Tumor-infiltrating lymphocytes for adoptive cell therapy

Abstract

To provide methods for ex vivo expansion of tumor-infiltrating lymphocytes for use in adoptive cell therapy (ACT).SOLUTION: The present invention provides a method that comprises culturing the tumor-infiltrating lymphocytes to produce expanded lymphocytes in a culture medium comprising a toll like receptor (TLR) agonist in an amount effective to improve the tumor-specificity of the expanded lymphocytes.SELECTED DRAWING: None

Description

Detailed Description of the Invention

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims priority to U.S. Provisional Application No. 61 / 955,970, filed March 20, 2014, and U.S. Provisional Application No. 61 / 973,002, filed March 31, 2014, which are hereby incorporated by reference in their entireties. [Background technology] Adoptive cell therapy (ACT) with tumor infiltrating lymphocytes (TILs) is a promising form of T cell-based immunotherapy. Preparation of TILs involves surgical resection and expansion of TILs derived from melanoma tumors. Once the TILs are sufficiently expanded, patients undergo lymphodepleting chemotherapy and adoptive transfer of TILs, followed by high doses of IL-2. The National Cancer Institute's Department of Surgery has pioneered this treatment for metastatic melanoma, reporting an approximate 50% response rate in treated patients, with approximately 20% of patients achieving a durable complete response (Rosenberg SA et al., Clinical cancer research: an official journal of the American Association for Cancer Research. 2011 17(13):4550-7). The excellent durability of the response of this ACT is a notable feature of this treatment and appears to be superior to existing therapies for melanoma. ACT depends on the infiltration of T cells into the tumor before harvesting, successful ex vivo expansion of TILs, and strong antitumor effector function after transfer. Although ACT of TILs is effective against melanoma, durable response rates require further improvement. Shortening the expansion period for initial TIL proliferation and improving the tumor specificity of expanded TILs may increase the response rate in patients treated with autologous TILs. [overview] Disclosed are compositions and methods for ex vivo expansion of tumor infiltrating lymphocytes for use in adoptive cell therapy (ACT). In some embodiments, the method comprises culturing the tumor infiltrating lymphocytes in a medium comprising a Toll-like receptor (TLR) agonist in an amount effective to improve tumor specificity of the expanded lymphocytes to produce the expanded lymphocytes. In some embodiments, the method comprises culturing the lymphocytes in a medium comprising a stimulatory peptide or peptidomimetic to produce the expanded lymphocytes. In some embodiments, the peptidomimetic is a peptoid or a peptide-peptoid hybrid. In some embodiments, the peptoid or peptide-peptoid hybrid is stabilized by a carbohydrate staple.

[0002] Also disclosed is a method of treating cancer using tumor-infiltrating lymphocytes expanded by the disclosed method. In some embodiments, the method includes obtaining autologous tumor-infiltrating lymphocytes from the subject, culturing the lymphocytes in a medium containing a Toll-like receptor (TLR) agonist to produce expanded lymphocytes, treating the subject with non-myeloablative lymphodepleting chemotherapy, and administering the expanded lymphocytes to the mammal.

[0003] In some embodiments, the cancer is a solid tumor. In some cases, the cancer is melanoma, ovarian cancer, breast cancer, and colon cancer. The cancer may be metastatic, recurrent, or a combination thereof.

[0004] The TLR agonist, in some embodiments, is a ligand for a TLR selected from the group consisting of TLR1, TLR2, TLR3, TLR4, and TLR9. For example, the TLR agonist may be a ligand selected from the group consisting of Pam3CSK4, Pam3CSK4, poly I:C, Ribomunyl, and CpG ODN.

[0005] Also disclosed are compositions and methods for identifying agents for ex vivo expansion of tumor infiltrating lymphocytes for use in ACT. The methods may include contacting tumor infiltrating lymphocytes with candidate peptides or peptidomimetics from a library of peptides or peptidomimetics for their ability to selectively bind to the tumor infiltrating lymphocytes. In some embodiments, the peptidomimetics are peptoids or peptide-peptoid hybrids. In some embodiments, the peptoids or peptide-peptoid hybrids are stabilized by carbohydrate staples. The methods may further include screening the effect of binding peptides or peptidomimetics on the proliferation of the tumor infiltrating lymphocytes. In some embodiments, identifying candidate peptides or peptidomimetics that increase the proliferation of the tumor infiltrating lymphocytes identifies agents for ex vivo expansion of tumor infiltrating lymphocytes for use in ACT. Agents identified by these methods may be used to expand tumor infiltrating lymphocytes for use in ACT.

