Compositions and systems for combination therapy containing fucosylated cells and immune checkpoint inhibitors, and their manufacture and use

Abstract

Disclosed are compositions, systems, and kits for adoptive cell therapy, comprising at least one immune checkpoint inhibitor and at least one immune cell type, wherein the at least one immune cell type is ex vivo fucosylated (fucoACT). Also disclosed are methods of making and using these compositions, systems, and kits.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 383,391, filed November 11, 2022, the entire contents of which are expressly incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not applicable. [Background technology]

[0003] Cancer is a significant cause of morbidity and mortality worldwide. Although treatment standards for many different types of cancer have improved significantly in recent years, current standards still fall short of the need for effective therapies to improve cancer treatment. The renaissance of cancer immunotherapy, which includes antibodies, vaccines, cytokines, oncolytic viruses, bispecific molecules, and cell therapies, is exemplified by the approval of several immunotherapeutic antibody agents, collectively referred to as immune checkpoint inhibitors (ICIs), targeting, for example (but not limited to), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed death receptor-1 (PD-1) and its ligand PD-L1, and lymphocyte-activation gene 3 (LAG-3). The renaissance of cancer immunotherapy is also exemplified by the approval of several cell-based immunotherapies, collectively referred to as adoptive cell therapy (ACT). ACT is created by isolating a patient's own immune cells, expanding them ex vivo, and then reinjecting them. The majority of adoptive cell therapy strategies utilize T cells isolated from tumors or peripheral blood, but some also utilize other immune cell subsets or cells derived from induced pluripotent stem cells (iPSCs). T cell therapies, including tumor-infiltrating lymphocytes (TILs), T cell receptor-transduced T cells (TCR-T cells), and chimeric antigen receptor-transduced T cells (CAR-T cells), act as "living drugs" because the infused cells proliferate, engraft, and persist in vivo, enabling long-term application and sustained remission in a subset of patients. Adoptive cell therapy has been less successful in the treatment of solid tumors due to the poor homing, proliferation, and survival of transplanted cells. [Brief explanation of the drawings]

[0004] [Figure 1] FIG. 1 is a diagram illustrating one non-limiting embodiment of a combination therapy constructed in accordance with the present disclosure, which includes the use of ex vivo fucosylated tumor-infiltrating lymphocytes and an immune checkpoint inhibitor. DETAILED DESCRIPTION OF THE INVENTION

[0005] Before describing in detail at least one embodiment of the present disclosure by way of illustrative language and results, it is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description. The present disclosure is capable of other embodiments or of being practiced or carried out in various ways. Therefore, the language used herein is intended to be accorded the broadest possible scope and meaning, and the embodiments are exemplary and not exhaustive. It is also to be understood that the phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting.

[0006] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. The nomenclature used in connection with surgery, anesthesia, wound healing, and infection control, as well as the medical procedures and techniques described herein, are those well known and commonly used in the art. Standard techniques are used for the diagnosis and treatment of infectious diseases.

[0007] All patents, published patent applications, and non-patent literature mentioned in this specification is indicative of the level of skill of those skilled in the art to which this disclosure pertains. All patents, published patent applications, and non-patent literature mentioned in any part of this application are expressly incorporated by reference herein in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0008] All of the articles, systems, kits, and / or methods disclosed herein can be made and executed without undue experimentation in light of the present disclosure. While the articles, systems, kits, and / or methods have been described in terms of specific embodiments, it will be apparent to those skilled in the art that variations may be applied to the articles, systems, kits, and / or methods, and to the steps or sequence of steps of the methods described herein, without departing from the concept, spirit, and scope of the present disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present disclosure as defined by the appended claims.

[0009] As utilized in accordance with the present disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0010] In the claims and / or specification, the use of the words "a" or "an," when used in conjunction with the word "comprising," can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more." Thus, the terms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, the phrase "a compound" can refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or any greater number of compounds. The term "plurality" means "two or more."

[0011] Use of the term "at least one" will be understood to include not only one, but any amount greater than one, including, but not limited to, two, three, four, five, ten, fifteen, twenty, thirty, forty, fifty, one hundred, etc. The term "at least one" can extend to one hundred or one thousand or more, depending on the term to which it is attached. Furthermore, the amounts of 100 or 1000 are not considered limiting, as higher limits may also provide satisfactory results. Furthermore, use of the term "at least one of X, Y, Z" will be understood to include X alone, Y alone, Z alone, and any combination of X, Y, and Z.

[0012] The use of ordinal terminology (i.e., "first," "second," "third," "fourth," etc.) is merely to distinguish between two or more items and does not imply, for example, any order, sequence, importance, or additional order to an item unless expressly stated otherwise.

[0013] The use of the word "or" in the claims is intended to mean an inclusive "and / or" unless explicitly stated to refer to alternatives only or unless the alternatives are mutually exclusive. For example, a condition "A or B" is satisfied by either A being true (or present) and B being false (or absent), A being false (or absent) and B being true (or present), or both A and B being true (or present).

[0014] As used herein, a reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment. For example, the appearances of the phrase "in some embodiments" or "in one example" in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, all references to one or more embodiments or examples should not be construed as limiting the scope of the claims.

[0015] Throughout this application, the term "about" is used to indicate that a value includes the variation of error inherent in the composition / device / instrument, the method employed to determine the value, or the variation that exists between test subjects. For example, and without limitation, when the term "about" is used, the specified value can vary plus or minus 20%, 15%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from the specified value, as appropriate for performing the disclosed methods and as understood by one of ordinary skill in the art.

[0016] As used in this specification and claims, the terms "comprising" (and any form of "comprising," such as "comprise" and "comprises"), "having" (any form of having, such as "have" and "has"), "including" (any form of including, such as "includes" and "include"), or "containing" (any form of including, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unlisted elements or method steps. For example, a process, method, article, or apparatus consisting of a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or elements inherent therein.

[0017] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed before that term. For example, "A, B, C, or combinations thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and, if order is important in the particular context, also includes BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations including one or more repeats of an item or term are expressly included, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. One of ordinary skill in the art will understand that there is typically no limit to the number of items or terms in any combination, unless otherwise clear from the context.

[0018] As used herein, the term "substantially" means that the subsequently described event or circumstance occurs entirely, or that the subsequently described event or circumstance occurs to a great extent or degree. For example (and not by way of limitation), when relating to a particular event or circumstance, the term "substantially" means that the subsequently described event or circumstance occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. The term "substantially adjacent" can mean that two items are 100% adjacent to each other, or that two items are close to each other but not 100% adjacent to each other, or that a portion of one of two items is close to the other item but not 100% adjacent to it.

[0019] As used herein, the phrases "associated with," "coupled to," and "connected to" include both direct association / couple / connection of two elements to one another and indirect association / couple / connection of two elements to one another. When two elements are indirectly associated / coupled / connected to one another, there may be one or more intervening elements between them (such as, but not limited to, a bridge).

[0020] All patents, applications, published applications, and other publications cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The chemical structures and formulas set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts. If there is a discrepancy between a drawn structure and the name given to that structure, the drawn structure should bear more weight. If the stereochemistry of a structure or portion of a structure is not shown in the drawn structure or portion of the drawn structure, the drawn structure is to be interpreted as encompassing all of its possible stereoisomers.

[0021] Methods, devices, and materials similar or equivalent to those described herein can be used in the practice of the present disclosure. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not intended to limit the scope of the present disclosure. In the event that there is a plurality of definitions for terms in this section, the definition in this section prevails unless stated otherwise. The headings used herein are for organizational purposes only and are not intended to limit the disclosure in any way.

[0022] The term "adoptive cell therapy" or "ACT" refers to the transplantation of cells into a patient, which cells can be derived from the patient, another individual, or from induced pluripotent stem cells (iPSCs).

[0023] The term "tumor-infiltrating lymphocytes" or "TILs" refers to any population of lymphocytes that have the ability to infiltrate tumor tissue. TIL therapy is a form of ACT in which infiltrating lymphocytes from tumors or circulating lymphocytes are collected, cultured and expanded in vitro, and then infused into one or more patients to treat a disease state or disorder.

[0024] The term "chimeric antigen receptor T cells" or "CAR-T cells" refers to T cells that have been genetically engineered to produce an artificial T-cell receptor for use in immunotherapy. CAR-T cell therapy is a form of ACT that involves collecting circulating lymphocytes from the patient, another donor, or iPSCs, genetically engineering the collected lymphocytes, culturing / expanding the genetically engineered cells in vitro, and infusing the cultured / expanded genetically engineered lymphocytes into one or more patients to treat a condition or disease.

[0025] The term "chimeric antigen receptor NK cells" or "CAR-NK cells" refers to NK cells that have been genetically engineered to produce an artificial NK cell receptor for use in immunotherapy. CAR-NK cell therapy is a form of ACT that involves harvesting circulating NK cells from the patient, another donor, or iPSCs, genetically engineering the NK cells, culturing / expanding the genetically engineered NK cells in vitro, and infusing the cultured / expanded genetically engineered NK cells into one or more patients for the treatment of a condition or disorder.

[0026] The term "PD-1 inhibitor" refers to a moiety (e.g., a compound, nucleic acid, polypeptide, antibody, or fragment thereof, etc.) that reduces, inhibits, blocks, neutralizes, or prevents the activity or expression of PD-1 (e.g., programmed cell death protein 1; PD-1 (CD279); GI:145559515), including variants, isoforms, species homologs (e.g., murine) of human PD-1, and analogs that share at least one epitope with PD-1. PD-1 inhibitors include, but are not limited to, molecules and macromolecules such as compounds, nucleic acids, polypeptides, antibodies, peptibodies, diabodies, minibodies, single-chain variable fragments (ScFv), and fragments or variants thereof. Thus, as used herein, a PD-1 inhibitor refers to any moiety that antagonizes PD-1 activity or expression. The efficacy of a PD-1 inhibitor can be measured, for example, by the 50% inhibitory concentration (half-maximal inhibitory concentration or IC 50 ) can be measured. PD-1 inhibitors include exemplary compounds and compositions described herein. PD-1 antibody refers to a PD-1 inhibitor that is a monoclonal or polyclonal antibody described herein.

[0027] The term "PD-L1 inhibitor" refers to a moiety (e.g., a compound, nucleic acid, polypeptide, antibody, or fragment thereof) that reduces, inhibits, blocks, neutralizes, or interferes with the activity of PD-L1 (e.g., programmed cell death protein ligand 1; PD-L1 (CD274); GI:30088843), the binding of PD-L1 to its receptor, PD-1, or expression, including human PD-L1 variants, isoforms, species homologs (e.g., murine), and analogs that share at least one epitope with PD-L1. PD-L1 inhibitors include, but are not limited to, molecules and macromolecules such as chemical compounds (small molecules), nucleic acids, polypeptides, antibodies, peptibodies, diabodies, minibodies, single-chain variable fragments (ScFv), and fragments or variants thereof. Therefore, as used herein, PD-L1 inhibitor refers to any moiety that antagonizes PD-L1 activity, binding to PD-1, or expression. The efficacy of PD-L1 inhibitors can be measured, for example, by measuring the 50% inhibitory concentration (half maximal inhibitory concentration or IC 50 ) can be measured. PD-L1 inhibitors include the exemplary compounds and compositions described herein. PD-L1 inhibitor antibody refers to a PD-L1 inhibitor that is a monoclonal or polyclonal antibody described herein.

[0028] The term "LAG-3 inhibitor" refers to a moiety (e.g., a compound, nucleic acid, polypeptide, antibody, or fragment thereof, etc.) that reduces, inhibits, blocks, neutralizes, or interferes with the activity or expression of LAG-3 (e.g., lymphocyte activation gene 3; LAG-3 (CD223); GI:251757512), including variants, isoforms, species homologs (e.g., murine) of human LAG-3, and analogs having at least one shared epitope with PD-1. LAG-3 inhibitors include, but are not limited to, molecules and macromolecules such as compounds, nucleic acids, polypeptides, antibodies, peptibodies, diabodies, minibodies, single-chain variable fragments (ScFv), and fragments or variants thereof. Thus, as used herein, a LAG-3 inhibitor refers to any moiety that antagonizes LAG-3 activity or expression. The efficacy of a LAG-3 inhibitor can be measured, for example, by the 50% inhibitory concentration (half-maximal inhibitory concentration or IC 50 ) can be measured. LAG-3 inhibitors include exemplary compounds and compositions described herein. LAG-3 antibody refers to a LAG-3 inhibitor that is a monoclonal or polyclonal antibody described herein.

[0029] The terms "polypeptide" and "protein" are used interchangeably herein to refer to any molecule containing at least two or more amino acids.

[0030] The term "effective amount" refers to the amount of a therapy (e.g., each active agent, combination of agents, or another active agent, e.g., an anti-cancer agent described herein) sufficient to achieve a stated purpose or effect for which it is administered. An effective amount may be sufficient to reduce and / or ameliorate the progression, onset, recurrence, severity, and / or duration of a given disease, disorder, or condition and / or its associated symptoms, or to reduce the level of activity or binding of a polypeptide (e.g., PD-1, PD-L1, or LAG-3). An effective amount may be a "therapeutically effective amount," which refers to an amount sufficient to reduce or ameliorate the progression or progression of a given disease, disorder, or condition, reduce or ameliorate the recurrence, onset, or occurrence of a given disease, disorder, or condition, and / or improve or enhance the prophylactic or therapeutic effect of another therapy, including, but not limited to, reducing or ameliorating the progression or progression of a given disease, disorder, or condition, reducing or ameliorating the recurrence, onset, or occurrence of a given disease, disorder, or condition, and / or improving or enhancing the prophylactic or therapeutic effect of another therapy. A therapeutically effective amount of one or more compositions described herein may enhance the therapeutic efficacy of another therapeutic agent.

[0031] The term "regimen" refers to a protocol for the administration and timing of one or more therapies (e.g., a combination described herein or another active agent, e.g., an anti-cancer agent described herein) to treat a disease, disorder, or condition described herein. A regimen can include periods of active administration and rest periods, as known in the art. A period of effective administration includes the duration of administration of the compositions and combinations described herein and the effectiveness of such combinations and compositions. Rest periods of the regimens described herein include periods during which the compounds are not actively administered and, in certain instances, also include periods during which the effectiveness of such compounds may be minimal. The combination of active administration and rest periods in the regimens described herein can increase the effectiveness and / or duration of administration of the compositions and combinations described herein.

[0032] As used herein, the terms "therapy" and "therapies" refer to any protocol, method, and / or agent that can be used to prevent, treat, manage, and / or ameliorate a disease, disorder, condition, or one or more symptoms thereof. In certain non-limiting examples, the term(s) refer to the administration of one or more active agents to a patient. In other non-limiting examples, the term(s) refer to one or more treatments administered to a patient.

[0033] The term "patient" or "subject" refers to a mammal, such as a human, cow, rat, mouse, dog, monkey, ape, goat, sheep, cattle, deer, etc. Generally, a patient as described herein is a human.

[0034] The terms "inhibition," "inhibit," and "inhibiting" refer to the reduction of polypeptide activity, binding, or expression, or the alleviation or amelioration of a disease, disorder, condition, or symptom thereof. As used herein, "inhibiting" includes partially or totally blocking a stimulus, reducing, preventing, or delaying activation or binding, or inactivating, desensitizing, or down-regulating protein or enzyme expression, activity, or binding.

