Nucleic acids and pharmaceutical compositions for immune cell adapters

Abstract

The present disclosure relates to novel nucleic acids for immune cell adapters, comprising a ribonucleic acid of formula I: R1-SP-R2-R3-R4-R5 (I) wherein R1 is a 5'non-transcriptional region (UTR); sP encodes a signal peptide; r2 encodes a first single chain variable fragment (scFv) that binds to a first extracellular protein or a heavy chain variable region (VH only) that binds to a first extracellular protein; r3 encodes a second scFv that binds to a second extracellular protein; r4 encodes a half-life extender; r < 5 > is 3 'UTR wherein R < 1 > to R < 5 > are in the 5' to 3 'direction for use in the treatment of cancer, including but not limited to hematological cancers, including multiple myeloma.

Description

[0001] Declaration of sequence list

[0002] The sequence list related to this application is provided in XML format in lieu of a paper copy, and is incorporated herein by reference. The XML file containing the sequence list is named "91016-412487-SL-updated.xml". This XML file is 27,576 bytes in size, was created on July 25, 2024, and is submitted electronically along with this application. Technical Field

[0003] This disclosure relates to a novel nucleic acid encoding a single-stranded variable fragment (scFv) that binds to a B-cell maturation antigen (BCMA) protein; and an immune cell adjuvant comprising the scFv and a second scFv that binds to immune cells. This disclosure also relates to novel methods for treating cancers (particularly hematologic malignancies such as multiple myeloma) using the novel scFv and the immune cell adjuvant. Background Technology

[0004] Despite advances in immunotherapy, treatment outcomes remain a concern. Furthermore, cancers such as blood cancers (e.g., multiple myeloma) remain among the leading causes of death worldwide. For example, in the United States, the lifetime risk of developing multiple myeloma is approximately 1 in 103 for men and 1 in 131 for women.

[0005] Antibody-based immunotherapy, which encodes nucleic acids, particularly mRNA, has revolutionized the treatment of many cancer types. Compared to traditional recombinant antibody manufacturing, mRNA-based approaches offer numerous advantages, as producing mRNA and using in vivo transcription and secretion is more economical and reduces development and manufacturing time, thus bringing new therapies to patients. mRNA therapies may also be easier to reuse and can provide more consistent and durable drug concentration profiles compared to recombinant therapies. Despite recent advances, patients with cancers such as multiple myeloma (MM) often try multiple different therapies to find one with sufficient efficacy; therefore, highly potent immunotherapies for cancers such as hematologic malignancies like multiple myeloma are an ideal option. Summary of the Invention

[0006] One embodiment described herein is a nucleic acid for use as an immune cell adjuvant, comprising ribonucleic acid of formula I: R1–SP–R2–R3–R4–R5(I).

[0007] in:

[0008] R1 is the 5' untranscribed region (UTR);

[0009] SP encodes a signal peptide;

[0010] R2 encodes the first single-stranded variable segment (scFv) that binds to the first extracellular protein or the heavy chain variable region (VH only) that binds to the first extracellular protein.

[0011] R3 encodes a second scFv that binds to a second extracellular protein;

[0012] R4 encodes a half-life extender;

[0013] R5 is 3'UTR, and

[0014] The directions of R1 to R5 are 5' to 3'.

[0015] In one aspect, R1 contains the hemoglobin subunit β (HBB) 5'UTR. In another aspect, SP contains IgG1SP.

[0016] In one respect, R2 encodes the first scFv, and the first scFv contains either Equation II or Equation III:

[0017] R6–R7(II), R7–R6(III),

[0018] in:

[0019] R6 encodes light chain variable segments (VL); and

[0020] R7 encodes heavy chain variable segments (VH).

[0021] In one respect, the nucleic acid encoding the first adaptor is located between R6 and R7. In another respect, the second scFv contains formula IV or formula V:

[0022] R8–R9(IV), R9–R8(V)

[0023] in:

[0024] R8 encoding of light chain variable segments (VL); and

[0025] R9 encoding heavy chain variable segments (VH).

[0026] In one aspect, the nucleic acid encoding the second adaptor is located between R8 and R9. In one aspect, R4 comprises a single-stranded crystallizable fragment (scFc) or a CH3 domain. In one aspect, R4 comprises an IgG1 single-stranded crystallizable fragment (scFc). In one aspect, compared to SEQ ID NO:7, scFc contains a leucine-to-alanine mutation at amino acids 234 and 235, or at the corresponding amino acid position in its homolog. In one aspect, compared to SEQ ID NO:7, scFc contains a leucine-to-alanine mutation at amino acids 234 and 235, a proline-to-glycine mutation at amino acid 329, or at the corresponding amino acid position in its homolog. In one aspect, R5 comprises the hemoglobin subunit β (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 cell is a hematologic malignancy. In one aspect, the cancer cell is a multiple myeloma cancer cell. 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 cell is a T cell. In one aspect, the second extracellular protein is differentiation cluster 3 (CD3). In one aspect, R1 comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:1. In one aspect, SP comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:2. In one aspect, R2 comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:3.

[0027] In one respect, any nucleic acid described herein also includes a nucleic acid encoding an adapter, wherein the adapter is located between R2 and R3.

[0028] In one respect, any nucleic acid described herein also includes a nucleic acid encoding an adapter, wherein the adapter is located between R3 and R4.

[0029] In one aspect, the adapter between R2 and R3 comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:4. In one aspect, R3 comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:5. In one aspect, the adapter between R3 and R4 comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:6. In one aspect, R4 comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:7. In one aspect, R5 comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:8.

[0030] In another embodiment, this document describes pharmaceutical compositions comprising any nucleic acid described herein and one or more pharmaceutically acceptable carriers, diluents, or excipients. In one aspect, one or more pharmaceutically acceptable carriers, diluents, or excipients comprise lipid nanoparticles.

[0031] In another embodiment, this document describes any nucleic acid or any pharmaceutical composition described herein for the manufacture or preparation of a medicament for the treatment of cancer.

[0032] In another embodiment, any nucleic acid or pharmaceutical composition described herein is used to manufacture or prepare a medicament for treating blood cancers.

[0033] In another embodiment, this document describes any of the nucleic acids or pharmaceutical compositions described herein for the manufacture or preparation of a medicament for the treatment of multiple myeloma.

[0034] In another embodiment, this document describes any nucleic acid or pharmaceutical composition described herein for manufacturing or preparing a medicament for binding immune cells.

[0035] In another embodiment, this document describes any nucleic acid or pharmaceutical composition described herein for manufacturing or preparing a medicament for binding to T cells.

[0036] In another embodiment, this document describes a method for treating cancer in a patient in need, the method comprising administering to the patient an effective amount of any of the nucleic acids or any of the pharmaceutical compositions described herein.

[0037] In another embodiment, this document describes a method for treating blood cancers in patients in need, the method comprising administering to the patient an effective amount of any of the nucleic acids or any of the pharmaceutical compositions described herein.

[0038] In another embodiment, this document describes a method for treating multiple myeloma in patients in need, the method comprising administering to the patient an effective amount of any of the nucleic acids or any of the pharmaceutical compositions described herein.

[0039] In another embodiment, this document describes a method for engaging a patient in need of immune cells, the method comprising administering to the patient an effective amount of any nucleic acid or any pharmaceutical composition described herein.

[0040] In another embodiment, this document describes a method for engaging T cells in a patient in need, the method comprising administering to the patient an effective amount of any nucleic acid or any pharmaceutical composition described herein.

[0041] In another embodiment, this document describes a single-stranded variable fragment (scFv) comprising a nucleic acid having at least 80% sequence identity with SEQ ID NO:3. Attached Figure Description

[0042] Figure 1 is a series of graphs describing the in vitro biochemical assessment of the binding affinity of various scFvs to BCMA.

[0043] Figure 2 Cytotoxicity assessments are described. Specifically, the percentage of viable cells (Viability%) is shown when cells expressing BCMA (BCMA+ target) and cells not expressing BCMA (BCMA- target) are treated with a T cell conjugation construct (TCE) and / or T cells.

[0044] Figures 3A-3D The assessment of dosage requirements is described. Figure 3A This is a schematic diagram of an experiment using lipid nanoparticles containing mRNA constructs to treat a humanized mouse model. The mouse model contains bioluminescent cancer cells for monitoring purposes. Figure 3B Exemplary bioluminescent images of a series of mice that have received various treatment regimens are described. Figure 3C It is a graph depicting tumor burden as measured by bioluminescence (BLI). Figure 3D It is a chart depicting the survival rates of mice treated with various treatment regimens.

[0045] Figures 4A-4B These are a series of exemplary schematic diagrams showing various embodiments of constructs of proteins encoded by mRNA constructs. Figure 4A This is an exemplary schematic diagram of the mRNA construct of the T cell conjugate (mTCE) and the resulting TCE protein ((scFV)2-Fc). Figure 4B This is a schematic diagram of an alternative embodiment of the TCE protein. Detailed Implementation

[0046] Immunotherapy options for treating cancer include immune cell adjuvants. Immune cell adjuvants are engineered proteins designed to bring tumor cells and immune effector cells closer together in a patient's body by binding to specific antigens on the surface of tumor cells and effector cells. Most immune cell adjuvants are antibody-based molecules with multiple arms. They can link tumor antigens to effector cell surface molecules (such as CD3 on T cells). The binding of cell surface molecules to effector cell molecules can activate the killing mechanisms of effector cells. For example, T cell adjuvants can bind to T cell (effector cell) surface molecules and activate and redirect T cells to recognize and kill cancer cells, such as hematologic malignancies or multiple myeloma cancer cells, by binding to target antigens expressed on cancer cells and triggering cell surface molecules (such as CD3) on T cells.

[0047] This article describes nucleic acids encoding BCMA-specific scFv, immune cell adaptors targeting BCMA, and methods for using them to treat cancer.