[0006] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief description of the drawings]

[0007] [Figure 1A] FIG. 1 shows examples of peptoids. [Figure 1B] FIG. 1 shows examples of peptoids. [Figure 1C] FIG. 1 shows examples of peptoids. [Figure 1D] FIG. 1 shows examples of peptoids. [Diagram 2] FIG. 1 shows a schematic diagram for the preparation of a peptoid entity comprising a linker attached to a resin and a second linker comprising a peptoid-peptoid sequence. [Diagram 3]FIG. 1 shows a schematic diagram for the preparation of a peptoid entity containing a linker attached to a resin and a second linker that is a β-turn promoter. [Figure 4A] FIG. 1 shows examples of stapled peptoid-peptide hybrids. [Figure 4B] FIG. 1 shows examples of stapled peptoid-peptide hybrids. [Figure 4C] FIG. 1 shows examples of stapled peptoid-peptide hybrids. [Diagram 5] FIG. 1 shows an example of a positional library scanning plate. [Figure 6] FIG. 1 shows an example of a reaction scheme for stabilizing a cyclic β-hairpin-like peptoid-peptide hybrid backbone with a meta-xylenyl group. [Figure 7] FIG. 1 shows an example of a reaction scheme for stabilizing a cyclic β-hairpin-like peptoid-peptide hybrid scaffold by stapling a propargyl side chain present on a more proximal peptide side chain. [Figure 8] FIG. 1 shows an example of a reaction scheme for stabilizing a cyclic β-hairpin-like peptoid-peptide hybrid scaffold using azides and alkynes. [Figure 9] FIG. 1 shows an example of a reaction scheme for stabilizing a cyclic β-hairpin-like peptoid-peptide hybrid scaffold using ring-closing metathesis (RCM). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] [Detailed Description] Adoptive cell transfer (ACT) is a highly effective form of immunotherapy that has been used to treat immune deficiencies with antitumor activity. ACT involves the transfer of immune cells into cancer patients. ACT is a therapeutic approach that involves the in vitro identification of lymphocytes with antitumor activity, the in vitro expansion of these cells to large numbers of cells, and the infusion of the cells into a cancer-bearing host. The lymphocytes used for adoptive transfer can be derived from the stroma of a resected tumor (tumor infiltrating lymphocytes, or TILs). The lymphocytes can also be blood-derived if they are genetically engineered to express antitumor T-cell receptors (TCRs) or chimeric antigen receptors (CARs), enriched in mixed lymphocyte tumor cell cultures (MLTCs), or cloned with autologous antigen-presenting cells and tumor-derived peptides. ACT derived from the cancer-bearing host into which the lymphocytes are infused is called autologous ACT. US2011 / 0052530 relates to methods of performing adoptive cell therapy to promote cancer regression, primarily for the treatment of patients with metastatic melanoma, and is incorporated by reference in its entirety for these methods.

[0009] Disclosed are compositions and methods for ex vivo expansion of tumor infiltrating lymphocytes (TILs) for use in ACT. In some embodiments, the method comprises culturing the tumor infiltrating lymphocytes in a medium containing a Toll-like receptor (TLR) agonist in an amount effective to improve tumor specificity of the expanded lymphocytes to produce the expanded lymphocytes. In some embodiments, the TLR agonist is a ligand for a TLR selected from the group consisting of TLR1, TLR2, TLR3, TLR4, and TLR9. For example, the TLR agonist may be a ligand selected from the group consisting of Pam3CSK4, Pam3CSK4, poly I:C, Ribomunyl, and CpG ODN.

[0010] In other embodiments, the method comprises culturing the tumor infiltrating lymphocytes in a medium containing a peptide or peptidomimetic in an amount effective to enhance tumor specificity of the expanded lymphocytes to produce the expanded lymphocytes. In some embodiments, the peptidomimetic is a peptoid or a peptide-peptoid hybrid. For example, in some embodiments, the peptoid or peptide-peptoid hybrid is stabilized by a carbohydrate staple.

[0011] The peptoid portion can confer resistance to proteolysis, and the peptide portion of the peptoid-peptide hybrid can confer the ability to acquire a β-hairpin-like secondary structure. These two contributions can lead to hybrids that are good drug candidates for therapies where proteolysis is a common limitation. As disclosed herein, these peptide-peptoid hybrids can also be used for ex vivo expansion of tumor infiltrating lymphocytes (TILs) for ACT.