[0035] Antibodies as described herein can be polyclonal or monoclonal, and include xenogeneic, allogeneic, or syngenic forms, and modified forms thereof (e.g., humanized or chimeric). An "antibody" is intended to mean a polypeptide product of B cells belonging to the immunoglobulin class of polypeptides, capable of binding to a specific molecular antigen, and composed of two identical pairs of polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), the amino-terminal portion of each chain containing a variable region of about 100 to about 130 or more amino acids, and each carboxy-terminal portion of each chain containing a constant region (see Borrebaeck (ed.) (1995) Antibody Engineering, Second Edition, Oxford University Press; Kuby (1997) Immunology, Third Edition, W.H. Freeman and Company, New York). Specific molecular antigens to which the antibodies described herein may bind include PD-1, PD-L1, LAG-3, etc., and epitopes thereof.

[0036] The term "monoclonal antibody" refers to a population of antibody molecules that contain one species of antigen-binding site capable of immunoreacting with a particular epitope of an antigen, and the term "polyclonal antibody" refers to a population of antibody molecules that contain multiple species of antigen-binding sites capable of interacting with a particular antigen. Monoclonal antibodies typically exhibit a single binding affinity for the particular antigen with which they immunoreact. For example, monoclonal antibodies for use in accordance with the present disclosure can be produced using, for example, hybridoma methods (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981); recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567); phage display techniques (e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 222:581-597 (1992)); al.,J.Mol.Biol.338(2):299-310(2004);Lee et al.,J.Mol.Biol.340(5):1073-1093(2004);Fellouse,Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004);and Lee et al. al., J. Immunol.Methods 284(1-2):119-132 (2004)); and techniques for producing human or human-like antibodies in animals that have some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998 / 24893, WO 1996 / 34096, WO 1996 / 33735, WO 1991 / 10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol.7:33(1993);USPat.Nos.5,545,807;5,545,806;5,569,825;5,625,126;5,633,425;and 5,661,016;Marks et al.,Bio / Technology 10:779-783(1992);Lon berg et al.,Nature 368:856-859(1994);Morrison,Nature 368:812-813(1994);Fishwild et al.,Nature Biotechnol.14:845-851(1996);Neuberger,Nature Biotechnol.14:826(1996);and Lonberg and See Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[0037] As used herein, monoclonal antibodies also include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical to or homologous to the corresponding sequences of antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to the corresponding sequences of antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,56; Morrison et al., Proc. Natl. Acad. Sci. USA, pp. 6851-6855 (1984)). "Humanized antibodies" can be considered a subset of the chimeric antibodies described herein.

[0038] The term "human" as used with respect to an antibody or functional fragment thereof (e.g., a "humanized antibody(ies)") refers to an antibody or functional fragment thereof having a human variable region or portion thereof that corresponds to human germline immunoglobulin sequences. Such human germline immunoglobulin sequences are described by Kabat et al. (Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242 (1991)). Human antibodies in the context of the present disclosure can include antibodies that bind to PD-1, PD-L1, LAG-3, etc., or variants thereof, as described herein.

[0039] In certain embodiments, a human antibody is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or an antibody produced using any of the techniques for producing human antibodies disclosed herein. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Human monoclonal antibodies can also be prepared using the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 2:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenoMouse, that have been engineered to produce such antibodies in response to antigen challenge, but in which the endogenous gene locus has been disabled (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584, regarding XenoMouse technology). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), regarding human antibodies generated via human B cell hybridoma technology.

[0040] A "humanized antibody" refers to an antibody produced by a non-human cell having variable regions, or variable and constant regions, modified to more closely resemble antibodies produced by a human cell. This can be achieved, for example, by modifying the amino acid sequence of a non-human antibody to incorporate amino acids found in human germline immunoglobulin sequences. The humanized antibodies of the present disclosure can include, for example, amino acid residues in the CDRs that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). Humanized antibodies can also include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences.

[0041] Humanized forms of non-human (e.g., murine) antibodies are antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues in a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and / or capacity. In some instances, framework ("FR") residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications can be made to further refine antibody performance, such as binding affinity. Generally, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulin sequence and all or substantially all of the FR regions are those of human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FRs is typically no more than 6 in the H chain and no more than 3 in the L chain. The humanized antibody may optionally also comprise at least a portion of an immunoglobulin constant region (Fc), which may be a human immunoglobulin. Exemplary methods and humanized antibodies include those described in Jones et al. Nature 321:522-525 (1986); Riechmann et al. Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992); Vaswani and Hamilton, Ann. Allergy. Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Burle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and US Pat. Nos. 6,982,321 and 7,087,409.

[0042] The term "functional fragment" as used with respect to antibodies refers to a portion of an antibody, including heavy or light chain polypeptides, that retains some or all of the binding activity of the antibody from which the fragment is derived. Such functional fragments can include, for example, Fd, Fv, Fab, F(ab'), F(ab)2, F(ab')2, single-chain Fv (ScFv), diabodies, triabodies, tetrabodies, and minibodies. Other functional fragments can include, for example, heavy chain polypeptides, light chain polypeptides, variable region polypeptides, CDR polypeptides, or portions thereof. Such antibody-binding fragments are described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molec. Biology and Biotechnology: A Comprehensive Desk Reference, New York: VCH Publishers, Inc.; Huston et al., Cell Biophysics, 22:189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day, ED., Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, NY (1990); Antibody Engineering, Second Edition, Oxford University Press, 1995.

[0043] The term "heavy chain" when used with respect to antibodies refers to a polypeptide chain of approximately 50 to 70 kDa, including an amino-terminal variable region of approximately 120 to 130 amino acids or more and a carboxy-terminal constant region. The constant region is one of five types, designated α (alpha), δ (delta), ε (epsilon), γ (gamma), or μ (mu), based on the amino acid sequence of the heavy chain constant region. Heavy chains vary in size: α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with light chains, these different types of heavy chains give rise to five well-known classes of antibodies: IgA, IgD, IgE, IgG, and IgM, each of which includes the four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4. The heavy chain may be (for example, but not limited to) a human heavy chain.

[0044] The term "light chain" when used in reference to an antibody refers to a polypeptide chain of approximately 25 kDa, including an amino-terminal portion containing a variable region of about 100 to about 110 or more amino acids, and a carboxy-terminal portion containing a constant region. The approximate length of a light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two different types, called kappa (κ) and lambda (λ). Light chain amino acid sequences are well known in the art. The light chain may be (for example, but not limited to) a human light chain.

[0045] The term "variable domain" or "variable region" generally refers to the amino-terminal portion of an antibody's light or heavy chain, approximately 120-130 amino acids in length in the heavy chain and approximately 100-110 amino acids in the light chain, and is used to determine the binding and specificity of each particular antibody to a specific antigen. Variable domains can vary significantly in sequence among different antibodies. Sequence variability is concentrated in the CDRs, while the less variable portions of variable domains are called framework regions (FRs). The CDRs of the light and heavy chains are primarily responsible for the antibody's interaction with the antigen. The numbering of amino acid positions used herein follows the EU index, as in Kabat et al. (1991) Sequences of proteins of immunological interest. (USDepartment of Health and Human Services, Washington, DC) 5th Ed. The variable region may be (for example, but not limited to) a human variable region.

[0046] A CDR refers to one of the three hypervariable regions (H1, H2, H3) within the non-framework region of the VH β-sheet framework of an immunoglobulin (Ig or antibody) or one of the three hypervariable regions (L1, L2, L3) within the non-framework region of the VL β-sheet framework of an antibody. CDRs are thus variable region sequences interspersed among framework region sequences. CDR regions are well known to those skilled in the art and have been defined, for example, by Kabat as the most hypervariable regions within antibody variable (V) domains (Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat, Adv. Prot. Chem. 32:1-75 (1978)). CDR region sequences have also been structurally defined by Chothia as residues that are not part of the conserved β-sheet framework and therefore can adopt different conformations (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). Both terms are well recognized in the art. The positions of CDRs within canonical antibody variable domains have been determined by comparison of numerous structures (Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); Morea et al., Methods 20:267-279 (2000)). Because the number of residues within hypervariable regions varies among antibodies, it is customary to designate additional residues relative to their canonical positions by numbering them with a, b, c, etc. next to the residue number in the canonical variable domain numbering scheme (Al-Lazikani et al., supra (1997)). Such nomenclature is also well known to those skilled in the art.

[0047] The term "cancer" refers to any physiological condition in a mammal characterized by unregulated cell growth. Cancers as described herein include solid tumors and hematologic (blood) cancers. "Hematologic cancer" refers to any cancer of blood origin, including, for example (but not limited to), myeloma, lymphoma, leukemia, and the like. "Solid tumor" or "tumor" refers to lesions and neoplastic cell growth and proliferation, and all precancerous and cancerous cells and tissues, whether malignant or benign, that result in abnormal tissue growth. As used herein, "neoplasia" refers to any form of unregulated or dysregulated cell growth that results in abnormal tissue growth, whether malignant or benign.

[0048] The term "treat" or "treatment" refers to an indication of successful or ameliorative measures of the progression, severity, and / or duration of a disease, pathology, or condition, including objective or subjective parameters such as relief, remission, alleviation of symptoms, or making the injury, pathology, or condition more tolerable to the patient, slowing the rate of degeneration or decline, making the end point of degeneration less debilitating, or improving the patient's physical or mental well-being.

[0049] The term "enhancement" refers to an increase or improvement in the function or activity of a protein or cell after administration or contact with a combination described herein compared to the protein or cell before such administration or contact.

[0050] The term "administration" refers to the act of delivering at least one composition or combination described herein to a subject by a route such as (for example, but not limited to) oral, mucosal, topical, suppository, intravenous, parenteral, intraperitoneal, intramuscular, intraanal, intrathecal, intranasal, or subcutaneous administration. Parenteral administration includes intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. Administration generally occurs after the onset of a disease, disorder, or condition, or a symptom thereof, but in certain instances can occur before the onset of a disease, disorder, or condition, or a symptom thereof (e.g., administration to a patient predisposed to such a disease, disorder, or condition).

[0051] The term "co-administration" refers to the administration of two or more agents (e.g., two active agents described herein and / or two active agents plus another active agent, such as (but not limited to) an anti-cancer agent described herein). The timing of co-administration depends in part on the combination and composition administered and may include administration simultaneously with, immediately before, or immediately after the administration of one or more additional therapies, e.g., cancer therapies such as chemotherapy, hormonal therapy, radiation therapy, or immunotherapy. The compositions of the present disclosure can be administered alone or co-administered to a patient. Co-administration is meant to include simultaneous or sequential administration of compounds individually or in combination. Thus, preparations can also be combined with other active substances (e.g., to reduce metabolic degradation) if desired. The compounds described herein can be used in combination with each other or with other active agents known to be useful in the treatment of cancer.

[0052] The term "anti-cancer agent" is used according to its plain and ordinary meaning to refer to a composition having anti-neoplastic properties or the ability to inhibit cell growth or proliferation. In certain non-limiting embodiments, the anti-cancer agent is a chemotherapeutic agent. In certain non-limiting embodiments, the anti-cancer agent is an agent identified herein that has utility in methods of treating cancer. In certain non-limiting embodiments, the anti-cancer agent is an agent approved by the FDA or a similar regulatory agency in a country other than the United States to treat cancer.

[0053] As used herein, "current good manufacturing practice" or "cGMP" refers to current good manufacturing practice regulations implemented by the U.S. Food and Drug Administration (FDA) or equivalent regulatory authorities in countries other than the United States. cGMP regulations establish systems that ensure the proper design, monitoring, and control of manufacturing processes and facilities. Compliance with cGMP regulations ensures the identity, strength, quality, and purity of drug products by requiring drug manufacturers to properly manage manufacturing operations. This includes establishing a strong quality control system, obtaining raw materials of appropriate quality, establishing robust operating procedures, detecting and investigating deviations in product quality, and maintaining reliable testing laboratories.

[0054] As used herein, the term "ex vivo expansion" or "expansion" refers to a method of growing a population of cells in tissue culture to increase the number of cells in that population. Cells that have been expanded ex vivo are said to be "expanded."

[0055] As used herein, the term "fucosylation" refers to treating a cell population with α1,3-fucosyltransferase and a fucose donor under conditions that increase the ability of the cells to bind to selectins or that increase the reactivity of the cells with antibodies known in the art to bind to sLeX, including, but not limited to, the ECA-452 monoclonal antibody. Cells treated with α1,3-fucosyltransferase and a fucose donor that exhibit increased binding to selectins, the ECA-452 monoclonal antibody, or another antibody specific for sLeX are said to be "fucosylated." As used herein, "fucosylation" can also refer to the level of sLeX present on a cell population.

[0056] Turning now to various concepts of the present invention, the present disclosure relates to a combination therapy comprising at least one immune checkpoint inhibitor with adoptive cellular immunotherapy, wherein isolated immune cells (and / or genetically modified versions thereof) are enhanced by cell surface modification using a fucosyltransferase enzyme that adds fucose to the immune cells and upregulates selectin ligands on the immune cells. The combination therapy is useful for treating cancer, including, but not limited to, reducing and / or preventing cancer metastasis. The combination is also useful for treating cancer, including those previously treated with either 1) an immune checkpoint inhibitor and / or 2) adoptive cellular immunotherapy.

[0057] Certain non-limiting embodiments of the present disclosure relate to compositions (e.g., pharmaceutical compositions) comprising at least one immune checkpoint inhibitor (ICI) and at least one ex vivo fucosylated isolated immune cell type (fucoACT). The composition may include one or more additional agents. For example, if the composition is a pharmaceutical composition, the composition may further include at least one pharmaceutically acceptable carrier. Furthermore, the composition may include one or more additional active agents, as described in more detail herein below.

[0058] Certain non-limiting embodiments of the present disclosure relate to a system comprising a composition comprising at least one immune checkpoint inhibitor (ICI) and a composition comprising at least one isolated immune cell type that is ex vivo fucosylated (fuco-ACT). The system (and the compositions contained therein) may further comprise one or more additional agents, as described in more detail herein below.

[0059] Any immune checkpoint inhibitor known in the art or otherwise contemplated herein may be utilized in accordance with the present disclosure. Non-limiting examples of immune checkpoint inhibitors that may be utilized include PD-1 inhibitors, PD-L1 inhibitors, LAG-3 inhibitors, CTLA-4 inhibitors, TIM-3 inhibitors, B7-H3 inhibitors, A2aR inhibitors, CD73 inhibitors, NKG2A inhibitors, PVRIG / PVRL2 inhibitors, CEACAM1 inhibitors, FAK inhibitors, CCL2 / CCR2 inhibitors, LIF inhibitors, CD47 / SIRPα inhibitors, CSF-1 inhibitors, IL-1 inhibitors, IL-8 inhibitors, SEMA4D inhibitors, Ang-2 inhibitors, CLEVER-1 inhibitors, phosphatylserine inhibitors, and the like, as well as any combination thereof.

[0060] An immune checkpoint inhibitor can be any molecule (or combination of molecules) capable of inhibiting, blocking, abrogating, or interfering with the activity or expression of any of the proteins disclosed herein. For example (but not limited to), an immune checkpoint inhibitor can be a small molecule compound, a nucleic acid, a polypeptide, an antibody, a peptibody, a diabody, a minibody, a single-chain variable fragment (ScFv), or a functional fragment or variant thereof. In one example, an immune checkpoint inhibitor is a small molecule compound (e.g., a compound having a molecular weight of less than about 1000 Da). In other examples, useful immune checkpoint inhibitors include nucleic acids and polypeptides. Furthermore, an immune checkpoint inhibitor can be a polypeptide (e.g., a macrocyclic polypeptide), such as those exemplified in U.S. Patent Application Publication No. 2014 / 0294898.

