Chimeric antigen receptor and antibody-nkg2d ligand domain fusion protein

Abstract

The disclosure provides a chimeric antigen receptor comprising a mutated NKG2D ectodomain, an lgG4 hinge domain, a transmembrane domain, an intracellular signaling domain, and a costimulatory domain. The disclosure further provides an antibody fusion protein comprising antibody heavy chains and antibody light chains, wherein the heavy chains or the light chains are fused at their N-terminus via an amino acid linker to an A1-A2 domain comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 11. The disclosure further provides a kit comprising one or more containers comprising the antibody fusion protein and / or comprising one or more containers comprising an immune cell, as well as methods of use, also are provided.

Description

CHIMERIC ANTIGEN RECEPTOR AND ANTIBODY-NKG2D LIGAND DOMAIN FUSION PROTEINFIELD OF THE INVENTION

[0001] The disclosure relates to a chimeric antigen receptor comprising a mutated NKG2D ectodomain, an antibody fusion protein comprising an A1-A2 domain of a non-natural NKG2D ligand, and related methods.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 483,747, filed February 7, 2023, the contents of which are hereby incorporated by reference.INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0003] Incorporated by reference in its entirety is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: 58808_SeqListing.xml; Size: 55,589 bytes; Created: January 18, 2024.BACKGROUND

[0004] The engineering of patient-derived T cells to express chimeric antigen receptors (CARs) has altered the landscape of adoptive cell therapies, providing scientists and clinicians the ability to harness the powerful cytolytic capabilities of T cells and direct them to specific antigen-expressing targets in an MHC-independent manner. Their initial application to treat hematologic malignancies has led to astounding responses and inspired a surge of research efforts to drive their effective use in non-hematologic indications. However, CAR-T cell therapies are limited by their utilization of a single-purpose targeting domain, lack of dose control which can contribute to cytokine release syndrome, inability to address tumor antigen loss leading to disease relapse, and immunogenicity of non-human targeting domains leading to lack of persistence. There is a need in the art for improved CAR-based cell therapies to address these limitations of current therapy options.SUMMARY

[0005] This summary is intended merely to introduce a simplified summary of some aspects of one or more implementations of the present disclosure. Further areas of applicability of the present disclosure will become apparent from the detailed description provided hereinafter. This summary is not an extensive overview, nor is it intended to identify key or critical elements of the present teachings, nor to delineate the scope of the disclosure. Rather, its purpose is merely to present one or more concepts in simplified form as a prelude to the detailed description below.

[0006] The disclosure provides a chimeric antigen receptor comprising a mutated NKG2D ectodomain, an lgG4 hinge domain, a transmembrane domain, an intracellular signaling domain, and a costimulatory domain. In various aspects, the mutated NKG2D ectodomain comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof, the variant comprising a substitution at amino acid position 118 within SEQ ID NO: 1. Optionally, the lgG4 hinge domain comprises an amino acid sequence comprising at least 85% identity (e.g., 100% identity)to SEQ ID NO: 2; the transmembrane domain comprises an amino acid sequence comprising at least 85% identity (e.g., 100% identity) to SEQ ID NO: 3; the intracellular signaling domain comprises an amino acid sequence comprising at least 85% identity (e.g., 100% identity) to SEQ ID NO: 5; and / or the costimulatory domain comprises an amino acid sequence comprising at least 85% identity (e.g., 100% identity) to SEQ ID NO: 4. A nucleic acid molecule comprising a nucleic acid sequence encoding the chimeric antigen receptor is provided, as well as an expression vector comprising the nucleic acid molecule and a cell comprising the expression vector.

[0007] The disclosure further provides an antibody fusion protein comprising antibody heavy chains and antibody light chains, wherein the heavy chains or the light chains are fused at their N-terminus via an amino acid linker to an A1-A2 domain comprising an amino acid sequence having at least 90% identity (e.g., 100% identity) to SEQ ID NO: 11. Optionally, the A1-A2 domain comprises the amino acid sequence of SEQ ID NO: 11 comprising an alanine or glutamine at one or more of positions 40, 54, and / or 84. In various aspects, the linker is 2-8 amino acids in length, 9-17 amino acids in length, or 18-30 amino acids in length. For example, in some aspects, each heavy chain is fused at the N-terminus to an A1-A2 domain via the amino acid linker, and the linker is optionally 9-17 amino acids in length or 18-30 amino acids in length. In alternative aspects, each light chain is fused at the N-terminus to an A1-A2 domain via the amino acid linker, and the linker is optionally 2-8 amino acids in length or 9-17 amino acids in length. A nucleic acid molecule comprising a nucleic acid sequence encoding the heavy and / or light chain is provided, as well as an expression vector comprising the nucleic acid molecule and a cell comprising the expression vector.

[0008] In addition, the disclosure provides a kit comprising one or more containers comprising the antibody fusion protein described herein and instructions for use. The kit may further comprise one or more containers comprising the immune cell described herein. A kit comprising one or more containers comprising the immune cell and instructions for use also is disclosed herein.

[0009] The disclosure further provides a method of treating a subject suffering from cancer, the method comprising administering to the subject (i) the antibody fusion protein described herein and (ii) an immune cell described herein, wherein the antibody fusion protein comprises an antibody which binds a cell surface antigen displayed on cancer cells. In various aspects, the cancer is a CD 19-positive cancer, and the antibody fusion protein comprises an anti-CD19 antibody.

[0010] It should be understood that, while various embodiments in the specification are presented using "comprising" language, under various circumstances, a related embodiment may also be described using "consisting of' or "consisting essentially of" language. The disclosure contemplates embodiments described as "comprising" a feature to include embodiments which "consist of" or "consist essentially of" the feature. The term "a" or "an" refers to one or more. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. The term "or" should be understood to encompass items in the alternative or together, unless context unambiguously requires otherwise.

[0011] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range and each endpoint, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein. However, the description also contemplates the same ranges in which the lower and / or the higher endpoint is excluded. When the term "about" is used, it means the recited number plus or minus 5%, 10%, or more of that recited number. The actual variation intended is determinable from the context.

[0012] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as”) provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. Only such limitations which are described herein as critical to the invention should be viewed as such; variations of the invention lacking limitations which have not been described herein as critical are intended as aspects of the invention.

[0013] Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, including the figures and detailed description, and all such features are intended as aspects of the invention. Likewise, features of the invention described herein can be re-combined into additional embodiments that also are intended as aspects of the invention, irrespective of whether the combination of features is specified as an aspect or embodiment of the invention. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein (even if described in separate sections) are contemplated, even if the combination of features is not found together in the same sentence, or paragraph, or section of this document.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a schematic of various formats of antibody fusion proteins comprising an A1-A2 domain fused to the N-terminus or C-terminus of antibody heavy chains or antibody light chains via an amino acid linker.

