BCMA VH only CAR

Abstract

Disclosed is a nucleic acid encoding a chimeric antigen receptor comprising an extracellular domain including an antigen recognition domain consisting of a fully human single variable heavy chain (VH) domain that binds to a first epitope on B-cell maturation antigen (BCMA), a transmembrane domain, and an intracellular domain including a signaling domain.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 422,711, filed November 4, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, and is incorporated herein by reference in its entirety. Said XML copy, created on October 23, 2023, is named 52095_766001WO_ST.xml and is 83 KB in size. [Background technology]

[0003] Chimeric antigen receptor (CAR) T-cell therapy has been a highly effective cancer therapy, particularly for treating B-cell acute lymphoblastic leukemia (ALL) and B-cell lymphoma. Despite advances in cancer treatment with CAR T-cell therapy, patients with hematological malignancies such as multiple myeloma (MM) and lymphoma still frequently experience relapse.

[0004] One factor in relapse after CAR T cell therapy is anti-CAR immunity. The patient's immune system can recognize the CAR as a foreign antigen, which in most CAR T cell therapies is a mouse-derived single-chain variable antibody fragment (scFv). Human anti-mouse antibodies target the mouse idiotopes used in CAR T cell therapy, which leads to neutralization or clearance of the administered CAR T cells. Mouse scFvs also bind to the CD19 epitope and induce human leukocyte antigen (HLA)-restricted T cell-mediated immunoregulatory responses, which can lead to reduced CAR T cell persistence in vivo and cancer recurrence (Wagner et al., Nat. Rev. Clin. Oncol. 18:379-393 (2021)).

[0005] Nanobodies, also called VH antibodies, are derived from the variable domains of heavy-chain-only antibodies (HCAbs), which lack the first constant CH1 domain in the light and heavy chains. Camelids (e.g., camels) naturally produce HCAb antibodies. However, camelid-based nanobodies are also susceptible to human immune responses when used in CAR T-cell therapy (Rossotti et al., FEBS J. 289:4304-4327 (2022)). Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there is a need to deliver effective CAR T-cell therapies that result in fewer anti-CAR immune responses. Such systems and methods of use would reduce CAR T-cell therapy relapse and improve patient outcomes. [Means for solving the problem]

[0007] A first aspect of the present disclosure is a nucleic acid containing a sequence encoding a chimeric antigen receptor (CAR), wherein the CAR contains an extracellular domain containing a first antigen-recognition domain composed solely of a fully human single variable heavy chain (VH) domain (i.e., a fully human VH-only antigen-recognition domain) that binds to an epitope on B-cell maturation antigen (BCMA), a transmembrane domain, and an intracellular domain containing a signaling domain. To date, there is no evidence that binding proteins based solely on fully human VHs can be produced and incorporated into CAR constructs for the treatment of cancer or autoimmune disease.

[0008] Another aspect of the present disclosure pertains to an expression vector containing (e.g., incorporated or cloned into) a nucleic acid sequence encoding a CAR.

[0009] Another aspect of the present disclosure is directed to genetically modified immune cells containing nucleic acid, including nucleic acid encoding a CAR.

[0010] Another aspect of the present disclosure relates to a method of producing genetically modified immune or hematopoietic cells, which involves introducing into a cell (i.e., an immune or hematopoietic cell) an expression vector containing a nucleic acid encoding a CAR.

[0011] Another aspect of the present disclosure is directed to a pharmaceutical composition containing a therapeutically effective number of cells expressing a CAR.

[0012] Another aspect of the present disclosure is directed to a method of treating cancer or autoimmune disease associated with aberrant BCMA activity, the method involving administering to a subject in need thereof a therapeutically effective number of genetically modified cells (i.e., immune cells or hematopoietic cells) that express a CAR.

[0013] As shown in the Examples herein, the inventors have found that incorporating a fully human VH-only antigen recognition domain into a CAR construct allows for efficient cell transduction, binding to BCMA, and CAR-specific killing of BCMA-expressing target cells. [Brief explanation of the drawings]

[0014] [Figure 1] Figures 1A-1D are a series of schematic diagrams showing antibody domains and regions of different antibody types. Figure 1A is a schematic diagram of a conventional mammalian (e.g., human) antibody, with two heavy chains and two light chains, and a total molecular weight of approximately 150 kDa. Figure 1B is a schematic diagram of a camelid HCAb, with two heavy chains, with a total molecular weight of approximately 95 kDa. Figure 1C is a schematic diagram of the human IgH locus showing recombination and translation to produce a heavy chain-only antibody. Figure 1D is a schematic diagram of a transgene containing both human and mouse sequences in a mouse background with a mouse heavy chain (HC) and kappa light chain knockout.

[0015] [Figure 2]Figures 2A-2D are a series of line plots showing that humanized HCAbs have good binding activity to human and cynomolgus monkey BCMA cell lines. Figure 2A is a line plot showing four antibodies binding to HEK 293T-hu BCMA cells. Figure 2B is a line plot showing two antibodies binding to HEK 293T-hu BCMA cells. Figure 2C is a line plot showing four antibodies binding to HEK 293T-cyno BCMA cells. Figure 2D is a line plot showing two antibodies binding to HEK 293T-cyno BCMA cells.

[0016] [Figure 3] 1 is a line plot showing binding of humanized HCAbs and isotype controls (iso) to NCI-H9292 cells.

[0017] [Figure 4] 1 is a line plot showing that humanized HCAbs blocked BAFF binding to BCMA cells.

[0018] [Figure 5] Figures 5A-5C are a series of line plots showing that HCAbs have strong binding affinity. Figure 5A is a line plot showing the binding of PR000943 to hBCMA-his-biotin (Acro, BCA-H82E4). Figure 5B is a line plot showing the binding of PR001046 to hBCMA-his-biotin. Figure 5C is a line plot showing the binding of PR000274 to hBCMA-his-biotin.

[0019] [Figure 6] Figures 6A-6C are a series of line plots showing that HCAbs have strong binding affinity. Figure 6A is a line plot showing the binding of PR001035 to hBCMA-his-biotin. Figure 6B is a line plot showing the binding of PR000940 to hBCMA-his-biotin. Figure 6C is a line plot showing the binding of PR0000274 to hBCMA-his-biotin.

[0020] [Figure 7] Figures 7A-7J are a series of schematics, photographs, and line plots showing the cytotoxic properties of VH-only anti-BCMA CAR cells. Figure 7A is a schematic of the CAR transgene (i.e., nucleic acid) and isolation, transduction, and killing assay procedures. Figure 7B is a series of photographs showing VH-only anti-BCMA CAR cells proliferating and killing effector cells. Figures 7C-7E are a set of bar graphs of VH-only anti-BCMA CAR T-cell clones quantifying target cell killing. Figures 7F-7I are a set of bar graphs showing the cytokine profiles of four VH-only anti-BCMA CAR T-cell clones after co-culture with BCMA-expressing target cells. Figure 7J is a bar graph of comparative cytokine secretion of VH-only anti-BCMA CAR T-cell clones.

[0021] [Figure 8] Figures 8A-8E are a series of schematics, flow cytometry, and bar plots showing low levels of tonic signaling in VH-only anti-BCMA CAR cells. Figure 8A is a schematic showing the detection of tonic signaling. Figure 8B is a set of flow cytometry plots showing low levels of tonic signaling. Figures 8C-8E are a series of bar plots quantifying the amount of tonic signaling in VH-only anti-BCMA CAR cell clones.

[0022] [Figure 9] Figures 9A-9B are a series of schematics, bar plots, and Kaplan-Meier plots demonstrating the in vivo efficacy of VH-only anti-BCMA CAR cells. Figure 9A is a schematic showing the injected mouse tumor model and a bar plot showing total bioluminescence tumor imaging (BLI) for each treatment group after tumor injection. Figure 9B is a Kaplan-Meier plot showing mouse survival after tumor injection.

[0023] [Figure 10]Figures 10A-10D are a series of microarrays showing VH-only anti-BCMA binder specificity. Figure 10A is a cell microarray showing binding of a VH-only anti-BCMA binding test antibody to selected human proteins. Figure 10B is a cell microarray showing binding of a negative control antibody to select human proteins. Figure 10C is a cell microarray showing binding of a rituximab biosimilar to selected human proteins. Figure 10D is a cell microarray showing binding of PBS, a secondary antibody negative control, to select human proteins. DETAILED DESCRIPTION OF THE INVENTION

[0024] definition 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 to which the subject matter of this specification belongs. As used in this specification and the appended claims, unless otherwise specified, the following terms have the meanings set forth to facilitate understanding of this disclosure.

[0025] As used in this specification and the appended claims, the singular forms "a," "an," and "the" mean "one or more," and thus include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "composition" includes mixtures of two or more such compositions, reference to an "inhibitor" includes mixtures of two or more such inhibitors, etc.

[0026] Unless otherwise specified, the term "about" is understood to be within normal tolerances in the art, e.g., within two standard deviations of the mean. "About" may be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about."

[0027] The term "about," as used herein, unless otherwise stated or clear from the context (except where such number exceeds 100% of possible values), refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of the stated reference value.

[0028] The transitional phrase "comprising" is synonymous with "include," "including," "contain," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. In contrast, the transitional phrases "consist of" and "consisting of" exclude any element or method step not specified in the claim (or the particular element or method step with which the phrase "consisting of" is associated). The transitional phrase "consisting essentially of" limits the scope of a claim to the particular elements and methods or steps of the claimed disclosure as well as "unrecited elements and method steps that do not materially affect the basic and novel characteristics."

[0029] nucleic acid In one aspect, the present disclosure provides a nucleic acid encoding a chimeric antigen receptor (CAR) composed of an extracellular domain containing a first antigen recognition domain composed only of a fully human single variable heavy chain (VH) domain that binds a B-cell maturation antigen (BCMA) epitope, a transmembrane domain, and an intracellular signaling domain containing a signaling domain. Because the antigen recognition domain "consists of" VH, it does not include any VL domain, CL domain, or CH1 domain.