[0061] In other embodiments, the immune checkpoint inhibitor is an antibody (i.e., a monoclonal antibody or a polyclonal antibody) or a functional fragment thereof, such as, but not limited to, a human antibody, a murine antibody, a chimeric antibody, a humanized antibody, or a chimeric-humanized antibody. In one non-limiting embodiment, the immune checkpoint inhibitor is a human antibody. In another non-limiting embodiment, the immune checkpoint inhibitor is a murine antibody. In yet another non-limiting embodiment, the immune checkpoint inhibitor is a chimeric antibody. In yet another non-limiting embodiment, the immune checkpoint inhibitor is a humanized antibody. In yet another non-limiting embodiment, the immune checkpoint inhibitor is a chimeric-humanized antibody.

[0062] The specificity of a PD-1 or PD-L1 or LAG-3 antibody or functional fragment thereof refers to the ability of a particular antibody or functional fragment thereof to react with only one antigen (e.g., a single epitope of PD-1 or PD-L1 or LAG-3). An antibody or functional fragment can be considered specific if it can distinguish between differences in the primary, secondary, or tertiary structure of the antigen or isomeric forms of the antigen.

[0063] Non-limiting examples of PD-1 inhibitors that can be utilized in accordance with the present disclosure include nivolumab (OPDIVO®, Bristol Meyers Squibb); pembrolizumab (KEYTRUDA®, Merck & Co.); cemiplimab (LIBTAYO®, Regeneron Pharmaceuticals); pidilizumab (Medivation); dostallimab (JEMPERLI®, GlaxoSmithKline); pimivalimab (Jounce Therapeutics); spartalizumab (Novartis); camrelizumab (AiRuiKa™, Jiangsu Hengrui Medicine); sintilimab (TYVYT®, Eli Lilly); ticerelizumab (BeiGene); toripalimab (Tuoyi™, Shanghai Junshi Bioscience); retifanlimab (Incyte); and the like, and combinations thereof.

[0064] Non-limiting examples of PD-L1 inhibitors that can be utilized in accordance with the present disclosure include atezolizumab (TECENTRIQ®, Genentech); durvalumab (IMFINZI®, Mediimmune / AstraZeneca); avelumab (BAVENCIO®, Pfizer); cosibelimab (Checkpoint Therapeutics); embafolimab (TRACON Pharmaceuticals); AUNP12 (Aurigene); socazolimab (Lee Pharmaceutical / Sorrento Therapeutics); STI-3031 (Sorrento Therapeutics), and the like, and combinations thereof.

[0065] Non-limiting examples of LAG-3 inhibitors that can be utilized in accordance with the present disclosure include yelamirimab (LAG525, Novartis); Sym022 (Symphogen); TSR-033 (GlaxoSmithKline); fianlimab (Regeneron); yelamirimab (Novartis); INCAGN2385-101 (Incyte Biosciences); favezelimab (Merck & Co.); BI754111 (Boehringer Ingelheim), and the like, and combinations thereof.

[0066] Other non-limiting examples of immune checkpoint inhibitors that may be utilized in accordance with the present disclosure include CTLA-4 inhibitors (ipilimumab (Yervoy); TIM-3 inhibitors (MBG453); B7-H3 inhibitors (MGC018); A2aR inhibitors (EOS100850); CD73 inhibitors (CPI-006); NKG2A inhibitors (monalizumab); PVRIG / PVRL2 inhibitors (COM701); CEACAM1 inhibitors (CM24); FAK inhibitors (defactinib); CCL 2 / CCR2 inhibitors (PF-04136309); LIF inhibitors (MSC-1); CD47 / SIRPα inhibitors (ALX148); CSF-1 inhibitors (lacnotuzumab); IL-1 inhibitors (canakinumab); IL-8 inhibitors (BMS-986253); SEMA4D inhibitors (pepinemab); Ang-2 inhibitors (trebananib); CLEVER-1 inhibitors (enapotamab); phosphatidylserine inhibitors (bavituximab), and combinations thereof.

[0067] The present disclosure also provides for the use of two or more immune checkpoint inhibitors in combination, for example (but not limited to), leratolimab (OPDUALAG™, Bristol Meyers Squibb) is a combination of a PD-1 inhibitor and a LAG-3 inhibitor.

[0068] Any immune cell capable of being fucosylated can be utilized in accordance with the present disclosure. In certain (but non-limiting) embodiments, the immune cell includes a tumor-infiltrating lymphocyte (TIL). Non-limiting examples of immune cell types that can be utilized in accordance with the present disclosure include cytotoxic T cells (CTL), regulatory T cells (Treg), helper T cells, NK cells, B cells, dendritic cells, and the like, as well as combinations thereof. Immune cells can be isolated from the patient (autologous), related or unrelated donors (allogeneic), induced pluripotent stem cells (iPSCs), or genetically modified. Non-limiting examples of genetically modified immune cells that can be utilized in accordance with the present disclosure include chimeric antigen receptor gene-transduced T cells (CAR-T cells), T cell receptor gene-transduced T cells (TCR-T cells), chimeric antigen receptor gene-transduced natural killer (NK) cells (CAR-NK cells), and the like. In yet another non-limiting embodiment, adoptive cellular immunotherapy is derived from T cells obtained from a patient, donor, or iPSCs that are stimulated in the laboratory with tumor cells or tumor cell products to produce tumor-selective immune cells.

[0069] Immune cells are fucosylated ex vivo using a fucosyltransferase enzyme that adds fucose to the immune cells and upregulates selectin ligands on the immune cells. Non-limiting examples of methods for fucosylating cells ex vivo that can be utilized in accordance with the present disclosure are disclosed in U.S. Pat. Nos. 7,332,334, 7,776,591, 8,084,255, 8,633,021, 9,511,095, and 10,799,538; and U.S. Patent Application Publication Nos. 2011 / 0091434, 2014 / 0161782, 2017 / 0121673, 2019 / 0017023, 2019 / 0062694, and 2023 / 0014609, among others.

[0070] In certain non-limiting embodiments, the method for fucosylating cells ex vivo includes one or more of the following steps: 1) identifying a cancer patient in need of adoptive cell therapy; 2) isolating immune cells from the patient's blood or cancer tumor; 3) expanding the immune cells ex vivo; and 4) contacting the expanded immune cells with a fucosyltransferase and at least one of fucose or GDP-fucose to provide fucosylated immune cells.

[0071] In certain (but non-limiting) embodiments, the method for fucosylating cells further comprises, after step (4), (4a) separating the fucosylated lymphocytes from other components, and step (5) is further defined as administering the specific fucosylated lymphocytes intravenously back to the patient. In certain (but non-limiting) embodiments, the method further comprises, after step (4a), (4b) separating the specific fucosylated lymphocytes from other fucosylated lymphocytes, and step (5) is further defined as administering the specific fucosylated lymphocytes intravenously back to the patient.

[0072] Any fucosyltransferase known in the art or contemplated herein can be utilized in accordance with the present disclosure. Non-limiting examples of fucosyltransferases that can be utilized in accordance with the present disclosure include α1,3-fucosyltransferase I (FUT1), α1,3-fucosyltransferase II (FUT2), α1,3-fucosyltransferase III (FUT3), α1,3-fucosyltransferase IV (FUT4), α1,3-fucosyltransferase V (FUT5), α1,3-fucosyltransferase VI (FUT6), α1,3-fucosyltransferase VII (FUT7), α1,3-fucosyltransferase I (FUT8), α1,3-fucosyltransferase VII (FUT9), α1,3-fucosyltransferase VIII (FUT10), α1,3-fucosyltransferase VIII (FUT11), α1,3-fucosyltransferase VIII (FUT12), α1,3-fucosyltransferase VIII (FUT13), α1,3-fucosyltransferase VIII (FUT14), α1,3-fucosyltransferase VIV (FUT15), α1,3-fucosyltransferase VIII (FUT16), α1,3-fucosyltransferase VIV (FUT17), α1,3-fucosyltransferase VIII (FUT18), α1,3-fucosyltransferase VIV (FUT19), α1,3-fucosyltransferase VIII (FUT19 ... Specific (but non-limiting) examples of fucosyltransferases that can be utilized in accordance with the present disclosure include α1,3-fucosyltransferase VIII (FUT8), α1,3-fucosyltransferase IX (FUT9), α1,3-fucosyltransferase X (FUT10), and α1,3-fucosyltransferase XI (FUT11), or any combination thereof. A specific (but non-limiting) example of a fucosyltransferase that can be utilized is FUT7 (product name TZ 102, Targazyme Inc., Carlsbad, CA).

[0073] Any fucose donor known in the art or contemplated herein can be utilized in accordance with the present disclosure, non-limiting examples of which include fucose and GDP-fucose.

[0074] The compositions and systems of the present disclosure may include any combination of immune checkpoint inhibitors and fucoACT disclosed herein or otherwise contemplated. Specific (but non-limiting) examples of such combinations include fucoACT in combination with a PD-1 inhibitor (such as, but not limited to, a PD-1 antibody); fucoACT in combination with a PD-L1 inhibitor (such as, but not limited to, a PD-L1 antibody); and fucoACT in combination with a LAG-3 inhibitor (such as, but not limited to, a LAG-3 antibody). In another non-limiting embodiment, the PD-1 or PD-L1 or LAG-3 inhibitor is a small molecule compound, nucleic acid, peptide, protein, antibody, peptibody, diabody, minibody, single-chain variable fragment (ScFv), or fragment or variant thereof.

[0075] The compositions of the present disclosure may be provided in any formulation known in the art or contemplated herein. In certain (but non-limiting) embodiments, the compositions contain one or more pharmaceutically acceptable carriers (and as such, the compositions may also be referred to as "pharmaceutical compositions"). Non-limiting examples of suitable pharmaceutically acceptable carriers include water; saline; glucose solution; fructose or mannitol; calcium carbonate; cellulose; ethanol; oils of animal, vegetable, or synthetic origin; carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low-molecular-weight proteins; surfactants; liposome carriers; nanocarriers; scaffolds capable of delayed drug release (such as, but not limited to, hydrogels); buffers such as sodium chloride, saline, phosphate-buffered saline, and / or other physiologically acceptable and / or safe substances for use; diluents; excipients such as polyethylene glycol (PEG); or any combination thereof. Suitable pharmaceutically acceptable carriers for pharmaceutical formulations are described, for example, in Remington: The Science and Practice of Pharmacy, 23rd ed. (2020).

[0076] Immune checkpoint inhibitors (such as, but not limited to, PD-1 or PD-L1 or LAG-3 antibodies) may be present in an amount based on the body weight of a patient in need thereof. For example (but not limited to), the immune checkpoint inhibitor may be present in an amount of about 0.1 mg / kg to about 30 mg / kg, 0.1 mg / kg to about 25 mg / kg, 0.1 mg / kg to about 20 mg / kg, 0.1 mg / kg to about 15 mg / kg, 0.1 mg / kg to about 10 mg / kg, 0.1 mg / kg to about 7.5 mg / kg, 0.1 mg / kg to about 5 mg / kg, 0.1 mg / kg to about 2.5 mg / kg, or about 0.1 mg / kg to about 1 mg / kg. The PD-1 or PD-L1 or LAG-3 antibody may be present in an amount of about 0.5 mg / kg to about 30 mg / kg, 0.5 mg / kg to about 25 mg / kg, 0.5 mg / kg to about 20 mg / kg, 0.5 mg / kg to about 15 mg / kg, 0.5 mg / kg to about 10 mg / kg, 0.5 mg / kg to about 7.5 mg / kg, 0.5 mg / kg to about 5 mg / kg, 0.5 mg / kg to about 2.5 mg / kg, or about 0.5 mg / kg to about 1 mg / kg. The immune checkpoint inhibitor may be present in an amount of about 0.5 mg / kg to about 5 mg / kg or about 0.1 mg / kg to about 10 mg / kg. The immune checkpoint inhibitor may be present in an amount of about 0.5 mg / kg to about 15 mg / kg or about 0.1 mg / kg to about 20 mg / kg.

[0077] In still other non-limiting embodiments, the immune checkpoint inhibitor can be present in an amount of about 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, or 30 mg / kg. The PD-1 antibody or PD-L1 antibody or LAG-3 antibody can be present in an amount of about 1 mg / kg, 2 mg / kg, 3 mg / kg, or 5 mg / kg.

[0078] The immune checkpoint inhibitor may be present in the combination in any amount including, but not limited to, 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, or 2000 mg. The immune checkpoint inhibitor may be present in the combination drug in an amount such as (but not limited to) about 1 mg to about 10 mg, about 10 mg to about 20 mg, about 25 mg to about 50 mg, about 30 mg to about 60 mg, about 40 mg to about 50 mg, about 50 mg to about 100 mg, about 75 mg to about 150 mg, about 100 mg to about 200 mg, about 200 mg to about 500 mg, about 500 mg to about 1000 mg, about 1000 mg to about 1200 mg, about 1000 mg to about 1500 mg, about 1200 mg to about 1500 mg, or about 1500 mg to about 2000 mg.

[0079] The immune checkpoint inhibitor may be present in the combination in an amount such as, but not limited to, about 0.1 mg / mL, about 0.5 mg / mL, about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 150 mg / mL, about 200 mg / mL, about 250 mg / mL, about 300 mg / mL, about 400 mg / mL, or about 500 mg / mL. In one non-limiting embodiment, the immune checkpoint inhibitor is present in the combination formulation in an amount of about 1 mg / mL to about 10 mg / mL, 5 mg / mL to about 10 mg / mL, 5 mg / mL to about 15 mg / mL, 10 mg / mL to about 25 mg / mL, 20 mg / mL to about 30 mg / mL, 25 mg / mL to about 50 mg / mL, or 50 mg / mL to about 100 mg / mL.

[0080] In certain non-limiting examples, a therapeutically effective amount of an immune checkpoint inhibitor is determined as the amount set forth in the package insert provided with the immune checkpoint inhibitor, which refers to instructions customarily accompanying commercial packaging of pharmaceutical products approved by the FDA (U.S. Food and Drug Administration) or similar regulatory body in countries other than the United States, and which contains, for example, information regarding the use, dosage, administration, contraindications, and / or warnings concerning the use of such pharmaceutical products.

[0081] The fucoACT may be present in the composition (and administered to a patient) in any amount that allows the fucoACT to function as described herein. Non-limiting examples of amounts within the scope of the present disclosure include amounts of at least about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 10 10 , about 10 11 , about 10 12 , about 10 13 , about 1014 etc., and ranges between two of the above values.

[0082] Certain non-limiting embodiments of the present disclosure include kits that include any of the compositions and / or systems disclosed or contemplated herein.

[0083] Certain non-limiting embodiments of the present disclosure relate to methods of adoptive cell therapy comprising the following steps: 1) identifying a cancer patient in need of adoptive cell therapy; 2) isolating immune cells from the patient's blood or cancer tumor, where the immune cells are any of the immune cells disclosed herein or otherwise contemplated; 3) expanding the immune cells ex vivo; 4) contacting the expanded immune cells with at least one fucosyltransferase and at least one fucose donor disclosed herein or otherwise contemplated to provide fucosylated immune cells; 5) returning the fucosylated immune cells to the patient intravenously; and 6) administering at least one immune checkpoint inhibitor disclosed herein or otherwise contemplated to the patient simultaneously with the fucosylated immune cells or sequentially, in whole or in part.