[0015] Figure 2A is a bar graph illustrating percent cytolysis (y-axis) of Ramos cells mediated by (I) anti-CD 19 antibody fusion proteins comprising A1-A2 domains fused at different locations on the fusion proteins and (ii) T cells displaying chimeric antigen receptors (CARs) comprising a modified NKG2D ectodomain, a CD8 hinge or an lgG4 hinge, a CD3zeta intracellular signaling domain, and a 4-1 BB costimulatory domain. The level of cytolysis mediated by the LC.S3 and HC.S3 antibody fusion protein formats, which comprise the A1-A2 domain fused to the C-terminus of the heavy or light chains, was inferior compared to formats comprising the A1-A2 domain fused to the N-termini. FMC63-S3med.LC corresponds to an N-terminal fusion to the antibody light chain with a "medium” linker comprising 11 amino acids in length. The experiment was performed using T cells comprising a CD8a-hinge (45 amino acid)-containing CAR as well as T cells comprising an lgG4-hinge (12 amino acid)-containing CAR. The N-terminally fused A1-A2 domain enhanced cytotoxicity with both the CD8-hinge and the lgG4-hinge CARs. The combination of the N-terminal fusion of the A1-A2 domain and the lgG4-hinge in the CAR construct produced superior results.

[0016] Figure 2B is a bar graph illustrating percent cytolysis (y-axis) of Ramos cells mediated by (I) anti-CD 19 antibody fusion proteins comprising A1-A2 domains fused at different locations on the fusion proteins and (ii) T cells displaying CARs comprising an NKG2D ectodomain, a CD8 hinge or an lgG4 hinge, a CD3zeta intracellular signaling domain, and a 4-1 BB costimulatory domain. Different linker lengths were used to fuse the A1-A2 domain to the N-terminus of the heavy or light chains of the anti-CD 19 antibody. S3short.LC corresponds to an N-terminal fusion to the antibody light chain with a "short” linker comprising five amino acids in length; S3med.LC corresponds to an N-terminal fusion to the antibody light chain with a "medium” linker comprising 11 amino acids in length; S3long.LC corresponds to an N-terminal fusion to the antibody light chain with a "long” linker comprising 18 amino acids in length; S3short.HC corresponds to an N-terminal fusion to the antibody heavy chain with a "short” linker comprising five amino acids in length; S3med.HC corresponds to an N-terminal fusion to the antibody heavy chain with a "medium” linker comprising 11 amino acids in length; and S3long.HC corresponds to an N-terminal fusion to the antibody heavy chain with a "long” linker comprising 18 amino acids in length. LC.S3 corresponds to a fusion comprising an A1-A2 domain fused to the C-termini of the light chains of the anti-CD9 antibody. The study was performed with T cells displaying a CD8a-hinge (45 amino acid) CAR as well as T cells displaying an lgG4-hinge (12 amino acid) CAR. All N-terminally fused HC molecules demonstrated cell killing activity. The constructs wherein the A1-A2 domain was N-terminally fused to light chains mediated more robust cytotoxicity, with formats comprising the "short” and "medium” linkers exhibiting superior activity.

[0017] Figure 3 is a median fluorescence intensity (MFI) curve generated by incubating Ramos cells overnight with various concentrations (x-axis, nM) of anti-CD 19 antibody fusion proteins comprising A1-A2 domains fused at different locations on the fusion proteins via linkers of various lengths. The graph demonstrates that the antibody fusion proteins retain the ability to bind target epitopes without significant loss of binding affinity.

[0018] Figure 4A illustrates the results of an in vivo study wherein 5 x 105Raji.luc cells were injected into mice, and the mice later received anti-CD 19 antibody fusion proteins comprising A1-A2 domains fused at different locations on the fusion proteins. The mice were subsequently administered 5 x 106INKG2D CAR-T cells comprising a CD8a hinge. Total flux (p / s; y-axis) represents luminescence output, which corresponds to tumor burden. For comparison purposes, a cohort of subjects was administered 5 x 106FMC63scFv CAR-T cells without the antibody fusion protein. Treatment with the antibody fusion protein proteins and INKG2D CAR-T cells comprising the CD8a hinge did not significantly control tumor burden; in contrast, the FMC63scFv CAR-T cell treatment controlled tumor growth.

[0019] Figure 4B illustrates the results of an in vivo study wherein 5 x 105Raji.luc cells were injected into mice, and the mice later received an anti-CD 19 antibody fusion protein comprising A1-A2 domains fused at the N-terminus of the light chains via a "medium” linker 11 amino acids in length and 3 x 106INKG2D CAR-T cells comprising an lgG4 hinge. For comparison purposes, a cohort of subjects was administered 3 x 106FMC63scFv CAR-T cells without the antibody fusion protein. The N-terminally fused format with the "medium” linkerdemonstrated a significant improvement in tumor control when administered with a CAR-T-cell comprising the lgG4 hinge. FMC63scFv CAR-T cell treatment also controlled tumor growth.

[0020] Figure 5 is a chart providing various sequences described herein. With respect to SEQ ID NOs: 1-10, 18, and 19, italicized portions of the sequence correspond to the A1-A2 domain, bolded and underlined portions of the sequence correspond to hinge sequence, bolded and italicized portions correspond to transmembrane domain sequences, italicized and underlined portions correspond to costimulatory domain sequences, and bold portions correspond to intracellular domain regions. Plain underlined regions correspond to signal sequences. SEQ ID NOs: 18 and 19 correspond to CAR constructs further comprising a glycine-serine linker (double underline) and the amino acid sequence of green fluorescence protein. In SEQ ID NOs: 21-44, dashed underlining denotes variable region sequences, dark (bold) underlining denotes constant region sequences, bold text with wavy underlining denotes linker sequences, and plain text without underlining denotes A1-A2 domain sequences. The disclosure contemplates the various domains as well as the full length construct as aspects of the disclosure.DETAILED DESCRIPTION

[0021] The following description of preferred aspects(s) of the disclosure is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses.

[0022] The instant disclosure provides a chimeric antigen receptor (CAR) comprising a mutated, non-natural NKG2D ectodomain, an I gG4 hinge domain, a transmembrane domain, an intracellular signaling domain, and a costimulatory domain, as well as immune cells which display the CAR on the cell surface. The disclosure further provides a fusion protein comprising an antibody (or other antigen binding protein) and the A1-A2 domain of a non-natural NKG2D ligand. The non-natural NKG2D ligand selectively binds a non-natural NKG2D receptor. In various aspects of the disclosure, the fusion protein is used in connection with the immune cell displaying the CAR comprising the non-natural NKG2D ectodomain to which the A1-A2 domain binds, thereby providing a powerful system for delivering a tailored CAR-T cell therapy which overcomes many of the disadvantages of current CAR-T cell-based therapeutics. The chimeric antigen receptor, antibody fusion protein, and system of the disclosure provides unexpectedly superior in vivo anti-tumor activity.

[0023] NKG2D is an activating receptor expressed as a type II homodimeric integral membrane protein on Natural Killer (NK) cells, some myeloid cells, and certain T cells. Human NKG2D has eight distinct natural MIC ligands (MICA, MICB, and ULBP1 through ULBP6) that are upregulated on the surface of cells in response to a variety of stresses and their differential regulation provides the immune system a means of responding to a broad range of emergency cues with minimal collateral damage. Groh et al., Proc. Natl. Acad. Sci. U.S.A. (1996) 93:12445-12450; Zwirner et al., Hum. Immunol. (1999) 60:323-330; and Spies et al., Nat. Immunol. (2008) 9: 1013-1015. The structure of the NKG2D ectodomain, several soluble ligands, and the bound complex of ligands to the ectodomain have been solved, revealing a saddle-like groove in the homodimer interface which engages the structurally conserved A1-A2 domains of the ligands that are otherwise of disparate amino acid identity. Li etal., Nat. Immunol. (2001) 2:443-451; Radaev et al., Immunity (2001) 15:1039-1049; and McFarland et al., Immunity (2003) 19:803-812. See also Culpepper et al., Mol. Immunol. (2011) 48: 516-523.