[0012] Examples of peptide-peptoid hybrids are described in WO2013 / 192628 by McLaughlin et al., which is incorporated herein by reference for the libraries of peptoid bodies described therein. The peptoid bodies in WO2013 / 192628 are cyclic peptoid-peptide hybrids that can be used in a pairwise combinatorial approach to provide scaffold libraries. The cyclic peptoid-peptide hybrids can adopt a β-hairpin-like secondary structure. This cyclic β-hairpin-like structure results from the alternation of peptide-peptoid subunits in two antiparallel β-strands. For example, the disclosed peptide-peptoid hybrids can be represented, for example, by the formula I

[0013] [ka] or a pharma- ceutically acceptable salt or hydrate form thereof, where R1-R6 are independently an organic group.

[0014] In some embodiments, the cyclic peptoid-peptide hybrid has the formula II

[0015] [ka] or a pharma- ceutically acceptable salt or hydrate form thereof, wherein each R group is independently an organic group; R' is an organic group or an organic bridging group to a resin or other substrate; and x is 1-3.

[0016] In some embodiments, the cyclic peptoid-peptide hybrid has the formula III

[0017] [ka] or a pharma- ceutically acceptable salt or hydrate form thereof, wherein each R group is independently an organic group; R' is an organic group or an organic bridging group to a resin or other base; and x is 1-3.

[0018] In some embodiments, the cyclic peptoid-peptide hybrid is represented by formula IV

[0019] [ka] or a pharma- ceutically acceptable salt or hydrate thereof, wherein each R group is independently an organic group; R' is an organic group or an organic bridging group to a resin or other substrate; and x is 1-3.

[0020] In some embodiments, the cyclic peptoid-peptide hybrid has the formula V

[0021] [ka] or a pharma- ceutically acceptable salt or hydrate thereof, wherein each R group is independently an organic group; R' is an organic group or an organic bridging group to a resin or other substrate; and x is 1-3.

[0022] In some embodiments, the cyclic peptoid-peptide hybrid is represented by formula VI

[0023] [ka] or a pharma- ceutically acceptable salt or hydrate thereof, wherein each R group is independently an organic group, each R' is independently an organic group or an organic bridging group to a resin or other substrate, and x is 1-3.

[0024] In some embodiments, the cyclic peptoid-peptide hybrid is represented by formula VII

[0025] [ka] or a pharma- ceutically acceptable salt or hydrate thereof, wherein each R group is independently an organic group, each R' is independently an organic group or an organic bridging group to a resin or other substrate, and x is 1-3.

[0026] In some embodiments, the cyclic peptoid-peptide hybrid is represented by Formula VIII

[0027] [ka] or a pharma- ceutically acceptable salt or hydrate thereof, wherein each R group is independently an organic group, each R' is independently an organic group or an organic bridging group to a resin or other substrate, and x is 1-3.

[0028] In some embodiments, the cyclic peptoid-peptide hybrid has the formula IX

[0029] [ka] or a pharma- ceutically acceptable salt or hydrate thereof, wherein each R group is independently an organic group; R' is an organic group or an organic bridging group to a resin or other substrate; and x is 1-3.

[0030] In some embodiments, the cyclic peptoid-peptide hybrid has the formula X

[0031] [ka] or a pharma- ceutically acceptable salt or hydrate thereof, wherein each R group is independently an organic group; R' is an organic group or an organic bridging group to a resin or other substrate; and x is 1 to 3.

[0032] In some embodiments, the R groups of at least two adjacent peptoid-glycine sequences are 4-piperidinyl groups, e.g., compounds of formula II where x is 2.

[0033] [ka] It is.

