability can include a mutation in the Ca transmembrane domain from the sequence LSVIGF (SEQ ID NO.:62) to the sequence LLVIVL (SEQ ID NO.:63) (“L-V-L” mutation; see Haga- Friedman et al., J Immunol 755:5538-5546 (2012), the TCR mutations and mutant TCR constant domain sequences of which are incorporated herein by reference). Also contemplated are embodiments wherein cognate TCR constant domains comprise mutations so that, for example, one TCR constant domain (e.g., one of Ca and CP) comprises an introduced “cavity” (e.g., obtainable by replacing one or more native amino acid with one or more amino acids having smaller side chains) and the other (e.g., the other of Ca and CP) comprises a compensatory “protuberance” (e.g., obtainable by replacing one or more native amino acid with one or more amino acids having larger side chains), similar to a “knob-into-hole” configuration used to promote preferential pairing of antibody heavy chains. Also contemplated are embodiments wherein TCR constant domain amino acids are mutated to introduce charge properties that favor pairing of the mutated constant domains. Examples of mutations that may be made in Ca and CP to promote specific pairing by a knobs-into-holes-type mechanism or by a charge-pairing mechanism are provided in Voss et al., J. Immunol 750(1 ): 391 -401 (2008) doi.org / 10.4049 / jimmunol.180.1.391; see also US Patent No. 9,062,127. The TCR constant domain mutations, mutated TCR constant domains, and methods used to identify sites for mutation, described in these documents, are incorporated herein by reference.

[0091] An example of a TCR Ca amino acid sequence is provided in UniProt KB P01848 (human TRAC): IQNPDPAVYQ LRDSKSSDKS VCLFTDFDSQ TNVSQSKDSD VYITDKTVLD MRSMDFKSNS AVAWSNKSDF ACANAFNNSI IPEDTFFPSP ESSCDVKLVE KSFETDTNLN FQNLSVIGFR ILLLKVAGFN LLMTLRLWSS (SEQ ID NO.:56)

[0092] An example of a TCR Ca amino acid sequence engineered to include threonine-to- cysteine and LVL mutations as described herein is provided in SEQ ID NO.: 57: IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSN SAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLR ILLLKVAGFNLLMTLRLWS S .

[0093] Two human TCR CP isoforms are TRBC1 and TRBC2. An example of a TRBC1 amino acid sequence is provided in UniProt KB P01850: DLNKVFPPEV AVFEPSEAEI SHTQKATLVC LATGFFPDHV ELSWWVNGKE VHSGVSTDPQ PLKEQPALND SRYCLSSRLR VS ATFWQNPR NHFRCQVQF Y GLSENDEWTQ DRAKPVTQIV SAEAWGRADC GFTSVSYQQG VLSATILYEI LLGKATLYAV LVSALVLMAM VKRKDF (SEQ ID NO.:58).

[0094] An example of a TRBC1 amino acid sequence engineered to include a serine-to-cysteine mutation is provided in SEQ ID NO.: 59: DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDP QPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVT QIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKD F.

[0095] An example of a TRBC2 amino acid sequence is provided in UniProt KB A0A5B9: DLKNVFPPKV AVFEPSEAEI SHTQKATLVC LATGFYPDHV ELSWWVNGKE VHSGVSTDPQ PLKEQPALND SRYCLSSRLR VS ATFWQNPR NHFRCQVQF Y GLSENDEWTQ DRAKPVTQIV SAEAWGRADC GFTSESYQQG VLSATILYEI LLGKATLYAV LVSALVLMAM VKRKDSRG (SEQ ID NO.: 60).

[0096] An example of a TRBC2 amino acid sequence engineered to include a serine-to-cysteine mutation is provided in SEQ ID NO : 61 : DLKNVFPPKV AVFEPSEAEI SHTQKATLVC LATGFYPDHV ELSWWVNGKE VHSGVCTDPQ PLKEQPALND SRYCLSSRLR VS ATFWQNPR NHFRCQVQF Y GLSENDEWTQ DRAKPVTQIV SAEAWGRADC GFTSESYQQG VLSATILYEI LLGKATLYAV LVSALVLMAM VKRKDSRG.

[0097] In any of the presently disclosed embodiments, a chTCR can comprise a TCR Ca having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs.: 56-57, and a TCR CP having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs.:58-61. In certain embodiments, a chTCR comprises a TCR Ca and a TCR CP having at least least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to, or comprising or consisting of, the amino acid sequences set forth in SEQ ID NOs.: (i) 56 and 58, respectively; (ii) 56 and 59, respectively; (iii) 56 and 60, respectively; (iv) 56 and 61, respectively; (v) 57 and 58, respectively; (vi) 57 and 59, respectively; (vii) 57 and 60, respectively; or (viii) 57 and 61, respectively.

[0098] In preferred embodiments, a variant TCR Ca or CP maintains the same or substantially the same length and / or number of amino acids as compared to a native TCR Ca or CP, respectively, such that, in certain embodiments, the variation does not comprise a truncation in the length thereof as compared to a native TCR Ca or CP, respectively. In certain embodiments, a variant of a TCR CP (TRBC1) maintains the intracellular sequence VKRKDF (SEQ ID NO.:64). In certain embodiments, a variant of a TCR CP (TRBC1) maintains the intracellular sequence MAMVKRKDSRG (SEQ ID NO. :65). Variant TCR constant domains of the present disclosure are capable of associating with a cognate TCR constant domain and with one or more CD3 proteins. In other words, in certain embodiments, chTCRs can be assimilated into a TCR complex on a host (e.g. T) cell surface that comprises the chTCR and CD3 proteins. In particular, chTCRs of the present disclosure, including those that comprise variant TCR constant domains, are capable of producing a TCR-CD3 complex signal in a host (e.g. T) cell when the chTCR expressed by the host cell binds to its target(s).

[0099] In some embodiments, the first polypeptide and / or the second polypeptide (preferably both) of a chTCR comprises an intracellular portion that consists essentially of or that consists of the intracellular portion of the respective TCR constant domain. In some embodiments, the first polypeptide and / or the second polypeptide (preferably both) does not comprise an intracellular signaling component (e.g. effector domain) from a CD3 protein, such as CD3(^. In some embodiments, the first polypeptide and / or the second polypeptide (preferably both) does not comprise an intracellular costimulatory domain from a costimulatory protein, such as CD28, 4- 1BB, ICOS, CD27, 0X40, DAP 10, or any combination thereof.

[0100] In some embodiments, the first polypeptide and / or the second polypeptide (preferably both) of a chTCR does not comprise an immunoglobulin CH2 domain and / or an immunoglobulin CH3 domain and / or an immunoglobulin light chain constant domain. In some embodiments, the first polypeptide and / or the second polypeptide (preferably both) of a chTCR does not comprise an immunoglobulin CH2 domain and / or an immunoglobulin CH3 domain and / or an immunoglobulin light chain constant domain disposed C-terminal to the TCR constant domain. In some embodiments, the polypeptide dimer does not comprise an immunoglobulin CH2-CH3 or an immunoglobulin CH2-CH3:CH2-CH3 dimer.

[0101] A polynucleotide can be codon-optimized for expression in a host cell. A polynucleotide can be comprised in a vector, such as, for example, a viral vector, such as a lentiviral vector or a retroviral vector. A polynucleotide or vector can include one or more additional features to facilitate desired expression of the encoded polypeptide(s), such as one or more promoter, one or more sequence encoding a signal peptide (also known as a leader peptide or leader sequence or transit peptide), one or more sequence encoding a furin cleavage sequence, one or more sequence encoding a self-cleaving peptide, or any combination thereof.

[0102] Non-limiting examples of promoters include an EFla promoter (SEQ ID NO.:7) and a MNDu3 promoter (SEQ ID NO.: 8).

[0103] Signal peptides target newly synthesized polypeptides to their appropriate location inside or outside the cell. A signal peptide may be removed, at least in part, from the polypeptide during or once localization or secretion is completed. Polypeptides that have a signal peptide are referred to herein as a "pre-protein" and polypeptides having their signal peptide removed are referred to herein as "mature" proteins or polypeptides. Signal peptides can be at the N-terminal or C-terminal end of an encoded polypeptide. Non-limiting examples of signal peptides include: the signal peptide MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO : 126; from GM-CSF); the signal peptide MALPVTALLLPLALLLHAARP (SEQ ID NO : 127; from CD8a); the signal peptide MRPRLWLLLAAQLTVLHGNSV (SEQ ID NO : 128; from CD8P); and the signal peptide METDTLLLWVLLLWVPGSTG (SEQ ID NO.: 150, from murine IgG, kappa light chain). It will be appreciated that any suitable naturally occurring or engineered signal peptide can be employed. In some contexts, a signal peptide that is native to the encoded polypeptide or ectodomain of a polypeptide is usedCertain signal peptides and characteristics of these are decribed in Owji et al., European Journal of Cell Biology 97(6):422-441 (2018), and in Ling et al. Front. Immunol. (2020) doi.org / 10.3389 / fimmu.2020.604318; the signal peptides of which are incorporated herein by reference. A furin cleavage sequence (also referred-to as a furin recognition site) can have a minimal cleavage site of R-X-X-R (SEQ ID NO.: 129). In some embodiments, a furin cleavage sequence has a minimal cleavage site of R-X-K / R-R (SEQ ID NO.: 130). In some embodiments, a furin cleavage sequence has a minimal cleavage site of RAKR (SEQ ID NO. : 131) or RARR (SEQ ID NO. : 132). In other embodiments, a polypeptide of the present disclosure does not comprise, or a polynucleotide of the present disclosure does not encode, a protease cleavage sequence, optionally a furin cleavage sequence, such as between a first polypeptide and a second polypeptide of a chTCR.

[0104] A nucleotide sequence encoding a self-cleaving peptide can be disposed between sequences encoding two polypeptides of interest. Self-cleavage of the peptide can separate a single-chain polypeptide into two polypeptides of interest. For example, expression of a TCR CP-containing polypeptide and a TCR Ca-containing polypeptide of a chTCR of the present disclosure can be coordinated by encoding both polypeptides as part of a fusion amino acid sequence that separates (such as by action of a self-cleaving peptide and optionally a furin cleavage sequence) following translation, permitting expression of the TCR CP-containing polypeptide and the TCR Ca-containing polypeptide as separate molecules at the cell surface. Non-limiting examples of self-cleaving peptides include: a porcine teschovirus-1 2A (P2A) selfcleaving peptide with N-terminal G-S-G linker (GSGATNFSLLKQAGDVEENPGP; SEQ ID NO.: 133); a Thoseaasigna virus 2A (T2A) self-cleaving peptide (LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO : 134); an Equine rhinitis A virus (ERAV) 2A (E2A) self-cleaving peptide (QCTNYALLKLAGDVESNPGP; SEQ ID NO : 135); and a Foot-and-Mouth disease virus 2A (F2A) self-cleaving peptide with N-terminal G-S-G linker (GSGVKQTLNFDLLKLAGDVESNPGP; SEQ ID NO : 136).

[0105] In some embodiments, a polynucleotide comprises nucleotide sequences encoding a first and a second TCR constant domain-containing polypeptide, and further comprises a nucleotide sequence encoding a self-cleaving peptide, a nucleotide sequence encoding a furin or other protease cleavage site, or both.

[0106] Certain embodiments of a chTCR or CCR further include one or more tag peptide, typically located in an extracellular component of the chTCR or CCR. An example of a tag peptide is a Flag tag (DYKDDDDK; SEQ ID NO. : 137) or a variant thereof e.g. DYKDEY; SEQ ID NO. : 138). Other non-limiting examples of tag peptides include a Strep tag (which refers the original Strep® tag, Strep® tag II, or any variant thereof; see, e.g., U.S. Patent No. 7,981,632, which Strep tags are incorporated herein by reference), His tag, Xpress tag, Avi tag, Calmodulin tag, Polyglutamate tag, an HA tag (YPYDVDPDYA; SEQ ID NO. : 139), Myc tag, Nus tag, S tag, SBP tag, Softag 1, Softag 3, V5 tag, CREB-binding protein (CBP), glutathione S-transferase (GST), maltose binding protein (MBP), green fluorescent protein (GFP), Thioredoxin tag, or any combination thereof. See, e.g., PCT Publication No. WO 2015 / 095895, which tag peptides and the amino acid sequences thereof are incorporated by reference herein. A combination of a Flag tag variant and a HA tag comprises the amino acid sequence DYKDEYYPYDVDPDYA (SEQ ID NO. : 140). Tag peptides can be useful for identifying, sorting, enriching, tracking, or isolating polypeptides that comprise the tag peptide(s), and cells that express the same. For example, antibodies or other proteins (e.g. Streptactin) having specificity for a tag peptide can be used for these purposes; such antibodies (or binding fragments thereof) or other proteins may be soluble or can be conjugated to beads, a cell culture plate, agarose, or any other solid surface matrix. Cells can be sorted, enriched, or isolated using an affinity column. In some contexts, an antibody having specificity for a tag peptide can be used to induce cell death (e.g. by ADCC or CDC or ADCP) of a cell expressing a tag peptide.

[0107] A polynucleotide (e.g., encoding a chTCR, a polypeptide of a chTCR, or any combination thereof) can be comprised in a vector, such as an expression vector comprising a lentiviral vector or a retroviral vector.

[0108] Also provided are fusion polypeptides that comprise a binding-domain-containing chTCR polypeptide of the present disclosure. Any of the presently disclosed first or second chTCR polypeptides may be provided as an isolated polypeptide, provided that the polypeptide comprises a binding domain, and not accompanied by a cognate chTCR polypeptide. Polynucleotides and vectors that encode the fusion polypeptides are also provided.

[0109] Also provided are host cells that express a presently disclosed chTCR. Also provided are host cells that comprise a polynucleotide or vector encoding a a presently disclosed chTCR. In certain embodiments, a host cell comprises a hematopoietic progenitor cell, an induced pluripotent stem cell, a hematopoeitic stem cell, or an immune system cell, such as a human immune system cell. In certain embodiments, an immune system cell comprises a T cell, a NK- T cell, or a macrophage. In certain embodiments, a T cell comprises a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, an ap+ T cell, a 76+ T cell, or any combination thereof. In certain embodiments, a T cell comprises a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof.

[0110] In some embodiments, a host cell comprises a chromosomal knockout of TIGIT, of a TCR locus e.g. TRAC, TRBC), of a CD8 locus, of a CD4 locus, of aPD-1 locus, of a LAGS locus, of a TIM3 locus, of an HLA locus e.g. a gene that encodes an al macroglobulin, an a2 macroglobulin, an a3 macroglobulin, a pi microglobulin, or a P2 microglobulin), of a TGFflRl locus, of a TGF / 3R2 locus, of a LAT locus, of an A2AR locus, of a Fas locus, of a FasL locus, of a B7-H3 locus, of a B7-H4 locus, of an IDO locus, of a VISTA locus, of a SIGLEC7 locus, of a SIGLEC9 locus, of a CBLB locus, of a RASA2 locus, of a UBASH3A locus, of a CISH locus, or of any combination thereof. In some embodiments, a host cell expresses a chTCR of the present disclosure and comprises a chromosomal knockout of the target(s) bound by the chTCR.

[0111] In certain embodiments, a host cell expressing or encoding a chTCR of the present disclosure is a T cell and comprises (1) a chromosomal knockout of the target(s) bound by the chTCR and (2) a chromosomal knockout of a CD8 locus and / or of a CD4 locus.

[0112] In certain embodiments, a host cell expressing or encoding a chTCR of the present disclosure is a T cell and comprises (1) a chromosomal knockout of the target(s) bound by the chTCR and (2) a chromosomal knockout of a TRAC locus and / or of a TRBC locus.

[0113] In certain embodiments, a host cell expressing or encoding a chTCR of the present disclosure is a T cell and comprises (1) a chromosomal knockout of the target(s) bound by the chTCR and (2) a chromosomal knockout of a TRAC locus and a chromosomal knockout of a TRBC locus. In some embodiments, a polynucleotide encoding the chTCR is not comprised in an endogenous TRAC locus or an endogenous TRBC locus of the host cell. In some embodiments, a chromosomal gene knockout comprises a point mutation. In some embodiments, a chromosomal gene knockout was introduced using base editing. In some embodiments, a chromosomal gene knockout comprises an introduced stop codon, an introduced splice site, or a disruption of an endogenous splice site. Non-limiting examples of sgRNA sequences targeting chromosomal TRAC and TRBC sequences are provided herein. Non-limiting examples of sgRNA sequences targeting chromosomal TIGIT sequences are provided herein.

[0114] Compositions that comprise the host cells (including any combination thereof), polynucleotides, chTCRs, or vectors, and optionally a pharmaceutically acceptable carrier, excipient, or diluent, are also provided. Also provided are methods of making a host cell, wherein the methods comprise introducing a polynucleotide or vector encoding a chTCR of the present disclosure. Also provided are methods of using any of the presently disclosed chTCRs , polypeptides, polynucleotides, vectors, host cells, and compositions to treat (preferably, in a human), or to prepare a medicament for the treatment of, a disease or disorder characterized by expression of CD 19 and / or BCMA, or CD 19 and / or CD20.

[0115] An extracellular component and an intracellular component of a polypeptide of the present disclosure are connected by a transmembrane domain. In some contexts, a "transmembrane domain" is a portion of a transmembrane protein that can insert into or span a cell membrane. Transmembrane domains have a three-dimensional structure that is thermodynamically stable in a cell membrane and generally range in length from about 15 amino acids to about 30 amino acids. The structure of a transmembrane domain may comprise an alpha helix, a beta barrel, a beta sheet, a beta helix, or any combination thereof. In certain embodiments, the transmembrane domain of a target-binding protein comprises or is derived from a known transmembrane protein (e.g., a CD4 transmembrane domain, a CD8 transmembrane domain, a CD27 transmembrane domain, a CD28 transmembrane domain, or any combination thereof), and can be a functional portion or variant thereof; / .< ., that retains or substantially retains a three-dimensional structure that is thermodynamically stable in a cell membrane and generally having a length from about 15 amino acids to about 30 amino acids. Preferably, chTCRs comprise (e.g. retain) the transmembrane domains of their respective TCR constant domains.

[0116] An intracellular component of a CCR can comprise a costimulatory domain or a functional portion or variant thereof.

[0117] In certain embodiments, the intracellular component of a CCR comprises a costimulatory domain or a functional portion thereof selected from CD27, CD28, 4-1BB (CD137), 0X40 (CD134), CD2, CD5, ICAM-1 (CD54), LFA-1 (CD1 la / CD18), ICOS (CD278), GITR, CD30, CD40, BAFF-R, HVEM, LIGHT, MKG2C, SLAMF7, NKp80, CD160, B7-H3, a ligand that specifically binds with CD83, or a functional variant thereof, or any combination thereof. In certain embodiments, the intracellular component comprises a CD28 costimulatory domain or a functional portion or variant thereof (which may optionally include a LL- GG mutation at positions 186-187 of the native CD28 protein (see Nguyen et al., Blood 102 A32Q, 2003)), a 4- 1BB costimulatory domain or a functional portion or variant thereof, or both.

[0118] In certain embodiments, one or more of an extracellular component, a binding domain, a linker, a transmembrane domain, an intracellular component, or a costimulatory domain or functional portion or variant thereof, of a target-binding protein can (or a fusion protein can) further comprise one or more junction amino acids. "Junction amino acids" or "junction amino acid residues" refer to one or more (e.g., about 2-20) amino acid residues between two adjacent domains, motifs, regions, modules, or fragments of a protein, such as between a binding domain and an adjacent linker, between a transmembrane domain and an adjacent extracellular or intracellular domain, or on one or both ends of a linker that links two domains, motifs, regions, modules, or fragments (e.g., between a linker and an adjacent binding domain or between a linker and an adjacent hinge). Junction amino acids may result from the construct design of a fusion protein (e.g., amino acid residues resulting from the use of a restriction enzyme site or self-cleaving peptide sequences during the construction of a polynucleotide encoding a fusion protein). For example, a transmembrane domain of a fusion protein may have one or more junction amino acids at the amino-terminal end, carboxy-terminal end, or both.

[0119] Protein tags are unique peptide sequences that are affixed or genetically fused to, or are a part of, a protein of interest and can be recognized or bound by, for example, a heterologous or non-endogenous cognate binding molecule or a substrate (e.g., receptor, ligand, antibody, carbohydrate, or metal matrix) or a fusion protein of this disclosure. Protein tags can be useful for detecting, identifying, isolating, tracking, purifying, enriching for, targeting, or biologically or chemically modifying tagged proteins of interest, particularly when a tagged protein is part of a heterogeneous population of cell proteins or cells (e.g., a biological sample like peripheral blood). In certain embodiments, a protein tag of a fusion protein or antigen-binding protein of this disclosure comprises a Myc tag, His tag, Flag tag, Xpress tag, Avi tag, Calmodulin tag, Polyglutamate tag, HA tag, Nus tag, S tag, X tag, SBP tag, Softag, V5 tag, CBP, GST, MBP, GFP, Thioredoxin tag, Strep tags (e.g., Strep-Tag; Strep-Tag II; and variants thereof, including those disclosed in, for example, Schmidt and Skerra, Nature Protocols, 2: 1528-1535 (2007), U.S. Patent No. 7,981,632; and PCT Publication No. WO 2015 / 067768, the strep-tag peptides, step-tag-peptide-containing polypeptides, and sequences of the same, are incorporated herein by reference), or any combination thereof.

[0120] Methods for making fusion proteins are described, for example, in U.S. Patent No. 6,410,319; U.S. Patent No. 7,446,191; U.S. Patent Publication No. 2010 / 065818; U.S. Patent No. 8,822,647; PCT Publication No. WO 2014 / 031687; U.S. Patent No. 7,514,537; Brentjens et al., 2007, Clin. Cancer Res. 13:5426, and Walseng et al., Scientific Reports 7: 10713, 2017, the techniques of which are herein incorporated by reference.

[0121] Methods useful for isolating and purifying recombinantly produced soluble fusion proteins and / or target-binding proteins, by way of example, may include obtaining supernatants from suitable host cell / vector systems that secrete the recombinant soluble fusion protein into culture media and then concentrating the media using a commercially available filter. Following concentration, the concentrate may be applied to a single suitable purification matrix or to a series of suitable matrices, such as an affinity matrix or an ion exchange resin. One or more reverse phase HPLC steps may be employed to further purify a recombinant polypeptide. These purification methods may also be employed when isolating an immunogen from its natural environment. Methods for large scale production of one or more of the isolated / recombinant soluble fusion protein described herein include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. Purification of the soluble fusion protein may be performed according to methods described herein and known in the art and that comport with laws and guidelines of domestic and foreign regulatory agencies. chTCRs and CCRs as described herein may be functionally characterized according to any of a large number of art-accepted methodologies for assaying host cell activity. For example, in the case of a host T cell, target-binding proteins can be functionally characterized by determination of T cell binding, activation or induction, as well as determination of T cell responses that are target (e.g., antigen)-specific. Examples include determination of T cell proliferation, T cell cytokine release, target-specific T cell stimulation, MHC-restricted T cell stimulation, CTL activity (e.g., by detecting51Cr or Europium release from pre-loaded target cells), changes in T cell phenotypic marker expression, and other measures of T-cell functions. Procedures for performing these and similar assays are found, for example, in Lefkovits (Immunology Methods Manual: The Comprehensive Sourcebook of Techniques, 1998). See, also, Current Protocols in Immunology, Weir, Handbook of Experimental Immunology, Blackwell Scientific, Boston, MA (1986); Mishell and Shigii (eds.) Selected Methods in Cellular Immunology, Freeman Publishing, San Francisco, CA (1979); Green and Reed, Science 281 : 1309 (1998) and references cited therein.

[0122] Levels of cytokines may be determined according to methods described herein and practiced in the art, including for example, ELISA, ELISPOT, intracellular cytokine staining, and flow cytometry and combinations thereof (e.g., intracellular cytokine staining and flow cytometry). Immune cell proliferation and clonal expansion resulting from an target-specific elicitation or stimulation of an immune response may be determined by isolating lymphocytes, such as circulating lymphocytes in samples of peripheral blood cells or cells from lymph nodes, stimulating the cells with antigen, and measuring cytokine production, cell proliferation and / or cell viability, such as by incorporation of tritiated thymidine or non-radioactive assays, such as MTT assays and the like. The effect of an immunogen described herein on the balance between a Thl immune response and a Th2 immune response may be examined, for example, by determining levels of Thl cytokines, such as IFN-y, IL-12, IL-2, and TNF-P, and Type 2 cytokines, such as IL-4, IL-5, IL-9, IL-10, and IL-13.

[0123] Polynucleotides., Vectors, and Host Cells

[0124] In certain aspects, nucleic acid molecules (also referred-to as polynucleotides) are provided that encode any one or more of the polypeptide dimers (chTCRs), or polypeptides described herein, or any combination thereof. In some embodiments, a polynucleotide comprises sequences encoding, in 5’ to 3’ direction: [chTCR CP-containing polypeptide - chTCR Ca- containing polypeptide]; or [chTCR Ca-containing polypeptide - chTCR CP-containing polypeptide]. It will be appreciated that polynucleotide sequences encoding the polypeptides can be separated by polynucleotides encoding: a furin or other protease cleavage site, a self-cleaving peptide, or both.

[0125] A polynucleotide encoding a desired fusion protein or polypeptide(s) of this disclosure can be inserted into an appropriate vector (e.g., viral vector or non-viral plasmid vector) for introduction into a host cell of interest (e.g., an immune cell, such as a T cell). A polynucleotide can further encode additional features, as described herein.

[0126] Exemplary markers (e.g., for transduction of a cell with a polynucleotide as provided herein) include green fluorescent protein, an extracellular domain of human CD2, a truncated human EGFR (huEGFRt, (see Wang et al., Blood 118 1255, 2011), a truncated human CD19 (huCD19t); a truncated human CD34 (huCD34t); or a truncated human NGFR (huNGFRt). In certain embodiments, an encoded marker comprises EGFRt, CD19t, CD34t, or NGFRt. A non- limiting example of an EGFRt amino acid sequence is provided in Figure 2A. In some embodiments, a cell surface marker may function as a safety switch for eliminating a host cell expressing a cell surface marker. In some embodiments, a polynucleotide or vector encoding a chTCR of the present disclosure further encodes, or a host cell expressing a chTCR of the present disclosure further expresses, EGFRtopt, as shown in Figure 2A. In some embodiments, a polynucleotide or vector encoding a chTCR of the present disclosure further encodes, or a host cell expressing a chTCR of the present disclosure further expresses a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 166 or SEQ ID NO:226. In some embodiments, a polynucleotide encoding SEQ ID NO:226 comprises the polynucleotide sequence shown in SEQ ID NO:225.

