Nucleic acids and pharmaceutical compositions for immune cell engagers

Abstract

The present disclosure relates to novel nucleic acids for an immune cell engagers, comprising a ribonucleic acid of formula I: R1 – SP – R2 – R3 – R4 – R5 (I), where R1 is a 5' untranslated region (UTR); SP encodes a signal peptide; R2 encodes a first single chain variable fragment (scFv) that binds a first extracellular protein or a variable region of a heavy chain (VH-only) that binds a first extracellular protein; R3 encodes a second scFv that binds a second extracellular protein; R4 encodes a half-life extender; R5 is a 3'UTR, and where R1 to R5 are oriented 5' to 3', for the treatment of cancers, including but not limited to hematological cancers, including multiple myeloma.

Description

DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 NUCLEIC ACIDS AND PHARMACEUTICAL COMPOSITIONS FOR IMMUNE CELL ENGAGERS STATEMENT REGARDING SEQUENCE LISTING

[0001] The sequence listing associated with this application is provided in XML format in lieu of paper copy, and is hereby incorporated by reference into the specification. The name of the XML file containing the sequence listing is “91016-428709_SeqListing.xml”. The XML file is 92,227 bytes, and was created on July 28, 2025, and is being submitted electronically, concurrent with the filing of this application. FIELD

[0002] The present disclosure relates to a novel nucleic acid encoding a single chain variable fragment (scFv) that binds to B-cell Maturation Antigen (BCMA) protein; and an immune cell engager, comprising the scFv and a second scFv that binds to an immune cell. The present disclosure is also related to novel methods of treating cancer, in particular hematological cancers such as multiple myeloma, using the novel scFv and immune cell engagers described herein. BACKGROUND

[0003] Despite the advances in the field of immunotherapies, treatment efficacy continues to be a problem. Furthermore, cancers, such as hematological cancers (e.g., multiple myeloma), remain among the most prevalent causes of death worldwide. For example, in the United States, the average lifetime risk of getting multiple myeloma is about 1 in 103 for men and about 1 in 131 for women.

[0004] Antibody-based immunotherapies, which can be encoded as nucleic acids, and in particular as mRNA, have revolutionized care for many cancer types. An mRNA-based approach has many advantages over conventional recombinant antibody manufacturing because producing mRNA and using in vivo translation and secretion is more economical and shortens the developmental and manufacturing timeline to bring novel therapies to patients. mRNA therapies may also be more easily multiplexed compared to recombinant therapies and can provide a more consistent and prolonged drug concentration profile. Despite recent advances, patients with cancer, such as multiple myeloma (MM), often try many different therapies searching for one with sufficient efficacy. Therefore,DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 immunotherapies with high efficacy for the treatment of cancer (e.g., hematological cancers, such as multiple myeloma) are desirable. SUMMARY

[0005] One embodiment described herein is a nucleic acid for an immune cell engager, comprising a ribonucleic acid of formula I: R1 – SP – R2 – R3 – R4 – R5 (I), wherein: R1 is a 5’ untranslated region (UTR); SP encodes a signal peptide; R2 encodes a first single chain variable fragment (scFv) that binds a first extracellular protein or a variable region of a heavy chain (VH-only) that binds a first extracellular protein; R3 encodes a second scFv that binds a second extracellular protein; R4 encodes a half-life extender; R5 is a 3’UTR, and wherein R1 to R5 are oriented 5’ to 3’.

[0006] In one aspect, R1 comprises a hemoglobin subunit beta (HBB) 5’UTR. In one aspect, SP comprises an IgG1 SP.

[0007] In one aspect, R2 encodes a first scFv and the first scFv comprises formula II or formula III: R6 – R7 (II), R7– R6 (III), wherein: R6 encodes a light chain variable fragment (VL); and R7 encodes a heavy chain variable fragment (VH).

[0008] In one aspect, a nucleic acid encoding a first linker is positioned between R6 and R7. In one aspect, the second scFv comprises formula IV or formula V: R8 – R9 (IV), R9 – R8 (V) wherein: R8 encodes a light chain variable fragment (VL); and R9 encodes a heavy chain variable fragment (VH).

[0009] In one aspect, a nucleic acid encoding a second linker is positioned between R8 and R9. In one aspect, R4 comprises a single chain crystallizable fragment (scFc) or a CH3DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 domain. In one aspect, R4 comprises an IgG1 single chain crystallizable fragment (scFc). In one aspect, the scFc comprises a leucine to alanine mutation at amino acids 234 and 235, as compared to SEQ ID NO: 7 or at corresponding amino acid positions in a homolog thereof. In one aspect, the scFc comprises a leucine to alanine mutation at amino acids 234 and 235, and a proline to glycine mutation at amino acid 329, as compared to SEQ ID NO: 7, or at corresponding amino acid positions in a homolog thereof. In one aspect, R5 comprises a hemoglobin subunit beta (HBB) 3’UTR. In one aspect, the first extracellular protein is expressed or overexpressed on cancer cells or tumor cells. In one aspect, the cancer cells are hematological cancer cells. In one aspect, the cancer cells are multiple myeloma cancer cells. In one aspect, the first extracellular protein is B-cell maturation antigen (BCMA). In one aspect, the second extracellular protein is expressed on immune cells. In one aspect, the immune cells are T cells. In one aspect, the second extracellular protein is cluster of differentiation 3 (CD3). In one aspect, R1 comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 1. In one aspect, SP comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 2. In one aspect, R2 comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 3.

[0010] In one aspect, any of the nucleic acids described herein further comprising a nucleic acid encoding a linker, wherein the linker is positioned between R2 and R3.

[0011] In one aspect, any of the nucleic acids described herein further comprising a nucleic acid encoding a linker, wherein the linker is positioned between R3 and R4.

[0012] In one aspect, the linker positioned between R2 and R3 comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 4. In one aspect, R3 comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 5. In one aspect, the linker positioned between R3 and R4 comprises a nucleic sequence having at least 80% sequence identity to SEQ ID NO: 6. In one aspect, R4 comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 7. In one aspect, R5 comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 8.

[0013] In another embodiment, described herein is pharmaceutical composition, comprising any of the nucleic acids described herein, and one or more pharmaceuticallyDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 acceptable carriers, diluents, or excipients. In one aspect, the one or more pharmaceutically acceptable carriers, diluents, or excipients comprises a lipid nanoparticle.

[0014] In another embodiment, described herein is any of the nucleic acids described herein or any of the pharmaceutical compositions described herein for the manufacture or preparation of a medicament for use in the treatment of cancer.

[0015] In another embodiment, described herein is any of the nucleic acids described herein or any of the pharmaceutical compositions described herein for the manufacture or preparation of a medicament for use in the treatment of a hematological cancer.

[0016] In another embodiment, described herein is any of the nucleic acids described herein or any of the pharmaceutical compositions described herein for the manufacture or preparation of a medicament for use in the treatment of multiple myeloma.

[0017] In another embodiment, described herein is any of the nucleic acids described herein or any of the pharmaceutical compositions described herein for the manufacture or preparation of a medicament for use in engaging immune cells.

[0018] In another embodiment, described herein is any of the nucleic acids described herein or any of the pharmaceutical compositions described herein for the manufacture or preparation of a medicament for use in engaging T cells.

[0019] In another embodiment, described herein is a method of treating cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of any of the nucleic acids described herein or any of the pharmaceutical compositions described herein.

[0020] In another embodiment, described herein is a method of treating hematological cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of any of the nucleic acids described herein or any of the pharmaceutical compositions described herein.

[0021] In another embodiment, described herein is a method of treating multiple myeloma in a patient in need thereof, the method comprising administering to the patient an effective amount of any of the nucleic acids described herein or any of the pharmaceutical compositions described herein.

[0022] In another embodiment, described herein is a method of engaging immune cells in a patient in need thereof, the method comprising administering to the patient an effectiveDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 amount of any of the nucleic acids described herein or any of the pharmaceutical compositions described herein.

[0023] In another embodiment, described herein is a method of engaging T cells in a patient in need thereof, the method comprising administering to the patient an effective amount of any of the nucleic acids described herein or any of the pharmaceutical compositions described herein.

[0024] In another embodiment, described herein is a single chain variable fragment (scFv) comprising a nucleic acid having at least 80% sequence identity to SEQ ID NO: 3. BRIEF DESCRIPTION OF THE FIGURES

[0025] Figures 1A-1D depict the binding affinities for the generated TCE binders for BCMA. Fig.1A shows the results of an in vitro biochemical assay of the binding affinities of various IgG format antibodies, containing the variable domains incorporated into the BCMA scFvs described herein [(binders 892-VHVL-scFv (mTCE120), 892-VLVH-scFv (mTCE- 121), 909-VHVL-scFv (mTCE114)], and a reference, mAb to BCMA (274) towards in a HEK293T-hu cell line expressing human BCMA. Fig.1B shows the results of an in vitro biochemical assessment of the binding affinities of various scFvs (binders 892-VHVL-scFv (mTCE120), 892-VLVH-scFv (mTCE-121) 909-VHVL-scFv (mTCE114), and a reference to BCMA in a HEK293T-cyano cell line. Fig.1C depicts the results of an in vitro biochemical assessments of the binding affinities of various scFvs (binders 892-VHVL-scFv (mTCE120), 892-VLVH-scFv (mTCE-121), 909-VHVL-scFv (mTCE114), and a reference to BCMA in a NCI-H929 cell line. Fig.1D shows an example of how the scFv is derived from the variable domains of the IgG.

[0026] Figure 2 describes a cytotoxicity assessment. Specifically, the percentage of viable cells (viability %) is shown when cells expressing BCMA (BCMA+ targets) and cells not expressing BCMA (BCMA- targets) were treated with the T-cell engager construct (TCE) and / or T-cells. The figure demonstrates that the cytotoxicity of the TCE proteins is specific to BCMA+ cells only in the presence of both TCE and T cells.

[0027] Figures 3A-3D describes an assessment of dosing requirements for mTCE09. Fig.3A is an experimental schematic for treating a humanized mouse model with a lipid nanoparticle (LNP) containing the mRNA constructs described herein. The mouse model contains bioluminescent cancer cells for monitoring purposes. Fig.3B depicts a series of exemplary bioluminescent images of mice that have undergone various treatment regimes.DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 Fig.3C shows a graph quantifying the tumor burden as measured by bioluminescence (BLI) at the treatment regimes for the mice treated in Fig.3B. Fig.3D shows a survival curve for the mice treated with the various treatment regimes shown in Fig.3B and quantified in Fig.3C.

[0028] Figures 4A-4B show a series of exemplary schematics depicting various embodiments of constructs of the protein encoded by the mRNA construct. Fig.4A depicts an exemplary schematic of one example mRNA construct of the T cell engager (mTCE) (top) and any one of the resulting TCE proteins ((scFv)2-Fc) (bottom). Fig.4B illustrates a schematic of alternative embodiments of any one of the TCE proteins incorporating various derivatives of an IgG Fc for the purpose of protein half-life extension.

[0029] Figures 5A-5C show in vivo use of the disclosed TCE proteins in an exemplary in vivo cancer model of multiple myeloma. Fig.5A exhibits representative BLI through day 49 for animals treated with mRNA mTCE120, mTCE121, mTCE114, and mTCE123. All animals in the mTCE123 treatment group had succumbed to progressive disease prior to day 49. Fig.5B shows quantification of the luminosity depicted in the respective mRNA treated mice at the specified day in Fig.5A. Fig.5C depicts long-term survival of the mice treated in Fig.5A and Fig.5B. DETAILED DESCRIPTION

[0030] Immunotherapies useful for treating cancer include immune cell engagers. An immune cell engager is an engineered protein designed to bring tumor cells and immune effector cells in close proximity to each other, in a patient’s body, by binding to specific antigens on both the surface of the tumor cell and the effector cell. Most immune cell engagers are antibody-based molecules with multiple arms. They may connect a tumor antigen(s) to an effector cell-surface molecule (such as CD3 on T cells). Binding of the cell- surface molecule on the effector cell molecule may activate the effector cell’s killing machinery. For example, “T cell engagers” (TCE) may bind a T cell (effector cell) cell- surface molecule and activate and redirect T cells to recognize and kill cancer cells, such as hematological cancer cells or multiple myeloma cancer cells, by binding to a target antigen expressed on the cancer cell and a trigger cell-surface molecule on T cells, such as CD3.DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709

[0031] Described herein are nucleic acid and amino acid sequences encoding an scFv specific for BCMA, and immune cell engagers targeting BCMA, and methods of treating cancer using the same.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0033] As used herein, the articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" can mean one element or more than one element.

[0034] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In some embodiments, the terms "about" or "approximately" when preceding a numerical value indicates the value plus or minus a range of 10%, 5%, or 1%.

[0035] As used herein, “nucleic acid” refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double and single stranded DNA, triplex DNA, as well as double and single stranded RNA, linear RNA, circular RNA and other RNA formats. It also includes modified forms, for example, by methylation and / or by capping, and unmodified forms of the polynucleotide. The term is also meant to include molecules that include non-naturally occurring or synthetic nucleotides as well as nucleotide analogs. Thus, while all sequences described herein are shown as DNA sequences, the corresponding RNA sequence is contemplated by the same sequence.

[0036] As used herein, the term “sequence identity” in the context of two nucleic acid sequences or amino acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window.DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981); by the alignment algorithm of Needleman and Wunsch, J Mal. Biol., 48:443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad Sci U.S.A., 85:2444 (1988); by computerized implementations of these algorithms (including, but not limited to CLUSTAL in the PC / Gene program by Intelligentics, Mountain View Calif, GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., U.S.A.); the CLUSTAL program is well described by Higgins and Sharp, Gene, 73:237-244 (1988) and Higgins and Sharp, CABIOS, 5:151-153 (1989); Corpet et al., Nucleic Acids Res., 16:10881-10890 (1988); Huang et al., Computer Applications in the Biosciences, 8:155-165 (1992); and Pearson et al., Methods in Molecular Biology, 24:307-331 (1994). Alignment is also often performed by inspection and manual alignment.