[0034] The R group of the cyclic peptoid-peptide hybrids of formulas I-XII above can be nearly any structure, so long as it does not contain moieties that interfere with the complementary hydrogen bonding of the β-sheet structures in the cyclic peptoid-peptide hybrids. The R group can correspond to the side chain of an amino acid, a non-amine portion of an amino acid, or a modified non-amine portion of an amino acid. The R group can be, but is not limited to, a sugar, such as a monosaccharide or disaccharide, or a fatty acid, or modified versions thereof. The R group can be any of the C1-C 12 Alkyl, C1-C 12 Hydroxyalkyl, C1-C 12 Aminoalkyl, C1-C 12Carboxylic Acid Substituted Alkyl, C2 ~C 12 Alkyloxyalkyl, C2-C 12 Alkenyl, C2-C 12 Hydroxyalkenyl, C2-C 12 Aminoalkenyl, C1-C 12 Carboxylic acid substituted alkenyl, C3-C 14 Alkyloxyalkenyl, C6-C 14 Aryl, C6-C 14 Hydroxyaryl, C6-C 14 Aminoaryl, C6-C 14 Carboxylic acid substituted aryl, C7-C 15 Alkyloxyaryl, C4-C 14 Heteroaryl, C4-C 14 Hydroxyheteroaryl, C4-C 14 Aminoheteroaryl, C4-C 14 Carboxylic Acid Substituted Heteroaryl, C5-C 15 Alkoxyheteroaryl, C7-C 15 Alkylaryl, C7-C 15 Hydroxyalkylaryl, C7-C 15 Aminoalkylaryl, C7-C 15 Carboxylic Acid Substituted Alkylaryl, C8-C 15It may be an alkoxyalkylaryl or the chemical transformation products of any of these R groups, such as an ester, thioester, thiol, amide, or sulfonamide, where the alkyl group can be linear, branched, multiple branched, cyclic, or polycyclic. For example, R may be selected from the group consisting of, but not limited to, 4-aminopiperidine, ethanolamine, allylamine, 1,4-diaminobutane, piperonylamine, 4,(2-aminoethyl)benzene, isobutylamine, tryptamine, 4-morpholinoaniline, 5-amino-2-methoxypyridine, (R)-methylbenzylamine, 1-(2-aminopropyl)-2-pyrrolidinone, furfurylamine, benzylamine, 4-chlorobenzylamine, 4-methoxybenzylamine, methoxyethylamine, 2-aminoadipic acid, N-ethylasparagine, 3-aminoadipic acid, hydroxylysine, β-alanine, allo-hydroxylysine, propionic acid, 2-aminobutyric acid, 3-hydroxypropylamine, 2-aminobutyric acid ... The cyclic peptoid-peptide hybrid may be a residue of a primary amine, which may be a group resulting from the incorporation of the following as a peptoid NR unit in the cyclic peptoid-peptide hybrid: 4-aminobutyric acid, 4-hydroxyprolinepiperidine acid, 6-aminocaproic acid, isodesmosine, 2-aminoheptanoic acid, allo-isoleucine, 2-aminoisobutyric acid, N-methylglycine, 3-aminoisobutyric acid, N-methylisoleucine, 2-aminopimelic acid, 6-N-methyllysine, 2,4-diaminobutyric acid, N-methylvaline, desmosine, norvaline, 2,2'-diaminopimelic acid, norleucine, 2,3-diaminopropionic acid, ornithine, N-ethylglycine, or a protected equivalent thereof.

[0035] In some embodiments, the peptoid-peptide hybrids can be formed on a solid support, such as a resin. The hybrids can then be cleaved from the support resin and used in the methods of the disclosure. Figures 1 and 2 illustrate examples of peptoid-peptide hybrids as resin-bound intermediates. Example peptoid-peptide hybrids Embodiment 1: A peptoid-peptide hybrid having a β-hairpin-like conformation comprising a plurality of alternating peptoid-peptide sequences, each of said sequences having at least one peptoid residue and an amino acid residue, said peptoid-peptide sequences forming at least two anti-parallel β-strands between a plurality of linkers, at least one linker being a β-turn promoter.

[0036] Embodiment 2: At least one of the linkers has the structure

[0037] [ka] wherein R′ is an organic group, or an organic bridging group that bonds to a resin or other substrate, and R″ is H or a carboxylic acid protecting group.

[0038] Embodiment 3: A hybrid according to embodiment 2, wherein R″ is t-butyl, allyl, or benzyl. Embodiment 4: A hybrid according to embodiment 2, wherein said organic linking group that binds to a resin or other substrate comprises a -NH(CH2)2- linking group.

[0039] Embodiment 5: A hybrid according to embodiment 1, wherein one of said linkers is two peptoid residues. Embodiment 6: The cyclic peptoid-peptide hybrid is

[0040] [ka] A hybrid according to embodiment 1, wherein R groups are independently an organic group, R' is independently an organic group or an organic bridging group that bonds to a resin, and x is 1-3.

[0041] Embodiment 7: R is independently C1 to C 12 Alkyl, C1-C 12 Hydroxyalkyl, C1-C 12Aminoalkyl, C1-C 12 Carboxylic Acid Substituted Alkyl, C2-C 12 Alkyloxyalkyl, C2-C 12 Alkenyl, C2-C 12 Hydroxyalkenyl, C2-C 12 Aminoalkenyl, C1-C 12 Carboxylic acid substituted alkenyl, C3-C 14 Alkyloxyalkenyl, C6-C 14 Aryl, C6-C 14 Hydroxyaryl, C6-C 14 Aminoaryl, C6-C 14 Carboxylic acid substituted aryl, C7-C 15 Alkyloxyaryl, C4-C 14 Heteroaryl, C4-C 14 Hydroxyheteroaryl, C4-C 14 Aminoheteroaryl, C4-C 14 Carboxylic acid substituted heteroaryls, C5~C 15 Alkoxyheteroaryl, C7-C 15 Alkylaryl, C7-C 15 Hydroxyalkylaryl, C7-C 15 Aminoalkylaryl, C7-C 15 Carboxylic Acid Substituted Alkylaryl, C8-C 15 A hybrid according to embodiment 6, which is an alkoxyalkylaryl, or any structure chemically converted therefrom.