[0127] In any of presently disclosed embodiments, a protein-encoding polynucleotide can further comprise a polynucleotide that encodes a marker and a polynucleotide that encodes a selfcleaving polypeptide, wherein the polynucleotide encoding the self-cleaving polypeptide is located between the polynucleotide encoding the protein and the polynucleotide encoding the marker. When the protein-encoding polynucleotide, marker-encoding polynucleotide, and selfcleaving polypeptide are expressed by a host cell, the (fusion or antigen-binding) protein and the marker will be present on the host cell surface as separate molecules. In certain embodiments, a self-cleaving polypeptide comprises a 2A peptide from porcine teschovirus-1 (P2A, Thoseaasigna virus (T2A, equine rhinitis A virus (E2A), or foot-and-mouth disease virus (F2A)). Exemplary nucleic acid and amino acid sequences of 2A peptides are set forth in, for example, Kim et al. (PLOS One 6:el8556, 2011, which 2A nucleic acid and amino acid sequences are incorporated herein by reference in their entirety).

[0128] In any of the presently disclosed embodiments, a self-cleaving polypeptide encoded by a polynucleotide of this disclosure comprises a P2A, a T2A, an E2A, or a F2A. A self-cleaving peptide can comprise a short linker sequence (e.g., G-S-G) disposed at the N-terminal end thereof.

[0129] In any of the embodiments described herein, a polynucleotide of the present disclosure may be codon-optimized for expression in a host cell (see, e.g, Scholten et al., Clin. Immunol. 779:135-145 (2006). Codon optimization can be performed using known techniques and tools, e.g., using the GenScript® OptimumGene™ tool, or the GeneArt™ / GeneOptimizer™ tools. Codon-optimized sequences include sequences that are partially codon-optimized (i.e., one or more of the codons is optimized for expression in the host cell) and those that are fully codon-optimized. In certain embodiments, polynucleotide encoding a polypeptide dimer, polypeptide, or fusion polypeptide further comprises a polynucleotide encoding a leader or signal sequence. An exemplary leader amino acid sequence is from GM-CSF, CD8a, or murine IgG kappa light chain.

[0130] In further aspects, expression constructs are provided, wherein the expression constructs comprise a polynucleotide of the present disclosure operably linked to an expression control sequence (e.g., a promoter). An exemplary promoter sequence includes an EFla promoter or a MNDu3 promoter. In certain embodiments, the expression construct is comprised in a vector. An exemplary vector may comprise a polynucleotide capable of transporting another polynucleotide to which it has been linked, or which is capable of replication in a host organism. Some examples of vectors include plasmids, viral vectors, cosmids, and others. Some vectors may be capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors), whereas other vectors may be integrated into the genome of a host cell or promote integration of the polynucleotide insert upon introduction into the host cell and thereby replicate along with the host genome (e.g., lentiviral vector, retroviral vector). Additionally, some vectors are capable of directing the expression of genes to which they are operatively linked (these vectors may be referred to as "expression vectors"). According to related embodiments, it is further understood that, if one or more agents (e.g., polynucleotides encoding fusion proteins as described herein) are co-administered to a subject, that each agent may reside in separate or the same vectors, and multiple vectors (each containing a different agent or the same agent) may be introduced to a cell or cell population or administered to a subject.

[0131] In certain embodiments, an expression construct encodes a chTCR of the present disclosure and at least one additional expression product (e.g., polypeptide) of interest. The expression construct comprises an EFla promoter (SEQ ID NO:7) and a MNDu3 promoter (SEQ ID NO:8), optionally on opposing polynucleotide strands and oriented in opposite directions. One of the EFla promoter and the MNDu3 promoter is operably linked to a polynucleotide encoding the chTCR, and the other of the EFla promoter and the MNDu3 promoter is operably linked to a polynucleotide encoding the at least one additional expression product of interest. In some embodiments, the EFla promoter is operably linked to the polynucleotide encoding the chTCR and the MNDu3 promoter is operably linked to the polynucleotide encoding the at least one expression product of interest. Non-limiting examples of expression construct architectures comprising an EFla promoter and a MNDu3 promoter are shown in certain of the Figures. In some embodiments, an expression construct is bidirectional. In other embodiments, the MNDu3 promoter is operably linked to the polynucleotide encoding the chTCR and the EFla promoter is operably linked to the polynucleotide encoding the at least one additional expression product of interest. As shown herein, an expression construct comprising the combination of a MNDu3 promoter and an EFla promoter effectively drives expression of a chTCR and at least one additional expression product of interest with advantages over expression constructs comprising a single promoter (e.g., EFla) driving expression of a plurality of expression products with interspersed 2A peptides or interspersed 2A peptides and RAKR sequences. The the combination of a MNDu3 promoter and an EFla promoter also has advantages over other combinations of promoters, including a combination of two copies of an EFla promoter. The at least one additional expression product of interest can comprise a polypeptide, an inhibitory oligonucleotide (e.g., an antisense oligonucleotide, a siRNA), a miRNA, or the like. In some embodiments, the at least one additional expression product of interest comprises a tag. In some embodiments, at least one additional expression product of interest comprises a truncated form of EGFR, NGFR, CD34, or CD 19. In some embodiments, the at least one additional expression product of interest comprises EGFRtopt(also shown as “fEGFRopt”). In some embodiments, the at least one additional expression product of interest comprises, consists essentially of, or consists of the amino acid sequences set forth in SEQ ID NO:226 or the amino acid sequence set forth in SEQ ID NO: 166. In some embodiments, the at least one additional expression product of interest comprises, consists essentially of, or consists of a human CD3 (zeta, gamma, delta, or epsilon) polypeptide or a fusion protein comprising the same.

[0132] In certain embodiments, polynucleotides of the present disclosure may be operatively linked to certain elements of a vector. For example, polynucleotide sequences that are needed to effect the expression and processing of coding sequences to which they are ligated may be operatively linked. Expression control sequences may include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and possibly sequences that enhance protein secretion. Expression control sequences may be operatively linked if they are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

[0133] In certain embodiments, the vector comprises a plasmid vector or a viral vector (e.g., a vector selected from lentiviral vector or a y-retroviral vector). Viral vectors include retrovirus, adenovirus (e.g., adeno-associated viruses), parvovirus, coronavirus, negative strand RNA viruses such as ortho-myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0134] "Retroviruses" are viruses having an RNA genome, which is reverse-transcribed into DNA using a reverse transcriptase enzyme, the reverse-transcribed DNA is then incorporated into the host cell genome. "Gammaretrovirus" refers to a genus of the retroviridae family. Examples of gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses.

[0135] "Lentiviral vector," as used herein, means HIV-based lentiviral vectors for gene delivery, which can be integrative or non-integrative, have relatively large packaging capacity, and can transduce a range of different cell types. Lentiviral vectors are usually generated following transient transfection of three (packaging, envelope and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter the target cell through the interaction of viral surface glycoproteins with receptors on the cell surface. On entry, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is a double-stranded linear viral DNA, which is the substrate for viral integration into the DNA of infected cells.

[0136] In certain embodiments, the viral vector can be a gammaretrovirus, e.g., Moloney murine leukemia virus (MLV)-derived vectors. In other embodiments, the viral vector can be a more complex retrovirus-derived vector, e.g., a lentivirus-derived vector. HIV-l-derived vectors belong to this category. Other examples include lentivirus vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (ovine lentivirus). Methods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian host cells with viral particles containing CAR transgenes are known in the art and have been previous described, for example, in: U.S. Patent 8,119,772; Walchli et al., PLoS One 6. 219 Q, 2011; Zhao et al., J. Immunol. 174 :4415, 2005; Engels et al., Hum. Gene Ther. 14:1155, 2003; Frecha et al., Mol. Ther. 18: 17^ , 2010; and Verhoeyen et al., Methods Mol. Biol. 506.97, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available. Other viral vectors also can be used for polynucleotide delivery including DNA viral vectors, including, for example adenovirus-based vectors and adeno-associated virus (AAV)- based vectors; vectors derived from herpes simplex viruses (HSVs), including amplicon vectors, replication-defective HSV and attenuated HSV (Krisky et al., Gene Ther. 5: 1517, 1998).

[0137] Other vectors developed for gene therapy uses can also be used with the compositions and methods of this disclosure. Such vectors include those derived from baculoviruses and CL- viruses. (Jolly, D J. 1999. Emerging Viral Vectors, pp 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as sleeping beauty or other transposon vectors).

[0138] When a viral vector genome comprises a plurality of polynucleotides to be expressed in a host cell as separate transcripts, the viral vector may also comprise additional sequences between the two (or more) transcripts allowing for bicistronic or multicistronic expression. Examples of such sequences used in viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptide, or any combination thereof.

[0139] Construction of an expression vector that is used for genetically engineering and producing a polypeptide dimer, polypeptide, or fusion polypeptide of interest can be accomplished by using any suitable molecular biology engineering techniques known in the art. To obtain efficient transcription and translation, a polynucleotide in each recombinant expression construct includes at least one appropriate expression control sequence (also called a regulatory sequence), such as a leader sequence and particularly a promoter operably (i.e., operatively) linked to the nucleotide sequence encoding the immunogen.

[0140] In certain embodiments, polynucleotides of the present disclosure are used to transfect / transduce a host cell (e.g., a T cell). A host cell encoding and / or expressing a fusion protein as disclosed herein is, in certain embodiments, useful in adoptive transfer therapy. Methods for transfecting / transducing T cells with desired nucleic acids have been described (e.g., U.S. Patent Application Pub. No. US 2004 / 0087025) as have adoptive transfer procedures using T cells of desired target-specificity (e.g., Schmitt et al., Hum. Gen. 20: 1240, 2009; Dossett et al., Mol. Ther. 17:742, 2009; Till et al., Blood 772:2261, 2008; Wang et al., Hum. Gene Ther. 18:712, 2007; Kuball et al., Blood 109:2331, 2007; US 2011 / 0243972; US 2011 / 0189141; Leen et al., Ann. Rev. Immunol. 25:243, 2007), such that adaptation of these methodologies to the presently disclosed embodiments is contemplated, based on the teachings herein, including those directed to fusion proteins of the present disclosure.

[0141] In certain embodiments, the host cell is a hematopoietic progenitor cell or a human immune system cell. A "hematopoietic progenitor cell", as referred to herein, is a cell that can be derived from hematopoietic stem cells or fetal tissue and is capable of further differentiation into mature cells types (e.g., immune system cells). Exemplary hematopoietic progenitor cells include those with a CD24LOLin CD117+phenotype or those found in the thymus (referred to as progenitor thymocytes).

[0142] As used herein, an "immune system cell" means any cell of the immune system that originates from a hematopoietic stem cell in the bone marrow, which gives rise to two major lineages, a myeloid progenitor cell (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) and a lymphoid progenitor cell (which give rise to lymphoid cells such as T cells, B cells, natural killer (NK) cells, and NK-T cells). Exemplary immune system cells include a CD4+T cell, a CD8+T cell, a CD4' CD8' double negative T cell, a yb T cell, a regulatory T cell, a stem cell memory T cell, a natural killer cell (e.g., aNK cell or a NK-T cell), a B cell, and a dendritic cell. Macrophages and dendritic cells may be referred to as "antigen presenting cells" or "APCs," which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with a TCR on the surface of a T cell.

[0143] A "T cell" or "T lymphocyte" is an immune system cell that matures in the thymus and produces T cell receptors (TCRs), though it will be understood that a T cell in which expression of a native TCR is (e.g., artificially) suppressed or abrogated is still a T cell. T cells can be naive (not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD 127, and CD45RA, and decreased expression of CD45RO as compared to TCM), memory T cells (TM) (antigen-experienced and long-lived), and effector cells (antigen-experienced, cytotoxic). TM can be further divided into subsets of central memory T cells (TCM, increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and decreased expression of CD45RA as compared to naive T cells) and effector memory T cells (TEM, decreased expression of CD62L, CCR7, CD28, CD45RA, and increased expression of CD127 as compared to naive T cells or TCM).

[0144] Effector T cells (TE) refer to antigen-experienced CD8+cytotoxic T lymphocytes that have decreased expression of CD62L, CCR7, CD28, and are positive for granzyme and perforin as compared to TCM. Helper T cells (TH) are CD4+cells that influence the activity of other immune cells by releasing cytokines. CD4+T cells can activate and suppress an adaptive immune response, and which of those two functions is induced will depend on presence of other cells and signals. T cells can be collected using known techniques, and the various subpopulations or combinations thereof can be enriched or depleted by known techniques, such as by affinity binding to antibodies, flow cytometry, or immunomagnetic selection. Other exemplary T cells include regulatory T cells, such as CD4+CD25+(Foxp3+) regulatory T cells and Tregl7 cells, as well as Tri, Th3, CD8+CD28‘, and Qa-1 restricted T cells.

[0145] "Cells of T cell lineage" refer to cells that show at least one phenotypic characteristic of a T cell, or a precursor or progenitor thereof that distinguishes the cells from other lymphoid cells, and cells of the erythroid or myeloid lineages. Such phenotypic characteristics can include expression of one or more proteins specific for T cells (e.g., CD3+, CD4+, CD8+), or a physiological, morphological, functional, or immunological feature specific for a T cell. For example, cells of the T cell lineage may be progenitor or precursor cells committed to the T cell lineage; CD25+immature and inactivated T cells; cells that have undergone CD4 or CD8 linage commitment; thymocyte progenitor cells that are CD4+CD8+double positive; single positive CD4+or CD8+; TCRaP or TCR y5; or mature and functional or activated T cells.

[0146] In certain embodiments, the immune system cell is a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a yb T cell, a natural killer cell (e.g., NK cell or NK-T cell), a dendritic cell, a B cell, or any combination thereof. In certain embodiments, the immune system cell is a CD4+ T cell. In certain embodiments, the T cell is a naive T cell, a central memory T cell, an effector memory T cell, a stem cell memory T cell, or any combination thereof.

[0147] A host cell may include any individual cell or cell culture which may receive a vector or the incorporation of nucleic acids or express proteins. The term also encompasses progeny of the host cell, whether genetically or phenotypically the same or different. Suitable host cells may depend on the vector and may include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells. These cells may be induced to incorporate the vector or other material by use of a viral vector, transformation via calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods. See, for example, Sambrook el al., Molecular Cloning: A Laboratory Manual 2d ed. (Cold Spring Harbor Laboratory, 1989).

[0148] In any of the foregoing embodiments, a host cell that comprises a heterologous polynucleotide encoding a polypeptide dimer, polypeptide, or fusion polypeptide can be an immune cell which is modified to reduce or eliminate expression of one or more endogenous genes that encode a polypeptide product selected from a CD4 gene locus, a CD8 gene locus, a TGFpRl gene locus, a TGFPR2 gene locus, a PD-1 gene locus, a CTLA4 gene locus, a LAT gene locus, a TIM-3 gene locus, a PD-L1 gene locus, a TIGIT gene locus, an A2AR gene locus, a Fas locus, a FasL gene locus, a B7-H3 gene locus, a B7-H4 gene locus, an IDO gene locus, a VISTA gene locus, a SIGLEC7 gene locus, a SIGLEC9 gene locus, a TRAC gene locus, a TRBC gene locus, a T cell receptor gene locus, a MHC (e.g. HLA) gene locus, a CBLB gene locus, a RASA2 gene locus, a UBASH3 A gene locus, a CISH gene locus, or any combination thereof. In some embodiments, a host cell is modified to reduce or eliminate expression of: TIGIT and one or both of TRAC and TRBC; TIGIT and one or both of CD4 and CD 8; one or both of TRAC and TRBC and one or both of CD4 and CD8; or TIGIT, one or both of TRAC and TRBC, and one or both of CD4 and CD8.

[0149] Without wishing to be bound by theory, certain endogenously expressed immune cell proteins may downregulate the immune activity of a modified immune host cell (e.g., PD-1, LAG-3, CTLA4, TIGIT, CBLB, RASA2, UBASH3A, CISH, Fas), or may compete with a disclosed polypeptide dimer, polypeptide, or fusion polypeptide for resources (e.g. Lek), or any combination thereof. Further, endogenous proteins (e.g., immune host cell proteins, such as an HLA) expressed on a donor immune cell to be used in a cell transfer therapy may be recognized as foreign by an allogeneic recipient, which may result in elimination or suppression of the donor immune cell by the allogeneic recipient.

[0150] Accordingly, decreasing or eliminating expression or activity of such endogenous genes or proteins can improve the activity, tolerance, and persistence of the host cells in an autologous or allogeneic host setting, and can allow universal administration of the cells (e.g., to any recipient regardless of HLA type). In certain embodiments, a modified host immune cell is a donor cell (e.g., allogeneic) or an autologous cell. In certain embodiments, a modified immune host cell of this disclosure comprises a chromosomal gene knockout of one or more of a gene that encodes PD-1, LAG-3, CTLA4, TIM3, TIGIT, CD4, CD8, an HLA component (e.g., a gene that encodes an al macroglobulin, an a2 macroglobulin, an a3 macroglobulin, a pi microglobulin, or a P2 microglobulin), or a TCR component (e.g., a gene that encodes a TCR variable region or a TCR constant region) (see, e.g., Torikai et al., Nature Sci. Rep. 6:21757 (2016); Torikai et al., Blood 119(2 )-.5697 (2012); and Torikai et al., Blood 722(8): 1341 (2013) the gene editing techniques, compositions, and adoptive cell therapies of which are herein incorporated by reference in their entirety), TGFpRl, TGFPR2, LAT, A2AR, Fas, FasL, B7-H3, B7-H4, IDO, VISTA, SIGLEC7, SIGLEC9, TRAC, TRBC, CBLB, RASA2, UBASH3A, and / or CISH. As used herein, the term "chromosomal gene knockout" refers to a genetic alteration in a host cell that prevents production, by the host cell, of a functionally active endogenous polypeptide product. Alterations resulting in a chromosomal gene knockout can include, for example, introduced nonsense mutations (including the formation of premature stop codons), missense mutations, gene deletion, and strand breaks, as well as the heterologous expression of inhibitory nucleic acid molecules that inhibit endogenous gene expression in the host cell.

[0151] In certain embodiments, a chromosomal gene knock-out or gene knock-in is made by chromosomal editing of a host cell. Chromosomal editing can be performed using, for example, endonucleases. As used herein "endonuclease" refers to an enzyme capable of catalyzing cleavage of a phosphodiester bond within a polynucleotide chain. In certain embodiments, an endonuclease is capable of cleaving a targeted gene thereby inactivating or "knocking out" the targeted gene. An endonuclease may be a naturally occurring, recombinant, genetically modified, or fusion endonuclease. The nucleic acid strand breaks caused by the endonuclease are commonly repaired through the distinct mechanisms of homologous recombination or non- homologous end joining (NHEJ). During homologous recombination, a donor nucleic acid molecule may be used for a donor gene "knock-in", for target gene "knock-out", and optionally to inactivate a target gene through a donor gene knock in or target gene knock out event. NHEJ is an error-prone repair process that often results in changes to the DNA sequence at the site of the cleavage, e.g., a substitution, deletion, or addition of at least one nucleotide. NHEJ may be used to "knock-out" a target gene. Examples of endonucleases include zinc finger nucleases, TALE-nucleases, CRISPR-Cas nucleases, meganucleases, and megaTALs.

[0152] As used herein, a "zinc finger nuclease" (ZFN) refers to a fusion protein comprising a zinc finger DNA-binding domain fused to a non-specific DNA cleavage domain, such as a Fokl endonuclease. Each zinc finger motif of about 30 amino acids binds to about 3 base pairs of DNA, and amino acids at certain residues can be changed to alter triplet sequence specificity see, e.g., Desjarlais et al., Proc. Natl. Acad. Sci. 90 2256-2260, 1993; Wolfe et al., J. Mol. Biol. 285: 1917-1934, 1999). Multiple zinc finger motifs can be linked in tandem to create binding specificity to desired DNA sequences, such as regions having a length ranging from about 9 to about 18 base pairs. By way of background, ZFNs mediate genome editing by catalyzing the formation of a site-specific DNA double strand break (DSB) in the genome, and targeted integration of a transgene comprising flanking sequences homologous to the genome at the site of DSB is facilitated by homology directed repair. Alternatively, a DSB generated by a ZFN can result in knock out of target gene via repair by non-homologous end joining (NHEJ), which is an error-prone cellular repair pathway that results in the insertion or deletion of nucleotides at the cleavage site. In certain embodiments, a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, made using a ZFN molecule.

[0153] As used herein, a "transcription activator-like effector nuclease" (TALEN) refers to a fusion protein comprising a TALE DNA-binding domain and a DNA cleavage domain, such as a FokI endonuclease. A "TALE DNA binding domain" or "TALE" is composed of one or more TALE repeat domains / units, each generally having a highly conserved 33-35 amino acid sequence with divergent 12th and 13th amino acids. The TALE repeat domains are involved in binding of the TALE to a target DNA sequence. The divergent amino acid residues, referred to as the Repeat Variable Diresidue (RVD), correlate with specific nucleotide recognition. The natural (canonical) code for DNA recognition of these TALEs has been determined such that an HD (histine-aspartic acid) sequence at positions 12 and 13 of the TALE leads to the TALE binding to cytosine (C), NG (asparagine-glycine) binds to a T nucleotide, NI (asparagineisoleucine) to A, NN (asparagine-asparagine) binds to a G or A nucleotide, and NG (asparagineglycine) binds to a T nucleotide. Non-canonical (atypical) RVDs are also known (see, e.g., U.S. Patent Publication No. US 2011 / 0301073, which atypical RVDs are incorporated by reference herein in their entirety). TALENs can be used to direct site-specific double-strand breaks (DSB) in the genome of T cells. Non- homologous end joining (NHEJ) ligates DNA from both sides of a double-strand break in which there is little or no sequence overlap for annealing, thereby introducing errors that knock out gene expression. Alternatively, homology directed repair can introduce a transgene at the site of DSB providing homologous flanking sequences are present in the transgene. In certain embodiments, a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, and made using a TALEN molecule.

[0154] As used herein, a "clustered regularly interspaced short palindromic repeats / Cas" (CRISPR / Cas) nuclease system refers to a system that employs a CRISPR RNA (crRNA)-guided Cas nuclease to recognize target sites within a genome (known as protospacers) via base-pairing complementarity and then to cleave the DNA if a short, conserved protospacer associated motif (PAM) immediately follows 3’ of the complementary target sequence. CRISPR / Cas systems are classified into three types (i.e., type I, type II, and type III) based on the sequence and structure of the Cas nucleases. The crRNA-guided surveillance complexes in types I and III need multiple Cas subunits. Type II system, the most studied, comprises at least three components: an RNA- guided Cas9 nuclease, a crRNA, and a trans-acting crRNA (tracrRNA). The tracrRNA comprises a duplex forming region. A crRNA and a tracrRNA form a duplex that is capable of interacting with a Cas9 nuclease and guiding the Cas9 / crRNA:tracrRNA complex to a specific site on the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA upstream from a PAM. Cas9 nuclease cleaves a double-stranded break within a region defined by the crRNA spacer. Repair by NHEJ results in insertions and / or deletions which disrupt expression of the targeted locus. Alternatively, a transgene with homologous flanking sequences can be introduced at the site of DSB via homology directed repair. The crRNA and tracrRNA can be engineered into a single guide RNA (sgRNA or gRNA) see, e.g., Jinek et al., Science 337:816-21, 2012). Further, the region of the guide RNA complementary to the target site can be altered or programed to target a desired sequence (Xie et al., P LOS One 9:el00448, 2014; U.S. Pat. Appl. Pub. No. US 2014 / 0068797, U.S. Pat. Appl. Pub. No. US 2014 / 0186843; U.S. Pat. No. 8,697,359, and PCT Publication No. WO 2015 / 071474; each of which is incorporated by reference). In certain embodiments, a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, and made using a CRISPR / Cas nuclease system or base editing system (Komor, A. C.; Kim, Y. B.; Packer, M. S.; Zuris, J. A.; Liu, D. R. Nature 533, 420-424 (2016). Briefly, base editing is a genomeediting approach that uses components from CRISPR systems together with other enzymes to directly introduce point mutations into cellular DNA or RNA without making double-stranded DNA breaks. Certain DNA base editors comprise a catalytically disabled nuclease fused to a nucleobase deaminase enzyme and, in some cases, a DNA glycosylase inhibitor. RNA base editors function similarly, using components that target RNA. Base editors directly convert one base or base pair into another, enabling the efficient installation of point mutations in nondividing cells without generating excess undesired editing by-products. See e.g. Rees H et al. Nature Reviews Genetics (2018). Base editing can be performed, for example, using a Cas9- based (NGG PAM) cytosine base editing enzyme, which can be introduced into a cell as mRNA. CBE4max (available from TriLink or Aldevron as mRNA), AncBE4max, or the like may be used. Cytidine and adenine base editors useful in accordance with the present disclosure include those described in, for example, Koblan et al., Nat Biotechnol. 2018 October; 36(9): 843-846. doi: 10.1038 / nbt.4172, the base editors, related methods, and amino acid and nucleic acid sequences of which (including in the supplemental materials thereof) are incorporated herein by reference. Base editing can be performed on T cells, for example, beginning at about 2 days following activation of the T cells with anti-CD3 / anti-CD28-coated beads.

[0155] Exemplary gRNA sequences and methods of using the same to knock out endogenous genes that encode immune cell proteins include those described in Ren et al., Clin. Cancer Res. 23(9):2255-2266 (2017), the gRNAs, CAS9 DNAs, vectors, and gene knockout techniques of which are hereby incorporated by reference in their entirety.

[0156] Alternative Cas nucleases may be used, including but not limited to, Cas 12, Cas 13, and Cas 14 nucleases, and variants thereof. For example, Cas nucleases disclosed in WO 2019 / 178427, which is hereby incorporated by reference in its entirety (including the Cas nucleases, CRISPR-Cas systems, and related methods disclosed therein), may be utilized.

[0157] As used herein, a "meganuclease," also referred to as a "homing endonuclease," refers to an endodeoxyribonuclease characterized by a large recognition site (double stranded DNA sequences of about 12 to about 40 base pairs). Meganucleases can be divided into five families based on sequence and structure motifs: LAGLID ADG (SEQ ID NO:210), GIY-YIG (SEQ ID NO:211), HNH, His-Cys box and PD-(DZE)XK (SEQ ID NO:212). Exemplary meganucleases include LScel, LCeuI, PI-PspI, Pl-Sce, LScelV, I-CsmI, LPanl, LScell, I-Ppol, LScelll, I-Crel, I-TevI, LTevII and I-TevIII, whose recognition sequences are known (see, e.g., U.S. Patent Nos. 5,420,032 and 6,833,252; Belfort et al., Nucleic Acids Res . 25:3379-3388, 1997; Dujon et al., Gene 52: 115-118, 1989; Perler et al., Nucleic Acids Res. 22: 1125-1127, 1994; Jasin, Trends Genet. 72:224-228, 1996; Gimble et al., J. Mol. Biol. 263: 163-180, 1996; Argast et al., J. Mol. Biol. 250:345-353, 1998).

