Patent Application
20240287202
Incorporated by reference in its entirety is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: 59,942 byte ASCII (Text) file named “56867_Seqlisting.txt”; created on Jun. 8, 2021.
The invention relates to antibodies fused to the A1-A2 domain of a non-natural NKG2D ligand that binds to non-natural NKG2D receptors.
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.
The disclosure provides an antibody fusion protein comprising (i) heavy chains comprising variable region sequences comprising the amino acid sequence of SEQ ID NO: 1 and (ii) light chains comprising variable region sequences comprising the amino acid sequence of SEQ ID NO: 8, wherein the light chains are fused at the C-terminus to an A1-A2 domain comprising the amino acid sequence of SEQ ID NO: 11. In various aspects, the heavy chains comprise constant domains comprising the amino acid sequence of SEQ ID NO: 3. Optionally, the A1-A2 domain is fused to the light chains via a linker comprising the amino acid sequence of SEQ ID NO: 10. In this regard, the light chains, in various aspects, comprise the amino acid sequence of SEQ ID NO: 13. In various aspects, the heavy chains comprising the amino acid sequence of SEQ ID NO: 7.
The disclosure further provides a nucleic acid molecule comprising a nucleotide sequence encoding the light chain of the antibody fusion protein (e.g., a light chain comprising variable region sequence comprising the amino acid sequence of SEQ ID NO: 8, wherein the light chains are fused at the C-terminus to an A1-A2 domain comprising the amino acid sequence of SEQ ID NO: 11). The disclosure further provides a composition comprising the nucleic acid molecule encoding a 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 described herein (e.g., a heavy chain comprising a variable region sequence comprising the amino acid sequence of SEQ ID NO: 1). Also provided is an expression vector comprising the nucleic acid molecule encoding the light chain of the antibody fusion protein described herein, optionally further comprising a nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain of the antibody fusion protein described herein. Further provided is a host cell comprising the expression vectors described herein. The disclosure provides a 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. A method of producing an antibody fusion protein is also provided, the method comprising culturing a 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, and recovering the antibody fusion protein.
Also provided is a kit comprising one or more containers comprising the antibody fusion protein described herein. Optionally, the kit further comprises one or more containers comprising a mammalian cell (e.g., human lymphocyte or a human macrophage) comprising a chimeric antigen receptor comprising SEQ ID NO: 15. In various aspects, the chimeric antigen receptor further comprises SEQ ID NOs: 16-18.
The disclosure further provides a method of treating a subject suffering from a CD20-positive cancer, the method comprising administering to the subject the antibody fusion protein described herein and a mammalian cell (e.g., human lymphocyte or a human macrophage) comprising a chimeric antigen receptor comprising SEQ ID NO: 15. Optionally, the chimeric antigen receptor further comprises SEQ ID NOs: 16-18. Use of the antibody fusion protein and mammalian cell to treat a CD20-positive cancer is provided, as well as use of the antibody fusion protein and mammalian cell in the preparation of medicaments to treat a CD20-positive cancer.
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.
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.
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.
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.
The instant disclosure provides a fusion protein comprising an antibody 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 CAR-T cells displaying the non-natural NKG2D receptor 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. Unlike currently available CAR-T cell-based therapies, the fusion protein and system of the disclosure allows for flexible targeting to direct T cell activity to antigen of choice, multiplex capabilities to reduce the potential for antigen-loss related relapse, dose control for differential engagement of CAR-T cells, and selective delivery of modulatory agents to CAR-expressing cells.
The disclosure provides an antibody fusion protein comprising (i) heavy chains comprising variable region sequences of SEQ ID NO: 1 and (ii) light chains comprising variable region sequences of SEQ ID NO: 8. The light chains are fused at the C-terminus to an A1-A2 domain comprising the amino acid sequence of SEQ ID NO: 11. The heavy chain variable region and light chain variable region of the instant antibody fusion protein are those of Rituximab, a chimeric monoclonal antibody (IgG1 kappa immunoglobulin) that binds CD20, a surface antigen displayed on B cells. Rituximab is further described in, e.g., U.S. Pat. Nos. 5,736,137; 5,776,456; and 5,843,439. B cells play a role in the pathogenesis of certain autoimmune diseases and cancers, and Rituximab is effective in targeting and killing B cells to achieve a beneficial effect in a variety of disorders. For example, Rituximab has shown efficacy in treating cancers, such as 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). Rituximab also demonstrated efficacy in treating autoimmune disorders, such as rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), chronic inflammatory demyelinating polyneuropathy, and autoimmune-associated anemias. Rituximab also has been approved for the treatment of Granulomatosis with Polyangiitis (GPA) (Wegener's Granulomatosis) and Microscopic Polyangiitis (MPA).
The term “antibody” as used 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 (IgG1, IgG2, IgG3, and IgG4), 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 IgG1 antibody, the constant region of which 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). In various aspects, the heavy chains of the antibody fusion protein comprise constant domains comprising the amino acid sequence of SEQ ID NO: 3. The disclosure also contemplates antibody fusion proteins wherein the heavy chains comprise a constant region comprising the amino acid sequence of SEQ ID NO: 2. The disclosure also contemplates antibody fusion proteins wherein the heavy chains comprise an amino acid sequence at least 90% identical or at least 95% identical to SEQ ID NO: 3 but wherein the amino acids at positions 234, 235, and 329 within SEQ ID NO: 3 are alanine. In some aspects, the antibody fusion protein comprises heavy chains of SEQ ID NO: 7. In this regard, the disclosure provides an antibody fusion protein comprising light chains of SEQ ID NO: 21 and heavy chains of SEQ ID NO: 7. In other aspects, the antibody fusion protein comprises heavy chains of SEQ ID NO: 6 In this regard, the disclosure contemplates an antibody fusion protein comprising light chains of SEQ ID NO: 21 and heavy chains of SEQ ID NO: 6.