[0024] "Chimeric antigen receptor" or "CAR" refers to an artificial immune cell receptor that is engineered to recognize and bind to an antigen expressed by a target cell, such as a tumor cell. Historically, CARs have been designed for a T cell and are chimeras of a signaling domain of the T cell receptor (TCR) complex and an antigen-recognizing domain (e.g., a single chain fragment (scFv) of an antibody or other antibody fragment). See, e.g., Enblad et al., Human Gene Therapy (2015) 26(8):498-505. The chimeric antigen receptor of the disclosure comprises, as the "antigen binding domain” of the CAR, a mutated NKG2D ectodomain that is incapable of engaging natural ligands. The mutated NKG2D is also referred to herein as "iNKG2D.” Because the CAR is inert, the CAR can only form a productive immunologic synapse with a target cell displaying the antigen and activate cytolysis when it is "armed” with its cognate antibody fusion protein noncovalently bound to its receptor. In various aspects, the iNKG2D ectodomain comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof. In this regard, the mutated NKG2D ectodomain optionally comprises an amino acid sequence comprising at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In various aspects, the mutated NKG2D ectodomain comprises a substitution at amino acid position 118 within SEQ ID NO: 1. For instance, the iNKG2D ectodomain optionally comprises an F at position 118 in place of the Y in SEQ ID NO: 1 . The mutated NKG2D ectodomain, in various aspects, comprises the amino acid sequence of SEQ ID NO: 1. Alternatively, the mutated NKG2D ectodomain comprises the amino acid sequence of SEQ ID NO: 10.

[0025] The ectodomain is associated with an lgG4 hinge, a transmembrane domain and an intracellular signaling domain. The CAR also preferably comprises a costimulatory domain. The hinge region of the CAR is an amino acid domain located between the antigen binding domain and the transmembrane domain. In various aspects, the lgG4 hinge domain comprises an amino acid sequence comprising at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 2. Optionally, the lgG4 hinge comprises the amino acid sequence of SEQ ID NO: 2.

[0026] The CARs disclosed herein comprise a transmembrane domain, an amino acid domain that connects the antigen binding domain to the intracellular signaling domain. The transmembrane domain is capable of spanning the plasma membrane of a cell (e.g., an immune cell). Examples of transmembrane domains include, but are not limited to, domains derived from CD28, CD3-epsilon, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, TCR-alpha, TCR-beta, TCR-zeta, H2-Kb, GITR, or CD3- zeta. In various aspects, the transmembrane domain is selected from CD3-zeta, CD28, or CD8 transmembrane domains. In a preferred aspect, the transmembrane domain is a CD8a transmembrane domain (e.g., an amino acid sequence of SEQ ID NO: 3). In various aspects, the transmembrane domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 3.

[0027] The intracellular signaling domain of a CAR produces stimulatory signals for proliferation and effector function when the CAR engages with a target antigen. Many intracellular signaling domains contain signaling motifs are known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of ITAM containing cytoplasmic signaling sequences include those derived from CD8, CD3 zeta, CD3 gamma, CD3 epsilon, CD32 (Fc gamma Rlla), DAP10, DAP12, CD79a, CD79b, FcyRly, FcyRllly, FcsRip (FCERI B), and FcsRIy (FCERIG). In various aspects, the intracellular signaling domain is a CD3zeta-derived domain, optionally comprising SEQ ID NO: 5. In various aspects, the intracellular signaling domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 5.

[0028] In various aspects, the CAR comprises a costimulatory domain. Costimulatory domains provide intracellular signals that, e.g., enhance immune cell proliferation, cytokine secretion, cytotoxic function, and the like. Examples of costimulatory domains include, but are not limited to, CD27, 4-1 BB, CD28, CD134, ICOS, 0X40, CD149, DAP10, CD30, IL2-R, IL7r6, IL21-R, NKp30, NKp44, CD40, CD137, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and DNAM-1 costimulatory domains. Constructs comprising two, three, or more costimulatory domains are also envisioned. Costimulatory domains are further described in, e.g., U.S. Patent No. 11,235,977, which is hereby incorporated by reference in its entirety and particularly with respect to the disclosure of CAR components. Optionally, the CAR of the disclosure comprises a 4-1 BB domain (e.g., SEQ ID NO: 4). In various aspects, costimulatory domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 4.

[0029] In exemplary aspects of the disclosure, the CAR comprises a mutated NKG2D ectodomain (e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 1), an lgG4 hinge (e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 2), a CD8a transmembrane domain (e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 3), a 4-1 BB domain (e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 4), and a CD3zeta intracellular signaling domain (e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 5). In various aspects, the CAR comprises or consists of the amino acid sequence of SEQ ID NO: 6. The disclosure further contemplates a CAR construction comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 6.

[0030] Immune cells modified to express the CAR and display the CAR on the cell surface also are provided. These cells are optionally obtained from the subject to be treated (i.e., are autologous). However, in some embodiments, donor cells (allogeneic) are used. Immune cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Immune cells can be obtained from blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. For example, cells from the circulating blood of an individual may be obtained by apheresis. In some embodiments, immune cells are isolated from peripheral blood lymphocytes by lysing the red blood cellsand depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of immune effector cells can be further isolated by positive or negative selection techniques. For example, immune effector cells can be isolated using a combination of antibodies directed to surface markers unique to the positively selected cells, e.g., by incubation with antibody- conjugated beads for a time period sufficient for positive selection of the desired immune effector cells. CAR- expressing cells are typically expanded by growth in a laboratory and then administered to the subject or patient, or another subject or patient. The CAR-expressing cells will recognize and kill cells (e.g., cancer cells) that express the targeted antigen on their surface.

[0031] Immune cells include, but are not limited to, lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combinations thereof. Examples of suitable immune cells for use in the context of the disclosure include, but are not limited to, Natural Killer (NK) cells, T cells (including cytotoxic T cells and / or regulatory T cells), monocytes, dendritic cells, and innate lymphoid cells (types 1-3).

[0032] In various aspects, the immune cell is a T cell. T cells or T lymphocytes can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. T helper cells (TH cells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response. These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH7, TH9, or TFH, which secrete different cytokines to facilitate a different type of immune response. Cytotoxic T cells (Tc cells or CTLs) destroy virally infected cells and tumor cells. These cells are also known as CD8+ T cells since they express the CD8 glycoprotein at their surface. These cells recognize their targets by binding to antigen associated with MHC class I molecules, which are present on the surface of all nucleated cells. Natural killer T (NKT) cells (not to be confused with natural killer (NK) cells) bridge the adaptive immune system with the innate immune system.Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigen presented by a molecule called CD1d. A population of T cells for modification may be enriched for one or more subsets based on cell surface expression. In some instances, the population of T cells expressing the CAR include cytotoxic CD8+ T lymphocytes and / or CD4+ T lymphocytes.