[0158] In certain embodiments, naturally occurring meganucleases may be used to promote sitespecific genome modification of a target selected from PD-1, LAG3, TIM3, CTLA4, TIGIT, an HLA-encoding gene, a CD4, a CD8, or a TCR component-encoding gene. In other embodiments, an engineered meganuclease having a novel binding specificity for a target gene is used for site-specific genome modification (see, e.g., Porteus et al., Nat. Biotechnol. 23:961-13, 2005; Sussman et al., J. Mol. Biol. 342:31-41, 2004; Epinat et al., Nucleic Acids Res. 31:2952- 62, 2003; Chevalier et al., Molec. Cell 70:895-905, 2002; Ashworth et al., Nature 441:656-659, 2006; Paques et al., Curr. Gene Ther. 7:49-66, 2007; U.S. Patent Publication Nos. US 2007 / 0117128; US 2006 / 0206949; US 2006 / 0153826; US 2006 / 0078552; and US 2004 / 0002092). In further embodiments, a chromosomal gene knockout is generated using a homing endonuclease that has been modified with modular DNA binding domains of TALENs to make a fusion protein known as a megaTAL. MegaTALs can be utilized to not only knockout one or more target genes, but to also introduce (knock in) heterologous or exogenous polynucleotides when used in combination with an exogenous donor template encoding a polypeptide of interest. A chromosomal gene knockout can be performed using base-editing, as known in the art and described herein. A splicing site or stop codon can be introduced into a host cell (e.g., T cell) chromosomal position (e.g., gene locus) using, for example, base editing as described herein. Base editing can be performed, for example, using a Cas9-based (NGG PAM) cytosine base editing enzyme, which can be introduced into a cell as mRNA. CBE4max (available from TriLink or Aldevron as mRNA), AncBE4max, or the like may be used. Cytidine and adenine base editors useful in accordance with the present disclosure include those described in, for example, Koblan et al., Nat Biotechnol. 2018 October; 36(9): 843-846. doi: 10.1038 / nbt.4172, the base editors, related methods, and amino acid and nucleic acid sequences of which (including in the supplemental materials thereof) are incorporated herein by reference. Base editing can be performed on T cells, for example, beginning at about 2 days following activation of the T cells with anti-CD3 / anti-CD28-coated beads.

[0159] In certain embodiments, a chromosomal gene knockout comprises an inhibitory nucleic acid molecule that is introduced into a host cell (e.g., an immune cell) comprising a heterologous polynucleotide encoding an antigen-specific receptor that specifically binds to a tumor associated antigen, wherein the inhibitory nucleic acid molecule encodes a target-specific inhibitor and wherein the encoded target-specific inhibitor inhibits endogenous gene expression (e.g., of PD-1, TIM3, LAG3, CTLA4, TIGIT, an HL A component, or a TCR component, a CD4, a CD8, or any combination thereof) in the host immune cell.

[0160] A chromosomal gene knockout can be confirmed directly by DNA sequencing of the host immune cell following use of the knockout procedure or agent. Chromosomal gene knockouts can also be inferred from the absence of gene expression (e.g., the absence of an mRNA or polypeptide product encoded by the gene) following the knockout.

[0161] Any of the foregoing gene-editing techniques can be used to introduce a polynucleotide of the present disclosure (e.g., encoding a fusion protein) into a host cell genome. In some embodiments, a heterologous polynucleotide is introduced into a locus encoding an endogenous TCR component, HLA component, PD-1, LAG-3, CTLA4, TIM3, or TIGIT, or a "safe harbor" locus such as Rosa26, AAVS1, CCR5, or the like.

[0162] In certain embodiments, a host cell (e.g., immune cell) of the present disclosure is engineered so that expression of polypeptide dimer, polypeptide, or fusion polypeptide is modulated (e.g., controlled) by binding of the host cell to a target (e.g. antigen) that is not the same target as the target to which the polypeptide dimer, polypeptide, or fusion polypeptide, respectively, binds. For example, a host cell can comprise (i) a polynucleotide encoding an engineered (z.e., synthetic) Notch receptor comprising (a) an extracellular component comprising a binding domain that binds to an antigen, which is a different antigen than the antigen to which the antigen-binding protein binds, (b) a Notch core domain, or a functional portion or variant thereof; and (c) an intracellular component comprising a transcriptional factor (z.e., a polypeptide capable of activating or increasing, or inhibiting, repressing or reducing, transcription of a target nucleotide sequence (e.g., a gene) or set of target nucleotide sequences); and (ii) the heterologous polynucleotide encoding a polypeptide dimer, polypeptide, or fusion polypeptide as disclosed herein and comprising an expression control sequence that can be recognized or bound by the transcriptional factor, wherein binding of the engineered Notch receptor to antigen leads to release of the transcriptional factor from the engineered Notch receptor (e.g., by protease- driven cleavage), which can, in turn, drive transcription of the polypeptide dimer, polypeptide, or fusion polypeptide. See, e.g, Morsut et al., Cell 164:780-791 (2016) and PCT Published Application No. WO 2016 / 138034A1, which synthetic Notch constructs are incorporated herein by reference. Briefly, such "logic-gated" expression systems may be useful to modulate expression of an antigen-binding protein of this disclosure so that the expression occurs only, or preferentially, when the host cell encounters a first antigen (i.e., that can be bound by the synthetic Notch receptor) that is only expressed by, or is principally expressed by, or has a higher expression level on diseased cells as compared to healthy cells. Such embodiments may reduce "on-target off-tissue" recognition by a fusion protein in circumstances where the target recognized by the antigen-binding protein is expressed by healthy cells.

[0163] In other aspects, kits are provided comprising (a) a vector or an expression construct as described herein and (b) reagents for transducing the vector or the expression construct into a host cell.

[0164] Uses

[0165] The present disclosure also provides methods for treating a disease or condition characterized by expression of CD 19 and / or BCMA, or CD 19 and / or CD20, wherein the methods comprise administering to a subject in need thereof an effective amount of a polypeptide dimer, polypeptide, polynucleotide, vector, host cell, composition, or unit dose of the present disclosure. In some embodiments, the disease or condition expresses or is otherwise associated with the target(s) (e.g., antigen(s)). Ins ome embodiments, a disease or condition comprises cells that express CD 19, BCMA, or CD 19 and BCMA, and and the disease or condition comprises an autoimmune disease or condition. In some embodiments, the disease or condition comprises systemic lupus erythematosus.

[0166] Ins ome embodiments, a disease or condition comprises cells that express CD 19, CD20, or CD 19 and CD20, and and the disease or condition comprises a cancer or a tumor, such as, for example, a hematological malignancy, or a solid cancer, wherein, optionally: (1) the cancer comprises a myeloma (e.g. multiple myeloma), a carcinoma, a sarcoma, a glioma, a lymphoma, a leukemia, a myeloma, or any combination thereof; and / or (2) the cancer comprises a cancer of the head or neck, melanoma, pancreatic cancer, cholangiocarcinoma, hepatocellular cancer, breast cancer such as triple-negative breast cancer (TNBC), gastric cancer, non-small-cell lung cancer, prostate cancer, esophageal cancer, mesothelioma, small-cell lung cancer, colorectal cancer, glioblastoma, or any combination thereof; and / or (3) the cancer comprises Askin's tumor, sarcoma botryoides, chondrosarcoma, Ewing's sarcoma, PNET, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma protuberans (DFSP), desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, undifferentiated pleomorphic sarcoma, malignant peripheral nerve sheath tumor (MPNST), neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, undifferentiated pleomorphic sarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, linitis plastic, vipoma, cholangiocarcinoma, hepatocellular carcinoma, adenoid cystic carcinoma, renal cell carcinoma, Grawitz tumor, ependymoma, astrocytoma, oligodendroglioma, brainstem glioma, optice nerve glioma, a mixed glioma, Hodgkin’s lymphoma, a B-cell lymphoma, non-Hodgkin’s lymphoma (NHL), Burkitt's lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, and mantle cell lymphoma, Waldenstrom's macroglobulinemia, CD37+ dendritic cell lymphoma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, extra-nodal marginal zone B- cell lymphoma of mucosa-associated (MALT) lymphoid tissue, nodal marginal zone B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, adult T-cell lymphoma, extranodal NK / T-cell lymphoma, nasal type, enteropathy-associated T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK cell lymphoma, Sezary syndrome, angioimmunoblastic T cell lymphoma, anaplastic large cell lymphoma, or any combination thereof. In some embodiments, the cancer comprises a solid tumor, wherein, optionally, the solid tumor is a sarcoma or a carcinoma, wherein, further optionally, the solid tumor is selected from: chondrosarcoma; fibrosarcoma (fibroblastic sarcoma); Dermatofibrosarcoma protuberans (DFSP); osteosarcoma; rhabdomyosarcoma; Ewing’s sarcoma; a gastrointestinal stromal tumor; Leiomyosarcoma; angiosarcoma (vascular sarcoma); Kaposi’s sarcoma; liposarcoma; pleomorphic sarcoma; or synovial sarcoma. In some embodiments, the solid tumor is selected from a lung carcinoma (e.g., Adenocarcinoma, Squamous Cell Carcinoma (Epidermoid Carcinoma); Squamous cell carcinoma; Adenocarcinoma; Adenosquamous carcinoma; anaplastic carcinoma; Large cell carcinoma; Small cell carcinoma; a breast carcinoma (e.g., Ductal Carcinoma in situ (non-invasive), Lobular carcinoma in situ (non-invasive), Invasive Ductal Carcinoma, Invasive lobular carcinoma, Non- invasive Carcinoma); a liver carcinoma (e.g., Hepatocellular Carcinoma, Cholangiocarcinomas or Bile Duct Cancer); Large-cell undifferentiated carcinoma, Bronchioalveolar carcinoma); an ovarian carcinoma (e.g., Surface epithelial-stromal tumor (Adenocarcinoma) or ovarian epithelial carcinoma (which includes serous tumor, endometrioid tumor and mucinous cystadenocarcinoma), Epidermoid (Squamous cell carcinoma), Embryonal carcinoma and choriocarcinoma (germ cell tumors)); a kidney carcinoma (e.g., Renal adenocarcinoma, hypernephroma, Transitional cell carcinoma (renal pelvis), Squamous cell carcinoma, Bellini duct carcinoma, Clear cell adenocarcinoma, Transitional cell carcinoma, Carcinoid tumor of the renal pelvis); an adrenal carcinoma (e.g., Adrenocortical carcinoma), a carcinoma of the testis (e.g., Germ cell carcinoma (Seminoma, Choriocarcinoma, Embryonal carciroma, Teratocarcinoma), Serous carcinoma); Gastric carcinoma (e.g., Adenocarcinoma); an intestinal carcinoma (e.g., Adenocarcinoma of the duodenum); a colorectal carcinoma; or a skin carcinoma (e.g., Basal cell carcinoma, Squamous cell carcinoma). In some embodiments, the solid tumor is an ovarian carcinoma, an ovarian epithelial carcinoma, a cervical adenocarcinoma or small cell carcinoma, a pancreatic carcinoma, a colorectal carcinoma (e.g., an adenocarcinoma or squamous cell carcinoma), a lung carcinoma, a breast ductal carcinoma, or an adenocarcinoma of the prostate.

[0167] In any of the presently disclosed embodiments, the host cell is an allogeneic cell, a syngeneic cell, or an autologous cell. Typically, the host cell will further express or encode an antigen-binding protein. Subjects that can be treated by the present invention are, in general, human and other primate subjects, such as monkeys and apes for veterinary medicine purposes. In any of the aforementioned embodiments, the subject may be a human subject. The subjects can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. Cells according to the present disclosure may be administered in a manner appropriate to the disease, condition, or disorder to be treated as determined by persons skilled in the medical art. In any of the above embodiments, a cell comprising a fusion protein as described herein is administered intravenously, intraperitoneally, intratumorally, into the bone marrow, into a lymph node, or into the cerebrospinal fluid so as to encounter the tagged cells to be ablated. An appropriate dose, suitable duration, and frequency of administration of the compositions will be determined by such factors as a condition of the patient; size, type, and severity of the disease, condition, or disorder; the undesired type or level or activity of the tagged cells, the particular form of the active ingredient; and the method of administration.

[0168] In any of the above embodiments, methods of the present disclosure comprise administering a host cell expressing a polypeptide dimer, polypeptide, or fusion polypeptide of the present disclosure. The amount of cells in a composition is at least one cell (for example, one fusion protein-modified CD8+T cell subpopulation; one fusion protein-modified CD4+T cell subpopulation) or is more typically greater than 102cells, for example, up to 106, up to 107, up to 108cells, up to 109cells, or more than IO10cells, such as about 1011cells / m2. In certain embodiments, the cells are administered in a range from about 105to about 1011cells / m2, preferably in a range of about 105or about 106to about 109or about IO10cells / m2. The number of cells will depend upon the ultimate use for which the composition is intended as well the type of cells included therein. For example, cells modified to contain a fusion protein specific for a particular antigen will comprise a cell population containing at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of such cells. For uses provided herein, cells are generally in a volume of a liter or less, 500 mis or less, 250 mis or less, or 100 mis or less. In embodiments, the density of the desired cells is typically greater than 104cells / ml and generally is greater than 107cells / ml, generally 108cells / ml or greater. The cells may be administered as a single infusion or in multiple infusions over a range of time. A clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 105, 106, 107, 108, 109, 1010, or 1011cells.

[0169] Unit doses are also provided herein which comprise a host cell (e.g., a modified immune cell comprising a polynucleotide of the present disclosure) or host cell composition of this disclosure. Typically, the host cell will further express or encoden an antigen-binding protein. In certain embodiments, a unit dose comprises (i) a composition comprising at least about 30% (e.g., including 30% or more), at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD4+T cells, combined with (ii) a composition comprising 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 85%, at least about 90%, or at least about 95% modified CD8+T cells, in about a 1 : 1 ratio (e.g., such as a 1 : 1 ratio), wherein the unit dose contains a reduced amount or substantially no naive T cells ( / .< ., has less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less then about 1% the population of naive T cells present in a unit dose as compared to a patient sample having a comparable number of PBMCs).

[0170] In some embodiments, a unit dose comprises (i) a composition comprising at least about 50% modified CD4+T cells, combined with (ii) a composition comprising at least about 50% modified CD8+T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells. In further embodiments, a unit dose comprises (i) a composition comprising at least about 60% modified CD4+T cells, combined with (ii) a composition comprising at least about 60% modified CD8+T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells. In still further embodiments, a unit dose comprises (i) a composition comprising at least about 70% modified CD4+T cells, combined with (ii) a composition comprising at least about 70% modified CD8+T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells. In some embodiments, a unit dose comprises (i) a composition comprising at least about 80% modified CD4+T cells, combined with (ii) a composition comprising at least about 80% modified CD8+T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells. In some embodiments, a unit dose comprises (i) a composition comprising at least about 85% modified CD4+T cells, combined with (ii) a composition comprising at least about 85% modified CD8+T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells. In some embodiments, a unit dose comprises (i) a composition comprising at least about 90% modified CD4+T cells, combined with (ii) a composition comprising at least about 90% modified CD8+T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells. In any of the embodiments described herein, a unit dose comprises equal, or approximately equal numbers of engineered CD45RA' CD3+CD8+and engineered CD45RA' CD3+CD4+TM cells.

[0171] Also contemplated are pharmaceutical compositions that comprise fusion proteins or cells expressing or encoding a fusion protein as disclosed herein, and a pharmaceutically acceptable carrier, diluents, or excipient. Suitable excipients include water, saline, dextrose, glycerol, or the like and combinations thereof. In embodiments, compositions comprising fusion proteins or host cells as disclosed herein further comprise a suitable infusion media. Suitable infusion media can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma- Lyte A (Baxter), 5% dextrose in water, Ringer's lactate can be utilized. An infusion medium can be supplemented with human serum albumin or other human serum components.

[0172] Pharmaceutical compositions may be administered in a manner appropriate to the disease or condition to be treated (or prevented) as determined by persons skilled in the medical art. An appropriate dose and a suitable duration and frequency of administration of the compositions will be determined by such factors as the health condition of the patient, size of the patient (i.e., weight, mass, or body area), the type and severity of the patient's condition, the undesired type or level or activity of the fusion protein-expressing cells, the particular form of the active ingredient, and the method of administration. In general, an appropriate dose and treatment regimen provide the composition(s) in an amount sufficient to provide therapeutic and / or prophylactic benefit (such as described herein, including an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and / or overall survival, or a lessening of symptom severity). For prophylactic use, a dose should be sufficient to prevent, delay the onset of, or diminish the severity of a disease associated with the target (e.g., antigen). Prophylactic benefit of the immunogenic compositions administered according to the methods described herein can be determined by performing pre-clinical (including in vitro and in vivo animal studies) and clinical studies and analyzing data obtained therefrom by appropriate statistical, biological, and clinical methods and techniques, all of which can readily be practiced by a person skilled in the art.

[0173] Certain methods of treatment or prevention contemplated herein include administering a host cell (which may be autologous, allogeneic or syngeneic) comprising a desired polynucleotide as described herein that is stably integrated into the chromosome of the cell. For example, such a cellular composition may be generated ex vivo using autologous, allogeneic or syngeneic immune system cells (e.g., T cells, antigen-presenting cells, natural killer cells) in order to administer a desired, fusion protein-expressing T-cell composition to a subject as an adoptive immunotherapy. In certain embodiments, the host cell comprises a hematopoietic progenitor cell or a human immune cell. In certain embodiments, the immune system cell comprises a CD4+T cell, a CD8+T cell, a CD4' CD8' double-negative T cell, a yb T cell, a natural killer cell, a dendritic cell, or any combination thereof. In certain embodiments, the immune system cell comprises a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof. In particular embodiments, the cell comprises a CD4+T cell. In particular embodiments, the cell comprises a CD8+T cell.

[0174] As used herein, administration of a composition refers to delivering the same to a subject, regardless of the route or mode of delivery. Administration may be effected continuously or intermittently, and parenterally. Administration may be for treating a subject already confirmed as having a recognized condition, disease or disease state, or for treating a subject susceptible to or at risk of developing such a condition, disease or disease state. Co-administration with an adjunctive therapy may include simultaneous and / or sequential delivery of multiple agents in any order and on any dosing schedule (e.g., fusion protein-expressing recombinant ( / .< ., engineered) host cells with one or more cytokines; immunosuppressive therapy such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low dose of a mycophenolic acid prodrug, or any combination thereof).

[0175] In certain embodiments, a plurality of doses of a recombinant host cell as described herein is administered to the subject, which may be administered at intervals between administrations of about two to about four weeks or more. In certain embodiments, the plurality of unit doses are administered at intervals between administrations of about two, three, four, five, six, seven, eight, or more weeks.

[0176] In still further embodiments, the subject being treated is further receiving immunosuppressive therapy, such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low dose of a mycophenolic acid prodrug, or any combination thereof. In yet further embodiments, the subject being treated has received a non-myeloablative or a myeloablative hematopoietic cell transplant, wherein the treatment may be administered at least two to at least three months after the non-myeloablative hematopoietic cell transplant.

[0177] An effective amount of a pharmaceutical composition (e.g., host cell, fusion protein, unit dose, or composition) refers to an amount sufficient, at dosages and for periods of time needed, to achieve the desired clinical results or beneficial treatment, as described herein. An effective amount may be delivered in one or more administrations. If the administration is to a subject already known or confirmed to have a disease or disease-state, the term "therapeutic amount" may be used in reference to treatment, whereas "prophylactically effective amount" may be used to describe administrating an effective amount to a subject that is susceptible or at risk of developing a disease or disease-state (e.g., recurrence) as a preventative course.

[0178] The level of a CTL immune response may be determined by any one of numerous immunological methods described herein and routinely practiced in the art. The level of a CTL immune response may be determined prior to and following administration of any one of the herein described fusion proteins expressed by, for example, a T cell. Cytotoxicity assays for determining CTL activity may be performed using any one of several techniques and methods routinely practiced in the art (see, e.g., Henkart et al., "Cytotoxic T-Lymphocytes" in Fundamental Immunology, Paul (ed.) (2003 Lippincott Williams & Wilkins, Philadelphia, PA), pages 1127-50, and references cited therein).

[0179] Target (e.g., antigen)-specific T cell responses are typically determined by comparisons of observed T cell responses according to any of the herein described T cell functional parameters (e.g, proliferation, cytokine release, CTL activity, altered cell surface marker phenotype, etc.) that may be made between T cells that are exposed to a cognate antigen in an appropriate context (e.g., the antigen used to prime or activate the T cells, when presented by immunocompatible antigen-presenting cells) and T cells from the same source population that are exposed instead to a structurally distinct or irrelevant control antigen. A response to the cognate antigen that is greater, with statistical significance, than the response to the control antigen signifies antigen-specificity.

[0180] A biological sample may be obtained from a subject for determining the presence and level of an immune response to a fusion protein or cell as described herein. A "biological sample" as used herein may be a blood sample (from which serum or plasma may be prepared), biopsy specimen, body fluids (e.g., lung lavage, ascites, mucosal washings, synovial fluid), bone marrow, lymph nodes, tissue explant, organ culture, or any other tissue or cell preparation from the subject or a biological source. Biological samples may also be obtained from the subject prior to receiving any immunogenic composition, which biological sample is useful as a control for establishing baseline (i.e., pre-immunization) data.

[0181] The pharmaceutical compositions described herein may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers may be frozen to preserve the stability of the formulation until. In certain embodiments, a unit dose comprises a recombinant host cell as described herein at a dose of about 105cells / m2to about 1011cells / m2. The development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., parenteral or intravenous administration or formulation.

[0182] If the subject composition is administered parenterally, the composition may also include sterile aqueous or oleaginous solution or suspension. Suitable non-toxic parenterally acceptable diluents or solvents include water, Ringer’s solution, isotonic salt solution, 1,3 -butanediol, ethanol, propylene glycol or polythethylene glycols in mixtures with water. Aqueous solutions or suspensions may further comprise one or more buffering agents, such as sodium acetate, sodium citrate, sodium borate or sodium tartrate. Of course, any material used in preparing any dosage unit formulation should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained- release preparation and formulations. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit may contain a predetermined quantity of recombinant cells or active compound calculated to produce the desired effect in association with an appropriate pharmaceutical carrier.

[0183] In general, an appropriate dosage and treatment regimen provides the active molecules or cells in an amount sufficient to provide therapeutic or prophylactic benefit. Such a response can be monitored by establishing an improved clinical outcome e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated subjects as compared to nontreated subjects. Increases in preexisting immune responses to a tumor protein generally correlate with an improved clinical outcome. Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which are routine in the art and may be performed using samples obtained from a subject before and after treatment.

[0184] In further aspects, kits are provided that comprise (a) a host cell, (b) a composition, or (c) a unit dose as described herein.

[0185] Methods according to this disclosure may further include administering one or more additional agents to treat the disease or disorder in a combination therapy. For example, in certain embodiments, a combination therapy comprises administering a polypeptide dimer, polypeptide, or fusion polypeptide (or an engineered host cell expressing the same) with (concurrently, simultaneously, or sequentially) an immune checkpoint inhibitor. In some embodiments, a combination therapy comprises administering fusion protein of the present disclosure (or an engineered host cell expressing the same) with an agonist of a stimulatory immune checkpoint agent. In further embodiments, a combination therapy comprises administering a polypeptide dimer, polypeptide, or fusion polypeptide of the present disclosure (or an engineered host cell expressing the same) with a secondary therapy, such as chemotherapeutic agent, a radiation therapy, a surgery, an antibody, or any combination thereof.

[0186] As used herein, the term "immune suppression agent" or "immunosuppression agent" refers to one or more cells, proteins, molecules, compounds or complexes providing inhibitory signals to assist in controlling or suppressing an immune response. For example, immune suppression agents include those molecules that partially or totally block immune stimulation; decrease, prevent or delay immune activation; or increase, activate, or up regulate immune suppression. Exemplary immunosuppression agents to target (e.g., with an immune checkpoint inhibitor) include PD-1, PD-L1, PD-L2, LAG3, CTLA4, B7-H3, B7-H4, CD244 / 2B4, HVEM, BTLA, CD160, TIM3, GAL9, KIR, PVR1G (CD112R), PVRL2, adenosine, A2aR, immunosuppressive cytokines (e.g., IL-10, IL-4, IL-IRA, IL-35), IDO, arginase, VISTA, TIGIT, LAIR1, CEACAM-1, CEACAM-3, CEACAM-5, Treg cells, or any combination thereof.

[0187] An immune suppression agent inhibitor (also referred to as an immune checkpoint inhibitor) may be a compound, an antibody, an antibody fragment or fusion polypeptide (e.g., Fc fusion, such as CTLA4-Fc or LAG3-Fc), an antisense molecule, a ribozyme or RNAi molecule, or a low molecular weight organic molecule. In any of the embodiments disclosed herein, a method may comprise administering a composition of the present disclosure (e.g., a fusion protein, polynucleotide, vector, an host cell, or pharmaceutical composition) with one or more inhibitor of any one of the following immune suppression components, singly or in any combination.

[0188] In certain embodiments, a composition is used in combination with a PD-1 inhibitor, for example a PD-1 -specific antibody or binding fragment thereof, such as pidilizumab, nivolumab (Keytruda, formerly MDX-1106), pembrolizumab (Opdivo, formerly MK-3475), MEDI0680 (formerly AMP-514), AMP-224, BMS-936558, or any combination thereof. In further embodiments, a composition is used in combination with a PD-L1 specific antibody or binding fragment thereof, such as BMS-936559, durvalumab (MEDI4736), atezolizumab (RG7446), avelumab (MSB0010718C), MPDL3280A, or any combination thereof.

[0189] In certain embodiments, a composition is used in combination with a LAG3 inhibitor, such as LAG525, IMP321, IMP701, 9H12, BMS-986016, or any combination thereof.

[0190] In certain embodiments, a composition is used in combination with an inhibitor of CTLA4. In particular embodiments, a composition is used in combination with a CTLA4 specific antibody or binding fragment thereof, such as ipilimumab, tremelimumab, CTLA4-Ig fusion proteins (e.g., abatacept, belatacept), or any combination thereof.

[0191] In certain embodiments, a composition is used in combination with a B7-H3 specific antibody or binding fragment thereof, such as enoblituzumab (MGA271), 376.96, or both. A B7-H4 antibody binding fragment may be a scFv or fusion protein thereof, as described in, for example, Dangaj et al., Cancer Res. 73:4820, 2013, as well as those described in U.S. Patent No. 9,574,000 and PCT Patent Publication Nos. WO / 201640724 Al and WO 2013 / 025779A1.