The light chains of the antibody comprise variable region sequences of SEQ ID NO: 8. Optionally, the light chains comprise a constant region comprising the amino acid sequence of SEQ ID NO: 9 (or a sequence at least about 90% identical or 95% identical to SEQ ID NO: 9). Thus, in various aspects, the light chains of the antibody fusion protein of the disclosure comprise SEQ ID NO: 8 and SEQ ID NO: 9 (SEQ ID NO: 21).
The light chains are fused at the C-terminus to an NKG2D ligand A1-A2 domain comprising the amino acid sequence of SEQ ID NO: 11. 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, 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. 93, 12445-12450 (1996); Zwirner et al., Hum. Immunol. 60, 323-330 (1999); and Spies et al., Nat. Immunol. 9, 1013-1015 (2008). 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 et al., Nat. Immunol. 2, 443-451 (2001); Radaev et al., Immunity 15, 1039-1049 (2001); Zuo et al., Sci Signal 10, (2017); and McFarland et al., Immunity 19, 803-812 (2003). 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 but 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 orthogonal A1-A2 domain, which is based on the U2S3 domain described in the Example, is fused to the light chain of the antibodies described herein to generate a bispecific antibody fusion protein which binds both CD20 and mutated NKG2D ectodomain. This format (antibodies fused to an A1-A2 domain) is also referred to herein as a “MicAbody.” As explained in more detail below, fusion of the A1-A2 domain to the C terminus of the light chain amino acid sequence resulted in superior activity compared to fusion of the A1-A2 domain on the heavy chains of the antibody of the disclosure. The superior properties of the placement of the domain on the antibody fusion protein described herein could not have been predicted prior to the study described in the Example.
In various aspects, the A1-A2 domain is fused to the C-terminus of the light chain via a linker, optionally a linker comprising (or consisting of) SEQ ID NO: 10. As explained in more detail below, the linker of SEQ ID NO: 10 produced a MicAbody which unexpectedly outperformed other antibody fusion constructs in terms of B cell cytotoxicity. Thus, in exemplary aspects of the disclosure, the antibody fusion protein of the disclosure comprises light chains comprising a variable region sequence of SEQ ID NO: 8 and the A1-A2 domain of SEQ ID NO: 11 fused to the C-terminus of the light chain via a linker sequence of SEQ ID NO: 10, optionally comprising the light chain constant region of SEQ ID NO: 9. In this regard, in various aspects of the disclosure, the light chains of the antibody fusion protein comprise the amino acid sequence of SEQ ID NO: 13.
The disclosure provides an antibody fusion protein comprising light chains of SEQ ID NO: 13 and heavy chains of SEQ ID NO: 7. The disclosure also provides an antibody fusion protein comprising light chains of SEQ ID NO: 13 and heavy chains of SEQ ID NO: 6. Methods of making antibodies and antibody fusion proteins are known in the art and described, e.g., in the Example below.
The disclosure also provides a kit comprising one or more containers comprising the antibody fusion protein 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, 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; (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 in the treatment of a disease or disorder (e.g., cancer). Also provided herein are compositions comprising the antibody fusion protein (and, in various aspects, mammalian cells expressing a CAR as described herein) and a pharmaceutically acceptable carrier, excipient or diluent. In exemplary aspects, the composition is a sterile composition.
The disclosure further provides a system or kit comprising components of a cell therapy regimen targeting CD20-displaying cells. In various aspects, the first component is the antibody fusion protein described herein, i.e., a bispecific, antibody-based fusion protein that binds both CD20 and a CAR comprising an NKG2D ectodomain. The second component is a mammalian cell (e.g., human cell) that is genetically modified to express a chimeric antigen receptor (CAR) that is itself inert (i.e., unarmed CAR-T). In various aspects, the mammalian 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 mammalian 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.
“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. Generally, a CAR is designed for a T cell and is a chimera 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. T cells and NK-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. Vol. 263: 68-89; Glienke et al., Front. Pharmacol. doi: 10.3389/fphar.2015.00021. There are various formats of CARs, each of which contains different components. “First generation” CARs join an antigen binding domain to the CD3zeta intracellular signaling domain of the T-cell receptor through hinge and transmembrane domains. “Second generation” CARs incorporate an additional domain, e.g., CD28, 4-1BB (41BB), or ICOS, to supply a costimulatory signal. “Third generation” CARs contain two costimulatory domains fused with the TCR CD3zeta chain. Third generation costimulatory domains may include, e.g., a combination of CD3zeta, CD27, CD28, 4-1BB, ICOS, or OX40. CARs so constructed can trigger, e.g., T cell activation upon binding the targeted antigen in a manner similar to an endogenous T cell receptor, but independent of the major histocompatibility complex (MHC).
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. Mutation of the NKG2D ectodomain is further described in, e.g., Culpepper et al., Mol. Immunol. 48, 516-523 (2011) and the Example. The mutated NKG2D is referred to herein as “INKG2D.” In various aspects, the iNKG2D domain comprises the amino acid sequence of SEQ ID NO: 15. The ectodomain is preferably associated with a transmembrane domain, an intracellular domain of a costimulatory molecule (e.g., 4-1BB or CD28), and/or a T cell receptor intracellular signaling domain. For example, in exemplary aspects of the disclosure, the iNKG2D ectodomain is fused to a CD8a hinge/transmembrane domain (e.g., comprising or consisting of the sequence of SEQ ID NO: 16), a 4-1BB domain (e.g., comprising or consisting of the sequence of SEQ ID NO: 17), and/or a CD3ζ domain (e.g., comprising or consisting of the sequence of SEQ ID NO: 18). In various aspects, the CAR comprises all of these components (e.g., SEQ ID NOs: 15-18 or SEQ ID NO: 19).