[0048] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of any conflict, this document (including the definitions) shall prevail. Methods and materials are described below, although similar or equivalent methods and materials may be used in practice or testing of this disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0049] As described in this article, the articles “a,” “one,” and “the” are used to refer to one or more (i.e., at least one) grammatical objects of the article. For example, “a part” can mean one part or more parts.

[0050] As described herein, the term “about” or “approximately” means that a quantity, level, value, number, frequency, percentage, size, volume, weight, or length differs from a reference quantity, level, value, number, frequency, percentage, size, volume, weight, or length by as much as 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, when the term “about” or “approximately” precedes a numerical value, it indicates a range of that value plus or minus 10%, 5%, or 1%.

[0051] As described herein, “nucleic acid” refers to a polymer of nucleotides of any length, which may be ribonucleotides or deoxyribonucleotides. The term refers only to the primary structure of the molecule. Therefore, the term includes double-stranded and single-stranded DNA, triple-stranded DNA, and double-stranded and single-stranded RNA, linear RNA, circular RNA, and other RNA formats. It also includes modified forms of polynucleotides (e.g., through methylation and / or capping) and unmodified forms. The term is also intended to include molecules containing non-naturally occurring or synthetic nucleotides and nucleotide analogues. Therefore, while all sequences described herein are shown as DNA sequences, corresponding RNA sequences are also considered by reference to the same sequence.

[0052] As described herein, the term “sequence identity” in the context of two nucleic acid sequences or amino acid sequences refers to the same residues in two sequences when a maximum correspondence is performed within a specified comparison window. Sequence alignment methods used for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed using methods such as: the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981); the alignment algorithm of Needleman and Wunsch, J. Mal. Biol., 48:443 (1970); the similarity search method of Pearson and Lipman, Proc. Nat. Acad. Sci US. A., 85:2444 (1988); and computerized implementations of such algorithms (including but not limited to CLUSTAL in the PC / Gene program of Intelligentics, Mountain View California, GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group (GCG), 575 Science). Dr., Madison, Wis., USA); the CLUSTAL procedure is described in detail in 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). Alignments are also frequently performed through inspection and manual comparison.

[0053] The term "coding sequence" as used herein refers to a polynucleotide fragment that encodes a protein or polypeptide. This region or sequence is bounded by a start codon near the 5' end and a stop codon near the 3' end. A coding sequence may also be referred to as an open reading frame. As is known to those skilled in the art, minor variations in a nucleic acid sequence do not necessarily alter the amino acid sequence encoding a polypeptide. This disclosure covers the degeneracy of codon usage as understood by those skilled in the art. For example, as is known in the art, different codons will encode the same amino acid.

[0054] As described in this article, the phrase “codon degenerate nucleic acid sequence” refers to a nucleic acid sequence that is different from a reference sequence but encodes a polypeptide with the same amino acid sequence as the reference sequence.

[0055] The term "effective dose" as used herein refers to a dose that alleviates disease symptoms through clinical testing and evaluation, patient observation, and other methods. "Effective dose" can also refer to a dose that causes a detectable change in biological or chemical activity. Those skilled in the art can detect and / or further quantify the detectable change in relation to the relevant mechanism or process. Furthermore, "effective dose" can refer to a dose that maintains a desired physiological state, i.e., a dose that reduces or prevents significant decline and / or promotes improvement in the target condition. "Effective dose" can also refer to a "therapeutic effective dose." As used herein, the term "effect cell" refers to the second target cell to which the immune cell adjuvant described herein binds. Effector cells can be any cell type, such as immune cells, i.e., T cells. Cell surface proteins or antigens on effector cells are targeted by the second scFv on the immune cell adjuvant. Without being bound by any theory, it is believed that the binding of the second scFv to the cell surface molecules of effector cells activates the effector cell. For example, if the second scFv binds to CD3 on the surface of a T cell, it is believed that this binding activates the T cell to kill infected cells or cancer cells.

[0056] As described herein, the terms “individual” and “subject” are generally used interchangeably to refer to any human or domestic animal that can be treated with the methods disclosed herein. Suitable subjects (e.g., patients) include humans and domestic animals or pets (e.g., cats or dogs). This includes non-human primates and human patients. In one embodiment, a subject may include a human patient diagnosed with cancer (e.g., blood cancers), including but not limited to multiple myeloma. As described herein, the term “patient” refers to a subject who can receive treatment for a disease or condition.

[0057] As described in this article, “treatment” means reversing, reducing, alleviating or slowing the progression of a condition or disease or symptoms associated with such a condition or disease, or inhibiting the progression of a condition or disease or symptoms associated with such a condition or disease, and is described in more detail in this article.

[0058] In describing the numerical ranges in this paper, each intermediate number with the same precision is explicitly considered. For example, for the range of 6-9, the numbers 7 and 8 are considered in addition to 6 and 9, while for the range of 6.0-7.0, the numbers 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 considered.

[0059] Nucleic acid encoding an immune cell adaptor may include a ribonucleic acid (RNA) construct. For example, the nucleic acid of an immune cell adaptor may include RNA encoding from 5' to 3', a 5' untranscribed region (UTR) (5'UTR), a signal peptide (SP), a variable domain of a first single-stranded variable fragment (scFv) or heavy chain (VH only) that binds a first extracellular protein, a second scFv that binds a second extracellular protein, a half-life extender, and a 3'UTR.

[0060] Therefore, in one embodiment described herein, the nucleic acid encoding an immune cell adaptor may include RNA of Formula I:

[0061] R1–SP–R2–R3–R4–R5(I),

[0062] Wherein: R1 is a 5' untranscribed region (5'UTR); SP encodes a signal peptide; R2 encodes a variable domain of a first single-stranded variable fragment (scFv) or heavy chain (VH only) that binds to a first extracellular protein; R3 encodes a second scFv that binds to a second extracellular protein; R4 encodes a half-life extender; and R5 is a 3' untranscribed region (3'UTR), wherein the orientation of R1 to R5 is 5' to 3'. Any nucleic acid sequence disclosed herein may also be codon-optimized for expression in mammalian cells (e.g., human cells).

[0063] In some respects, R1 or the 5' untranscribed region (also known as the 5' UTR, leader sequence, transcription leader, or leader RNA) is a region in messenger RNA (mRNA) located directly upstream of the start codon of the coding sequence. This region is important for the transcriptional regulation of RNA transcripts. In some respects, the 5' UTR is the 5' UTR of the hemoglobin subunit β (HBB gene), but may originate from other genes. In some respects, R1 contains 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) with SEQ ID NO:1 (ATCCACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGGGCGGCCGCCACC), or a codon-degenerate nucleic acid sequence thereof. In other respects, R1 contains a nucleic acid sequence having at least 98.2% sequence identity with SEQ ID NO:1. In some aspects, R1 comprises a nucleic acid sequence having at least 85% sequence identity with SEQ ID NO:1. In some aspects, R1 comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:1. In some aspects, R1 comprises a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO:1. In some aspects, R1 comprises a nucleic acid sequence having at least 96% sequence identity with SEQ ID NO:1. In some aspects, R1 comprises a nucleic acid sequence having at least 97% sequence identity with SEQ ID NO:1. In some aspects, R1 comprises a nucleic acid sequence having at least 98% sequence identity with SEQ ID NO:1. In other aspects, R1 comprises a nucleic acid sequence having at least 99% sequence identity with SEQ ID NO:1. In some aspects, R1 comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO:1. In some aspects, R1 comprises a nucleic acid sequence having at least 98.2% sequence identity with SEQ ID NO:1.

[0064] 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). 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 that differs from SEQ ID NO:1 by no more than two nucleotide substitutions. In some aspects, R1 comprises a nucleic acid sequence that differs from SEQ ID NO:1 by no more than one nucleotide substitution. In some aspects, R1 comprises the nucleic acid sequence of SEQ ID NO:1.

[0065] A signal peptide (SP) (sometimes referred to as a signal sequence, target signal, localization signal, localization sequence, transport peptide, leader sequence, or leader peptide) is a short peptide, for example, about 16-30 amino acids long, most commonly found at the N-terminus (or occasionally at the C-terminus or interior) of a newly synthesized protein destined for the secretory pathway. Signal peptides prompt cells to translocate proteins onto the cell membrane in the endoplasmic reticulum. In some respects, SP is the signal peptide of the IgG kappa light chain. In other respects, SP is the signal peptide of the albumin gene (ALB). In any nucleic acid encoding the immune cell adjuvant described herein, the signal peptide is operatively linked to an RNA construct comprising at least R2-R3-R4-R5, causing the encoded polypeptide to be secreted from the host cell. In some aspects, 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%) with respect to SEQ ID NO:2 (ATGGTGTTGCAGACCCAGGTCTTCATTTCTCTGTTGCTCTGGATCTCTGGTGCCTACGGG) or its codon-degenerate nucleic acid sequence. In other aspects, SP comprises a nucleic acid sequence having at least 98.3% sequence identity with SEQ ID NO:2. In some aspects, SP comprises a nucleic acid sequence having at least 85% sequence identity with SEQ ID NO:2. In some aspects, SP comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:2. In some aspects, SP comprises a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO:2. In some aspects, SP comprises a nucleic acid sequence having at least 96% sequence identity with SEQ ID NO:2. In some aspects, SP comprises a nucleic acid sequence having at least 97% sequence identity with SEQ ID NO:2. In some respects, SP contains a nucleic acid sequence having at least 98% sequence identity with SEQ ID NO:2. In other respects, SP contains a nucleic acid sequence having at least 99% sequence identity with SEQ ID NO:2. In some respects, SP contains a nucleic acid sequence having at least 91% sequence identity with SEQ ID NO:2. It also contains 100% sequence identity with SEQ ID NO:2. In some respects, SP contains a nucleic acid sequence having at least 98.3% sequence identity with SEQ ID NO:2.

[0066] 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). 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, SP 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.