[0037] The term “coding sequence” as used herein refers to a segment of a polynucleotide that encodes for protein or polypeptide. The region or sequence is bounded nearer the 5’ end by a start codon and nearer the 3’ end with a stop codon. Coding sequences may also be referred to as open reading frames. As will be appreciated by the skilled practitioner, slight changes in nucleic acid sequence do not necessarily alter the amino acid sequence of the encoded polypeptide. This disclosure embraces the degeneracy of codon usage as would be understood by one of ordinary skill in the art. For example, as known in the art, different codons will code for the same amino acid.

[0038] As used herein, the phrase “codon degenerate nucleic acid sequence” when used with reference to a nucleic acid sequence refers to a nucleic acid sequence that differs from the referenced sequence, but that encodes a polypeptide having the same amino acid sequence as that encoded by the referenced sequence.

[0039] As used herein, "an effective amount" refers to an amount that causes relief of symptoms of a disorder or disease as noted through clinical testing and evaluation, patient observation, and / or the like. An "effective amount" may further designate a dose that causes a detectable change in biological or chemical activity. The detectable changes may be detected and / or further quantified by one skilled in the art for the relevant mechanism or process. Moreover, an "effective amount" may designate an amount that maintains aDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 desired physiological state, i.e., reduces or prevents significant decline and / or promotes improvement in the condition of interest. An "effective amount" may further refer to a “therapeutically effective amount”.

[0040] As used herein, the term “effector cell” refers to a second target cell for the immune cell engager described herein to bind to. Effector cells may be any cell type, for example, immune cells, i.e. T cells. Cell-surface proteins or antigens on the effector cells are targeted by the second scFv on the immune cell engager. Without being bound by any theory, it is believed that binding of the second scFv on the cell surface molecule of the effector cell activates the effector cell. For example, if the second scFv binds CD3 on a T-cell surface, the binding is believed to activate the T cell to kill infected or cancer cells.

[0041] As used herein, the term "individual" and "subject" are often used interchangeably and refer to any human or domestic animal that may be treated with the methods disclosed herein. Suitable subjects (e.g., patients) include humans and domestic animals or pets (such as a cat or dog). Non–human primates and human patients are included. In one embodiment, subjects may include human patients that have been diagnosed with cancer, such as hematological cancer, including but not limited to: multiple myeloma. As used herein, the term "patient" refers to a subject that may receive a treatment of a disease or condition.

[0042] As used herein, "treatment", "treat", and "treating" refer to reversing, alleviating, mitigating, or slowing the progression of, or inhibiting the progress of, a disorder or disease or symptoms associated with such disorder or disease, and as described in more detail herein.

[0043] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0- 7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0044] Nucleic acids encoding immune cell engagers may comprise ribonucleic acid (RNA) constructs. For example, a nucleic acid for an immune cell engager may include an RNA encoding, from 5’ to 3’, a 5’ untranslated region (UTR) (5’UTR), a signal peptide (SP), a first single chain variable fragment (scFv) or variable domain of a heavy chain (VH-only) thatDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 binds a first extracellular protein, a second scFv that bind a second extracellular protein, a half-life extender, and a 3’UTR.

[0045] Thus, in one embodiment described herein is a nucleic acid encoding an immune cell engager may include an RNA of formula I: R1 – SP – R2 – R3 – R4 – R5 (I), wherein: R1 is a 5’ untranslated region (5’UTR); SP encodes a signal peptide; R2 encodes a first single chain variable fragment (scFv) or variable domain of a heavy chain (VH-only) that binds a first extracellular protein; R3 encodes a second scFv that binds a second extracellular protein; R4 encodes a half-life extender; R5 is a 3’ untranslated region (3’UTR), and wherein R1 to R5 are oriented 5’ to 3’. Any of the nucleic acid sequences disclosed herein may also be codon-optimized for expression in a mammalian cell (e.g., a human cell).

[0046] In some aspects, the R1 or the 5′ untranslated region (also known as 5′ UTR, leader sequence, transcript leader, or leader RNA) is the region of a messenger RNA (mRNA) that is directly upstream from the initiation codon of a coding sequence. This region is important for the regulation of translation of an RNA transcript. In some aspects, the 5’UTR is 5’UTR of the hemoglobin subunit beta (HBB gene) but may be derived from other genes. In some aspects, R1 comprises a nucleic acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 1 (ATCCACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGGGC GGCCGCCACC), or a codon degenerate nucleic acid sequence thereof. In other aspects R1 comprises a nucleic acid sequence having at least 98.2% sequence identity) to SEQ ID NO: 1. In some aspects, R1 comprises a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 1. In some aspects, R1 comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 1. In some aspects, R1 comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 1. In some aspects, R1 comprises a nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 1. In some aspects, R1 comprises a nucleic acid sequence having at least 97% sequence identity to SEQ ID NO: 1. In some aspects, R1 comprises a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO: 1. In other aspects, R1 comprises a nucleic acid sequence having at least 99% sequence identity to SEQ IDDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 NO: 1. In some aspects, R1 comprises a nucleic acid sequence having 100% sequence identity to SEQ ID NO: 1. In some aspects, R1 comprises a nucleic acid sequence having at least 98.2% sequence identity to SEQ ID NO: 1.

[0047] In some aspects, R1 comprises a nucleic acid sequence differing from SEQ ID NO: 1 by no more than 11 nucleotide substitutions (e.g., no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide substitution(s)). In some aspects, R1 comprises a nucleic acid sequence differing from SEQ ID NO: 1 by no more than 10 nucleotide substitutions. In some aspects, R1 comprises a nucleic acid sequence differing from SEQ ID NO: 1 by no more than 9 nucleotide substitutions. In some aspects, R1 comprises a nucleic acid sequence differing from SEQ ID NO: 1 by no more than 8 nucleotide substitutions. In some aspects, R1 comprises a nucleic acid sequence differing from SEQ ID NO: 1 by no more than 7 nucleotide substitutions. In some aspects, R1 comprises a nucleic acid sequence differing from SEQ ID NO: 1 by no more than 6 nucleotide substitutions. In some aspects, R1 comprises a nucleic acid sequence differing from SEQ ID NO: 1 by no more than 5 nucleotide substitutions. In some aspects, R1 comprises a nucleic acid sequence differing from SEQ ID NO: 1 by no more than 4 nucleotide substitutions. In some aspects, R1 comprises a nucleic acid sequence differing from SEQ ID NO: 1 by no more than 3 nucleotide substitutions. In some aspects, R1 comprises a nucleic acid sequence differing from SEQ ID NO: 1 by no more than 2 nucleotide substitutions. In some aspects, R1 comprises a nucleic acid sequence differing from SEQ ID NO: 1 by no more than 1 nucleotide substitution. In some aspects, R1 comprises the nucleic acid sequence of SEQ ID NO: 1.

[0048] The signal peptide (SP) (sometimes referred to as signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide) is a short peptide, for example, about 16-30 amino acids long, that is most often present at the N-terminus (or occasionally at the C-terminus or internally) of newly synthesized proteins that are destined toward the secretory pathway. Signal peptides prompt a cell to translocate the protein to the cellular membrane in the endoplasmic reticulum. In some aspects, the SP is the signal peptide of the IgG kappa light chain. In some aspects, the SP is the signal peptide of the albumin gene (ALB). In any of the nucleic acids encoding an immune cell engager described herein, the signal peptide is operably linked to a RNA construct that includes at least R2-R3-R4-R5 such that the encodedDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 polypeptide is secreted from a host cell. In some aspects, the SP comprises a nucleic acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) to SEQ ID NO: 2 (ATGGTGTTGCAGACCCAGGTCTTCATTTCTCTGTTGCTCTGGATCTCTGGTGCCTACG GG) or a codon degenerate nucleic acid sequence thereof. In other aspects, the SP comprises a nucleic acid sequence having at least 98.3% sequence identity) to SEQ ID NO: 2. In some aspects, the SP comprises a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 2. In some aspects, the SP comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 2. In some aspects, the SP comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 2. In some aspects, the SP comprises a nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 2. In some aspects, the SP comprises a nucleic acid sequence having at least 97% sequence identity to SEQ ID NO: 2. In some aspects, the SP comprises a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO: 2. In other aspects, the SP comprises a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 2. In some aspects, the SP comprises a nucleic acid sequence having 100% sequence identity to SEQ ID NO: 2. In some aspects, the SP comprises a nucleic acid sequence having at least 98.3% sequence identity to SEQ ID NO: 2.

[0049] In some aspects, SP comprises a nucleic acid sequence differing from SEQ ID NO: 2 by no more than 12 nucleotide substitutions (e.g., no more than 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide substitution(s)). In some aspects, SP comprises a nucleic acid sequence differing from SEQ ID NO: 2 by no more than 11 nucleotide substitutions. In some aspects, SP comprises a nucleic acid sequence differing from SEQ ID NO: 2 by no more than 10 nucleotide substitutions. In some aspects, SP comprises a nucleic acid sequence differing from SEQ ID NO: 2 by no more than 9 nucleotide substitutions. In some aspects, SP comprises a nucleic acid sequence differing from SEQ ID NO: 2 by no more than 8 nucleotide substitutions. In some aspects, SP comprises a nucleic acid sequence differing from SEQ ID NO: 2 by no more than 7 nucleotide substitutions. In some aspects, SP comprises a nucleic acid sequence differing from SEQ ID NO: 2 by no more than 6 nucleotide substitutions. In some aspects, SP comprises a nucleic acid sequence differing from SEQ ID NO: 2 by no more than 5 nucleotide substitutions. In some aspects, SPDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 comprises a nucleic acid sequence differing from SEQ ID NO: 2 by no more than 4 nucleotide substitutions. In some aspects, SP comprises a nucleic acid sequence differing from SEQ ID NO: 2 by no more than 3 nucleotide substitutions. In some aspects, SP comprises a nucleic acid sequence differing from SEQ ID NO: 2 by no more than 2 nucleotide substitutions. In some aspects, SP comprises a nucleic acid sequence differing from SEQ ID NO: 2 by no more than 1 nucleotide substitution. In some aspects, SP comprises the nucleic acid sequence of SEQ ID NO: 2.

[0050] R2 encodes an antigen binding domain that specifies one of the antigen targets of the immune cell engager (e.g., an antigen target on a multiple myeloma cancer cell). In some aspects, R2 may encode a single chain variable fragment (scFv) or variable domain of a heavy chain (VH-only) that binds a first extracellular protein. A scFv is a molecule that contains a variable region of the light chain (VL) and a variable region of the heavy chain (VH) of an antibody, joined by a linker, which enables the two variable regions to be synthesized as a single polypeptide chain (see, e.g., Bird et al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad Sci. USA, 85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16: 778 (1998)). In other aspects, R2 may encode a variable region of a heavy chain (VH-only) that binds a first extracellular protein. In other aspects, R2 may encode a variable region of a heavy chain (VH-like) that binds a first extracellular protein.

[0051] Binding of a domain of a polypeptide to an extracellular protein (e.g., a first extracellular protein or a second extracellular protein) can be assessed using any appropriate method. For example, an enzyme-linked immunosorbent assay (ELISA) or flow cytometry may be used to assess binding of a domain of a polypeptide encoded by an mRNA described herein to an extracellular protein.

[0052] The first extracellular protein may be an antigen target on a cancer cell, such as an antigen target on a hematological cancer (e.g., an antigen target on a multiple myeloma cancer cell). In some aspects, the first extracellular protein is expressed or overexpressed on cancer or tumor cells. For example, in some aspects, the first extracellular protein is expressed or overexpressed on hematological cancer cells, such as multiple myeloma cancer cells. In some aspects, the first extracellular protein is BCMA. As used herein, “BCMA” refers to the B-cell Maturation Antigen protein, which is also called Tumor Necrosis Factor Receptor Superfamily Member 17 or TNFRSF17. BCMA is a member of the TNF- receptor superfamily. This protein is expressed in mature B lymphocytes and plasma cells,DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 and influences B cell development and autoimmune responses. BCMA has been shown to specifically bind to the tumor necrosis factor (ligand) superfamily, member 13b (TNFSF13B / TALL-1 / BAFF), and to lead to NF-kappaB and MAPK8 / JNK activation. This protein also binds to various TRAF family members and thus may transduce signals for cell survival and proliferation. It has been shown that BCMA is highly expressed on the surface of multiple myeloma cancer cells. See, for example, Shah et al. (2020) Leukemia 34, 985– 1005. The amino acid and nucleic acid sequences of BCMA from various species can be found in public databases, such as GenBank, UniProt, and Swiss-Prot. These sequences include, but are not limited to, the human BCMA protein sequence (e.g., UniProt / Swiss-Prot Accession No. Q02223) and its corresponding nucleotide sequence (e.g., NCBI Reference Sequence: NP_001183.2), as well as their counterparts in other species.