[0042] Embodiment 8: The hybrid according to embodiment 7, wherein said chemically transformed structure comprises an ester, a thioester, a thiol, an amide, or a sulfonamide. Embodiment 9: R is independently a residue of a primary amine: 4-aminopiperidine, ethanolamine, allylamine, 1;4-diaminobutane, piperonylamine, 4;(2-aminoethyl)benzene, isobutylamine, tryptamine, 4-morpholinoaniline, 5-amino-2-methoxypyridine, (R)-methylbenzylamine, 1-(2-aminopropyl)-2-pyrrolidinone, furfurylamine, benzylamine, 4-chlorobenzylamine, 4-methoxybenzylamine, methoxyethylamine, 2-aminoadipic acid, N-ethylasparagine, 3-aminoadipic acid, hydroxylysine, β-alanine, allo-hydroxylamine, β-aminopyridine ... 7. The hybrid according to embodiment 6, which is silicic acid propionic acid, 2-aminobutyric acid, 3-hydroxyproline, 4-aminobutyric acid, 4-hydroxyproline piperidinic acid, 6-aminocaproic acid, isodesmosine, 2-aminoheptanoic acid, allo-isoleucine, 2-aminoisobutyric acid, N-methylglycine, 3-aminoisobutyric acid, N-methylisoleucine, 2-aminopimelic acid, 6-N-methyllysine, 2,4-diaminobutyric acid, N-methylvaline, desmosine, norvaline, 2,2'-diaminopimelic acid, norleucine, 2,3-diaminopropionic acid, ornithine, N-ethylglycine, or any protected equivalent thereof.

[0043] Embodiment 10: A hybrid according to embodiment 6, wherein at least one R is a residue of 4-aminopiperidine. Embodiment 11: A hybrid according to embodiment 1, wherein all of said amino acid residues are glycine residues.

[0044] Peptoid-peptide hybrids can be further stabilized by bridges between amino acid side chains and / or N-substituents on glycines, and / or by backbone cyclization. Such peptoids are referred to herein as "stapled peptoids," whereas such peptoid-peptide hybrids are referred to herein as "stapled peptoid-peptide hybrids."

[0045] Stapling of peptoids or peptoid-peptide hybrids involves side chain-to-side chain linkages and / or backbone cyclization to stabilize the peptoid or peptoid-peptide hybrid, and thus the present invention extends the approach of stabilizing peptides using stabilized side chain linkages to the stabilization of peptoids or peptoid-peptide hybrids.

[0046] For the purposes of this invention, the term "side chain" encompasses the side chain on an amino acid as well as the moiety attached to the N-atom of an N-substituted glycine. Several possible side chain-to-side chain linkages (hereafter referred to as intramolecular bridges) can be designed. An intramolecular bridge between two side chains of a peptoid or peptoid-peptide hybrid of the invention can be mediated by a chemical reaction between said side chains, which may involve additional chemical agents.

[0047] For example, the intramolecular bridge may be mediated by a chemical moiety that is not part of the side chains, where the side chains are linked to each other via the chemical moiety. Examples of the chemical moieties that form the intramolecular bridges are described in Figures 6-9.

[0048] In some embodiments, the intramolecular bridge is established by the RCM (ring-closing metathesis) approach by Aileron, or by a click reaction (e.g., copper-catalyzed 3+2 cycloaddition) as described in U.S. Patent No. 5,811,515, which is incorporated herein by reference in its entirety. An example of an intramolecular bridge mediated by the RCM approach is depicted in FIG.

[0049] In a further embodiment, the intramolecular bridge between the two side chains is constructed by the formation of a chemical bond between the functional groups present on the side chains, for example, by a condensation reaction. A condensation reaction is a chemical reaction in which two molecules or moieties (functional groups) join together through a chemical bond, with the loss of one or more smaller molecules. Examples of condensation reactions between side chains that can be used to generate intramolecular bridges according to the present invention are well known to those skilled in the art, and such embodiments are within the scope of the present invention.