[0192] In certain embodiments, a composition is used in combination with an inhibitor of CD244. In certain embodiments, a composition is used in combination with an inhibitor of BLTA, HVEM, CD 160, or any combination thereof. Anti CD- 160 antibodies are described in, for example, PCT Publication No. WO 2010 / 084158. In certain embodiments, a composition is used in combination with an inhibitor of TIM3. In certain embodiments, a composition is used in combination with an inhibitor of Gal9. In certain embodiments, a composition is used in combination with an inhibitor of adenosine signaling, such as a decoy adenosine receptor. In certain embodiments, a composition is used in combination with an inhibitor of A2aR. In certain embodiments, a composition is used in combination with an inhibitor of KIR, such as lirilumab (BMS-986015). In certain embodiments, a composition is used in combination with an inhibitor of an inhibitory cytokine (typically, a cytokine other than TGFP) or Treg development or activity. In certain embodiments, a composition is used in combination with an IDO inhibitor, such as levo-l-methyl tryptophan, epacadostat (INCB024360; Liu etal., Blood 775:3520-30, 2010), ebselen (Terentis et al. , Biochem. 49:591-600, 2010), indoximod, NLG919 (Mautino et al., American Association for Cancer Research 104th Annual Meeting 2013; Apr 6-10, 2013), 1- methyl-tryptophan (l-MT)-tira-pazamine, or any combination thereof. In certain embodiments, a composition is used in combination with an arginase inhibitor, such as N(omega)-Nitro-L- arginine methyl ester (L-NAME), N-omega-hydroxy-nor-l-arginine (nor-NOHA), L-NOHA, 2(S)-amino-6-boronohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine (BEC), or any combination thereof. In certain embodiments, a composition of the present disclosure (or an engineered host cell expressing the same) is used in combination with an inhibitor of VISTA, such as CA-170 (Curis, Lexington, Mass.). In certain embodiments, a composition is used in combination with an inhibitor of TIGIT such as, for example, COM902 (Compugen, Toronto, Ontario Canada), an inhibitor of CD155, such as, for example, COM701 (Compugen), or both. In certain embodiments, a composition is used in combination with an inhibitor of PVRIG, PVRL2, or both. Anti-PVRIG antibodies are described in, for example, PCT Publication No. WO 2016 / 134333. Anti-PVRL2 antibodies are described in, for example, PCT Publication No. WO 2017 / 021526.

[0193] In certain embodiments, a composition is used in combination with a LAIR1 inhibitor. In certain embodiments, a composition is used in combination with an inhibitor of CEACAM-1, CEACAM-3, CEACAM-5, or any combination thereof. In certain embodiments, a composition is used in combination with an agent that increases the activity ( / .< ., is an agonist) of a stimulatory immune checkpoint molecule. For example, a composition of the present disclosure can be used in combination with a CD137 (4-1BB) agonist (such as, for example, urelumab), a CD134 (OX-40) agonist (such as, for example, MEDI6469, MEDI6383, or MEDI0562), lenalidomide, pomalidomide, a CD27 agonist (such as, for example, CDX-1127), a CD28 agonist (such as, for example, TGN1412, CD80, or CD86), a CD40 agonist (such as, for example, CP-870,893, rhuCD40L, or SGN-40), a CD122 agonist (such as, for example, IL-2) an agonist of GITR (such as, for example, humanized monoclonal antibodies described in PCT Patent Publication No. WO 2016 / 054638), an agonist of ICOS (CD278) (such as, for example, GSK3359609, mAb 88.2, JTX-2011, Icos 145-1, Icos 314-8, or any combination thereof). In any of the embodiments disclosed herein, a method may comprise administering a composition with one or more agonist of a stimulatory immune checkpoint molecule, including any of the foregoing, singly or in any combination.

[0194] In certain embodiments, a combination therapy comprises a composition and a secondary therapy comprising one or more of: an antibody or antigen binding-fragment thereof that is specific for an antigen, a radiation treatment, a surgery, a chemotherapeutic agent, a cytokine, RNAi, or any combination thereof.

[0195] In certain embodiments, a combination therapy method comprises administering a composition and further administering a radiation treatment or a surgery. Radiation therapy is well-known in the art and includes X-ray therapies, such as gamma-irradiation, and radiopharmaceutical therapies. Surgeries and surgical techniques appropriate to treating a given disease or disorder in a subject are well-known to those of ordinary skill in the art.

[0196] In certain embodiments, a combination therapy method comprises administering composition and further administering a chemotherapeutic agent. A chemotherapeutic agent includes, but is not limited to, an inhibitor of chromatin function, a topoisomerase inhibitor, a microtubule inhibiting drug, a DNA damaging agent, an antimetabolite (such as folate antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), a DNA synthesis inhibitor, a DNA interactive agent (such as an intercalating agent), and a DNA repair inhibitor. Illustrative chemotherapeutic agents include, without limitation, the following groups: anti- metabolites / anti-proliferative agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2- chlorodeoxyadenosine (cladribine)); antiproliferative / antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, Cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, temozolamide, teniposide, triethylenethiophosphoramide and etoposide (VP 16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L- asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative / antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates -busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes — dacarbazinine (DTIC); antiproliferative / antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti -angiogenic compounds (TNP470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti-sense oligonucleotides; antibodies (trastuzumab, rituximab); chimeric antigen receptors; cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11) and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prenisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers, toxins such as Cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, or diphtheria toxin, and caspase activators; and chromatin disruptors.

[0197] Cytokines can be used to manipulate host immune response. See, e.g., Floros & Tarhini, Semin. Oncol. 42(4):539-548, 2015. Cytokines useful for some thereapeutic applications include, for example, IFN-a, IL-2, IL-3, IL-4, IL-10, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-24, and GM-CSF, singly or in any combination with the binding proteins or cells expressing the same of this disclosure.

[0198] In certain embodiments, the subject is receiving, has received, or will receive one or more of: (i) chemotherapy; (ii) radiation therapy; (iii) an inhibitor of an immune suppression component; (iv) an agonist of a stimulatory immune checkpoint agent; (v) RNAi; (vi) a cytokine; (vii) a surgery; (viii) a monoclonal antibody and / or an antibody-drug conjugate; or (ix) any combination of (i)-(viii), in any order.

[0199] Also provided herein are uses of any of the presently disclosed polypeptide dimers, polypeptides, polynucleotides, vectors, host cells, compositions, or unit doses, for use in the treatment of a disease or disorder in a subject, wherein the disease or condition is optionally characterized by expression of CD 19 and / or BCMA, preferably CD 19 and BCMA, or wherein the disease or condition is optionally characterized by expression of CD 19 and / or CD20, preferably CD 19 and CD20.

[0200] Also provided herein are uses of any of the presently disclosed polypeptide dimers, polypeptides, fusion polypeptides, polynucleotides, vectors, host cells, compositions, or unit doses, for use in the manufacture of a medicament for the treatment of a disease or condition in a subject, wherein the disease is characterized by expression of CD 19 and / or BCMA, preferably CD 19 and BCMA, or wherein the disease or condition is optionally characterized by expression of CD 19 and / or CD20, preferably CD 19 and CD20.

[0201] The present disclosure also provides the following non-limiting enumerated Embodiments.

[0202] Embodiment 1. A polypeptide dimer comprising a first polypeptide and a second polypeptide, wherein: the first polypeptide comprises: a BCMA-specific single-chain variable fragment (scFv) comprising the heavy chain variable domain (VH) amino acid sequence QIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPA YAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSS, and the light chain variable domain (VL) amino acid sequence DIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTG VP ARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIK, wherein, optionally, the scFv is in a VH-linker-VL orientation; and a T cell receptor (TCR) constant domain; and the second polypeptide comprises: a CD19-specific single-chain variable fragment (scFv) comprising the VL amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPS RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT, and the VH amino acid sequence EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYY NSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVT VSS, wherein, optionally, the scFv is in a VL-linker-VH orientation; and a T cell receptor (TCR) constant domain, wherein (1) the TCR constant domain of the first polypeptide is a human TCR alpha chain constant domain (Ca) or an engineered variant thereof, and the TCR constant domain of the second polypeptide is a human TCR beta chain constant domain (CP) or an engineered variant thereof, or (2) the TCR constant domain of the first polypeptide is a human TCR beta chain constant domain (CP) or an engineered variant thereof and the TCR constant domain of the second polypeptide is a human TCR alpha chain constant domain (Ca) or an engineered variant thereof.

[0203] Embodiment 2. The polypeptide dimer of Embodiment 1, wherein the amino acid sequence of the scFv of the first polypeptide is QIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPA YAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSS GGGGSGGGGSGGGGSDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPG QPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGT KLEIK (SEQ ID NO:213).

[0204] Embodiment 3. The polypeptide dimer of Embodiment 1 or 2, wherein the amino acid sequence of the scFv of the second polypeptide is DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPS RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEG STKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSS.

[0205] Embodiment 4. The polypeptide dimer of any one of Embodiments 1-3, wherein the amino acid sequence of the TCR constant domain of the first polypeptide is

[0206] DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSG VCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDR AKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAM VKRKDF, and the amino acid sequence of the TCR constant domain of the second polypeptide is

[0207] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSM DFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNL LVI VLRI LLLK V AGFN LLMTLRLWS S .

[0208] Embodiment 5. The polypeptide dimer of any one of Embodiments 1-3, wherein the amino acid sequence of the TCR constant domain of the second polypeptide is

[0209] DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSG VCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDR AKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAM VKRKDF, and the amino acid sequence of the TCR constant domain of the first polypeptide is

[0210] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSM DFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNL LVI VLRI LLLK V AGFN LLMTLRLWS S .

[0211] Embodiment 6. The polypeptide of any one of Embodiments 1-5, wherein: the first polypeptide comprises, consists essentially of, or consists of the amino acid sequence QIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTE TREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTS VTVSSGGGGSGGGGSGGGGSDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWY QQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRT FGGGTKLEIKDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGK EVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDE WTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALV LMAMVKRKDF (SEQ ID NO:214); and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPS RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEG STKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRS MDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQN LLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:209).

[0212] Embodiment 7. The polypeptide of any one of Embodiments 1-5, wherein: the first polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0213] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLH SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKP GSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGV IWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMD YWGQGTSVTVSSDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVN GKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEN DEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSA LVLMAMVKRKDF (SEQ ID NO:215); and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequence QIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPA YAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSS GGGGSGGGGSGGGGSDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPG QPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGT KLEIKIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSM DFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNL LVI VLRI LLLK V AGFN LLMTLRLWS S .

[0214] Embodiment 8. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence

[0215] MLLLVTSLLLCELPHPAFLLIPQIQLVQSGPELKKPGETVKISCKASGYTFTDYSIN WVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTAT YFCALDYSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVLTQSPPSLAMSLGKR ATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTID PVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKDLNKVFPPEVAVFEPSEAEISHTQKATLV CLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQ NPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSAT ILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATNFSLLKQAGDVEENPGPM LLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKP DGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGG TKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSW IRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTA1YYCA KHYYYGGSYAMDYWGQGTSVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVS QSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSC DVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS.

[0216] Embodiment 9. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence

[0217] MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNW YQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLP YTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLP DYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDD TAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDLNKVFPPEVAVFEPSEAEISHTQKAT LVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVL SATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATNFSLLKQAGDVEENP GPMLLLVTSLLLCELPHPAFLLIPQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWV KRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYF CALDYSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVLTQSPPSLAMSLGKRATI SCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPV EEDDVAVYYCLQSRTIPRTFGGGTKLEIKIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT NVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSP ESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS.

[0218] Embodiment 10. The polypeptide of Embodiment 8 or 9, further comprising, fused to the C-terminal end thereof, the amino acid sequence RAKRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIG EFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLL IQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKN LCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCR NVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYID GPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIP SIATGMVGALLLLLVVALGIGLFMRRR.

[0219] Embodiment 11. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence

[0220] MLLLVTSLLLCELPHPAFLLIPQIQLVQSGPELKKPGETVKISCKASGYTFTDYSIN WVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTAT YFCALDYSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVLTQSPPSLAMSLGKR ATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTID PVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKDLNKVFPPEVAVFEPSEAEISHTQKATLV CLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQ NPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSAT ILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATNFSLLKQAGDVEENPGPM LLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKP DGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGG TKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSW IRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCA KHYYYGGSYAMDYWGQGTSVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVS QSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSC DVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSSRAKRLEGGGEGRG SLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKH FKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAF ENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLF GTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCN LLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVM GENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLL VVALGIGLFMRRR.

[0221] Embodiment 12. A polypeptide dimer comprising a first polypeptide and a second polypeptide, wherein: the first polypeptide comprises: a CD20-specific single-chain variable fragment (scFv) comprising the heavy chain variable domain (VH) amino acid sequence EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGD TSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGA GTTVTVSS, and the light chain variable domain (VL) amino acid sequence DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPA RFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIK; and a T cell receptor (TCR) constant domain; and the second polypeptide comprises: a CD19-specific single-chain variable fragment (scFv) comprising the VL amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPS RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT, and the VH amino acid sequence EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYY NSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVT VSS; and a T cell receptor (TCR) constant domain, wherein (1) the TCR constant domain of the first polypeptide is a human TCR alpha chain constant domain (Ca) or an engineered variant thereof, and the TCR constant domain of the second polypeptide is a human TCR beta chain constant domain (CP) or an engineered variant thereof, or (2) the TCR constant domain of the first polypeptide is a human TCR beta chain constant domain (CP) or an engineered variant thereof and the TCR constant domain of the second polypeptide is a human TCR alpha chain constant domain (Ca) or an engineered variant thereof. Embodiment 13. The polypeptide dimer of Embodiment 12, wherein the amino acid sequence of the scFv of the first polypeptide is: DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPA RFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSGGGSG GGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIY PGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFD VWGAGTTVTVSS; or EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGD TSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGA GTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMD WYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSF NPPTFGGGTKLEIK.

[0222] Embodiment 14. The polypeptide dimer of Embodiment 12 or 13, wherein the amino acid sequence of the scFv of the second polypeptide is: DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPS RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEG STKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSS; or EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYY NSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVT VSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQK PDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGG GTKLEIT.

[0223] Embodiment 15. The polypeptide dimer of any one of Embodiments 12-14, wherein the amino acid sequence of the TCR constant domain of the first polypeptide is

[0224] DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSG VCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDR AKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAM VKRKDF, and the amino acid sequence of the TCR constant domain of the second polypeptide is IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSM DFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNL LVI VLRI LLLK V AGFN LLMTLRLWS S .

[0225] Embodiment 16. The polypeptide dimer of any one of Embodiments 12-14, wherein the amino acid sequence of the TCR constant domain of the second polypeptide is

[0226] DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSG VCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDR AKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAM VKRKDF, and the amino acid sequence of the TCR constant domain of the first polypeptide is

[0227] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSM DFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNL LVI VLRI LLLK V AGFN LLMTLRLWS S .

[0228] Embodiment 17. The polypeptide of any one of Embodiments 12-16, wherein:

[0229] (i) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0230] DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLAS GVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSG GGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI GAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSY WFFDVWGAGTTVTVSSDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELS WWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFY GLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYA VLVSALVLMAMVKRKDF; and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0231] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLH SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKP GSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGV IWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMD YWGQGTSVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKC VLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDT NLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS; (ii) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0232] DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLAS GVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSG GGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI GAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSY WFFDVWGAGTTVTVSSDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELS WWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFY GLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYA VLVSALVLMAMVKRKDF; and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0233] EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNW YQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLP YTFGGGTKLEITIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCV LDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTN LNFQNLL VIVLRILLLKVAGFNLLMTLRLWS S;

[0234] (iii) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0235] EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYP GNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDV WGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPAILSASPGEKVTMTCRASSSVN YMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQ WSFNPPTFGGGTKLEIKDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELS WWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFY GLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYA VLVSALVLMAMVKRKDF; and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0236] EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE

[0237] TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNW YQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLP YTFGGGTKLEITIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCV LDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTN LNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS; or

[0238] (iv) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0239] EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYP GNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDV WGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPAILSASPGEKVTMTCRASSSVN YMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQ WSFNPPTFGGGTKLEIKDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELS WWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFY GLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYA VLVSALVLMAMVKRKDF; and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0240] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLH SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKP GSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGV IWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMD YWGQGTSVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKC VLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDT NLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS.

[0241] Embodiment 18. The polypeptide of any one of Embodiments 12-16, wherein:

[0242] (i) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0243] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLH SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKP GSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGV IWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMD YWGQGTSVTVSSDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVN GKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEN DEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSA

[0244] LVLMAMVKRKDF; and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0245] DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLAS GVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSG GGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI GAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSY WFFDVWGAGTTVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYI TDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSF ETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS;

[0246] (ii) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0247] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLH SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKP GSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGV IWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMD YWGQGTSVTVSSDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVN GKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEN DEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSA LVLMAMVKRKDF; and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0248] EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYP GNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDV WGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPAILSASPGEKVTMTCRASSSVN YMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQ WSFNPPTFGGGTKLEIKIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYI TDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSF ETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS;

[0249] (iii) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequence EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNW YQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLP YTFGGGTKLEITDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVN GKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEN DEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSA LVLMAMVKRKDF; and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0250] EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYP GNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDV WGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPAILSASPGEKVTMTCRASSSVN YMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQ WSFNPPTFGGGTKLEIKIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYI TDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSF ETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS; or

[0251] (iv) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0252] EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNW YQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLP YTFGGGTKLEITDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVN GKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEN DEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSA LVLMAMVKRKDF; and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0253] DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLAS GVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSG GGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI GAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSY WFFDVWGAGTTVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYI

[0254] TDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSF ETDTNLNFQNLLVIVLRILLLK VAGFNLLMTLRLWS S .

[0255] Embodiment 19. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence

[0256] MLLLVTSLLLCELPHPAFLLIPDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDW YQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNP PTFGGGTKLEIKGSTSGGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTF TSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLT SEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSDLNKVFPPEVAVFEPSEAEISHTQ KATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVS ATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQ GVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATNFSLLKQAGDVE ENPGPMLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLN WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL PYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSL PDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYC AKHYYYGGS YAMDYWGQGTS VT VS SIQNPDP AVYQLRDSKS SDKS VCLFTD FDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDT FFPSPES SCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWS S .

[0257] Embodiment 20. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence

[0258] MLLLVTSLLLCELPHPAFLLIPDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDW YQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNP PTFGGGTKLEIKGSTSGGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTF TSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLT SEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSDLNKVFPPEVAVFEPSEAEISHTQ KATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVS ATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQ GVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATNFSLLKQAGDVE ENPGPMLLLVTSLLLCELPHPAFLLIPEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGV SWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYY CAKHYYYGGSYAMDYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSAS LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLT ISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITIQNPDPAVYQLRDSKSSDKSVCLFTDFD SQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFF PSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS.

[0259] Embodiment 2E A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence

[0260] MLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYN MHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDS ADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPA ILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGT SYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKDLNKVFPPEVAVFEPSEAEISHT QKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRV SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQ QGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATNFSLLKQAGDV EENPGPMLLLVTSLLLCELPHPAFLLIPEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYG VSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIY YCAKHYYYGGSYAMDYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSA SLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSL TISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITIQNPDPAVYQLRDSKSSDKSVCLFTDF DSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTF FPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS.

[0261] Embodiment 22. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence

[0262] MLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYN MHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDS ADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPA ILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGT SYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKDLNKVFPPEVAVFEPSEAEISHT QKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRV SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQ QGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATNFSLLKQAGDV EENPGPMLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLN WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL PYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSL

[0263] PDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYC AKHYYYGGS YAMDYWGQGTS VT VS SIQNPDP AVYQLRDSKS SDKS VCLFTD FDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDT FFPSPES SCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWS S .

[0264] Embodiment 23. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence

[0265] MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNW

[0266] YQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLP YTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLP DYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDD TAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDLNKVFPPEVAVFEPSEAEISHTQKAT

[0267] LVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVL SATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATNFSLLKQAGDVEENP GPMLLLVTSLLLCELPHPAFLLIPDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQ

[0268] KKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPT FGGGTKLEIKGSTSGGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTS YNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSE DSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSIQNPDPAVYQLRDSKSSDKSVCLFT

[0269] DFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPED TFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS.

[0270] Embodiment 24. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence

[0271] MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNW

[0272] YQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLP YTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLP DYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDD TAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDLNKVFPPEVAVFEPSEAEISHTQKAT

[0273] LVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVL SATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATNFSLLKQAGDVEENP

[0274] GPMLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMH WVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAD YYCARSNYYGSSYWFFDVWGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPAILS ASPGEKVTMTCRAS SS VNYMDWYQKKPGS SPKPWIYATSNL ASGVP ARF SGSGSGTS Y SLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKIQNPDPAVYQLRDSKSSDKSVCLF TDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPE DTFFPSPES SCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWS S .

[0275] Embodiment 25. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence

[0276] MLLLVTSLLLCELPHPAFLLIPEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGV SWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYY CAKHYYYGGSYAMDYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSAS LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLT ISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITDLNKVFPPEVAVFEPSEAEISHTQKATL VCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFW QNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLS ATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATNFSLLKQAGDVEENPG PMLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHW VKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADY YCARSNYYGSSYWFFDVWGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPAILSA SPGEKVTMTCRASS S VNYMDWYQKKPGS SPKPWIYATSNL ASGVP ARF SGSGSGTS YSL TISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKIQNPDPAVYQLRDSKSSDKSVCLFTD FDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDT FFPSPES SCDVKLVEKSFETDTNLNFQNLL VIVLRILLLKVAGFNLLMTLRLWS S .

[0277] Embodiment 26. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence

[0278] MLLLVTSLLLCELPHPAFLLIPEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGV SWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYY CAKHYYYGGSYAMDYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSAS LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLT ISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITDLNKVFPPEVAVFEPSEAEISHTQKATL VCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFW QNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLS ATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATNFSLLKQAGDVEENPG PMLLLVTSLLLCELPHPAFLLIPDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQK

[0279] KPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTF

[0280] GGGTKLEIKGSTSGGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTS

[0281] YNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSE

[0282] DSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSIQNPDPAVYQLRDSKSSDKSVCLFT

[0283] DFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPED TFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS.

[0284] Embodiment 27. The polypeptide of any one of Embodiments 19-26, further comprising, fused to the C-terminal end thereof, the amino acid sequence RAKRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIG

[0285] EFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLL

[0286] IQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKN

[0287] LCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCR

[0288] NVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYID

[0289] GPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIP

[0290] SIATGMVGALLLLLVVALGIGLFMRRR.

[0291] Embodiment 28. A polypeptide dimer comprising a first polypeptide (Polypeptide 1) that binds human CD20 and a second polypeptide (Polypeptide 2) that binds human CD 19, wherein Polypeptide 1 and Polypeptide 2 are according to Format 1, Format 2, Format 3, Format 4, Format 5, Format 6, Format 7, or Format 8, as shown in the following table: wherein:

[0292] (i) for the anti-CD20 scFv, VL is

[0293] DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYAT SNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGG

[0294] TKLEIK;

[0295] (ii) for the anti-CD20 scFv, VH is

[0296] EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI

[0297] GAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCA RSNYYGSSYWFFDVWGAGTTVTVSS;

[0298] (iii) for the anti-CD19 scFv, VL is

[0299] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHT SRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGT KLEIT; (iv) for the anti-CD19 scFv, VH is EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGV IWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYY YGGSYAMDYWGQGTSVTVSS; and

[0300] (v) the C-terminus of the anti-CD20 scFv of Polypeptide 1 is directly fused to the N- terminus of the TCR constant domain of Polypeptide 1, and the C-terminus of the anti-CD19 scFv of Polypeptide 2 is directly fused to the N-terminus of the TCR constant domain of Polypeptide 2, and wherein, optionally:

[0301] (1) each linker is independently selected from: G4S linker, as shown in Figure 1 A;

[0302] Linker 218, as shown in Figure IB; and Linker GST, as shown in Figure 3B, wherein, further optionally, the linker of the scFv of Polypeptide 1 is Linker GST, and the linker of the scFv of

[0303] Polypeptide 2 is Linker 218, or the linker of the scFv of Polypeptide 1 is Linker 218, and the linker of the scFv of Polypeptide 2 is Linker GST; and

[0304] (2) the Ca is a human Ca or an engineered variant thereof, further optionally comprising the TRAC (S to C mut) amino acid sequence shown in Figure IB, and the CP is a human CP or an engineered variant thereof, further optionally comprising the TRBC (S to C mut) amino acid sequence shown in Figure 1 A.

[0305] Embodiment 29. A polynucleotide encoding the polypeptide dimer of any one of

[0306] Embodiments 1-7, 12-18 and 28 or the polypeptide of any one of Embodiments 8-11 and 19-27 or the amino acid sequence shown in any one of Figures 1C, ID, 2B, and 3A-3H, wherein, optionally, the polynucleotide is codon-optimized for expression in a human T cell, and / or wherein the polynucleotide comprises DNA, RNA, mRNA, circRNA, saRNA, or any combination thereof.

[0307] Embodiment 30. A vector comprising the polynucleotide of Embodiment 29.

[0308] Embodiment 31. The vector of Embodiment 30, wherein the vector is a viral vector.

[0309] Embodiment 32. The vector of Embodiment 31, wherein the viral vector is a lentiviral vector or a y-retroviral vector.

[0310] Embodiment 33. A host cell expressing: (i) the polypeptide dimer of any one of Embodiments 1-7, 12-18 and 28, wherein, optionally, the host cell further expresses the polypeptide EGFRtopt, as shown in Figure 2A; or (ii) the polypeptide of any one of Embodiments 8-11 and 19-27. Embodiment 34. A host cell comprising the polynucleotide of Embodiment 29, optionally wherein the host cell comprises, in an endogenous TRAC locus and in an endogenous TRBC locus, a splice site mutation, stop codon, or missense mutation, preferably a stop codon or a splice site mutation, reducing or preferably eliminating expression by the host cell of the endogenous TRAC and the endogenous TRBC.

[0311] Embodiment 35. A host cell comprising the vector of any one of Embodiments 30- 32.

[0312] Embodiment 36. The host cell of any one of Embodiments 33-35, wherein the host cell comprises a hematopoietic progenitor cell, an induced pluripotent stem cell, an embryonic stem cell, a hematopoietic stem cell, a peripheral blood mononuclear cell, or a human immune system cell.

[0313] Embodiment 37. The host cell of any one of Embodiments 33-36, wherein the host cell comprises a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a yb T cell, a natural killer cell, a natural killer T cell, a monocyte, or any combination thereof.

[0314] Embodiment 38. The host cell of Embodiment any one of Embodiments 33-37, wherein the host cell comprises a T cell.

[0315] Embodiment 39. The host cell of Embodiment 38, wherein the T cell comprises a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof.