[0030] As known in the art, the term "nucleic acid" as used herein refers to a polymer of nucleotides, each of which is an organic molecule composed of a nucleoside (a nucleic acid base and a pentose sugar) and a phosphate. The term nucleotide, unless otherwise stated or clear from the context, includes nucleosides with a ribose sugar (i.e., ribonucleotides that form ribonucleic acids, RNA) or a 2'-deoxyribose sugar (i.e., deoxyribonucleotides that form deoxyribonucleic acids, DNA). Nucleotides serve as monomer units of nucleic acid polymers or polynucleotides. The four nucleic acid bases in DNA are guanine (G), adenine (A), cytosine (C), and thymine (T). The four nucleic acid bases in RNA are guanine (G), adenine (A), cytosine (C), and uracil (U). Nucleic acids are linear chains of nucleotides (e.g., at least three nucleotides) chemically joined by a series of ester bonds between the phosphoryl group of one nucleotide and the hydroxyl group of the sugar (i.e., ribose or 2'-deoxyribose) in the adjacent nucleotide. In this context, nucleic acids are understood to be exogenous to the cells into which they can be introduced.

[0031] Fully human heavy chain-only antibodies are known in the art. Roovers et al., Cancer Immunol. Immunother. 56(3):303-317 (2007), Tang et al., Mol. Cancer Ther. 12:416-26 (2013), Sanchez-Martin et al., Proc. Natl. Acad. Sci. USA 110(34):13791-6 (2013), Rouet et al. al., J. Biol. Chem. 290:11905-17 (2015), Ingram et al., Proc. Natl. Acad. Sci. USA 115(15):3912-3917 (2018), Zhu et al., Protein Expr. Purif. 157:57-62 (2019), and Wang et al., Antibodies (Basel) 8(1):25 (2019).

[0032] BCMA, also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17), is a cell surface receptor that recognizes B-cell activating factor (BAFF). BCMA is primarily expressed in mature B lymphocytes. BCMA has been used as a CAR target in clinical trials; however, these therapies have several drawbacks, such as short CAR cell persistence of less than 6 months (Brudno et al., J. Clin. Oncol. 36:2267-2280 (2018), Raje et al., N. Engl. J. Med. 380:1726-1737 (2019), Berdeja et al., Lancet 398:314-324 (2021)).

[0033] The VH domain derived from a heavy chain-only antibody can be subdivided into seven interconnecting regions (or motifs), four heavy chain framework regions (FWRs), and three heavy chain complementarity determining regions (CDRs). The FWRs have conserved sequences and constitute approximately 85% of the variable region of an antibody. The FWRs form a scaffold for the CDRs, maintaining the overall structure of the variable region. The CDRs have highly variable sequences and are responsible for the specific binding of the antibody to the epitope of the antigen.

[0034] The fully human VH-only BCMA recognition domain contains heavy chain framework regions FWR1, FWR2, FWR3, and FWR4, and heavy chain complementarity determining regions CDR1, CDR2, and CDR3.

[0035] In some embodiments, the antigen recognition domain has the amino acid sequence shown below (SEQ ID NO:1) (derived from the VH domain designated herein as PR000940), and includes all seven motifs (or "regions") from 5' to 3' shown in Table 1: JPEG2025537156000002.jpg17169

[0036] In some embodiments, the antigen recognition domain has the following codon-optimized nucleic acid sequence (SEQ ID NO:88), which encodes, from 5' to 3', SEQ ID NO:1: JPEG2025537156000003.jpg34169 [Table 1]

[0037] In some embodiments, the antigen recognition domain has, from 5' to 3', the amino acid sequence set forth below (SEQ ID NO:9) (derived from the VH domain designated herein as PR000943), which includes all seven motifs shown in Table 2: JPEG2025537156000005.jpg13170

[0038] In some embodiments, the antigen recognition domain has the codon-optimized nucleic acid sequence shown below (SEQ ID NO:89), which encodes SEQ ID NO:9 from 5' to 3'. JPEG2025537156000006.jpg30169 [Table 2]

[0039] In some embodiments, the antigen recognition domain has, from 5' to 3', the amino acid sequence set forth below (SEQ ID NO: 17) (derived from the VH domain designated herein as PR001035), which includes all seven motifs shown in Table 3: JPEG2025537156000008.jpg14169

[0040] In some embodiments, the antigen recognition domain has the codon-optimized nucleic acid sequence shown below (SEQ ID NO:90), which encodes, from 5' to 3', SEQ ID NO:17. JPEG2025537156000009.jpg30169 [Table 3]

[0041] In some embodiments, the antigen recognition domain has, from 5' to 3', the amino acid sequence set forth below (SEQ ID NO:25) (derived from the VH domain designated herein as PR001046), which contains all seven motifs shown in Table 4: JPEG2025537156000011.jpg17169

[0042] In some embodiments, the antigen recognition domain has the codon-optimized nucleic acid sequence shown below (SEQ ID NO:91), which encodes, from 5' to 3', SEQ ID NO:25. JPEG2025537156000012.jpg30169 [Table 4]

[0043] In some embodiments, the antigen recognition domain is a variant of (and therefore differs from) any one of the BCMA recognition domains of SEQ ID NOs: 1, 9, 17, and 25. In some embodiments, a variant of any one of SEQ ID NOs: 1, 9, 17, and 25 retains at least about 95% identity with the corresponding non-variant SEQ ID NOs: 1, 9, 17, and 25, respectively. In some embodiments, a variant of any one of SEQ ID NOs: 1, 9, 17, and 25 retains at least about 98%, e.g., 99%, identity with the corresponding non-variant SEQ ID NOs: 1, 9, 17, and 25, respectively. The term "identity" refers to the percentage of residues that are identical in two sequences when the sequences are optimally aligned. If, in optimal alignment, a position in a first sequence is occupied by the same amino acid as the corresponding position in a second sequence, the sequences exhibit identity with respect to that position. The percentage of identity determines the number of identical residues over a specified length in a given alignment. Thus, the level of identity or ("percent sequence identity") between two sequences is measured as the ratio of the number of identical positions shared by the sequences to the number of positions compared (i.e., percent sequence identity = number of identical positions / total number of positions compared) x 100). Gaps, i.e., positions in the alignment where a residue is present in one sequence but not the other, are considered positions with non-identical residues and are counted as compared positions.

[0044] Variants are well understood by those of skill in the art. In the context of the present fully human VH-only BCMA binding domains, they may contain amino acid sequence modifications that typically fall into one or more of three classes: substitutional, insertional, or deletional variants. For the purposes of this disclosure, variants are "functional" in the sense that they retain binding to BCMA.

[0045] The term "substitutional variant" refers to at least one amino acid in the native or starting sequence of a polypeptide that has been removed and a different amino acid inserted in its place. The substitutions may be single, where only one amino acid in the polypeptide molecule is substituted, or multiple, where two or more amino acids are substituted in the same polypeptide molecule.

[0046] In some embodiments, the variant contains one or more amino acid substitutions that are essentially conservative. The term "conservative substitution" refers to the replacement of at least one amino acid in the native or starting sequence of a polypeptide with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the replacement of a non-polar (hydrophobic) residue with another non-polar residue, such as isoleucine, valine, or leucine. Similarly, examples of conservative substitutions include the replacement of one polar (hydrophilic) residue with another, such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Further examples of conservative substitutions include the replacement of a basic amino acid, such as lysine, arginine, or histidine, with another, or the replacement of one acidic residue, such as aspartic acid or glutamic acid, with another. Further examples of conservative substitutions include the replacement of amino acid residues with similar chemical properties, such as between serine and threonine, where each residue has a hydroxyl group (-OH).

[0047] The term "insertion variant" refers to a variant having one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native or starting sequence. As used herein, the term "directly adjacent to" an amino acid refers to attachment of the amino acid via either the α-carboxy or α-amino functional group of the amino acid.

[0048] As used herein, the term "deletion variant" refers to a variant in which one or more amino acids in a native or starting amino acid sequence have been removed. Typically, deletion variants have one or more amino acids deleted in a particular region of the molecule.

[0049] In some embodiments, variants of antigen recognition domains having the amino acid sequence of any one of SEQ ID NOs: 1, 9, 17, and 25 may have one or more amino acid substitutions. Representative amino acid substitutions in SEQ ID NOs: 1, 9, 17, and 25 are illustrated in the alignments below, with a downward caret ("v") and bolded amino acid residues for substitution variants, and dashes for deletion or insertion variants. JPEG2025537156000014.jpg42169 JPEG2025537156000015.jpg48169

[0050] In some embodiments, the substitution mutant is SEQ ID NO:1 with a I35S substitution. In some embodiments, the substitution mutant is SEQ ID NO:9 with a S31D substitution. In some embodiments, the substitution mutant is SEQ ID NO:17 with a S75A substitution. In some embodiments, SEQ ID NO:25 has an A74S substitution. In some embodiments, the deletion mutant is SEQ ID NO:1 with a glutamic acid (E) deletion at position 53 (E53del). In some embodiments, the substitution mutant is SEQ ID NO:9 with an aspartic acid (D) deletion at position 105 (D105del). In some embodiments, the substitution mutant is SEQ ID NO:17 with an aspartic acid (D) deletion at position 105 (D105del). In some embodiments, the deletion mutant is SEQ ID NO:25 with a threonine (T) and arginine I deletion at positions 104-105 (T104_L105del). In some embodiments, the insertion variant is SEQ ID NO: 9 with a glutamic acid (E) inserted between the phenylalanine (F) at position 52 and the serine (S) at position 53 (F52_S53insE). In some embodiments, the substitution variant is SEQ ID NO: 17 with a leucine (L) and an arginine (R) inserted between the alanine (A) at position 102 and the glycine (G) at position 103 (A102_G103insLR). In some embodiments, the substitution variant is SEQ ID NO: 9 with a leucine (L) inserted between the aspartic acid (D) at position 102 and the glycine (G) at position 103. In some embodiments, the substitution variant is SEQ ID NO: 17 with an arginine (R) inserted between the alanine (A) at position 102 and the glycine (G) at position 103.

[0051] In some embodiments, the extracellular domain comprises a second antigen recognition domain that is also composed solely of (i.e., consists of) a fully human single VH domain that binds to a second BCMA epitope. The BCMA epitopes bound by the first and second antigen recognition domains can be the same epitope or different epitopes.

[0052] In some embodiments, the first and second antigen recognition domains are connected by a linker. In some embodiments, the linker has the amino acid sequence of GGGX (SEQ ID NO: 33), GGGGX (SEQ ID NO: 34), or GSSGSX (SEQ ID NO: 35), where X is either cysteine ​​(C) or serine (S). In some embodiments, the linker has a repeating sequence of SEQ ID NOs: 33-35. In some embodiments, the linker has the amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 36), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 37), KESGSVSSEQLAQFRSLD (SEQ ID NO: 38), EGKSSGSGSESKST (SEQ ID NO: 39), or GSAGSAAGSGEF (SEQ ID NO: 40).