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. The CAR-expressing cell is referred to herein as “convertibleCAR.” An example of the system is illustrated in
A CAR cellular therapy may be an immunotherapy utilizing a subject or a patient's own immune cells that are engineered to be able to produce a particular CAR(s) on their surface. In some situations, cells (e.g., T cells) are collected from the body of a subject or a patient via apheresis. The cells (e.g., T cells) collected from the body are then genetically engineered to produce a particular chimeric antigen receptor on their surface. The CAR-expressing cells are 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. The cells may be isolated from the subject which will be recipient of the therapy, or may be isolated from a donor subject that is not ultimate recipient of the therapy. In various aspects, the cells are autologous CD4+ and CD8+ T cells.
The disclosure further provides a method of treating a subject for a disease or disorder associated with cells expressing CD20, such as cancer (CD20-positive cancers). 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. 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).
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.
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.
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.
The disclosure also provides (i) nucleic acid molecules (i.e., isolated nucleic acids) encoding the light chain 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). 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 also include complementary nucleic acids. In some instances, the sequences will be fully complementary (no mismatches) when aligned. In other instances, there may be up to about a 20% mismatch in the sequences. The disclosure 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 can be cloned into an expression vector, such as a plasmid, cosmid, bacmid, phage, artificial chromosome (BAC, 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 the 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.
Recombinant host cells comprising the nucleic acid molecules (optionally contained in expression vectors) also are provided. The recombinant 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.
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 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.
The following example is given merely to illustrate the present invention and not in any way to limit its scope.
This Example describes exemplary methods of producing an antibody fusion protein of the disclosure and NKG2D ectodomain-comprising CAR-T cells. The Example further demonstrates the ability of an antibody fusion protein comprising the variable region sequences from Rituximab and comprising an A1-A2 domain fused to the C-terminus of the light chain to selectively bind a CAR-T cell comprising the amino acid sequences of SEQ ID NOs: 15-18, and the ability of the antibody fusion protein and CAR-T cell combination to kill CD20-bearing cancer cells in vivo.
Cloning, expression, and purification: The wild-type ectodomain of NKG2D (UniProtKB P26718, residues 78-216; https://www.uniprot.org) was expressed as a fusion to the C-terminus of human IgG1 Fc via a short factor Xa recognizable Ile-Glu-Gly-Arg linker (Fc-wtNKG2D). Inert NKG2D variants comprising either a single Y152A (iNKG2D.YA) or double Y152A/Y199A substitution (INKG2D.AF) were generated by PCR-mediated mutagenesis or synthesized (gBlocks®, IDT). DNA constructs for Fc-NKG2D molecules were expressed in Expi293™ cells (Thermo Fisher Scientific) and dimeric secreted protein was purified by Protein A affinity chromatography (Pierce™ #20334, Thermo Fisher). Eluted material was characterized and further purified by size-exclusion chromatography (SEC) on an ÄKTA Pure system using Superdex 200 columns (GE Life Sciences). Correctly assembled, size-appropriate monomeric material was fractionated into phosphate-buffered saline (PBS).
The A1-A2 domains of human MICA*001 (UniProtKB Q29983, residues 24-205), MICB (UniProtKB Q29980.1, 24-205), ULBP1 (UniProtKB Q9BZM6, 29-212), ULBP2 (UniProtKB Q9BZM5, 29-212), ULBP3 (UniProtKB Q9BZM4, 30-212), ULBP5 (NCBI accession NP_001001788.2, 29-212), and ULBP6 (UniProtKB, 29-212) were cloned with a C-terminal 6×-His tag. Monomeric protein was purified from Expi293™ supernatants Ni-NTA resin (HisPur™, Thermo Fisher) and eluted material exchanged into PBS with Sephadex G-25 in PD-10 Desalting Columns (GE Life Sciences).
MIC ligands and orthogonal variants were cloned by ligation-independent assembly (HiFi DNA Assembly Master Mix, NEB #E2621) as fusions to the C-terminus of either the kappa light-chain or the heavy-chain of human IgG1 antibodies via either an APTSSSGGGGS or GGGS linker, respectively. Additionally, D265A/N297A (Kabat numbering) mutations were introduced into the CH2 domain of the heavy chain of all antibody and MicAbody clones to eliminate antibody-dependent cell cytotoxicity (ADCC) function. Heavy-and light-chain plasmid DNAs (in the mammalian expression vector pD2610-V12 (ATUM) for a given antibody clone were co-transfected into Expi293™ cells and purified by Protein A. For any monoclonal antibody fusion generated, the appropriate VL or VH domains were swapped into either the kappa light-chain or an ADCC-deficient IgG1 heavy-chain.
Inert NKG2D and orthogonal ligand engineering: Bio-layer interferometry (BLI) with the ForteBio Octet system (Pall ForteBio LLC) was implemented to validate loss of wild-type MIC ligand binding by iNKG2D. Fc-wtNKG2D, Fc-INKG2D.YA, or Fc-INKG2D.AF was captured on anti-human IgG Fc capture (AHC) biosensor tips and association/dissociation kinetics monitored in a titration series of monomeric MIC-His ligands. Additionally, ELISA (enzyme-linked immunosorbent assay) binding assays were performed with MICA-Fc, MICB-Fc, ULBP1-Fc, ULBP2-Fc, ULBP3-Fc, or ULBP4-Fc (R&D Systems) coated onto microtiter plates, a titration of biotinylated Fc-wtNKG2D or Fc-iNKG2D.YA, detected with streptavidin-HRP (R&D Systems #DY998), and developed with 1-Step Ultra TMB ELISA (Thermo Fisher #34208).