[0316] Embodiment 40. The host cell of any one of Embodiments 36-39, comprising a chromosomal gene knockout or a mutation of: a TGFpRl gene locus, a TGFPR2 gene locus, a PD-1 gene locus, a CTLA4 gene locus, a LAT gene locus, a TIM-3 gene locus, a PD-L1 gene locus, a TIGIT gene locus, an A2AR gene locus, a Fas locus, a FasL gene locus, a B7-H3 gene locus, a B7-H4 gene locus, an IDO gene locus, a VISTA gene locus, a SIGLEC7 gene locus, a SIGLEC9 gene locus, a TRAC gene locus, a TRBC gene locus, a T cell receptor gene locus, a MHC (e.g. HLA) gene locus, a CBLB gene locus, a RASA2 gene locus, a UBASH3 A gene locus, a CISH gene locus, a CD4 gene locus, a CD8 gene locus, or any combination thereof, such as a TIGIT locus, a TRAC gene locus, a TRBC gene locus, and / or one or both of a CD4 gene locus and a CD8 gene locus, wherein, optionally, the host cell comprises (1) an introduced splicing site or stop codon that disrupts expression of a TRAC and is further optionally an introduced splicing site or stop codon in a TRAC gene locus, (2) an introduced splicing site or stop codon that disrupts expression of a TRBC and is further optionally an introduced splicing site or stop codon in a TRBC gene locus, or (3) both (1) and (2). Embodiment 41. The host cell of any one of Embodiments 33-40, wherein the host cell is modified (e.g., having a chromosomal knockout mutation and / or a chromosomal missense mutation and / or a chromosomal splice junction mutation; encoding an inhibitory nucleic acid such as an siRNA or an antisense oligonucleotide) to have reduced protein expression (including null expression), of an endogenous TRAC, an endogenous TRBC, an endogenous TIGIT, endogenous CD4, endogenous CD8, or any combination thereof, as compared to the unmodified host cell, wherein, optionally, the host cell comprises (1) an introduced splicing site or stop codon that disrupts expression of a TRAC and is further optionally an introduced splicing site or stop codon in a TRAC gene locus, (2) an introduced splicing site or stop codon that disrupts expression of a TRBC and is further optionally an introduced splicing site or stop codon in a TRBC gene locus, or (3) both (1) and (2).

[0317] Embodiment 42. A composition comprising: (i) the polypeptide dimer of any one of Embodiments 1-7, 12-18 and 28; and / or (ii) the polypeptide of any one of Embodiments 8-11 and 19-27, optionally comprised in a lipid composition; and / or (iii) the polynucleotide of Embodiment 29; and / or (iv) the vector of any one of Embodiments 30-32; and / or (vi) the host cell of any one of Embodiments 16-24 and 33-41, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0318] Embodiment 43. The composition of Embodiment 42, comprising (i) a composition comprising at least about 30% CD4+ T host cells, combined with (ii) a composition comprising at least about 30% CD8+ T host cells, in about a 1 : 1 ratio.

[0319] Embodiment 44. A method of treating a disease or condition characterized by expression of CD 19 and / or BCMA, preferably both CD 19 and BCMA, in a subject, the method comprising administering to the subject an effective amount of: (i) the polypeptide dimer of any one of Embodiments 1-7 and / or (ii) the polypeptide of any one of Embodiments 8-11; and / or (iii) the polynucleotide of Embodiment 29, optionally comprised in a lipid formulation or composition; and / or (iv) the vector of any one of Embodiments 30-32; and / or (v) the host cell of any one of Embodiments 33-41; and / or (vi) the composition of Embodiment 42 or 43.

[0320] Embodiment 45. The method of Embodiment 44, wherein the disease or condition comprises an autoimmune disease or condition, optionally wherein the subject does not receive lymphodepleting therapy (e.g., lymphodepleting chemotherapy) or has not received lymphodepleting therapy (e.g., lymphodepleting chemotherapy), prior to the administering.

[0321] Embodiment 46. The method of Embodiment 44, wherein the disease or condition comprises systemic lupus erythematosus (SLE). Embodiment 47. The method of Embodiment 44, wherein the disease or condition comprises a cancer or a tumor and is optionally a hematological malignancy.

[0322] Embodiment 48. A method of treating a disease or condition characterized by expression of CD 19 and / or CD20, preferably both CD 19 and CD20, in a subject, the method comprising administering to the subject an effective amount of: (i) the polypeptide dimer of any one of Embodiments 12-18 and 28 and / or (ii) the polypeptide of any one of Embodiments 19-27; and / or (iii) the polynucleotide of Embodiment 29, optionally comprised in a lipid formulation or composition; and / or (iv) the vector of any one of Embodiments 30-32; and / or (v) the host cell of any one of Embodiments 33-41; and / or (vi) the composition of Embodiment 42 or 43.

[0323] Embodiment 49. The method of Embodiment 48, wherein the disease or condition comprises a cancer or a tumor and is optionally a hematological malignancy.

[0324] Embodiment 50. Any one or more for the following for use as a medicament, or for use in the manufacture of a medicament for treating a disease or disorder characterized by expression of CD 19, BCMA, CD20, CD 19 and BCMA, or CD 19 and CD20: (i) the polypeptide dimer of any one of Embodiments 1-7, 12-18, and 28 and / or (ii) the polypeptide of any one of Embodiments 8-11 and 19-27; and / or (iii) the polynucleotide of Embodiment 29, optionally comprised in a lipid formulation or composition; and / or (iv) the vector of any one of Embodiments 30-32; and / or (v) the host cell of any one of Embodiments 33-41; and / or (vi) the composition of Embodiment 42 or 43.

[0325] Embodiment la. A polypeptide dimer comprising a first polypeptide and a second polypeptide, wherein: the first polypeptide comprises: a BCMA-specific single-chain variable fragment (scFv) comprising the heavy chain variable domain (VH) amino acid sequence (SEQ ID NO: 153), and the light chain variable domain (VL) amino acid sequence (SEQ ID NO: 155), wherein, optionally, the scFv is in a VH-linker-VL orientation; and a T cell receptor (TCR) constant domain; and the second polypeptide comprises: a CD19-specific single-chain variable fragment (scFv) comprising the VL amino acid sequence (SEQ ID NO: 159), and the VH amino acid sequence (SEQ ID NO: 161), wherein, optionally, the scFv is in a VL-linker-VH orientation; and a T cell receptor (TCR) constant domain, wherein (1) the TCR constant domain of the first polypeptide is a human TCR alpha chain constant domain (Ca) or an engineered variant thereof, and the TCR constant domain of the second polypeptide is a human TCR beta chain constant domain (CP) or an engineered variant thereof, or (2) the TCR constant domain of the first polypeptide is a human TCR beta chain constant domain (CP) or an engineered variant thereof and the TCR constant domain of the second polypeptide is a human TCR alpha chain constant domain (Ca) or an engineered variant thereof, optionally wherein a polynucleotide encoding the polypeptide dimer encodes the amino acid sequence set forth in SEQ ID NO:228.

[0326] Embodiment 2a. The polypeptide dimer of Embodiment la, wherein the amino acid sequence of the scFv of the first polypeptide is (SEQ ID NO:213).

[0327] Embodiment 3 a. The polypeptide dimer of Embodiment la or 2a, wherein the amino acid sequence of the scFv of the second polypeptide is (SEQ ID NO:208).

[0328] Embodiment 4a. The polypeptide dimer of any one of Embodiments la-3 a, wherein the amino acid sequence of the TCR constant domain of the first polypeptide is (SEQ ID NO:59), and the amino acid sequence of the TCR constant domain of the second polypeptide is (SEQ ID NO:57).

[0329] Embodiment 5a. The polypeptide dimer of any one of Embodiments la-3a, wherein the amino acid sequence of the TCR constant domain of the second polypeptide is (SEQ ID NO:59), and the amino acid sequence of the TCR constant domain of the first polypeptide is (SEQ ID NO:57).

[0330] Embodiment 6a. The polypeptide of any one of Embodiments la-5a, wherein: the first polypeptide comprises, consists essentially of, or consists of the amino acid sequence (SEQ ID NO:214); and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequence (SEQ ID NO:209).

[0331] Embodiment 7a. The polypeptide of any one of Embodiments la-5a, wherein: the first polypeptide comprises, consists essentially of, or consists of the amino acid sequence (SEQ ID NO:215); and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequence (SEQ ID NO:216).

[0332] Embodiment 8a. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence

[0333] (SEQ ID NO: 163), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO: 163.

[0334] Embodiment 9a. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence (SEQ ID NO: 164), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO: 164. Embodiment 10a. The polypeptide of Embodiment 8a or 9a, further comprising, fused to the C-terminal end thereof, the amino acid sequence (SEQ ID NO:217), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO :217.

[0335] Embodiment I la. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence (SEQ ID NO: 167), or a variant thereof wherein one or both instances of the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO: 167.

[0336] Embodiment 12a. A polypeptide dimer comprising a first polypeptide and a second polypeptide, wherein: the first polypeptide comprises: a CD20-specific single-chain variable fragment (scFv) comprising the heavy chain variable domain (VH) amino acid sequence EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGD TSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGA GTTVTVSS (SEQ ID NO:205), and the light chain variable domain (VL) amino acid sequence DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPA RFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIK (SEQ ID NO:203); and a T cell receptor (TCR) constant domain; and the second polypeptide comprises: a CD19-specific single-chain variable fragment (scFv) comprising the VL amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPS RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT (SEQ ID NO: 159), and the VH amino acid sequence EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYY NSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVT VSS (SEQ ID NO: 161); and a T cell receptor (TCR) constant domain, wherein (1) the TCR constant domain of the first polypeptide is a human TCR alpha chain constant domain (Ca) or an engineered variant thereof, and the TCR constant domain of the second polypeptide is a human TCR beta chain constant domain (CP) or an engineered variant thereof, or (2) the TCR constant domain of the first polypeptide is a human TCR beta chain constant domain (CP) or an engineered variant thereof and the TCR constant domain of the second polypeptide is a human TCR alpha chain constant domain (Ca) or an engineered variant thereof.

[0337] Embodiment 13 a. The polypeptide dimer of Embodiment 12a, wherein the amino acid sequence of the scFv of the first polypeptide is: (SEQ ID NO:206); or (SEQ ID NO:218).

[0338] Embodiment 14a. The polypeptide dimer of Embodiment 12a or 13 a, wherein the amino acid sequence of the scFv of the second polypeptide is: (SEQ ID NO:208); or (SEQ ID NO:220).

[0339] Embodiment 15a. The polypeptide dimer of any one of Embodiments 12a-14a, wherein the amino acid sequence of the TCR constant domain of the first polypeptide is (SEQ ID NO: 156), and the amino acid sequence of the TCR constant domain of the second polypeptide is (SEQ ID NO: 162).

[0340] Embodiment 16a. The polypeptide dimer of any one of Embodiments 12a- 14a, wherein the amino acid sequence of the TCR constant domain of the second polypeptide is (SEQ ID NO: 156), and the amino acid sequence of the TCR constant domain of the first polypeptide is IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSM

[0341] DFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNL LVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 162).

[0342] Embodiment 17a. The polypeptide dimer of any one of Embodiments 12a-16a, wherein:

[0343] (i) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0344] (SEQ ID NO:207); and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0345] (SEQ ID NO:209);

[0346] (ii) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0347] (SEQ ID NO:207); and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0348] (SEQ ID NO:220);

[0349] (iii) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0350] (SEQ ID NO:221); and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0351] (SEQ ID NO:220); or

[0352] (iv) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0353] (SEQ ID NO:221); and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0354] (SEQ ID NO:209).

[0355] Embodiment 18a. The polypeptide dimer of any one of Embodiments 12a- 16a, wherein:

[0356] (i) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0357] (SEQ ID NO:215); and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0358] (SEQ ID NO:222);

[0359] (ii) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0360] (SEQ ID NO:215); and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0361] (SEQ ID NO:223);

[0362] (iii) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0363] (SEQ ID NO:224); and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0364] (SEQ ID NO:223); or

[0365] (iv) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequence

[0366] (SEQ ID NO:224); and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequence (SEQ ID NO:222).

[0367] Embodiment 19a. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence (SEQ ID NO: 195), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO: 195.

[0368] Embodiment 20a. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence (SEQ ID NO: 196), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO: 196.

[0369] Embodiment 21a. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence (SEQ ID NO: 197), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO: 197.

[0370] Embodiment 22a. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence (SEQ ID NO: 198), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO: 198.

[0371] Embodiment 23a. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence (SEQ ID NO: 199), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO: 199.

[0372] Embodiment 24a. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence (SEQ ID NO:200), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO:200.

[0373] Embodiment 25a. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence (SEQ ID NO:201), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO:201.

[0374] Embodiment 26a. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence (SEQ ID NO:202), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO:202.

[0375] Embodiment 27a. The polypeptide of any one of Embodiments 19a-26a, further comprising, fused to the C-terminal end thereof, the amino acid sequence RAKRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIG EFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLL IQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKN LCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCR NVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYID GPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIP SIATGMVGALLLLLVVALGIGLFMRRR (SEQ ID NO:217), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO:217.

[0376] Embodiment 28a. A polypeptide dimer comprising a first polypeptide (Polypeptide 1) that binds human CD20 and a second polypeptide (Polypeptide 2) that binds human CD 19, wherein Polypeptide 1 and Polypeptide 2 are according to Format 1, Format 2, Format 3, Format 4, Format 5, Format 6, Format 7, or Format 8, as shown in the following table: wherein:

[0377] (vi) for the anti-CD20 scFv, VL is

[0378] DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYAT SNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGG TKLEIK (SEQ ID NO:203);

[0379] (vii) for the anti-CD20 scFv, VH is

[0380] EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI GAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCA RSNYYGSSYWFFDVWGAGTTVTVSS (SEQ ID NO:205);

[0381] (viii) for the anti-CD19 scFv, VL is

[0382] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHT SRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGT KLEIT (SEQ ID NO: 159);

[0383] (ix) for the anti-CD19 scFv, VH is

[0384] EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGV IWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYY YGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 161); and

[0385] (x) the C-terminus of the anti-CD20 scFv of Polypeptide 1 is directly fused to the N- terminus of the TCR constant domain of Polypeptide 1, and the C-terminus of the anti-CD19 scFv of Polypeptide 2 is directly fused to the N-terminus of the TCR constant domain of Polypeptide 2, and wherein, optionally:

[0386] (1) each linker is independently selected from: G4S linker, as shown in Figure 1 A; Linker 218, as shown in Figure IB; and Linker GST, as shown in Figure 3B, wherein, further optionally, the linker of the scFv of Polypeptide 1 is Linker GST, and the linker of the scFv of Polypeptide 2 is Linker 218, or the linker of the scFv of Polypeptide 1 is Linker 218, and the linker of the scFv of Polypeptide 2 is Linker GST; and

[0387] (2) the Ca is a human Ca or an engineered variant thereof, further optionally comprising the TRAC (S to C mut) amino acid sequence shown in Figure IB, and the CP is a human CP or an engineered variant thereof, further optionally comprising the TRBC (S to C mut) amino acid sequence shown in Figure 1 A.

[0388] Embodiment 29a. A polynucleotide encoding the polypeptide dimer of any one of Embodiments la-7a, 12a-18a and 28a or the polypeptide of any one of Embodiments 8a-l la and 19a-27a or the amino acid sequence shown in any one of Figures 1C, ID, 2B, and 3A-3H, wherein, optionally, the polynucleotide is codon-optimized for expression in a human T cell, and / or wherein the polynucleotide comprises DNA, RNA, mRNA, circRNA, saRNA, or any combination thereof.

[0389] Embodiment 30a. A polynucleotide encoding the polypeptide dimer of any one of Embodiments la-7a, 12a-18a, and 28a, wherein the encoded first polypeptide, the encoded second polypeptide, or both, further comprises the signal peptide MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO:229) N-terminal to the scFv.

[0390] Embodiment 31a. A polynucleotide encoding a variant of the polypeptide of any one of Embodiments 9a-l la and 19a-27a, wherein the variant comprises the signal peptide MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO:229) in place of one or more of the signal peptide(s) in the amino acid sequence.

[0391] Embodiment 32a. A polynucleotide encoding the polypeptide dimer of any one of Embodiments la-7a, 12a-18a, and 30a, wherein the polynucleotide does not encode a protease cleavage site, optionally a furin cleavage site, further optionally a RAKR sequence, disposed between a polynucleotide encoding a sequence comprising the first polypeptide and a polynucleotide encoding a sequence comprising the second polypeptide, wherein, optionlly, the polynucleotide encoding a sequence comprising the first polypeptide and the polynucleotide encoding a sequence comprising the second polypeptide are separated by a GSG-2A sequence, optionally the amino acid sequence set forth in SEQ ID NO: 158.

[0392] Embodiment 33a. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence MDMRVPAQLLGLLLLWLRGARCQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINW VKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATY FCALDYSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVLTQSPPSLAMSLGKRAT ISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPV EEDDVAVYYCLQSRTIPRTFGGGTKLEIKDLNKVFPPEVAVFEPSEAEISHTQKATLVCL ATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNP RNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATIL YEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQAGDVEENPGPMDMRVP AQLLGLLLLWLRGARCDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGT VKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKL EITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQ PPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKH YYYGGSYAMDYWGQGTSVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQS KDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDV KLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:228).

[0393] Embodiment 34a. A polynucleotide encoding the amino acid sequence set forth in SEQ ID NO:228, wherein the polynucleotide comprises the polynucleotide sequence set forth in SEQ ID NO:227, and optionally further encodes the amino acid sequence set forth in SEQ ID NO:226 and comprises the polynucleotide sequence set forth in SEQ ID NO:225.

[0394] Embodiment 35a. An expression construct encoding (i) the polypeptide dimer of any one of Embodiments la-7a, 12a-18a, and 28a, or the polypeptide of any one of Embodiments 9a- 11a, 19a-27a, and 33a, and (ii) at least one additional expression product of interest, wherein: the expression construct comprises an EFla promoter (SEQ ID NO:7) operably linked to a polynucleotide encoding (i) and a MNDu3 promoter (SEQ ID NO: 8) operably linked to a polynucleotide encoding (ii); or the expression construct comprises a MNDu3 promoter (SEQ ID NO:8) operably linked to a polynucleotide encoding (i) and an EFla promoter (SEQ ID NO: 7) operably linked to a polynucleotide encoding (ii), wherein, optionally, the EFla promoter and the MNDu3 promoter are disposed on opposite strands of the expression construct, and are preferably oriented in opposite directions, wherein, further optionally, the at least one additional expression product of interest comprises, consists essentially of, or consists of, a tag (e.g., SEQ ID NO: 166 or SEQ ID NO:226), a CD3 polypeptide, or a CD3 fusion polypeptide.

[0395] Embodiment 36a. A vector comprising the polynucleotide of any one of Embodiments 30a-32a and 34, or the expression construct of Embodiment 35a.

[0396] Embodiment 37a. The vector of Embodiment 36a, wherein the vector is a viral vector.

[0397] Embodiment 38a. The vector of Embodiment 37a, wherein the viral vector is a lentiviral vector or a y-retroviral vector.

[0398] Embodiment 39a. A host cell expressing: (i) the polypeptide dimer of any one of Embodiments la-7a, 12a-18a and 28a, and 29a, wherein, optionally, the host cell further expresses the polypeptide EGFRtopt, as shown in Figure 2A; or (ii) the polypeptide of any one of Embodiments 8a-l la and 19a-27a.

[0399] Embodiment 40a. A host cell comprising the polynucleotide of any one of

[0400] Embodiments 30a-32a and 34a, optionally wherein the host cell comprises, in an endogenous

[0401] TRAC locus and in an endogenous TRBC locus, a splice site mutation, stop codon, or missense mutation, preferably a stop codon or a splice site mutation, reducing or preferably eliminating expression by the host cell of the endogenous TRAC and the endogenous TRBC.

[0402] Embodiment 41a. A host cell comprising the expression construct of Embodiment

[0403] 35a.

[0404] Embodiment 42a. A host cell comprising the vector of any one of Embodiments 36a-

[0405] 38a.

[0406] Embodiment 43 a. The host cell of any one of Embodiments 39a-42a, wherein the host cell comprises a hematopoietic progenitor cell, an induced pluripotent stem cell, an embryonic stem cell, a hematopoietic stem cell, a peripheral blood mononuclear cell, or a human immune system cell.

[0407] Embodiment 44a. The host cell of any one of Embodiments 39a-43a, wherein the host cell comprises a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a yb T cell, a natural killer cell, a natural killer T cell, a monocyte, or any combination thereof. Embodiment 45a. The host cell of any one of Embodiments 39a-44a, wherein the host cell comprises a T cell, preferably a human T cell, optionally a human CD8+ T cell, a human CD4 + T cell, or both.

[0408] Embodiment 46a. The host cell of Embodiment 45a, wherein the T cell comprises a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof.

[0409] Embodiment 47a. The host cell of any one of Embodiments 33a-46a, comprising a chromosomal gene knockout or a mutation of: a TGFpRl gene locus, a TGFPR2 gene locus, a PD-1 gene locus, a CTLA4 gene locus, a LAT gene locus, a TIM-3 gene locus, a PD-L1 gene locus, a TIGIT gene locus, an A2AR gene locus, a Fas locus, a FasL gene locus, a B7-H3 gene locus, a B7-H4 gene locus, an IDO gene locus, a VISTA gene locus, a SIGLEC7 gene locus, a SIGLEC9 gene locus, a TRAC gene locus, a TRBC gene locus, a T cell receptor gene locus, a MHC (e.g. HLA) gene locus, a CBLB gene locus, a RASA2 gene locus, a UBASH3 A gene locus, a CISH gene locus, a CD4 gene locus, a CD8 gene locus, or any combination thereof, such as a TIGIT locus, a TRAC gene locus, a TRBC gene locus, and / or one or both of a CD4 gene locus and a CD8 gene locus, wherein, optionally, the host cell comprises (1) an introduced splicing site or stop codon that disrupts expression of a TRAC and is further optionally an introduced splicing site or stop codon in a TRAC gene locus, (2) an introduced splicing site or stop codon that disrupts expression of a TRBC and is further optionally an introduced splicing site or stop codon in a TRBC gene locus, or (3) both (1) and (2).

[0410] Embodiment 48a. The host cell of any one of Embodiments 33a-47a, wherein the host cell is modified (e.g., having a chromosomal knockout mutation and / or a chromosomal missense mutation and / or a chromosomal splice junction mutation; encoding an inhibitory nucleic acid such as an siRNA or an antisense oligonucleotide) to have reduced protein expression (including null expression), of an endogenous TRAC, an endogenous TRBC, an endogenous TIGIT, endogenous CD4, endogenous CD8, or any combination thereof, as compared to the unmodified host cell, wherein, optionally, the host cell comprises (1) an introduced splicing site or stop codon that disrupts expression of a TRAC and is further optionally an introduced splicing site or stop codon in a TRAC gene locus, (2) an introduced splicing site or stop codon that disrupts expression of a TRBC and is further optionally an introduced splicing site or stop codon in a TRBC gene locus, or (3) both (1) and (2). Embodiment 49a. A composition comprising:

[0411] (i) the polypeptide dimer of any one of Embodiments la-7a, 12a-18a and 28a; and / or (ii) the polypeptide of any one of Embodiments 8a-l la, 19a-27a, 29a, and 33a; and / or (iii) the polynucleotide of any one of Embodiments 30a-32a and 34a, optionally comprised in a lipid composition; and / or (iv) the expression construct of Embodiment 35a, optionally comprised in a lipid composition;

[0412] (v) the vector of any one of Embodiments 36a-38a; and / or

[0413] (vi) the host cell of any one of Embodiments 39a-48a, and a pharmaceutically acceptable carrier, excipient, or diluent. Embodiment 50a. The composition of Embodiment 49a, comprising (i) a composition comprising at least about 30% CD4+ T host cells, combined with (ii) a composition comprising at least about 30% CD8+ T host cells, in about a 1 : 1 ratio.

[0414] Embodiment 51a. A method of treating a disease or condition characterized by expression of CD 19 and / or BCMA, preferably both CD 19 and BCMA, in a (preferably, human) subject, the method comprising administering to the subject an effective amount of: (i) the polypeptide dimer of any one of Embodiments la-7a, 12a-18a and 28a; and / or (ii) the polypeptide of any one of Embodiments 8a-l la, 19a-27a, 29a, and 33a; and / or (iii) the polynucleotide of Embodiment any one of Embodiments 30a-32a and 34a, optionally comprised in a lipid composition; and / or (iv) the expression construct of Embodiment 35, optionally comprised in a lipid composition; (v)the vector of any one of Embodiments 36-38; and / or (vi) the host cell of any one of Embodiments 39-48; and / or (vii) the composition of Embodiment 49 or 50.

[0415] Embodiment 52a. The method of Embodiment 51a, wherein the disease or condition comprises an autoimmune disease or condition, optionally wherein the subject does not receive lymphodepleting therapy (e.g., lymphodepleting chemotherapy) or has not received lymphodepleting therapy (e.g., lymphodepleting chemotherapy), prior to the administering.

[0416] Embodiment 53a. The method of Embodiment 52a, wherein the disease or condition comprises systemic lupus erythematosus (SLE).

[0417] Embodiment 54a. The method of Embodiment 51a, wherein the disease or condition comprises a cancer or a tumor and is optionally a hematological malignancy.

[0418] Embodiment 55a. A method of treating a disease or condition characterized by expression of CD 19 and / or CD20, preferably both CD 19 and CD20, in a (preferably, human) subject, the method comprising administering to the subject an effective amount of: (i) the polypeptide dimer of any one of Embodiments la-7a, 12a-18a and 28a; and / or (ii) the polypeptide of any one of Embodiments 8-11, 19-27, 29, and 33; and / or (iii) the polynucleotide of Embodiment any one of Embodiments 30-32 and 34, optionally comprised in a lipid composition; and / or (iv) the expression construct of Embodiment 35, optionally comprised in a lipid composition; (iv) the vector of any one of Embodiments 36-38; and / or (vi) the host cell of any one of Embodiments 39-48; and / or (vii) the composition of Embodiment 49 or 50.

[0419] Embodiment 56a. The method of Embodiment 55a, wherein the disease or condition comprises a cancer or a tumor and is optionally a hematological malignancy.

[0420] Embodiment 57a. Any one or more for the following for use as a medicament, or for use in the manufacture of a medicament for treating a disease or disorder characterized by expression of CD 19, BCMA, CD20, CD 19 and BCMA, or CD 19 and CD20: (i) the polypeptide dimer of any one of Embodiments la-7a, 12a-18a and 28a; and / or (ii) the polypeptide of any one of Embodiments 8a-l la, 19a-27a, 29a, and 33a; and / or (iii) the polynucleotide of Embodiment any one of Embodiments 30a-32a and 34a, optionally comprised in a lipid composition; and / or (iv) the expression construct of Embodiment 35a, optionally comprised in a lipid composition; (v)the vector of any one of Embodiments 36a-38a; and / or (vi) the host cell of any one of Embodiments 39a-48a; and / or (vii) the composition of Embodiment 49a or 50a.