[0053] The antigen recognition domain can be derived from a heavy chain-only fully human antibody according to known procedures. To be "fully human," the entire sequence of the antigen recognition domain (e.g., the antibody or antibody fragment from which the antigen recognition domain is derived) is derived from human variable gene segments, diversity gene segments, and linker gene segments. Such fully human antigen recognition domains derived from antibodies can be produced in transgenic non-human mammals that have endogenous antibody heavy and light chain loci knocked out or inactivated (thus rendering the mammal unable to produce endogenous antibodies) and provide one or more human heavy chain loci. Such transgenic non-human mammals with synthetic configurations of only human heavy chains can produce single-domain binding antibodies that bind to antigens without light chains. See U.S. Patent No. 10,638,735 and U.S. Patent Application Publication No. 2023 / 0322953. In some embodiments, the antigen recognition domain is derived from a heavy chain-only antibody generated from such a transgenic mouse. An antigen recognition domain derived from a heavy chain-only antibody does not include a light chain domain (ie, a VL domain or a VH domain).

[0054] The transmembrane domain of the CAR connects the extracellular domain (including the antigen recognition domain) to the intracellular signaling region. In some embodiments, the transmembrane domain is directly connected to the extracellular domain.

[0055] In some embodiments, the transmembrane domain is derived from CD3α, CD3β, CD3γ, CD3ζ, CD3ε, CD4, CD5, CD8α, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137 (4-1BB or TNF receptor superfamily member 9 (TNFRSF9)), CD154, FcεRIα, FcεRIβ, FcεRIγ, ICOS, KIR2DS2, MHC class I, MHC class II, or NKG2D. In some embodiments, the transmembrane domain is derived from CD3ζ, CD4, CD8α, CD28, or CD137, representative sequences of which are listed in Table 5. [Table 5]

[0056] The amino acid sequence of a naturally occurring transmembrane domain can be modified by amino acid substitution to prevent binding of such regions to the transmembrane domain of the same or a different surface membrane protein and minimize interaction with other members of the receptor complex. See, e.g., U.S. Patent Application Publication No. 2021 / 0101954; Soudais et al., Nat Genet 3:77-81 (1993); Muller et al., Front. Immunol. 12:639818-13 (2021); and Elazar et al., eLife 11:e75660-29 (2022).

[0057] In some embodiments, the CAR further comprises a hinge domain disposed between the antigen-recognition domain and the transmembrane domain. The hinge domain provides flexibility in that it allows the antigen-recognition domain to obtain an optimal orientation for antigen binding, which may enhance the anti-tumor activity of cells expressing the CAR.

[0058] In some embodiments, the hinge domain is derived from IgA, IgD, IgE, IgG, or IgM. In some embodiments, the hinge domain is derived from CD3ζ, CD4, CD8α, CD28, IgG1, IgG2, or IgG4, representative sequences of which are listed in Table 6. [Table 6]

[0059] The intracellular domain contains a signaling domain that enables intracellular signaling and immune cell function. The signaling domain may include a primary signaling domain and / or a costimulatory signaling domain. In some embodiments, the intracellular domain contains one or more phosphorylatable intracellular motifs (ITAMs) that can deliver an immune activation signal. In some embodiments, the intracellular domain can deliver a signal that approximates the signal of natural ligation of an ITAM-containing molecule or receptor complex, such as a TCR receptor complex.

[0060] In some embodiments, the intracellular signaling domain is selected from a plurality, e.g., two or three, of the costimulatory signaling domains described herein, e.g., 4-1BB, CD28, CD27, ICOS, and OX40. In some embodiments, the intracellular signaling domain can include a CD3ζ domain as the primary signaling domain and any of the following pairs of costimulatory signaling domains from the extracellular to intracellular direction: 4-1BB-CD27; CD27-4-1BB; 4-1BB-CD28; CD28-4-1BB; OX40-CD28; CD28-OX40; CD28-4-1BB and 4-1BB-CD28. In some embodiments, the primary signaling domain is derived from CD3ζ, CD27, CD28, CD40, KIR2DS2, MyD88, or OX40. In some embodiments, the costimulatory signaling domain is derived from one or more of CD3γ, CD3δ, CD3ε, CD3ζ, CD27, CD40, CD28, CD72, CD80, CD86, CLEC-1, 4-1BB, TYROBP (DAP12), Dectin-1, FcαRI, FcγRI, FcγRII, FcγRIII, FcεRI, IL-2RB, ICOS, KIR2DS2, MyD88, OX40, and ZAP70. Representative sequences of signaling domains are listed in Table 7. [Table 7-1] [Table 7-2]

[0061] In some embodiments, the signaling domain is derived from CD3ζ and the costimulatory domain is derived from 4-1BB. In some embodiments, the stimulatory domain is derived from CD3ζ and the costimulatory domain is derived from CD28. In some embodiments, the stimulatory domain is derived from CD3ζ and the costimulatory domain is derived from 4-1BB and CD28.

[0062] The sequences of 4-1BB and CD28 are shown in Table 7, and further isoforms of CD28 are shown in Table 8. [Table 8]

[0063] In some embodiments, the CAR further comprises a signal peptide. As used herein, the term "signal peptide" refers to a short (e.g., 5-30 or 10-100 amino acids in length) stretch of amino acids, typically at the N-terminus of a protein, that directs protein transport. The signal peptide enables association of mRNA and ribosomes with the endoplasmic reticulum, insertion of the newly translated protein into the translocon, translocation, and transport of the protein product to the plasma membrane. Signal peptides are often cleaved during post-translational modification of proteins by the cell. Signal peptides can be evaluated in silico to ensure efficient functional cleavage.

[0064] In some embodiments, the signal peptide is derived from albumin, CD8α, CD33, erythropoietin, IL-2, human or mouse Ig-kappa chain V-III (IgK VIII), tissue plasminogen activator (tPA), secreted alkaline phosphatase (SEAP), as well as synthetic sequences. Representative sequences of signal peptides are listed in Table 9. [Table 9]

[0065] In some embodiments, the sequence of the nucleic acid encoding the CAR can be codon-optimized to balance codon usage in the organism and cell type (e.g., human immune cells) in which the CAR is expressed. Efficient codon usage increases the elongation rate. In some embodiments, the nucleic acid sequence is designed to exclude stretches of 20 nucleic acids of direct repeats or inverted repeats. A direct repeat is a nucleotide sequence consisting of two or more repeats of a specific sequence, such that the repeat exists in multiple copies of a larger sequence. Generally, a direct repeat occurs when a sequence is repeated downstream in the same pattern. An inverted repeat is a single-stranded sequence of nucleotides followed downstream by its reverse complement.

[0066] In some embodiments, the sequence of the nucleic acid encoding the CAR sequence is designed to exclude putative alternative splice sites. See Wang et al., Gene 366(2):219-27 (2006), Lee et al., Annu. Rev. Biochem. 84:291-323 (2015), Baharlou et al., Sci. Rep. 8:5063-11 (2018), Jaganathan et al., Cell 176:535-548 (2019). In some embodiments, the 3' end is modified to ensure the presence of a proper transcription termination consensus sequence.

[0067] In some embodiments, the amino acid sequence of the complete human VH domain is reverse-translated to generate a non-optimized DNA sequence, which is then manually codon-optimized. A nucleic acid insert can be generated by adding a short overlapping nucleic acid sequence from the 3' insertion site of an expression vector to the 5' end of the optimized nucleic acid encoding the complete human VH domain, and adding a short overlapping nucleic acid sequence from the 5' insertion site of an expression vector to the 3' end of the optimized nucleic acid encoding the complete human VH domain. Furthermore, a restriction enzyme recognition site (e.g., a NotI site) and a start site (e.g., a consensus Kozak sequence) can be added to the 5' end of the optimized nucleic acid encoding the complete human VH domain, and a spacer (e.g., 15 nucleic acid base pairs (bp)) and a restriction enzyme recognition site (e.g., an RsrII site) can be added to the 3' end of the optimized nucleic acid encoding the complete human VH domain.

[0068] The nucleic acid insert can then be analyzed for the presence of restriction enzyme recognition sites for selected restriction enzymes (e.g., NotI and RsrII) using commercially available software (Snapgene, GSL Biotech LLC, San Diego, CA). Identified internal restriction enzyme recognition sites can be removed by generating silent mutations according to codon usage. After the nucleic acid insert no longer contains internal restriction enzyme recognition sites, hidden or alternative splice sites can be identified using bioinformatics tools, such as alternative splice site predictors (see (ASSP) Wang et al., Gene 366(2):219-227(2006)). High-scoring constitutive splice sites can be removed by generating silent mutations according to codon usage and rechecked for the presence of both internal restriction enzyme recognition sites (e.g., NotI and RsrII) and new alternative splice sites. The nucleic acid insert can then be checked for unwanted regions of homology or long stretches (less than 20 nucleic acid bp) of direct or inverted repeat sequences via DNA dot plot tools, such as YASS and IDT's E-block™ "Test Complexity" tool (see Noe et al., Nucleic Acids Research 33(2):W540-W543 (2005)). Unwanted regions of homology longer than 20 nucleic acid bp can be removed by generating silent mutations according to codon usage and rechecked for the presence of both internal restriction enzyme recognition sites and new alternative splice sites. After removal of internal restriction enzyme recognition sites, unwanted regions of homology, and high-scoring constitutive splice sites, the entire open reading frame (ORF) can be translated and queried by BLAST to ensure that all protein elements are encoded by the expected start codon and transcription termination consensus sequence (i.e., AATAAA).

[0069] Expression vector The nucleic acid encoding CAR can be introduced into immune cells by suitable expression vector.The expression vector has the elements that any expression vector can have, so as to transport the nucleic acid encoding CAR in immune cells and cause its expression.Such elements include: replication origin, polyA tail sequence, selection marker, and one or more suitable sites for inserting nucleic acid (for example, multi-cloning site (MCS)), one or more suitable promoters (each promoter is operably linked to the insertion site of nucleic acid and selection marker).

[0070] As used herein, the term "promoter" refers to a non-coding nucleic acid that directly or indirectly regulates the transcription of a corresponding nucleic acid coding sequence to which it is operably linked, which in the context of this disclosure is a CAR. A promoter may function alone to regulate transcription or may act in concert with one or more other regulatory sequences (e.g., enhancers or silencers, or regulatory elements that may be present in a gene construct or expression vector). A promoter is located near the transcription start site of a gene, on the same strand of DNA, and upstream (toward the 5' region of the sense strand). Promoters typically range in length from about 100 to 1,000 base pairs.