[0084] In one specific (but non-limiting) embodiment, the method further comprises, after step (4), (4a) separating the fucosylated lymphocytes from other components, and step (5) is further defined as administering the specific fucosylated lymphocytes intravenously back to the patient. In another specific (but non-limiting) embodiment, the method further comprises, after step (4a), (4b) separating the specific fucosylated lymphocytes from other fucosylated lymphocytes, and step (5) is further defined as administering the specific fucosylated lymphocytes intravenously back to the patient.

[0085] Any step of the method may be repeated one or more times. For example (but not limited to), at least step (5) may be repeated one or more times and / or at least step (6) may be repeated one or more times.

[0086] At least one immune checkpoint inhibitor may be administered before, simultaneously with, and / or after at least one fucosylated immune cell type (fucoACT) for adoptive cell therapy.

[0087] Certain non-limiting embodiments of the present disclosure relate to methods of treating one or more cancers and / or reducing the incidence or severity of metastases in a cancer patient. The method comprises administering to a cancer patient at least one of any ICI disclosed herein or otherwise contemplated and at least one of any fuco-ACT disclosed herein or otherwise contemplated, either simultaneously or in whole or in part sequentially. In certain (but non-limiting) embodiments, the ICI(s) and fuco-ACT(s) are present in the same composition; or, the ICI(s) and fuco-ACT(s) are present in separate compositions and are administered simultaneously or in whole or in part sequentially (either composition is administered first). In addition, each composition may be administered to a patient one or more times.

[0088] The methods of the present disclosure can be utilized to treat any cancer, including, but not limited to, prostate cancer; skin cancer; ovarian cancer; breast cancer (such as, but not limited to, triple-negative breast cancer); cancer of non-lymphoid parenchymal organs such as the heart, placenta, skeletal muscle, and lung; cancer of the head and neck, including various lymphomas (such as, but not limited to, mantle cell lymphoma, non-Hodgkin's B-cell lymphoma, PTCL, adenoma, squamous cell carcinoma, laryngeal carcinoma, salivary gland carcinoma, thymoma, and thymic carcinoma); leukemia, retinal cancer, esophageal cancer, multiple myeloma, melanoma, colorectal cancer, lung cancer, cervical cancer, endometrial cancer, gallbladder cancer, liver cancer, follicular thyroid cancer, gastric cancer, non-small cell lung cancer, glioma, urothelial carcinoma, bladder cancer, prostate cancer, renal cell carcinoma, invasive ductal carcinoma of the breast, and glioblastoma multiforme; and combinations thereof.

[0089] The methods of the present disclosure can optionally include one or more additional steps. For example (but not limited to), the methods can include administering at least one additional treatment to the primary tumor of the cancer patient, wherein the at least one additional treatment is selected from the group consisting of radiation therapy, surgery, chemotherapy, immunotherapy, targeted therapy, hormone therapy, stem cell transplant, cryotherapy, laser therapy, precision medicine, and combinations thereof.

[0090] In certain (but non-limiting) embodiments, the patient being treated may have a primary tumor such as, but not limited to, breast, lung, bladder, skin, intestine, colon, kidney, ovary, pancreas, prostate, brain, stomach, thyroid, head and neck, gastroesophageal tract, connective tissue or other non-epithelial tissue, lymphoid cell, or uterine tumor.

[0091] In the methods of the present disclosure, treating cancer may result in one or more of: (i) reducing or slowing tumor metastasis; (ii) preventing or delaying cancer recurrence; (iii) increasing disease-free or tumor-free survival; (iv) increasing overall survival; (v) reducing the frequency of treatments; (vi) alleviating one or more symptoms of cancer; and / or (vii) reducing tumor burden.

[0092] If metastases are present and are being reduced, the reduced metastases may be metastases of one or more of the adrenal gland, brain and / or spinal cord, bone, lung, liver and / or pleura, gastrointestinal tract, peritoneum, muscle, lymph nodes, skin, etc. If metastases are present or are prevented, the primary or secondary tumor of the subject being treated may be a cancer of the breast, lung, bladder, skin, intestine, colon, kidney, ovary, pancreas, prostate, liver, brain, stomach, thyroid, head and neck, gastroesophageal, myeloid, lymphatic, connective tissue, or other non-epithelial tissue, uterus, etc.

[0093] In certain (but non-limiting) embodiments, the primary tumor is an advanced metastatic breast cancer, possibly a triple-negative breast cancer, and the method further comprises administering a histone deacetylase inhibitor (HDACi) to prime the tumor prior to treatment with the ICI / FucoACT combination therapy (e.g., the priming period is about, but not limited to, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 1 week or more, 2 weeks, 2 weeks or more, 3 weeks, or 3 weeks or more). In certain (but non-limiting) embodiments, the HDACi is administered every other week or every three weeks for a period before treatment with the combination begins.

[0094] Certain non-limiting embodiments of the present disclosure relate to a method for treating cancer or reducing the incidence or severity of metastasis by improving immune cell trafficking to solid tumors during adoptive cell therapy (ACT), comprising administering to a subject with a solid tumor at least one ICI disclosed herein or otherwise contemplated and at least one fucoACT disclosed herein or otherwise contemplated. In certain (but non-limiting) embodiments, the ICI(s) and fucoACT(s) are present in the same composition; alternatively, the ICI(s) and fucoACT(s) are present in separate compositions and are administered simultaneously or, in whole or in part, sequentially (either composition is administered first). In addition, each composition may be administered to a patient one or more times.

[0095] Certain non-limiting embodiments of the present disclosure relate to methods for enhancing natural killer (NK), cytotoxic T cell, or Treg cell activity in cancer patients, comprising administering at least one of any ICI disclosed herein or otherwise contemplated and at least one of any fucoACT disclosed herein or otherwise contemplated. In certain (but non-limiting) embodiments, the ICI(s) and fucoACT(s) are present in the same composition; alternatively, the ICI(s) and fucoACT(s) are present in separate compositions and are administered simultaneously or, in whole or in part, sequentially (either composition is administered first). In addition, each composition may be administered to a patient one or more times.

[0096] Certain non-limiting embodiments of the present disclosure relate to methods for enhancing antibody-dependent cellular cytotoxicity in cancer patients, comprising administering to the cancer patient at least one of any ICI disclosed herein or otherwise contemplated and at least one of any fuco-ACT disclosed herein or otherwise contemplated. In certain (but non-limiting) embodiments, the ICI(s) and fuco-ACT(s) are present in the same composition; alternatively, the ICI(s) and fuco-ACT(s) are present in separate compositions and are administered simultaneously or, in whole or in part, sequentially (either composition is administered first). In addition, each composition may be administered to the patient one or more times.

[0097] Certain non-limiting embodiments of the present disclosure relate to methods of treating diseases, disorders, or alleviating or eliminating symptoms of diseases and disorders, comprising administering to a subject in need of treatment at least one of any of the ICIs disclosed herein or otherwise contemplated and at least one of any of the fuco-ACTs disclosed herein or otherwise contemplated, wherein the compounds are administered simultaneously or within 24 hours of each other. In certain (but non-limiting) embodiments, the ICI(s) and fuco-ACT(s) are present in the same composition; or, the ICI(s) and fuco-ACT(s) are present in separate compositions and are administered simultaneously or, in whole or in part, sequentially (either composition is administered first). In addition, each composition may be administered to a patient one or more times.

[0098] Certain non-limiting embodiments of the present disclosure relate to methods of improving the ability of immune cells to resist cancer cells by first contacting the immune cells with a fucosyltransferase and then ex vivo GDP-fucose such that 50% or more of the immune cells so contacted are fucosylated. The immune cells may be any of the immune cells disclosed or otherwise contemplated herein, such as, but not limited to, tumor-infiltrating lymphocytes (TILs). Fucosylation of TILs is thought to result in: (i) increased adhesion to endothelial cell selectins during circulation; (ii) increased surface expression of the trafficking molecule CD162 / PSGL-1, the chemotactic receptor CD183 (CXCR3), and the stimulatory coregulatory molecule CD137 (41BB); (iii) enhanced cytotoxic T cell (CTL) activity, as measured by expression of key components of the cytotoxic machinery, including at least one of FasL / CD95L, perforin, granzymes, or the ability to kill tumor cells; and / or (iv) an increased frequency of fuco-TILs forming complexes with target cells compared to nonfucosylated TILs. [Example]

[0099] The following examples are provided. However, it should be understood that the present disclosure is not limited to the specific experiments, results, or experimental procedures disclosed herein. Rather, the examples are provided merely as one of various embodiments and are intended to be illustrative rather than exhaustive.

[0100] Example 1 In this example, TILs were fucosylated with fucosyltransferase-VII (also known as FuT7; TZ 102, Targazyme Inc., Carlsbad, CA) to produce sialyl Lewis X (sLeX) expression will be restored and its ability to enhance TIL binding to E- and P-selectin will be tested.

[0101] Ex vivo expanded TILs are suspended in a fucosylation solution consisting of 1 μg / mL of FuT7 in 1 mmol / L GDP fucose in PBS containing 1% human serum albumin (PBS / HSA). The cell suspension is then incubated at room temperature for 30 minutes. The cells are washed twice with PBS / HSA and then resuspended in PBS.

[0102] The TIL suspension is stained with FITC-conjugated HECA-452 antibody (BD Biosciences). The HECA-452 antibody targets the cutaneous lymphocyte antigen (CLA), which has been shown to be sLeX-positive after fucosylation. TIL fucosylation is confirmed using a suitable flow cytometer (e.g., LSR Fortessa; BD Biosciences).

[0103] As a result of this procedure, TIL expression of sLeX significantly increases from a baseline level of 0-5% to 90-100%.

[0104] The functional consequences of sLeX's increased TIL expression are measured using rolling and adhesion assays. E-, L-, and P-selectin are bound or immobilized to an appropriate substrate (e.g., a microscopy chamber). Fucosylated TILs (fuco-TILs) suspended in PBS / HSA are applied to a substrate coated with E-, L-, or P-selectin, incubated, and then washed with PBS / HSA. Non-fucosylated TILs (non-fuco-TILs) are used as a control. TIL adhesion is measured using standard microscopy assays.

[0105] Example 2 In this example, fuco-TILs from Example 1 are examined for phenotypic changes, particularly changes in T cell surface markers, some of which indicate an activated state and some of which indicate enhanced lymphocyte trafficking.

[0106] For phenotypic analysis, 1.5 × 10 cells were cultured within 2 hours after fucosylation for molecules regulating T cell trafficking, including CD49d (e.g., clone 9F10; BioLegend), CD162 (also known as PSGL-1; e.g., clone KPL-1; BioLegend), CD183 (also known as CXCR3; e.g., clone 1C6 / CXCR3; BD Biosciences), and CD195 (also known as CCR5; e.g., clone 2D7 / CCR; BD Biosciences), and molecules involved in costimulation / inhibition, including CD137 (also known as 41BB; e.g., clone 5F4; BioLegend), CD279 (also known as PD1; e.g., clone EH12.2H7; BioLegend), and CD357 (also known as GITR; e.g., eBioGITR; eBioscience). 6Fuco-TILs and negative control non-fuco-TILs are stained. Cell viability is assessed using the LIVE / DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies). Flow cytometry is performed on live cells using an appropriate flow cytometer (e.g., LSR Fortessa; BD Biosciences). Data are analyzed using accepted flow cytometry analysis software (e.g., FlowJo; FlowJo, LLC).

[0107] Differences in cell surface marker expression after fucosylation are measured by cell surface staining and flow cytometry. Fucosylated TILs show increased surface expression of the trafficking molecule CD162 / PSGL-1 and the chemotactic receptor CD183 (CXCR3), as well as the stimulatory coregulatory molecule CD137 (41BB). No other statistically significant changes were detected, including those of the immune checkpoint receptor CD195 (PD-1) or the regulatory T cell receptor GITR.

[0108] Example 3 In this example, fuco-TILs are evaluated for their activation status, particularly cytotoxic T cell (CTL) activation status as measured by the expression of key components of the cytotoxic machinery (i.e., FasL / CD95L, perforin, and granzyme B), as this is a key feature of successful TIL therapy. TIL (fuco-TILs and non-fuco-TILs) activation is analyzed by measuring the expression of Fas ligand (FasL (also known as CD95L)), perforin, and granzyme B. Fuco-TILs and non-fuco-TILs are co-cultured overnight at 37°C with autologous tumor cells obtained from the same host as the TILs at a 1:1 ratio. At the end of the incubation period, cells are stained with fluorescently conjugated antibodies targeting CD3, CD8, FasL (BioLegend, CLA, and Ghost Dye Violet 510). After staining for cell surface markers, cells are permeabilized and stained with fluorescently conjugated antibodies targeting granzyme B and perforin (BioLegend). Staining is analyzed using flow cytometry (BD LSR Fortessa).

[0109] The mechanism of CTL cytotoxicity after fucosylation was investigated by analyzing the intracellular expression of granzyme B and perforin, and the surface expression of FasL after co-culture of fuco- and nonfuco-TILs with tumor targets. The analysis demonstrates an increased percentage of CTLs expressing all three markers after fucosylation. The effect of fucosylation on CTL-target cell binding was also determined by analyzing synapse formation between CTLs and tumor target cells. The data show that fucosylation increases the frequency of fuco-TIL CTLs forming conjugates with target cells compared with nonfuco-TIL CTLs.

[0110] Example 4 In this example, fucoTILs are evaluated for antitumor cytotoxicity against autologous tumor cell targets. To do so, TILs are first passed through a negative selection column (MACS Miltenyi Biotec-CD8 T Cell Isolation Kit) to isolate CD8-positive CTLs from the TIL population. These are then treated with TZ 102 as described above to fucosylate the CTLs and compare them with nonfucoTILs. Tumor-specific cytotoxicity is then assessed in a standard 4-hour calcein-AM release assay. Tumor target cells are fluorescently labeled with calcein-AM (Invitrogen) for 15 minutes at 37°C and washed with RPMI-1640 to remove free calcein-AM. These cells are seeded into 60-well Tarasaki plates at multiple effector-to-target (E:T) ratios for 4 hours at 37°C. The reaction is quenched with trypan blue, and fluorescence is measured using a microplate fluorescence reader (BioTek Cytation3). The percentage of specific cytotoxicity is calculated using the following formula: ([ 1 - fluorescence target + effector - fluorescence media ] ÷ [fluorescence target alone - fluorescence media ])

[0111] Analysis of the data demonstrates that fucosylation increases the antitumor cytotoxic activity of CD8-positive cells within TILs against their corresponding autologous tumor cells. This enhanced tumor-killing activity is observed both by an increase in absolute tumor cytotoxicity and by a shift in the effector:target ratio required for the level of killing from the higher effector:target ratio required for non-fuco TILs to a much lower effector:target ratio required for the same level of killing (e.g., 50% killing) for fuco TIL CD*-positive cells (e.g., 50% killing).