[0067] R2 encodes an antigen-binding domain that designates one of the antigen targets of an immune cell adaptor (e.g., an antigen target on multiple myeloma cancer cells). In some respects, R2 may encode a single-chain variable fragment (scFv) or a heavy-chain variable domain (VH only) that binds to a first extracellular protein. The scFv is a molecule containing an antibody light-chain variable region (VL) and a heavy-chain variable region (VH) linked by a linker, which allows the two variable regions to be synthesized into a single polypeptide chain (see, for example, Bird et al., Science, 242:423-426 (1988); Huston et al., Proc. Natl. Acad. USA, 85:5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16:778 (1998)). In other respects, R2 may encode a heavy-chain variable region (VH only) that binds to a first extracellular protein. In other respects, R2 can encode the variable region (VH-like) of the heavy chain, which binds to the first extracellular protein.

[0068] Any appropriate method can be used to assess the binding of peptide domains to extracellular proteins (e.g., the first or second extracellular protein). For example, enzyme-linked immunosorbent assay (ELISA) or flow cytometry can be used to assess the binding of peptide domains encoded by the mRNA described herein to extracellular proteins.

[0069] The first extracellular protein can be an antigenic target on cancer cells, such as on blood cancers (e.g., on multiple myeloma cancer cells). In some respects, the first extracellular protein is expressed or overexpressed on cancer or tumor cells. For example, in some respects, the first extracellular protein is expressed or overexpressed on blood cancer cells (e.g., multiple myeloma cancer cells). In some respects, the first extracellular protein is BCMA. As described herein, "BCMA" refers to the B cell maturation antigen protein, also known as 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 and influences B cell development and autoimmune responses. Studies have shown that BCMA can specifically bind to tumor necrosis factor (ligand) superfamily member 13b (TNFSF13B / TALL-1 / BAFF) and lead to activation of NF-kappaB and MAPK8 / JNK. This protein also binds to various TRAF family members and may therefore transmit signals for cell survival and proliferation. Studies have shown that BCMA is highly expressed on the surface of multiple myeloma cancer cells. For example, see Shah et al. (2020) Leukemia 34,985–1005. BCMA amino acid and nucleic acid sequences from different species can be found in public databases such as GenBank, UniProt, and SwissProt, including but not limited to the human BCMA protein sequence (e.g., UniProt / SwissProt Accession No. Q02223) and its corresponding nucleotide sequence (e.g., NCBI reference sequence: NP_001183.2), as well as corresponding sequences from other species.

[0070] This article describes BCMA-specific scFvs (e.g., BCMA-specific scFvs encoded by R2) having the components described in Table 1 or their codon-degenerate nucleic acid sequences. Any BCMA-specific scFv described herein may contain the complementarity-determining regions (CDRs) described in Table 1.

[0071] Table 1. BCMA-specific scFv characteristics

[0072]

[0073]

[0074] This document also discloses single-stranded variable fragments (scFvs) comprising 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) with respect to SEQ ID NO:3 or its codon-degenerate nucleic acid sequence. In other aspects, the scFv comprises a nucleic acid sequence having at least 99.5% or 99.8% sequence identity with SEQ ID NO:3. In some aspects, the scFv comprising a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:3 binds to BCMA. This document describes BCMA-specific scFvs having the components described in Table 1 (e.g., BCMA-specific scFvs encoded by R2). Any BCMA-specific scFv described herein may contain the complementarity-determining regions (CDRs) described in Table 1.

[0075] In some aspects, R2 comprises an scFv containing a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:3 (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.8% sequence identity). In some aspects, the scFv of R2 contains a nucleic acid sequence having at least 85% sequence identity with SEQ ID NO:3. In some aspects, the scFv of R2 contains a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:3. In some aspects, the scFv of R2 contains a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO:3. In some aspects, the scFv of R2 contains a nucleic acid sequence having at least 96% sequence identity with SEQ ID NO:3. In some aspects, the scFv of R2 contains a nucleic acid sequence having at least 97% sequence identity with SEQ ID NO:3. In some aspects, the scFv of R2 contains a nucleic acid sequence having at least 98% sequence identity with SEQ ID NO:3. In some aspects, the scFv of R2 contains a nucleic acid sequence having at least 99% sequence identity with SEQ ID NO:3. In some aspects, the scFv of R2 contains a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:3. In some aspects, the scFv of R2 contains a nucleic acid sequence having 100% sequence identity with SEQ ID NO:3. In some aspects, the scFv of R2 contains a nucleic acid sequence having at least 99.5% sequence identity with SEQ ID NO:3. In some aspects, the scFv of R2 contains a nucleic acid sequence having at least 99.8% sequence identity with SEQ ID NO:3.

[0076] In some aspects, the scFv of R2 contains a nucleic acid sequence that differs 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). In some aspects, the scFv of R2 contains a nucleic acid sequence that differs from SEQ ID NO:3 by no more than 15 nucleotide substitutions. In some aspects, the scFv of R2 contains a nucleic acid sequence that differs from SEQ ID NO:3 by no more than 10 nucleotide substitutions. In some aspects, the scFv of R2 contains a nucleic acid sequence that differs from SEQ ID NO:3 by no more than 9 nucleotide substitutions. In some aspects, the scFv of R2 contains a nucleic acid sequence that differs from SEQ ID NO:3 by no more than 8 nucleotide substitutions. In some aspects, the scFv of R2 contains a nucleic acid sequence that differs from SEQ ID NO:3 by no more than 7 nucleotide substitutions. In some aspects, the scFv of R2 contains a nucleic acid sequence that differs from SEQ ID NO:3 by no more than 6 nucleotide substitutions. In some aspects, the scFv of R2 contains a nucleic acid sequence differing from SEQ ID NO:3 by no more than 5 nucleotide substitutions. In some aspects, R2 contains a nucleic acid sequence differing from SEQ ID NO:3 by no more than 4 nucleotide substitutions. In some aspects, R2 contains a nucleic acid sequence differing from SEQ ID NO:3 by no more than 3 nucleotide substitutions. In some aspects, R2 contains a nucleic acid sequence differing from SEQ ID NO:3 by no more than 2 nucleotide substitutions. In some aspects, R2 contains a nucleic acid sequence differing from SEQ ID NO:3 by no more than 1 nucleotide substitution. In some aspects, R2 contains the nucleic acid sequence of SEQ ID NO:3.

[0077] Any R2 domain disclosed herein may also include additional subdomains in various orientations. For example, the scFv of R2 may also include domains according to Formula II or Formula III: R6-R7(II), R7-R6(III), where R6 encodes the VL domain and R7 encodes the VH domain. In another example, in some aspects, from the 5' to 3' orientation, the scFv of R2 may also include Formula II: R6-R7(II). In other aspects, from the 5' to 3' orientation, 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 subdomains from the scFv sequence of R2, such as the nucleic acid sequence of SEQ ID NO: 3.

[0078] In some respects, R6 comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:15 (see Table 1) or its codon-degenerate nucleic acid sequence (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity). In other respects, R6 comprises a nucleic acid sequence having at least 99.5% or 99.8% sequence identity with SEQ ID NO:15 (see Table 1).

[0079] In some respects, R7 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) with respect to SEQ ID NO:10 (see Table 1) or its codon-degenerate nucleic acid sequence. In other respects, R7 comprises a nucleic acid sequence having at least 99.5% or 99.8% sequence identity with respect to SEQ ID NO:10 (see Table 1).

[0080] R3 encodes a second antigen-binding domain, or second scFv, which is specific for effector cells (e.g., cell surface antigen targets on T cells). In some respects, R3 encodes an scFv that binds to a second extracellular protein. The second extracellular protein can be an antigen target on effector cells (e.g., T cells). In some respects, the second extracellular protein is expressed on immune cells. In some respects, the second extracellular protein is expressed on T cells. In some respects, the second extracellular protein is CD3. As described herein, "CD3" refers to the differentiation cluster 3 protein complex and T cell co-receptor. It is involved in activating cytotoxic T cells (CD8+). + Immature T cells) and T helper cells (CD4+) + The TCR complex (a complex of naive T cells) consists of four distinct chains. In mammals, this complex comprises a CD3γ chain, a CD3δ chain, and two CD3ε chains. These chains bind to the T cell receptor (TCR) and CD3-zeta (ζ chain), generating activation signals in T lymphocytes. The TCR, CD3-zeta, and other CD3 molecules together constitute the TCR complex. Amino acid and nucleic acid sequences of CD3ε from different species can be found in public databases such as GenBank, UniProt, and SwissProt. Such sequences include, but are not limited to, the human CD3ε protein sequence (e.g., UniProt / SwissProt accession number P07766) and its corresponding nucleotide sequence (e.g., NCBI reference sequence: NM_000733.3), and its counterparts in other species.

[0081] 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) with respect to SEQ ID NO:5 or its codon-degenerate nucleic acid sequence. In other aspects, R3 comprises a nucleic acid sequence having at least 99.5% or 99.8% sequence identity with SEQ ID NO:5. In some aspects, R3 comprises a nucleic acid sequence having at least 85% sequence identity with SEQ ID NO:5. In some aspects, R3 comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:5. In some aspects, R3 comprises a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO:5.In some aspects, R3 comprises a nucleic acid sequence having at least 96% sequence identity with SEQ ID NO:5. In some aspects, R3 comprises a nucleic acid sequence having at least 97% sequence identity with SEQ ID NO:5. In some aspects, R3 comprises a nucleic acid sequence having at least 98% sequence identity with SEQ ID NO:5. In some aspects, R3 comprises a nucleic acid sequence having at least 99% sequence identity with SEQ ID NO:5. In some aspects, R3 comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO:5. In some aspects, R3 comprises a nucleic acid sequence having at least 99.5% sequence identity with SEQ ID NO:5. In some aspects, R3 comprises a nucleic acid sequence having at least 99.8% sequence identity with SEQ ID NO:5.

[0082] 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). 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.