Phage display was employed to identify orthogonal ULBP2 A1-A2 variants that exhibited exclusive binding to either INKG2D.YA or INKG2D.AF. Synthetic NNK (where N=A/C/G/T and K=G/T, resulting in representation of all 20 amino acids without stop codons) DNA libraries were generated targeting the codons of helix 2 (residues 74-78, numbering based upon mature protein) or helix 4 (residues 156-160) that in the bound state are positioned in close proximity to the Y152 positions on the natural NKG2D receptor45. Müller et al., PLOS Pathog. 6, e1000723 (2010). Libraries exploring helix 2 alone, helix 4 alone, or the combination were cloned as fusions to the pIII minor coat protein of M13 phage, and phage particles displaying the mutagenized A1-A2 domain variants were produced in SS320 E. coli cells according to standard methods. These A1-A2 phage libraries were captured with either biotinylated Fc-INKG2D.YA or Fc-iNKG2D.AF protein (EZ-Link™ NHS-Biotin Kit, Thermo Fisher #20217) and enriched by cycling through four rounds of selection with increasing concentrations of non-biotinylated Fc-wtNKG2D competitor. Positive phage clones were verified for preferential binding to plate-bound Fc-iNKG2D.YA or Fc-iNKG2D.AF versus Fc-wtNKG2D by spot ELISA and bound phage detected with biotinylated M13 phage coat protein monoclonal antibody E1 (Thermo Fisher #MA1-34468) followed by incubation with streptavidin-HRP.
Phage variants were sequenced then cloned as human IgG1 monoclonal antibody fusions for additional validation. To confirm that selectivity of orthogonal variants was maintained in the bivalent MicAbody format, ELISA wells were coated with 1 ug/mL Fc-wtNKG2D, Fc-iNKG2D. YA, or Fc-iNKG2D.AF, and bound MicAbody was detected with an HRP-conjugated mouse-anti-human kappa chain antibody (Abcam #ab79115). Affinity of both monomeric and antibody-fused ULBP2 variants was also determined by Octet analysis as described above.
Generation of convertibleCAR-T cells: Human-codon optimized DNA (GeneArt, Thermo Fisher) comprising the CD8α-chain signal sequence, NKG2D variant, CD8α hinge and transmembrane domains, 4-1BB, CD3ζ, and eGFP were cloned into the pHR-PGK transfer plasmid for second generation Pantropic VSV-G pseudotyped lentivirus production along with packaging plasmids pCMVdR8.91 and pMD2.G48. The VH and VL domains of Rituximab separated by a (GGGGS)3 linker were substituted for the NKG2D module to generate the rituximab scFv-based CAR (RITscFv-CAR). For each batch of lentivirus produced, 6×106 Lenti-X 293T (Takara Bio #632180) cells were seeded in a 10 cm dish the day prior to transfection. Then 12.9 μg pCMVdR8.91, 2.5 μg pMD2.G and 7.2 μg of the pHR-PGK-CAR constructs were combined in 720 μl Opti-MEM™ (Thermo Fisher #31985062), then mixed with 67.5 μl of Fugene HD (Promega Corp. E2311), briefly vortexed, and incubated at room temperature for 10 minutes before adding to the dish of cells. After two days, supernatants were collected by centrifugation and passed through 0.22 μm filters. 5× concentrated PEG-6000 and NaCl were added to achieve final concentrations of 8.5% PEG-6000 (Hampton Research #HR2-533) and 0.3 M NaCl, incubated on ice for two hours, then centrifuged at 4° C. for 20 minutes. Concentrated viral particles were resuspended in 0.01 volume of PBS, and stored frozen at −80° C.
For primary human T cell isolation, a Human Peripheral Blood Leuko Pak (Stemcell Technologies #70500.1) from an anonymous donor was diluted with an equivalent volume of PBS+2% FBS, then centrifuged at 500×g for 10 minutes at room temperature. Cells were resuspended at 5×107 cells/ml in PBS+2% FBS and CD4+ or CD8+ cells enriched by negative selection (Stemcell EasySep™ Human CD4 T Cell Isolation Kit #17952 or EasySep Human CD8 T Cell Isolation Kit #17953) by addition of 50 μl of isolation cocktail per ml of cells and incubating for five minutes at room temperature. Subsequently, 50 μl of RapidSpheres™ were added per ml of cells and samples topped off (to each 21 mL cells, 14 mL of PBS). Cells were isolated for 10 minutes with an EasySEP™ magnet followed by removal of buffer while maintaining the magnetic field. Enriched cells were transferred into new tubes with fresh buffer and the magnet reapplied for a second round of enrichment after which cells were resuspended, counted, and cryopreserved at 10-15×106 cells/cryovial (RPMI-1640, Corning #15-040-CV; 20% human AB serum, Valley Biomedical #HP1022; 10% DMSO, Alfa Aesar #42780).
To generate CAR-T cells, one vial of cryopreserved cells was thawed and added to 10 ml T cell medium “TCM” (TexMACS medium, Miltenyi 130-097-196; 5% human AB serum, Valley Biomedical #HP1022; 10 mM neutralized N-acetyl-L-Cysteine; 1× 2-mercaptoethanol, Thermo Fisher #21985023, 1000×; 45 IUe/ml human IL-2 IS “rhIL-2”, Miltenyi #130-097-746) added at time of addition to cells. Cells were centrifuged at 400× g for 5 minutes then resuspended in 10 ml TCM and adjusted to 1×106/ml and plated at 1 ml/well in a 24 well plate. After an overnight rest 20 μL of Dynabeads™ Human T-Activator CD3/CD28 (Thermo Fisher #1131D) were added per well and incubated for 24 hours. Concentrated lentiviral particles (50 μL) were added per well, cells incubated overnight, then transferred to T25 flasks with an added 6 ml TCM. After three days of expansion, Dynabeads were removed (MagCellect magnet, R&D Systems MAG997), transduction efficiency assessed by flow cytometry for GFP, back-diluted to 5×105 cells/mL, and cell density monitored daily to ensure they did not exceed 4×106 cells/ml. When necessary, surface expression of iNKG2D was correlated with GFP expression using a MicAbody and detecting with PE-anti-human kappa chain (Abcam #ab79113) or by directly conjugating the Rituximab-MicAbody to Alexa Fluor 647 (Alexa Fluor Protein Labeling Kit #A20173, Thermo Fisher). The amount of iNKG2D expression on the surface of convertible CAR-CD8 cells was quantified using Alexa Fluor 647 conjugated Rituximab-MicAbody, and median fluorescence intensity was correlated with Quantum™ MESF 647 beads (Bangs Laboratories #647). All flow cytometry was performed on either Bio-Rad S3e Cell Sorter or Miltenyi MACSQuant Analyzer 10 instruments.