[0421] LIST OF SEQUENCES

[0422] SEQ ID NOs.:l-6 [Reserved]

[0423] Sequence promoters:

[0424] (SEQ ID NO.:7) EFla promoter ggatctgcgatcgctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtg cctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtag tcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacagctgaagcttcgaggggctcgcatctctccttcacgcgcccgccgccctacctga ggccgccatccacgccggttgagtcgcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtctaggtaagtttaaagctcaggtcga gaccgggcctttgtccggcgctcccttggagcctacctagactcagccggctctccacgctttgcctgaccctgcttgctcaactctacgtctttgtttcgtttt ctgttctgcgccgtTACAGATCCAAGCTGTGACCGGCGCCTAC

[0425] (SEQ ID NO.: 8) MNDu3 promoter tgaaagaccccacctgtaggtttggcaagctaggatcaaggttaggaacagagagacagcagaatatgggccaaacaggatatctgtggtaagcagttc ctgccccggctcagggccaagaacagttggaacagcagaatatgggccaaacaggatatctgtggtaagcagttcctgccccggctcagggccaaga acagatggtccccagatgcggtcccgccctcagcagtttctagagaaccatcagatgtttccagggtgccccaaggacctgaaatgaccctgtgccttatt tgaactaaccaatcagttcgcttctcgcttctgttcgcgcgcttctgctccccgagctcaataaaagagcccacaacccctcactcggc

[0426] SEQ ID NOs.:9-55 [Reserved]

[0427] Other Sequences

[0428] (SEQ ID NO.:56) human TRAC from UniProt KB P01848

[0429] IQNPDPAVYQ LRDSKSSDKS VCLFTDFDSQ TNVSQSKDSD VYITDKTVLD MRSMDFKSNS

[0430] AVAWSNKSDF ACANAFNNSI IPEDTFFPSP ESSCDVKLVE KSFETDTNLN FQNLSVIGFR ILLLKVAGFN LLMTLRLWSS

[0431] (SEQ ID NO.:57) human TRAC with Thr->Cys and LVL mutations

[0432] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAW

[0433] SNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNL LMTLRLWSS

[0434] (SEQ ID NO.:58) human TRBC1 from UniProt KB P01850

[0435] DLNKVFPPEV AVFEPSEAEI SHTQKATLVC LATGFFPDHV ELSWWVNGKE VHSGVSTDPQ

[0436] PLKEQPALND SRYCLSSRLR VSATFWQNPR NHFRCQVQFY GLSENDEWTQ DRAKPVTQIV

[0437] SAEAWGRADC GFTSVSYQQG VLSATILYEI LLGKATLYAV LVSALVLMAM VKRKDF

[0438] (SEQ ID NO.:59) human TRBC1 with Ser— >Cys mutation

[0439] DLNKVFPPEV AVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKE

[0440] QPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGR

[0441] ADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF

[0442] (SEQ ID NO.:60) human TRBC2 from UniProt KB A0A5G9

[0443] DLKNVFPPKV AVFEPSEAEI SHTQKATLVC LATGFYPDHV ELSWWVNGKE VHSGVSTDPQ

[0444] PLKEQPALND SRYCLSSRLR VSATFWQNPR NHFRCQVQFY GLSENDEWTQ DRAKPVTQIV

[0445] SAEAWGRADC GFTSESYQQG VLSATILYEI LLGKATLYAV LVSALVLMAM VKRKDSRG

[0446] (SEQ ID NO.:61) human TRBC2 with Ser— >Cys mutation

[0447] DLKNVFPPKV AVFEPSEAEI SHTQKATLVC LATGFYPDHV ELSWWVNGKE VHSGVCTDPQ PLKEQPALND SRYCLSSRLR VSATFWQNPR NHFRCQVQFY GLSENDEWTQ DRAKPVTQIV SAEAWGRADC GFTSESYQQG VLSATILYEI LLGKATLYAV LVSALVLMAM VKRKDSRG

[0448] (SEQ ID NO.:62) TRAC sequence motif

[0449] LSVIGF

[0450] (SEQ ID NO.:63) TRAC sequence motif with L-V-L mutations

[0451] LLVIVL

[0452] (SEQ ID NO.:64) TRBC1 intracellular sequence

[0453] VKRKDF

[0454] (SEQ ID NO.:65) TRBC2 intracellular sequence

[0455] MAMVKRKDSRG

[0456] (SEQ ID NOs.:66-151) [reserved]

[0457] (SEQ ID NO.: 152) GM-CSF signal peptide

[0458] MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO.: 153) VH BCMA scFv

[0459] QIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDF

[0460] RGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSS

[0461] (SEQ ID NO.: 154) G4S linker

[0462] GGGGSGGGGSGGGGS

[0463] (SEQ ID NO.: 155) VL BCMA scFv

[0464] DIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFS

[0465] GSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIK

[0466] (SEQ ID NO.: 156) TRBC (S to C mut)

[0467] DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKE

[0468] QPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGR

[0469] ADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF

[0470] (SEQ ID NO.: 157) Furin site

[0471] RAKR

[0472] (SEQ ID NO.: 158) GSG-P2A

[0473] GSGATNFSLLKQAGDVEENPGP

[0474] (SEQ ID NO.: 159) VL CD19 scFv

[0475] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSG SGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT

[0476] (SEQ ID NO.: 160) Linker 218

[0477] GSTSGSGKPGSGEGSTKG

[0478] (SEQ ID NO.: 161) VH CD19 scFv

[0479] EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKS RLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

[0480] (SEQ ID NO.: 162) TRAC (S to C mut)

[0481] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAW SNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNL LMTLRLWSS

[0482] (SEQ ID NO.:163) BiChTCR _CD19 TRAC BCMA TRBC

[0483] MLLLVTSLLLCELPHPAFLLIPQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKG LKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYW GQGTSVTVSSGGGGSGGGGSGGGGSDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWY QQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGT KLEIKDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDP QPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAE AWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATN FSLLKQAGDVEENPGPMLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDI SKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL

[0484] PYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVS WIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHY YYGGSYAMDYWGQGTSVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYI TDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTN LNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS*

[0485] (SEQ ID NO.:164) BiChTCR BCMA TRAC_CD19 TRBC MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTV KLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTS GSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVI WGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGT SVTVSSDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTD PQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA EAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGAT NFSLLKQAGDVEENPGPMLLLVTSLLLCELPHPAFLLIPQIQLVQSGPELKKPGETVKISCKASGY TFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDT ATYFCALDYSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVLTQSPPSLAMSLGKRATISC RASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAV YYCLQSRTIPRTFGGGTKLEIKIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYIT DKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNL NFQNLLVIVLRILLLKVAGFNLLMTLRLWSS*

[0486] (SEQ ID NO.: 165) T2A

[0487] LEGGGEGRGSLLTCGDVEENPGPR

[0488] (SEQ ID NO.: 166) EGFRtOPT

[0489] RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEI TGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKN LCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRG RECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPA GVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVV ALGIGLFMRRR

[0490] (SEQ ID NO.:167) BiChTCR _CD19 TRAC BCMA TRBC_EGFRtOPT

[0491] MLLLVTSLLLCELPHPAFLLIPQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKG LKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYW GQGTSVTVSSGGGGSGGGGSGGGGSDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWY QQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGT KLEIKDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDP QPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAE AWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATN FSLLKQAGDVEENPGPMLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDI SKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL PYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVS WIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHY YYGGSYAMDYWGQGTSVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYI TDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTN LNFQNLLVIVLRILLLKVAGFNLLMTLRLWSSRAKRLEGGGEGRGSLLTCGDVEENPGPRMLLL VTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTH TPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGL

[0492] RSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGC WGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCI QCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPK IPSIATGMVGALLLLLVV ALGIGLFMRRR*

[0493] Other sequences Sequence sgRNAs targeting TRAC and TRBC chains:

[0494] (SEQ ID NO.: 168) sgRNA CRISPR TRAC 1

[0495] AGAGTCTCTCAGCTGGTACA

[0496] (SEQ ID NO.: 169) sgRNA CRISPR TRAC 2

[0497] GAGAATCAAAATCGGTGAAT (SEQ ID NO.: 170) sgRNA CRISPR_TRBC 1

[0498] CAAACACAGCGACCTTGGGT

[0499] (SEQ ID NO.:171) sgRNA CRISPR_TRBC 2

[0500] GGCTCTCGGAGAATGACGAG

[0501] (SEQ ID NO.:172) sgRNA_Base_Editor_TRAC_l TTCGTATCTGTAAAACCAAG

[0502] (SEQ ID NO.: 173) sgRNA_ Base Editor TRAC 2 CTTACCTGGGCTGGGGAAGA

[0503] (SEQ ID NO.:174) sgRNA_ Base Editor TRBC l AGGTCCTCTGGAAAGGGAAG

[0504] (SEQ ID NO.:175) sgRNA_ Base Editor TRBC 2

[0505] CCACTCACCTGCTCTACCCC

[0506] Sequence sgRNAs targeting TIGIT :

[0507] (SEQ ID NO.:176) sgRNA_ Base Editor TIGIT l

[0508] GGTACCTTCCACAAGATCGG

[0509] (SEQ ID NO.: 177) sgRNA_ Base Editor TIGIT 2 GGTGGTACCTTCCACAAGAT

[0510] (SEQ ID NO.: 178) sgRNA_ Base Editor TIGIT 3 CAGGAATACCTGAGCTTTCT

[0511] (SEQ ID NO.: 179) sgRNA_ Base Editor TIGIT 4 CTGGGCCcAGGGGCTGAGGC

[0512] (SEQ ID NO.: 180) sgRNA_ Base Editor TIGIT 5 AGGTTCCAGATTCCATTGCT

[0513] (SEQ ID NO.: 181) sgRNA_ Base Editor TIGIT 6 AAGATCGGTGGCTTGTACAC

[0514] (SEQ ID NO.: 182) sgRNA_ Base Editor TIGIT 7 TCTGCTTCCTGTAGGCCCTC

[0515] (SEQ ID NO.: 183) sgRNA_ Base Editor TIGIT 8 TCTCCTCCTGATCTGGGCCC

[0516] (SEQ ID NO.: 184) sgRNA_ Base Editor TIGIT 9

[0517] CCACTCGATCCTTGAAGGAT

[0518] Other Sequences

[0519] (SEQ ID NO.: 185) CD8a leader sequence

[0520] MALPVTALLLPLALLLHAARP

[0521] (SEQ ID NO.: 186) CD8 leader sequence MRPRLWLLLAAQLTVLHGNSV

[0522] (SEQ ID NO.: 187) consensus minimal furin cleavage site sequence R-X-X-R

[0523] (SEQ ID NO.: 188) consensus minimal furin cleavage site sequence R-X-K / R-R

[0524] (SEQ ID NO.: 189) minimal furin cleavage site sequence RAKR

[0525] (SEQ ID NO.: 190) minimal furin cleavage site sequence

[0526] RARR

[0527] (SEQ ID NO.: 191) Porcine teschovirus-1 2A (P2A) self-cleaving peptide with N-terminal GSG linker GSGATNFSLLKQAGDVEENPGP

[0528] (SEQ ID NO.:192)Thoseaasigna virus 2A (T2A) self-cleaving peptide LEGGGEGRGSLLTCGDVEENPGPR

[0529] (SEQ ID NO.:193)Equine rhinitis A virus (ERAV) 2A (E2A) self-cleaving peptide QCTNYALLKLAGDVESNPGP

[0530] (SEQ ID NO.:194) Foot-and-Mouth disease virus 2A (F2A) self-cleaving peptide with N-terminal G-S-G linker

[0531] GSGVKQTLNFDLLKLAGDVESNPGP

[0532] (SEQ ID NO.: 195) BiChTCR _CD20 (VL / VH)_TRBC_CD19 (VL / VH)_TRAC

[0533] MLLLVTSLLLCELPHPAFLLIPDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPK PWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTS GGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI GAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDV WGAGTTVTVSSDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHS GVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV TQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAK RGSGATNFSLLKQAGDVEENPGPMLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTIS CRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVS LPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIY YCAKHYYYGGSYAMDYWGQGTSVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQS KDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEK SFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS*

[0534] (SEQ ID NO.: 196) BiChTCR _CD20 (VL / VH)_TRBC_CD19 (VH / VL)_TRAC

[0535] MLLLVTSLLLCELPHPAFLLIPDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPK PWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTS GGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI GAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDV WGAGTTVTVSSDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHS GVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV TQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAK RGSGATNFSLLKQAGDVEENPGPMLLLVTSLLLCELPHPAFLLIPEVKLQESGPGLVAPSQSLSVT CTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQ TDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSL SASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISN LEQEDIATYFCQQGNTLPYTFGGGTKLEITIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQS KDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEK SFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS*

[0536] (SEQ ID NO.: 197) BiChTCR _CD20 (VH / VL)_TRBC_CD19 (VH / VL)_TRAC

[0537] MLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPG QGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSY WFFDVWGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPAILSASPGEKVTMTCRASSSVN YMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNP PTFGGGTKLEIKDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHS GVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV TQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAK RGSGATNFSLLKQAGDVEENPGPMLLLVTSLLLCELPHPAFLLIPEVKLQESGPGLVAPSQSLSVT

[0538] CTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQ TDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSL SASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISN LEQEDIATYFCQQGNTLPYTFGGGTKLEITIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQS KDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEK SFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS*

[0539] (SEQ ID NO.: 198) BiChTCR _CD20 (VH / VL)_TRBC_CD19 (VL / VH)_TRAC

[0540] MLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPG QGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSY WFFDVWGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPAILSASPGEKVTMTCRASSSVN YMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNP

[0541] PTFGGGTKLEIKDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHS GVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV TQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAK RGSGATNFSLLKQAGDVEENPGPMLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTIS CRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVS LPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIY YCAKHYYYGGSYAMDYWGQGTSVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQS

[0542] KDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEK SFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS*

[0543] (SEQ ID NO.: 199) BiChTCR_ CD 19 (VL / VH)_TRBC_ CD20 (VL / VH)_TRAC

[0544] MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTV KLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTS GSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVI WGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGT SVTVSSDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTD

[0545] PQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA EAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGAT NFSLLKQAGDVEENPGPMLLLVTSLLLCELPHPAFLLIPDIVLTQSPAILSASPGEKVTMTCRASSS VNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSF NPPTFGGGTKLEIKGSTSGGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSY NMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYY CARSNYYGSSYWFFDVWGAGTTVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSK DSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKS

[0546] FETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS* (SEQ ID N0.:200) BiChTCR_ CD 19 (VL / VH)_TRBC_ CD20 (VH / VL)_TRAC

[0547] MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTV KLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTS GSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVI WGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGT SVTVSSDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTD PQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA EAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGAT NFSLLKQAGDVEENPGPMLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASG YTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSE DSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPAILSA SPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVE AEDAATYYCQQWSFNPPTFGGGTKLEIKIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSK DSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKS FETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS*

[0548] (SEQ ID NO.:201) BiChTCR_ CD19 (VH / VL)_TRBC_ CD20 (VH / VL)_TRAC

[0549] MLLLVTSLLLCELPHPAFLLIPEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKG LEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAM DYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLN WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFG GGTKLEITDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCT DPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS AEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGA TNFSLLKQAGDVEENPGPMLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKAS GYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTS EDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPAILS ASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRV EAEDAATYYCQQWSFNPPTFGGGTKLEIKIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQS KDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEK SFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS*

[0550] (SEQ ID NO.:202) BiChTCR_ CD 19 (VH / VL)_TRBC_ CD20 (VL / VH)_TRAC

[0551] MLLLVTSLLLCELPHPAFLLIPEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKG LEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAM DYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLN WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFG GGTKLEITDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCT DPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS AEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGA TNFSLLKQAGDVEENPGPMLLLVTSLLLCELPHPAFLLIPDIVLTQSPAILSASPGEKVTMTCRASS SVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWS FNPPTFGGGTKLEIKGSTSGGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTS YNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADY YCARSNYYGSSYWFFDVWGAGTTVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQS KDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEK SFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS*

[0552] (SEQ ID NO.:203) VL anti-CD20 scFv Leu 16

[0553] DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSG SGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIK (SEQ ID NO.:204) Linker GST

[0554] GSTSGGGSGGGSGGGGSS

[0555] (SEQ ID NO.:205) VH anti-CD20 scFv Leu 16

[0556] EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQ KFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSS

[0557] (SEQ ID NO.:206) anti-CD20 scFv (Leul6 VL-linker-Leul6 VH)

[0558] DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSG SGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSGGGSGGGGSSEVQLQQ SGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKA TLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSS

[0559] (SEQ ID NO.:207) anti-CD20 scFv (Leul6 VL-linker-Leul6 VH) TRBC

[0560] DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSG SGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSGGGSGGGGSSEVQLQQ SGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKA

[0561] TLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSDLNKVFPPEV AVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYC LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSY QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF

[0562] (SEQ ID NO.:208) anti-CD19 scFv (FMC63 VL-linker-FMC63 VH)

[0563] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSG SGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQES GPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKD NSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

[0564] (SEQ ID NO.:209) anti-CD19 scFv (FMC63 VL-linker-FMC63 VH) TRAC

[0565] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSG SGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQES GPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKD NSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSIQNPDPAVYQLRDS KSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFN NSIIPEDTFFPSPES SCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWS S

[0566] (SEQ ID NOs: 210-224) see Sequence Listing

[0567] (SEQ ID NO.:225) polynucleotide encoding tEGFR Opt with signal peptide

[0568] Atgctgctgctggtcacatctctgctgctgtgcgagctgccccatcctgcctttctgctgatccccagaaaagtgtgcaacggcatcggcatcggagagtt caaggacagcctgagcatcaacgccaccaacatcaagcacttcaagaactgcaccagcatcagcggcgacctgcacattctgcctgtggcctttagagg cgacagcttcacccacacacctccactggatccccaagagctggacatcctgaaaaccgtgaaagagatcaccggatttctgttgatccaagcttggccc gagaaccggacagatctgcacgccttcgagaacctggaaatcatcagaggccggaccaagcagcacggccagttttctctggctgtggtgtccctgaac atcaccagcctgggcctgagaagcctgaaagaaatcagcgacggcgacgtgatcatctccggcaacaagaacctgtgctacgccaacaccatcaactg gaagaagctgttcggcaccagcggccagaaaacaaagatcatcagcaaccggggcgagaacagctgcaaggctacaggccaagtgtgccacgctct gtgtagccctgaaggctgttggggacccgagcctagagattgcgtgtcctgcagaaacgtgtcccggggcagagaatgcgtggacaagtgcaatctgc tggaaggcgagccccgcgagttcgtggaaaacagcgagtgcatccagtgtcaccccgagtgtctgccccaggccatgaacattacctgtaccggcaga ggccccgacaactgtattcagtgcgcccactacatcgacggccctcactgcgtgaaaacatgtcctgctggcgtgatgggagagaacaacaccctcgtg tggaagtatgccgacgccggacatgtgtgccacctgtgtcaccctaattgcacctacggctgtaccggccctggcctggaaggctgtccaacaaacgga cctaagatcccctctatcgccaccggcatggttggagccctgctgcttctgctggtggtggcccttggaatcggcctgtttatgcgacggagatga

[0569] (SEQ ID NO.:226) tEGFR Opt with signal peptide MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGD SFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITS LGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPE GCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPD NCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTN GPKIPSIATGMVGALLLLLVVALGIGLFMRRR

[0570] (SEQ ID NO.:227) polynucleotide encoding CD19 / BCMA Bi-ChTCR CllD5.3scFv-TRBC_ FMC63scFv-TRAC atggacatgagagtgcccgctcaactgctgggactgctgctgctttggctgagaggtgccagatgccagatccagctggtgcagtctggacccgagctg aagaaacctggcgagacagtgaagatcagctgcaaggccagcggctacaccttcaccgactacagcatcaactgggtcaagagagcccctggcaag ggcctgaaatggatgggctggatcaacaccgaaaccagagagcccgcctacgcctacgacttcagaggcagattcgccttcagcctggaaaccagcg ccagcacagcctacctgcagatcaacaacctgaagtacgaggacaccgccacctacttttgcgccctggattacagctacgccatggactattggggcc agggcacaagcgtgacagtctcttctggtggcggaggatctggcggaggtggaagcggcggaggcggatctgatattgtgctgacacagagccctcc aagcctggccatgtctctgggaaagagagccaccatcagctgtagagccagcgagagcgtgacaatcctgggctctcacctgatccactggtatcagca gaagcccggccagcctcctacactgctgattcagctggcctccaatgtgcagacaggcgtgccagccagattttctggcagcggcagcagaaccgactt caccctgacaatcgaccccgtggaagaggacgatgtggccgtgtactactgcctgcagagccggacaatccccagaacatttggcggaggcaccaag ctggaaatcaaggacctgaacaaggtgttccctccagaggtggcagtgttcgagccttctgaggccgagatcagccacacacagaaagccacactcgt gtgcctggccaccggctttttccccgatcacgtggaactgtcttggtgggtcaacggcaaagaggtgcacagcggcgtctgtaccgatcctcagcctctg aaagagcagcccgctctgaacgacagcagatactgcctgagcagcagactgagagtgtccgccaccttctggcagaaccccagaaaccacttcaggt gccaggtgcagttctacggcctgagcgagaacgatgagtggacccaggatagagccaagcctgtgacacagatcgtgtctgccgaagcctggggcag agccgattgtggctttaccagcgtgtcctatcagcagggcgtgctgtctgccaccatcctgtatgagatcctgctgggcaaagccactctgtacgccgtgct ggtgtctgccctggtgctgatggccatggtcaagaggaaggattttggctccggcgccaccaatttcagcctgcttaaacaggccggcgacgtggaaga aaaccccggacctatggatatgcgggtgccagctcagctgctcggccttcttcttttgtggctgcggggagccagatgtgacatccagatgacccagacc accagcagcctgagcgcctctctgggagatagagtgaccatctcttgccgggccagccaggacatcagcaagtacctgaattggtatcaacaaaagcct gacggcaccgtgaagctgctgatctaccacaccagcaggctgcactctggcgtgccctctagattttccggctccggaagcggcaccgattacagcctg accatcagcaacctggaacaagaggatatcgctacctacttctgtcagcaaggcaacaccctgccttacacctttggcggcggaacaaagctcgagatc accggcagcacaagcggcagcggaaaacctggaagcggagagggctctaccaagggcgaagtgaaactgcaagagtctggccctggactggtggc cccatctcagtctctgtctgtgacctgtaccgtcagcggagtgtccctgcctgattatggcgtgtcctggatcagacagcctcctcggaaaggcctggaatg gctgggagtgatctggggcagcgagacaacctactacaacagcgccctgaagtcccggctgacaatcatcaaggacaactccaagagccaggtgttcc tgaagatgaacagcctgcagaccgacgataccgccatctactactgcgccaagcactactactacggcggctcctatgccatggattactggggacaag ggacctccgtgaccgtgtccagcattcagaatcccgatcctgccgtgtaccagctgagggacagcaagagcagcgacaagagcgtgtgtctgttcacc gatttcgacagccagaccaacgtgtcccagagcaaggacagcgacgtgtacatcaccgataagtgcgtgctggacatgcggagcatggactcaagag caactccgccgtggcctggtccaacaagagcgatttcgcctgcgccaacgcctcaacaacagcatatccccgaggacacattctcccaagtcctgag agcagctgtgatgtgaagctggtggaaaagagctcgagacagacaccaacctgaacttccagaacctgctggtcatcgtgctgcgcatcctgctgctga aagtggccggcttcaacctgctgatgaccctgagactgtggtccagctga

[0571] (SEQ ID NO.:228) CD19 / BCMA Bi-ChTCR CllD5.3scFv-TRBC_ FMC63scFv-TRAC

[0572] MDMRVPAQLLGLLLLWLRGARCQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPG KGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMD YWGQGTSVTVSSGGGGSGGGGSGGGGSDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIH WYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFG GGTKLEIKDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVC TDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIV SAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNF SLLKQAGDVEENPGPMDMRVPAQLLGLLLLWLRGARCDIQMTQTTSSLSASLGDRVTISCRASQ DISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNT LPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGV SWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHY YYGGSYAMDYWGQGTSVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYI TDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTN LNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS

[0573] (SEQ ID NO.:229) Human Ig kappa signal peptide

[0574] MDMRVPAQLLGLLLLWLRGARC EXAMPLES

[0575] EXAMPLE 1

[0576] DESIGN OF CHTCRS TARGETING BCMA AND CD 19 chTCR constructs (also referred to as ChTCRs or chimeric TCRs) are described in, e.g., PCT Publication WO / 2023 / 215725 (filed as PCT / US2023 / 066466).

[0577] Expression constructs encoding bispecific chTCR constructs targeting BCMA and CD 19 were generated using amino acid sequences shown in Figures 1-2B.

[0578] EXAMPLE 2

[0579] ANTI-CD19 x ANTI-BCMA CHTCRS FOR SYSTEMIC LUPUS ERYTHEMATOSUS

[0580] Background: Systemic lupus erythematosus (SLE) is a life-threatening autoimmune disease characterized by the dysregulated activation of the adaptive immune system resulting in organ inflammation. Abnormal B-cell activation plays a role in SLE development, and depletion of humoral immune cells represents a potential therapeutic strategy to induce disease remission1'4The adoptive transfer of T cells expressing a Chimeric Antigen Receptor (CAR) targeting CD 19, or administration bispecific T cell engagers specific for these molecules has been shown to resolve SLE and other autoimmune conditions, in small human studies4'8. However, longer follow-up is needed to determine durability of responses, and whether it is necessary to eliminate both B cells and plasma cells, which are a source of pathogenic autoantibodies, to prevent disease recurrence9. Analysis of the “autoreactome” — antibodies reactive with human peptides — in cancer patients treated with BCMA or CD19 CAR T cells has shown that targeting BCMA profoundly reduces the autoantibody repertoire, whereas targeting CD 19 has minimal effects10'13. Without wishing to be bound by theory, targeting both mature B cells and plasma cells14,15may be necessary to eliminate all potentially pathogenic cells and sustain remission in SLE. The safety of such a strategy is of paramount importance. Current mono- and bispecific CAR designs incorporate costimulation in an unnatural architecture that causes excessive cytokine release potentially leading to life-threatening toxicities, which are undesirable in autoimmunity. Moreover, currently approved CAR T cells lack a mechanism for their elimination after efficacy is achieved, rendering patients deficient in humoral immunity16'20. Additionally, bispecific CARs are less sensitive for each antigen than monospecific CARs, leading to less effective elimination of targeted cell populations14,15. Bispecific receptors that are highly sensitive for each target antigen and don’t require unnatural costimulation may be beneficial in SLE.