[0083] Any R3 domain disclosed herein may also include additional subdomains. For example, the second scFv of R3 may also include subdomains having Formula IV or Formula V: R8–R9(IV), R9–R8(V), where R8 encodes the VL domain and R9 encodes the 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, it may also include Formula III: R9–R8(V). In other instances, the VL sequence of R7 and the VH sequence of R8 may be subdomains of sequences derived from R3, such as the nucleic acid sequence of SEQ ID NO:5. In some respects, 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) with respect to SEQ ID NO:21 (ATTCAATTGACTCAATCACCCGCTATTATGTCCGCGTCCCCTGGGGAGAAGGTTA CTATGACGTGTCGGGCGTCTAGCTCCGTGTCTTATGAATTGGTACCAACAGAAGTCTGGCACCTCCCCGAAAAGGTGGATATACGATACAAGCAAGGTCGCCTCCGGAGTTCCATATCGCTTTTCCGGTAGTGGGTCTGGGACCAGCTATTCCTTGACTATTTCTTCAATGGAAGCCGAGGATGCTGCCACCTACTATTGTCAACAATGGTCTTCCAACCCTTTGACGTTCGGCGCTGGTACGAAACTGGAGCTGAAG) or a codon-degenerate nucleic acid sequence thereof. In other respects, R8 contains a nucleic acid sequence that has at least 99.5% or 99.8% sequence identity with SEQ ID NO:21.In some aspects, R9 contains the same sequence as SEQ ID NO: 22 (ATTAAGCTCCAACAATCCGGCGCCGAACTGGCCAGACCCGGCGCTTCCGTCAAAATGTCCTGCAAAACTTCAGGCTATACTTTCACCCGGTACACAATGCACTGGGTGAACAAAGGCCCGGTCAAGGTCTTGAATGGATCGGATACATTAACCCAAGCCGCGGATATACTAATTATAATCAAAAGTTCAAAGATAAGGCTACGCTGACCACG The nucleic acid sequence GATAAATCCAGTAGCACAGCATATATGCAGCTGTCATCTCTCACTTCCGAGGACAGCGCAGTTTATTATTGTGCAAGATACTACGACGATCACTACTGCCTCGACTACTGGGGACAAGGAACCACGTTGACGGTGTCTAGC or its codon degenerate nucleic acid sequence has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity). In other respects, R9 comprises a nucleic acid sequence having at least 99.5% or 99.8% sequence identity with SEQ ID NO:22.

[0084] R4 encodes a half-life extender. Half-life extenders are modifications of immune cell adjuvants that prolong the duration of their activity in the body. Several methods can be used to extend the half-life of immune cell adjuvants. For example, R4 can encode a single-chain crystallizable region (scFc) or a CH3 domain. The scFc is part of the antibody tail region, for example, comprising a CH2 domain and a CH3 domain 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 in the complement system. Without being bound by any particular theory, it is thought that when an antibody binds to an Fc receptor, it activates the immune system. For example, this interaction can lead to opsonization (enhanced phagocytosis) or antibody-dependent cell-mediated cytotoxicity. The Fc region may contain two polypeptide fragments from the second and third constant domains of the antibody's two heavy chains and remains unchanged in a class of antibodies in each species. The CH3 domain is the third domain of the antibody's constant region (Fc region). The Fc region is the portion of an antibody molecule with the most conserved amino acid sequence among different antibodies of the same class (e.g., IgG, IgM, IgA). The Fc region may include CH1, CH2, and CH3 domains in the heavy chain. These regions may confer antibody stability, effector function, and interactions with other components of the immune system. The CH3 domain promotes Fc region dimerization, which can increase the interaction between the antibody and the nascent Fc receptor (FcRn), thereby limiting lysosomal degradation.

[0085] In some aspects, R4 encodes immunoglobulin 1 (IgG1) scFc. If the CH3 domains of the IgG1 polypeptide and the IgG1 polypeptide are linked as a single polypeptide, then the IgG1 scFc may include a CH2 domain. In some aspects, R4 contains the same as SEQ ID NO:7

[0086] (GATAAAACCCACACTTGCCCTCCGTGTCCCGCGCCTGAGGCTGCCGGTGGAC) or a codon-degenerate 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). In other aspects, R4 comprises a nucleic acid sequence having at least 99.5% or 99.8% sequence identity with SEQ ID NO:7. In some aspects, R4 comprises a nucleic acid sequence having at least 85% sequence identity with SEQ ID NO:7. In some aspects, R4 comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:7. In some aspects, R4 comprises a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO:7. In some aspects, R4 comprises a nucleic acid sequence having at least 96% sequence identity with SEQ ID NO:7.In some aspects, R4 comprises a nucleic acid sequence having at least 97% sequence identity with SEQ ID NO:7. In some aspects, R4 comprises a nucleic acid sequence having at least 98% sequence identity with SEQ ID NO:7. In some aspects, R4 comprises a nucleic acid sequence having at least 99% sequence identity with SEQ ID NO:7. In some aspects, R4 comprises a nucleic acid sequence having at least 99% sequence identity with SEQ ID NO:7. It also comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO:7. In some aspects, R4 comprises a nucleic acid sequence having at least 99.5% sequence identity with SEQ ID NO:7. In some aspects, R4 comprises a nucleic acid sequence having at least 99.8% sequence identity with SEQ ID NO:7.

[0087] 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). 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.

[0088] In some aspects, R4 encodes a modified scFc, for example, a modified scFc is an IgG1 scFc. As described herein, a “modified scFc” is an scFc that may contain one or more (e.g., one, two, three, four or more) nucleotide modifications (e.g., nucleotide substitutions compared to a reference sequence). In some aspects, compared to the amino acid sequence of the wild-type IgG1 scFc (SEQ ID NO:24), R4 contains a leucine-to-alanine mutation at amino acids 234 and 235, or a leucine-to-alanine mutation at the corresponding amino acid position in its homolog (referred to as the “LALA mutation”). In some aspects, compared to SEQ ID NO:24, R4 contains a proline-to-glycine mutation at amino acid 329, or a proline-to-glycine mutation at the corresponding amino acid position in its homolog (referred to as the “PG mutation”). In some respects, R4 further includes two modifications: the first is a leucine-to-alanine mutation at amino acids 234 and 235, and the second is a proline-to-glycine mutation at amino acid 329 (referred to as the "LALAPG mutation") compared to the corresponding amino acid position in SEQ ID NO:24 or its homologs.

[0089] IgG1 scFc wild-type amino acid sequence (SEQ ID NO:24)

[0090] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW

[0091] YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK

[0092] TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY

[0093] KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKR5 encodes the 3'UTR region. The 3' untranscribed region (3'UTR) of messenger RNA (mRNA) immediately follows the transcription stop codon. It can affect mRNA localization, stability, export, and transcription efficiency. Within the 3'UTR, regulatory portions may include microRNA response sites and AU-enriched portions (AREs). Any suitable 3'UTR can be used. In some respects, R5 encodes the 3'UTR of hemoglobin subunit β (HBB gene), but the 3'UTR may originate from other genes.

[0094] In some respects, R5 contains elements related to SEQ ID NO:8

[0095] (CTCGAGGGCGCGCCGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTT GTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAtCATCTG GATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAG) or a codon-degenerate 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). In other aspects, R5 comprises a nucleic acid sequence having at least 99.3% sequence identity with SEQ ID NO:8. In some aspects, R5 comprises a nucleic acid sequence having at least 85% sequence identity with SEQ ID NO:8. In some aspects, R5 comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:8. In some aspects, R5 comprises a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO:8. In some aspects, R5 comprises a nucleic acid sequence having at least 96% sequence identity with SEQ ID NO:8. In some aspects, R5 comprises a nucleic acid sequence having at least 97% sequence identity with SEQ ID NO:8. In some aspects, R5 comprises a nucleic acid sequence having at least 98% sequence identity with SEQ ID NO:8. In some aspects, R5 comprises a nucleic acid sequence having at least 99% sequence identity with SEQ ID NO:8. In some aspects, R5 comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:8. In some aspects, R5 comprises a nucleic acid sequence having at least 91% sequence identity with SEQ ID NO:8. It also has at least 100% sequence identity with SEQ ID NO:8. In some aspects, R5 comprises a nucleic acid sequence having at least 99.3% sequence identity with SEQ ID NO:8.

[0096] In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO:8 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). In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO:8 by no more than 15 nucleotide substitutions. In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO:8 by no more than 10 nucleotide substitutions. In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO:8 by no more than 9 nucleotide substitutions. In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO:8 by no more than 8 nucleotide substitutions. In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO:8 by no more than 7 nucleotide substitutions. In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO:8 by no more than 6 nucleotide substitutions. In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO:8 by no more than 5 nucleotide substitutions. In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO:8 by no more than 4 nucleotide substitutions. In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO:8 by no more than 3 nucleotide substitutions. In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO:8 by no more than 2 nucleotide substitutions. In some aspects, R5 comprises a nucleic acid sequence differing from SEQ ID NO:8 by no more than 1 nucleotide substitution. In some aspects, R5 comprises the nucleic acid sequence of SEQ ID NO:8.

[0097] Nucleic acids encoding a poly(A) tail may be contained within the 3' UTR or at the 3' end of the 3' UTR. A poly(A) tail is an adenine nucleotide (A) base added to the 3' end of mature eukaryotic messenger RNA (mRNA). It can enhance mRNA stability, prevent enzymatic degradation, facilitate nuclear export, and promote transcription in the cytoplasm. In some cases, a poly(A) tail contains a nucleic acid sequence of approximately 10 to approximately 300 nucleotide bases (e.g., approximately 150 to approximately 250 nucleotide bases). In other cases, nucleotides other than adenine may also be included in the poly(A) tail to promote mRNA synthesis.