[0050] Further examples of peptoids stapled by certain intramolecular bridges are shown in Figures 4A-4C. Further examples of crosslinks in peptides are disclosed in U.S. Patent Nos. 8,592,377, 8,324,428, 8,198,405, 7,786,072, 7,723,469, and 7,192,713, the contents of which are incorporated herein by reference in their entireties. One of skill in the art can envision the use of the various crosslinks described in these patent documents in preparing the stapled peptoids and stapled peptoid-peptide hybrids of the present invention, and such embodiments are within the scope of the present invention.

[0051] Thus, the present invention provides a stapled peptoid comprising a plurality of N-substituted glycines, at least two of which are linked to each other by an intramolecular bridge, the length and geometry of which confers stability to the peptoid.

[0052] In some cases, the stapled peptoid-peptide hybrid can include a plurality of amino acids and a plurality of N-substituted glycines, where at least two residues from the plurality of amino acids and the plurality of N-substituted glycines are bound to each other by an intramolecular bridge, the length and geometry of which confers stability to the peptoid-peptide hybrid.

[0053] In one embodiment, the two N-substituted glycine residues or the two amino acid residues are linked to each other by a bridge. In another embodiment, the N-substituted glycine residue is linked to the amino acid residue by a bridge.

[0054] In further embodiments, the stapled peptoid-peptide hybrid is a cyclic peptoid-peptide hybrid comprising a plurality of alternating peptoid-peptide sequences, each of which has at least one peptoid residue and an amino acid residue, and the peptoid-peptide sequences form at least two anti-parallel β-strands.

[0055] In some embodiments, the intramolecular bridges are all hydrocarbon bridges. In some embodiments, the peptoid or peptoid-peptide hybrid contains multiple intramolecular bridges, eg, 2, 3, or 4 intramolecular bridges.

[0056] In some embodiments, the bridge is between two or more amino acid residues that are located on the same side of a β-sheet, or N-substituted glycine residues, thereby forming a bridge between the peptoid or In further embodiments, the intramolecular bridge is between two or more amino acid residues located on residues of a β-sheet or N-substituted glycine residues, thereby conferring stability to the peptoid or peptoid-peptide hybrid.

[0057] In some embodiments, the side chain can be selected from cyclic or acyclic, branched or unbranched, substituted cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkylene, cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkenylene, cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkynylene, cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkylene, cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkenylene, cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkynylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or substituted or unsubstituted acylene.

[0058] Further examples of side chains and crosslinks applicable in the present invention are described, for example, in U.S. Pat. No. 8,592,377, column 37, line 26 to column 43, line 14; U.S. Pat. No. 8,198,405, column 3, line 54 to column 10, line 2 and column 25, line 14 to column 26, line 21; U.S. Pat. No. 7,786,072, column 5, line 44 to column 9, line 43 and column 11, line 16 to column 12, line 8; U.S. Pat. No. 7,723,469, column 5, line 30 to column 9, line 12 and column 24, line 60 to column 26, line 3; and U.S. Pat. No. 7,192,713, column 4, line 26 to column 9, line 45 and column 11, line 23 to column 12, line 18.

[0059] The stapled peptoids, peptoid-peptide hybrids, and stapled cyclic peptoid-peptide hybrids of the invention can also optionally contain substituents in the side chains of the amino acid residues and / or N-substituted glycine residues which further stabilize the peptoids, peptoid-peptide hybrids, and cyclic peptoid-peptide hybrids. Non-limiting examples of various substitutions that can be used in the present invention are set forth in Table 2.

[0060] The production of tumor infiltrating lymphocytes (TILs) is a two-step process: 1) a pre-REP (Rapid Expansion) stage in which the cells are cultured in standard laboratory medium such as RPMI and the TILs are treated with irradiated feeder cells and reagents such as anti-CD3 antibodies to achieve the desired effect, and 2) a REP stage in which the TILs are expanded in a culture volume large enough for patient treatment. The REP stage requires cGMP grade reagents and 30-40 L of medium. However, the pre-REP stage can utilize laboratory grade reagents (provided that the laboratory grade reagents are diluted during the REP stage), which facilitates the adoption of alternative strategies to improve TIL production. Thus, in some embodiments, the pre-REP stage can include the disclosed TLR agonists and / or peptides or peptidomimetics in the medium.

[0061] ACT can be performed by (i) obtaining autologous lymphocytes from a mammal, (ii) culturing the autologous lymphocytes to produce expanded lymphocytes, and (ii) administering the expanded lymphocytes to the mammal. Preferably, the lymphocytes are tumor-derived, i.e., the lymphocytes are TILs, and are isolated from the mammal to be treated, i.e., autologous.