[0053] Described herein is a BCMA-specific scFv (e.g., a BCMA-specific scFv encoded by R2) having the components described in Table 1 or codon degenerate nucleic acid sequences thereof. Any of the BCMA-specific scFvs described herein may comprise the complementarity-determining regions (CDRs) described in Table 1. Table 1. BCMA molecules (CDRs shown in bold) SEQ G A G G A A G A G C T A T T A A E V S Q YDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 Table 1. BCMA molecules (CDRs shown in bold) SEQ Molecule G A G G A A G A E V G C T G A A G LI Y C g G A G G A A G A a C T C C C C G cDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 Table 1. BCMA molecules (CDRs shown in bold) SEQ Molecule gg ac ta a g tt att tc g ga A C T C G C G G G A C A T A A A g T T C T D Q C E S S D AI G L R V T L PDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 Table 1. BCMA molecules (CDRs shown in bold) SEQ Molecule a ga C g G A C C C G C C G C C G G G C G a cc aa g tc a gg a ca g at G C T G A C G A T G G T C C G T T cc A ADFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 Table 1. BCMA molecules (CDRs shown in bold) SEQ Molecule T T D Q C F G R D M V E T V T L P C g G C A A A C A G G T G A A A C G a cc aa g tc a gg a ca g at G C T G ADFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 Table 1. BCMA molecules (CDRs shown in bold) SEQ Molecule C G A T G G T C C G T T cc A A T SI S Q W Y S S D AI G L R V T L P G A C C C G C C G C C G G G C G E MDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 Table 1. BCMA molecules (CDRs shown in bold) SEQ Molecule S K Y G C A A A C A G G T G A A A C G LI F R K L G A C C C G C C E M G C A A A C G G C A ADFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 Table 1. BCMA molecules (CDRs shown in bold) SEQ Molecule A C A LI F C g G C A A A C G A T T G A A A T C g cc a a tg ta a ct g ctDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 Table 1. BCMA molecules (CDRs shown in bold) SEQ Molecule ttt G A G G G A A A G G G C G A T G gc T C tC SI S V V C G N Y E G G V L E F D G C A A A C G A T T G A A ADFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 Table 1. BCMA molecules (CDRs shown in bold) SEQ Molecule T C LI T S R L G C T A G A T G E V G C A A A C G LI L G C G G C GDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 Table 1. BCMA molecules (CDRs shown in bold) SEQ Molecule C A A C A T A A G G G G T G G G C C C T C G T A C H L Q L G F K K G L C C G G C G C A A C A T A A GDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 Table 1. BCMA molecules (CDRs shown in bold) SEQ Molecule G G G T G G G C C C T C G T A C H L Q L G F K K G L C C G G C G C A A C A T A A H L Q L

[0054] Also disclosed herein is a single chain variable fragment (scFv) comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%,DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 3 or a codon degenerate nucleic acid sequence thereof. In other aspects, the scFv comprises a nucleic acid sequence having at least 99.5%, or 99.8% to SEQ ID NO: 3. In some aspects, the scFv comprising a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 3 binds to BCMA. Described herein is a BCMA-specific scFv (e.g., a BCMA-specific scFv encoded by R2) having the components described in Table 1. Any of the BCMA-specific scFvs described herein may comprise the complementarity-determining regions (CDRs) described in Table 1. In some aspects, the single chain variable fragment (scFv) encodes an amino acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 9.

[0055] In some aspects, R2 comprises an scFv comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.8% sequence identity) to SEQ ID NO: 3 In some aspects, the scFv of R2 comprises a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 3. In some aspects, the scFv of R2 comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 3. In some aspects, the scFv of R2 comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 3. In some aspects, the scFv of R2 comprises a nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 3. In some aspects, the scFv of R2 comprises a nucleic acid sequence having at least 97% sequence identity to SEQ ID NO: 3. In some aspects, the scFv of R2 comprises a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO: 3. In some aspects, the scFv of R2 comprises a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 3. In some aspects, the scFv of R2 comprises a nucleic acid sequence having 100% sequence identity to SEQ ID NO: 3. In some aspects, the scFv of R2 comprises a nucleic acid sequence having at least 99.5% sequence identity to SEQ ID NO: 3. In some aspects, the scFv of R2 comprises a nucleic acid sequence having at least 99.8% sequence identity to SEQ ID NO: 3. In some aspects, the scFv of R2 encodes an amino acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 9.

[0056] In some aspects, the scFv of R2 comprises a nucleic acid sequence differing from SEQ ID NO: 3 by no more than 20 nucleotide substitutions (e.g., no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide substitution(s)). In some aspects, the scFv of R2DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 comprises a nucleic acid sequence differing from SEQ ID NO: 3 by no more than 15 nucleotide substitutions. In some aspects, the scFv of R2 comprises a nucleic acid sequence differing from SEQ ID NO: 3 by no more than 10 nucleotide substitutions. In some aspects, the scFv of R2 comprises a nucleic acid sequence differing from SEQ ID NO: 3 by no more than 9 nucleotide substitutions. In some aspects, the scFv of R2 comprises a nucleic acid sequence differing from SEQ ID NO: 3 by no more than 8 nucleotide substitutions. In some aspects, the scFv of R2 comprises a nucleic acid sequence differing from SEQ ID NO: 3 by no more than 7 nucleotide substitutions. In some aspects, the scFv of R2 comprises a nucleic acid sequence differing from SEQ ID NO: 3 by no more than 6 nucleotide substitutions. In some aspects, the scFv of R2 comprises a nucleic acid sequence differing from SEQ ID NO: 3 by no more than 5 nucleotide substitutions. In some aspects, R2 comprises a nucleic acid sequence differing from SEQ ID NO: 3 by no more than 4 nucleotide substitutions. In some aspects, R2 comprises a nucleic acid sequence differing from SEQ ID NO: 3 by no more than 3 nucleotide substitutions. In some aspects, R2 comprises a nucleic acid sequence differing from SEQ ID NO: 3 by no more than 2 nucleotide substitutions. In some aspects, R2 comprises a nucleic acid sequence differing from SEQ ID NO: 3 by no more than 1 nucleotide substitution. In some aspects, R2 comprises the nucleic acid sequence of SEQ ID NO: 3.

[0057] Any of the R2 domains disclosed herein may also include additional subdomains in various orientations. For example, the scFv of R2 may further comprise domains according to formula II or formula III: R6 – R7 (II), R7 – R6 (III), where R6 encodes a VL domain and R7 encodes a VH domain. In another example, in some aspects, from the 5’ to 3’ direction, the scFv of R2 may also include formula II: R6 – R7 (II). In other aspects, from the 5’ to 3’ direction, the scFv of R2 may also include formula III: R7 – R6 (III). In yet another example, the VL sequence of R6 and the VH sequence of R7 may be a subdomain from the scFv sequence of R2, such as the nucleic acid sequence of SEQ ID NO: 3.

[0058] In some aspects, R6 comprises a VL domain having a nucleic acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 15 (See Table 1) or a codon degenerate nucleic acid sequence thereof. In other aspects, R6 comprises a nucleic acid sequence comprising at least 99.5%, or 99.8% sequence identity) to SEQ ID NO: 15 (See Table 1). In some aspects, the R6 comprises a VL domain encoding an amino acid sequence of at least 80%DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 16. In other aspect, R6 comprises a VL-CDR1 of SEQ ID NO: 17, a VL-CDR2 of SEQ ID NO: 18, and a VL-CDR3 of SEQ ID NO: 19.

[0059] In some aspects, R7 comprises a VH sequence having a nucleic acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 10 (See Table 1) or a codon degenerate nucleic acid sequence thereof. In other aspects, R7 comprises a nucleic acid sequence having at least 99.5% or 99.8% sequence identity to SEQ ID NO: 10 (See Table 1). In some aspects, the R7 comprises a VL sequence encoding an amino acid sequence of at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 11. In other aspect, R7 comprises a VL- CDR1 of SEQ ID NO: 12, a VL-CDR2 of SEQ ID NO: 13, and a VL-CDR23 of SEQ ID NO: 14.

[0060] In some aspects, R6 comprises a VL domain having a nucleic acid sequence comprising SEQ ID NO: 15 (See Table 1) or a codon degenerate nucleic acid sequence thereof. In some aspects, R6 comprises a VL domain encoding an amino acid sequence of at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 16. In other aspect, the amino acid sequence of R6 comprises a VL-CDR1 of SEQ ID NO: 17, a VL-CDR2 of SEQ ID NO: 18, and a VL- CDR3 of SEQ ID NO: 19.

[0061] In some aspects, R7 comprises a VH sequence having a nucleic acid sequence comprising SEQ ID NO: 10 (See Table 1) or a codon degenerate nucleic acid sequence thereof. In some aspects, R7 comprises a VH sequence encoding an amino acid sequence of at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 11. In other aspect, the amino acid sequence of R7 comprises a VH-CDR1 of SEQ ID NO: 12, a VH-CDR2 of SEQ ID NO: 13, and a VH-CDR3 of SEQ ID NO: 14.

[0062] In some aspects, R6 comprises a VL domain having a nucleic acid sequence comprising SEQ ID NO: 38 (See Table 1) or a codon degenerate nucleic acid sequence thereof e.g. SEQ ID NO: 65. In some aspects, R6 comprises a VL domain encoding an amino acid sequence of at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 39. In otherDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 aspects, the amino acid sequence of R6 comprises a VL-CDR1 of SEQ ID NO: 17, a VL- CDR2 of SEQ ID NO: 18, and a VL-CDR3 of SEQ ID NO: 43.

[0063] In some aspects, R7 comprises a VH sequence having a nucleic acid sequence comprising SEQ ID NO: 36 (See Table 1) or a codon degenerate nucleic acid sequence thereof. In some aspects, R7 comprises a VH sequence encoding an amino acid sequence of at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 37. In other aspect, the amino acid sequence of R7 comprises a VL-CDR1 of SEQ ID NO: 40, a VL-CDR2 of SEQ ID NO: 41, and a VL-CDR23 of SEQ ID NO: 42.

[0064] In some aspects, the R6-R7 subdomain comprises a nucleic acid sequence of SEQ ID NO: 35, or a codon degenerate nucleic acid sequence thereof. In some aspects, the R6-R7 encodes an amino acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 63 (See Table 1).In other aspects, the R7-R6 subdomain comprises a nucleic acid sequence of SEQ ID NO: 34, or a codon degenerate nucleic acid sequence thereof. In some aspects, the R6-R7 subdomain encodes an amino acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 64 (See Table 1).

[0065] In some aspects, R6 comprises a VL domain having a nucleic acid sequence comprising SEQ ID NO: 49 (See Table 1) or a codon degenerate nucleic acid sequence thereof. In some aspects, R6 encodes a VL domain having an amino acid sequence of at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 50. In other aspects, the amino acid sequence of R6 comprises a VL-CDR1 of SEQ ID NO: 17, a VL-CDR2 of SEQ ID NO: 18, and a VL- CDR3 of SEQ ID NO: 54.

[0066] In some aspects, R7 comprises a VH sequence having a nucleic acid sequence comprising SEQ ID NO: 47 (See Table 1) or a codon degenerate nucleic acid sequence thereof. In some aspects, R7 encodes a VH sequence having an amino acid sequence of at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 48. In other aspect, the amino acid sequence of R7 comprises a VH-CDR1 of SEQ ID NO: 51, a VH-CDR2 of SEQ ID NO: 52, and a VH- CDR3 of SEQ ID NO: 53.DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709

[0067] In some aspects, the R6-R7 subdomain comprises a nucleic acid sequence of SEQ ID NO: 62, or a codon degenerate nucleic acid sequence thereof. In some aspects, the R6-R7 subdomain encodes an amino acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 61 (See Table 1).

[0068] In other aspects, the R7-R6 subdomain comprises a nucleic acid sequence of SEQ ID NO: 34, or a codon degenerate nucleic acid sequence thereof. In some aspects, the R6-R7 subdomain encodes an amino acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 64 (See Table 1).

[0069] R3 encodes a second antigen binding domain, or a second scFv, that is specific for an effector cell (e.g., an cell-surface antigen target on a T cell). In some aspects, R3 encodes a scFv that binds a second extracellular protein. In some aspects, within the context of Formula (1), R2 may be oriented 5ʹ to R3. In other aspects, R3 may be oriented 5ʹ to R2, such that the positions of R2 and R3 within the context of Formula (1) may be reversed. In other aspects, R4 may be positioned between R2 and R3. The second extracellular protein may be an antigen target on an effector cell, such as a T cell. In some aspects, the second extracellular protein is expressed on immune cells. In some aspects, the second extracellular protein is expressed on T cells. In some aspects, the second extracellular protein is CD3. As used herein, “CD3” refers to the cluster of differentiation 3 protein complex and T cell co-receptor. It is involved in activating both the cytotoxic T cell (CD8+naive T cells) and T helper cells (CD4+naive T cells) and is composed of four distinct chains. In mammals, the complex contains a CD3γ chain, a CD3δ chain, and two CD3ε chains. These chains associate with the T-cell receptor (TCR) and the CD3- zeta (ζ-chain) to generate an activation signal in T lymphocytes. The TCR, CD3-zeta, and the other CD3 molecules together constitute the TCR complex. The amino acid and nucleic acid sequences of CD3ε from various species can be found in public databases, such as GenBank, UniProt, and Swiss-Prot. These sequences include, but are not limited to, the human CD3ε protein sequence (e.g., UniProt / Swiss-Prot Accession No. P07766) and its corresponding nucleotide sequence (e.g., NCBI Reference Sequence: NM_000733.3), as well as their counterparts in other species.DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709

[0070] In some aspects, R3 comprises a nucleic acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 5 (GATATTAAGCTCCAACAATCCGGCGCCGAACTGGCCAGACCCGGCGCTTCCGTCAAA ATGTCCTGCAAAACTTCAGGCTATACTTTCACCCGGTACACAATGCACTGGGTGAAAC AAAGGCCCGGTCAAGGTCTTGAATGGATCGGATACATTAACCCAAGCCGCGGATATA CTAATTATAATCAAAAGTTCAAAGATAAGGCTACGCTGACCACGGATAAATCCAGTAGC ACAGCATATATGCAGCTGTCATCTCTCACTTCCGAGGACAGCGCAGTTTATTATTGTGC AAGATACTACGACGATCACTACTGCCTCGACTACTGGGGACAAGGAACCACGTTGAC GGTGTCTAGCGTGGAGGGTGGTAGTGGAGGCTCTGGCGGGAGCGGTGGGTCCGGT GGAGTTGACGACATTCAATTGACTCAATCACCCGCTATTATGTCCGCGTCCCCTGGGG AGAAGGTTACTATGACGTGTCGGGCGTCTAGCTCCGTGTCTTATATGAATTGGTACCA ACAGAAGTCTGGCACCTCCCCGAAAAGGTGGATATACGATACAAGCAAGGTCGCCTC CGGAGTTCCATATCGCTTTTCCGGTAGTGGGTCTGGGACCAGCTATTCCTTGACTATT TCTTCAATGGAAGCCGAGGATGCTGCCACCTACTATTGTCAACAATGGTCTTCCAACC CTTTGACGTTCGGCGCTGGTACGAAACTGGAGCTGAAG) or a codon degenerate nucleic acid sequence thereof. In other aspects, R3 comprises a nucleic acid sequence having at least 99.5%, or 99.8% sequence identity to SEQ ID NO: 5. In some aspects, R3 comprises a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 5. In some aspects, R3 comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 5. In some aspects, R3 comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 5. In some aspects, R3 comprises a nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 5. In some aspects, R3 comprises a nucleic acid sequence having at least 97% sequence identity to SEQ ID NO: 5. In some aspects, R3 comprises a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO: 5. In some aspects, R3 comprises a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 5. In some aspects, R3 comprises a nucleic acid sequence having 100% sequence identity to SEQ ID NO: 5. In some aspects, R3 comprises a nucleic acid sequence having at least 99.5% sequence identity to SEQ ID NO: 5. In some aspects, R3 comprises a nucleic acid sequence having at least 99.8% sequence identity to SEQ ID NO: 5.DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709