[0098] Any nucleic acid disclosed herein for use as an immune cell adjuvant may also include a nucleic acid encoding an adapter (e.g., any suitable adapter). An adapter is a short polypeptide that connects two or more domains of a polypeptide. In some aspects, the adapter is located between R2 and R3. In some aspects, the adapter is located between R3 and R4. In some aspects, a first adapter is located between R2 and R3, and a second adapter is located between R3 and R4. In some aspects, the adapter is located between R6 and R7. In some aspects, the adapter is located between R8 and R9. In some aspects, a first adapter is located between R6 and R7, and a second adapter is located between R8 and R9. The first and second adapters can be the same nucleic acid encoding the same polypeptide, different nucleic acids encoding the same polypeptide (e.g., conserved nucleotide mutations), or different nucleic acids encoding different polypeptides. In some aspects, the first adapter is located between the R2 subdomains of R6 and R7, the second adapter is located between the R3 subdomains of R8 and R9, the third adapter is located between R2 and R3, the fourth adapter is located between R3 and R4, or any combination thereof. Additional adapters can exist in any nucleic acid, which are used by immune cell adaptors to connect additional domains and / or subdomains.

[0099] In some aspects, the adapter comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:4 (ACCggtgggggaggaagtgat) or its codon degenerate nucleic acid sequence (e.g., at least 85%, 90%, 95%, or 100%). In some aspects, the adapter comprises a nucleic acid sequence having at least 85% sequence identity with SEQ ID NO:4. In some aspects, the adapter comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:4. In some aspects, the adapter comprises a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO:4. In some aspects, the adapter comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO:4.

[0100] In some aspects, the adapter 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). In some aspects, the adapter comprises a nucleic acid sequence differing from SEQ ID NO:4 by no more than 4 nucleotide substitutions. In some aspects, the adapter comprises a nucleic acid sequence differing from SEQ ID NO:4 by no more than 3 nucleotide substitutions. In some aspects, the adapter comprises a nucleic acid sequence differing from SEQ ID NO:4 by no more than 2 nucleotide substitutions. In some aspects, the adapter comprises a nucleic acid sequence differing from SEQ ID NO:4 by no more than 1 nucleotide substitution. In some aspects, the adapter comprises the nucleic acid sequence of SEQ ID NO:4. In some aspects, the adapter located 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 with SEQ ID NO:4 (e.g., at least 85%, 90%, 95%, or 100%), 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).

[0101] In some respects, the connector contains elements consistent with SEQ ID NO:6

[0102] (GGCGGTGGTGGCGCCGGCGGAGGTGGCGCTAGC) or a codon-degenerate nucleic acid sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, or 100%). In some aspects, the adapter comprises a nucleic acid sequence having at least 96% sequence identity with SEQ ID NO:6. In some aspects, the adapter comprises a nucleic acid sequence having at least 85% sequence identity with SEQ ID NO:6. In some aspects, the adapter comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:6. In some aspects, the adapter comprises a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO:6. In some aspects, the adapter comprises a nucleic acid sequence having at least 96% sequence identity with SEQ ID NO:6. In some aspects, the adapter comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO:6.

[0103] In some aspects, the adapter 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). In some aspects, the adapter comprises a nucleic acid sequence differing from SEQ ID NO:6 by no more than 5 nucleotide substitutions. In some aspects, the adapter comprises a nucleic acid sequence differing from SEQ ID NO:6 by no more than 4 nucleotide substitutions. In some aspects, the adapter comprises a nucleic acid sequence differing from SEQ ID NO:6 by no more than 3 nucleotide substitutions. In some aspects, the adapter comprises a nucleic acid sequence differing from SEQ ID NO:6 by no more than 2 nucleotide substitutions. In some aspects, the adapter comprises a nucleic acid sequence differing from SEQ ID NO:6 by no more than 1 nucleotide substitution. In some aspects, the adapter comprises the nucleic acid sequence of SEQ ID NO:6. In some aspects, the connector located between R3 and R4 (e.g., a second connector) contains the nucleic acid sequence of SEQ ID NO:6, contains a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:6 (e.g., at least 85%, 90%, 95%, 100%), or contains a nucleic acid sequence that differs from SEQ ID NO:6 by no more than 6 nucleotide substitutions (e.g., 5, 4, 3, 2 or 1 nucleotide substitution).

[0104] Any nucleic acid disclosed herein may include a 5' RNA cap. A 5' RNA cap is a modified nucleotide structure located at the 5' end of mRNA. It consists of 7-methylguanosine (m7G) linked to the first nucleotide via a triphosphate bridge. The 5' RNA protects mRNA from degradation, facilitates transcription, and marks mRNA as "self-RNA" of the innate immune system. Other suitable RNA caps are also considered in this paper.

[0105] Any nucleic acid disclosed herein may include additional design tags to facilitate production, manufacturing, or purification. Any suitable design tag may be included, including but not limited to multihistidine tags (His tags), glutathione S-transferase tags (GST tags), maltose-binding protein tags (MBP tags), chitin-binding protein tags (CBP tags), hemagglutinin tags (HA tags), flag tags, and polyglutamate tags. For example, any suitable tag may be included at any suitable location in any nucleic acid construct. In some aspects, the design tag may be located between R4 and R5. In some aspects, the design tag located between R4 and R5 has the same characteristics as SEQ ID NO:23.

[0106] (GCGATCGCCGATTACAAGGATGACGATGACAAGGAGAATCTCTACTTCCAATCT GAGCAGAAACTAATATCAGAGGAGGATCTAGGGGGAGGAAGTCACCACCATCAC CACCATCCTCGA) or a codon-degenerate nucleic acid sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 98%, 99%, or 100% sequence identity).

[0107] In some aspects, the nucleic acid of the immune cell adaptor may include RNA of Formula I: R1-SP-R2-R3-R4-R5 (I), wherein: R1 is a 5' untranscribed region (UTR); SP encodes a signal peptide; R2 encodes a single-stranded variable fragment (scFv) that binds BCMA; R3 encodes a CD3-binding scFv; R4 encodes a half-life extender; R5 is a 3' UTR, wherein the orientation of R1 to R5 is 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, the nucleic acid of the immune cell adaptor of Formula I may also include a linker located between R2 and R3, encoded by the nucleic acid sequence of SEQ ID NO:4. In some respects, the nucleic acid of the immune cell adaptor of Formula I may also include a connector located between R3 and R4 encoded by the nucleic acid sequence of SEQ ID NO:6. Therefore, in some respects, the nucleic acid of the immune cell adaptor may include RNA of formula I: R1–SP–R2–R3–R4–R5 (I), wherein: R1 is a 5' untranscribed region (UTR); SP encodes a signal peptide; R2 encodes a single-stranded variable fragment (scFv) that binds BCMA; R3 encodes a scFv that binds CD3; R4 encodes a half-life extender; R5 is a 3' UTR, wherein the orientation of R1 to R5 is 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, the adapter encoded by the nucleic acid sequence of SEQ ID NO:4 is located between R2 and R3, and the second adapter encoded by the nucleic acid sequence of SEQ ID NO:6 is located between R3 and R4.

[0108] In some respects, nucleic acids used as immune cell adaptors may include those compatible with SEQ ID NO:20

[0109]

[0110] Any nucleic acid encoding the immune cell adjuvant described herein can also be used to achieve the desired pharmacological effect by administration in a suitably formulated pharmaceutical composition to a patient in need. For the purposes of this disclosure, a patient is a domestic animal or human requiring treatment for a specific condition or disease. Therefore, pharmaceutical compositions for immune cell adjuvants are also disclosed herein, comprising any nucleic acid encoding the immune cell adjuvant described herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0111] "Pharmaceutical acceptable" means that the carrier, diluent, or excipient must be compatible with other components in the formulation (including nucleic acids encoding immune cell adjuvants) and will not cause harm to the recipient. "Pharmaceutical acceptable carrier" means any carrier that is relatively non-toxic and harmless to the patient at concentrations consistent with the effective activity of the active ingredient (e.g., nucleic acids encoding immune cell adjuvants), so that any side effects attributable to the carrier do not impair the beneficial effects of the active ingredient.

[0112] This article considers suitable pharmaceutical compositions comprising nucleic acids encoding immune cell adjuvants, and is based in part on the specific tissue and cell types involved. Therefore, pharmaceutical compositions suitable for encoding nucleic acids encoding the immune cell adjuvants of this disclosure can be formulated according to any method known in the art. (See, for example: Remington Pharmaceutical Journal, 15th Edition, Chapter 33; Gagliardi et al., 2021; or Hammond et al., 2021).

[0113] Lipid nanoparticles (LNPs) encapsulating nucleic acids encoding immune cell adjuvants are considered a method to protect these nucleic acids from degradation and facilitate cellular delivery. Therefore, LNPs offer the opportunity to formulate compositions containing nucleic acids encoding immune cell adjuvants that are less likely to trigger an immune response and subsequently be cleared. This is likely due to the composition of the lipid particles themselves. Therefore, this disclosure contemplates the use of lipid nanoparticles to encapsulate nucleic acids encoding the immune cell adjuvants described herein. See, for example, Wang et al. J Biomed Sci 30,84 (2023). Thus, in one aspect described herein, there is a pharmaceutical composition comprising any nucleic acid encoding an immune cell adjuvant described herein, a pharmaceutically acceptable salt thereof, and lipid nanoparticles. In some aspects, the lipid nanoparticles are cationic nanoparticles. LNPs can direct a payload (e.g., a nucleic acid encoding an immune cell adjuvant) to a preferred cell type in a patient. For example, LNPs can target hepatocytes by binding to specific ligands exhibiting hepatic orientation or by surface modifications. Such modifications enable LNPs to interact with receptors on the hepatocyte membrane, thereby facilitating cellular uptake and payload delivery. The liver's natural affinity for lipid 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. LPNs can be formulated according to the method described by Wang et al. in J Biomed Sci 30, 84 (2023).