[0062] Autologous ACT as described herein also includes (i) culturing autologous lymphocytes to produce expanded lymphocytes, (ii) administering non-myeloablative lymphodepleting chemotherapy to the mammal, and (iii) administering to the mammal, following administration of non-myeloablative lymphodepleting chemotherapy, It can also be performed by administering the lymphocytes expanded as described above. Autologous TIL can be obtained from the stroma of resected tumor. A tumor sample is obtained from the patient to obtain a single cell suspension. The single cell suspension can be obtained by any suitable method, for example, mechanically (e.g., using a gentleMACS™ separator, Miltenyi Biotec, Auburn, Calif., to disperse the tumor) or enzymatically (e.g., collagenase or DNase).

[0063] Expansion of lymphocytes, including tumor infiltrating lymphocytes such as T cells, can be accomplished by any of several methods known in the art. For example, T cells can be rapidly expanded using non-specific T cell receptor stimulation in the presence of feeder lymphocytes and interleukin-2 (IL-2), IL-7, IL-15, IL-21, or a combination thereof. The non-specific T cell receptor stimulation can include, for example, about 30 ng / ml of OKT3, a murine monoclonal anti-CD3 antibody (available from Ortho-McNeil®, Raritan, NJ, or Miltenyi Biotec, Bergisch Gladbach, Germany). Alternatively, T cells can be cultured in vitro in the presence of one or more T cell growth factors, such as IL-2 or IL-15, preferably IL-2, at about 200-400 Ill / ml, e.g., 300 IU / ml. The T cells can be rapidly expanded by stimulation of peripheral blood mononuclear cells (PBMCs) with several antigens (including antigenic portions of the antigen, such as epitope(s), or optionally expressed from a vector, such as human leukocyte antigen A2 (HLA-A2) binding peptides, such as MART-1:26-35 (27L) or gp 100:209-217 (210M), at approximately 0.3 μM). In vitro induced T cells can be rapidly expanded by restimulation with the same cancer antigen(s) pulsed onto antigen presenting cells expressing HLA-A2. Alternatively, the T cells can be restimulated with, for example, irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0064] In some embodiments, the mammal is administered non-myeloablative lymphodepleting chemotherapy before the mammal is administered the expanded tumor-infiltrating lymphocytes. The purpose of lymphodepletion is to make way for the infused lymphocytes, in particular by removing regulatory T cells and other non-specific T cells that compete for homeostatic cytokines. The non-myeloablative lymphodepleting chemotherapy can be any and suitable such therapy and can be administered by any and suitable route known to those skilled in the art. The non-myeloablative lymphodepleting therapy can include, for example, administration of cyclophosphamide and fludarabine, especially when the cancer is melanoma, which may be metastatic. The preferred route for administering cyclophosphamide and fludarabine is intravenous administration. Similarly, any and suitable doses of cyclophosphamide and fludarabine can be administered. In particular, when the cancer is melanoma, about 40 to 80 mg / kg, for example about 60 mg / kg, of cyclophosphamide is administered for about 2 days, followed by about 15 to 35 mg / m 2 , for example about 25 mg / m 2 Preferably, one dose of fludarabine is administered for about 5 days.

[0065] The specific tumor reactivity of the expanded TILs can be tested by any method known in the art, for example, by measuring cytokine release (e.g., interferon-gamma) after co-culture with tumor cells. In one embodiment, the autologous ACT method includes enriching the cultured TILs for CD8+ T cells prior to rapid expansion of the cells. Following culture of the TILs in IL-2, for example, CD8 microbead separation (e.g., CliniMACS <plus>T cells are depleted of CD4+ cells and enriched for CD8+ cells using a CD8 microbead system (Miltenyi Biotec). In one embodiment of the method, a T cell growth factor that promotes proliferation and activation of autologous T cells is administered to the mammal either simultaneously with the autologous T cells or autologous T cells. The T cell growth factor is administered either before or after autologous T cells. The T cell growth factor may be any suitable growth factor that promotes the proliferation and activation of autologous T cells. Examples of suitable T cell growth factors include interleukin (IL)-2, IL-7, IL-15, IL-12, and IL-21, which may be used alone or in various combinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL-2. IL-12 is a preferred T cell growth factor.

[0066] The expanded lymphocytes produced by these methods are preferably administered as an intra-arterial or intravenous infusion lasting preferably about 30 to about 60 minutes. Other examples of routes of administration include intraperitoneal, intrathecal, and intralymphatic. Similarly, any suitable dose of lymphocytes can be administered. In one embodiment, about 1×10 10 Lymphocytes ~ approx. 15 x 10 10 of lymphocytes are administered.