[0071] In some aspects, R3 comprises a nucleic acid sequence differing from SEQ ID NO: 5 by no more than 20 nucleotide substitutions (e.g., no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide substitution(s)). In some aspects, R3 comprises a nucleic acid sequence differing from SEQ ID NO: 5 by no more than 15 nucleotide substitutions. In some aspects, R3 comprises a nucleic acid sequence differing from SEQ ID NO: 5 by no more than 10 nucleotide substitutions. In some aspects, R3 comprises a nucleic acid sequence differing from SEQ ID NO: 5 by no more than 9 nucleotide substitutions. In some aspects, R3 comprises a nucleic acid sequence differing from SEQ ID NO: 5 by no more than 8 nucleotide substitutions. In some aspects, R3 comprises a nucleic acid sequence differing from SEQ ID NO: 5 by no more than 7 nucleotide substitutions. In some aspects, R3 comprises a nucleic acid sequence differing from SEQ ID NO: 5 by no more than 6 nucleotide substitutions. In some aspects, R3 comprises a nucleic acid sequence differing from SEQ ID NO: 5 by no more than 5 nucleotide substitutions. In some aspects, R3 comprises a nucleic acid sequence differing from SEQ ID NO: 5 by no more than 4 nucleotide substitutions. In some aspects, R3 comprises a nucleic acid sequence differing from SEQ ID NO: 5 by no more than 3 nucleotide substitutions. In some aspects, R3 comprises a nucleic acid sequence differing from SEQ ID NO: 5 by no more than 2 nucleotide substitutions. In some aspects, R3 comprises a nucleic acid sequence differing from SEQ ID NO: 5 by no more than 1 nucleotide substitution. In some aspects, R3 comprises the nucleic acid sequence of SEQ ID NO: 5.

[0072] Any of the R3 domains disclosed herein may also include additional subdomains. For example, the second scFv of R3 may further comprise a subdomain having formula IV or formula V: R8 – R9 (IV), R9 – R8 (V) where R8 encodes a VL domain and R9 encodes a VH domain. For example, in some aspects, from the 5’ to 3’ direction, R3 may also include formula II: R8 – R9 (IV). In other aspects, from the 5’ to 3’ direction, may also include formula III: R9 – R8 (V). In other examples, the VL sequence of R7 and the VH sequence of R8 may be subdomains from the sequence of R3, such as the nucleic acid sequence of SEQ ID NO: 5. In some aspects, R8 comprises a nucleic acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 21 (GACATTCAATTGACTCAAT CACCCGCTATTATGTCCGCGTCCCCTGGGGAGAAGGTTACTATGACGTGTCGGGCGT CTAGCTCCGTGTCTTATATGAATTGGTACCAACAGAAGTCTGGCACCTCCCCGAAAAGDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 GTGGATATACGATACAAGCAAGGTCGCCTCCGGAGTTCCATATCGCTTTTCCGGTAGT GGGTCTGGGACCAGCTATTCCTTGACTATTTCTTCAATGGAAGCCGAGGATGCTGCCA CCTACTATTGTCAACAATGGTCTTCCAACCCTTTGACGTTCGGCGCTGGTACGAAACT GGAGCTGAAG) or a codon degenerate nucleic acid sequence thereof. In other aspects, R8 comprises a nucleic acid sequence having at least 99.5%, or 99.8% sequence identity) to SEQ ID NO: 21 In some aspects, R9 comprises a nucleic acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 22 (GATATTAAGCTCCAACAATCCGGCGCCGAACTGGCCAGACCCGGCGCTTCCGTCAAA ATGTCCTGCAAAACTTCAGGCTATACTTTCACCCGGTACACAATGCACTGGGTGAAAC AAAGGCCCGGTCAAGGTCTTGAATGGATCGGATACATTAACCCAAGCCGCGGATATA CTAATTATAATCAAAAGTTCAAAGATAAGGCTACGCTGACCACGGATAAATCCAGTAGC ACAGCATATATGCAGCTGTCATCTCTCACTTCCGAGGACAGCGCAGTTTATTATTGTGC AAGATACTACGACGATCACTACTGCCTCGACTACTGGGGACAAGGAACCACGTTGAC GGTGTCTAGC) or a codon degenerate nucleic acid sequence thereof. In other aspects, R9 comprises a nucleic acid sequence having at least 99.5%, or 99.8% sequence identity) to SEQ ID NO: 22.

[0073] R4 encodes a half-life extender. A half-life extender is a modification to the immune cell engager that prolongs the time the immune cell engager is active in the body of a patient. A variety of methods may extend the half-life of an immune cell engager. For example, R4 may encode a single chain crystallizable region (scFc), a double scFc (double Fc) where the scFc is duplicated via a linker, or a CH3 domain. A scFc is a portion of the tail region of an antibody, for example, including a CH2domain and CH3domain linked as a single polypeptide. In the context of an antibody, the crystallizable region (Fc) interacts with cell surface receptors called Fc receptors and certain proteins of the complement system. Without being bound by any theory, it is believed that when binding to Fc receptors, antibodies activate the immune system. For example, this interaction can lead to opsonization (enhancing phagocytosis) or antibody-dependent cell-mediated cytotoxicity. The Fc region may contain two polypeptide fragments derived from the second and third constant domains of the antibody’s two heavy chains and remains constant within a class of antibodies for each species. A CH3 domain is the third domain of the constant region (Fc region) of an antibody. The Fc region refers to the part of the antibody molecule has aDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 mostly conserved amino acid sequence across different antibodies of the same class (e.g., IgG, IgM, IgA). The Fc region may include the CH2, and CH3 domains in the heavy chain. These regions may confer antibody stability, effector functions, and interactions with other immune system components. The CH3 domain facilitates dimerization of the Fc region, which can increase the interaction between an antibody and the neonatal Fc receptor (FcRn), thereby limiting degradation by the lysosome.

[0074] In some aspects, R4 encodes an immunoglobulin 1 (IgG1) scFc. An IgG1 scFc may include the a CH2 domain if the IgG1 polypeptide and CH3 domain of the IgG1 polypeptide linked as a single polypeptide In some aspects, R4 comprises a nucleic acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 7 (GATAAAACCCACACTTGCCCTCCGTGTCCCGCGCCTGAGGCTGCCGGTGGACCATC CGTGTTTTTGTTCCCTCCCAAACCGAAGGACACTCTGATGATCTCAAGAACTCCAGAG GTGACGTGCGTTGTAGTTGACGTATCACACGAGGAcCCAGAAGTCAAGTTTAATTGGT ATGTGGATGGAGTGGAAGTGCACAACGCTAAAACAAAACCCAGAGAGGAACAATATAA TTCTACCTACAGAGTAGTATCTGTATTGACGGTGCTTCATCAAGACTGGTTGAATGGCA AGGAATATAAATGTAAGGTATCAAATAAAGCCCTTGGCGCCCCGATCGAAAAAACTATT TCCAAGGCCAAGGGGCAGCCGCGCGAACCCCAGGTATATACGCTTCCACCGTCCCG CGACGAGCTGACGAAGAACCAGGTGTCATTGACGTGCCTGGTGAAGGGTTTTTATCC CTCCGACATCGCCGTGGAGTGGGAGAGTAACGGCCAGCCCGAGAACAACTACAAGA CCACCCCCCCTGTGCTGGACTCAGATGGCAGTTTTTTCCTTTACTCAAAACTCACCGT GGATAAGAGTAGATGGCAACAAGGAAATGTGTTTTCATGTTCTGTTATGCACGAAGCT CTTCACAACCATTACACACAGAAGTCCCTGTCTCTCtccCCTGGCAAA) or a codon degenerate nucleic acid sequence thereof. In other aspects, R4 comprises a nucleic acid sequence having at least 99.5%, or 99.8% sequence identity to SEQ ID NO: 7 In some aspects, R4 comprises a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 7. In some aspects, R4 comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 7. In some aspects, R4 comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 7. In some aspects, R4 comprises a nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 7. In some aspects, R4 comprises a nucleic acid sequence having at least 97% sequence identity to SEQ ID NO: 7. In some aspects, R4 comprises a nucleic acid sequence havingDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 at least 98% sequence identity to SEQ ID NO: 7. In some aspects, R4 comprises a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 7. In some aspects, R4 comprises a nucleic acid sequence having 100% sequence identity to SEQ ID NO: 7. In some aspects, R4 comprises a nucleic acid sequence having at least 99.5% sequence identity to SEQ ID NO: 7. In some aspects, R4 comprises a nucleic acid sequence having at least 99.8% sequence identity to SEQ ID NO: 7. In some aspects, R4 comprises a nucleic acid sequence differing from SEQ ID NO: 7 by no more than 20 nucleotide substitutions (e.g., no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide substitution(s)). In some aspects, R4 comprises a nucleic acid sequence differing from SEQ ID NO: 7 by no more than 15 nucleotide substitutions. In some aspects, R4 comprises a nucleic acid sequence differing from SEQ ID NO: 7 by no more than 10 nucleotide substitutions. In some aspects, R4 comprises a nucleic acid sequence differing from SEQ ID NO: 7 by no more than 9 nucleotide substitutions. In some aspects, R4 comprises a nucleic acid sequence differing from SEQ ID NO: 7 by no more than 8 nucleotide substitutions. In some aspects, R4 comprises a nucleic acid sequence differing from SEQ ID NO: 7 by no more than 7 nucleotide substitutions. In some aspects, R4 comprises a nucleic acid sequence differing from SEQ ID NO: 7 by no more than 6 nucleotide substitutions. In some aspects, R4 comprises a nucleic acid sequence differing from SEQ ID NO: 7 by no more than 5 nucleotide substitutions. In some aspects, R4 comprises a nucleic acid sequence differing from SEQ ID NO: 7 by no more than 4 nucleotide substitutions. In some aspects, R4 comprises a nucleic acid sequence differing from SEQ ID NO: 7 by no more than 3 nucleotide substitutions. In some aspects, R4 comprises a nucleic acid sequence differing from SEQ ID NO: 7 by no more than 2 nucleotide substitutions. In some aspects, R4 comprises a nucleic acid sequence differing from SEQ ID NO: 7 by no more than 1 nucleotide substitution. In some aspects, R4 comprises the nucleic acid sequence of SEQ ID NO: 7.

[0075] In some aspects, R4 encodes a modified scFc, for example, a modified scFc is an IgG1 scFc. As used herein a “modified scFc” is an scFc that may contain one or more (e.g., one, two, three, four or more) nucleotide modifications, such as a nucleotide substitution compared to a reference sequence. In some aspects, R4 comprises a leucine to alanine mutation at amino acids 234 and 235 as compared to the wild type IgG1 scFc amino acid sequence (SEQ ID NO: 24), or at corresponding amino acid positions in a homolog thereofDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 (referred to as a “LALA mutation”). In some aspects, R4 comprises a proline to glycine mutation at amino acid 329 as compared to SEQ ID NO: 24 or at corresponding amino acid positions in a homolog thereof (referred to as a “PG mutation”). In some aspects, R4 comprises two modifications, the first modification a leucine to alanine mutation at amino acids 234 and 235, and the second modification a proline to glycine mutation at amino acid 329 as compared to SEQ ID NO: 24 or at corresponding amino acid positions in a homolog thereof (referred to as a “LALAPG mutation”).

[0076] IgG1 scFc wild type amino acid sequence (SEQ ID NO: 24) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0077] In some aspects, R4 comprises a nucleic acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 55, or a codon degenerate sequence. In other aspects, R4 encodes an amino acid sequence comprising at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 60.

[0078] In some aspects, R4 comprises a nucleic acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 56, or a codon degenerate sequence. In other aspects, R4 encodes an amino acid sequence comprising at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 59.

[0079] In some aspects, R4 comprises a nucleic acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 57, or a codon degenerate sequence. In other aspects, R4 encodes an amino acid sequence comprising at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 58.

[0080] R5 encodes a 3’UTR region. The 3’ untranslated region (3’UTR) of messenger RNA (mRNA) immediately follows the translation termination codon. It may influence mRNADFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 localization, stability, export, and translation efficiency. Within the 3’UTR, regulatory elements may include microRNA response sites and AU-rich elements (AREs). Any appropriate 3’UTR can be used. In some aspects, R5 encodes a hemoglobin subunit beta (HBB gene) 3’UTR, but the 3’UTR may be derived from other genes.

[0081] In some aspects, R5 comprises a nucleic acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 8 (CTCGAGGGCGCGCCGCTCGCTTTCTTGCTGTCCA ATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGA AGGGCCTTGAtCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAG) or a codon degenerate nucleic acid sequence thereof. In other aspects, R5 comprises a nucleic acid sequence having at least 99.3% sequence identity to SEQ ID NO: 8. In some aspects, R5 comprises a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 8. In some aspects, R5 comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 8. In some aspects, R5 comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 8. In some aspects, R5 comprises a nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 8. In some aspects, R5 comprises a nucleic acid sequence having at least 97% sequence identity to SEQ ID NO: 8. In some aspects, R5 comprises a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO: 8. In some aspects, R5 comprises a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 8. In some aspects, R5 comprises a nucleic acid sequence having at least 100% sequence identity to SEQ ID NO: 8. In some aspects, R5 comprises a nucleic acid sequence having at least 99.3% sequence identity to SEQ ID NO: 8.