[0071] As used herein, the term "operably linked" should be understood to mean that the nucleic acid encoding the CAR is spatially positioned or arranged in the expression vector relative to the promoter to drive expression of the nucleic acid coding sequence encoding the CAR.

[0072] In some embodiments, the expression vector contains a strong mammalian promoter, such as the cytomegalovirus (CMV) promoter, the simian virus 40 (SV40) early promoter, or the promoter of the β-actin or factor EF1α gene. In some embodiments, the promoter may have a core region located near the transcription start site (TSS) and an enhancer, typically located further upstream of the TSS. In some embodiments, the promoter is modified. One modification involves the removal of methylation-sensitive sites (e.g., a cytosine nucleotide followed by a guanine nucleotide, or "CpG"). Another modification involves the addition of regulatory sequences that bind to DNA methylation-repressing transcription factors. In some embodiments, the expression vector contains an A / T-rich nuclear matrix-interacting sequence known as a scaffold matrix attachment region (S / MAR), which may enhance transformation efficiency and improve the stability of transgene expression.

[0073] In some embodiments, the expression vector is a viral vector, such as a retroviral vector, a lentiviral vector, an adenoviral vector, a herpesvirus vector, an adenovirus, or an adeno-associated virus (AAV) vector. As used herein, the term "lentiviral vector" is intended to mean an infectious lentiviral particle. The Lentivirinae subfamily (lentivirus) is an enveloped subfamily of the Retrovirinae subfamily (retrovirus) and can be distinguished from other viruses by virion structure, host range, and pathological effects. Infectious lentiviral particles can invade target host cells, including infecting and transducing non-dividing cells and immune cells.

[0074] In some embodiments, expression vector is non-integrating and non-replicating recombinant lentiviral vector.The construction of lentiviral vector is described in, for example, U.S. Patent No. 5,665,577, U.S. Patent No. 5,981,276, U.S. Patent No. 6,013,516, U.S. Patent No. 7,090,837, U.S. Patent No. 8,119,119 and U.S. Patent No. 10,954,530.Lentiviral vector comprises defective lentiviral genome, in which at least one of gag, pol and env of lentiviral genome is deleted or otherwise inactivated.

[0075] In other embodiments, the expression vector is a non-viral vector, representative examples of which include plasmids, mRNA, linear single-stranded (ss) DNA or linear double-stranded (ds) DNA, minicircles, and transposon-based vectors such as Sleeping Beauty (SB)-based vectors and piggyBac (PB)-based vectors. In yet other embodiments, the vector may contain both viral and non-viral elements.

[0076] In some embodiments, the expression vector is a plasmid.In addition to the promoter operably linked to the nucleic acid encoding CAR, the plasmid may also contain other elements that facilitate the transport and expression of the nucleic acid encoding CAR, for example, in immune cells.The plasmid can be linearized with a restriction enzyme, transcribed in vitro to produce mRNA, and then modified with a 5' cap and a 3' poly A tail.

[0077] In some embodiments, the carrier encapsulates the expression vector. The carrier may be lipid-based, for example, a lipid nanoparticle (LNP), liposome, lipid vesicle, or lipoplex.

[0078] In some embodiments, the carrier is LNP. In certain embodiments, the LNP comprises two or more concentric bilayers separated by aqueous compartments. The lipid bilayers can be functionalized and / or crosslinked with other lipid bilayers. The lipid bilayers can comprise one or more ligands, proteins, or channels.

[0079] Lipid carriers, e.g., LNPs, may contain one or more cationic / ionizable lipids, one or more polymer-conjugated lipids, one or more structured lipids, and / or one or more phospholipids. "Cationic" refers to a positively charged lipid or a lipid capable of retaining a positive charge. Cationic lipids contain one or more amine groups that have a positive charge depending on the pH. "Polymer-bound lipids" refer to lipids with an attached polymer moiety. Polymer-bound lipids include PEGylated lipids, which are lipids attached to polyethylene glycol. "Structured lipids" refer to non-cationic lipids that have no net charge at physiological pH. Exemplary structured lipids include cholesterol, fecosterol, sitosterol, ergosterol, campesterol, etc. "Phospholipids" refer to lipids containing a triester of glycerol with two fatty acids and one phosphate ion. The phospholipids in LNPs assemble the lipids into one or more lipid bilayers. LNPs, their preparation, formulation, and methods of delivery are disclosed, for example, in U.S. Patent Application Publication Nos. 2004 / 0142025, 2007 / 0042031, and 2020 / 0237679, and U.S. Patent Nos. 9,364,435, 9,518,272, 10,022,435, and 11,191,849.

[0080] Lipoplexes, liposomes, and lipid nanoparticles may contain lipid molecular combinations of cationic lipids, neutral lipids, anionic lipids, polypeptide-lipid conjugates, and other stabilizing components. Exemplary stabilizing components include antioxidants, surfactants, and salts. The compositions and methods of preparing lipoplexes, liposomes, and lipid nanoparticles are known in the art. See, e.g., U.S. Patent Nos. 8,058,069, 8,969,353, 9,682,139, 10,238,754, U.S. Patent Application Publication Nos. 2005 / 0064026 and 2018 / 0291086, as well as Lasic, Trends Biotechnol. 16(7):307-21 (1998), Lasic et al., FEBS Lett. 312(2-3):255-8 (1992), and Drummond et al., Pharmacol. Rev. 51(4):691-743 (1999).

[0081] cell One aspect of the present disclosure is a genetically modified immune or hematopoietic cell that expresses one or more anti-BCMA CARs.

[0082] As used herein, the term "immune cell" refers to a cell of hematopoietic origin that is functionally involved in the initiation and / or execution of innate and / or adaptive immune responses. As used herein, the term "hematopoietic cell" refers to a multipotent cell that can develop into all types of blood cells, including myeloid, lymphoid, and erythroid cells. Representative examples of immune cells include T cells, natural killer (NK) cells, macrophages, and dendritic cells. Representative examples of T cells include cytotoxic lymphocytes, T cells, cytotoxic T cells (CD8+ T cells), T helper cells (CD4+ T cells), αβ T cells and / or γδ T cells, Th17 T cells, and NK T (NKT) cells. In some embodiments, the immune cell is a CD8+ T cell. In some embodiments, the immune cell is a CD4+ T cell. In some embodiments, the immune cell is a central memory T cell. In some embodiments, the immune cell is a stem cell-like central memory T cell. In some embodiments, the immune cell is an NK cell. In some embodiments, the immune cell is a macrophage, hi some embodiments, the immune cell is a dendritic cell.

[0083] Immune cells include cells derived from stem cells. Stem cells can be adult stem cells (e.g., induced pluripotent stem cells (iPSCs)), embryonic stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells. In some embodiments, immune cells are derived from peripheral blood mononuclear cells (PBMCs), cell lines, or cell bank cells. Collection, isolation, purification, and differentiation of cells from body fluids and tissues are known in the art. For example, Brown et al., PloS One 5:e11373-9 (2010), Rivera et al., Curr. Protoc. Stem Cell Biol. 54:e117-21 (2020), Seki et al., Cell Stem Cell 7:11-4 (2010), Takahashi et al., Cell, 126:663-76 (2006), Fusaki et al., Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 85:348-62 (2009), Park et al., Nature 451:141-6 (2008), and U.S. Patent Nos. 10,214,722, 10,370,452, 10,428,309, 10,844,356, 11,141,471, 11,162,076, and 11,193,108, and U.S. Patent Application Publication Nos. 2012 / 0121544, 2018 / 0362927, 2019 / 0112577, and 2021 / 0015859.

[0084] Hematopoietic cells can be obtained from peripheral blood, bone marrow, and umbilical cord blood. Hematopoietic cells are distinguished by their ability to self-renew (i.e., generate more hematopoietic cells without differentiation) and to undergo specific and extensive differentiation into cells of various lineages. Hematopoietic cells can differentiate into one of several intermediate progenitor cells, including multipotent progenitor cells, oligopotent progenitor cells, and lineage-restricted progenitor cells (Seita and Weissman, Wiley Interdiscip. Rev. Syst. Biol. Med. 2(6):640-653 (2010)). Mature effector cells into which hematopoietic cells can differentiate include platelets, erythrocytes, granulocytes, macrophages, dendritic cells, B cells, T cells, and NK cells. Hematopoietic cells obtained from different sources typically have different differentiation potentials. For example, hematopoietic cells obtained from bone marrow can differentiate into any lineage of blood cells, whereas hematopoietic cells obtained from peripheral blood are in a state of blood myelosuppression. Therefore, hematopoietic cells in peripheral blood may not readily form myeloid cells, but instead contribute to the repair of damaged tissues (Lee and Hong, Int. J. Stem Cells 13(1):1-12 (2020)).

[0085] In some embodiments, the cells (immune cells or hematopoietic cells) are autologous to the subject receiving the cells. In some embodiments, the cells are allogeneic to the subject receiving the cells, i.e., the cells have a full or at least partial HLA match with the subject. For example, the cells, or their progenitors, can be obtained from one subject and administered to the same subject (autologous) or a differently compatible subject (allogeneic). In some embodiments, the cells contain one or more genetic modifications. In some embodiments, the cells are genetically modified by knocking out components of the T cell receptor (TCR), including one or more of T cell receptor alpha constant (TRAC), T cell receptor beta constant (TRBC)1, TRBC2, CD3γ, CD3δ, and CD3ε. In some embodiments, the cells are genetically modified by knocking out one or more of beta-2-microglobulin (B2MG), class II major histocompatibility complex transactivator (CIITA), HLA class I, and HLA class II. In some embodiments, cells are genetically modified by knocking in (i.e., adding a transgene for) an inhibitory molecule. Exemplary inhibitory molecules include major histocompatibility complex, class I, E (HLA-E) and CD47.

[0086] In some embodiments, the nucleic acid encodes more than one CAR, e.g., a first and a second CAR, where each CAR contains an antigen recognition domain composed solely of (i.e., consisting of) a fully human single VH domain that binds BCMA, but they bind to different BCMA epitopes.

[0087] Methods for introducing an expression vector containing a nucleic acid encoding a CAR into a cell are known in the art and are described in detail in Example 1. In some embodiments, the cell is transduced with a lentiviral expression vector.