Cell lines and in vitro assays: Ramos human B cell lymphoma cells (ATCC #CRL-1596) were cultured in RPMI supplemented with 20 mM HEPES and 10% FBS. The mouse colon carcinoma line CT26 transfected to express human Her2 were also used. No additional mycoplasma testing nor authentication was performed except to verify by flow cytometry that target antigens were expressed.
For calcein-release assays, tumor cells were centrifuged and resuspend in 4 mM probenecid (MP Biomedicals #156370)+25 UM calcein-AM (Thermo Fisher #C1430) in T cell medium at 1-2×106 cells/ml for one hour at 37° C., washed once, and adjusted to 8×105 cells/ml. CD8+ CAR-T cells were pelleted and resuspended in 4 mM probenecid with 60 IUe/ml IL-2 in TCM at 4×106 cells/mL then adjusted according to the desired effector:target ratio (unadjusted for transduction efficiency). 25 UL target cells were plated followed by 25 UL medium or diluted MicAbody. Then 100 μL medium (minimum lysis), medium +3% Triton-X 100 (maximum lysis), or CAR-T cells were added and plates incubated at 37° C. for two hours. Cells were pelleted and 75 μL supernatant transferred to black clear-bottom plates and fluorescence (excitation 485 nm, emission cutoff 495 nm, emission 530 nm, 6 flashes per read) acquired on a Spectramax M2e plate reader (Molecular Devices). For experiments with armed convertibleCAR-CD8+s, T cells were pre-incubated at 37° C., with either saturating (5 nM) or a titration of MicAbody for 30 minutes before washing to remove unbound MicAbody and co-culturing with calcein-loaded target cells.
In order to quantify the target-dependent activation of T-cells, experiments were set up as described above except that calcein-preloading was omitted and assays set up in T cell medium without IL-2 supplementation. After 24 hours co-culture, supernatants were harvested and stored at −80° C. until the amount of liberated cytokine could be quantified by ELISA MAX™ Human IL-2 or Human IFN-g detection kits (BioLegend #431801 and #430101).
The MicAbody binding curve data were generated by ProMab Biotechnologies, Inc. (Richmond, CA). 3×105 convertibleCAR-CD8+ cells were plated in 96-wells V-bottom plates and incubated with labeled Alexa Fluor 647 labeled Rituximab. LC-U2S3 MicAbody for 30 minutes at room temperature in a final volume of 100 μL RPMI+1% FBS with a titration curve starting at 200 nM. Cells were then rinsed and median fluorescence intensity determined for each titration point by flow cytometry.
Animal studies: For PK analysis of serum levels of MicAbodies, six-week old female NSG mice (NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ, The Jackson Laboratory #005557) were injected intravenously (IV) with 100 μg of either parent rituximab antibody (ADCC-defective), heavy-chain U2S3 fusion of rituximab (Rituximab.HC-U2S3), or light-chain fusion (Rituximab.LC-U2S3). Collected sera were subjected to ELISA by capturing with human anti-rituximab idiotype antibody (HCA186, Bio-Rad Laboratories), detected with rat-anti-rituximab-HRP antibody (MCA2260P, Bio-Rad), and serum levels interpolated using either a rituximab or Ritxumab-U2S3 standard curve. PK analysis of U2S3-hFc-mutIL2 was performed in NSG mice by IP injection of 60 μg followed by regular serum collection. Samples were examined by ELISA capturing with Fc-iNKG2D and detecting with biotinylated rabbit-anti-human IL-2 polyclonal antibody (Peprotech #500-P22BT) followed by incubation with streptavidin-HRP. Half-lives were calculated in GraphPad Prism based upon the β-phase of the curve using a nonlinear regression analysis, exponential, one-phase decay analysis with the plateau constrained to zero.
For disseminated Raji B cell lymphoma studies, six-week old female NSG mice were implanted IV with Raji cells (ATCC #CCL-86) stably transfected to constitutively express luciferase from Luciola italica (Perkin Elmer RediFect Red-FLuc-GFP #CLS960003). Initiation of treatment administration is detailed in each in vivo study figure. For all experiments, CD4 and CD8 primary human T cells were independently transduced, combined post-expansion at a 1:1 mixture of CD4:CD8 cells without normalizing for transfection efficiency between cell types or CAR constructs, and the mixture validated by flow cytometry prior to IV injection. Administration of MicAbody or control antibody was by the intraperitoneal (IP) route unless otherwise specified, and in vivo imaging for bioluminescence was performed with a Xenogen IVIS system (Perkin Elmer). Animals were bled regularly to monitor human T cell dynamics by flow cytometry, staining with APC Anti-Human CD3 (clone OKT3, #20-0037-T100, Tonbo Biosciences), monitoring GFP, and examining cell-associated MicAbody levels with biotinylated Anti-Human F(ab′)2 (#109-066-097, Jackson ImmunoResearch Laboratories Inc.) followed by Streptavidin-PE detection (BD #554061). Serum ELISAs to monitor MicAbody levels was performed as described above.