[0033] Natural-killer (NK) cells are CD56+CD3- large granular lymphocytes that kill virally infected and transformed cells and constitute a critical cellular subset of the innate immune system (Godfrey et al. Leuk Lymphoma (2012) 53:1666-1676). Unlike cytotoxic CD8+ T lymphocytes, NK cells launch cytotoxicity without the requirement for prior sensitization, and can also eradicate MHC-l-negative cells (Nami-Mancinelli et al. Int Immunol (2011) 23:427-431). In some embodiments, the immune cell displaying the CAR of the disclosure is an NK cell. In some aspects, the immune cell is a macrophage. Immune cells, such as T cells, macrophages, andNK cells, can be modified using gene transfer techniques to directly and stably express on their surface transmembrane signaling receptors that confer novel antigen specificities. See, e.g., Gill & June, Immunological Reviews (2015) 263: 68-89; Glienke et al., Front. Pharmacol. (2015) 6, DOI=10.3389 / fphar.2015.00021.

[0034] The disclosure also provides an antibody fusion protein comprising antibody heavy chains and antibody light chains, wherein the heavy chains or the light chains are fused at their N-terminus via an amino acid linker to an A1-A2 domain. In various aspects of the disclosure, the A1-A2 domain comprises an amino acid sequence having at least 90% identity (e.g., at least 95% or at least 99% identity) to SEQ ID NO: 11 (e.g., 100% identity to SEQ ID NO: 11). The combination of elements of the antibody fusion protein bestow particularly advantageous properties to the construct, and results in a bispecific fusion protein which binds both a target antigen and mutated NKG2D ectodomain. This format (antibodies fused to an A1-A2 domain) is also referred to as a "MicAbody.” The A1-A2 domain of the instant disclosure is not a naturally occurring A1-A2 domain, but comprises an amino acid sequence which binds a mutated version of an NKG2D ectodomain and which does not bind wild-type NKG2D (wtNKG2D) (or at least does not bind wtNKG2D in such a manner to be biologically relevant in vivo). This A1-A2 domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 11 (e.g., at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 11). In various aspects, the A1-A2 domain comprises an alanine or glutamine one or more of positions 40, 54, and / or 84 of SEQ ID NO: 11. Substitutions at these positions, e.g., reduce N-glycosy lation in certain host cell types (such as HEK 293 cells and / or CHO cells), thereby reducing undesirable heterogeneity in the final product. In this regard, the A1-A2 domain may comprise a glutamine at position 40, an alanine at position 40, a glutamine at position 54, an alanine at position 54, an alanine at position 40 and a glutamine at position 54 (optionally with a glutamine or alanine at position 84), an alanine at position 40 and an alanine at position 54 (optionally with a glutamine or alanine at position 84), a glutamine at position 40 and an alanine at position 54 (optionally with a glutamine or alanine at position 84), a glutamine at position 40 and a glutamine at position 54 (optionally with a glutamine or alanine at position 84), an alanine at position 40 and a glutamine at position 84, an alanine at position 40 and an alanine at position 84, a glutamine at position 40 and an alanine at position 84, a glutamine at position 40 and a glutamine at position 84, an alanine at position 54 and a glutamine at position 84, an alanine at position 54 and an alanine at position 84, a glutamine at position 54 and an alanine at position 84, or a glutamine at position 54 and a glutamine at position 84, wherein the position is in reference to the amino acid position within SEQ ID NO: 11. For example, the disclosure provides an A1-A2 domain peptide having at least 95% identity to SEQ ID NO: 11, wherein the peptide comprises glutamine residues at positions 40 and 54 with respect to the sequence of SEQ ID NO: 11. Optionally, the A1-A2 domain comprises a glutamine at position 84 of SEQ ID NO: 11. Alternatively, the A1-A2 domain may, in various aspects, comprise an alanine at position 84 of SEQ ID NO: 11. In various aspects, the A1-A2 domain peptide comprises (or consists of) SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14. In various aspects, the A1-A2 domain peptide comprises (or consists of) an amino acid sequence having at least 90% identity (e.g., at least 91% identity, atleast 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity) to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14. A1-A2 domains are further described in, e.g., U.S. Patent Nos. 8,658,765; 10,259,858; and 11,453,713; and U.S. Patent Publication No. 2019 / 0300594, each of which are incorporated by reference in their entireties and in particular with respect to disclosure relating to A1-A2 domains.

[0035] The term "antibody” herein refers to immunoglobulins with full length heavy chains and light chains. The antibody of the disclosure is an IgG antibody, which includes four highly conserved subclasses (I gG 1 , lgG2, lgG3, and lgG4), which generally differ in their constant regions (e.g., in the hinge and / or CH2 domain). Optionally, the antibody fusion protein of the disclosure comprises an lgG1 antibody. The constant region of an antibody (e.g., an lgG1 antibody) may be modified to reduce or inactivate the antibody's ability to trigger antibody-dependent cell cytolysis (ADCC) (e.g., by introducing D265A / D297A substitutions into the Fc domain).

[0036] In various aspects, the antibody is an anti-CD 19 antibody. CD19 is a type I transmembrane glycoprotein belonging to the immunoglobulin superfamily. The glycoprotein is expressed on B cells and follicular dendritic cells and plays a role in B cell cancers and autoimmune diseases. Examples of B cell-related cancers include, but are not limited to, leukemias (e.g., Hairy Cell Leukemia (HCL) and Chronic Lymphocytic Leukemia (CLL)) and lymphomas (e.g., Non-Hodgkins Lymphoma (NHL, such as Diffuse Large B-cell Lymphoma (DLBCL), Burkitt Lymphoma (BL), Mantel cell Lymphoma (MCL), and follicular lymphoma). Examples of CD19- associated autoimmune disease include, e.g., multiple sclerosis (MS), rheumatoid arthritis (RA), systemic lupus erythematosus and graft-versus-host (GVH) disease. Examples of anti-CD 19 antibodies include, but are not limited to, inebilizumab (MEDI-551), an afucosylated anti-human CD19 monoclonal antibody (described further in Herbst et al., B-cell depletion in vitro and in vivo with an afucosylated anti-CD 19 antibody. J Pharmacol Exp Ther. (2010) 335:213-22); MDX-1342, a human anti-CD 19 antibody (described further in Cardarelli et al., A nonfucosylated human antibody to CD19 with potent B-cell depletive activity for therapy of B-cell malignancies. Cancer Immunol Immunother. (2010) 59:257-65); tafasitamab, an anti-CD 19 antibody comprising amino acid substitutions S239D / I332E that impact effector cell recruitment (described further in Rosskopf et al., Enhancing CDC and ADCC of CD19 Antibodies by Combining Fc Protein-Engineering With Fc Glyco-Engineering.Antibodies (2020) 9(4):63); and FMC63, an lgG2a monoclonal antibody specific for CD19 (described further in Zola et al., Preparation and characterization of a chimeric CD19 monoclonal antibody, Immunol Cell Biol (1991) 69(Pt 6):411-22). Methods of making antibodies and antibody fusion proteins are known in the art.