[0088] In other embodiments, the method involves the use of gammaretroviral vectors.See, for example, U.S. Patent Nos. 9,669,049, 11,065,311, and 11,230,719.In some embodiments, the method involves the use of CRISPR to integrate (knock in) expression vector-delivered nucleic acids.In some embodiments, the method involves the use of adeno-associated virus (AAV), dsRNA, ssDNA, or dsRNA to deliver CRISPR and CAR-encoding nucleic acids.See, for example, U.S. Patent No. 10,563,226, and U.S. Patent Application Publication Nos. 2019 / 0225991, 2020 / 0080108, and 2022 / 0186263.

[0089] In some embodiments, the method involves ex vivo or in vivo delivery of linear, circular, or self-amplifying mRNA. See, e.g., U.S. Patent Nos. 7,442,381, 7,332,322, 9,822,378, 9,254,265, 10,532,067, and 11,291,682. In some embodiments, the method involves the use of a transposase to integrate an expression vector-delivered nucleic acid into the genome of a cell. See, e.g., U.S. Patent Nos. 7,985,739, 10,174,309, 11,186,847, and 11,351,272. In some embodiments, the method involves the use of a self-replicating episomal nanovector. See, for example, U.S. Patent Nos. 5,624,820, 5,674,703, and 9,340,775.

[0090] In some embodiments, the plasmid carrying the nucleic acid encoding CAR is transfected into cells.The nucleic acid (for example, plasmid, mRNA, linear ssDNA or dsDNA) that encodes CAR can be delivered to cells by electroporation or by being incorporated into LNP or exosome.In some embodiments, the expression vector carrying nucleic acid is delivered to cells by lipofection.Lipofection is described in, for example, United States Patent No. 5,049,386, United States Patent No. 4,946,787 and United States Patent No. 4,897,355.

[0091] Pharmaceutical Composition The pharmaceutical compositions of the present disclosure comprise a therapeutically effective number of genetically modified cells (immune or hematopoietic cells) and a pharmaceutically acceptable carrier. The compositions may be provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid carriers may also include aqueous or non-aqueous carriers. Representative examples of liquid carriers include saline, phosphate-buffered saline, soluble proteins, dimethyl sulfoxide (DMSO), polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. In some embodiments, the liquid carrier contains a protein dissolved or dispersed therein, representative examples of which include serum albumin (e.g., human serum albumin, recombinant human albumin), gelatin, and casein. The compositions are typically isotonic, i.e., have the same osmotic pressure as blood. Sodium chloride and an isotonic electrolyte solution (e.g., Plasma-Lyte®) can be used to achieve the desired isotonicity. Depending on the carrier and cells, other excipients may be added, such as wetting, dispersing or emulsifying agents, gelling and viscosity enhancing agents, preservatives, etc., as known in the art.

[0092] The pharmaceutical composition may contain more than one type of cell. Thus, a combination of at least two different genetically modified cells can be used, where each type of cell is modified with the same or different CAR-encoding nucleic acid. In some embodiments, the cells are a combination of immune cells and hematopoietic cells. In some embodiments, the cells are CD8 + T cells and CD4 + In some embodiments, the cells are a combination of T cells and NK cells.

[0093] How to use In some embodiments, the present disclosure is directed to treating cancer or autoimmune diseases associated with abnormal BCMA activity in a subject. The method involves administering to a subject in need thereof a therapeutically effective number of genetically modified cells carrying a nucleic acid encoding one of the CARs described herein. The term "aberrant," when used in the context of BCMA gene product (RNA or protein) activity, refers to decreased BCMA gene expression, increased BCMA gene expression ("overexpression"), BCMA mutation, altered cellular location, or altered tissue location compared to a normal, non-cancerous or non-disease physiological state.

[0094] As used herein, the term "subject" (or "patient") includes all members of the animal kingdom susceptible to (or prone to) or afflicted with the indicated cancer or autoimmune disease. In some embodiments, the subject is a human. Thus, a subject "having cancer," "having an autoimmune disease," or "in need of treatment" according to the present disclosure broadly encompasses positively diagnosed subjects, including subjects with active disease who have previously undergone one or more treatments, and subjects who are not currently receiving treatment (e.g., in remission) but may still be at risk of recurrence, and subjects who have not been positively diagnosed but who are predisposed to cancer or autoimmune disease (e.g., based on previous medical history and / or family history, or who have one or more risk factors that may lead a medical professional to reasonably suspect that the subject is predisposed to cancer or autoimmune disease).

[0095] As used herein, the terms "therapeutically effective number of immune cells" and "therapeutically effective number of hematopoietic cells" (each indirectly comprising a corresponding amount of a CAR) refer to a sufficient number of genetically modified cells containing a nucleic acid encoding a CAR to provide a desired effect.

[0096] The effective number of genetically modified cells for a given patient varies depending on one or more factors, which may include age, weight, type, location, severity, and overall health of the cancer or autoimmune disease. Ultimately, the attending physician will determine the appropriate dose and dosing regimen. Typically, cells are administered in a single dose. In some embodiments, the effective number of genetically modified cells is about 1 x 10 per subject. 5 ~Approx. 1×10 10 In some embodiments, the effective number of genetically modified cells is about 1 x 10 cells per kg of subject body weight. 5 ~Approx. 6×10 8 Each cell is an individual cell.

[0097] As used herein, the terms "treat," "treating," and "treatment" refer to any type of intervention, process, or administration of genetically modified cells performed on a subject in need thereof for therapeutic purposes (a "therapeutic effect") to reverse, alleviate, ameliorate, inhibit, reduce, slow, halt, stabilize, or prevent the onset, progression, occurrence, severity, or recurrence of symptoms, complications, or conditions, or biochemical manifestations associated with cancer or autoimmune diseases involving aberrant BCMA activity.

[0098] In some embodiments, the methods involve treating a subject with an autoimmune disease associated with aberrant BCMA activity. Exemplary autoimmune diseases that may be treatable according to the present disclosure include lupus, myasthenia gravis, immune thrombocytopenia (ITP), relapsed or refractory ITP, scleroderma, immune nephritis, Sjogren's syndrome, systemic lupus erythematosus (SLE), relapsed or refractory SLE, POEMS syndrome, pemphigus vulgaris, amyloidosis, autoimmune hemolytic anemia, and vasculitis.

[0099] In some embodiments, the methods involve treating a subject having a cancer associated with aberrant BCMA activity. Exemplary cancers that may be treatable according to the present disclosure include hematological cancers and carcinomas.

[0100] Representative hematological cancers that may be treatable according to the present disclosure include plasma cell neoplasms (e.g., myeloma, multiple myeloma, relapsed or refractory multiple myeloma, high-risk multiple myeloma, plasma cell myeloma, extramedullary multiple myeloma, monoclonal gammopathy of undetermined significance (MUGS), asymptomatic smoldering multiple myeloma, or solitary plasmacytoma), lymphomas (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, plasmablastic lymphoma, plasmacytoid lymphoma, or diffuse large B-cell lymphoma), leukemias (e.g., relapsed or refractory acute B-lymphocytic leukemia, or relapsed or refractory acute lymphoblastic leukemia).

[0101] Representative carcinomas that may be treatable according to the present disclosure include Waldenstrom's macroglobulinemia and glioblastoma (astrocytoma). In these embodiments, the therapeutic effect may include one or more art-recognized indicia of therapeutic efficacy, representative examples of which include prevention or prolongation of metastasis, improved survival, and overall / complete or partial remission of the cancer, e.g., no detectable cancer cells and few tumor cells or a small tumor, a reduction in tumor cell count, or a state of minimal residual disease (MRD), respectively. MRD is a condition in which a cancer patient has a small number of cancer cells remaining in the body after treatment. In some embodiments, the cancer is relapsed or refractory. A relapsed cancer is a cancer that has been treated, the treatment has been discontinued, and then the cancer recurs after a disease-free period. A refractory cancer is a cancer that has been previously treated, responded to the treatment, and then stopped responding to the treatment. Relapsed and refractory cancers may be in remission, under control, or in a state of MRD.

[0102] Administration Compositions containing a therapeutically effective number of genetically modified cells (immune or hematopoietic cells) can be administered to a subject by any medically acceptable route for the treatment of cancer or autoimmune diseases with aberrant BCMA activity. The genetically modified cells are typically delivered intravenously, but may also be introduced at other convenient sites (e.g., diseased organs or tissues) or in other ways, as determined by the attending physician. Growth and differentiation agents can be provided before, during, or after administration of the genetically modified cells (e.g., T cells and NK cells) to increase differentiation, proliferation, or persistence.

[0103] Combination therapy In some embodiments, the methods may include the co-administration of another anti-cancer agent.

[0104] The term "co-administration" includes sequential, substantially simultaneous administration, for example, as part of the same treatment regimen or by a sequential treatment regimen, in the same or separate dosage forms. Thus, when given sequentially, at the start of administration of the second therapy, the first of the two therapies may still be detectable at an effective concentration at the treatment site. The order and time intervals may be determined so that they can act together (e.g., synergistically, to provide increased benefit than if they were administered separately). For example, therapeutic agents may be administered sequentially in any order at the same time or at different times, but if not administered simultaneously, they may be administered sufficiently close in time to provide the desired therapeutic effect, which may be in a synergistic manner. Thus, this term is not limited to the administration of active agents at exactly the same time.

[0105] Anti-cancer agents that can be used in combination with the cells of the present invention are known in the art. See, for example, U.S. Patent No. 9,101,622 (Section 5.2). An "anti-cancer" agent can negatively affect cancer in a subject by, for example, killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to tumors or cancer cells, promoting an immune response to cancer cells or tumors, preventing or inhibiting the progression of cancer, or extending the lifespan of a subject with cancer. More generally, these other compositions are provided in a combined amount effective to kill or inhibit the growth of cancerous cells. This process can involve simultaneously contacting the cancer cells with the recipient cells and the agent or multiple factors. This can be achieved by contacting the cancer cells with a single composition or pharmacological formulation containing both agents, or by simultaneously contacting the cancer cells with two different compositions or formulations, where one composition contains the recipient cells and the other contains the second agent.

[0106] In some embodiments, the genetically modified cells of the present disclosure are used in combination with chemotherapy, radiation therapy, immunotherapy intervention, targeted therapy, pro-apoptotic therapy, or cell cycle modulation therapy. In some embodiments, the genetically modified cells of the present disclosure are administered after the subject has undergone lymphodepleting chemotherapy. In some embodiments, the lymphodepleting chemotherapy is melphalan. In some embodiments, the lymphodepleting chemotherapy includes one or both of fludarabine (Flu) and cyclophosphamide (Cy). In some embodiments, the subject undergoes stem cell transplantation after lymphodepleting chemotherapy.