[0082] In some aspects, R5 comprises a nucleic acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 69 (GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTA CTAAACTGGGGGATATTATGAAGGGCCTTGAtCATCTGGATTCTGCCTAATAAAAAACA TTTATTTTCATTGCAAG) or a codon degenerate nucleic acid sequence thereof. In other aspects, R5 comprises a nucleic acid sequence having at least 99.3% sequence identity to SEQ ID NO: 69. In some aspects, R5 comprises a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 69. In some aspects, R5 comprises a nucleic acidDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 sequence having at least 90% sequence identity to SEQ ID NO: 69. In some aspects, R5 comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 69. In some aspects, R5 comprises a nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 69. In some aspects, R5 comprises a nucleic acid sequence having at least 97% sequence identity to SEQ ID NO: 69. In some aspects, R5 comprises a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO: 69. In some aspects, R5 comprises a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 69. In some aspects, R5 comprises a nucleic acid sequence having at least 100% sequence identity to SEQ ID NO: 69. In some aspects, R5 comprises a nucleic acid sequence having at least 99.3% sequence identity to SEQ ID NO: 69.

[0083] In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO: 8 or SEQ ID NO: 69 by no more than 20 nucleotide substitutions (e.g., no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide substitution(s)). In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO: 8 or SEQ ID NO: 69 by no more than 15 nucleotide substitutions. In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO: 8 or SEQ ID NO: 69 by no more than 10 nucleotide substitutions. In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO: 8 or SEQ ID NO: 69 by no more than 9 nucleotide substitutions. In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO: 8 or SEQ ID NO: 69 by no more than 8 nucleotide substitutions. In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO: 8 or SEQ ID NO: 69 by no more than 7 nucleotide substitutions. In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO: 8 or SEQ ID NO: 69 by no more than 6 nucleotide substitutions. In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO: 8 or SEQ ID NO: 69 by no more than 5 nucleotide substitutions. In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO: 8 or SEQ ID NO: 69 by no more than 4 nucleotide substitutions. In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO: 8 or SEQ ID NO: 69 by no more than 3 nucleotide substitutions. In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO: 8 or SEQ ID NO: 69 by no more than 2 nucleotide substitutions. In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO: 8 or SEQ ID NO: 69 by no more than 1 nucleotide substitution. In some aspects, R5 comprises the nucleic acid sequence of SEQ ID NO: 8 or SEQ ID NO: 69.DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709

[0084] A nucleic acid encoding a poly(A) tail may be included inside the 3’UTR or 3’ to the 3’UTR. A poly(A) tail is a stretch of adenine nucleotide bases (A) added to the 3’ end of mature eukaryotic messenger RNA (mRNA). It may enhance mRNA stability, protect against enzymatic degradation, aid in nuclear export, and facilitate translation in the cytoplasm of a cell. In some aspects, a poly(A) tail comprises a nucleic acid sequence of about 10 to about 300 nucleotide bases in length (e.g., from about 150 to about 250 nucleotide bases in length). In some cases, nucleotides other than adenine may be included in the polyA tail to facilitate synthesis of the mRNA.

[0085] Any of the nucleic acids for an immune cell engager disclosed herein may also include a nucleic acid encoding a linker (e.g., any appropriate linker). A linker is a short polypeptide that links two or more domains of a polypeptide. In some aspects, a linker is positioned between R2 and R3. In some aspects, a linker is positioned between R3 and R4. In some aspects, a first linker is positioned between R2 and R3 and a second linker is positioned between R3 and R4. In some aspects, a linker is positioned between R6 and R7. In some aspects, a linker is positioned between R8 and R9. In some aspects, a first linker is positioned between R6 and R7, and a second linker is positioned between R8 and R9. The first linker and the second linker may be the same nucleic acid encoding the same polypeptide, a different nucleic acid encoding the same polypeptide (e.g., conservative nucleotide mutations), or a different nucleic acid encoding a different polypeptide. In some aspects, a first linker is positioned between the R2 subdomains of R6 and R7, a second linker is positioned between the R3 subdomains of R8 and R9, a third linker is positioned between R2 and R3, and a fourth linker is positioned between R3 and R4, or any combination thereof. Additional linkers may be present in any of the nucleic acids for an immune cell engager to link additional domains and / or subdomains.

[0086] In some aspects, a linker comprises a nucleic acid sequence having at least 80% sequence identity (e.g., at least 85%, 90% 95%, or 100%) to SEQ ID NO: 4 (ACCggtgggggaggaagt) or a codon degenerate nucleic acid sequence thereof. In some aspects, a linker comprises a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 4. In some aspects, a linker comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 4. In some aspects, a linker comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 4. In someDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 aspects, a linker comprises a nucleic acid sequence having 100% sequence identity to SEQ ID NO: 4.

[0087] In some aspects, a linker comprises a nucleic acid sequence differing from SEQ ID NO: 4 by no more than 5 nucleotide substitutions (e.g., no more than 5, 4, 3, 2, or 1 nucleotide substitution(s)). In some aspects, a linker comprises a nucleic acid sequence differing from SEQ ID NO: 4 by no more than 4 nucleotide substitutions. In some aspects, a linker comprises a nucleic acid sequence differing from SEQ ID NO: 4 by no more than 3 nucleotide substitutions. In some aspects, a linker comprises a nucleic acid sequence differing from SEQ ID NO: 4 by no more than 2 nucleotide substitutions. In some aspects, a linker comprises a nucleic acid sequence differing from SEQ ID NO: 4 by no more than 1 nucleotide substitution. In some aspects, a linker comprises the nucleic acid sequence of SEQ ID NO: 4. In some aspects, a linker positioned between R2 and R3 comprises the nucleic acid sequence of SEQ ID NO: 4, comprises a nucleic acid sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, or 100%) to SEQ ID NO: 4, or comprises a nucleic acid sequence differing from SEQ ID NO: 4 by no more than 5 nucleotide substitutions (e.g., 4, 3, 2, or 1 nucleotide substitution(s)).

[0088] In some aspects, a linker comprises a nucleic acid sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, or 100%) to SEQ ID NO: 6 (GGCGGTGGTGGCGCCGGCGGAGGTGGCGCTAGC) or a codon degenerate nucleic acid sequence thereof. In some aspects, a linker comprises at least 96% sequence identity to SEQ ID NO: 6. In some aspects, a linker comprises a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 6. In some aspects, a linker comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 6. In some aspects, a linker comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 6. In some aspects, a linker comprises a nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 6. In some aspects, a linker comprises a nucleic acid sequence having 100% sequence identity to SEQ ID NO: 6.

[0089] In some aspects, a linker comprises a nucleic acid sequence differing from SEQ ID NO: 6 by no more than 6 nucleotide substitutions (e.g., no more than 6, 5, 4, 3, 2, or 1 nucleotide substitution(s)). In some aspects, a linker comprises a nucleic acid sequence differing from SEQ ID NO: 6 by no more than 5 nucleotide substitutions. In some aspects, a linker comprises a nucleic acid sequence differing from SEQ ID NO: 6 by no more than 4DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 nucleotide substitutions. In some aspects, a linker comprises a nucleic acid sequence differing from SEQ ID NO: 6 by no more than 3 nucleotide substitutions. In some aspects, a linker comprises a nucleic acid sequence differing from SEQ ID NO: 6 by no more than 2 nucleotide substitutions. In some aspects, a linker comprises a nucleic acid sequence differing from SEQ ID NO: 6 by no more than 1 nucleotide substitution. In some aspects, a linker comprises the nucleic acid sequence of SEQ ID NO: 6. In some aspects, a linker (e.g., a second linker) positioned between R3 and R4 comprises the nucleic acid sequence of SEQ ID NO: 6, comprises a nucleic acid sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 100%) to SEQ ID NO: 6, or comprises a nucleic acid sequence differing from SEQ ID NO: 6 by no more than 6 nucleotide substitutions (e.g., 5, 4, 3, 2, or 1 nucleotide substitution(s)).

[0090] Any of the nucleic acids disclosed herein may include a 5’ RNA cap. A 5’ RNA cap is a modified nucleotide structure found at the 5’ end of mRNA. It includes 7-methylguanosine (m7G) connected by a triphosphate bridge to the first nucleotide. The 5’ RNA may protect the mRNA from degradation, promote translation, and marks the mRNA as “self RNA” to the innate immune system. Other suitable RNA caps are also contemplated herein.

[0091] Any of the nucleic acids disclosed herein may include additional design tags to facilitate production, manufacturing, or purification. Any appropriate design tag may be included, include, but not limited to, a polyhistidine tag (His tags), a glutathione S- transferase tag (GST tag), a maltose-binding protein tag (MBP tag), a chitin binding protein tag (CBP tag) a hemagglutinin tag (HA tag), a Flag tag, and a polyglutamate tag. For example, any appropriate tag may be included in any appropriate location in any of the nucleic acid constructs. In some aspects, a design tag may be positioned between R4 and R5. In some aspects, the design tag positioned between R4 and R5 has a nucleic acid sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 23 (GCGATCGCCGATTACAAGGATGACGA TGACAAGGAGAATCTCTACTTCCAATCTGAGCAGAAACTAATATCAGAGGAGGATCTA GGGGGAGGAAGTCACCACCATCACCACCATCCTCGA), SEQ ID NO: 29 (GATTACAAGGAtGACGATGACAAGGAGAATCTCTACTTCCAATCTgagcagaaactaatatcag aggaggatctagggggaggaagtcaccaccatcaccaccatcctcga), or a codon degenerate nucleic acid sequence thereof.DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709

[0092] In some aspects, the tag comprises a nucleic acid sequence comprising SEQ ID NO: 29 (See Table 1) or a codon degenerate nucleic acid sequence thereof. In some aspects, the (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 66.

[0093] In some aspects, a linker comprises a nucleic acid sequence having at least 80% sequence identity (e.g., at least 85%, 90% 95%, or 100%) to SEQ ID NO: 27 (ggtgggggaggaagt) or a codon degenerate nucleic acid sequence thereof. In some aspects, a linker comprises a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 27. In some aspects, a linker comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 27. In some aspects, a linker comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 27. In some aspects, a linker comprises a nucleic acid sequence having 100% sequence identity to SEQ ID NO: 27.

[0094] In some aspects, the linker comprises a nucleic acid sequence comprising SEQ ID NO: 27 (See Table 1) or a codon degenerate nucleic acid sequence thereof. In some aspects, the linker comprises an amino acid sequence of at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 68.

[0095] In some aspects, a linker comprises a nucleic acid sequence differing from SEQ ID NO: 27 by no more than 5 nucleotide substitutions (e.g., no more than 5, 4, 3, 2, or 1 nucleotide substitution(s)). In some aspects, a linker comprises a nucleic acid sequence differing from SEQ ID NO: 27 by no more than 4 nucleotide substitutions. In some aspects, a linker comprises a nucleic acid sequence differing from SEQ ID NO: 27 by no more than 3 nucleotide substitutions. In some aspects, a linker comprises a nucleic acid sequence differing from SEQ ID NO: 27 by no more than 2 nucleotide substitutions. In some aspects, a linker comprises a nucleic acid sequence differing from SEQ ID NO: 27 by no more than 1 nucleotide substitution. In some aspects, a linker comprises the nucleic acid sequence of SEQ ID NO: 27. In some aspects, a linker positioned between R2 and R3 comprises the nucleic acid sequence of SEQ ID NO: 27, comprises a nucleic acid sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, or 100%) to SEQ ID NO: 27, or comprises a nucleic acid sequence differing from SEQ ID NO: 27 by no more than 5 nucleotide substitutions (e.g., 4, 3, 2, or 1 nucleotide substitution(s)).DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709

[0096] In some aspects, a linker comprises a nucleic acid sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, or 100%) to SEQ ID NO: 28 (GGCGGTGGTGGCGCCGGCGGAGGTGGC) or a codon degenerate nucleic acid sequence thereof. In some aspects, a linker comprises at least 96% sequence identity to SEQ ID NO: 28. In some aspects, a linker comprises a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 28. In some aspects, a linker comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 28. In some aspects, a linker comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 28. In some aspects, a linker comprises a nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 28. In some aspects, a linker comprises a nucleic acid sequence having 100% sequence identity to SEQ ID NO: 28.

[0097] In some aspects, a linker comprises a nucleic acid sequence differing from SEQ ID NO: 28 by no more than 6 nucleotide substitutions (e.g., no more than 6, 5, 4, 3, 2, or 1 nucleotide substitution(s)). In some aspects, a linker comprises a nucleic acid sequence differing from SEQ ID NO: 28 by no more than 5 nucleotide substitutions. In some aspects, a linker comprises a nucleic acid sequence differing from SEQ ID NO: 28 by no more than 4 nucleotide substitutions. In some aspects, a linker comprises a nucleic acid sequence differing from SEQ ID NO: 28 by no more than 3 nucleotide substitutions. In some aspects, a linker comprises a nucleic acid sequence differing from SEQ ID NO: 28 by no more than 2 nucleotide substitutions. In some aspects, a linker comprises a nucleic acid sequence differing from SEQ ID NO: 28 by no more than 1 nucleotide substitution. In some aspects, a linker comprises the nucleic acid sequence of SEQ ID NO: 28. In some aspects, a linker (e.g., a second linker) positioned between R3 and R4 comprises the nucleic acid sequence of SEQ ID NO: 28, comprises a nucleic acid sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 100%) to SEQ ID NO: 28, or comprises a nucleic acid sequence differing from SEQ ID NO: 28 by no more than 6 nucleotide substitutions (e.g., 5, 4, 3, 2, or 1 nucleotide substitution(s)).