[0581] Improved receptors for safe and effective therapy of SLE: Bispecific chimeric T cell receptors (Bi-ChTCRs) were designed, comprising two different scFv binding domains (antiCD 19, anti-CD22; Fig. 4) linked to T cell receptor a and b constant domains, respectively (Fig. 4). Bi-ChTCRs assemble with all CD3 chains, are more sensitive than CARs, form classical immune synapses and exhibit improved proximal signaling compared to bispecific CARs. CD19 / CD22 Bi-ChTCR T cells do not produce excessive cytokines and are more effective for eliminating heterogeneous tumors that express low levels of antigen than two 4-1 / BBz CAR T cell products21. Following initial work seeking to reduce escape of antigen negative / low tumors after CAR T cell therapy for cancer14,22'25’ the potential for Bi-ChTCRs in therapy of autoimmune diseases, including SLE2’3’5’6’26’27, was explored. An important issue for autoimmune applications of CAR T cells is how to achieve timely immune reconstitution after T cell therapy. EGFRt was developed as a safety switch for eliminating CAR T cells28, and an improved version of the molecule (EGFRtopt) was generated to achieve higher cell surface expression29. In preclinical models, CAR T cells expressing EGFRtoptwere rapidly and completely eliminated by administering Cetuximab (anti-EGFR Mab)29.

[0582] Lentiviral vectors are designed that encode a CD19 / BCMA Bi-ChTCR (representative sequences shown in Figures 1 A-2B; example schematic in Figure 5) and EGFRtopt(Figure 2A) as a safety switch. Vectors are expressed in primary T cells and recognition of B cells and plasma cells is evaluated.

[0583] Humanized mouse model of SLE: Mouse models that support development of a functional human immune system (Hu-mice) have important applications in human immunology30. Hu-mice that reproduce clinical and immunologic features of SLE could inform clinical translation of novel therapeutics, including T cell therapy. In the “MISTRG6” mouse, a human / mouse homolog gene replacement strategy was used in Rag2 / gc knockout mice to provide physiologic expression of molecules that support human immune cell engraftment31’32. An alternative Hu-mouse model, the THX mouse, has been shown to have robust reconstitution of adaptive and innate immune cells, and to develop features of SLE after pristane injection33. Safety and efficacy studies of Bi-ChTCR and monospecific CD19 or BCMA CAR T cells are performed for eliminating CD 19+ B-cells and / or BCMA+ plasma cells, resolving autoantibody production, and reversing SLE pathology in THX mice. The ability to eliminate Bi-ChTCR T cells using EGFRtoptand reconstitute humoral immunity is evaluated in this model. This work shows that T cells engineered with novel Bi-ChTCRs can provide rapid and safe depletion of both B cells and plasma cells, reduce SLE pathology and eliminate autoantibodies in a humanized mouse model of SLE. Further studies investigate whether lymphodepleting chemotherapy (LD-CTX) is necessary for treatment efficacy. Certain embodiments of Bi-ChTCR constructs include EGFRtoptenabling subsequent in vivo depletion of engineered T cells to hasten reconstitution of B cells, plasma cells, and response to immunization.

[0584] Results a. Design of Bi-ChTCRs: Monospecific ChTCRs have superior Ag sensitivity compared to CARs28,34’35. Bi-ChTCRs specific for CD 19 and CD22 were constructed in which one scFv was fused to TRAC and a second scFv was fused to TRBC (Fig. 4). To avoid mispairing of the ChTCR with endogenous TCR chains and competition for CD3 molecules, base editing with optimized gRNAs was used to knock-out (ko) both endogenous TCR chains in >90% of T cells concurrent with lentiviral transduction (Fig.4). The ChTCR construct was not knocked into the TRAC locus since this would leave the potential for mispairing with TCRb35. ChTCRs were expressed in T cells as well as monospecific CD 19 and CD22 4-lBB / z CARs, and a bispecific CD19 / CD22 CAR (“Loop” CAR) (Fig. 4). Functional studies showed that T cells expressing CD19 / CD22 Bi-ChTCRs produced lower levels of proinflammatory cytokines compared to monospecific CAR T cells in response to tumor cells, which should reduce the risk of cytokine release syndrome. Although Bi-ChTCR T cells exhibit sensitive recognition of target cells expressing either target antigen, the lack of costimulation (CD28 or 4- IBB) in the receptor raised possibility that these T cells may not persist in vivo. To investigate this, Nalm-6 tumor cells that lack CD80 and CD86 and were heterogeneous for CD 19 or CD22 expression were engrafted in NSG mice. T cells engineered with the Bi-ChTCR, with monospecific CD19 or CD22 4-lBBz CARs, or with a bispecific CD19 / CD22 CAR14were adoptively transferred and tumor burden was monitored. The CD19 / CD22 Bi-ChTCR provided superior tumor elimination compared to all CAR T treated groups despite the lacking receptor intrinsic costimulation (Fig. 4). The Bi- ChTCR design proved to be resilient as a BCMA / SLAMF7 Bi-ChTCR demonstrated similar attributes, including lower cytokine levels in response to antigen high target cells, sensitive recognition of each target antigen, and superior antitumor activity against heterogenous tumors compared to monospecific and bispecific CAR T cells21. These receptors hold promise for improving the outcome of monospecific CAR T cell therapies in B cell malignancies and myeloma where outgrowth of antigen low / negative tumor is frequent14,15. b. Design of a CD19 / BCMA Bi-ChTCR: A Bi-ChTCR was designed that simultaneously targets CD 19 and BCMA to potentially eliminate B cells and plasma cells for autoimmune applications. A Bi-ChTCR construct was generated that encodes a CD 19 scFv fused to TRBC, a P2A element (immediately preceded by a RAKR sequence followed by a GSG linker), and a BCMA scFv fused to TRAC, all under control of the EFla promoter. The CD19 / BCMA Bi- ChTCR was expressed in human T cells and bound recombinant CD 19 and BCMA.

[0585] Experimental Setup

[0586] The initial CD19 / BCMA Bi-ChTCR construct did not include EGFRtopt. To provide a safety switch for SLE therapy, EGFRtoptwas added downstream of the TRBC with a P2A element (with upstream GSG linker and RAKR sequence) to separate the transgenes (Fig 5). Binding of BCMA and CD 19 by T cells expressing the Bi-ChTCR was confirmed by flow cytometry after staining with recombinant CD 19 and BCMA (Fig. 5). In additional embodiments, the MND U3 promoter is used as an alterative to EFla36.

[0587] Investigating sensitivity of CD19 / BCMA Bi-ChTCR T cells compared to monospecific CAR T cells

[0588] Normal donor T cells are transduced with the CD19 / BCMA Bi-ChTCR and with monospecific CD 19 and BCMA CARs described previously37,38. Normal B cells and plasma cells are purified from donor blood by magnetic selection using CD20 and CD138 microbeads respectively, to avoid masking of CD 19 and BCMA, cocultured in a 1 : 1 ratio with Bi-ChTCR, CD 19 and BCMA CAR T cells, and recognition measured by cytokine (IL-2, IFN-g) production. Antigen sensitivity of Bi-ChTCR and CAR T cells for CD 19 is determined using Nalm-6 cells engineered to express CD 19 at wild type (-24000 molecules / cell), medium (-2600 molecules / cell) and low (-230 molecules / cell) levels21. Antigen sensitivity of Bi-ChTCR and CAR T cells for BCMA is determined using Nalm-6 cells that express high, medium, and low levels of BCMA after transfection and single cell cloning. For these experiments cytotoxicity, cytokine release, and T cell proliferation assays are used to evaluate Bi-ChTCR and CAR T cell recognition.

[0589] To investigate whether EGFRtoptconfers susceptibility to antibody dependent cellular cytotoxicity (ADCC) in vitro, natural killer cells are isolated from normal blood using the EasySep Human NK Cell Kit (STEMCELL Technologies). To activate cytolytic function before use, NK cells are cultured in media with 10 ng / ml IL-15 overnight28,29. CD19 / BCMA Bi-ChTCR and Bi-ChTCR / EGFRtopttransduced T cells are counted, added to a 96 well plate, and incubated with cetuximab or media. IL-15 primed NK cells are added at a 10:1 ratio of NK:Bi-ChTCR T cells and the plate is gently centrifuged to bring effector and target cells together. After coculture, any remaining Bi-ChTCR T cells are identified by flow cytometry after staining with anti-CD3, CD19-Fc, and BCMA-Fc in conditions treated or not treated with cetuximab.

[0590] Bi-ChTCR T cells produce lower amounts of proinflammatory cytokines than T cells expressing monospecific CARs in response to CD 19 or BCMA antigen high target cells but respond better to CD 19 and BCMA antigen low target cells. This phenotype predicts both for efficient elimination of B cells and plasma cells in vivo and a lower risk of cytokine release syndrome. EGFRtoptconfers sensitivity of Bi-ChTCR T cells to cetuximab in vitro as observed with CAR T cells29.

[0591] Studies compare the safety and efficacy of mono- and bi-specific ChTCR and CAR T cells in humanized mice with SLE and the tempo of immune reconstitution after removal of T cells with Cetuximab.

[0592] SLE can be induced in NSG mice repopulated with a human immune system (Hu-mice) by pristane injection39,40. THX mice in which non-irradiated genetically myeloablated NSGW41 (NOD .C -Kit4 l.JTyr prkdcscidIL2rgrm lWjH\\Qm]) mutant mice are engrafted with human CD34+ cells followed by treatment with 17b (17beta) estradiol to promote immune cell differentiation have been shown to reconstitute high levels of human B (hu-B) and T cells (hu- T), develop marginal zone and germinal center B cells, lymph nodes, Peyer’s patches, and human thymic epithelial cells33. The BCR huV(D)J and huTCRa and huTCRb repertoires are similar to that of humans, and THX mice mount T cell dependent and independent antibody responses to pathogens33. Importantly Clinical, pathologic, and serologic features of SLE are induced in THX mice by pristane injection, and THX mice have a long life-span to allowing studies of the durability of treatment effects33.

[0593] Experimental Setup

[0594] Characterization of humanized (THX) mice: THX mice are generated according to an established protocol and confirm reconstitution of human immune cell subsets33. NSGW41 neonates are engrafted with fetal human CD34+ cells through intracardiac injection and given 17p estradiol beginning at 14 weeks of age to support engraftment and differentiation of hematopoietic stem cells, lymphoid and myeloid immune cells. After 4 weeks of 17|3 estradiol, blood samples are analyzed for hu-B, hu-T, hu-NK, hu-monocyte, and hu-dendritic cell numbers, and for levels of circulating huIgM and huIgG. To assess immune competence, THX mice are immunized at 20 wks of age with ovalbumin in FCA and analyzed 14 days later for ova-specific antibodies. Flow cytometry is used to confirm high level (>95%) engraftment of huCD45+ cells and engraftment of CD 19+ B cells, CD3+ T cells, CD 138+ plasma cells, NK cells, and myeloid cell populations, and mice are immunized with a model antigen to confirm immune competence. In some contexts, human CD45+ cell engraftment is less than 80% or mice do not respond to immunization, and higher doses of CD34+ cells are administered to ensure diverse, high level immune reconstitution before inducing lupus with pristane.

[0595] Studies investigate what Bi-ChTCR and CAR T cell doses and level of lymphodepletion are required for engraftment and functional depletion of B cells and / or plasma cells in THX mice. i). Transduction of T cells from THX mice: To avoid alloreactive T cell responses in adoptive transfer experiments, Bi-ChTCR and CAR T cells are produced from blood obtained by sacrificing a subset (n=2-3) of a larger cohort of THX mice engrafted with CD34+ cells from a single fetal donor. T cells (2-5xl05) are stimulated with aCD3 / aCD28 and transduced with lentiviruses encoding the CD 19 / BCMA Bi-ChTCR, CD19 / 4-lBBz, or BCMA / 4-lBBz CAR. After 7-10 days of expansion, T cells are adoptively transferred into THX mice engrafted with CD34+ cells from the same donor. Donor units for CD34+ cells are selected to ensure a large cohort (n~50) of mice can be generated from one unit, permitting generation T cells from a subset of mice for treatment of mice in the same cohort. ii. Investigating whether Bi-ChTCR or CAR T cells engraft and function in THX mice in the absence of lymphodepleting chemotherapy.

[0596] Lymphodepleting chemotherapy (LD-CTX) improves CAR T cell engraftment and antitumor activity in cancer patients41. Preferably, LD-CTX could be avoided in autoimmune indications to limit toxicity since higher normal B cell numbers may be sufficient to drive Bi- ChTCR or CAR T cell expansion in vivo. To test the necessity of LD-CTX, mock transduced, Bi-ChTCR and CD 19 CAR T cells are administered without preceding LD-CTX to mice (n=6) at two dose levels (2xl06and 10xl06). Serum cytokines (IFN-g, IL-6, MCP-1), frequency of EGFRtopt T cells in blood, and levels of CD19+ B cells are measured at day 1, 7, 14, and 28 to assess T cells’ engraftment, proliferation, and depletion of endogenous B cells.

[0597] Cytokine levels and symptoms of CRS are less with Bi-ChTCR T cells than with CAR T cells. Fepletion of CD19+ B cells is measured as a surrogate for function of Bi-ChTCR and CAR T cells. A subset of THX mice in which B cell depletion is observed are sacrificed at 1 month and measure the frequency of CD 138+ plasma cells in blood, bone marrow, and spleen to evaluate Bi-ChTCR depletion of plasma cells. SLE pathology in THX mice. THX mice with SLE are generated by injecting 16-week- oldmale and female THX mice (n = 12) once i.p. with 500 pl pristane33. Control THX mice (n =7) are injectedwith 500 pl PBS. Mice are followed for signs of lupus, which is typically first manifested by a malar rash 3 weeks following pristane. Serum Hu-IgG and Hu-IgA, anti-dsDNA and anti-histone autoantibodies are measured by ELISA 6 weeks after pristane in both cohorts. Lupus nephritis also develops in THX mice33. To confirm lupus nephritis, two mice per group are sacrificed 8 weeks after pristane, and renal pathology is evaluated by H&E staining and anti-Hu-IgG immunofluorescence. Pristane-treated and control mice are followed for survival.

[0598] Kidney immunopathology is performed at euthanasia for SLE mice. Pristane injection induces SLE in THX mice and recapitulates clinical features observed in SLE patients.

[0599] Safety and efficacy of adoptive transfer of Bi-ChTCR and monospecific CD 19 or BCMA CAR T cells. To investigate the safety and efficacy of Bi-ChTCR, CD19 and BCMA CAR T cells, 16-week-old mice THX mice (n=50) are injected with pristane. Two weeks later, Bi- ChTCR and monospecific CD 19, BCMA or Mock CAR T cells are generated from 2-3 control THX mice as described above. THX mice that develop SLE as indicated by a malar rash are separated into 4 groups of 12 animals. Animals receive LD-CTX or not, and each group is treated with Bi-ChTCR or CAR T cells at a pre-determined dose (Fig. 6). Blood and serum are collected from treated mice on days 1, 3, 7 and 14, and cytokines (IFN-g, IL-6, MCP-1) associated with CRS are measured by ELISA. Absolute numbers of transferred T cells and B cells and plasma cells in blood are determined by flow cytometry. Because plasma cells are rare in blood, 2 mice per group are euthanized to measure CD 138+ plasma cells in spleen and bone marrow 3 weeks after T cell therapy. SLE resolution is determined by reduction in serum Hu- IgG, Hu-IgA, antidsDNA, and anti-histone autoantibodies by ELISA. Kidney function is measured in serum and immunopathology is performed on 2 animals sacrificed 8 weeks after T cell therapy to determine the presence of Hu-IgG deposition. The remaining cohorts of 8 mice / group are followed for survival and disease recurrence.

[0600] Experiments are repeated with mice engrafted with CD34+ cells from at least 2 donors to ensure robustness of findings.

[0601] CAR T cells produce higher cytokine levels and toxicity than Bi-ChTCR T cells. CD 19 CAR T cells deplete B cells but not plasma cells that secrete autoantibodies, while BCMA CAR T cells deplete only plasma cells. Bi-ChTCR therapy eliminates B cells, plasma cells, and autoantibodies. All groups of mice are followed for evidence of SLE resolution and maintenance of clinical benefit, which provides insight into the role of B cells and plasma cells respectively in SLE severity.

[0602] Eliminating Bi-ChTCR or CAR T cells with cetuximab and restoring functional humoral immunity. B cell depletion in cancer patients treated with CD 19 CAR T increases the risk of infections, which are the main cause for non-relapse mortality43. All tested CAR and Bi-ChTCR constructs contain EGFRtopt, allowing depletion of transferred cells in THX mice following SLE resolution by administering Cetuximab29. Three cohorts of mice (n=10 each) are injected with pristane to induce SLE and then treated with Bi-ChTCR or CAR T cells. Six to 8 weeks after resolution of SLE and reduction of autoantibodies, Bi-ChTCR (or CAR) T cells are depleted from two cohorts of animals by administering Cetuximab (100 pg / animal). The other cohort is left untreated. Depletion of EGFRtoptT cells and B cell and plasma cell recovery is determined by flow cytometry in mouse blood on day 0, 1, 3, 7 and 14 following cetuximab and compared with the control cohort. Functional recovery of humoral immunity is assessed by immunization with ova in FCA (2a). Mice are followed for recurrence of SLE by clinical signs and serum levels of Hu-IgG and autoantibodies, and for survival.

[0603] Cetuximab depletes EGFRtoptT cells in THX mice. Statistical Analysis: Replicate experiments are performed with engineered T cells from 2 donors. Outcome measures including T, B, and plasma cell frequency, cytokine levels, and survival are compared between treated groups at each cell dose using a t-test for single comparisons and a linear mixed model for comparing the data over all time points. Group sizes for individual experiments are determined to power pairwise testing of continuous variables between multiple groups, and statistical tests appropriate for comparing multiple groups are used.

[0604] References

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[0651] EXAMPLE 3

[0652] DESIGN A D TESTING OF CHTCRS TARGETING CD20 AND CD 19

[0653] Expression constructs encoding bispecific chTCR constructs targeting CD20 and CD 19 were generated using amino acid sequences shown in 3 A-3H. The chTCR constructs comprise an anti-CD20 scFv comprising VH and VL of antibody Leul6 fused to one TCR constant domain and an anti-CD19 scFv comprising VH and VL of antibody FMC63 fused to a cognate TCR constant domain.

[0654] Bi-ChTCRs are expressed in primary T cells. Binding to CD 19 and CD20 (MFI ) by Bi- ChTCR is compared to that of a bispecific CD19xCD20 CAR (Zah et al., Cancer Immunol Res. 2016 Jun;4(6):498-508. doi: 10.1158 / 2326-6066.CIR-15-0231).

[0655] In vitro cytotoxicity, cytokine production, and T cell proliferation against CD19 / CD20 + / - and High / Low tumor cells is assessed to demonstrate bi-specificity and sensitivity of Bi- ChTCRs. Raji cells (CD19high and CD22high), Nalm6 CD19high and CD221ow) and engineered Nalm6 cell lines (CD19high / Low / Ko and CD22high / Low / Ko) are used.

[0656] Function of Bi-ChTCRs is compared to that of the above-referenced bispecific CAR, as well as to monospecific CD 19 (FMC63 scFv fused to TRAC) or CD20 (Leu 16 scFv fused to TRBC) chTCR.

[0657] In vivo activity of Bi-TCR-T cells is investigated in NSG mice engrafted with tumor cells high CD19+ / CD20+, + / - , - / + and N6 Low model (+ / + high and + / + low).

[0658] Additional experiments were performed using chTCRs targeting CD 19 and CD20, as shown and described for Figures 7-9. EXAMPLE 4

[0659] ANTI-BCMA x ANTI-CD19 CHTCR CONSTRUCTS WITH IMPROVED EXPRESSION IN T CELLS

[0660] Further engineering produced additional constructs for expression of Cl 1D5.3 scFv x FMC63_scFv chTCRs. In one approach, the RAKR (furin cleavage site) sequence was removed, resulting in improved expression as compared to expression (from separate experiments) of the RAKR-containing counterpart. In another approach, a different signal peptide (MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO.:229)) was used for both chains of the chTCR, also resulting in improved expression as compared to data from separate experiments for the comparator containing GM-CSF signal peptide (SEQ ID NO.: 152) on both chains of the chTCR.

[0661] These approaches were combined to generate further chTCR expression constructs. The following construct was selected for further studies:

[0662] MDMRVPAQLLGLLLLWLRGARCQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINW VKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATY FCALDYSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVLTQSPPSLAMSLGKRAT ISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPV EEDDVAVYYCLQSRTIPRTFGGGTKLEIKDLNKVFPPEVAVFEPSEAEISHTQKATLVCL ATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNP RNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATIL YEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQAGDVEENPGPMDMRVP AQLLGLLLLWLRGARCDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGT VKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKL EITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQ PPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKH YYYGGSYAMDYWGQGTSVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQS KDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDV

[0663] KLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO.:228).

[0664] In certain embodiments where the chTCR is co-expressed with a tag (e.g., tEGFRopt), the construct comprises the signal peptide MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO.:229) at the N-terminus of the tag. EXAMPLE 5

[0665] IN VIVO ANTITUMOR ACTIVITY OF CHTCR-T CELLS TARGETING BCMA AND CD 19

[0666] An in vivo experiment using NSG mice engrafted with Nalm6 tumor cells and treated with BCMA x CD19-specific chTCR T cells was performed, as shown and described for Figures 10 and 11. The chTCR construct encoded the amino acid sequence set forth in SEQ ID NO:228.

[0667] These data show that Bi-ChTCR comprisng a scFv comprising the VH and VL of Cl 1D5.3 shows early tumor control in a BCMA antigen-low mouse model.

[0668] EXAMPLE 6

[0669] DUAL PROMOTER EXPRESSION CONSTRUCTS

[0670] Some T cell engineering strategies involve transducing T cells with a vector (e.g., a lentiviral or retroviral vector) encoding a plurality of polypeptide expression products, for example: an antigen-binding protein (e.g., a CAR) and a tag (e.g., an EGFR-derived, NGFR- derived, CD19-derived, or CD34-derived tag); an antigen-binding protein and a cytokine or cytokine fusion protein; a first antigen-binding protein (e.g., a CAR) and a second antigenbinding protein (e.g., a PD-L1 -binding antibody or antibody fragment); a TCR and a CD8 or CD4 co-receptor. For contemporaneous and equimolar expression, a single expression cassette comprising one promoter is often employed, with self-cleaving peptide (e.g., P2A, T2A, E2A, or F2A, typically preceded by a short GSG linker) and RAKR furin cleavage sequences disposed between distinct polypeptide molecules for physical separation following translation, though some data (not shown) indicates that favorable expression may be achieved without inclusion of the RAKR sequence.

[0671] T cells expressing an antigen-binding protein (e.g., a chTCR) can benefit from the further expression of a costimulatory polypeptide (e.g., a wild-type or ITAM-engineered CD3 polypeptide, or CD3 fusion polypeptide (comprising a human CD3(^, CD36, CD3s, or CD3y fused to a costimulatory domain from, e.g., human 4- IBB, CD28, CD226, CD2, or 0X40, or a functional variant thereof) and / or of a tag useful to identify, sort, track, and / or target the T cell for ablation.

[0672] Using a single expression cassette-approach wherein each polypeptide is separated by a RAKR sequence and a GSG-P2A self-cleaving peptide sequence, reduced expression of chTCR on primary human CD8+ T cells was observed when the chTCR was co-expressed with a costimulatory polypeptide or a tag. To potentially improve expression of the chTCR when a second molecule is expressed in the same vector, alternative designs were explored. A bi-directional, two-promoter expression vector was developed wherein an EFla promoter (SEQ ID NO: 7) drives expression of the chTCR and, disposed on the opposite DNA strand and oriented in the opposite direction, a second promoter drives expression of a tag or a CD3 polypeptide. Non-limiting examples of expression vectors are shown in Figures 12, 13, 14, and 18. As shown in Figure 14, multiple candidate promoters were tested for driving expression of tEGFR. Primary human CD8+ T cells were transduced with lentivirus comprising the expression vectors. The MNDU3 promoter (SEQ ID NO: 8) was selected as the best candidate for pairing with EFla promoter due to the combination of high transduction efficiency and expression (Figures 16A-17).

[0673] Again using the EFla promoter to drive expression of the chTCR, a second copy of the EFla promoter or the MND promoter was used for driving expression of wild-type human CD3(^ (Figure 18). As shown in Figure 19-20, the EFla / MNDU3 dual promoter combination has superior high receptor expression and improved transduction efficiency over vectors comprising multiple P2A sequences, and the vector with two copies of EFla had poor expression. Vector comprising the EFla / MND dual promoter combination is used for delivery of chTCR with CD3 polypeptide or tag.

[0674] The EFla / MNDU3 dual promoter combination also successfully drives expression of a bispecific chTCR (scFvs derived from Cl 1D5.3 and FMC63; specifically, the expression product shown in SEQ ID NO:228) and tEGFR (Figure 21).

[0675] Swapping the position of the two promoters ( / .< ., with MNDU3 driving expression of receptor and EFla driving expression of a tag) did not result in an observable difference in chTCR expression (data not shown). Specifically, chTCR expression was equivalent as between (1) primary human CD8+ T cells transduced with vector comprising: EFla chTCR / MNDU3 tEGFR and (2) primary human CD8+ T cells transduced with vector comprising: MNDU3 chTCR / EFla Thyl.l.

[0676] Dual-promoter constructs also improved co-expression of: fEGFRoptwith an anti-CD19- 4-lBBz CAR; fEGFRoptwith an anti-CD19-28z CAR; and tEGFRoptwith a NY-ESO-1 :pMHC- specific TCR, relative to constructs driving expression of these molecules with a single promoter and 2 A peptide(s).