[0037] The A1-A2 domain is fused to the N-terminus of the heavy chains or the light chains of the antibody (e.g., an anti-CD 19 antibody) via an amino acid linker. The linker may be 2-8 amino acids in length, 9-17 amino acids in length, or 18 or more (e.g., 18-30 or 18-25) amino acids in length. The linker is optionally rich in glycine, as well as serine, threonine, or charged amino acids. Examples of linkers include [GGGS (SEQ ID NO: 45)]n, wherein n is 1-5, such as GGGS (SEQ ID NO: 45); [GGGGS (SEQ ID NO: 15)]n, wherein n is 1-5, such as GGGGS (SEQ ID NO: 15); APTSSSGGGGS (SEQ ID NO: 16); and KESGSVSSEQLAQFRSLD (SEQ ID NO: 17). Other linkers include, e.g., (GGS)n, wherein n is 1-5, such as GGSGGS (SEQ ID NO: 46). In variousaspects, each heavy chain of the antibody of the fusion protein is fused at the N-terminus to an A1-A2 domain via the amino acid linker, and the linker is 9-17 amino acids in length (e.g., SEQ ID NO: 16) or 18 or more amino acids in length (such as 18-30 or 18-25 amino acids in length) (e.g., SEQ ID NO: 17). In various alternative aspects of the disclosure, each light chain of the antibody of the fusion protein is fused at the N-terminus to an A1-A2 domain via the amino acid linker, and the linker is 2-8 amino acids in length (e.g., SEQ ID NO: 15) or 9-17 amino acids in length (e.g., SEQ ID NO: 16).

[0038] While the disclosure above, in various aspects, focuses on anti-CD 19 antibodies as the fusion partner to an A1-A2 domain peptide, it will be appreciated that the A1-A2 domain peptide of the disclosure may be fused to other antibodies or antigen-binding fragments of antibodies. The disclosure above with respect to the structure of anti-CD 19 antibodies also applies to other antigen binding proteins and antibodies (i.e., antibodies that bind other targets). The antibody may be a monoclonal antibody or multispecific antibody (e.g., bispecific antibody). Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments. The antibody or fragment thereof may target any other suitable antigen, such as antigens expressed on the surface of cancer cells. Examples of antigens include, but are not limited to CD20, BMCA, HER2, EGFR, EpCAM, CEA, BCMA, PSMA, CD 19, CD20, CD22, CD33, CD37, CD38, CD 123, CD276 (B7-H3), GPC2, GPC3, GPRC5D, WT-1, NY- ESO-1 , CLDN4, CLDN6, CLDN18.2, PSCA, and TSPAN8.

[0039] The disclosure also provides a kit comprising one or more containers comprising the antibody fusion protein described herein and / or the CAR-displaying immune cell described herein. The kit may further comprise instructions and written information on indications and usage of the antibody fusion protein. Syringes, e.g., single use or pre-filled syringes, sterile sealed containers, e.g., vials, bottle, vessel, and / or kits or packages comprising the antibody fusion protein or immune cell, optionally with suitable instructions for use, are also contemplated. In a further aspect, the disclosure provides an article of manufacture, or unit dose form, comprising: (a) a composition of matter comprising the antibody fusion protein described herein or the immune cell described herein; (b) a container containing said composition; and (c) a label affixed to said container, or a package insert included in said container referring to the use of said antibody fusion protein and / or immune cell in the treatment of a disease or disorder (e.g., cancer). Also provided herein are compositions comprising the antibody fusion protein and / or cells expressing a CAR as described herein and a pharmaceutically acceptable carrier, excipient, or diluent. In exemplary aspects, the composition is a sterile composition.

[0040] The disclosure contemplates a system or kit comprising components of a cell therapy regimen. In various aspects, the first component is the antibody fusion protein described herein, i.e., a bispecific, antibodybased fusion protein that binds both (I) a cell surface antigen (e.g., a cancer cell surface antigen, such as CD19) and (ii) a CAR described herein. The second component is a mammalian cell (e.g., human cell, such as a human immune cell) that is genetically modified to express the chimeric antigen receptor (CAR) described herein, i.e., a CAR comprising a mutated NKG2D ectodomain (e.g., comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof comprising a substitution at amino acid position 118 within SEQ ID NO: 1), an lgG4 hinge domain (e.g., comprising the amino acid sequence of SEQ ID NO: 2), a transmembrane domain (e.g., aCD8a transmembrane domain, such as a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 3), an intracellular signaling domain (e.g., a CD3zeta intracellular signaling domain, such as an intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 5), and a costimulatory domain (e.g., a 4-1 BB costimulatory domain, such as a costimulatory domain comprising the amino acid sequence of SEQ ID NO: 4). In various aspects, the cell is a lymphocyte or a macrophage, e.g., a human lymphocyte (such as human T cell) or a human macrophage. In various aspects, the second component is a human NK (natural killer) cell (e.g., an autologous human NK cell); disclosure herein with reference to T cells also applies to NK cells. The kit comprises one or more containers comprising cells expressing the CAR and one or more containers comprising the antibody fusion protein. A kit may further comprise instructions and written information on indications and usage of the components described herein.

[0041] The disclosure further provides a method of treating a disease or disorder in a subject in need thereof. In this regard, the disclosure contemplates the antibody fusion protein and the immune cell for use as a medicament, as well as use of the antibody fusion protein and the immune cell in the preparation of medicaments for treating a disease or disorder in a subject in need thereof. The antibody fusion protein comprises an antibody which binds a cell surface antigen on a target cell, such as a cell surface antigen displayed on cancer cells. Optionally, the disease or disorder is a cancer, although the disclosure also contemplates treatment of other diseases or disorders, including disease or disorders associated with CD19- expressing cells. The method comprises administering to the subject the CAR-expressing cell described herein (e.g., a T cell or NK cell expressing the INKG2D-based CAR described herein) and administering to the subject the antibody fusion protein described herein. Optionally, the cancer is a CD 19-positive cancer, and the antibody fusion protein comprises an anti-CD 19 antibody. Also optionally, each heavy chain of the antibody of the fusion protein administered is fused at the N-terminus to an A1-A2 domain via the amino acid linker 9-17 amino acids in length (e.g., SEQ ID NO: 16) or 18-30 amino acids in length (e.g. SEQ ID NO: 17). Alternatively, each light chain of the antibody of the fusion protein administered to the subject may be fused at the N-terminus to an A1-A2 domain via the amino acid linker 2-8 amino acids in length (e.g., SEQ ID NO: 15) or 9-17 amino acids in length (e.g., SEQ ID NO: 16). Examples of cancers include, but are not limited to, leukemias and lymphomas, such as Hairy Cell Leukemia, Chronic Lymphocytic Leukemia, and Non-Hodgkins Lymphoma (e.g., Diffuse Large B-cell Lymphoma, Burkitt Lymphoma, Mantel cell Lymphoma, and follicular lymphoma).

[0042] The antibody fusion protein described herein is capable of activating INKG2D-CAR-expressing cells (e.g., T cells) only in the presence of cells expressing a target antigen recognized by the antibody fusion protein. When used with additional antibody fusion proteins that target other antigens (i.e., antibody fusion proteins having different variable regions that bind different cell surface antigens), the immune cells can be targeted to different antigens simultaneously or sequentially to mediate cytolysis; this approach can help address, e.g., tumor resistance and escape as a result of target antigen loss without having to create, expand and infuse multiple different autologous CAR cells. This highly modular system expands the potential of adoptive celltherapies and overcomes many of disadvantages of existing cell therapies, including severe systemic toxicity, antigen escape, and limited and uncontrolled persistence of current CAR-T and CAR-NK cell therapeutics.