[0112] Example 5 In this example, fuco-TILs are directly compared with non-fuco-TILs for their ability to transport and infiltrate syngeneic B16-F10 tumors growing in vivo in immunocompetent C57BL / 6 mice. This experiment also includes a vehicle-treated group to serve as a no-intervention control for analyzing tumor homing and invasion. Additionally, a non-fuco-TIL group is included as a comparison for the fuco-TIL group. To provide a source of B16-F10 TILs, B16-F10 tumor cells are implanted into 8-week-old C57BL / 6 mice (Charles River Laboratories) with a body weight (BW) range of 15.4-22.0 g on study day 1. Tumors are measured twice weekly until study termination on day 10, at which point they are excised. TILs are enriched from tumors using standard tumor dissociation and TIL enrichment methods known in the art. After at least one week of growth in mice, tumors are excised and separated into approximately 3-4 mm tissue. 2 Dice the tumor into small pieces. Wash each tumor piece with fresh medium and seed 2 mL of medium containing 6000 IU IL-2 / mL into each well of a 24-well plate. After 3 weeks of culture, TILs are harvested and the T cell phenotype is measured. Overall, the frequencies of CD4+ and CD8+ T cells vary.

[0113] To assess TIL tumor homing and invasion, C57BL / 6 mice were implanted with B16-F10 tumor cells and 100 mm 3 Tumors were allowed to grow until they reached an average tumor volume of 1 × 10. Tumor-bearing mice were randomly assigned to different treatment groups, and after 1 day, tumors were 7 Fuco-TILs or non-fuco-TILs are injected into the tumors. Tumors are measured twice a week until the end of the study on day 10. Each animal is injected with a tumor measuring 1000 mm 3Mice are euthanized when the tumor reaches the endpoint tumor volume of 0.01 mm Hg or on the final day of the study, whichever comes first. TILs are enriched from tumors using standard tumor dissociation and TIL enrichment methods known in the art. TILs are stained with mCD3, mCD4, mCD8 (BioLegend), mCD45, CD90.1 (eBioscience), and Ghost Dye Violet 510 (Tonbo Biosciences) and analyzed by flow cytometry. CD8+ T cells obtained from growing B16-F10 tumors are identified as mCD3+, mCD8+, mCD45+, and CD90.1+.

[0114] Data analysis revealed that homing of fucosylated B16-F10 tumor-derived TILs to B16-F10 tumors was significantly increased compared to non-fucosylated TILs.

[0115] Example 6 In this example, fuco-TILs are directly compared with non-fuco-TILs for their ability to traffic and infiltrate human triple-negative breast cancer (TNBC) tumors growing in vivo in NSG mice. This experiment also includes a vehicle-treated group to serve as a no-intervention control for analyzing tumor homing and invasion. Additionally, a non-fuco-TIL group is included as a comparison for the fuco-TIL group. Both tumor cells and paired autologous TILs were obtained from Yale University.

[0116] To assess TIL tumor homing and invasion, NSG mice were implanted with human TNBC tumor cells and grown to a mean tumor volume of 100 mm. 3 Tumor-bearing mice were randomly assigned to different treatment groups, and after 1 day, tumors were grown to 1 × 10 7 Each animal is injected with either human TNBC-derived fuco-TILs or non-fuco-TILs. Tumors are measured twice weekly until the end of the study on day 10. Each animal is injected with a tumor measuring 1000 mm 3Mice are euthanized when the tumor reaches the endpoint tumor volume of 100 μg / mL or on the final day of the study, whichever comes first. TILs are enriched from tumors using standard tumor dissociation and TIL enrichment methods known in the art. TILs are stained with hCD3, hCD4, hCD8 (BioLegend), hCD45, hCD90.1 (eBioscience), and Ghost Dye Violet 510 (Tonbo Biosciences) and analyzed by flow cytometry. CD8+ T cells obtained from growing B16-F10 tumors are identified as hCD3+, hCD8+, hCD45+, and hCD90.1+.

[0117] Data analysis revealed that homing of fucosylated TNBC tumor-derived TILs to TNBC tumors was significantly increased compared to non-fucosylated TNBC tumors.

[0118] Example 7 In this example, fuco-TILs are directly compared to non-fuco-TILs for in vivo antitumor efficacy in syngeneic B16-F10 model I immunocompetent mice. A vehicle-treated group was included in the experiment to serve as a no-intervention control for analysis. Additionally, a non-fuco-TIL group was included as a comparison for the fuco-TIL group. To provide a source of B16-F10 TILs, B16-F10 tumor cells were implanted into 8-week-old C57BL / 6 mice (Charles River Laboratories) with a body weight (BW) range of 15.4-22.0 g on study day 1. Tumors were measured twice weekly until study termination on day 10, at which point they were excised. TILs were enriched from the tumors using standard tumor dissociation and TIL enrichment methods known in the art. After at least one week of growth in the mice, tumors were excised and separated into approximately 3-4 mm2. 2 Dice the tumor into small pieces. Wash each tumor piece with fresh medium and seed 2 mL of medium containing 6000 IU IL-2 / mL into each well of a 24-well plate. After 3 weeks of culture, TILs are harvested and the T cell phenotype is measured. Overall, the frequencies of CD4+ and CD8+ T cells vary.

[0119] To evaluate the antitumor effect of B16-F10 TILs, B16-F10 tumor cells were implanted into C57BL / 6 mice until the mean tumor volume reached 100 mm 3 Tumor-bearing mice were randomly assigned to different treatment groups and 1 day later (day 1), tumors were grown to 1 × 10 7 Each animal is injected with fuco-TILs or non-fuco-TILs derived from B16-F10 tumors. Tumors are measured twice weekly until the end of the study. Each animal receives a tumor-grown tumor at an endpoint tumor volume of 1500 mm. 3 At the time of tumor volume increase, mice were euthanized and the time to endpoint (TTE) was calculated for each mouse. Treatment efficacy was determined primarily by analysis of mean tumor volume. Treatment efficacy was secondarily determined by analysis of percent tumor growth delay (%TGD), defined as the percentage increase in median time to endpoint (TTE) between treated and control mice, and by log-rank significance of differences in survival rates and regression responses between groups.

[0120] Three groups of B16-F10 tumor-bearing C57BL / 6 mice are administered according to the protocol shown in Table 1. [Table 1]

[0121] Tumors were measured twice weekly with a caliper, and tumor volumes for each animal were measured at 1500 mm 3 Animals will be euthanized when they reach a tumor volume endpoint or at the end of the study, whichever comes first. Animals that terminate the study at tumor volume endpoint will be recorded as euthanized due to tumor progression (TP) along with the date of euthanasia. The time to endpoint (TTE) for analysis will be calculated for each mouse using the following formula: %TGD = [(TC) ÷ C] × 100. where T = median TTE for the treatment group and C = median TTE for the designated control group.

[0122] The efficacy of treatment can be determined by the tumor volume of animals remaining in the study on the final day. MTV(n) is defined as the median tumor volume on the final day of the study for the remaining number of animals (n) whose tumors have not reached the endpoint volume. Treatment efficacy can also be determined by the incidence and size of regression responses observed during the study. Treatment results in partial regression (PR) or complete regression (CR) of the animal's tumor. A PR response is when the tumor volume measured three consecutive times during the study is 50% or less of the volume on day 1. A CR response is when the tumor volume is less than the tumor volume on day 1. Animals that show a CR response at the end of the study are further classified as tumor-free survivors (TFS). Animals are monitored for regression responses.

[0123] Treatment efficacy can also be graphed using a "spider plot" that shows the progression of tumor volume over time for each animal, clearly showing the number of animals experiencing different categories of response: CR, PR, SD, and NR.

[0124] Prism (GraphPad) was used for graphical display and statistical analysis. The log-rank test to assess overall survival was used to analyze the significance of differences between TTE values ​​in the two groups. The log-rank analysis included data from all animals in a group except for those assessed as NTR deaths. Two-sided statistical analysis was performed at a significance level of P = 0.05. Median tumor volumes were plotted as a function of time. If an animal discontinued the study due to tumor burden, the final tumor volume recorded for that animal was included in the data used to calculate the median volume at subsequent time points. Kaplan-Meier plots show the survival rate of animals in each group over time. graph: 1. Mean tumor volume vs. time, standard deviation (SD) 2. Individual tumor volume vs. time 3. Kaplan-Meier table: 1. Median TTE and %TGD

[0125] In this example, mice administered fuco-TILs showed superior tumor regression (as CR) and enhanced tumor control (as PR, SD) compared to mice administered non-fuco-TILs.

[0126] Example 8 In this example, fuco-TILs were directly compared with non-fuco-TILs for their antitumor efficacy against human TNBC tumors growing in vivo in NSG mice. A vehicle-treated group served as a no-intervention control for the analysis. Additionally, a non-fuco-TIL group was included as a comparison for the fuco-TIL group. Autologous TILs paired with tumor cells were both obtained from Yale University. TILs were enriched from human TNBC tumors using standard tumor dissociation and TIL enrichment methods known in the art.

[0127] To evaluate the antitumor efficacy of TNBC TILs, NSG mice were implanted with human TNBC tumor cells and treated with TNBC TILs until the mean tumor volume reached 100 mm. 3 Tumor-bearing mice were randomly assigned to different treatment groups, and after 1 day, tumors were grown to 1 × 10 7 Each animal is injected with either human TNBC-derived fuco- or non-fuco-TILs. Tumors are measured twice weekly until the end of the study. Each animal receives a tumor-grown tumor at an endpoint tumor volume of 1500 mm. 3 At the time of tumor volume increase, mice were euthanized and the time to endpoint (TTE) was calculated for each mouse. Treatment efficacy was determined primarily by analysis of mean tumor volume. Treatment efficacy was secondarily determined by analysis of percent tumor growth delay (%TGD), defined as the percentage increase in median time to endpoint (TTE) between treated and control mice, and by log-rank significance of differences in survival rates and regression responses between groups.

[0128] Three groups of TNBC tumor-bearing NSG mice are administered according to the protocol shown in Table 2. [Table 2]

[0129] Tumors were measured twice weekly with a caliper, and tumor volumes for each animal were measured at 1500 mm 3 Animals will be euthanized when they reach a tumor volume endpoint or at the end of the study, whichever comes first. Animals that terminate the study at tumor volume endpoint will be recorded as euthanized due to tumor progression (TP) along with the date of euthanasia. The time to endpoint (TTE) for analysis will be calculated for each mouse using the following formula: %TGD = [(TC) ÷ C] × 100. where T = median TTE for the treatment group and C = median TTE for the designated control group.

[0130] The efficacy of treatment can be determined by the tumor volume of animals remaining in the study on the final day. MTV(n) is defined as the median tumor volume on the final day of the study for the remaining number of animals (n) whose tumors have not reached the endpoint volume. Treatment efficacy can also be determined by the incidence and size of regression responses observed during the study. Treatment results in partial regression (PR) or complete regression (CR) of the animal's tumor. A PR response is when the tumor volume measured three consecutive times during the study is 50% or less of the volume on day 1. A CR response is when the tumor volume is less than the tumor volume on day 1. Animals that show a CR response at the end of the study are further classified as tumor-free survivors (TFS). Animals are monitored for regression responses.

[0131] Treatment efficacy can also be graphed using a "spider plot" that shows the progression of tumor volume over time for each animal, clearly showing the number of animals experiencing different categories of response: CR, PR, SD, and NR.

[0132] Prism (GraphPad) was used for graphical display and statistical analysis. The log-rank test to assess overall survival was used to analyze the significance of differences between TTE values ​​in the two groups. The log-rank analysis included data from all animals in a group except for those assessed as NTR deaths. Two-sided statistical analysis was performed at a significance level of P = 0.05. Median tumor volumes were plotted as a function of time. If an animal discontinued the study due to tumor burden, the final tumor volume recorded for that animal was included in the data used to calculate the median volume at subsequent time points. Kaplan-Meier plots show the survival rate of animals in each group over time. graph: 1. Mean tumor volume vs. time, standard deviation (SD) 2. Individual tumor volume vs. time 3. Kaplan-Meier table: 1. Median TTE and %TGD

[0133] In this example, mice administered fuco-TILs showed tumor regression (as CR) and enhanced tumor control (as PR, SD) compared to mice administered non-fuco-TILs.

[0134] Example 9 In this example, we directly compare the in vivo antitumor efficacy of fuco-TILs with non-fuco-TILs in syngeneic B16-F10 immunocompetent mice. Furthermore, we evaluated the potential additive or synergistic antitumor activity of this combination, including the effect of combining fuco-TILs with an immune checkpoint inhibitor (ICI) antibody. The experiment included both a vehicle-treated group and an ICI-only treated group, which served as control groups for analysis. Additionally, a non-fuco-TIL group was included as a comparison for the fuco-TIL group, and a non-fuco-TIL + ICI group was included as a comparison for the fuco-TIL + ICI group. To provide a source of B16-F10 TILs, B16-F10 tumor cells were implanted into 8-week-old C57BL / 6 mice (Charles River Laboratories) with a body weight (BW) range of 15.4-22.0 g on study day 1. Tumors were measured twice weekly until study termination on day 10, at which point tumors were excised. TILs are enriched from tumors using standard tumor dissociation and TIL enrichment methods known in the art. After at least one week of growth on mice, tumors are excised and diced into approximately 3-4 mm2 fragments. Each tumor fragment is washed with fresh medium and seeded into each well of a 24-well plate with 2 mL of medium containing 6000 IU IL-2 / mL. After 3 weeks of culture, TILs are harvested and T cell phenotypes are measured. Overall, the frequencies of CD4+ and CD8+ T cells vary.

[0135] To evaluate the antitumor effect of B16-F10 TILs, B16-F10 tumor cells were implanted into C57BL / 6 mice until the mean tumor volume reached 100 mm 3 Tumor-bearing mice were randomly assigned to different treatment groups and 1 day later (day 1), tumors were grown to 1 × 10 7 Each animal is injected with fuco-TILs or non-fuco-TILs derived from B16-F10 tumors. Tumors are measured twice weekly until the end of the study. Each animal receives a tumor-grown tumor at an endpoint tumor volume of 1500 mm. 3At the time of tumor volume increase, mice were euthanized and the time to endpoint (TTE) was calculated for each mouse. Treatment efficacy was determined primarily by analysis of mean tumor volume. Treatment efficacy was secondarily determined by analysis of percent tumor growth delay (%TGD), defined as the percentage increase in median time to endpoint (TTE) between treated and control mice, and by log-rank significance of differences in survival rates and regression responses between groups.

[0136] Six groups of B16-F10 tumor-bearing C57BL / 6 mice are treated according to the protocol shown in Table 3. As above, both fuco- and non-fuco-TILs are administered at 10 x 10 per mouse. 7 The ICI antibody in this study is anti-mouse PD-1, administered ip at 5 mg / kg every other week for three doses. [Table 3]

[0137] Tumors were measured twice weekly with a caliper, and tumor volumes for each animal were measured to be 1500 mm 3 Animals will be euthanized when they reach a tumor volume endpoint or at the end of the study, whichever comes first. Animals that terminate the study at tumor volume endpoint will be recorded as euthanized due to tumor progression (TP) along with the date of euthanasia. The time to endpoint (TTE) for analysis will be calculated for each mouse using the following formula: %TGD = [(TC) ÷ C] × 100. where T = median TTE for the treatment group and C = median TTE for the designated control group.

[0138] The efficacy of treatment can be determined by the tumor volume of animals remaining in the study on the final day. MTV(n) is defined as the median tumor volume on the final day of the study for the remaining number of animals (n) whose tumors have not reached the endpoint volume. Treatment efficacy can also be determined by the incidence and size of regression responses observed during the study. Treatment results in partial regression (PR) or complete regression (CR) of the animal's tumor. A PR response is when the tumor volume measured three consecutive times during the study is 50% or less of the volume on day 1. A CR response is when the tumor volume is less than the tumor volume on day 1. Animals that show a CR response at the end of the study are further classified as tumor-free survivors (TFS). Animals are monitored for regression responses.