[0107] In some embodiments, the genetically modified cells of the present disclosure are used in conjunction with an additional active agent that targets myeloma cells. In some embodiments, the additional active agent binds to CD3, CD16A, CD16B, CD38, CD44, CD138, CD229, SLAM family member 7 (SLAMF7), integrin β7 (ITGB7), natural killer group 2D (NKG2D), NK cell-activating receptor (NKp44), also known as natural cytotoxicity triggering receptor 2 (NCR2) and CD336, NKp46, also known as NCR1 and CD335, or cereblon E3 ligase. In some embodiments, the additional active agent is an anti-BCMA and anti-CD16 bispecific antibody, such as AFM26 manufactured by Affimed Therapeutics or RO7297089 manufactured by Genentech. In some embodiments, the additional active agent is a trispecific antibody, e.g., the trispecific anti-BCMA, anti-CD200, and anti-CD16 antibody aTriFlex manufactured by Affimed Therapeutics. In some embodiments, the additional active agent is the trifunctional natural killer (NK) cell engager SAR443579 manufactured by Sanofi and Innate Pharma, which targets CD123 and co-engages NKp46 and CD16A on NK cells.

[0108] In some embodiments, the additional active agent is one or more of bortezomib, carfilzomib, ixazomib, lenalidomide, pomalidomide, thalidomide, dexamethasone, prednisone, elotuzumab, daratumumab, isatuximab, and mezigdomide, ibeldomide, talquetamab, monalizumab, AMG420, and AMG701.

[0109] In some embodiments, the genetically modified cells of the present disclosure are combined with a therapeutically effective amount of an additional active agent that targets lymphoma cells. In some embodiments, the additional active agent is one or more of rituximab, mosunetuzumab, and blinatumomab.

[0110] In some embodiments, the genetically modified cells of the present disclosure are used in combination with a therapeutically effective amount of a gamma secretase inhibitor, in some embodiments, the gamma secretase inhibitor is one or more of Avagacestat, Begacestat, Crenigacestat, Iminostilbene, Itanapraced, Nirogacestat, L-685458, Semagacestat, and Tarenflurbil.

[0111] immunotherapy Immunotherapy, including immune checkpoint inhibitors, can be used to treat diagnosed cancer. Examples of immune checkpoint molecules include PD1, CTLA4, KIR, TIGIT, TIM-3, LAG-3, BTLA, VISTA, CD47, and NKG2A. Clinically available examples of immune checkpoint inhibitors include durvalumab (Imfinzi®), atezolizumab (Tecentriq®), and avelumab (Bavencio®). Clinically available examples of PD1 inhibitors include nivolumab (Opdivo®), pembrolizumab (Keytruda®), and cemiplimab (Libtayo®).

[0112] chemotherapy Anti-cancer therapy also includes a variety of combination therapies with both chemo- and radiation-based treatments, including, for example, Abraxane®, altretamine, docetaxel, Herceptin®, methotrexate, Novantrone®, Zoladex®, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosoureas, dactinomycin, daunorubicin, doxorubicin, bromide, thiazolinone ... These include leomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, Taxol®, gemcitabine, Navelbine®, farnesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate, or any analog or derivative variant of the foregoing, and combinations thereof.

[0113] Radiation therapy Anticancer therapy also includes radiation-based DNA damage therapy.Combined radiation therapy includes gamma rays, commonly known as X-rays, and / or the directed delivery of radioisotopes to tumor cells, which cause widespread damage to DNA, DNA replication and repair, and chromosome assembly and maintenance.The dose range of radioisotopes varies widely and depends on the half-life of the isotope, the strength and type of radiation emitted, and uptake by tumor cells, and is determined by the attending physician.

[0114] Radiation therapy can include external or internal radiation therapy. External radiation therapy involves a radiation source outside the subject's body that sends radiation toward the area of ​​the cancer inside the body. Internal radiation therapy uses radioactive material enclosed in needles, seeds, wires, or catheters that are placed directly into or near the cancer.

[0115] These and other aspects of the present disclosure will be further understood in light of the following examples, which are intended to illustrate particular embodiments of the present disclosure but are not intended to limit its scope, as defined by the claims. [Example]

[0116] Example 1: Materials and Methods Cell lines and donor T cells. The human MM cell line OPM2 was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DMSZ) and maintained in RPMI and 10% fetal bovine serum (FBS) (Gibco, Life Technologies, Gaithersburg, MD). NIH 3T3 fibroblasts were maintained in DMEM and 10% FBS (Gibco, Life Technologies). To generate GFP / luciferase-positive target cells or to express BCMA in NIH 3T3, cells were stably transduced with a gammaretrovirus expressing a cDNA. These target cells were then sorted into single-cell clones in 96-well plates and expanded to generate clonal populations that were confirmed by flow cytometry for use in all experiments. Human T cells were obtained from the peripheral blood of a healthy donor (MGB Crimson Core Blood Bank; study number T0761).

[0117] T cells were stimulated with CD3 / CD28 TransAct (Miltenyi, #130-111-160) at a ratio of 1:100 for 48 hours and expanded in the presence of IL-2 ± IL-7 and IL-15 (NIH BRB Preclinical Biologics Repository).

[0118] Lentiviral plasmid construction and virus production and transduction. The amino acid sequence of the BCMA binder was cloned into a modular bicistronic lentiviral CAR backbone containing 4-1BB / CD3ζ and a dsRedE2 fluorescent reporter separated by a T2A sequence after codon optimization and confirmation of predicted alternative splice sites. All generated plasmids were sequence-verified by whole-plasmid sequencing (plasmidsaurus) or Sanger sequencing (Genewiz).

[0119] To design and generate optimal anti-BCMA CAR DNA constructs from the discovered anti-BCMA single-domain binders, the amino acid sequence of the fully human anti-BCMA VH binder was first reverse-translated to generate a non-optimized DNA sequence. After reverse translation, this non-optimized DNA sequence of the fully human anti-BCMA VH binder was manually codon-optimized. A short overlap sequence for the CAR recipient expression vector containing a NotI restriction enzyme recognition site, a consensus Kozak sequence, and an Igκ signal peptide was added to the 5' end of the newly optimized fully human anti-BCMA VH binder sequence, and a short overlap region for the CAR recipient expression vector encoding a 15-bp spacer and an RsrII restriction enzyme recognition site was added to the 3' end to generate the CAR insert. The CAR insert was then analyzed for the presence of recognition sites for NotI and RsrII—selected restriction enzymes used for subcloning into the CAR recipient expression vector—using commercially available software (Snapgene, GSL Biotech LLC, San Diego, CA). Any internal restriction enzyme recognition sites were eliminated by generating silent mutations according to codon usage.

[0120] After the CAR insert contained no internal restriction enzyme recognition sites, cryptic or alternative splice sites were identified using the Alternative Splice Site Predictor (ASSP) bioinformatics tool (see Wang et al., Gene 366(2):219-227 (2006)). Any high-scoring constitutive splice sites were removed by generating silent mutations according to codon usage, and the insert was rechecked for the presence of both internal NotI and RsrII recognition sites and new alternative splice sites. The insert was then checked for unwanted homology regions or long stretches (longer than 20 bp) of direct or inverted repeat sequences via DNA dot plot tools, YASS, and IDT's E-block™ "Test Complexity" tool (see Noe et al., Nucleic Acids Research 33(2):W540-W543 (2005)). Unwanted homologous regions longer than 20 bp were removed by generating silent mutations according to codon usage, and the sequences were rechecked for the presence of both internal restriction sites and novel alternative splice sites. After removal of internal restriction sites, undesired homologous regions, and high-scoring constitutive splice sites, the entire ORF was translated and queried by BLAST to confirm that all protein elements were encoded with the expected initiation codon and transcription termination consensus sequence (i.e., AATAAA). Optimized inserts free of predicted sequence variations were synthesized as eBlock™ gene fragments by IDT (Coralville, IA) and subcloned into CAR recipient expression vectors to generate CAR plasmids.

[0121] Lentivirus was packaged and produced based on a protocol adapted from a previously described publication (see Salmon and Trono, Curr. Protoc. Hum. Genet. 54(1) Chapter 12:12-10). Briefly, 293-based packaging cells were seeded onto 150 mm tissue culture-treated plates for 24 hours at 8 x 10 6 The cells / plate were then transfected with a CAR-encoding plasmid, pMD.2G encoding the VSV-G envelope, and the packaging expression vector psPAX2 using the transfection reagent, polyethyleneimine (PEI, Polysciences, #23966) according to the manufacturer's instructions. Viral supernatants were collected at 24 and 48 hours, filtered, and concentrated by ultracentrifugation at 25,000 rpm for 2 hours at 4°C. The supernatant was discarded, and the pellet was resuspended in 100 μL of serum-free medium and shaken overnight at 4°C. Aliquots were then stored at -80°C.

[0122] Two days after their activation, T cells were spun at 2000 × g for 1 h with lentivirus (1% by volume) and Lentiboost-B (Sirion Biotech). Four days after transduction, transduction rates were determined by flow cytometry analysis of the fluorescent reporter DsRedE2 with Northern Lights (Cytek Bio). All experiments were performed using CAR + Normalized for viable cells.

[0123] Cytotoxicity. BCMA-overexpressing 3T3 cells stably transduced with ffLuc via retrovirus as described above were used as target cells. 10,000 target cells were plated in a 96-well plate containing CAR + Plated in triplicate with T cells at the indicated effector-to-target (E:T) ratios and incubated for 24 hours. ATP-dependent assays were performed, where % cytotoxicity = (BLI モック -BLI 試料 ) / BLI モック , BLI モック= average target cell alone value for that experiment. Bioluminescence was read on a BioTek Cytation 5.

[0124] Tonic signaling assay. The Jurkat T-cell leukemia Nur77-GFP reporter cell line was generated by inserting the 2A-GFP sequence in-frame with the endogenous Nur77 gene via homologous recombination. This Jurkat Nur77-GFP line was further engineered to express various anti-BCMA CAR-2A-RFP bicistronic constructs. Cells were plated alone or in a 2:1 ratio with 3T3 BCMA cells for 20 hours. Antigen-independent (tonic signaling) and antigen-dependent activation were assessed by measuring changes in GFP expression by flow cytometry. Signaling in transduced cells was monitored by GFP + RFP + Total cellular RFP + Calculated as a ratio to cells.