[0043] As used herein, the term "treat," as well as words related thereto, do not necessarily imply 100% or complete treatment or remission. Rather, there are varying degrees of treatment of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the methods of treating a disease or disorder can provide any amount or any level of treatment. Furthermore, the treatment provided by the method may include treatment of one or more conditions or symptoms or signs of the disease being treated. For instance, the treatment method of the present disclosure may inhibit one or more symptoms of the disease. Also, the treatment provided by the methods of the present disclosure may encompass slowing the progression of the disease.

[0044] Treatment for cancer may be determined by any of a number of ways. Any improvement in the subject's wellbeing is contemplated (e.g., at least or about a 10% reduction, at least or about a 20% reduction, at least or about a 30% reduction, at least or about a 40% reduction, at least or about a 50% reduction, at least or about a 60% reduction, at least or about a 70% reduction, at least or about an 80% reduction, at least or about a 90% reduction, or at least or about a 95% reduction of any parameter described herein). For example, a therapeutic response would refer to one or more of the following improvements in the disease: (1) a reduction in the number of neoplastic cells; (2) an increase in neoplastic cell death; (3) inhibition of neoplastic cell survival; (5) inhibition (i.e., slowing to some extent, preferably halting) of tumor growth or appearance of new lesions; (6) decrease in tumor size or burden; (7) absence of clinically detectable disease, (8) decrease in levels of cancer markers; (9) an increased patient survival rate; and / or (10) some relief from one or more symptoms associated with the disease or condition (e.g., pain). In addition, treatment efficacy also can be characterized in terms of responsiveness to other immunotherapy treatment or chemotherapy. In various aspects, the methods of the disclosure further comprise monitoring treatment in the subject.

[0045] In various aspects, the subject is a mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits, mammals from the order Carnivora, including Felines (cats) and Canines (dogs), mammals from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). In some aspects, the mammal is of the order Primate, Ceboid, or Simoid (monkey) or of the order Anthropoid (humans and apes). In some aspects, the mammal is a human. Therapeutic compositions may be delivered to a subject using any of a variety of routes, including parenteral, topical, oral, intrathecal, or local administration. Indeed, a composition may be administered subcutaneously, intracutaneously, intradermally, intravenously, intraarterially, intratumorally, parenterally, intraperitoneally, intramuscularly, intraocularly, intraosteally, epidurally, intradurally, intratumorally and the like.

[0046] The disclosure also provides nucleic acid molecules encoding the chimeric antigen receptor described herein. The disclosure also provides (I) nucleic acid molecules (i.e., isolated nucleic acids) encoding the lightchain of the antibody fusion protein described herein and (ii) nucleic acid molecules (i.e. , isolated nucleic acids) encoding the heavy chain of the antibody fusion protein described herein, as well as compositions comprising (I) and / or (ii). The disclosure further provides nucleic acid molecules comprising nucleic acid sequences encoding both a heavy chain and a light chain of an antibody fusion protein of the disclosure. Nucleic acids of the disclosure include nucleic acids encoding any of the amino acid sequences disclosed herein, as well as nucleic acids comprising nucleotide sequences having at least 80%, more preferably at least about 90%, more preferably at least about 95%, and most preferably at least about 98% identity to nucleic acids of the disclosure (i.e., the nucleic acid sequences set forth in the sequence listing). Nucleic acids of the disclosure include nucleic acids encoding any of the amino acid sequences disclosed herein, as well as nucleic acids encoding amino acid sequences having at least 80%, more preferably at least about 90%, more preferably at least about 95%, and most preferably at least about 98% identity to the amino acid sequences of the disclosure (i.e., the amino sequences set forth in the sequence listing.

[0047] Nucleic acids of the disclosure can be cloned into an expression vector, such as a plasmid, cosmid, bacmid, phage, artificial chromosome (BAG, YAC) or virus, into which another genetic sequence or element (either DNA or RNA) may be inserted so as to bring about the replication of the attached sequence or element. In some embodiments, the expression vector contains a constitutively active promoter segment (such as but not limited to CMV, SV40, Elongation Factor or LTR sequences) or an inducible promoter sequence such as the steroid inducible pIND vector (Invitrogen), where the expression of the nucleic acid can be regulated. Expression vectors of the disclosure may further comprise regulatory sequences, for example, an internal ribosomal entry site. A secretory signal peptide sequence can also, optionally, be encoded by the expression vector, operably linked to the coding sequence of interest, so that the expressed polypeptide can be secreted by a recombinant host cell, for more facile isolation of the polypeptide of interest from the cell. The expression vector can be introduced into a cell by transfection, for example.

[0048] Host cells comprising the nucleic acid molecules (optionally contained in expression vectors) also are provided. The host cell may be a prokaryotic cell, for example an E. coli cell, or a eukaryotic cell, for example a mammalian cell or a yeast cell. Yeast cells include, e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris cells. Mammalian cells include, for example, VERO, HeLa, Chinese hamster Ovary (CHO), W138, baby hamster kidney (BHK), COS-7, MDCK, human embryonic kidney line 293, African green monkey kidney cells, and COS cells. Recombinant protein-producing cells of the disclosure also include any insect expression cell line known, such as for example, Spodoptera frugiperda cells. In one embodiment, the cells are mammalian cells, such as CHO cells. In the context of the antibody fusion proteins of the disclosure, the disclosure provides (I) a host cell comprising a nucleic acid molecule comprising a nucleotide sequence encoding the antibody heavy chain fused at the N-terminus to the A1 A2 domain and a nucleic acid molecule comprising a nucleotide sequence encoding the light chain of the antibody fusion protein, as well as (ii) a host cell comprising a nucleic acid molecule comprising a nucleotide sequence encoding the antibody light chainfused at the N-terminus to the A1 A2 domain and a nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain of the antibody fusion protein.

[0049] A method of producing an antibody fusion protein further is provided by the disclosure. The method comprises culturing a host cell (an isolated host cell) comprising a nucleic acid molecule comprising a nucleotide sequence encoding the light chain of the antibody fusion protein and a nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain of the antibody fusion protein. The method further comprises recovering the antibody fusion protein. Culture conditions and methods for generating recombinant proteins, such as antibody proteins, are known in the art. Similarly, protein purification methods are known in the art and utilized herein for recovery of recombinant proteins from cell culture media. In some aspects, methods for protein and antibody purification include filtration, affinity column chromatography, cation exchange chromatography, anion exchange chromatography, and concentration. Optionally, the method comprises formulating the antibody fusion protein.

[0050] A method of producing an immune cell displaying the CAR of the disclosure also is provided herein. The method comprises introducing a nucleic acid molecule encoding the CAR into an immune cell under conditions which allow expression and display of the CAR on the cell surface (i.e., allow processing of the CAR protein and incorporation into the cell membrane such that the ectodomain is presented on the cell surface). Optionally, the method comprises formulating the immune cell.

[0051] The following example is given merely to illustrate the present invention and not in any way to limit its scope.EXAMPLE

[0052] This example and other implementations described herein are exemplary and not intended to be limiting in describing the full scope of compositions and methods of this disclosure. Equivalent changes, modifications and variations of specific implementations, materials, compositions and methods may be made within the scope of the present disclosure, with substantially similar results.