[0098] In some aspects, the linker comprises a nucleic acid sequence comprising SEQ ID NO: 28 (See Table 1) or a codon degenerate nucleic acid sequence thereof. In some aspects, the linker comprises an amino acid sequence of at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 67.DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709

[0099] In some aspects, a nucleic acid for an immune cell engager may include an RNA of formula I: R1 – SP – R2 – R3 – R4 – R5 (I), wherein: R1 is a 5’ untranslated region (UTR); SP encodes a signal peptide; R2 encodes a single chain variable fragment (scFv) that binds BCMA; R3 encodes a scFv that binds CD3; R4 encodes a half-life extender; R5 is a 3’UTR, and wherein R1 to R5 are oriented 5’ to 3’, R1 is encoded by the nucleic acid of SEQ ID NO: 1, SP is encoded by the nucleic acid of SEQ ID NO: 2, R2 is encoded by the nucleic acid of SEQ ID NO: 3, R3 is encoded by the nucleic acid of SEQ ID NO: 5, R4 is encoded by the nucleic acid of SEQ ID NO: 7, and R5 is encoded by the nucleic acid of SEQ ID NO: 8. In some aspects, a nucleic acid for an immune cell engager of formula I may also include a linker encoded by the nucleic acid sequence of SEQ ID NO: 4 that is positioned between R2 and R3. In some aspects, a nucleic acid for an immune cell engager of formula I may also include a linker encoded by the nucleic acid sequence of SEQ ID NO: 6 that is positioned between R3 and R4. Therefore, in some aspects, a nucleic acid for an immune cell engager may include an RNA of formula I: R1 – SP – R2 – R3 – R4 – R5 (I), wherein: R1 is a 5’ untranslated region (UTR); SP encodes a signal peptide; R2 encodes a single chain variable fragment (scFv) that binds BCMA; R3 encodes a scFv that binds CD3; R4 encodes a half-life extender; R5 is a 3’UTR, and wherein R1 to R5 are oriented 5’ to 3’, R1 is encoded by the nucleic acid of SEQ ID NO: 1, SP is encoded by the nucleic acid of SEQ ID NO: 2, R2 is encoded by the nucleic acid of SEQ ID NO: 3, R3 is encoded by the nucleic acid of SEQ ID NO: 5, R4 is encoded by the nucleic acid of SEQ ID NO: 7, R5 is encoded by the nucleic acid of SEQ ID NO: 8, a linker encoded by the nucleic acid sequence of SEQ ID NO: 4 is positioned between R2 and R3, and a second linker encoded by the nucleic acid sequence of SEQ ID NO: 6 is positioned between R3 and R4.

[0100] In some aspects, a nucleic acid for an immune cell engager may include at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity) to SEQ ID NO: 20 (ATCCACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGGGC GGCCGCCACCATGGTGTTGCAGACCCAGGTCTTCATTTCTCTGTTGCTCTGGATCTCT GGTGCCTACGGGCAGGTCCAACTGGTTGAGTCTGGGGGAGGCGTTGTTCAGCCGGG GAGGTCACTTCGGCTCTCATGCGCGGCTACTGGTTTTACGTTCAGTACATACGGCATG TATTGGGTCAGGCAGGCGCCCGGTAAGGGCCTCGAATGGGTCGCGGCCATCTGGAA CGATGGGTCCAACAAGTATTATGCTGATTCCGTCAAAGGACGGTTCACAATAAGCCGGDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 GACGATTCCAAAAATACTCTGTATGTTCAGATGAACTCACTCAGAGCTGAGGACACAG CCGTTTATTACTGTGCACGCGAGCGAATCCCGATGGCCAGCTTGAGGTATTTCGATTG GTTGGGCGTAATGGACGCATGGGGTCAGGGAGCCAGCGTAACAGTAAGTTCAGGTG GCGGAGGATCTGGCGGCGGAGGAAGCGGAGGCGGCGGATCTGACATACAAATGACA CAGAGTCCTTCCACCTTGTCTGCTTCTGTGGGTGACAGGGTTACGATAACTTGTAGGG CATCACAAAGTATCTCCTCATGGCTTGCTTGGTACCAGCAGAAGCCAGGACAAGCACC GAAGTTGCTTATTTACAAGGCGTCTAGCCTTGAAAGTGGAGTCCCTTCAAGGTTTAGC GGCTCTGGATCTGGCACTGAATTTACGCTGACCATTAGCAGTCTTCAGCCGGATGACT TCGCCACATACTACTGCCAACAATACTATTCATACCTCTACACTTTTGGCCAAGGCACG AAACTTGAGATCAAGACCGGTGGGGGAGGAAGTGATATTAAGCTCCAACAATCCGGC GCCGAACTGGCCAGACCCGGCGCTTCCGTCAAAATGTCCTGCAAAACTTCAGGCTAT ACTTTCACCCGGTACACAATGCACTGGGTGAAACAAAGGCCCGGTCAAGGTCTTGAAT GGATCGGATACATTAACCCAAGCCGCGGATATACTAATTATAATCAAAAGTTCAAAGAT AAGGCTACGCTGACCACGGATAAATCCAGTAGCACAGCATATATGCAGCTGTCATCTC TCACTTCCGAGGACAGCGCAGTTTATTATTGTGCAAGATACTACGACGATCACTACTG CCTCGACTACTGGGGACAAGGAACCACGTTGACGGTGTCTAGCGTGGAGGGTGGTA GTGGAGGCTCTGGCGGGAGCGGTGGGTCCGGTGGAGTTGACGACATTCAATTGACT CAATCACCCGCTATTATGTCCGCGTCCCCTGGGGAGAAGGTTACTATGACGTGTCGG GCGTCTAGCTCCGTGTCTTATATGAATTGGTACCAACAGAAGTCTGGCACCTCCCCGA AAAGGTGGATATACGATACAAGCAAGGTCGCCTCCGGAGTTCCATATCGCTTTTCCGG TAGTGGGTCTGGGACCAGCTATTCCTTGACTATTTCTTCAATGGAAGCCGAGGATGCT GCCACCTACTATTGTCAACAATGGTCTTCCAACCCTTTGACGTTCGGCGCTGGTACGA AACTGGAGCTGAAGGGCGGTGGTGGCGCCGGCGGAGGTGGCGCTAGCGATAAAACC CACACTTGCCCTCCGTGTCCCGCGCCTGAGGCTGCCGGTGGACCATCCGTGTTTTTG TTCCCTCCCAAACCGAAGGACACTCTGATGATCTCAAGAACTCCAGAGGTGACGTGC GTTGTAGTTGACGTATCACACGAGGACCCAGAAGTCAAGTTTAATTGGTATGTGGATG GAGTGGAAGTGCACAACGCTAAAACAAAACCCAGAGAGGAACAATATAATTCTACCTA CAGAGTAGTATCTGTATTGACGGTGCTTCATCAAGACTGGTTGAATGGCAAGGAATAT AAATGTAAGGTATCAAATAAAGCCCTTGGCGCCCCGATCGAAAAAACTATTTCCAAGG CCAAGGGGCAGCCGCGCGAACCCCAGGTATATACGCTTCCACCGTCCCGCGACGAG CTGACGAAGAACCAGGTGTCATTGACGTGCCTGGTGAAGGGTTTTTATCCCTCCGACA TCGCCGTGGAGTGGGAGAGTAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 CCTGTGCTGGACTCAGATGGCAGTTTTTTCCTTTACTCAAAACTCACCGTGGATAAGA GTAGATGGCAACAAGGAAATGTGTTTTCATGTTCTGTTATGCACGAAGCTCTTCACAAC CATTACACACAGAAGTCCCTGTCTCTCTCCCCTGGCAAAGCGATCGCCGATTACAAGG ATGACGATGACAAGGAGAATCTCTACTTCCAATCTGAGCAGAAACTAATATCAGAGGA GGATCTAGGGGGAGGAAGTCACCACCATCACCACCATCCTCGATAACTCGAGGGCGC GCCGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAA CTACTAAACTGGGGGATATTATGAAGGGCCTTGATCATCTGGATTCTGCCTAATAAAAA ACATTTATTTTCATTGCAAG) or a codon degenerate nucleic acid sequence thereof.

[0101] In some aspects, a nucleic acid for an immune cell engager comprises the nucleic acid sequence SEQ ID NO: 25, or a codon degenerate sequence thereof. In some aspects, an amino acid sequence for an immune cell engager comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity) to SEQ ID NO: 26.

[0102] In some aspects, a nucleic acid for an immune cell engager comprises the nucleic acid sequence of SEQ ID NO: 30, or a codon degenerate sequence thereof. In some aspects, an amino acid for an immune cell engager comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity) to SEQ ID NO: 31.

[0103] In some aspects, a nucleic acid for an immune cell engager comprises the nucleic acid sequence of SEQ ID NO: 32, or a codon degenerate sequence thereof. In some aspects, an amino acid for an immune cell engager comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity) to SEQ ID NO: 33.In some aspects, a nucleic acid for an immune cell engager comprises the nucleic acid sequence of SEQ ID NO: 44, or a codon degenerate sequence thereof. In some aspects, an amino acid for an immune cell engager comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity) to SEQ ID NO: 45. Any of the nucleic acids encoding immune cell engagers described herein may also be used to achieve the desired pharmacological effect by administration to a patient in need thereof in an appropriately formulated pharmaceutical composition. A patient, for the purpose of this disclosure, is a domestic animal or a human, in need of treatment for a particular condition or disease. Therefore, also disclosed hereinDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 are pharmaceutical compositions for immune cell engagers comprising any of the nucleic acids encoding immune cell engagers described herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0104] By “pharmaceutically acceptable” it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation, including nucleic acids encoding immune cell engagers, and not deleterious to the recipient thereof. A “pharmaceutically acceptable carrier” is any carrier which is relatively non-toxic and innocuous to a patient at concentrations consistent with effective activity of the active ingredient (e.g., a nucleic acid encoding immune cell engagers) so that any side effects ascribable to the carrier do not impair the beneficial effects of the active ingredient.

[0105] Appropriate pharmaceutical compositions comprising the nucleic acids encoding immune cell engagers are contemplated herein, and are based partly on the specific tissues, and cell types involved. Pharmaceutical compositions appropriate for nucleic acids encoding immune cell engagers of the instant disclosure may be thus be formulated according to any means known in the art. (See for example: Remington's Pharmaceutical Sciences, 15th Edition, chapter 33; Gagliardi et al., 2021; or Hammond et al., 2021).

[0106] Lipid nanoparticle (LNP) encapsulation of the nucleic acids encoding immune cell engagers is believed to be an approach for protecting the nucleic acids encoding immune cell engagers from degradation and facilitating cellular delivery. LNP’s thus provide an opportunity to formulate compositions comprising nucleic acids encoding immune cell engagers that are less susceptible to eliciting an immune response and subsequent clearance. This may be due to the components of the lipid particle itself. The present disclosure thus contemplates the use of lipid nanoparticles to encapsulate the nucleic acids encoding immune cell engagers described herein. See, for example, Wang et al. J Biomed Sci 30, 84 (2023). Thus, in one aspect described herein, is a pharmaceutical composition comprising any of the nucleic acids encoding immune cell engagers described herein, a pharmaceutically acceptable salt thereof, and a lipid nanoparticle. In some aspects, the lipid nanoparticle is a cationic nanoparticle. The LNP may direct the payload (e.g., nucleic acids encoding immune cell engagers) to a preferred cell type in the patient. For example, LNPs may target hepatocytes by incorporating specific ligands or surface modifications that exhibit a liver tropism. These modifications allow LNPs to interact with receptors on hepatocyte membranes, facilitating cellular uptake and payload delivery. The liver’s naturalDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 affinity for lipid-based carriers enhances the selective accumulation of LNPs in hepatocytes, making them an effective delivery system for liver-targeted therapies. See, for example, Chu et al. J. Mater. Chem. B, (2024), 12, 4759-4784. LNPs may be formulated as described in Example 3. LPNs may be formulated as described in Wang et al. J Biomed Sci 30, 84 (2023).

[0107] The pharmaceutical compositions for the administration of the nucleic acids of this disclosure may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredients (e.g., nucleic acids encoding immune cell engagers) into association with a carrier which constitutes one or more accessory ingredients. In general, the pharmaceutical compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. In the pharmaceutical composition, the active object compound is included in an amount sufficient to produce the desired effect upon the process or condition of diseases.

[0108] The nucleic acids encoding immune cell engagers described herein may be administered with a pharmaceutically acceptable carrier as a pharmaceutical composition using any effective conventional dosage unit forms, including, for example, immediate and timed-release preparations, injectable, parenterally, topically or the like. In some aspects, any of the nucleic acids encoding immune cell engagers or pharmaceutical compositions described herein is administered intravenously, intra-arterially, intra-tumorally, inter- dermally, subcutaneously, topically, ex vivo, extra-corporally, or via intraperitoneal administration, or as local, regional, systemic, or continual administration. In some aspects, any of the nucleic acids encoding immune cell engagers or pharmaceutical compositions described herein is administered intravenously.

[0109] The nucleic acids encoding immune cell engagers described herein may use multiple binding regions (e.g., R2 and R3) to simultaneously bind an effector cell and a cell overexpressing the first extracellular protein. For example, nucleic acids described herein can use multiple binding regions (e.g., R2 and R3) to simultaneously bind an immune cell and a cell overexpressing BCMA.

[0110] The nucleic acids encoding immune cell engagers described herein may use multiple binding regions (e.g., R2 and R3) to simultaneously bind an effector cell and aDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 cancer or tumor cell. Thus, the immune cell engagers encoded by the nucleic acids described herein bring effector cells that may have anti-tumor or anti-cancer cell properties into close proximity to the cancer or tumor cells. Therefore, administering the nucleic acids encoding the immune cell engagers described herein or any of the pharmaceutical compositions described herein facilitates targeting cancer or tumor cells, such as multiple myeloma cells, by anti-cancer effector cells, such as T cells. Thus, also described herein are methods of treating cancer, such as methods of treating a hematological cancer (e.g., methods of treating multiple myeloma), and method of engaging effector cells, such as methods of engaging T cells. It is contemplated that any method or pharmaceutical composition described herein may be implemented with respect to any other method or pharmaceutical composition described herein.

[0111] In one aspect of the present disclosure is a method of treating a cancer in a patient in need thereof. A method of treating cancer in a patient in need thereof comprises administering to the patient an effective amount of any of the nucleic acids encoding an immune cell engager described herein or any of the pharmaceutical compositions described herein. In another aspect, a method of treating cancer in a patient in need thereof comprises administering to the patient an effective amount of any of the nucleic acids encoding an immune cell engager described herein or any of the pharmaceutical compositions described herein.