[0139] Treatment efficacy can also be graphed using a "spider plot" that shows the progression of tumor volume over time for each animal, clearly showing the number of animals experiencing different categories of response: CR, PR, SD, and NR.

[0140] Prism (GraphPad) was used for graphical display and statistical analysis. The log-rank test to assess overall survival was used to analyze the significance of differences between TTE values ​​in the two groups. The log-rank analysis included data from all animals in a group except for those assessed as NTR deaths. Two-sided statistical analysis was performed at a significance level of P = 0.05. Median tumor volumes were plotted as a function of time. If an animal discontinued the study due to tumor burden, the final tumor volume recorded for that animal was included in the data used to calculate the median volume at subsequent time points. Kaplan-Meier plots show the survival rate of animals in each group over time. graph: 1. Mean tumor volume vs. time, standard deviation (SD) 2. Individual tumor volume vs. time 3. Kaplan-Meier table: 1. Median TTE and %TGD

[0141] In this example, mice receiving fuco-TILs again showed superior tumor regression (as CR) and enhanced tumor control (as PR, SD) compared with mice receiving non-fuco-TILs. Although the addition of ICI only slightly enhanced the activity of non-fuco-TILs, the addition of ICI to fuco-TILs resulted in complete responses (tumor regression) in all mice.

[0142] Example 10 In this example, we directly compared fuco-TILs with non-fuco-TILs for their antitumor effects against human TNBC tumors growing in vivo in NSG mice. This experiment included a vehicle-treated group, which served as a no-intervention control for analysis. Additionally, a non-fuco-TIL group was included as a comparison for the fuco-TIL group. Furthermore, the effect of combining fuco-TILs with an immune checkpoint inhibitor (ICI) antibody was also included to evaluate the potential additive or synergistic antitumor activity of this combination. This experiment included both a vehicle-treated group and an ICI-only treated group, which served as control groups for analysis. Additionally, a non-fuco-TIL group was included as a comparison for the fuco-TIL group, and a non-fuco-TIL + ICI group was included as a comparison for the fuco-TIL + ICI group. Autologous TILs paired with tumor cells were both obtained from Yale University. TILs were enriched from human TNBC tumors using standard tumor dissociation and TIL enrichment methods known in the art.

[0143] To evaluate the antitumor efficacy of TNBC TILs, NSG mice were implanted with human TNBC tumor cells and treated with TNBC TILs until the mean tumor volume reached 100 mm. 3 Tumor-bearing mice were randomly assigned to different treatment groups, and after 1 day, tumors were grown to 1 × 10 7 Each animal is injected with either human TNBC-derived fuco- or non-fuco-TILs. Tumors are measured twice weekly until the end of the study. Each animal receives a tumor-grown tumor at an endpoint tumor volume of 1500 mm. 3At the time of tumor volume increase, mice were euthanized and the time to endpoint (TTE) was calculated for each mouse. Treatment efficacy was determined primarily by analysis of mean tumor volume. Treatment efficacy was secondarily determined by analysis of percent tumor growth delay (%TGD), defined as the percentage increase in median time to endpoint (TTE) between treated and control mice, and by log-rank significance of differences in survival rates and regression responses between groups.

[0144] Six groups of TNBC tumor-bearing NSG mice are administered according to the protocol shown in Table 4.

[0145] As described above, both fuco- and non-fuco-TILs were cultured at 10 × 10 per mouse. 7 The ICI antibody in this study is anti-mouse PD-1, administered ip at 5 mg / kg every other week for three doses. [Table 4]

[0146] Tumors were measured twice weekly with a caliper, and tumor volumes for each animal were measured at 1500 mm 3 Animals will be euthanized when they reach a tumor volume endpoint or at the end of the study, whichever comes first. Animals that terminate the study at tumor volume endpoint will be recorded as euthanized due to tumor progression (TP) along with the date of euthanasia. The time to endpoint (TTE) for analysis will be calculated for each mouse using the following formula: %TGD = [(TC) ÷ C] × 100. where T = median TTE for the treatment group and C = median TTE for the designated control group.

[0147] The efficacy of treatment can be determined by the tumor volume of animals remaining in the study on the final day. MTV(n) is defined as the median tumor volume on the final day of the study for the remaining number of animals (n) whose tumors have not reached the endpoint volume. Treatment efficacy can also be determined by the incidence and size of regression responses observed during the study. Treatment results in partial regression (PR) or complete regression (CR) of the animal's tumor. A PR response is when the tumor volume measured three consecutive times during the study is 50% or less of the volume on day 1. A CR response is when the tumor volume is less than the tumor volume on day 1. Animals that show a CR response at the end of the study are further classified as tumor-free survivors (TFS). Animals are monitored for regression responses.

[0148] Treatment efficacy can also be graphed using a "spider plot" that shows the progression of tumor volume over time for each animal, clearly showing the number of animals experiencing different categories of response: CR, PR, SD, and NR.

[0149] Prism (GraphPad) was used for graphical display and statistical analysis. The log-rank test to assess overall survival was used to analyze the significance of differences between TTE values ​​in the two groups. The log-rank analysis included data from all animals in a group except for those assessed as NTR deaths. Two-sided statistical analysis was performed at a significance level of P = 0.05. Median tumor volumes were plotted as a function of time. If an animal discontinued the study due to tumor burden, the final tumor volume recorded for that animal was included in the data used to calculate the median volume at subsequent time points. Kaplan-Meier plots show the survival rate of animals in each group over time. graph: 1. Mean tumor volume vs. time, standard deviation (SD) 2. Individual tumor volume vs. time 3. Kaplan-Meier table: 1. Median TTE and %TGD

[0150] In this example, mice receiving fuco-TILs again showed superior tumor regression (as CR) and enhanced tumor control (as PR, SD) compared with mice receiving non-fuco-TILs. Although the addition of ICI only slightly enhanced the activity of non-fuco-TILs, the addition of ICI to fuco-TILs resulted in complete responses (tumor regression) in all mice.

[0151] Example 11 FIG. 1 is a schematic diagram of one non-limiting embodiment of a combinatorial therapeutic method constructed in accordance with the present disclosure, which includes the use of ex vivo fucosylated tumor-infiltrating lymphocytes and immune checkpoint inhibitors.

[0152] In this method, at least a portion of at least one tumor is excised from the patient and sent to a laboratory for processing. Immune cells, such as tumor-infiltrating leukocytes (TILs), are extracted from the tumor and may be subjected to one or more optional steps, including, but not limited to, priming / activation, proliferation, and / or selection steps (such as exposure to IL-2). These cells are then exposed to TZ 102 (FUT7) at room temperature for at least about 30 minutes to fucosylate the cells and provide fucoACTs, thereby enhancing the cells' trafficking and tumor-infiltrating properties. The fucoACTs may be transported directly to a clinical center for infusion into the patient. Optionally, the fucoACTs may be frozen for transportation and / or storage purposes before delivery to the clinical center. Upon arrival at the clinical center, the fucoACTs are infused back into the patient. The patient may then be administered one or more ICIs either before, simultaneously with, or after administration of the fucoACTs.

[0153] Prior to the injection step, the patient may optionally be conditioned for the transplantation of the fucoACT by administering one or more chemotherapy methods known in the art, such as, but not limited to, cyclophosphamide (CY), fludarabine (FLU), and / or total body irradiation (TBI). Additionally, after injection of the fucoACT, the patient may optionally be administered an immunotherapeutic dose of high-dose IL-2 (HD IL-2).

[0154] Example 12 Several breakthroughs in cancer immunotherapy have been achieved in recent years, including stimulating immune cell proliferation signals (such as, but not limited to, CD28, IL-2, and / or interferon (IFN)); removing immunosuppression by checkpoint inhibitors (such as, but not limited to, inhibitors of CTLA-4 and PD1:PD-L1 interactions); and adoptive cell therapy (such as, but not limited to, CAR-T cells, TCR-T cells, TILs, NK cells, dendritic cells).

[0155] However, despite the promise of immunotherapy, challenges remain. Immune cells do not traffic to solid tumors and infiltrate them poorly. In fact, less than 3% of immune cells traffic to tumors and infiltrate the tumor core. Immune checkpoint inhibitors have limited efficacy in most cancer patients with solid tumors. Immune checkpoint inhibitor (ICI)-mediated antitumor responses depend on the infiltration of T cells that can recognize and kill tumor cells. However, ICIs are ineffective in "cold" tumors, characterized by a lack or poor T cell infiltration. "Cold" tumors include immune-excluded tumors (i.e., CD8+ T cells are localized only at the infiltrating margin and do not efficiently infiltrate the tumor) and immune-desert tumors (i.e., CD8+ T cells are absent from the tumor and its periphery). In contrast, "hot" tumors refer to immunoinflammatory tumors characterized by high immune cell infiltration, increased IFNγ signaling, and high (relative) PD-L1 expression. In contrast, adoptive cell therapy is ineffective in most cancer patients with solid tumors, as only 3–5% of the introduced T cells reach the tumor microenvironment.

[0156] Cancer immunotherapy, including TIL therapy and / or checkpoint inhibitors, is promising, however, with limited efficacy in cold cancer tumor indications, as shown in Table 5 below. [Table 5]

[0157] Immune checkpoint inhibitors are most responsive to hot tumors than to cold tumors, and therefore one goal of the present disclosure is to convert cold tumors into hot tumors.

[0158] The present disclosure found that ex vivo fucosylation of TILs promotes immune cell priming / activation, immune cell proliferation, and tumor trafficking and infiltration, thereby converting cold tumors into hot tumors and enhancing checkpoint inhibitor immunity. In particular, ex vivo fucosylated TILs (e.g., TILs isolated from patients, donors, or iPSCs, or genetically engineered cells, and then exposed to TZ102 (or other fucosyltransferase) and a fucose donor) are activated and expanded during the manufacturing process, accompanied by cell selection. These ex vivo fucosylated TILs demonstrated a 2.5-fold improvement in tumor trafficking and tumor infiltration. Additionally, ex vivo production of fucosylated TILs results in the delivery of several antitumor effector cell types with antigen receptor diversity for improved solid tumor killing. Furthermore, the observed effects are not limited to CD8+ effector cells; ex vivo fucosylation also promotes the trafficking and infiltration of various cancer-killing cells (including, but not limited to, NK cells, dendritic cells, B cells, etc.) into tumors, thereby improving overall efficacy.

[0159] Ex vivo, fucosylated immune cells enter the tumor microenvironment in 200% to 500% more numbers and infiltrate tumors, thereby enhancing the resistance of these transported / infiltrated immune cells to cancer and improving the efficacy of co-administered immune checkpoint inhibitors.

[0160] The specific cell surface modification of fucosylation has been shown to increase T cell tumor targeting and efficacy. Fucosylation is a post-translational modification that "turns on" T cell homing and infiltration. Fucosylation allows T cells to adhere to and pass through blood vessel walls, thereby infiltrating not only blood vessel walls but also tumor masses.

[0161] For example (but not limited to), exposure of TCR-T cells to TZ102 increased fucosylation levels from 25% to 92%. Subsequently, TCR-T homing and penetration measurements in an A549 lung cancer model demonstrated that TZ102-treated T cells increased lung tumor homing by 200%, demonstrating that fucosylation doubled the number of TCR-T cells infiltrating tumors. Furthermore, fucosylation increased the proportion of both CD3+ and CD8+ TCR-T cells in tumors by 3.3-fold and 4.7-fold, respectively. This confirms that TZ102-treated TCR-T cells trafficked to and infiltrated lung cancers 330-470% more effectively than untreated TZ102-treated T cells.

[0162] Al-Atrash et al. (Clinical Cancer Research (2019) 25(8):2610-2620) demonstrated that in a SKBR3 breast cancer model, TZ102-treated T cells increased their trafficking and infiltration rate into breast tumors by 200% and reduced breast tumor mass by 70% compared to untreated TZ102-treated T cells. This reference also demonstrated that in a B16-F10 melanoma animal model, TZ102-treated T cells increased their trafficking and infiltration rate into melanomas by 250% and reduced melanoma mass by 77% compared to untreated TZ102-treated T cells. Fucosylation increased TME homing, penetration, and antitumor cytotoxicity, resulting in increased TCR-T cell antitumor efficacy.

[0163] Example 13 A Phase IIa, 2:1, randomized, double-blind study was conducted to demonstrate the safety and efficacy of TZ102-fucosylated TILs in combination with immune checkpoint inhibitors (ICIs) in patients with stage III and IV metastatic melanoma. Approximately 78,000 cases of stage III and IV metastatic melanoma are diagnosed annually, with over 9,000 deaths annually. The 5-year survival rate is 5-19%, with a median overall survival of 5.3 months. Therefore, there is an unmet medical need for treatment of these patients.

[0164] Up to 33 subjects with stage III or IV unresectable melanoma whose cancer has progressed despite immune checkpoint therapy and / or BRAF / MEK inhibitors (if BRAF mutations) will be included. Up to 20 subjects will receive TZ102-TIL + ICI combination therapy, up to 3 subjects will receive TZ102-TIL monotherapy, and up to 10 subjects will receive standard of care + ICI therapy. Lymphocyte-reducing conditioning will be performed before the infusion of TZ102-fucosylated TILs, and IL-2 will be administered after the infusion.

[0165] Efficacy endpoints will be measured primarily as objective response rate (ORR), secondarily as duration of response, progression-free survival, and overall survival. Safety endpoints will also be assessed based on the number and percentage of all adverse and serious events (AEs) and related AEs classified by severity, as well as the number and percentage of clinically significant abnormal laboratory values.

[0166] This study demonstrates the synergistic combination of TZ102-fucosylated TILs with ICIs, as well as the safety and efficacy of TZ102-fucosylated TILs in combination with ICIs in patients with stage III and IV metastatic melanoma.

[0167] Example 14 We will conduct a Phase IIa, 2:1, randomized, double-blind study to demonstrate the safety and efficacy of TZ102-fucosylated TILs in combination with immune checkpoint inhibitors (ICIs) in patients with metastatic breast cancer. Metastatic breast cancer is the second leading cause of cancer death worldwide, with approximately 284,200 new cases and over 44,000 deaths per year. The 5-year survival rate is approximately 11%, and the median overall survival is 12-18 months. Therefore, there is an unmet medical need for the treatment of these patients.

[0168] Up to 33 subjects with pretreated metastatic triple-negative breast cancer whose cancer has progressed despite standard of care will be included. Up to 20 subjects will receive TZ102-TIL + ICI combination therapy, up to 3 subjects will receive TZ102-TIL monotherapy, and up to 10 subjects will receive standard of care + ICI therapy. Lymphocyte-reducing conditioning will be performed before the infusion of TZ102-fucosylated TILs, and IL-2 will be administered after the infusion.

[0169] Efficacy endpoints will be measured primarily by objective response rate (ORR), and secondarily by duration of response, progression-free survival, and overall survival. Safety endpoints will also be evaluated to characterize the safety profile of TIL as monotherapy in patients with metastatic triple-negative breast cancer, as measured by the incidence of treatment-emergent adverse events (TEAEs) greater than grade 3.