[0053] This example demonstrates the ability of an antibody fusion protein comprising an A1-A2 domain fused to the N- or C-terminus of the antibody heavy chains or antibody light chains to bind a CAR-T cell comprising the chimeric antigen receptor of the disclosure, and the ability of the antibody fusion protein and CAR-T cell combination to kill cancer cells in vivo. The antibody fusion protein comprising the A1-A2 domain fused to the N- terminus of both light chains of the parent antibody demonstrated surprisingly superior activity.

[0054] Antibody fusion proteins were constructed using the anti-CD 19 antibody FMC63 (SEQ ID NOs: 21 and 22 comprising signal sequences, SEQ ID NOs: 23 and 24 lacking signal sequences) as the parent antibody. The A1-A2 domain of SEQ ID NO: 11 was fused to the N-terminus or the C-terminus of either the kappa light-chain or the heavy-chain of the parent antibody. Constructs were generated wherein A1-A2 domains were fused to the heavy or light chains via a representative "short” linker (GGGGS; SEQ ID NO: 15), a representative "medium”linker (APTSSSGGGGS; SEQ ID NO: 16), or a representative "long” linker (KESGSVSSEQLAQFRSLD; SEQ ID NO: 17). Additionally, D265A / N297A (Kabat numbering) mutations were introduced into the CH2 domain of the heavy chain of the antibody eliminate antibody-dependent cell cytotoxicity (ADCC) function. The sequences of the constructs are set forth in SEQ ID NOs: 30, 32, 34, 36, 38, 40, 42, and 44.

[0055] CAR-T cells also were generated comprising the CAR of the disclosure. CARs were constructed which comprise the CD8a-chain signal sequence, NKG2D variant ectodomain (also referred to herein as "INKG2D”), CD8a hinge or lgG4 hinge, CD8a transmembrane domains, 4-1 BB costimulatory domain, CD3zeta intracellular signaling domain. Constructs were generated which included the CAR sequence attached to eGFP (green fluorescent protein). The sequences of the domains and the full-length construct are set forth in SEQ ID NOs: 1- 9. The VH and VL domains of FMC63 were substituted for the NKG2D ectodomain to generate the FMC63 scFv-based CAR (FMC63scFv CAR) (SEQ ID NO: 20).

[0056] The ability of various constructs to kill cancer cells was examined. Ramos human B cell lymphoma cells (ATCC #CRL-1596) were engineered to express firefly luciferase and were co-cultured with INKG2D CAR-T cells comprising either the CD8a-hinge or the lgG4-hi nge in the CAR construct. The cells were incubated overnight with an increasing concentration of the anti-CD 19 antibody fusion proteins. The amount of luciferase signal was quantified and the data normalized to the signal observed with CAR-T cell treatment only (0 nM antibody fusion protein) control. Error bars correspond to the standard deviation for triplicate samples in the assay.

[0057] Figure 2A illustrates the direct comparison of the three antibody fusion protein formats with CAR-T cells of different hinge lengths (CD8a-hinge (45 amino acid) vs. lgG4-hinge (12 amino acid)). The level of cytolysis mediated by the LC.S3 and HC.S3 antibody fusion protein formats, which comprise the A1-A2 domain fused to the C-terminus of the heavy or light chains, was inferior compared to formats comprising the A1-A2 domain fused to the N-termini. FMC63-S3med.LC corresponds to the antibody fusion protein wherein the A1-A2 domain is fused to the N-terminus of the light chains via a "medium” linker comprising 11 amino acids in length. The N- terminally fused A1-A2 domain enhanced cytotoxicity with both the CD8-hinge and the lgG4-hinge CARs. The combination of the N-terminal fusion of the A1-A2 domain and the lgG4-hinge in the CAR construct produced superior results. Figure 2B compares antibody fusion proteins with N-terminal A1-A2 domain fusions across the two different CAR-T hinge lengths. All N-terminally fused HC molecules demonstrated cell killing activity. The constructs wherein the A1-A2 domain was N-terminally fused to light chains mediated more robust cytotoxicity, with formats comprising the "short” and "medium” linkers exhibiting superior activity.

[0058] A study also was performed to confirm the ability of the various antibody fusion proteins to target cells. Ramos human B cell lymphoma cells were incubated overnight with FMC63-based antibody fusion proteins at varying concentrations, and bound antibody was detected the next day with an anti-human-Fc antibody. Figure 3 shows the median fluorescence intensity (MFI) curves for each format. EC50 values were calculated in GraphPad Prism. Fusion of an exogenous domain to the N-terminus of an antibody may block or impede theability of the Fv region to bind its epitope. The reduction in binding may or may not impact cytolysis. The data of this study demonstrated that the majority of antibody fusion proteins tested were capable of engaging their target epitope without significant loss of binding affinity.

[0059] The ability of the system of the disclosure to control tumor burden in vivo also was examined. Mice were injected IV with 5 x 105Raji B cell lymphoma cells (ATCC #CCL-86) stably transfected to constitutively express luciferase (Perkin Elmer RediFect Red-FLuc-GFP #CLS960003). Antibody fusion proteins were administered two days later at 20 ug per mouse, followed by intravenous infusion of CAR-T cells the next day (day 0 of study, 5 x 106INKG2D CAR-T cells for Figure 4A or 3 x 106INKG2D CAR-T cells for Figure 4B). In the groups that received antibody fusion protein, mice were re-dosed every two days for a total of five doses. For comparison purposes, a cohort of subjects was administered FMC63scFv CAR-T cells without the antibody fusion protein (3 x 106FMC63scFv CAR-T cells for Figure 4A or 3 x 106FMC63scFv CAR-T cells for Figure 4B). Treatment with the antibody fusion protein proteins and INKG2D CAR-T cells comprising the CD8a hinge did not significantly control tumor burden. See Figure 4A. In contrast, the N-terminally fused antibody fusion format (here, with the "medium” linker) demonstrated a significant improvement in tumor control when administered with the INKG2D CAR-T-cell comprising the lgG4 hinge.

[0060] The example describes the engineering of a privileged CAR-ligand pairing (I NKG2D. lgG4 hinge and antibody fusion protein comprising a modified A1-A2 domain fused at the N-terminus of heavy or light chains via a linker) which provides a versatile and broadly controllable platform for cell therapy. Each component of the system - the INKG2D-based CAR receptor and the antibody fusion protein - are functionally inert on their own. When paired, the system mediates potent cytolysis of cancer cells. The disclosure identifies a CAR construct and antibody fusion format which is unexpectedly superior to other constructs and formats, particularly when utilized together. The system described herein has demonstrated enhanced ability to control tumor burden (here, CD19-expressing cancer cells) in vivo.

[0061] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. The following patent publications are expressly incorporated by reference in their entireties, including their sequence listings: U.S. Patent Nos. 8,658,765; 10,259,858; 11,440,948; and 11,453,713, and International Patent Publication Nos. WO 2019 / 191243, WO 2020 / 097064, and WO 2022 / 261121.

[0062] While the present invention has been described with reference to several embodiments, which embodiments have been set forth in considerable detail for the purposes of making a complete disclosure of the invention, such embodiments are merely exemplary and are not intended to be limiting or represent an exhaustive enumeration of all aspects of the invention. The scope of the invention is to be determined from the claims appended hereto. Further, it will be apparent to those of skill in the art that numerous changes may be made in such details without departing from the spirit and the principles of the invention.