[0114] Pharmaceutical compositions for administering the nucleic acids of this disclosure can be conveniently presented in unit doses and can be prepared by any method well known in the pharmaceutical field. All methods include the step of binding the active ingredient (e.g., a nucleic acid encoding an immune cell adjuvant) to a carrier constituting one or more auxiliary components. Typically, pharmaceutical compositions are prepared by uniformly and tightly binding the active ingredient to a liquid carrier or a finely fragmented solid carrier, or both, and then, if desired, shaping the product into the desired formulation. In the pharmaceutical composition, the amount of the active target compound is sufficient to produce the desired effect on the course or condition of a disease.

[0115] The nucleic acids encoding immune cell adjuvants described herein can be administered as pharmaceutical compositions with pharmaceutically acceptable carriers, using any effective and conventional dosage unit form, including, for example, immediate-release and time-release formulations, injections, parenteral formulations, topical formulations, etc. In some aspects, either the nucleic acids encoding immune cell adjuvants or any of the pharmaceutical compositions described herein can be administered intravenously, intraarterially, intratumorally, intradermally, subcutaneously, locally, ex vivo, in vitro, or intraperitoneally, or as local, regional, systemic, or continuous administration. In some aspects, either the nucleic acids encoding immune cell adjuvants or any of the pharmaceutical compositions described herein can be administered intravenously.

[0116] The nucleic acid encoding the immune cell adaptor described herein can use multiple binding regions (e.g., R2 and R3) to simultaneously bind to effector cells and cells overexpressing the first extracellular protein. For example, the nucleic acid described herein can use multiple binding regions (e.g., R2 and R3) to simultaneously bind to immune cells and cells overexpressing BCMA.

[0117] The nucleic acids encoding immune cell adjuvants described herein can simultaneously bind effector cells and cancer or tumor cells using multiple binding regions (e.g., R2 and R3). Therefore, the nucleic acids encoding immune cell adjuvants described herein bring effector cells, which may possess anti-tumor or anti-cancer cell properties, closer to cancer or tumor cells. Thus, administration of the nucleic acids encoding immune cell adjuvants described herein, or any pharmaceutical composition described herein, facilitates the targeting of anti-cancer effector cells (e.g., T cells) to cancer or tumor cells (e.g., multiple myeloma cells). Therefore, methods for treating cancer, such as methods for treating hematologic malignancies (e.g., methods for treating multiple myeloma), and methods for conjugating effector cells, such as methods for conjugating T cells, are also described herein. It is anticipated that any method or pharmaceutical composition described herein can be implemented relative to any other method or pharmaceutical composition described herein.

[0118] One aspect of this disclosure is a method of treating cancer in patients in need. The method of treating cancer in patients in need includes administering to the patient an effective amount of any nucleic acid encoding an immune cell adjuvant described herein or any pharmaceutical composition described herein. Another aspect of this disclosure is that the method of treating cancer in patients in need includes administering to the patient an effective amount of any nucleic acid encoding an immune cell adjuvant described herein or any pharmaceutical composition described herein.

[0119] For example, any nucleic acid encoding an immune connective as described herein can be used in a method of treating blood cancers, comprising administering to a patient an effective amount of any nucleic acid encoding an immune cell connective as described herein or any pharmaceutical composition described herein. Exemplary blood 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.

[0120] One aspect of this disclosure is a method for treating multiple myeloma in patients in need. For example, this document discloses a method for treating multiple myeloma in patients in need, comprising administering to the patient an effective amount of any nucleic acid encoding an immune cell connective agent described herein or any pharmaceutical composition described herein. Multiple myeloma is a blood cancer that originates from plasma cells. These plasma cells normally produce antibodies to fight infection. However, in patients with multiple myeloma, abnormal plasma cells accumulate in the bone marrow, crowding out healthy plasma cells. This can lead to a variety of symptoms, including, but not limited to, bone pain, especially in the chest, spine, pelvis, and ribs; fatigue; muscle weakness; nausea; constipation; hypercalcemia (elevated blood calcium levels, leading to thirst, confusion, and constipation); elevated kidney protein levels; anemia; and a weakened immune system, making them more susceptible to infection. Risk factors for multiple myeloma include family history, age (more common in older adults), sex (more common in men), race (higher incidence in African Americans), and obesity.

[0121] In one aspect, the compositions and methods described herein are used as first-line therapy (sometimes referred to as primary therapy). In another aspect, the compositions and methods described herein are used as second-line therapy. In some aspects, the compositions and methods described herein are used as third-line therapy or as more advanced therapy (e.g., as fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more advanced therapy). In some aspects, the compositions and methods described herein are used as salvage therapy. The term "salvage therapy" refers to a therapeutic agent that can be taken with any regimen after a subject's initial treatment regimen has failed or after a subject's condition has become unresponsive to the initial treatment. In another aspect, the compositions and methods described herein are used as salvage therapy. In one embodiment, the composition is used as a salvage agent to counteract the effect of the initial treatment. In one embodiment, the composition is used as a salvage agent administered to a subject who has developed resistance to standard or initial treatment. Common first-line treatments for multiple myeloma include, but are not limited to, chemotherapy (e.g., immunomodulatory imide drugs such as lenalidomide or pomalidomide) and proteasome inhibitors (e.g., bortezomib and carfilzomib), corticosteroids (e.g., prednisone or dexamethasone), bone marrow transplantation, and radiation therapy.

[0122] On one hand, the patient has received at least one prior treatment. On another hand, the patient has received at least two, at least three, or at least four or more prior treatments. On the other hand, the prior treatment is lenalidomide or pomalidomide. On the other hand, the prior treatment is bortezomib or carfilzomib. On the other hand, the prior treatment is lenalidomide, pomalidomide, bortezomib, or carfilzomib. On the other hand, the patient may have received radiation therapy. On the other hand, the cancer no longer responds to lenalidomide, pomalidomide, bortezomib, carfilzomib, bone marrow transplantation, radiation therapy, or combinations thereof. In one aspect, the method described herein includes the administration of a second treatment agent, including an anticancer agent, bone marrow transplantation, or radiation therapy. In one aspect, the second treatment agent is an anticancer agent selected from lenalidomide, pomalidomide, bortezomib, carfilzomib, or combinations thereof. Therefore, another embodiment described herein is a method for treating cancer (e.g., blood cancer) in patients in need (e.g., a method for treating multiple myeloma), comprising: (a) administering a first treatment, comprising any pharmaceutical composition described herein or any nucleic acid encoding an immune cell binder described herein or any pharmaceutical composition described herein; and (b) administering a second therapeutic agent (e.g., an anticancer agent, bone marrow transplantation, or radiation therapy).

[0123] In any of the treatments described herein, the pharmaceutical composition or nucleic acid encoding an immune cell adjuvant described herein may be administered to the subject according to an infrequent dosing regimen (e.g., once weekly or less frequently). In another aspect, the composition may be administered to the subject once weekly. In another aspect, the composition may be administered to the subject once every two weeks. In one aspect, the composition may be administered to the subject once every four weeks. In one aspect, the composition may be administered to the subject twice weekly. In one aspect, the composition may be administered to the subject once every three weeks. In one aspect, the composition may be administered to the subject according to a frequent dosing regimen (e.g., more than once daily or more than once weekly). In one aspect, the composition may be administered to the subject in repeated cycles of once daily, twice daily, once weekly, once every two weeks, once every three weeks, once every four weeks, or combinations thereof. Any suitable dosing regimen is considered herein.

[0124] As described in this article, the articles “a,” “one,” and “the” are used to refer to one or more (i.e., at least one) grammatical objects of the article. For example, “a part” can mean one part or more parts.

[0125] As described herein, the term “about” or “approximately” means that a quantity, level, value, number, frequency, percentage, size, volume, weight, or length differs from a reference quantity, level, value, number, frequency, percentage, size, volume, weight, or length by as much as 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, when the term “about” or “approximately” precedes a numerical value, it indicates a range of that value plus or minus 10%, 5%, or 1%.

[0126] As used herein, “nucleic acid” refers to a polymer of nucleotides of any length, which may be ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Therefore, the term includes double-stranded and single-stranded DNA, triple-stranded DNA, and double-stranded and single-stranded RNA. Any RNA format is acceptable as long as the described amino acid sequence can be produced. It also includes modified forms of polynucleotides (e.g., through methylation and / or capping) and unmodified forms. The term is also intended to include molecules containing non-naturally occurring or synthetic nucleotides, as well as nucleotide analogs.

[0127] As described herein, the term “sequence identity” in the context of two nucleic acid sequences or amino acid sequences refers to the same residues in two sequences when a maximum correspondence is performed within a specified comparison window. Sequence alignment methods used for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed using methods such as: the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981); the alignment algorithm of Needleman and Wunsch, J. Mal. Biol., 48:443 (1970); the similarity search method of Pearson and Lipman, Proc. Nat. Acad. Sci US. A., 85:2444 (1988); and computerized implementations of such algorithms (including but not limited to CLUSTAL in the PC / Gene program of Intelligentics, Mountain View California, GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group (GCG), 575 Science). Dr., Madison, Wis., USA); the CLUSTAL procedure is described in detail in 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). Alignments are also frequently performed through inspection and manual comparison.

[0128] The term "effective amount" as used herein refers to an amount that can alleviate disease symptoms through clinical testing and evaluation, patient observation, and other methods. "Effective amount" can also refer to a dose that causes a detectable change in biological or chemical activity. Those skilled in the art can detect and / or further quantify the detectable change in relation to the relevant mechanism or process. Furthermore, "effective amount" can refer to an amount that maintains a desired physiological state, i.e., an amount that reduces or prevents significant decline and / or promotes improvement in the target condition. "Effective amount" can also refer to a "therapeutic effective amount." As used herein, the terms "individual" and "subject" are generally used interchangeably to refer to any human or domestic animal that can be treated with the methods disclosed herein. Suitable subjects (e.g., patients) include humans and domestic animals or pets (e.g., cats or dogs). This includes non-human primates and human patients. In one embodiment, a subject may include a human patient diagnosed with cancer (e.g., blood cancer), including but not limited to: multiple myeloma. As used herein, the term "patient" refers to a subject who can receive treatment for a disease or condition.