[0112] For example, any of the nucleic acids encoding immune engagers described herein can be used in a method of treating a hematological cancer, the method comprising administering to the patient an effective amount of any of the nucleic acids encoding an immune cell engager described herein or any of the pharmaceutical compositions described herein. Exemplary hematological cancers include, but are not limited to myeloma, leukemia, and lymphoma. All three types affect the growth and function of white blood cells. Leukemia affects immature white blood cells, lymphoma affects lymphocytes, and myeloma affects plasma cells.

[0113] In one aspect of the present disclosure is a method of treating a myeloma in a patient in need thereof. For example, disclosed herein is a method of treating multiple myeloma in a patient in need thereof, the method comprising administering to the patient an effective amount of any of the nucleic acids encoding an immune cell engager described herein or any of the pharmaceutical compositions described herein. Multiple myeloma is aDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 type of hematological cancer that originates in plasma cells. These plasma cells typically produce antibodies to fight infections. However, patients within multiple myeloma, abnormal plasma cells accumulate in the bone marrow, crowding out healthy plasma cells. This leads to several symptoms include, but not limited to, bone pain, especially in the chest, spine, pelvis, and ribs, fatigue, muscle weakness, nausea, constipation, hypercalcemia (elevated blood calcium levels, causing thirst, confusion, and constipation), increased protein levels in the kidneys, anemia, and a weakened immune system resulting in greater susceptibility to infections. Risk factors of multiple myeloma include family history, age (more common in older adults), gender (more common in males), ethnicity (higher incidence in African Americans), and obesity.

[0114] In one aspect, the compositions and methods described herein are used as a first- line therapy (sometimes called primary therapy). In another aspect, the compositions and methods described herein are used as a second- line therapy. In some aspects, the compositions and methods described herein are used as third-line therapy or as a more advanced-line therapy (e.g., as a fourth-line, fifth-line, sixth-line, seventh-line, eight-line, ninth-line, tenth-line or more advanced-line therapy). In some aspects, the compositions and methods described herein are used as a salvage therapy. The term “salvage therapy” means a therapeutic agent that can be taken with any regimen after a subject’s initial treatment regimen has failed or after the subject’s condition has not responded to an initial treatment. In another aspect, the compositions and methods described herein are used as a rescue therapy. In one embodiment, the compositions are used as a rescue agent to counteract the action of an initial treatment. In one embodiment, the compositions are used as rescue agent that is administered to a subject who has developed resistance to a standard or an initial treatment. Common first line treatments for multiple myeloma include, but are not limited to, treatment with chemotherapy drugs such as an immunomodulatory imide drug (e.g., lenalidomide or pomalidomide), and proteasome inhibitors such as bortezomib and carfilzomib, treatment with corticosteroids such as prednisone or dexamethasone bone marrow transplantation, and radiation.

[0115] In one aspect, the patient has received at least one prior therapeutic agent. In one aspect the patient has received at least two, at least three, or at least four or more prior therapeutic agents. In another aspect, the prior therapeutic agent is lenalidomide or pomalidomide. In another aspect, the prior therapeutic agent is bortezomib or carfilzomib.DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 In another aspect, the prior therapeutic agent is lenalidomide, pomalidomide, bortezomib or carfilzomib. In another aspect, the patient may have been previously treated with radiation. In one aspect, the cancer no longer responds to treatment lenalidomide, pomalidomide, bortezomib, carfilzomib, bone marrow transplantation, radiation, or a combination thereof.

[0116] In one aspect, the methods described herein include administration of a second therapeutic agent including an anti-cancer agent, bone marrow transplantation, or radiation. In aspect, the second therapeutic agent is an anti-cancer agent selected from lenalidomide, pomalidomide, bortezomib, carfilzomib, or combinations thereof. Thus, another embodiment described herein is a method of treating cancer, such as a hematological cancer (e.g., a method of treating multiple myeloma) in a patient in need thereof comprising: (a) administering a first treatment comprising any of the pharmaceutical compositions described herein or any of the nucleic acids encoding an immune cell engager described herein or any of the pharmaceutical compositions described herein; and (b) administering a second therapeutic agent (e.g., an anti-cancer agent, bone marrow transplantation, or radiation).

[0117] In any of the methods of treatment described herein the pharmaceutical composition or nucleic acids encoding immune cell engagers described herein may be administered to a subject according to an infrequent dosing regimen (e.g., administered once per week or less frequently). In another aspect, the composition is administered to a subject once weekly. In another aspect, the composition is administered to a subject once every two weeks. In one aspect, the composition is administered to a subject once every four weeks. In one aspect, the composition is administered to a subject twice a week. In one aspect, the composition is administered to a subject once every three weeks. In one aspect, the composition is administered to a subject according to a frequent dosing regimen (e.g., administered more than once per day or more than once per week). In one aspect, the composition is administered to a subject in a repeated cycle of once daily, twice daily, once weekly, once every two weeks, once every three weeks, once every four weeks or combinations thereof. Any appropriate dosing regimen is contemplated herein.

[0118] As used herein, the articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" can mean one element or more than one element.DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709

[0119] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In some embodiments, the terms "about" or "approximately" when preceding a numerical value indicates the value plus or minus a range of 10%, 5%, or 1%.

[0120] As used herein, “nucleic acid” refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double and single stranded DNA, triplex DNA, as well as double and single stranded RNA. Any RNA format is contemplated so long as it results in the described amino acid sequences. It also includes modified forms, for example, by methylation and / or by capping, and unmodified forms of the polynucleotide. The term is also meant to include molecules that include non-naturally occurring or synthetic nucleotides as well as nucleotide analogs.

[0121] As used herein, the term “sequence identity” in the context of two nucleic acid sequences or amino acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981); by the alignment algorithm of Needleman and Wunsch, J Mal. Biol., 48:443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad Sci U.S.A., 85:2444 (1988); by computerized implementations of these algorithms (including, but not limited to CLUSTAL in the PC / Gene program by Intelligentics, Mountain View Calif, GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., U.S.A.); the CLUSTAL program is well described by Higgins and Sharp, Gene, 73:237-244 (1988) and Higgins and Sharp, CABIOS, 5:151-153 (1989); Corpet et al., Nucleic Acids Res., 16:10881-10890 (1988); Huang et al., Computer Applications in the Biosciences, 8:155-165 (1992); and Pearson et al., Methods in Molecular Biology, 24:307-331 (1994). Alignment is also often performed by inspection and manual alignment.DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709

[0122] As used herein, "an effective amount" refers to an amount that causes relief of symptoms of a disorder or disease as noted through clinical testing and evaluation, patient observation, and / or the like. An "effective amount" may further designate a dose that causes a detectable change in biological or chemical activity. The detectable changes may be detected and / or further quantified by one skilled in the art for the relevant mechanism or process. Moreover, an "effective amount" may designate an amount that maintains a desired physiological state, i.e., reduces or prevents significant decline and / or promotes improvement in the condition of interest. An "effective amount" may further refer to a “therapeutically effective amount”.

[0123] As used herein, the term "individual" and "subject" are often used interchangeably and refer to any human or domestic animal that may be treated with the methods disclosed herein. Suitable subjects (e.g., patients) include humans and domestic animals or pets (such as a cat or dog). Non–human primates and human patients are included. In one embodiment, subjects may include human patients that have been diagnosed with cancer, such as hematological cancer, including but not limited to: multiple myeloma. As used herein, the term "patient" refers to a subject that may receive a treatment of a disease or condition.

[0124] As used herein, "treatment", "treat", and "treating" refer to reversing, alleviating, mitigating, or slowing the progression of, or inhibiting the progress of, a disorder or disease or symptoms associated with such disorder or disease, and as described in more detail herein.

[0125] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0126] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this disclosure that certainDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results. EQUIVALENTS

[0127] Various aspects of the present disclosure may be used alone, in combination, or in a variety of arrangements not specifically discussed in the embodiments described in the foregoing and is, therefore, not limited in its application to the details and arrangement of components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments. EXAMPLES Example 1 - Binding of fully human H2L2 antibodies against BCMA

[0128] H2L2 mice (Harbor Antibodies) produced humanized, tetrameric antibodies comprising two identical heavy chains and two identical light chains. H2L2 mice (transgenic for human VH and human VL) were utilized to produce fully-human variable domain H2L2 antibodies. Single B / plasma cells were isolated from mice, production and specificity of antibody confirmed at the single cell level, followed by single-cell sequencing. Recombinant antibodies were produced and characterized including PR000892 (892), PR000897 (897), PR00903 (903), PR00909 (909), as well as the reference antibody PR000274 (274). The humanized H2L2s are described in PCT Patent Publication WO2021147941. The reference, positive control antibody PR0000274 is clone CA8-J6M0. It is described in U.S. Patent 9,273,141 and is a clinically validated anti-BCMA antibody. The newly raised H2L2s antibodies bound to human cell lines expressing BCMA (HEK293T-hu BCMA), with comparable binding strength as the reference antibody PR000274 (Fig.1A). All antibodies exhibited cross-reactivity (Fig.1B), exemplified by binding to HEK293T cells that express the cynomolgus (cyno) monkey BCMA protein (HEK293T-cyno BCMA). Furthermore, the fully human H2L2 antibodies bound strongly to NCI-H9292 myeloma cells (Fig.1C). A model for the binding of the various TCE proteins is shown in Fig.1D. Example 2 – Assessing In vitro cytotoxicity of T cell engagers (TCEs)DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709

[0129] NIH 3T3 Fibroblast cells were maintained in DMEM and 10% FBS (Gibco, Life Technologies). To generate GFP / luciferase–positive target cells or to express BCMA or TROPS (irrelevant target) in NIH- 3T3s, cells were stably transduced with gamma retrovirus expressing the cDNA. These target cells were then sorted into single-cell clones in 96-well plates, and expanded to generate clonal populations, which were confirmed by flow cytometry for use in all experiments.

[0130] Expi293 (Thermo Fisher) served as the producer cell line for TCEs. Cells were transduced per manufacturer instruction with plasmid DNA, and supernatant collected. Supernatant was then concentrated and buffer exchanged to PBS via size exclusion (Amicon Ultra, Millipore). TCE concentration was quantified via ELISA.

[0131] Human T cells were obtained from the peripheral blood of healthy donors (MGB Crimson Core Blood Bank; study #T0761).1 × 104human CD3+T cells as effector cells were stimulated with CD3 / CD28 TransAct (Miltenyi, #130-111-160) at a 1:100 ratio for 48 hours and grown in the presence of IL-2 ± IL-7 and IL-15 (NIH BRB Preclinical Biologics Repository), plated together with 5 × 103luciferase-transduced target cells into white 96- well flat-bottom plates (Costar). TCE was added at 1 nM per well. Plates were incubated for 24 h for effector cells to lyse cancer cells. End point lysis was a measurement of viable luciferase positive cells according to a ONE-Glo™ Luciferase Assay System (BD Biosciences). Conditions were plated in triplicate at the indicated effector-to-target (E:T) ratios. Bioluminescence was read on a BioTek Cytation 5. % cytotoxicity = (BLI Mock- BLI Sample) / BLI Mock, BLI Mock = mean target cell alone value of that experiment.

[0132] Results are shown in Fig.2. Example 3 – Assessing In vivo efficacy dose of TCEs

[0133] mRNA synthesis: DNA template plasmids were linearized with EcorI (NEB) and purified (Nucleospin PCR clean up, Takara Bio). mRNA IVT was performed with using HiScribe T7 High-Yield RNA synthesis kit (NEB). The UTP in the reaction was fully replaced with 1-methylpseudouridine triphosphate (N1-methylpseudouridine-5′- triphosphate, m1ΨTP) (TriLink). m7G5′ppp5′G2´-O-Met-capped (cap1) IVT mRNAs were generated using vaccinia virus guanylyltransferase and 2´-O-methyltransferase (NEB). mRNAs were polyadenylated with E.coli Poly(A) polymerase then cleaned using Monarch RNA Cleanup Kit (NEB). Quality of the purified IVT mRNA wereDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 assessed by electrophoresis on a 2100 Bioanalyzer (Agilent) and concentration determined by UV spectroscopy. mRNA was aliquoted and stored at -80oC.

[0134] Lipids and LNP formulation: Ionizable lipid SM-102 was obtained from Cayman Chemical (Cat#33474).1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Cat# 850365P) and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG200, Cat# 880150) were obtained from Avanti Polar Lipids. Cholesterol (Cat# C8667) was purchased from Sigma-Aldrich. All lipids were dissolved in absolute ethanol (Cat# BP2818, Fisher Scientific).

[0135] For IV administration in in vivo studies, mRNA was encapsulated in lipid nanoparticles by NanoAssembler Ignite microfluidic cartridge technology (Precision Nanosystems). In brief, lipids were mixed at a molar ratio of 50:10:38.5:1.5 (SM102:DSPC:cholesterol:DMG-PEG) and mRNA was prepared in 10^mM citrate buffer (pH 3.0) at 0.1^mg / mL. Then the lipid mixture and mRNA were mixed at an N / P ratio of 4, flow rate of 12^mL / min and a flow rate ratio of 3:1 (aqueous phase:organic phase). After formulation, the mRNA-LNP was dialyzed twice through a 10K MWCO Slide-A-Lyzer dialysis cassette (Cat# 66380, Thermo Scientific) 50^μg / ml in 10% sucrose PBS solution (w / v). The mRNA encapsulation efficiency and concentration were determined by Quant-iT TM RiboGreen assay (Cat# R11490, Life Technologies). Aliquots were stored at -20oC.

[0136] The human multiple myeloma (MM) cell line OPM2 was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DMSZ) and maintained in RPMI and 10% fetal bovine serum (FBS) (Gibco, Life Technologies, Gaithersburg, MD). To generate GFP / luciferase–positive target cells, as before these were stably transduced with gamma retrovirus expressing the cDNA. Cells were then sorted into single-cell clones in 96-well plates, and expanded to generate clonal populations, which were confirmed by flow cytometry for use in all experiments. Human T cells were prepared as before.