Claims

WHAT IS CLAIMED:1 . A chimeric antigen receptor comprising a mutated NKG2D ectodomain, an lgG4 hinge domain, a transmembrane domain, an intracellular signaling domain, and a costimulatory domain.

2. The chimeric antigen receptor of claim 1, wherein the mutated NKG2D ectodomain comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof, the variant comprising a substitution at amino acid position 118 within SEQ ID NO: 1.

3. The chimeric antigen receptor of claim 1, wherein the mutated NKG2D ectodomain comprises an amino acid sequence comprising at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1.

4. The chimeric antigen receptor of any one of claims 1-3, wherein the lgG4 hinge domain comprises an amino acid sequence comprising at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 2.

5. The chimeric antigen receptor of any one of claims 1-4, wherein the transmembrane domain is a CD8a transmembrane domain.

6. The chimeric antigen receptor of any one of claims 1-5, wherein the transmembrane domain comprises an amino acid sequence comprising at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 3.

7. The chimeric antigen receptor of any one of claims 1-6, wherein the intracellular signaling domain comprises an amino acid sequence comprising at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 5.

8. The chimeric antigen receptor of any one of claims 1-7, wherein the costimulatory domain comprises an amino acid sequence comprising at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 4.

9. A nucleic acid molecule comprising a nucleic acid sequence encoding the chimeric antigen receptor of any one of claims 1-8.

10. An expression vector comprising the nucleic acid molecule of claim 9.

11. A cell comprising the expression vector of claim 10.

12. An immune cell displaying the chimeric antigen receptor of any one of claims 1-8.

13. The immune cell of claim 12, which is a T cell, a macrophage, or a natural killer (NK) cell.

14. An antibody fusion protein comprising antibody heavy chains and antibody light chains, wherein the heavy chains or the light chains are fused at their N-terminus via an amino acid linker to an A1-A2 domain comprising an amino acid sequence comprising at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 11.

15. The antibody fusion protein of claim 14, wherein the A1-A2 domain comprises an amino acid sequence comprising at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 11 and further comprising an alanine or glutamine at one or more of positions 40, 54, and / or 84.

16. The antibody fusion protein of claim 15, wherein the A1-A2 domain comprises glutamine residues at positions 40 and 54 of SEQ ID NO: 11 .

17. The antibody fusion protein of claim 15 or claim 16, wherein the A1-A2 domain comprises a glutamine at position 84 of SEQ ID NO: 11.

18. The antibody fusion protein of claim 15 or claim 16, wherein the A1-A2 domain comprises an alanine at position 84 of SEQ ID NO: 11.

19. The antibody fusion protein of claim 14, wherein the peptide comprises SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.

20. The antibody fusion protein of claim 14, wherein the A1-A2 domain comprises SEQ ID NO: 11.

21. The antibody fusion protein of any one of claims 14-20, wherein the linker is 2-8 amino acids in length.

22. The antibody fusion protein of any one of claims 14-20, wherein the linker is 9-17 amino acids in length.

23. The antibody fusion protein of any one of claims 14-20, wherein the linker is 18-30 amino acids in length.

24. The antibody fusion protein of any one of claims 14-20, wherein linker comprises the amino acid sequence of SEQ ID NO: 16.

25. The antibody fusion protein of any one of claims 14-20, wherein linker comprises the amino acid sequence of SEQ ID NO: 15.

26. The antibody fusion protein of any one of claims 14-20, wherein the linker comprises the amino acid sequence of SEQ ID NO: 17.

27. The antibody fusion protein of any one of claims 14-26, wherein the antibody is an anti-CD19 antibody.

28. The antibody fusion protein of any one of claims 14-27, wherein each heavy chain is fused at the N-terminus to an A1-A2 domain via the amino acid linker.

29. The antibody fusion protein of claim 28, wherein the linker is 9-17 amino acids in length or 18- 30 amino acids in length.

30. The antibody fusion protein of any one of claims 14-27, wherein each light chain is fused at the N-terminus to an A1-A2 domain via the amino acid linker.

31. The antibody fusion protein of claim 30, wherein the linker is 2-8 amino acids in length or 9-17 amino acids in length.

32. A nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain of the antibody fusion protein of claim 28.

33. A nucleic acid molecule comprising a nucleotide sequence encoding the light chain of the antibody fusion protein of claim 30.

34. An expression vector comprising the nucleic acid molecule of claim 32 or claim 33.

35. A composition comprising the nucleic acid molecule of claim 32 and a nucleic acid molecule comprising a nucleotide sequence encoding the light chain of the antibody fusion protein of any one of claims 14- 31.

36. A composition comprising the nucleic acid molecule of claim 33 and a nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain of the antibody fusion protein of any one of claims 14-31.

37. A host cell comprising the nucleic acid molecule of claim 32 and a nucleic acid molecule comprising a nucleotide sequence encoding the light chain of the antibody fusion protein of any one of claims 14- 31.

38. A host cell comprising the nucleic acid molecule of claim 33 and a nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain of the antibody fusion protein of any one of claims 14-31.

39. A method of producing an antibody fusion protein, the method comprising culturing the host cell of claim 37 or claim 38, and recovering said antibody fusion protein.

40. A kit comprising one or more containers comprising the antibody fusion protein of any one of claims 14-31 and instructions for use.41 . The kit of claim 40, further comprising one or more containers comprising the immune cell of claim 12 or claim 13.

42. A kit comprising one or more containers comprising the immune cell of claim 12 or claim 13 and instructions for use.

43. The kit of any one of claims 40-42, wherein the chimeric antigen receptor comprises SEQ ID NOs: 1-5.

44. A method of treating a subject suffering from cancer, the method comprising administering to the subject (I) the antibody fusion protein of any one of claims 14-31 and (ii) an immune cell of claim 12 or claim 13, wherein the antibody fusion protein comprises an antibody which binds a cell surface antigen displayed on cancer cells.

45. The method of claim 44, wherein the cancer is a CD19-positive cancer, and the antibody fusion protein comprises an anti-CD19 antibody.

46. The antibody fusion protein of any one of claims 14-31 and the immune cell of claim 12 or claim 13 for use as a medicament.

47. The antibody fusion protein of any one of claims 14-31 and the immune cell of claim 12 or claim 13 for use in treating cancer in a subject in need thereof, wherein the antibody fusion protein comprises an antibody which binds a cell surface antigen displayed on cancer cells.

48. The antibody fusion protein and the immune cell of claim 47, wherein the cancer is a CD19- positive cancer, and the antibody fusion protein comprises an anti-CD 19 antibody.

49. Use of the antibody fusion protein of any one of claims 14-31 and the immune cell of claim 12 or claim 13 as a medicament.

50. Use of the antibody fusion protein of any one of claims 14-31 and the immune cell of claim 12 or claim 13 for treating cancer in a subject in need thereof.

51. The use of claim 50, wherein the cancer is a CD19-positive cancer, and the antibody fusion protein comprises an anti-CD 19 antibody.

52. Use of the antibody fusion protein of any one of claims 14-31 and the immune cell of claim 12 or claim 13 in the preparation of medicaments for treating cancer in a subject in need thereof.

53. The use of claim 52, wherein the cancer is a CD 19-positive cancer, and the antibody fusion protein comprises an anti-CD 19 antibody.

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