[0125] In vivo studies. Studies were conducted in accordance with the Dana Farber Cancer Institutional Animal Care and Use Committee approved protocol (20-010). Six-week-old NSG (NOD.Cg-Prkdc) mice were used. scid Il2rg tm1Wjl / SzJ) mice (Jackson Labs; Bar Harbor, ME) were inoculated with the myeloid-tropic cell line OPM2-ffLuc at 1 × 10 6 Cells were injected via the tail vein at a dose of 0.5 x 10 cells. Tumor engraftment was confirmed by baseline bioluminescence imaging prior to cell therapy. Two weeks after tumor engraftment, 0.5 x 10 cells were injected via the tail vein at a dose of 0.5 x 10 cells. 6A single dose of human anti-BCMA CAR T cells was injected via the tail vein. In vivo imaging was performed after injection of D-luciferin (Millipore-Sigma; Darmstadt, Germany) and analyzed with Living Image software (PerkinElmer; Waltham, MA). To reproducibly observe statistically significant differences, studies were designed with a minimum number of animals per treatment group (n = 5 per experiment). Statistical significance between Kaplan-Meier curves was determined using the log-rank (Mantel-Cox) test. Statistical significance of BLI quantification was determined using the Student's t-test.

[0126] Example 2: Generation of fully human HCAb antibodies against BCMA We used Harbour HCAb mice to produce HCAb antibodies, which produce humanized dimeric antibodies composed of two heavy chains, each with a VH, CH2, and CH3 domain.

[0127] HCAb antibodies were isolated from mice and characterized. Characterized antibodies include PR000940, PR000943, PR001035, PR001046, and the reference antibody PR000274. Humanized HCAbs are described in U.S. Patent Application Publication No. 2023 / 0322953. For reference, the positive control antibody PR0000274 is clone CA8-J6M0, described in U.S. Patent No. 9,273,141. Clone CA8-J6M0 is a clinically validated anti-BCMA antibody. The antibody sequences were determined and are listed in Tables 1-4. The newly generated HCAb antibodies bind to human BCMA cell lines with binding strength comparable to that of the reference antibody PR000274 (Figures 2A-B). Two antibodies, PR000943 and PR001046, had good cross-reactivity, as exemplified by binding to HEK 293T cells expressing cynomolgus monkey (cyno) BCMA protein (Figure 2C-D).

[0128] Furthermore, the fully human HCAb antibodies PR000940, PR001035, and PR001046 bound more strongly to NCI-H9292 cells compared to the PR000274 reference (Figure 3). As shown in Figure 4 and quantified in Table 10, the HCAb antibodies PR000940, PR000943, PR001035, and PR001046 also blocked BAFF binding to BCMA cells. [Table 10]

[0129] HCAb antibodies have strong binding affinity to the BMCA target epitope. The binding affinities of PR000943 and PR001046 compared to reference PR000274 are shown in Figures 5A-5C and quantified in Table 11. The binding affinities of HCAb antibodies PR000940, PR001035, and reference PR000274 are shown in Figures 6A-6C and quantified in Table 12. The sensor was hBCMA-his-biotin from Acro catalog number BCA-H82E4 and streptavidin (SA) loaded with 50 nM antibody with a 1:2 dilution factor. The association time in Figures 5A-5C was 600 s, and the dissociation time was 900 s, respectively. The fitted curves were for concentrations of 12.5 nM, 6.25 nM, 3.13 nM, 1.56 nM, and 0.78 nM. [Table 11] [Table 12]

[0130] Example 3: Cytotoxicity of VH-only BCMA CAR To confirm the cytotoxicity of VH-only anti-BCMA CAR cells, T cells were isolated from healthy donor PBMCs, transduced with the CAR construct, and then subjected to transduction confirmation, co-culture, and CAR T cell killing detection (Figure 7A). CAR T cells containing antigen recognition domains derived from PR000940, PR000943, PR001035, and PR001046 (abbreviated as BCMA CAR 940, BCMA CAR 943, BCMA CAR 1035, and BCMA CAR 1046, respectively) were generated. Transduction was confirmed by flow cytometry for direct BCMA CAR expression or by markers (e.g., dsRed2).

[0131] VH-only anti-BCMA CAR cells were incubated with mouse embryonic fibroblast 3T3 cell line target cells at a 1:1 effector:target (E:T) ratio in 96-well plates for 24 hours. Cytotoxicity was confirmed by clearance of GFP+ target cells. Representative images show that VH-only anti-BCMA CAR cells killed 3T3 target cells, as seen by the loss of GFP+ cells after 2 days of coculture (Figure 7B, left two images). Proliferation of VH-only anti-BCMA CAR cells was also observed (red). Cells expressing a negative control, a CAR with deleted signaling domains (abbreviated as Del CAR), did not kill 3T3 cells (Figure 7B, right two images). Del CAR cells did not proliferate.

[0132] Quantification of the percentage of target cells remaining after the killing assay is shown in Figure 7C. All four VH-only anti-BCMA CAR cell groups tested effectively killed 3T3 target cells (first four bars, from left to right). Negative control CAR-deleted cells (abbreviated as BCMA Del) did not kill target cells. Positive control or benchmark anti-BCMA cells also killed target cells. The positive control CAR cells have a BCMA-binding single-chain variable fragment antigen recognition domain, as described in Works et al., Mol. Cancer Ther. 18:2246-2257 (2019) and U.S. Patent Application Publication No. 2021 / 0324100.

[0133] BCMA VH-only CARs induce effective cytotoxicity at low E:T ratios. Quantification of the percentage of target cells remaining after the killing assay is shown in Figures 7D and 7E. The VH-only anti-BCMA CAR cell groups tested effectively killed either OPM2 (left) or MM.1S (right) target myeloma cells. An irrelevantly targeted (CD19) negative control CAR did not kill target cells. Positive control anti-BCMA CAR T cells, described in Works et al., supra, and in U.S. Patent Application Publication No. 2021 / 0324100, possess an extracellular BCMA-binding single-chain variable fragment antigen recognition domain and also killed target cells.

[0134] BCMA VH-only CARs produce cytokines when cocultured with target cells. The cytokine profiles of the four novel VH-only anti-BCMA CAR T cells are shown in Figures 7F-7I. VH-only anti-BCMA CAR T cells produced GM-CSF, IFN-γ, IL-2, IL-18, and to a lesser extent IL-5 and TNF-α after coculture with endogenously BCMA-expressing OPM2 myeloma cells at a 1:1 E:T ratio (Figures 7F-7I). Figure 7J shows the comparative cytokine secretion of the four novel BCMA CAR T cells cocultured with the endogenously BCMA-expressing OPM2 myeloma cell line at a 1:1 E:T ratio. Control CAR T cells expressing a CAR containing the antigen recognition domain derived from the reference antibody PR00274 produced cytokines at levels comparable to the VH-only anti-BCMA CAR T cells.

[0135] Example 4: Tonic Signaling Some VH-only anti-BCMA CAR cells exhibited minimal persistent (antigen-independent) signaling while maintaining the ability to induce antigen-specific signaling. We generated a tonic signaling reporter cell line that can identify CAR cells with high antigen-independent signaling. Most studies have demonstrated that antigen-independent (persistent) signaling contributes to activation-induced cell death (AICD) or premature dysfunction, potentially limiting the in vivo or clinical efficacy of CAR candidates. A reporter cell line for CAR signaling was developed by knocking in GFP in-frame with the nuclear receptor subfamily 4, group A, member 1 (NR4A1) gene, also known as nuclear hormone receptor 77 (Nur77). NR4A1 is transcribed early downstream of CD3ζ signaling. In this reporter line, RFP signal indicates successful CAR transduction into cells, and GFP signal indicates downstream CAR signaling (Figure 8A). GFP is co-transcribed with NF4A1, an early indicator of CD3ζ signaling. The RFP+GFP+ double-positive population indicates CAR-induced signaling, reflecting tonic signaling in the absence of target cells. Representative flow cytometry plots (Figure 8B, left) show minimal tonic signaling in the effector-only group, with only 3.93% of cells RFP+GFP+ double-positive. When VH-only anti-BCMA CAR effector cells were cocultured with target cells, robust antigen-dependent signaling was observed, with 46.6% of cells expressing both RFP+ and GFP+ (Figure 8B, center). CD3 / CD28 beads represent a positive control for T cell activation, with 89.4% of cells expressing both RFP+ and GFP+ (Figure 8B, right).

[0136] We investigated the sustained antigen-independent signaling of four VH-only anti-BCMA CAR cells. Only one, PR000943 VH-only anti-BCMA CAR cell, exhibited RFP+GFP+ tonic signaling cells when incubated with effector cells alone (Figure 8C). VH-only anti-BCMA CAR cells were confirmed to be functional when incubated with target cells at a 2:1 effector:target ratio (Figure 8D) and when incubated with CD3 / CD28 beads (TransAct) (Figure 8E). PR001046 VH-only anti-BCMA CAR cells exhibited the highest antigen-specific signaling after co-culture with target cells (Figure 8D).

[0137] Example 5: VH-only anti-BCMA CAR cells in myeloid MM xenografts Four VH-only anti-BCMA CARs, each incorporating a different VH-only clone (940, 943, 1035, and 1046), were investigated for tumor control in a myeloid-tropic MM xenograft mouse model. Mice were injected as shown in Figure 9A. All four VH-only anti-BCMA CARs were shown to be highly active compared to a negative control CAR (41BB / CD3z signaling-deficient (BCMA Del)), quantified as bioluminescence tumor imaging mean (Figure 9A). Figure 9B shows Kaplan-Meier curves of mouse survival. The VH-only anti-BCMA clone 1046 CAR had the highest antigen-specific activation and the most rapid (day 20) disease control (Figure 9A). Similarly, mice treated with VH-only anti-BCMA clone 1046 CAR T cells also had the greatest percentage of durable rejection when compared to other VH-only CAR T cells in the positive rejection control CAR T cells (Figure 9B). Thus, although CAR T cells containing the VH-only anti-BCMA clone 1046 had the highest in vivo survival (Figure 9B), they also had the lowest cytokine release (Figure 7F), an attribute that may uniquely result in a high therapeutic window.