[0137] Six to eight week-old NSG DKO (NOD.Cg-Prkdcscid H2-K1b-tm1Bpe H2- Ab1g7-em1Mvw H2-D1b-tm1Bpe Il2rgtm1Wjl / SzJ) mice (Jackson Labs; Bar Harbor, ME) were injected with the bone marrow tropic cell line OPM2-ffLuc, at a dose of 1×106cells via tail vein. Tumor engraftment was confirmed by baseline bioluminescent imaging. Twelve days after tumor engraftment, mice were humanizedDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 with a single dose of 10 ×106human T cells injected via tail vein. The following day animals were injected IV with mRNA-LNP according to the dose schedule depicted. In vivo imaging was preformed after injection of D- luciferin (Millipore-Sigma; Darmstadt, Germany) and analyzed with Living Image software (PerkinElmer; Waltham, MA). Studies were planned with the minimum number of animals per treatment group to reproducibly observe statistically significant differences (n = 4-5 per group).

[0138] Results are shown in Figs.3A-3D. Example 4 – Exemplary TCEs

[0139] The mTCE sequence as outlined in Fig.4A is composed of a region encoding a RNA polymerase promoter, capping signal, 5 UTR, signal peptide, a tumor associated antigen binding domain (scFv (further comprised of a VH linked to VL) or VH domain), linker, immune cell engaging domain (again a scFv (further comprised of a VH linked to VL) or VH domain) linked to a half-life extension domain and required termination sequences, with or without an encoded poly-A tail, finalized by 3’ UTR.

[0140] The 5’UTR may be selected from the mRNA sequences of naturally occurring proteins or be synthetically derived. The term signal peptide as used herein refers to a short (e.g., 5-30 or 10-100 amino acids long) stretch of amino acids typically at the N-terminus of a protein that directs the transport of the protein. The signal peptide allows for the association of the mRNA and ribosome with the endoplasmic reticulum, insertion of the newly translated protein into the translocon, translocation, and trafficking of the protein product to the plasma membrane. Often, the signal peptide is cleaved off during the post-translational modification of a protein by a cell. The signal peptide was evaluated in silico to ensure efficient functional cleavage. VH and VL amino acid sequences were raised as described in Example 1. The anti-CD3 VH and VL sequences and their bridging linker from blinatumomab. See, for example, PCT Patent Publication No. WO2010037838.

[0141] Half-life extension domain (scFc) was derived from conserved human IgG1 sequences (IMGT). Point mutations were introduced into the Fc region for silencing ofDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 typical Fc mediated immune effector functions (e.g., complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity).

[0142] In some embodiments, the sequence of the nucleic acid encoding the TCE may be codon-optimized to balance expression in human cells, avoid sequence liabilities, and / or improve synthesis and engineering. The use of efficient codons may increase elongation rate.

[0143] In some embodiments, the fully human VH and VL domain amino acid sequences are reverse translated to generate an unoptimized DNA sequence, and then manually codon optimized. Additionally, an initiation site (e.g., a consensus Kozak Sequence) may be appended to the 5’ end of the optimized nucleic acid encoding the TCE and restriction enzyme recognition sites (e.g., an AgeI site) may be used throughout the entire nucleotide sequence transcribed as the mTCE.

[0144] Alternatively, the nucleic acid insert may then be analyzed for the presence of restriction enzyme recognition sites for the chosen linearization restriction enzyme or other cloning enzymes (e.g., EcoRI) using commercially available software (Snapgene, GSL Biotech LLC, San Diego, CA). In some embodiments, the nucleic acid sequence is designed to exclude stretches of 20 nucleic acids of direct or inverted repeat sequences using a DNA dot plot tool, e.g., YASS as well as IDTs E-blockTMTest Complexity tool. Direct repeats are nucleotides sequences that consists of two or more repeats of a specific sequence, such that the repeats are present in multiple copies of a larger sequence. Generally, a direct repeat occurs when a sequence is repeated with the same pattern downstream. An inverted repeat is a single stranded sequence of nucleotides followed downstream by its reverse complement. Removal of repeated nucleotides sequences increase efficiency ultimately of expression.

[0145] Internal restriction enzyme recognition sites identified may be removed by generating silent mutations. Cryptic or alternative splice sites may be identified using a bioinformatic tool e.g., the Alternative Splice Site Predictor (ASSP). Highscoring constitutive splice sites may be removed by generating silent mutations.

[0146] In some embodiments, 3’ ends are modified to ensure the presence of functional transcription termination consensus sequences and screened to avoid the presence of type I and type II termination sequences prior to the desired terminationDFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 site. The sequence may also be screened for and certain slow translation codon pairings removed.

[0147] As a final check, the sequence is once again reviewed for high scoring splice sites, as well as for the exclusion of non-desirable restriction enzyme recognition sites and inclusion of desired sites, as well as appropriate start and stop codons.

[0148] The sequence encoding the TCE was cloned into a vector additionally incorporating a T7 polymerase site, as well as non-coding elements, prior to an EcoRI restriction site. All the generated plasmids were sequence verified by whole plasmid sequencing.

[0149] Additional exemplary TCEs are shown in Fig.4B. Example 5

[0150] Six to eight week-old NSG DKO (NOD.Cg-Prkdcscid H2-K1b-tm1Bpe H2-Ab1g7- em1Mvw H2-D1b-tm1Bpe Il2rgtm1Wjl / SzJ) mice (Jackson Labs; Bar Harbor, ME) were injected with the bone marrow tropic cell line OPM2-ffLuc, at a dose of 1×106cells via tail vein. Tumor engraftment was confirmed by baseline bioluminescent imaging and animals randomized for signal between groups. Thirteen days after tumor engraftment, mice were humanized with a single dose of 10 ×106human T cells injected via tail vein. The following day animals were injected IV with mRNA-LNP according to the dosed weekly with 3 µg mRNA-LNP times 3 doses. In vivo imaging was preformed after injection of D-luciferin (Millipore-Sigma; Darmstadt, Germany) and analyzed with Living Image software (PerkinElmer; Waltham, MA) (Fig.5A). Studies were planned with the minimum number of animals per treatment group to reproducibly observe statistically significant differences (n = 4-5 per group).

[0151] In the tumor xenograft model of multiple myeloma (MM), the imaging of the tumor burden of the mice via bioluminescence (BLI) is shown in Fig.5A. As seen in Fig.5A, following 17 days of treatment with the various TCE proteins, the tumor burden of the mice decreases, except for mTCE123, which was used as a negative control. The quantification of the bioluminescence shown visually in Fig.5A is quantified in Fig.5B at each day time point and for the various TCE proteins. The long-term survival of the mice following treatment with the various TCE proteins is shown in Fig.5C.DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709

[0152] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results. References provided throughout are meant to be incorporated by reference only with regard to such background teaching that they refer to.

Claims

DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 WHAT IS CLAIMED:

1. A nucleic acid for an immune cell engager, comprising a ribonucleic acid of formula I: R1 – SP – R2 – R3 – R4 – R5 (I), wherein: R1 is a 5’ untranslated region (UTR); SP encodes a signal peptide; R2 encodes a first single chain variable fragment (scFv) that binds a first extracellular protein or a variable region of a heavy chain (VH-only) that binds a first extracellular protein; R3 encodes a second scFv that binds a second extracellular protein; R4 encodes a half-life extender; R5 is a 3’UTR, and wherein R1 to R5 are oriented 5’ to 3’.

2. The nucleic acid of claim 1, wherein R1 comprises a hemoglobin subunit beta 5’UTR.

3. The nucleic acid of claim 1 or claim 2, wherein SP comprises a IgG1 SP.

4. The nucleic acid of any one of claims 1-3, further comprising a nucleic acid encoding a linker, wherein the linker is positioned between R2 and R3.

5. The nucleic acid of any one of claims 1-4, further comprising a nucleic acid encoding a linker, wherein the linker is positioned between R3 and R4.

6. The nucleic acid of any one of claims 1-5, wherein R2 encodes a first scFv and the first scFv comprises formula II or formula III: R6 – R7 (II), R7– R6 (III), wherein: R6 encodes a light chain variable fragment (VL); and R7 encodes a heavy chain variable fragment (VH).DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 7. The nucleic acid of claim 6, wherein a nucleic acid encoding a first linker is positioned between R6 and R7.

8. The nucleic acid of claim 6, wherein R6 comprises a VL domain encoding three variable light-complementarity determining regions (VL-CDRs), VL-CDR1, VL-CDR2, VL-CDR3, wherein VL-CDR1 comprises an amino acid sequence according to SEQ ID NO: 17; VL-CDR2 comprises an amino acid sequence according to SEQ ID NO: 18; VL-CDR3 comprises an amino acid sequence according to SEQ ID Nos: 19, 43 or 54; and wherein R7 comprises a VH domain comprising on or more variable heavy - complementarity determining regions (VH-CDRs), VH-CDR1, VH-CDR2, VH-CDR3, wherein VH-CDR1 comprises an amino acid sequence according to any one of SEQ ID NOs: 12, 40, or 51; VH-CDR2 comprises an amino acid sequence according to any one of SEQ ID NOs: 13, 41, or 52; VH-CDR3 comprises an amino acid sequence according to any one of SEQ ID NOs: 14, 42 and 55.

9. The nucleic acid of claim 1, comprising the sequence selected from SEQ ID Nos: 20, 25, 30, 32, or 44.

10. The nucleic acid of any one of claims 1-9, wherein the second scFv comprises formula IV or formula V: R8 – R9 (IV), R9 – R8 (V) wherein: R8 encodes a light chain variable fragment (VL); and R9 encodes a heavy chain variable fragment (VH).DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 11. The nucleic acid of claim 10, wherein a nucleic acid encoding a second linker is positioned between R8 and R9.

12. The nucleic acid of any one of claims 1-11, wherein R4 comprises a single chain crystallizable fragment (scFc) or a CH3 domain.

13. The nucleic acid of claim 12, wherein R4 comprises an IgG1 single chain crystallizable fragment (scFc).

14. The nucleic acid of claim 13, wherein the scFc comprises a leucine to alanine mutation at amino acids 234 and 235, as compared to SEQ ID NO: 24 or at corresponding amino acid positions in a homolog thereof.

15. The nucleic acid of claim 13, wherein the scFc comprises a leucine to alanine mutation at amino acids 234 and 235, and a proline to glycine mutation at amino acid 329, as compared to SEQ ID NO: 24, or at corresponding amino acid positions in a homolog thereof.

16. The nucleic acid of any one of claims 1-15, wherein R5 comprises a hemoglobin subunit beta 3’UTR.

17. The nucleic acid of any one of claims 1-16, wherein the first extracellular protein is expressed or overexpressed on cancer cells or tumor cells.

18. The nucleic acid of claim 17, wherein the cancer cells are hematological cancer cells.

19. The nucleic acid of claim 17, wherein the cancer cells are multiple myeloma cancer cell.

20. The nucleic acid of any one of claims 1-19, wherein the first extracellular protein is B-cell maturation antigen (BCMA).DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 21. The nucleic acid of any one of claims 1-20, wherein the second extracellular protein is expressed on immune cells.

22. The nucleic acid of claim 21, wherein the immune cells are T cells.

23. The nucleic acid of any one of claims 1-12, wherein the second extracellular protein is cluster of differentiation 3 (CD3).

24. The nucleic acid of any one of claims 1-13, wherein R1 comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:

1.

25. The nucleic acid of claim 1, wherein SP comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:

2.

26. The nucleic acid of any one of claims 1-25, wherein R2 comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:

3.

27. The nucleic acid of any one of claims 4-26, wherein the linker positioned between R2 and R3 comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:

4.

28. The nucleic acid of any one of claims 1-27, wherein R3 comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:

5.

29. The nucleic acid of any one of claims 5-28, wherein the linker positioned between R3 and R4 comprises a nucleic sequence having at least 80% sequence identity to SEQ ID NO:

6.

30. The nucleic acid of any one of claims 1-29, wherein R4 comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 7.DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 31. The nucleic acid of any one of claims 1-17, wherein R5 comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:

8.

32. The nucleic acid of claim 1, comprising any one of SEQ ID NOs: 20, 25, 30, 32, or 44.

33. A pharmaceutical composition, comprising the nucleic acid of any one of claims 1- 32, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

34. The pharmaceutical composition of claim 33, wherein the one or more pharmaceutically acceptable carriers, diluents, or excipients comprises a lipid nanoparticle.

35. The nucleic acid of any one of claims 1-32 or the pharmaceutical composition of claims 33-34 for the manufacture or preparation of a medicament for use in the treatment of cancer.

36. The nucleic acid of any one of claims 1-32 or the pharmaceutical composition of claims 33-34 for the manufacture or preparation of a medicament for use in the treatment of a hematological cancer.

37. The nucleic acid of any one of claims 1-32 or the pharmaceutical composition of claims 33-34 for the manufacture or preparation of a medicament for use in the treatment of multiple myeloma.

38. The nucleic acid of any one of claims 1-32 or the pharmaceutical composition of claims 33-34 for the manufacture or preparation of a medicament for use in engaging immune cells.

39. The nucleic acid of any one of claims 1-32 or the pharmaceutical composition of claims 33-34 for the manufacture or preparation of a medicament for use in engaging T cells.DFCI IP No.3541.W01.WO / Atty Ref.: 91016-428709 40. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of the nucleic acid of any one of claim 1-32 or the pharmaceutical composition of claims 33-34.

41. A method of treating hematological cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of the nucleic acid of any one of claim 1-32 or the pharmaceutical composition of claims 33-34.

42. A method of treating multiple myeloma in a patient in need thereof, the method comprising administering to the patient an effective amount of the nucleic acid of any one of claim 1-32 or the pharmaceutical composition of claims 33-34.

43. A method of engaging immune cells in a patient in need thereof, the method comprising administering to the patient an effective amount of the nucleic acid of any one of claim 1-32 or the pharmaceutical composition of claims 33-34.

44. A method of engaging T cells in a patient in need thereof, the method comprising administering to the patient an effective amount of the nucleic acid of any one of claim 1-32 or the pharmaceutical composition of claims 33-34.

45. A single chain variable fragment (scFv) comprising a nucleic acid having at least 80% sequence identity to SEQ ID NO: 3.

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