[0129] As described in this article, “treatment” means reversing, reducing, alleviating or slowing the progression of a condition or disease or symptoms associated with such a condition or disease, or inhibiting the progression of a condition or disease or symptoms associated with such a condition or disease, and is described in more detail in this article.

[0130] 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 any conflict, this document (including the definitions) shall prevail. Methods and materials are described below, although similar or equivalent methods and materials may be used in practice or testing of this disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0131] Although the above disclosure has been described in detail with illustrations and examples for ease of understanding, it will be readily apparent to those skilled in the art, based on the teachings of this disclosure, that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided for illustration only and not as limitations. Those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to produce substantially similar results.

[0132] equivalent

[0133] The various aspects of this disclosure can be used individually, in combination, or in various arrangements not specifically discussed in the above embodiments. Therefore, its application is not limited to the details and arrangements set forth in the above description or shown in the accompanying drawings. For example, an aspect described in one embodiment can be combined in any way with aspects described in other embodiments.

[0134] Example

[0135] Example 1 - Binding of fully human H2L2 antibody to BCMA

[0136] Humanized tetrameric antibodies were generated from H2L2 mice (Harbor Antibodies) consisting of two identical heavy chains and two identical light chains. Fully human variable-domain H2L2 antibodies were generated using H2L2 mice (transgenic human VH and human VL). Single B / plasma cells were isolated from the mice, and antibody production and specificity were confirmed at the single-cell level, followed by single-cell sequencing. Recombinant antibodies, including PR000892, PR000897, PR00903, PR00909, and the reference antibody PR000274, were generated and characterized. Humanized H2L2 is described in PCT patent publication WO2021147941. The reference positive control antibody PR0000274 is a clone of CA8-J6M0. It is described in US Patent 9,273,141 as a clinically validated anti-BCMA antibody. The newly prepared H2L2s antibody bound to the human cell line expressing BCMA (HEK293T-hu BCMA), and the binding strength was comparable to that of the reference antibody PR000274. Figure 1A All antibodies exhibited cross-reactivity. Figure 1B For example, it binds to HEK293T cells expressing macaque (cyno) BCMA protein (HEK293T-cyno BCMA). Furthermore, the fully human H2L2 antibody exhibits strong binding affinity to NCI-H9292 myeloma cells. Figure 1C ).

[0137] Example 2 – Evaluation of the in vitro cytotoxicity of T cell adaptors (TCEs)

[0138] NIH 3T3 fibroblasts were cultured in DMEM and 10% FBS (Gibco, Life Technologies). To generate GFP / luciferase-positive target cells or cells expressing BCMA or TROPS (irrelevant targets) in NIH-3T3, cells were stably transduced with cDNA-expressing gamma retroviruses. 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 to be usable for all experiments. Expi293 (ThermoFisher) was used as the cell line for TCE production. Cells were transduced with plasmid DNA according to the manufacturer's instructions, and the supernatant was collected. The supernatant was then concentrated and the buffer was exchanged for PBS via size exclusion (Amicon Ultra, Millipore). TCE concentration was quantified by ELISA.

[0139] Human T cells were obtained from peripheral blood of healthy donors (MGB Crimson Core Blood Bank; Study No.: T0761). 1×10 4 Human CD3+ T cells, acting as effector cells, were stimulated for 48 hours with CD3 / CD28 TransAct (Miltenyi, catalog number: 130-111-160) at a ratio of 1:100 and grown in the presence of IL-2±IL-7 and IL-15 (NIH BRB preclinical biologics library). These cells were then seeded together with 5×10³ luciferase-transduced target cells into white 96-well flat-bottomed plates (Costar). 1 nM TCE was added to each well. The plates were incubated for 24 hours to induce lysis of cancer cells by effector cells. Intense lysis was determined according to ONE-Globe assays. TM Measurement of active luciferase-positive cells using a luciferase detection system (BD Biosciences). Three replicates were performed at the indicated effector cell to target cell (E:T) ratio. Bioluminescence was read on a BioTek Cytation 5. % Cytotoxicity = (BLI Mock - BLI Sample) / BLI Mock, where BLI Mock = the average value of individual target cells in this experiment.

[0140] The results are as follows Figure 2 As shown.

[0141] Example 3 – Evaluation of the effective in vivo dose of TCE

[0142] mRNA synthesis: The DNA template plasmid was linearized and purified using EcoI (NEB) (Nucleospin PCR purification, Takara Bio). mRNA IVT was performed using the HiScribe T7 High-Yield RNA Synthesis Kit (NEB). UTP in the reaction was completely replaced by 1-methylpseudouridine triphosphate (N1-methylpseudouridine-5′-triphosphate, m1ΨTP) (TriLink). m7G5′ppp5′G2′-O-Met-capped (cap1) IVT mRNA was generated using vaccinia virus guanylate transferase and 2′-O-methyltransferase (NEB). The mRNA was polyadenylated using E. coli Poly(A) polymerase and then purified using the Monarch RNA Purification Kit (NEB). The quality of the purified IVT mRNA was assessed using electrophoresis on a 2100 Bioanalyzer (Agilent), and the concentration was determined by UV spectroscopy. The mRNA was aliquoted and stored at -80°C.

[0143] Lipid and LNP formulation: Ionizable lipid SM-102 was purchased from Cayman Chemical (Cat#33474). 1,2-Distearayoyl-sn-glycerol-3-phosphocholine (DSPC, Cat#850365P) and 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG200, Cat#880150) were purchased from Avanti Polar Lipids. Cholesterol (Cat#C8667) was purchased from Sigma-Aldrich. All lipids were dissolved in anhydrous ethanol (Cat#BP2818, Fisher Scientific).

[0144] For intravenous administration in in vivo studies, mRNA was encapsulated in lipid nanoparticles using NanoAssembler Ignite microfluidic cassette technology (Precision Nanosystems). Briefly, lipids were mixed at a molar ratio of 50:10:38.5:1.5 (SM102:DSPC:cholesterol:DMG-PEG), and mRNA was prepared to 0.1 mg / ml in 10 mM citrate buffer (pH 3.0). The lipid mixture and mRNA were then mixed at an N / P ratio of 4, a flow rate of 12 ml / min, and a flow ratio of 3:1 (aqueous phase: organic phase). After preparation, mRNA-LNP was dialyzed twice through a 10 KMWCO Slide-A-Lyzer dialysis kit (Cat#66380, ThermoScientific) to a concentration of 50 μg / ml in 10% sucrose PBS solution (w / v). mRNA encapsulation efficiency and concentration were measured using Quant-iT... TMDetermined using the RiboGreen assay (Cat#R11490, Life Technologies). Store at -20°C after aliquoting.

[0145] The human multiple myeloma (MM) cell line OPM2 was obtained from the German Center for Microbiology and Cell Culture (DMSZ) and cultured in RPMI and 10% fetal bovine serum (FBS) (Gibco, Life Technologies, Gaithersburg, MD). To generate GFP / luciferase-positive target cells, these cells were stably transduced with a cDNA-expressing gamma retrovirus, as before. Cells were then sorted into single-cell clones in 96-well plates and expanded to generate clonal populations, which were confirmed by flow cytometry to be usable for all experiments. Human T cells were prepared as before.

[0146] Six to eight-week-old NSG DKO (NOD.Cg-Prkdcscid H2-K1b-tm1Bpe H2-Ab1g7-em1MvwH2-D1b-tm1Bpe Il2rgtm1Wjl / SzJ) mice (Jackson Labs; Bar Harbor, ME) were injected intravenously with the bone marrow hyperactivity cell line OPM2-ffLuc at a dose of 1×10⁻⁶. 6 10 × 10⁶ cells. Tumor implantation was confirmed by baseline bioluminescence imaging. Twelve days after tumor implantation, a single dose of 10 × 10⁶ cells was administered via tail vein injection. 6 Human T cells were used to humanize mice. The following day, mRNA-LNP was intravenously injected into the animals according to the prescribed dosage regimen. In vivo imaging was performed after injection of D-luciferin (Millipore-Sigma; Darmstadt, Germany), and analysis was performed using Living Image software (PerkinElmer; Waltham, MA). The study planned to use a minimum number of animals in each treatment group to reproduce statistically significant differences (n = 4–5 per group).

[0147] The results are as follows Figures 3A-3D As shown.

[0148] Example 4 – Typical TCEs

[0149] like Figure 4AThe mTCE sequence shown consists of a region encoding an RNA polymerase promoter, a capping signal, a 5UTR, a signal peptide, a tumor-associated antigen-binding domain (scFv (further composed of a VH domain linked to VL) or a VH domain), a linker, an immune cell-binding domain (again scFv (further composed of a VH domain linked to VL) or a VH domain), a half-life extension domain, and a desired termination sequence, with or without a poly-A tail, and is ultimately determined by the 3'UTR.

[0150] The 5'UTR can be selected from the mRNA sequence of naturally occurring proteins or synthesized. As used herein, the term signal peptide refers to a short (e.g., 5-30 or 10-100 amino acid long) amino acid sequence typically located at the N-terminus of a protein, used to guide protein transport. The signal peptide allows mRNA and ribosomes to bind to the endoplasmic reticulum, inserting newly transcribed proteins into transporters, transporting and delivering protein products to the plasma membrane. Typically, the signal peptide is cleaved during post-transcriptional modifications of the protein by the cell. The signal peptide is computer-evaluated to ensure efficient functional cleavage. The VH and VL amino acid sequences were generated as described in Example 1. The anti-CD3 VH and VL sequences and their bridging linkers were derived from blinatumomab. See, for example, PCT patent publication number WO2010037838.

[0151] The extended half-life domain (scFc) is derived from the conserved human IgG1 sequence (IMGT). Point mutations are introduced into the Fc region to suppress typical Fc-mediated immune effector functions (such as complement-dependent cytotoxicity or antibody-dependent cytotoxicity).