For subcutaneous tumor studies 1×106 Raji cells were implanted in matrigel on the right flank of six-week old female NSG mice and therapy initiated when tumors reached 70-100 mm3. For the cohort that received armed convertibleCAR-T cells, the cells were incubated with 5 nM Rituximab.LC-U2S3 MicAbody ex vivo for 30 minutes at room temperature before washing and final mixing to achieve the desired 1:1 CD4:CD8 ratio and cell concentration. Arming was confirmed by flow cytometry with the biotinylated Anti-Human F(ab′)2 antibody and revealed a strong correlation between GFP and F(ab′)2 MFIs. These mice did not receive a separate MicAbody administration. Caliper measurements were regularly taken to estimate tumor volume (L×W×W×0.5=mm3), and terminal tumor masses were weighed.
Complement-mediated ablation of INKG2D.AF-CAR cells: To generate Fc reagents with enhanced complement binding and targeted delivery to the T cells expressing iNKG2D.AF, the orthogonal ligand was cloned as a fusion to either the N-(U2R-Fc) or C-terminus (Fc-U2R) of human IgG1 Fc via a GGGS linker with the Fc including the hinge, CH2, and CH3 domains. In addition to the wild-type Fc, the K326A/E333A21 (Kabat numbering, “AA”) and S267E/H268F/S324T/G236A/1332E20 (“EFTAE”) C1q-enhanced binding mutation sets were explored. All were expressed in Expi293T cells, purified, and fractionated as described above. Confirmatory ELISAs were performed by capturing with Fc-NKG2D.AF followed by binding U2R/Fc-variant fusions at 1 μg/mL concentration, titrating in human-C1q protein (Abcam #ab96363), then detecting with polyclonal sheep-anti-C1q-HRP antibody (Abcam #ab46191). Complement-dependent cytotoxicity (CDC) assays were performed by iQ Biosciences (Berkeley, CA). Briefly, 5×104 CD8+ cells from an NKG2D.AF-CAR transduction were plated in 96-well plates and incubated for three hours with a serial dilution of each U2R/Fc-variant fusion, in triplicate, in the presence of normal human serum complement (Quidel Corporation) at a final concentration of 10% (v/v). Cells were then harvested and resuspended with SYTOX™ Red dead cell stain (Thermo Fisher) at a final concentration of 5 μg/mL and analyzed by flow cytometry. EC50 values for cytotoxicity were calculated in GraphPad prism fitted to a non-linear regression curve.
Delivery of mutant-IL2 to T cells expressing INKG2D-CAR: To generate a reagent that was monomeric for the U2S3 ligand, monomeric for a mutant IL-2 with the inability to bind IL-2Rx (mutIL2, R38A/F42K) (Heaton et al., Cancer Res. 53, 2597-2602 (1993); Sauve et al., Proc. Natl. Acad. Sci. U.S.A. 88, 4636-4640 (1991) yet retained serum stability, a heterodimeric Fc strategy was employed. Gunasekaran et al., J. Biol. Chem. 285, 19637-19646 (2010). U2S3 was fused to the N-terminus of the Fc-hinge of one chain with K392D/K409D (Kabat numbering) mutations while the mutIL2 was fused to the C-terminus of the second Fc-chain which harbored E356K/D399K mutations. Additionally, D265A/N297A mutations were introduced in both Fc chains to render the Fc ADCC-deficient. Expression in Expi293T cells and purification was as described above. Appropriately assembled U2S3-hFc-mutIL2 material was fractionated by SEC and the presence of individual size-appropriate polypeptides was confirmed by denaturing SDS-PAGE. A direct fusion between orthogonal ligand and mutIL2 expressed as a single polypeptide with a linker comprising glycine-serine linkages, a FLAG tag, and a 6×His tag was also generated and purified by Ni-NTA exchange chromatography. Ghasemi et al., Nat Commun 7, 12878 (2016). Determination of IUe activity equivalents was based on the calculation that a 4.4 M solution of wild-type IL-2 has the equivalent of 1000 IU/μL. IL-15 with a V49D mutation, which reduced binding to IL-15Ra but retained bioactivity26, was similarly formatted with U2S3. Bernard et al., J. Biol. Chem. 279, 24313-24322 (2004).
CAR-T cell proliferation in response to various cytokines or U2S3-cytokine fusions was quantified with the WST-1 Cell Proliferation Reagent (Millipore Sigma #5015944001). Briefly, CAR-T cells were pelleted and resuspended in T cell media without IL-2, dispensed into 96-well plates at 4×104 cells/well, and the appropriate amount of diluted U2S3-cytokine fusions was added to achieve 30 IUe/mL or higher concentration as needed in a final assay volume of 100 μL per well. Recombinant-human IL2 and IL 15 (Peprotech #200-02 and #200-15) were included as controls. After incubation for three days at 37° C., 10 μL of WST-1 was added to each well and allowed to incubate for 30-60 minutes before quantifying intensity of color development on a plate reader. Changes in the proportion of GFP+ CAR-expressing cells in response to U2S3-cytokine fusion were monitored by flow cytometry. To monitor activation of STAT3 or STAT5 upon cytokine-fusion engagement cells were rested overnight in TCM media without IL-2 supplementation then treated with 150 IUe/mL IL-2, IL-15, U2S3-hFc-mutIL2, or U2S3-hFc-mutIL 15 for two hours before fixing and staining for intracellular phospo-STAT3 (Biolegend PE anti-STAT3 Tyr705 clone 13A3-1) and -STAT5 (BD Alexa Fluor 647 anti-STAT5 pY694 clone 47). To monitor a temporal response, treated convertibleCAR-CD8 T cells were fixed at 0, 30, 60, and 120 minutes after exposure to cytokines or U2S3-hFc-cytokine fusions then stained.