[0170] This study demonstrates the synergistic combination of TZ102-fucosylated TILs with ICIs, as well as the safety and efficacy of TZ102-fucosylated TILs in combination with ICIs against metastatic breast cancer.

[0171] Non-limiting exemplary embodiments Exemplary embodiment 1. A pharmaceutical composition for adoptive cell therapy comprising at least one isolated immune cell type that is fucosylated ex vivo and at least one immune checkpoint inhibitor.

[0172] Exemplary Embodiment 1a. The composition of Exemplary Embodiment 1, further comprising at least one pharmaceutically acceptable carrier.

[0173] Exemplary Embodiment 2. The composition of Exemplary Embodiment 1 or 1a, wherein the at least one isolated immune cell type is selected from the group consisting of cytotoxic T cells (CTLs), regulatory T cells (Tregs), helper T cells, NK cells, B cells, dendritic cells, genetically modified versions thereof, and combinations thereof.

[0174] Exemplary Embodiment 2a. The composition of Exemplary Embodiment 2, wherein the genetically modified version of the immune cell type is selected from the group consisting of chimeric antigen receptor gene-transduced T cells (CAR-T cells), T cell receptor gene-transduced T cells (TCR-T cells), chimeric antigen receptor gene-transduced natural killer (NK) cells (CAR-NK cells), and combinations thereof.

[0175] Exemplary Embodiment 3. The composition of Exemplary Embodiment 1 or 2, wherein the at least one immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor, a CTLA-4 inhibitor, a TIM-3 inhibitor, a B7-H3 inhibitor, an A2aR inhibitor, a CD73 inhibitor, an NKG2A inhibitor, a PVRIG / PVRL2 inhibitor, a CEACAM1 inhibitor, a FAK inhibitor, a CCL2 / CCR2 inhibitor, a LIF inhibitor, a CD47 / SIRPα inhibitor, a CSF-1 inhibitor, an IL-1 inhibitor, an IL-8 inhibitor, a SEMA4D inhibitor, an Ang-2 inhibitor, a CLEVER-1 inhibitor, a phosphatylserine inhibitor, and combinations thereof.

[0176] Exemplary Embodiment 4. The composition of Exemplary Embodiment 3, wherein the at least one immune checkpoint inhibitor comprises at least one PD-1 inhibitor and at least one LAG-3 inhibitor.

[0177] Exemplary Embodiment 4a. The composition of any one of Exemplary Embodiments 1-4, wherein the composition comprises a therapeutically effective amount of at least one immune checkpoint inhibitor and / or the composition comprises a therapeutically effective amount of at least one isolated immune cell that is ex vivo fucosylated.

[0178] Exemplary Embodiment 4b. The composition of any one of Exemplary Embodiments 1-4a, wherein the at least one isolated immune cell type that is fucosylated ex vivo is produced by a method comprising the following steps: 1) identifying a cancer patient in need of adoptive cell therapy; 2) isolating immune cells from the patient's blood or cancer tumor; 3) expanding the immune cells ex vivo; and 4) contacting the expanded immune cells with a fucosyltransferase and at least one of fucose or GDP-fucose to provide fucosylated immune cells.

[0179] Exemplary Embodiment 4c. The composition of Exemplary Embodiment 4b, wherein the fucosyltransferase (FUT) is selected from the group consisting of FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9, FUT10, FUT11, and combinations thereof.

[0180] Exemplary Embodiment 4d. The composition of Exemplary Embodiment 4c, wherein the fucosyltransferase is selected from the group consisting of FUT3, FUT5, FUT6, FUT7, FUT9, FUT10, and combinations thereof.

[0181] Exemplary Embodiment 4e. The composition of any one of Exemplary Embodiments 4b-4d, wherein the method further comprises, after step (4), (4a) separating the fucosylated lymphocytes from other components, and wherein step (5) is further defined as returning the fucosylated lymphocytes to the patient intravenously.

[0182] Exemplary Embodiment 4f. The composition of Exemplary Embodiment 4e, wherein the method comprises, after step (4a), (4b) separating the specific fucosylated lymphocytes from other fucosylated lymphocytes, and wherein step (5) is further defined as returning the specific fucosylated lymphocytes to the patient intravenously.

[0183] Exemplary embodiment 5. A system comprising a composition comprising at least one immune checkpoint inhibitor (ICI), and comprising at least one fucosylated immune cell type for adoptive cell therapy (fucoACT).

[0184] Exemplary Embodiment 6. The system of Exemplary Embodiment 5, wherein the at least one immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor, a CTLA-4 inhibitor, a TIM-3 inhibitor, a B7-H3 inhibitor, an A2aR inhibitor, a CD73 inhibitor, an NKG2A inhibitor, a PVRIG / PVRL2 inhibitor, a CEACAM1 inhibitor, a FAK inhibitor, a CCL2 / CCR2 inhibitor, a LIF inhibitor, a CD47 / SIRPα inhibitor, a CSF-1 inhibitor, an IL-1 inhibitor, an IL-8 inhibitor, a SEMA4D inhibitor, an Ang-2 inhibitor, a CLEVER-1 inhibitor, a phosphatylserine inhibitor, and combinations thereof.

[0185] Exemplary Embodiment 7. The system of exemplary embodiment 6, wherein the at least one immune checkpoint inhibitor comprises at least one PD-1 inhibitor and at least one LAG-3 inhibitor.

[0186] Exemplary Embodiment 8. The system of exemplary embodiment 6 or 7, wherein the at least one immune cell type is selected from the group consisting of cytotoxic T cells (CTLs), regulatory T cells (Tregs), helper T cells, NK cells, B cells, dendritic cells, genetically modified versions thereof, and combinations thereof.

[0187] Exemplary Embodiment 8a. The system of Exemplary Embodiment 8, wherein the genetically modified version of the immune cell type is selected from the group consisting of chimeric antigen receptor gene-transduced T cells (CAR-T cells), T cell receptor gene-transduced T cells (TCR-T cells), chimeric antigen receptor gene-transduced natural killer (NK) cells (CAR-NK cells), and combinations thereof.

[0188] Exemplary Embodiment 8b. The system of any one of Exemplary Embodiments 6-8a, wherein the composition provides a therapeutically effective amount of an immune checkpoint inhibitor.

[0189] Exemplary Embodiment 8c. The system of any one of Exemplary Embodiments 6-8b, wherein the composition provides a therapeutically effective amount of a fucoACT.

[0190] Exemplary Embodiment 8d. The system of any one of Exemplary Embodiments 6-8c, wherein the system is further defined as a kit.

[0191] Exemplary Embodiment 8e. The system of any one of Exemplary Embodiments 6-8d, wherein the fucoACT is produced by a method comprising the following steps: 1) identifying a cancer patient in need of adoptive cell therapy; 2) isolating immune cells from the patient's blood or cancer tumor; 3) expanding the immune cells ex vivo; and 4) contacting the expanded immune cells with a fucosyltransferase and at least one of fucose or GDP-fucose to provide fucosylated immune cells.

[0192] Exemplary Embodiment 8f. The system of exemplary embodiment 8e, wherein the fucosyltransferase (FUT) is selected from the group consisting of FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9, FUT10, FUT11, and combinations thereof.

[0193] Exemplary Embodiment 8g. The system of Exemplary Embodiment 8f, wherein the fucosyltransferase is selected from the group consisting of FUT3, FUT5, FUT6, FUT7, FUT9, FUT10, and combinations thereof.

[0194] Exemplary embodiment 9. A method of adoptive cell therapy, comprising the steps of: 1) identifying a cancer patient in need of adoptive cell therapy; 2) isolating immune cells from the patient's blood or cancer tumor; 3) expanding the immune cells ex vivo; 4) contacting the expanded immune cells with a fucosyltransferase and at least one of fucose or GDP-fucose to provide fucosylated immune cells; 5) returning the fucosylated immune cells intravenously to the patient; and 6) administering at least one immune checkpoint inhibitor to the patient simultaneously with, or sequentially in whole or in part with, the fucosylated immune cells.

[0195] Exemplary Embodiment 10. The method of exemplary embodiment 9, further comprising, after step (4), (4a) separating the fucosylated lymphocytes from other components, and wherein step (5) is further defined as returning the fucosylated lymphocytes to the patient intravenously.

[0196] Exemplary Embodiment 11. The method of exemplary embodiment 10, further comprising, after step (4a), (4b) isolating the specific fucosylated lymphocytes from other fucosylated lymphocytes, and wherein step (5) is further defined as returning the specific fucosylated lymphocytes to the patient intravenously.

[0197] Exemplary Embodiment 12. The method of any one of exemplary embodiments 9 to 11, wherein the fucosyltransferase (FUT) is selected from the group consisting of FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9, FUT10, FUT11, and combinations thereof.

[0198] Exemplary Embodiment 12a. The method of Exemplary Embodiment 12, wherein the fucosyltransferase is selected from the group consisting of FUT3, FUT5, FUT6, FUT7, FUT9, FUT10, and combinations thereof.

[0199] Exemplary Embodiment 12b. The method of any one of Exemplary Embodiments 9-12a, wherein at least step (6) is repeated one or more times.

[0200] Exemplary Embodiment 12c. The method of any one of Exemplary Embodiments 9-12b, wherein at least step (5) is repeated one or more times.

[0201] Exemplary Embodiment 12d. The method of any one of Exemplary Embodiments 9-12c, wherein at least one immune checkpoint inhibitor is administered prior to the administration of at least one fucosylated immune cell type for adoptive cellular therapy (FucoACT).

[0202] Exemplary Embodiment 12e. The method of any one of Exemplary Embodiments 9-12d, wherein at least one immune checkpoint inhibitor is administered simultaneously with at least one fucosylated immune cell type for adoptive cellular therapy (fucoACT).

[0203] Exemplary Embodiment 12f. The method of any one of Exemplary Embodiments 9-12e, wherein at least one fucosylated immune cell type for adoptive cellular therapy (FucoACT) is administered prior to at least one immune checkpoint inhibitor.

[0204] Exemplary embodiment 13. A method of treating one or more cancers and reducing the incidence or severity of metastases in a cancer patient, comprising administering to the cancer patient, simultaneously or in whole or in part sequentially, a combination of at least one ICI and at least one fucosylated immune cell-type adoptive cell therapy (fucoACT).

[0205] Exemplary Embodiment 13a. The method of Exemplary Embodiment 13, wherein the at least one ICI and at least one fucosylated immune cell type for adoptive cellular therapy (FucoACT) are present in the same pharmaceutical composition.

[0206] Exemplary Embodiment 13b. The method of Exemplary Embodiment 13, wherein the at least one ICI and the at least one fucosylated immune cell type for adoptive cellular therapy (FucoACT) are present in separate pharmaceutical compositions.

[0207] Exemplary Embodiment 14. The method of any one of Exemplary Embodiments 9-13b, wherein the at least one immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor, a CTLA-4 inhibitor, a TIM-3 inhibitor, a B7-H3 inhibitor, an A2aR inhibitor, a CD73 inhibitor, an NKG2A inhibitor, a PVRIG / PVRL2 inhibitor, a CEACAM1 inhibitor, a FAK inhibitor, a CCL2 / CCR2 inhibitor, a LIF inhibitor, a CD47 / SIRPα inhibitor, a CSF-1 inhibitor, an IL-1 inhibitor, an IL-8 inhibitor, a SEMA4D inhibitor, an Ang-2 inhibitor, a CLEVER-1 inhibitor, a phosphatylserine inhibitor, and combinations thereof.

[0208] Exemplary Embodiment 15. The method of any one of Exemplary Embodiments 9-14, wherein the at least one immune checkpoint inhibitor comprises at least one PD-1 inhibitor and at least one LAG-3 inhibitor.

[0209] Exemplary Embodiment 16. The method of any one of Exemplary Embodiments 9-15, wherein at least one immune cell type is selected from the group consisting of cytotoxic T cells (CTLs), regulatory T cells (Tregs), helper T cells, NK cells, B cells, dendritic cells, genetically modified versions thereof, and combinations thereof.

[0210] Exemplary Embodiment 16a. The method of Exemplary Embodiment 16, wherein the genetically modified version of the immune cell type is selected from the group consisting of chimeric antigen receptor gene-transduced T cells (CAR-T cells), T cell receptor gene-transduced T cells (TCR-T cells), chimeric antigen receptor gene-transduced natural killer (NK) cells (CAR-NK cells), and combinations thereof.

[0211] Exemplary embodiment 17. The method of any one of exemplary embodiments 9 to 16, wherein the cancer is selected from the group consisting of prostate cancer, skin cancer, ovarian cancer, breast cancer, non-lymphoid parenchymal organ cancer; head and / or neck cancer; leukemia; retinal cancer; esophageal cancer; multiple myeloma; melanoma; colorectal cancer; lung cancer; cervical cancer; endometrial cancer; gallbladder cancer; liver cancer; thyroid follicular cancer; gastric cancer; non-small cell lung cancer; glioma; urothelial carcinoma; bladder cancer; prostate cancer; renal cell carcinoma; invasive ductal carcinoma; glioblastoma multiforme; and combinations thereof.

[0212] Exemplary Embodiment 18. The method of any one of Exemplary Embodiments 9-17, further comprising administering to the cancer patient at least one additional treatment, wherein the at least one additional treatment is selected from the group consisting of radiation therapy, surgery, chemotherapy, immunotherapy, targeted therapy, hormone therapy, stem cell transplant, cryotherapy, laser therapy, precision medicine, and combinations thereof.

[0213] Exemplary Embodiment 18a. The method of any one of exemplary embodiments 9-18, wherein the administration of at least one ICI is repeated one or more times.

[0214] Exemplary Embodiment 18b. The method of any one of Exemplary Embodiments 9-18a, wherein the administration of at least one fucosylated immune cell type for adoptive cellular therapy (fucoACT) is repeated one or more times.

[0215] Exemplary Embodiment 18c. The method of any one of Exemplary Embodiments 9-18b, wherein at least one immune checkpoint inhibitor is administered prior to the administration of at least one fucosylated immune cell type for adoptive cellular therapy (fucoACT).

[0216] Exemplary Embodiment 18d. The method of any one of Exemplary Embodiments 9-18c, wherein at least one immune checkpoint inhibitor is administered simultaneously with at least one fucosylated immune cell type for adoptive cellular therapy (fucoACT).

[0217] Exemplary Embodiment 18e. The method of any one of Exemplary Embodiments 9-18d, wherein at least one fucosylated immune cell type for adoptive cellular therapy (FucoACT) is administered prior to at least one immune checkpoint inhibitor.

[0218] Exemplary Embodiment 19. The method of any one of exemplary embodiments 9-18e, wherein the patient has a primary tumor selected from the group consisting of a breast, lung, bladder, skin, intestine, colon, kidney, ovary, pancreas, prostate, brain, stomach, thyroid, head and neck, gastroesophageal tract, connective tissue or other non-epithelial tissue, lymphoid cell, or uterine tumor.

[0219] Exemplary embodiment 20. The method of exemplary embodiment 19, wherein the primary tumor is advanced metastatic breast cancer, optionally triple-negative breast cancer, and further comprising administering a histone deacetylase inhibitor to the patient to prime the tumor about 1 day to about 3 weeks prior to treatment with the ICI / fucoACT combination.

[0220] Exemplary Embodiment 21. The method of any one of exemplary embodiments 9-20, wherein treating cancer results in one or more of: (i) reducing or delaying tumor metastasis; (ii) preventing or delaying cancer recurrence; (iii) increasing disease-free or tumor-free survival; (iv) increasing overall survival; (v) reducing the frequency of treatment; (vi) alleviating one or more symptoms of said cancer; and (vii) reducing tumor burden.