[0152] In some embodiments, the nucleic acid sequence encoding TCE can be codon-optimized to balance expression in human cells, avoid sequence defects, and / or improve synthesis and engineering. Using efficient codons can increase elongation.

[0153] In some embodiments, the complete human VH and VL domain amino acid sequences are reverse transcribed to generate an unoptimized DNA sequence, which is then manually codon-optimized. Furthermore, a start site (e.g., a consistent Kozak sequence) can be appended to the 5' end of the optimized nucleic acid encoding the TCE, and a restriction enzyme recognition site (e.g., an AgeI site) can be used to transcribe the entire nucleotide sequence into the mTCE.

[0154] Commercially available software (Snapgene, GSL Biotech LLC, San Diego, CA) can also be used to analyze the presence of restriction enzyme recognition sites for selected linearized restriction enzymes or other cloning enzymes (e.g., EcoRI). In some embodiments, nucleic acid sequences are designed using DNA dot mapping tools (e.g., YASS and IDTs E-block™ complexity testing tools) to exclude 20 nucleic acid fragments of direct or inverted repeat sequences. Direct repeats are nucleotide sequences consisting of two or more repetitions of a specific sequence, such that the repeat exists in multiple copies of a larger sequence. Direct repeats typically occur when sequences are repeated downstream in the same pattern. Inverted repeats are single-stranded nucleotide sequences whose downstream is their inverted complementary sequence. Removing repetitive nucleotide sequences ultimately increases expression efficiency.

[0155] Identified internal restriction enzyme recognition sites can be removed by generating silent mutations. Bioinformatics tools (such as Alternate Splice Site Predictors (ASSPs)) can be used to identify cryptic or alternative splice sites. High-scoring constitutive splice sites can be removed by generating silent mutations.

[0156] In some embodiments, the 3' end is modified to ensure the presence of a functional transcription termination consensus sequence, and screening is performed to avoid the presence of type I and type II termination sequences before the desired termination site. This sequence may also be screened to remove certain slow transcription codon pairs.

[0157] As a final check, high-scoring splicing sites in the sequence are checked again, and undesirable restriction enzyme recognition sites are excluded, along with the desired sites and appropriate start and stop codons.

[0158] The sequence encoding TCE was cloned into a vector that also contained the T7 polymerase site and a non-coding region preceding the EcoRI restriction site. All generated plasmids were validated by whole-plasmid sequencing.

[0159] Figure 4B Other exemplary TCEs are shown.

[0160] Predictive Implementation

[0161] Future experiments will continue to investigate the scFv and immune cell adjuvants described in this paper.

[0162] The patient was diagnosed with multiple myeloma using any appropriate method, including blood tests, urine tests, imaging studies, bone marrow biopsy, or serum antibody testing. An effective amount of a pharmaceutical composition comprising a nucleic acid for an immune cell adaptor, said immune cell adaptor having RNA of formula I: R1–SP–R2–R3–R4–R5 (I), wherein: R1 is a 5' untranscribed region (UTR); SP encodes a signal peptide; R2 encodes a single-stranded variable fragment (scFv) that binds BCMA; R3 encodes a CD3-binding scFv; R4 encodes a half-life extender; R5 is a 3' UTR, wherein R1 to R5 are in the 5' to 3' orientation, 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, wherein the adapter encoded by the nucleic acid sequence of SEQ ID NO:4 is located between R2 and R3, and is encoded by SEQ ID NO:8. The second connector encoded by the NO:6 nucleic acid sequence is located between R3 and R4, and said nucleic acid is encapsulated in lipid nanoparticles. After receiving the pharmaceutical composition, patients experience a reduction in the severity of multiple myeloma disease or a reduction in the severity of multiple myeloma symptoms. The severity of multiple myeloma disease can be measured by any appropriate test, including blood tests, urine tests, imaging studies, bone marrow biopsy, or serum antibody testing.

[0163] Although the above disclosure has been described in detail by way of illustration and example for ease of understanding, those skilled in the art will readily understand, based on 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. Such examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize that various non-critical parameters may be changed or modified to produce substantially similar results. The references provided throughout are intended only to incorporate the background teachings cited therein.

Claims

1. A nucleic acid for use in immune cell conjugates, comprising ribonucleic acid of formula I: R1–SP–R2–R3–R4–R5(I), in: R1 is the 5' untranscribed region (UTR); SP encodes a signal peptide; R2 encodes the first single-stranded variable segment (scFv) that binds to the first extracellular protein or the heavy chain variable region (VH only) that binds to the first extracellular protein. R3 encodes a second scFv that binds to a second extracellular protein; R4 encodes a half-life extender; R5 is 3'UTR, and The directions of R1 to R5 are 5' to 3'.

2. The nucleic acid of claim 1, wherein R1 comprises the hemoglobin subunit β5'UTR.

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

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

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

6. The nucleic acid according to 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), in: R6 encodes light chain variable segments (VL); and R7 encodes heavy chain variable segments (VH).

7. The nucleic acid of claim 6, wherein the nucleic acid encoding the first adapter is located between R6 and R7.

8. The nucleic acid according to any one of claims 1-7, wherein the second scFv comprises formula IV or formula V: R8–R9 (IV), R9–R8 (V) in: R8 encoding of light chain variable segments (VL); and R9 encoding heavy chain variable segments (VH).

9. The nucleic acid of claim 8, wherein the nucleic acid encoding the second adapter is located between R8 and R9.

10. The nucleic acid of any one of claims 1-9, wherein R4 comprises a single-stranded crystallizable fragment (scFc) or a CH3 domain.

11. The nucleic acid of claim 10, wherein R4 comprises an IgG1 single-stranded crystallizable fragment (scFc).

12. The nucleic acid of claim 11, wherein scFc contains a mutation from leucine to alanine at amino acids 234 and 235 compared to the corresponding amino acid positions in SEQ ID NO:24 or its homologs.

13. The nucleic acid of claim 11, wherein scFc contains a leucine-to-alanine mutation at amino acids 234 and 235 and a proline-to-glycine mutation at amino acid 329, compared to the corresponding amino acid positions in SEQ ID NO:24 or its homologs.

14. The nucleic acid of any one of claims 1-13, wherein R5 comprises the hemoglobin subunit β3'UTR.

15. The nucleic acid according to any one of claims 1-14, wherein the first extracellular protein is expressed or overexpressed on cancer cells or tumor cells.

16. The nucleic acid of claim 15, wherein the cancer cell is a hematologic malignancy.

17. The nucleic acid of claim 15, wherein the cancer cell is a multiple myeloma cancer cell.

18. The nucleic acid of any one of claims 1-17, wherein the first extracellular protein is a B cell maturation antigen. (BCMA).

19. The nucleic acid of any one of claims 1-18, wherein the second extracellular protein is expressed on immune cells.

20. The nucleic acid of claim 19, wherein the immune cell is a T cell.

21. The nucleic acid of any one of claims 1-10, wherein the second extracellular protein is differentiation cluster 3 (CD3).

22. The nucleic acid of any one of claims 1-11, wherein R1 comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:

1.

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

2.

24. The nucleic acid of any one of claims 1-23, wherein R2 comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:

3. The nucleic acid of any one of claims 4-24, wherein the linker between R2 and R3 comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:

4.

25. The nucleic acid of any one of claims 1-25, wherein R3 comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:

5.

26. The nucleic acid of any one of claims 5-26, wherein the linker between R3 and R4 comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:

6.

27. The nucleic acid of any one of claims 1-26, wherein R4 comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:

7.

28. The nucleic acid of any one of claims 1-15, wherein R5 comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:

8.

29. A pharmaceutical composition, characterized in that, It comprises the nucleic acid as described in any one of claims 1-29, and one or more pharmaceutically acceptable carriers, diluents or excipients.

30. The pharmaceutical composition of claim 30, wherein one or more pharmaceutically acceptable carriers, diluents, or excipients comprise lipid nanoparticles.

31. Use of the nucleic acid according to any one of claims 1-29 or the pharmaceutical composition according to claims 30-31 in the manufacture or preparation of a medicament for treating cancer.

32. The use of the nucleic acid according to any one of claims 1-29 or the pharmaceutical composition according to claims 30-31 in the manufacture or preparation of a medicament for treating hematologic malignancies.

33. The use of the nucleic acid according to any one of claims 1-29 or the pharmaceutical composition according to claims 30-31 in the manufacture or preparation of a medicament for the treatment of multiple myeloma.

34. The nucleic acid of any one of claims 1-29 or the pharmaceutical composition of claims 30-31, for manufacturing or preparing a medicament for binding immune cells.

35. The nucleic acid of any one of claims 1-29 or the pharmaceutical composition of claims 30-31, for manufacturing or preparing a drug for conjugating T cells.

36. A method of treating cancer in a patient in need, the method comprising administering to the patient an effective amount of any one of claims 1-29 of a nucleic acid or any one of claims 30-31 of a pharmaceutical composition.

37. A method for treating blood cancers in a patient in need, the method comprising administering to the patient an effective amount of any one of claims 1-29, or a pharmaceutical composition of claims 30-31.

38. A method for treating multiple myeloma in patients in need, the method comprising administering to the patient an effective amount of any one of claims 1-29 of the nucleic acid or any one of claims 30-31 of the pharmaceutical composition.

39. A method of engaging immune cells in a patient in need, the method comprising administering to the patient an effective amount of any one of claims 1-29 of the nucleic acid or any one of claims 30-31 of the pharmaceutical composition.

40. A method for engaging T cells in a patient in need, the method comprising administering to the patient an effective amount of the nucleic acid of any one of claims 1-29 or the pharmaceutical composition of claims 30-31.

41. A single-stranded variable fragment (scFv) containing a nucleic acid having at least 80% sequence identity with SEQ ID NO:3.

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