[0677] These data show that co-expression of a chTCR of the present disclosure with another product of interest (e.g., a tag, a CD3 polypeptide, or a CD3 fusion polypeptide) can be improved by using a dual promoter EFla / MND expression construct. The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and / or listed in the Application Data Sheet, including U.S. provisional patent applications 63 / 717,886, filed November 7, 2024, and 63 / 736,574, filed December 19, 2024, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments. These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

CLAIMSWhat is claimed is:

1. A polypeptide dimer comprising a first polypeptide and a second polypeptide, wherein: the first polypeptide comprises: a BCMA-specific single-chain variable fragment (scFv) comprising the heavy chain variable domain (VH) amino acid sequence QIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPA YAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSS (SEQ ID NO: 153), and the light chain variable domain (VL) amino acid sequence DIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTG VPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIK (SEQ ID NO: 155), wherein, optionally, the scFv is in a VH-linker-VL orientation; and a T cell receptor (TCR) constant domain; and the second polypeptide comprises: a CD19-specific single-chain variable fragment (scFv) comprising the VL amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPS RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT (SEQ ID NO: 159), and the VH amino acid sequence EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYY NSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVT VSS (SEQ ID NO: 161), wherein, optionally, the scFv is in a VL-linker-VH orientation; and a T cell receptor (TCR) constant domain, wherein (1) the TCR constant domain of the first polypeptide is a human TCR alpha chain constant domain (Ca) or an engineered variant thereof, and the TCR constant domain of the second polypeptide is a human TCR beta chain constant domain (CP) or an engineered variant thereof, or (2) the TCR constant domain of the first polypeptide is a human TCR beta chain constant domain (CP) or an engineered variant thereof and the TCR constant domain of the second polypeptide is a human TCR alpha chain constant domain (Ca) or an engineered variantthereof, optionally wherein a polynucleotide encoding the polypeptide dimer encodes the amino acid sequence set forth in SEQ ID NO:228.

2. The polypeptide dimer of claim 1, wherein the amino acid sequence of the scFv of the first polypeptide is QIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPA YAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSS GGGGSGGGGSGGGGSDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPG QPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGT KLEIK (SEQ ID NO:213).

3. The polypeptide dimer of claim 1 or 2, wherein the amino acid sequence of the scFv of the second polypeptide is DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPS RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEG STKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSS (SEQ ID NO:208).

4. The polypeptide dimer of any one of claims 1-3, wherein the amino acid sequence of the TCR constant domain of the first polypeptide isDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSG VCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDR AKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAM VKRKDF (SEQ ID NO:59), and the amino acid sequence of the TCR constant domain of the second polypeptide isIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSM DFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNL LVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:57).

5. The polypeptide dimer of any one of claims 1-3, wherein the amino acid sequence of the TCR constant domain of the second polypeptide isDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSG VCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDR AKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAM VKRKDF (SEQ ID NO:59), and the amino acid sequence of the TCR constant domain of the first polypeptide isIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSM DFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNL LVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:57).

6. The polypeptide of any one of claims 1-5, wherein: the first polypeptide comprises, consists essentially of, or consists of the amino acid sequenceQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTE TREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTS VTVSSGGGGSGGGGSGGGGSDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWY QQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRT FGGGTKLEIKDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGK EVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDE WTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALV LMAMVKRKDF (SEQ ID NO:214); and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPS RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEG STKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRS MDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQN LLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:209).

7. The polypeptide of any one of claims 1-5, wherein: the first polypeptide comprises, consists essentially of, or consists of the amino acid sequenceDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLH SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKP GSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGV IWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMD YWGQGTSVTVSSDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVN GKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEN DEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSA LVLMAMVKRKDF (SEQ ID NO:215); and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequence QIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPA YAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSS GGGGSGGGGSGGGGSDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPG QPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGT KLEIKIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSM DFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNL LVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:216).

8. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequenceMLLLVTSLLLCELPHPAFLLIPQIQLVQSGPELKKPGETVKISCKASGYTFTDYSIN WVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTAT YFCALDYSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVLTQSPPSLAMSLGKR ATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTID PVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKDLNKVFPPEVAVFEPSEAEISHTQKATLV CLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQ NPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSAT ILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATNFSLLKQAGDVEENPGPM LLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKP DGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGG TKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSW IRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCA KHYYYGGSYAMDYWGQGTSVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSC DVI<LVEI<SFETDTNLNFQNLLVIVLRILLLI<VAGFNLLMTLRLWSS (SEQ ID NO: 163), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO: 163.

9. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequenceMLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNW YQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLP YTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLP DYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDD TAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDLNKVFPPEVAVFEPSEAEISHTQKAT LVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVL SATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATNFSLLKQAGDVEENP GPMLLLVTSLLLCELPHPAFLLIPQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWV KRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYF CALDYSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVLTQSPPSLAMSLGKRATI SCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPV EEDDVAVYYCLQSRTIPRTFGGGTKLEIKIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT NVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSP ESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 164), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO: 164.

10. The polypeptide of claim 8 or 9, further comprising, fused to the C-terminal end thereof, the amino acid sequence RAKRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIG EFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLL IQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKN LCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCR NVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYID GPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLL VVALGIGLFMRRR (SEQ ID NO:217), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO:217.

11. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequenceMLLLVTSLLLCELPHPAFLLIPQIQLVQSGPELKKPGETVKISCKASGYTFTDYSIN WVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTAT YFCALDYSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVLTQSPPSLAMSLGKR ATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTID PVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKDLNKVFPPEVAVFEPSEAEISHTQKATLV CLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQ NPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSAT ILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATNFSLLKQAGDVEENPGPM LLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKP DGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGG TKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSW IRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCA KHYYYGGSYAMDYWGQGTSVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVS QSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSC DVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSSRAKRLEGGGEGRG SLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKH FKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAF ENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLF GTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCN LLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVM GENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLL VVALGIGLFMRRR (SEQ ID NO: 167), or a variant thereof wherein one or both instances of the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO: 167.

12. A polypeptide dimer comprising a first polypeptide and a second polypeptide, wherein:the first polypeptide comprises: a CD20-specific single-chain variable fragment (scFv) comprising the heavy chain variable domain (VH) amino acid sequence EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGD TSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGA GTTVTVSS (SEQ ID NO:205), and the light chain variable domain (VL) amino acid sequence DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPA RFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIK (SEQ ID NO:203); and a T cell receptor (TCR) constant domain; and the second polypeptide comprises: a CD19-specific single-chain variable fragment (scFv) comprising the VL amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPS RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT (SEQ ID NO: 159), and the VH amino acid sequence EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYY NSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVT VSS (SEQ ID NO: 161); and a T cell receptor (TCR) constant domain, wherein (1) the TCR constant domain of the first polypeptide is a human TCR alpha chain constant domain (Ca) or an engineered variant thereof, and the TCR constant domain of the second polypeptide is a human TCR beta chain constant domain (CP) or an engineered variant thereof, or (2) the TCR constant domain of the first polypeptide is a human TCR beta chain constant domain (CP) or an engineered variant thereof and the TCR constant domain of the second polypeptide is a human TCR alpha chain constant domain (Ca) or an engineered variant thereof.

13. The polypeptide dimer of claim 12, wherein the amino acid sequence of the scFv of the first polypeptide is: DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPA RFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSGGGSG GGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSS (SEQ ID NO:206); orEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMD WYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIK (SEQ ID NO:218).

14. The polypeptide dimer of claim 12 or 13, wherein the amino acid sequence of the scFv of the second polypeptide is:DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSS (SEQ ID NO:208); orEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQK PDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGG GTKLEIT (SEQ ID NO:220).

15. The polypeptide dimer of any one of claims 12-14, wherein the amino acid sequence of the TCR constant domain of the first polypeptide isDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 156), and the amino acid sequence of the TCR constant domain of the second polypeptide isIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNL LVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 162).

16. The polypeptide dimer of any one of claims 12-14, wherein the amino acid sequence of the TCR constant domain of the second polypeptide isDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSG VCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDR AKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAM VKRKDF (SEQ ID NO: 156), and the amino acid sequence of the TCR constant domain of the first polypeptide isIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSM DFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNL LVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 162).

17. The polypeptide dimer of any one of claims 12-16, wherein:(i) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequenceDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLAS GVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSG GGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI GAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSY WFFDVWGAGTTVTVSSDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELS WWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFY GLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYA VLVSALVLMAMVKRKDF (SEQ ID NO:207); and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequenceDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLH SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKP GSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGV IWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMD YWGQGTSVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKC VLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDT NLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:209);(ii) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequenceDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLAS GVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSG GGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI GAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSY WFFDVWGAGTTVTVSSDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELS WWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFY GLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYA VLVSALVLMAMVKRKDF (SEQ ID NO:207); and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequenceEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNW YQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLP YTFGGGTKLEITIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCV LDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTN LNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:220);(iii) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequenceEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYP GNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDV WGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPAILSASPGEKVTMTCRASSSVN YMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQ WSFNPPTFGGGTKLEIKDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELS WWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFY GLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYA VLVSALVLMAMVKRKDF (SEQ ID NO:221); and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequenceEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNW YQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCV LDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTN LNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:220); or(iv) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequenceEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYP GNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDV WGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPAILSASPGEKVTMTCRASSSVN YMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQ WSFNPPTFGGGTKLEIKDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELS WWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFY GLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYA VLVSALVLMAMVKRKDF (SEQ ID NO:221); and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequenceDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLH SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKP GSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGV IWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMD YWGQGTSVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKC VLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDT NLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:209).

18. The polypeptide dimer of any one of claims 12-16, wherein:(i) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequenceDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLH SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKP GSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGV IWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMD YWGQGTSVTVSSDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVN GKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSA LVLMAMVKRKDF (SEQ ID NO:215); and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequenceDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLAS GVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSG GGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI GAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSY WFFDVWGAGTTVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYI TDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSF ETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:222);(ii) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequenceDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLH SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKP GSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGV IWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMD YWGQGTSVTVSSDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVN GKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEN DEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSA LVLMAMVKRKDF (SEQ ID NO:215); and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequenceEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYP GNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDV WGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPAILSASPGEKVTMTCRASSSVN YMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQ WSFNPPTFGGGTKLEIKIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYI TDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSF ETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:223);(iii) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequenceEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNW YQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLP YTFGGGTKLEITDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVN GKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEN DEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSA LVLMAMVKRKDF (SEQ ID NO:224); and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequenceEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYP GNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDV WGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPAILSASPGEKVTMTCRASSSVN YMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQ WSFNPPTFGGGTKLEIKIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYI TDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSF ETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:223); or(iv) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequenceEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNW YQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLP YTFGGGTKLEITDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVN GKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEN DEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSA LVLMAMVKRKDF (SEQ ID NO:224); and the second polypeptide comprises, consists essentially of, or consists of the amino acid sequenceDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLAS GVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSG GGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI GAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:222).

19. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequenceMLLLVTSLLLCELPHPAFLLIPDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDW YQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNP PTFGGGTKLEIKGSTSGGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTF TSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLT SEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSDLNKVFPPEVAVFEPSEAEISHTQ KATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVS ATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQ GVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATNFSLLKQAGDVE ENPGPMLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLN WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL PYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSL PDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYC AKHYYYGGS YAMDYWGQGTS VT VS SIQNPDP AVYQLRDSKS SDKS VCLFTD FDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDT FFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 195), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO: 195.

20. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequenceMLLLVTSLLLCELPHPAFLLIPDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDW YQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNP PTFGGGTKLEIKGSTSGGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTF TSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLT SEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSDLNKVFPPEVAVFEPSEAEISHTQ KATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVS ATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATNFSLLKQAGDVE ENPGPMLLLVTSLLLCELPHPAFLLIPEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGV SWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYY CAKHYYYGGSYAMDYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSAS LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLT ISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITIQNPDPAVYQLRDSKSSDKSVCLFTDFD SQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFF PSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 196), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO: 196.

21. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequenceMLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYN MHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDS ADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPA ILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGT SYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKDLNKVFPPEVAVFEPSEAEISHT QKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRV SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQ QGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATNFSLLKQAGDV EENPGPMLLLVTSLLLCELPHPAFLLIPEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYG VSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIY YCAKHYYYGGSYAMDYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSA SLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSL TISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITIQNPDPAVYQLRDSKSSDKSVCLFTDF DSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTF FPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 197), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO: 197.

22. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequenceMLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYN MHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDS ADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPA ILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGT SYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKDLNKVFPPEVAVFEPSEAEISHT QKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRV SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQ QGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATNFSLLKQAGDV EENPGPMLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLN WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL PYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSL PDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYC AKHYYYGGS YAMDYWGQGTS VT VS SIQNPDP AVYQLRDSKS SDKS VCLFTD FDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDT FFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 198), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO: 198.

23. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequenceMLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNW YQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLP YTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLP DYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDD TAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDLNKVFPPEVAVFEPSEAEISHTQKAT LVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVL SATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATNFSLLKQAGDVEENP GPMLLLVTSLLLCELPHPAFLLIPDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQ KKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPT FGGGTKLEIKGSTSGGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTS YNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSE DSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPED TFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 199), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO: 199.

24. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequenceMLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNW YQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLP YTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLP DYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDD TAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDLNKVFPPEVAVFEPSEAEISHTQKAT LVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVL SATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATNFSLLKQAGDVEENP GPMLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMH WVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAD YYCARSNYYGSSYWFFDVWGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPAILS ASPGEKVTMTCRAS SS VNYMDWYQKKPGS SPKPWIYATSNL ASGVPARF SGSGSGTS Y SLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKIQNPDPAVYQLRDSKSSDKSVCLF TDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPE DTFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:200), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO:200.

25. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequenceMLLLVTSLLLCELPHPAFLLIPEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGV SWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYY CAKHYYYGGSYAMDYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSAS LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLT ISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITDLNKVFPPEVAVFEPSEAEISHTQKATL VCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLS ATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATNFSLLKQAGDVEENPG PMLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHW VKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADY YCARSNYYGSSYWFFDVWGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPAILSA SPGEKVTMTCRASS S VNYMDWYQKKPGS SPKPWIYATSNL ASGVPARF SGSGSGTS YSL TISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKIQNPDPAVYQLRDSKSSDKSVCLFTD FDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDT FFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:201), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO:201.

26. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequenceMLLLVTSLLLCELPHPAFLLIPEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGV SWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYY CAKHYYYGGSYAMDYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSAS LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLT ISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITDLNKVFPPEVAVFEPSEAEISHTQKATL VCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFW QNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLS ATILYEILLGKATLYAVLVSALVLMAMVKRKDFRAKRGSGATNFSLLKQAGDVEENPG PMLLLVTSLLLCELPHPAFLLIPDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQK KPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTF GGGTKLEIKGSTSGGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTS YNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSE DSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSIQNPDPAVYQLRDSKSSDKSVCLFT DFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPED TFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:202), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO:202.

27. The polypeptide of any one of claims 19-26, further comprising, fused to the C- terminal end thereof, the amino acid sequence RAKRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIG EFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLL IQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKN LCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCR NVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYID GPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIP SIATGMVGALLLLLVVALGIGLFMRRR (SEQ ID NO:217), or a variant thereof wherein the amino acid sequence RAKR is not present but the amino acid sequence is otherwise identical to SEQ ID NO:217.

28. A polypeptide dimer comprising a first polypeptide (Polypeptide 1) that binds human CD20 and a second polypeptide (Polypeptide 2) that binds human CD 19, wherein Polypeptide 1 and Polypeptide 2 are according to Format 1, Format 2, Format 3, Format 4, Format 5, Format 6, Format 7, or Format 8, as shown in the following table:wherein:(xi) for the anti-CD20 scFv, VL isDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYAT SNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGG TKLEIK (SEQ ID NO:203);(xii) for the anti-CD20 scFv, VH isEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWI GAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCA RSNYYGSSYWFFDVWGAGTTVTVSS (SEQ ID NO:205);(xiii) for the anti-CD19 scFv, VL isDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHT SRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGT KLEIT (SEQ ID NO: 159);(xiv) for the anti-CD19 scFv, VH isEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYY YGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 161); and(xv) the C-terminus of the anti-CD20 scFv of Polypeptide 1 is directly fused to the N- terminus of the TCR constant domain of Polypeptide 1, and the C-terminus of the anti-CD19 scFv of Polypeptide 2 is directly fused to the N-terminus of the TCR constant domain of Polypeptide 2, and wherein, optionally:(1) each linker is independently selected from: G4S linker, as shown in Figure 1 A; Linker 218, as shown in Figure IB; and Linker GST, as shown in Figure 3B, wherein, further optionally, the linker of the scFv of Polypeptide 1 is Linker GST, and the linker of the scFv of Polypeptide 2 is Linker 218, or the linker of the scFv of Polypeptide 1 is Linker 218, and the linker of the scFv of Polypeptide 2 is Linker GST; and(2) the Ca is a human Ca or an engineered variant thereof, further optionally comprising the TRAC (S to C mut) amino acid sequence shown in Figure IB, and the CP is a human CP or an engineered variant thereof, further optionally comprising the TRBC (S to C mut) amino acid sequence shown in Figure 1 A.

29. A polynucleotide encoding the polypeptide dimer of any one of claims 1-7, 12-18 and 28 or the polypeptide of any one of claims 8-11 and 19-27 or the amino acid sequence shown in any one of Figures 1C, ID, 2B, and 3A-3H, wherein, optionally, the polynucleotide is codon- optimized for expression in a human T cell, and / or wherein the polynucleotide comprises DNA, RNA, mRNA, circRNA, saRNA, or any combination thereof.

30. A polynucleotide encoding the polypeptide dimer of any one of claims 1-7, 12-18, and 28, wherein the encoded first polypeptide, the encoded second polypeptide, or both, further comprises the signal peptide MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO:229) N- terminal to the scFv.

31. A polynucleotide encoding a variant of the polypeptide of any one of claims 9-11 and 19-27, wherein the variant comprises the signal peptide MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO:229) in place of one or more of the signal peptide(s) in the amino acid sequence.

32. A polynucleotide encoding the polypeptide dimer of any one of claims 1-7, 12-18, and 30, wherein the polynucleotide does not encode a protease cleavage site, optionally a furin cleavage site, further optionally a RAKR sequence, disposed between a polynucleotide encoding a sequence comprising the first polypeptide and a polynucleotide encoding a sequence comprising the second polypeptide, wherein, optionlly, the polynucleotide encoding a sequence comprising the first polypeptide and the polynucleotide encoding a sequence comprising the second polypeptide are separated by a GSG-2A sequence, optionally the amino acid sequence set forth in SEQ ID NO: 158.

33. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence MDMRVPAQLLGLLLLWLRGARCQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINW VKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATY FCALDYSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVLTQSPPSLAMSLGKRAT ISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPV EEDDVAVYYCLQSRTIPRTFGGGTKLEIKDLNKVFPPEVAVFEPSEAEISHTQKATLVCL ATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNP RNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATIL YEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQAGDVEENPGPMDMRVP AQLLGLLLLWLRGARCDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGT VKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKL EITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQ PPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKH YYYGGSYAMDYWGQGTSVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQS KDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDV KLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:228).

34. A polynucleotide encoding the amino acid sequence set forth in SEQ ID NO:228, wherein the polynucleotide comprises the polynucleotide sequence set forth in SEQ ID NO:227, and optionally further encodes the amino acid sequence set forth in SEQ ID NO:226 and comprises the polynucleotide sequence set forth in SEQ ID NO:225.

35. An expression construct encoding (i) the polypeptide dimer of any one of claims 1-7, 12-18, and 28, or the polypeptide of any one of claims 9-11, 19-27, and 33, and (ii) at least one additional expression product of interest, wherein: the expression construct comprises an EFla promoter (SEQ ID NO:7) operably linked to a polynucleotide encoding (i) and a MNDu3 promoter (SEQ ID NO: 8) operably linked to a polynucleotide encoding (ii); or the expression construct comprises a MNDu3 promoter (SEQ ID NO:8) operably linked to a polynucleotide encoding (i) and an EFla promoter (SEQ ID NO: 7) operably linked to a polynucleotide encoding (ii), wherein, optionally, the EFla promoter and the MNDu3 promoter are disposed on opposite strands of the expression construct, and are preferably oriented in opposite directions, wherein, further optionally, the at least one additional expression product of interest comprises, consists essentially of, or consists of, a tag (e.g., SEQ ID NO: 166 or SEQ ID NO:226), a CD3 polypeptide, or a CD3 fusion polypeptide.

36. A vector comprising the polynucleotide of any one of claims 30-32 and 34, or the expression construct of claim 35.

37. The vector of claim 36, wherein the vector is a viral vector.

38. The vector of claim 37, wherein the viral vector is a lentiviral vector or a y- retroviral vector.

39. A host cell expressing: (i) the polypeptide dimer of any one of claims 1-7, 12-18 and 28, and 29, wherein, optionally, the host cell further expresses the polypeptide EGFRtopt, as shown in Figure 2A; or (ii) the polypeptide of any one of claims 8-11 and 19-27.

40. A host cell comprising the polynucleotide of any one of claims 30-32 and 34, optionally wherein the host cell comprises, in an endogenous TRAC locus and in an endogenous TRBC locus, a splice site mutation, stop codon, or missense mutation, preferably a stop codon or a splice site mutation, reducing or preferably eliminating expression by the host cell of the endogenous TRAC and the endogenous TRBC.

41. A host cell comprising the expression construct of claim 35.

42. A host cell comprising the vector of any one of claims 36-38.

43. The host cell of any one of claims 39-42, wherein the host cell comprises a hematopoietic progenitor cell, an induced pluripotent stem cell, an embryonic stem cell, a hematopoietic stem cell, a peripheral blood mononuclear cell, or a human immune system cell.

44. The host cell of any one of claims 39-43, wherein the host cell comprises a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a yb T cell, a natural killer cell, a natural killer T cell, a monocyte, or any combination thereof.

45. The host cell of any one of claims 39-44, wherein the host cell comprises a T cell, preferably a human T cell, optionally a human CD8+ T cell, a human CD4 + T cell, or both.

46. The host cell of claim 45, wherein the T cell comprises a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof.

47. The host cell of any one of claims 33-46, comprising a chromosomal gene knockout or a mutation of: a TGFpRl gene locus, a TGFPR2 gene locus, a PD-1 gene locus, a CTLA4 gene locus, a LAT gene locus, a TIM-3 gene locus, a PD-L1 gene locus, a TIGIT gene locus, an A2AR gene locus, a Fas locus, a FasL gene locus, a B7-H3 gene locus, a B7-H4 gene locus, an IDO gene locus, a VISTA gene locus, a SIGLEC7 gene locus, a SIGLEC9 gene locus, a TRAC gene locus, a TRBC gene locus, a T cell receptor gene locus, a MHC (e.g. HLA) gene locus, a CBLB gene locus, a RASA2 gene locus, a UBASH3 A gene locus, a CISH gene locus, a CD4 gene locus, a CD8 gene locus, or any combination thereof, such as a TIGIT locus, a TRAC gene locus, a TRBC gene locus, and / or one or both of a CD4 gene locus and a CD8 gene locus, wherein, optionally, the host cell comprises (1) an introduced splicing site or stop codon that disrupts expression of a TRAC and is further optionally an introduced splicing site or stop codon in a TRAC gene locus, (2) an introduced splicing site or stop codon that disrupts expression of a TRBC and is further optionally an introduced splicing site or stop codon in a TRBC gene locus, or (3) both (1) and (2).

48. The host cell of any one of claims 33-47, wherein the host cell is modified (e.g., having a chromosomal knockout mutation and / or a chromosomal missense mutation and / or a chromosomal splice junction mutation; encoding an inhibitory nucleic acid such as an siRNA or an antisense oligonucleotide) to have reduced protein expression (including null expression), of an endogenous TRAC, an endogenous TRBC, an endogenous TIGIT, endogenous CD4, endogenous CD8, or any combination thereof, as compared to the unmodified host cell, wherein, optionally, the host cell comprises (1) an introduced splicing site or stop codon that disrupts expression of a TRAC and is further optionally an introduced splicing site or stop codon in a TRAC gene locus, (2) an introduced splicing site or stop codon that disrupts expression of a TRBC and is further optionally an introduced splicing site or stop codon in a TRBC gene locus, or (3) both (1) and (2).

49. A composition comprising: (i) the polypeptide dimer of any one of claims 1-7, 12- 18 and 28; and / or (ii) the polypeptide of any one of claims 8-11, 19-27, 29, and 33; and / or (iii) the polynucleotide of any one of claims 30-32 and 34, optionally comprised in a lipid composition; and / or (iv) the expression construct of claim 35, optionally comprised in a lipid composition; (v) the vector of any one of claims 36-38; and / or (vi) the host cell of any one of claims 39-48, and a pharmaceutically acceptable carrier, excipient, or diluent.

50. The composition of claim 49, comprising (i) a composition comprising at least about 30% CD4+ T host cells, combined with (ii) a composition comprising at least about 30% CD8+ T host cells, in about a 1 : 1 ratio.

51. A method of treating a disease or condition characterized by expression of CD 19 and / or BCMA, preferably both CD 19 and BCMA, in a (preferably, human) subject, the method comprising administering to the subject an effective amount of: (i) the polypeptide dimer of any one of claims 1-7, 12-18 and 28; and / or (ii) the polypeptide of any one of claims 8-11, 19-27, 29, and 33; and / or (iii) the polynucleotide of claim any one of claims 30-32 and 34, optionally comprised in a lipid composition; and / or (iv)the expression construct of claim 35, optionallycomprised in a lipid composition; (v)the vector of any one of claims 36-38; and / or (vi) the host cell of any one of claims 39-48; and / or (vii) the composition of claim 49 or 50.

52. The method of claim 51, wherein the disease or condition comprises an autoimmune disease or condition, optionally wherein the subject does not receive lymphodepleting therapy (e.g., lymphodepleting chemotherapy) or has not received lymphodepleting therapy (e.g., lymphodepleting chemotherapy), prior to the administering.

53. The method of claim 52, wherein the disease or condition comprises systemic lupus erythematosus (SLE).

54. The method of claim 51, wherein the disease or condition comprises a cancer or a tumor and is optionally a hematological malignancy.

55. A method of treating a disease or condition characterized by expression of CD19 and / or CD20, preferably both CD 19 and CD20, in a (preferably, human) subject, the method comprising administering to the subject an effective amount of: (i) the polypeptide dimer of any one of claims 1-7, 12-18 and 28; and / or (ii) the polypeptide of any one of claims 8-11, 19-27, 29, and 33; and / or (iii) the polynucleotide of claim any one of claims 30-32 and 34, optionally comprised in a lipid composition; and / or (iv)the expression construct of claim 35, optionally comprised in a lipid composition; (iv) the vector of any one of claims 36-38; and / or (vi) the host cell of any one of claims 39-48; and / or (vii) the composition of claim 49 or 50.

56. The method of claim 55, wherein the disease or condition comprises a cancer or a tumor and is optionally a hematological malignancy.

57. Any one or more for the following for use as a medicament, or for use in the manufacture of a medicament for treating a disease or disorder characterized by expression of CD19, BCMA, CD20, CD19 and BCMA, or CD19 and CD20: (i) the polypeptide dimer of any one of claims 1-7, 12-18 and 28; and / or (ii) the polypeptide of any one of claims 8-11, 19-27, 29, and 33; and / or (iii) the polynucleotide of claim any one of claims 30-32 and 34, optionally comprised in a lipid composition; and / or (iv)the expression construct of claim 35, optionallycomprised in a lipid composition; (v)the vector of any one of claims 36-38; and / or (vi) the host cell of any one of claims 39-48; and / or (vii) the composition of claim 49 or 50.

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