Human PBMC stimulation and immune-phenotyping studies were performed. Briefly, normal PBMCs from three donors were seeded in 96-well plates at 1×105 cells/well and exposed to a 10-fold dilution series of either U2S3-hFc-mutIL2 or U2S3-hFc-wtIL2 (wild-type IL2) for four days at 37° C., with 5% CO2. Positive controls included wells coated with anti-human CD3 (OKT2) at 2 μg/mL and rhIL-2 at 300 IUe/mL. After incubation, cells were treated with TruStain FcX block (BioLegend #422301) followed by staining with BioLegend antibody panels for proliferating T cells (CD8 clone RPA-T8 #301050, CD4 clone OKT4 #317410, CD3 clone OKT3 #300430, KI-67 #350514), and Treg cells (Fox3 clone 206D #320106, CD4 clone OKT4, CD3 clone OKT3, KI-67).
Engineering an orthogonal NKG2D-ligand interaction: Two central tyrosine residues in each NKG2D monomer have critical roles in driving receptor-ligand interactions. Culpepper et al., Mol. Immunol. 48, 516-523 (2011). Mutations at these residues were heavily explored, with the Y152A mutant (“INKG2D.YA”) and the Y152A/Y199F double mutant (“iNKG2D.AF”) selected for further study and confirmed by biolayer interferometry (BLI) (
Candidate orthogonal variants were similarly identified for iNKG2D.AF and ELISAs comparing rituximab-LC fusions to Fc-wtNKG2D, Fc-iNKG2D. YA, and Fc-INKG2D.AF identified four variants that selectively bound only iNKG2D.AF (
Expression of INKG2D. YA as a chimeric antigen receptor. Lentiviral transduction of iNKG2D.YA fused to 4-1BB, CD3ζ, and eGFP into primary human T cells efficiently generated convertibleCAR-T cells with robust transgene expression on par with a rituximab-scFv based CAR construct (RITscFv-CAR) with the same hinge, transmembrane, and intracellular architecture (
Staining of convertibleCAR-CD8+ cells with a fluorescently labeled Rituximab.LC-U2S3 MicAbody revealed saturation of total iNKG2D. YA-CAR receptors at 5 nM (
convertibleCAR-T cells inhibit disseminated B-cell lymphomas: The pharmacokinetics of both the HC and LC Rituximab-U2S3 MicAbodies (
Rituximab.LC-U2S3 (Rit-S3; the antibody fusion protein wherein the U2S3 A1-A2 domain is fused to the light chain of the antibody) was deployed in further experiments exploring dosing parameters for lymphoma control. An intermediate Rit-S3 dose of 20 μg was shown to be the most efficacious as high concentrations may result in over saturation of receptors on the CAR cells and antigens on the tumor cells, thereby interfering with productive engagement. Additionally, a higher frequency of Rit-S3 administration of every two days versus every four days paired with a higher dose (10×106) of convertibleCAR-T cells resulted in the greatest suppression of tumor growth. Rit-S3 alone was ineffective at tumor control while a graft-vs-tumor effect was consistently observed in both untransduced and convertibleCAR only cohorts. Rit-S3 was detectable in the serum of mice throughout the course of the study with peak levels appearing earlier with more frequent dosing.
A Raji disseminated lymphoma model with optimized convertibleCAR-T dosing was performed with 20 μg Rit-S3 dosing every two days comparing 5×106 (5M) to 15×106 (15M) convertibleCAR-T cells. As a positive control, RITscFv-CAR cells were also included which have in vitro Ramos killing potency comparable to convertibleCAR-T cells (
convertibleCAR-T cells inhibit subcutaneous lymphomas: Raji B-cells were implanted subcutaneously to assess the ability of the convertibleCAR system to suppress growth of a solid tumor mass. Once tumors were established at 10 days, either 7×106 (7M) or 35×106 (35M) convertibleCAR-Ts were administered after a single IV dose of 60 μg Rit-S3. Additionally, one cohort received 35M cells that were pre-armed with a saturating concentration of Rit-S3 prior to administration but no additional MicAbody introduced injections. Administration of 7M convertibleCAR-T cells along with Rit-S3 (7M+Rit-S3) resulted in reduced tumor size relative to convertibleCAR-T cells alone (
Selective delivery of biomolecules to convertibleCAR-T cells: The privileged interaction between iNKG2D variants and their orthogonal ligands enables the selective delivery of agents to iNKG2D-CAR expressing cells simply by fusing them as payloads to the orthogonal ligands themselves. To demonstrate the utility of this feature two disparate applications were explored: targeted ablation utilizing the complement system and selective delivery of activating cytokines. In the first application, the U2R variant was fused to either the N- or C-terminus of the wild-type human IgG1 Fc-domain or to mutant Fc domains previously described as enhancing C1q binding-S267E/H268F/S324T/G236A/1332E (“EFTAE”)20 and K326A/E333A (“AA”)21 (
The potential ability of orthogonal ligands to deliver cytokines selectively to iNKG2D-CAR expressing cells has advantages to not only promote their expansion but also potentially leverage differential cytokine signaling to control T cell phenotype and function. As a general design principle, mutant cytokines with reduced binding to their natural receptor complexes were employed to reduce their engagement with immune cells not expressing the CAR and to minimize toxicity associated with wild-type cytokines. Additionally, cytokine fusions were kept monovalent to eliminate avidity-enhanced binding and signaling. To this end, the R38A/F42K mutations in IL-2 (mutIL2)25 and the V49D mutation in IL-15 (mutIL 15) dramatically reduce binding to each cytokine's respective Ra subunit while maintaining IL-2Rβ/γ complex engagement. Initial experiments using the iNKG2D.YA orthogonal variant U2S2 fused to either mutIL2 or mutIL 15 promoted proliferation of INKG2D.YA-CAR expressing cells but not those expressing wtNKG2D-CAR (
U2S3-hFc-mutIL2 was shown to have an in vivo PK half-life of a few days (
The effect of U2S3-hFc-mutIL2 on normal human PBMCs from three donors was explored in vitro by exposure to increasing concentrations of the agent for four days followed by flow-based quantification of cells positive for the proliferative marker Ki-67 (
Comparison of A1-A2 domain location and linkers: In addition to the studies above, constructs comprising different linkers connecting the antibody heavy or light chains to A1-A2 domains were studied. See
The disclosure describes the engineering of a privileged receptor-ligand (iNKG2D.YA and U2S3) pairing comprised of human components for a highly adaptable CAR, resulting in a versatile and broadly controllable platform. The iNKG2D.YA-CAR receptor itself is held invariant on T cells with CAR function readily directed to potentially any antigen of interest by virtue of attaching the orthogonal ligand to the appropriate antigen-recognizing antibody. In this manner, the same convertibleCAR-T cells can be retargeted as needed if, for example, the original tumor antigen becomes downregulated during the course of therapy. This targeting flexibility is not limited to sequential engagement of antigens, but can also be multiplexed to simultaneously direct T cells to more than one antigen in order to reduce the likelihood of tumor escape by antigen loss, address the issue of heterogeneity of intratumoral antigen expression, or even simultaneously target tumor and suppressive cellular components of the tumor microenvironment. Traditional scFv-CAR cells are generally committed to a fixed expression level of a receptor which reduces their ability to discriminate between antigen levels present on healthy versus aberrant cells. The use of switch/adaptor strategies, like MicAbodies with convertibleCAR-T cells, may provide an opportunity to differentially engage CAR-Ts to achieve a therapeutic index that reduces the risk of severe adverse events.