[0221] Exemplary Embodiment 22. The method of any one of exemplary embodiments 13-21, wherein the metastases reduced are metastases of one or more of the adrenal gland, brain and / or spinal cord, bone, lung, liver and / or pleura, gastrointestinal tract, peritoneum, muscle, lymph nodes, and skin.

[0222] Exemplary Embodiment 23. The method of any one of exemplary embodiments 13-22, wherein the reduction or prevention of metastasis is when the primary or secondary tumor in the subject being treated is a cancer of the breast, lung, bladder, skin, intestine, colon, kidney, ovary, pancreas, prostate, liver, brain, stomach, thyroid, head and neck, gastroesophageal, bone marrow, lymphatic system, connective or other non-epithelial tissue, and uterus.

[0223] Exemplary embodiment 24 The method of any one of exemplary embodiments 9-23, wherein the cancer is triple-negative breast cancer.

[0224] Exemplary Embodiment 25. A method for treating cancer or reducing the incidence or severity of metastases by improving the trafficking of immune cells to solid tumors during adoptive cell therapy (ACT), comprising administering to a subject having a solid tumor a composition described in any one of exemplary embodiments 1-4d or a system described in any one of exemplary embodiments 5-8d.

[0225] Exemplary Embodiment 26. The method of exemplary embodiment 25, wherein ACT comprises: 1) identifying a cancer patient in need of ACT; 2) isolating immune cells from the patient's blood or cancer tumor; 3) expanding the immune cells ex vivo; 4) contacting the expanded immune cells with a fucosyltransferase and fucose or GDP-fucose; and 5) administering the fucosylated immune cells intravenously back to the patient.

[0226] Exemplary embodiment 27. A method according to exemplary embodiment 25 or 26, wherein the ACT utilizes a tumor infiltrating lymphocyte (TIL)-based therapy that requires cell surface modification using a fucosyltransferase and fucose or GDP-fucose, or a genetically engineered or otherwise engineered product, to add fucose to the cells and upregulate selectin ligands on the cells.

[0227] Exemplary Embodiment 28. The method of any one of exemplary embodiments 25-27, wherein the ACT utilizes chimeric antigen receptor gene-transduced T cell (CAR-T)-based therapy enhanced by cell surface modification using a fucosyltransferase and fucose or GDP-fucose, or a genetically engineered or otherwise engineered product, to add fucose to the cells and upregulate selectin ligands on the cells.

[0228] Exemplary Embodiment 29. The method of any one of exemplary embodiments 25-28, wherein the ACT utilizes T cell receptor gene transduced T cell (TCR-T) based therapy enhanced by cell surface modification using a fucosyltransferase and fucose or GDP-fucose, or a genetically engineered or otherwise engineered product, to add fucose to the cells and upregulate selectin ligands on the cells.

[0229] Exemplary Embodiment 30. The method of any one of exemplary embodiments 25-29, wherein the ACT utilizes chimeric antigen receptor gene-transduced natural killer (NK) cell (CAR-NK)-based therapy enhanced by cell surface modification using a fucosyltransferase and fucose or GDP-fucose, or a genetically engineered or otherwise engineered product, to add fucose to the cells and upregulate selectin ligands on the cells.

[0230] Exemplary embodiment 31. The method of any one of exemplary embodiments 25-30, wherein the ACT utilizes T cells harvested from a patient, donor, or iPSCs and stimulated in vitro with tumor cells or tumor cell products to produce tumor-selective immune cells, and enhanced by cell surface modification, such as with fucosyltransferase and fucose or GDP-fucose, or a genetically engineered product, to add fucose to the cells and upregulate selectin ligands on the cells.

[0231] Exemplary Embodiment 32 The method of any one of Exemplary Embodiments 9-31, wherein the at least one immune checkpoint inhibitor comprises at least one agent selected from the group consisting of a nucleic acid, a peptide, a protein, an antibody, a peptibody, a diabody, a minibody, a single-chain variable fragment (ScFv), a fragment or variant thereof, and any combination thereof.

[0232] Exemplary Embodiment 33. The method of any one of Exemplary Embodiments 9-32, wherein the at least one immune checkpoint inhibitor comprises at least one agent selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, pidilizumab, dostarimab, pimivalimab, spartalizumab, canrelizumab, sintilimab, tislelizumab, toripalimab, retifanlimab, and combinations thereof.

[0233] Exemplary Embodiment 34. The method of any one of Exemplary Embodiments 9-33, wherein the at least one immune checkpoint inhibitor comprises at least one agent selected from the group consisting of atezolizumab, durvalumab, avelumab, cosibelimab, embafolimab, AUNP12, sosazolimab, STI-3031, and combinations thereof.

[0234] Exemplary Embodiment 35. The method of any one of Exemplary Embodiments 9-34, wherein the at least one immune checkpoint inhibitor comprises at least one agent selected from the group consisting of yelamirimab, Sym022, TSR-033, fianlimab, yelamirimab, INCAGN2385-101, favezelimab, BI754111, and combinations thereof.

[0235] Exemplary Embodiment 36. A method of treating cancer in a patient, comprising administering a therapeutically effective amount of the composition of any one of exemplary embodiments 1-4d or the system of any one of exemplary embodiments 5-8d to a patient in need thereof.

[0236] Exemplary Embodiment 37. A method of enhancing the activity of natural killer (NK), cytotoxic T cell, or Treg cell activity in a cancer patient, comprising administering a therapeutically effective amount of a composition described in any one of exemplary embodiments 1-4d or a system described in any one of exemplary embodiments 5-8d.

[0237] Exemplary Embodiment 38. A method for enhancing antibody-dependent cell-mediated cytotoxicity in a cancer patient, comprising administering a therapeutically effective amount of a composition described in any one of exemplary embodiments 1-4d or a system described in any one of exemplary embodiments 5-8d.

[0238] Exemplary Embodiment 39. A method of treating a disease, disorder, or alleviating or eliminating symptoms of a disease or disorder, comprising administering to a subject in need of treatment a therapeutically effective amount of a combination of at least one fucosylated immune cell type for adoptive cellular therapy (FucoACT) and at least one of a PD-1 inhibitor, a PD-L1 inhibitor, and / or a LAG-3 inhibitor, wherein said compounds are administered simultaneously or within 24 hours of each other.

[0239] Exemplary embodiment 40. A method of improving the resistance of immune cells to cancer cells by first contacting the immune cells ex vivo with a fucosyltransferase and UDP-fucose, such that greater than 50% of the immune cells so contacted are fucosylated.

[0240] Exemplary embodiment 41 The method of exemplary embodiment 40, wherein the immune cells are tumor-infiltrating lymphocytes (TILs).

[0241] Exemplary Embodiment 42 The method of exemplary embodiment 41, wherein fucosylation of TILs results in increased adhesion to endothelial cell selectins during circulation.

[0242] Exemplary embodiment 43. The method of exemplary embodiment 41 or 42, wherein fucosylation of TILs results in increased surface expression of the transport molecule CD162 / PSGL-1 and the chemotactic receptor CD183 (CXCR3) and the stimulatory coregulatory molecule CD137 (41BB).

[0243] Exemplary embodiment 44. The method of any one of exemplary embodiments 41-43, wherein fucosylation of TILs results in enhanced activity of cytotoxic T cells (CTLs) as measured by expression of key components of the cytotoxic machinery, including at least one of FasL / CD95L, perforin, granzymes, or the ability to kill tumor cells.

[0244] Exemplary embodiment 45. The method of any one of exemplary embodiments 41 to 44, wherein fucosylation of TILs exhibits a higher frequency of fuco-TIL CTLs forming conjugates with target cells compared to non-fucosylated TIL CTLs.

[0245] Exemplary embodiment 46. The method of any one of exemplary embodiments 40-45, wherein injection of autologous tumor-derived fuco-TILs into a patient resulted in significant tumor regression and enhanced tumor control.

[0246] While the accompanying disclosure describes the concepts of the present invention in conjunction with the specific experiments, results, and statements set forth below, it should be understood that many alternatives, modifications, and variations will be apparent to those skilled in the art, and accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of this disclosure.

Claims

1. at least one isolated immune cell type that is fucosylated ex vivo; at least one immune checkpoint inhibitor; and at least one pharmaceutically acceptable carrier.

2. 2. The composition of claim 1, wherein the at least one isolated immune cell type is selected from the group consisting of cytotoxic T cells (CTLs), regulatory T cells (Tregs), helper T cells, NK cells, B cells, dendritic cells, genetically modified versions thereof, and combinations thereof.

3. 2. The composition of claim 1, wherein the at least one immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor, a CTLA-4 inhibitor, a TIM-3 inhibitor, a B7-H3 inhibitor, an A2aR inhibitor, a CD73 inhibitor, an NKG2A inhibitor, a PVRIG / PVRL2 inhibitor, a CEACAM1 inhibitor, a FAK inhibitor, a CCL2 / CCR2 inhibitor, a LIF inhibitor, a CD47 / SIRPα inhibitor, a CSF-1 inhibitor, an IL-1 inhibitor, an IL-8 inhibitor, a SEMA4D inhibitor, an Ang-2 inhibitor, a CLEVER-1 inhibitor, a phosphatylserine inhibitor, and combinations thereof.

4. 4. The composition of claim 3, wherein the at least one immune checkpoint inhibitor comprises at least one PD-1 inhibitor and at least one LAG-3 inhibitor.

5. a composition comprising at least one immune checkpoint inhibitor (ICI); a composition comprising at least one fucosylated immune cell type for adoptive cell therapy (fucoACT); A system including:

6. 6. The system of claim 5, wherein the at least one immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor, a CTLA-4 inhibitor, a TIM-3 inhibitor, a B7-H3 inhibitor, an A2aR inhibitor, a CD73 inhibitor, an NKG2A inhibitor, a PVRIG / PVRL2 inhibitor, a CEACAM1 inhibitor, a FAK inhibitor, a CCL2 / CCR2 inhibitor, a LIF inhibitor, a CD47 / SIRPα inhibitor, a CSF-1 inhibitor, an IL-1 inhibitor, an IL-8 inhibitor, a SEMA4D inhibitor, an Ang-2 inhibitor, a CLEVER-1 inhibitor, a phosphatylserine inhibitor, and combinations thereof.

7. 7. The system of claim 6, wherein the at least one immune checkpoint inhibitor comprises at least one PD-1 inhibitor and at least one LAG-3 inhibitor.

8. 7. The system of claim 6, wherein the at least one isolated immune cell type is selected from the group consisting of cytotoxic T cells (CTLs), regulatory T cells (Tregs), helper T cells, NK cells, B cells, dendritic cells, genetically modified versions thereof, and combinations thereof.

9. 1. A method of adoptive cell therapy comprising the steps of: 1) identifying cancer patients in need of adoptive cell therapy; 2) isolating immune cells from the patient's blood or cancer tumor; 3) expanding the immune cells ex vivo; 4) contacting the expanded immune cells with a fucosyltransferase and at least one of fucose or GDP-fucose to provide fucosylated immune cells; 5) returning the fucosylated immune cells to the patient intravenously; and 6) administering to said patient at least one immune checkpoint inhibitor simultaneously with, or wholly or partially sequentially with, said fucosylated immune cells.

10. 10. The method of claim 9, further comprising the step of (4a) separating the fucosylated lymphocytes from other components after step (4), and wherein step (5) is further defined as returning the fucosylated lymphocytes to the patient by intravenous administration.

11. 11. The method of claim 10, further comprising, after step (4a), (4b) isolating the specific fucosylated lymphocytes from other fucosylated lymphocytes, and step (5) is further defined as returning the specific fucosylated lymphocytes to the patient by intravenous administration.

12. 10. The method of claim 9, wherein the fucosyltransferase (FUT) is selected from the group consisting of FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9, FUT10, FUT11, and combinations thereof.

13. 10. The method of claim 9, wherein the at least one immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor, a CTLA-4 inhibitor, a TIM-3 inhibitor, a B7-H3 inhibitor, an A2aR inhibitor, a CD73 inhibitor, an NKG2A inhibitor, a PVRIG / PVRL2 inhibitor, a CEACAM1 inhibitor, a FAK inhibitor, a CCL2 / CCR2 inhibitor, a LIF inhibitor, a CD47 / SIRPα inhibitor, a CSF-1 inhibitor, an IL-1 inhibitor, an IL-8 inhibitor, a SEMA4D inhibitor, an Ang-2 inhibitor, a CLEVER-1 inhibitor, a phosphatylserine inhibitor, and combinations thereof.

14. 14. The method of claim 13, wherein the at least one immune checkpoint inhibitor comprises at least one PD-1 inhibitor and at least one LAG-3 inhibitor.

15. 10. The method of claim 9, wherein the immune cells are selected from the group consisting of cytotoxic T cells (CTLs), regulatory T cells (Tregs), helper T cells, NK cells, B cells, dendritic cells, genetically modified versions thereof, and combinations thereof.

16. 1. A method of treating one or more cancers and reducing the incidence of metastases in a cancer patient, comprising: A method comprising administering to said cancer patient, simultaneously or wholly or partially sequentially, a combination of at least one ICI and at least one fucosylated immune cell-based adoptive cell therapy (fucoACT).

17. 17. The method of claim 16, wherein the at least one immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor, a CTLA-4 inhibitor, a TIM-3 inhibitor, a B7-H3 inhibitor, an A2aR inhibitor, a CD73 inhibitor, an NKG2A inhibitor, a PVRIG / PVRL2 inhibitor, a CEACAM1 inhibitor, a FAK inhibitor, a CCL2 / CCR2 inhibitor, a LIF inhibitor, a CD47 / SIRPα inhibitor, a CSF-1 inhibitor, an IL-1 inhibitor, an IL-8 inhibitor, a SEMA4D inhibitor, an Ang-2 inhibitor, a CLEVER-1 inhibitor, a phosphatylserine inhibitor, and combinations thereof.

18. 18. The method of claim 17, wherein the at least one immune checkpoint inhibitor comprises at least one PD-1 inhibitor and at least one LAG-3 inhibitor.

19. 17. The method of claim 16, wherein the at least one isolated immune cell type is selected from the group consisting of cytotoxic T cells (CTLs), regulatory T cells (Tregs), helper T cells, NK cells, B cells, dendritic cells, genetically modified versions thereof, and combinations thereof.

20. 17. The method of claim 16, wherein the cancer is selected from the group consisting of prostate cancer, skin cancer, ovarian cancer, breast cancer, non-lymphoid solid organ cancer; head and / or neck cancer; leukemia; retinal cancer; esophageal cancer; multiple myeloma; melanoma; colorectal cancer; lung cancer; cervical cancer; endometrial cancer; gallbladder cancer; liver cancer; thyroid follicular cancer; gastric cancer; non-small cell lung cancer; glioma; urothelial cancer; bladder cancer; prostate cancer; renal cell carcinoma; invasive ductal carcinoma; glioblastoma multiforme; and combinations thereof.

21. 17. The method of claim 16, further comprising administering to the cancer patient at least one additional treatment, wherein the at least one additional treatment is selected from the group consisting of radiation therapy, surgery, chemotherapy, immunotherapy, targeted therapy, hormone therapy, stem cell transplant, cryotherapy, laser therapy, precision medicine, and combinations thereof.

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