[0138] Example 6: On-target VH-only anti-BCMA CAR specificity To screen the on-target specificity of one of the VH-only anti-BCMA-targeting CARs, we used the Retrogenix Cell Microarray Technology platform, a high-throughput platform for screening binding interactions between test molecules and approximately 6,100 human surfacesome proteins. We used a CAR VH-only binder reformatted as a VH-only human IgG1, known as PR001046. Preliminary analysis using untransfected HEK293 cells and BCMA-overexpressing HEK293 cells indicated that 2.5 μg / mL of the PR001046 VH-only human IgG1 test antibody was an appropriate concentration for subsequent full library screening. The AlexaFluor 647 anti-hIgGFc detection antibody was used as the secondary antibody.

[0139] In a library screen, the CAR-derived PR001046 VH-only human IgG1 test antibody was screened for binding to fixed human HEK293 cells individually expressing 6105 full-length human plasma membrane (PM), membrane-embedded (M), secreted (S), and cell surface-tethered secreted (TS) proteins plus an additional 400 human heterodimers (HD). The screen identified 21 library interactions, primarily strong interactions with BCMA (also known as TNF receptor superfamily member 17 (TNFRSF17)) and weak interactions with members of the Fc gamma receptor (FcγR) family (Table 13). Each library interaction was re-expressed with two control receptors and retested with the CAR-derived PR001046 VH-only human IgG1 test antibody and controls (i.e., a rituximab biosimilar, a clinical anti-CD20 IgG1 positive control benchmark known to be specific, and a second format-specific VH-only binder heavy chain antibody (hcAb) control binding to an unrelated target antigen). This was performed on both fixed and live cells (Figures 11A-11D). The results are summarized in Table 13. The PR001046 VH-only human IgG1 test antibody demonstrated significant specific interactions with its primary target, BCMA (TNFRSF17), on both fixed and live cell microarrays. No other interactions were specifically identified for the CAR-derived PR001046 VH-only human IgG1 test antibody (i.e., none with rituximab or the format-specific controls), thus demonstrating the high specificity of PR001046 for its primary target, BCMA (TNFRSF17). [Table 13-1] [Table 13-2] [Table 13-3] [Table 13-4]

[0140] All patent publications and non-patent literature are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All these publications, including any specific portions thereof referenced, are herein incorporated by reference to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference. Although the disclosure herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. Accordingly, it is to be understood that numerous modifications may be made to the exemplary embodiments and that other arrangements may be devised without departing from the spirit and scope of the present disclosure, as defined by the appended claims.

Claims

1. A nucleic acid comprising a sequence encoding a first chimeric antigen receptor (CAR), said first CAR comprising: an extracellular domain comprising a first antigen recognition domain consisting of a fully human single variable heavy (VH) domain that binds to a first epitope on B-cell maturation antigen (BCMA); a transmembrane domain; and Intracellular domain containing signaling domain A nucleic acid comprising:

2. 2. The nucleic acid of claim 1, wherein the extracellular domain further comprises a second antigen recognition domain consisting of a fully human single variable heavy (VH) domain that binds to a second epitope on BCMA, and the first BCMA epitope and the second BCMA epitope are different.

3. The nucleic acid of claim 2, wherein the first or second antigen recognition domain has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17 or SEQ ID NO:

25.

4. 3. The nucleic acid of claim 2, wherein the first or second antigen recognition domain comprises a variant of any of SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17, or SEQ ID NO: 25 that binds to BCMA.

5. The nucleic acid of claim 4, wherein the variant of the first or second antigen recognition domain has an amino acid sequence having at least about 95% identity to any one of SEQ ID NOs: 1, 9, 17, or 25.

6. The nucleic acid of claim 4, wherein the variant of the first or second antigen recognition domain has an amino acid sequence having at least about 98% identity to any one of SEQ ID NOs: 1, 9, 17, or 25.

7. The nucleic acid of claim 2 , wherein the first and second antigen recognition domains are connected by a linker.

8. 8. The nucleic acid of claim 7, wherein the linker comprises the amino acid GGGX (SEQ ID NO: 33), GGGGX (SEQ ID NO: 34), or GSSGSX (SEQ ID NO: 35), where X is either C or S.

9. The nucleic acid of claim 7, wherein the linker has the amino acids GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 36), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 37), KESGSVSSEQLAQFRSLD (SEQ ID NO: 38), EGKSSGSGSESKST (SEQ ID NO: 39), or GSAGSAAGSGEF (SEQ ID NO: 40).

10. The nucleic acid of claim 1, wherein the transmembrane domain is derived from CD3, CD8α, CD28, or CD137.

11. The nucleic acid of claim 1 , wherein the extracellular domain further comprises a hinge domain disposed between the antigen recognition domain and the transmembrane domain.

12. The nucleic acid of claim 11 , wherein the hinge domain is derived from IgG1 or IgG4.

13. 2. The nucleic acid of claim 1, wherein the signaling domain comprises a primary signaling domain, a costimulatory signaling domain, or both a primary signaling domain and a costimulatory signaling domain.

14. The nucleic acid of claim 13, wherein the intracellular domain comprises a CD3ζ primary signaling domain and a 4-1BB costimulatory signaling domain, a CD3ζ primary signaling domain and a CD28 costimulatory signaling domain, or both a CD3ζ primary signaling domain and a 4-1BB costimulatory signaling domain and a CD28 costimulatory signaling domain.

15. The nucleic acid of claim 13, wherein the intracellular domain comprises a CD3ζ primary signaling domain and a CD28 costimulatory signaling domain.

16. The nucleic acid of claim 13, wherein the intracellular domain comprises a CD3ζ primary signaling domain and a 4-1BB costimulatory signaling domain.

17. 14. The nucleic acid of claim 13, wherein the intracellular domain comprises a CD3ζ primary signaling domain and a 4-1BB and CD28 costimulatory signaling domain.

18. The nucleic acid of claim 1, wherein the first CAR further comprises a signal peptide.

19. The nucleic acid of claim 18, wherein the signal peptide is derived from albumin, CD8α, EPO, IgGκ, or IL-2.

20. The nucleic acid of claim 1, which is codon-optimized for expression in human immune cells.

21. 2. The nucleic acid of claim 1, wherein the nucleic acid does not contain stretches of direct or inverted repeat sequences of 20 base pairs or more.

22. The nucleic acid of claim 1 , wherein the nucleic acid does not contain alternative splice sites.

23. An expression vector encoding the nucleic acid sequence of claim 1.

24. The expression vector of claim 23, which is a viral vector.

25. The expression vector of claim 23, which is a non-viral vector.

26. 26. The expression vector of claim 25, wherein the non-viral vector is a plasmid.

27. 24. The expression vector of claim 23 , encapsulated in a lipid-based carrier.

28. A method for producing genetically modified immune cells or genetically modified hematopoietic cells, A method comprising introducing the expression vector of claim 23 into an immune cell or a hematopoietic cell.

29. An immune cell or hematopoietic cell comprising the nucleic acid of claim 1.

30. 30. The cell of claim 29, wherein the immune cell is a T cell.

31. The cell of claim 30, wherein the immune cell is a CD8+ T cell.

32. The cell of claim 30, wherein the immune cell is a central memory T cell or a stem cell-like central memory T cell.

33. 30. The cell of claim 29, wherein the immune cell is a NK cell.

34. 30. The cell of claim 29, wherein the immune cell is a macrophage or a dendritic cell.

35. 30. The cell of claim 29, wherein the immune cell or the hematopoietic cell is derived from a hematopoietic stem cell.

36. 30. The cell of claim 29, wherein the immune cell or the hematopoietic cell is derived from an induced pluripotent stem cell.

37. 30. The cell of claim 29, further comprising a second CAR, wherein the second CAR has an extracellular domain comprising an antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, wherein the antigen recognition domain consists of a fully human single VH domain that binds to a third BCMA epitope, and wherein the first and third BCMA epitopes are different.

38. 30. A pharmaceutical composition comprising a therapeutically effective number of the cells of claim 29.

39. 1. A method of treating cancer or an autoimmune disease associated with aberrant BCMA activity, comprising:

40. A method comprising administering the pharmaceutical composition of claim 38 to a subject in need thereof.

40. 40. The method of claim 39, wherein the cells are allogeneic and have a full or partial HLA match with the subject.

41. 40. The method of claim 39, wherein the cells are autologous.

42. 40. The method of claim 39, wherein the cancer is a hematological cancer.

43. 43. The method of claim 42, wherein the hematological cancer is multiple myeloma.

44. 44. The method of claim 43, further comprising administering to the subject a therapeutically effective amount of an additional active agent that binds to a surface antigen on a myeloma cell.

45. 45. The method of claim 44, wherein the additional active agent binds to CD3, CD16, CD38, CD44, CD138, CD229, SLAMF7, integrin β7 (ITGB7), natural killer group 2D (NKG2D), NKp44, NKp46, or cereblon E3 ligase.

46. 45. The method of claim 44, wherein the additional active agent is one or more of bortezomib, carfilzomib, ixazomib, lenalidomide, pomalidomide, thalidomide, dexamethasone, prednisone, elotuzumab, daratumumab, isatuximab, and mezigdomide, ibeldomide, talquetamab, monalizumab, AMG 420, and AMG 701.

47. 43. The method of claim 42, wherein the cancer is lymphoma.

48. 48. The method of claim 47, further comprising administering to the subject a therapeutically effective amount of an additional active agent that binds to a surface antigen on a lymphoma cell.

49. 49. The method of claim 48, wherein the additional active agent is one or more of rituximab, mosunetuzumab, and blinatumomab.

50. 40. The method of claim 39, wherein the autoimmune disease comprises lupus, systemic lupus erythematosus (SLE), myasthenia gravis, immune thrombocytopenia (ITP), scleroderma, immune nephritis, Sjogren's syndrome, POEMS syndrome, pemphigus vulgaris, amyloidosis, autoimmune hemolytic anemia, or vasculitis.

51. 40. The method of claim 39, further comprising administering to the subject a therapeutically effective amount of a gamma secretase inhibitor.

52. 52. The method of claim 51 , wherein the gamma secretase inhibitor comprises one or more of Avagacestat, Begacestat, Crenigacestat, Iminostilbene, Itanapraced, Nirogacestat, L-685458, Semagacestat, and Tarenflurbil.

53. 40. The method of claim 39, wherein the cancer is recurrent or refractory.

54. 40. The method of claim 39, further comprising administering lymphocyte-depleting chemotherapy to the subject prior to administration of the cells.

55. 55. The method of claim 54, wherein the lymphocyte-depleting chemotherapy comprises administering a therapeutically effective amount of melphalan.

56. 55. The method of claim 54, further comprising administering a stem cell transplant to the subject after the lymphodepleting chemotherapy.

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