The use of the privileged receptor-ligand interaction for delivery of payloads specifically to iNKG2D-bearing cells without additional cellular engineering is another advantage. The capability of harnessing interleukin functions to drive expansion and activation, prevent exhaustion, or even promote suppression in a controlled and targeted manner could have beneficial consequences for efficacy and safety. Introduction of cytokine-ligand fusions during CAR manufacturing could address qualitative and quantitative limitations of patient T cells and their administration post-CAR infusion could expand the number of CAR-T cells and their persistence which, with CD19-CAR therapies, is correlated positively with response rates. Most CAR therapies require a preconditioning lymphodepletion regimen to promote engraftment and expansion of CAR cells, one rationale being that it provides a more verdant immunological setting for CARs to expand. Robust and controllable convertibleCAR-T expansion in patients may supplant the need for lymphodepletion, allowing for retention of endogenous immune functions that are fully competent to support the initial convertibleCAR-mediated anti-tumor activity. Another clinical strategy might be to deliver cytokine-ligand fusions to bolster convertibleCAR-T function, possibly with a cycling regimen to reduce T cell exhaustion and promote the maintenance of memory T cells. And lastly, as CARs have been demonstrated to persist in humans for years post-infusion, the ability to recall resident convertibleCAR-Ts to attack primary or secondary malignancies (either with the original targeting MicAbody or a different one) without having to re-engineer or generate a new batch of CAR cells should be highly advantageous. Unlike scenarios where CARs have been engineered to constitutively express cytokines, delivery of cytokines exclusively to convertibleCAR-T cells can be modulated depending upon the manufacturing or clinical needs.
By design, each component of the convertibleCAR system—the iNKG2D-based CAR receptor and the MicAbody (which is ADCC-deficient)—are functionally inert on their own. This has advantages during manufacturing, particularly in the context of indications such as T cell malignancies where traditional scFv-based CARs encounter expansion hurdles due to fratricide. Additionally, it provides enhanced control of CAR function during treatment. The disclosure demonstrates that convertibleCAR-T cells can be armed with MicAbody prior to administration to provide an initial burst of anti-tumor activity on par with traditional scFv-CARs. In addition to activation-induced replication, these cells also internalize their engaged CAR receptors in a manner consistent with what has been observed with other 4-1BB/CD3zeta scFv-CARs. As a consequence of these two processes, convertibleCAR-T cells will rapidly disarm after initial expansion and target engagement, which then provides an opportunity rearm and re-engage in a manner controlled by MicAbody dosing.
In addition to the iNKG2D-U2S3 pairing based upon ULBP2, the disclosure identifies high-affinity orthogonal MicA and ULBP3 variants to iNKG2D.YA that are non-redundant in their amino acid compositions through the helix 4 domain. Additionally, a completely independent iNKG2D.AF and U2R pairing is described. Having mutually exclusive receptor-ligand pairs enables, for example, their introduction into distinct cell populations (e.g., CD4 and CD8 T-cells) to differentially engage them as needed. Furthermore, within the same cell, the two iNKG2D variants could be expressed with split intracellular signaling domains to provide dual antigen-dependent activation to enhance on-tumor selectivity. Alternatively, the two iNKG2D variants could be differentially linked to either activating or immunosuppressive domains to enhance the discriminatory power of the T cells between tumors or healthy tissue, respectively.
In summary, the system described herein has demonstrated capabilities to not only be readily targeted to different cell-surface antigens but can also be selectively engaged exogenously to drive cell expansion. The privileged receptor-ligand interaction that has been developed is agnostic to cell type and can be engineered into any cell of interest as long as the cell-appropriate signaling domains are provided. Additionally, the adoptive cellular therapy field is aggressively pursuing the development of allogeneic cells to bring down the time, complexity, and cost of manufacturing to provide a more consistent, readily accessible product. A highly adaptable CAR system would be powerfully synergistic with allogenic efforts and once a truly universal allogeneic CAR system has been validated, the therapeutic field then becomes characterized by the relative ease of developing and implementing a library of adaptor molecules from which personalized selections can be made. This strategy also broadens the potential areas of application to any pathogenic call with a targetable surface antigen.
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.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US21/36461 | 6/8/2021 | WO |