[0067] Cancers that may be treated by the disclosed compositions and methods include acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, osteosarcoma, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, intrahepatic bile duct cancer, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the vulva, chronic lymphocytic leukemia, chronic bone marrow cancer, cervical cancer, glioma, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, The cancer may be any cancer, including lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, ovarian cancer, peritoneal, omental, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, soft tissue cancer, testicular cancer, thyroid cancer, ureteral cancer, bladder cancer, and gastrointestinal cancer, such as, for example, esophageal cancer, gastric cancer, pancreatic cancer, stomach cancer, small intestine cancer, gastrointestinal carcinoid tumors, oral cancer, colon cancer, and hepatobiliary cancer.

[0068] The cancer may be a recurrent cancer. Preferably, the cancer is a solid cancer. Preferably, the cancer is melanoma, ovarian cancer, breast cancer and colon cancer, and even more preferably, the cancer is melanoma, in particular metastatic melanoma. definition The term "subject" refers to any individual who is the target of administration or treatment. A subject may be a vertebrate, e.g., a mammal. Thus, a subject may be a human or veterinary patient. The term "patient" refers to a subject under the care of a clinician, e.g., a physician.

[0069] The term "therapeutically effective" refers to an amount of a composition used that is sufficient to ameliorate the cause or symptoms of one or more diseases or disorders. Such amelioration need only reduce or alter, not necessarily eliminate.

[0070] The term "treatment" refers to the medical management of a patient with the goal of curing, ameliorating, stabilizing, or preventing a disease, condition, or disorder. The term encompasses active treatment, i.e., treatment specifically directed to ameliorating the disease, condition, or disorder, and also causal treatment, i.e., treatment directed to eliminating the cause of the associated disease, condition, or disorder. The term further encompasses palliative treatment, i.e., treatment designed to alleviate symptoms rather than cure the disease, condition, or disorder; preventive treatment, i.e., treatment directed to minimizing or partially or completely preventing the onset of the associated disease, condition, or disorder; and supportive treatment, i.e., treatment used to complement another specific therapy directed to ameliorating the associated disease, condition, or disorder.

[0071] The terms "peptide," "protein," and "polypeptide" are used interchangeably and refer to natural or synthetic molecules containing two or more amino acids linked by the carboxyl group of one amino acid to the α-amino group of another.

[0072] As used herein, "peptidomimetics" refers to mimics of peptides that contain some modifications of the normal peptide chemical structure. Peptidomimetics generally improve some properties of the original peptide, such as increased stability, increased efficacy, improved delivery, increased half-life, etc. Methods for making peptidomimetics based on known polypeptide sequences are described, for example, in U.S. Patent Nos. 5,631,280, 5,612,895, and 5,579,250. The use of peptidomimetics can involve the incorporation of non-amino acid residues at given positions through non-amide bonds. One embodiment of the present invention is a peptidomimetic, where the compound has bonds, peptide backbones, or amino acid components replaced with appropriate mimetics. Some non-limiting examples of unnatural amino acids that can be suitable amino acid mimetics include β-alanine, L-α-aminobutyric acid, L-γ-aminobutyric acid, L-α-aminoisobutyric acid, L-ε-aminocaproic acid, 7-aminoheptanoic acid, L-aspartic acid, L-glutamic acid, N-ε-Boc-N-α-CBZ-L-lysine, N-ε-Boc-N-α-Fmoc-L-lysine, L-methionine sulfone, L-norleucine, L-norvaline, N-α-Boc-N-δCBZ-L-ornithine, N-δ-Boc-N-α-CBZ-L-ornithine, Boc-p-nitro-L-phenylalanine, Boc-hydroxyproline, and Boc-L-thioproline.

[0073] The term "peptoid" refers to a class of peptidomimetics in which side chains are attached to nitrogen atoms of the peptide backbone rather than to the α-carbon (as in amino acids). The term "tumor infiltrating lymphocytes" or "TILs" refers to white blood cells that have left the bloodstream and migrated into a tumor.

[0074] The term "regression" does not necessarily mean 100% or complete regression. Rather, there are various degrees of regression that one of skill in the art recognizes as having their potential beneficial or therapeutic effect. The term also encompasses delaying the onset of a disease, or its symptoms or conditions.

[0075] The term "staple" or "hydrocarbon staple" as used herein refers to the use of carbohydrates to stabilize the secondary structure of a synthetic peptide or peptidomimetic.

[0076] Although several embodiments of the invention have been described, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. [Example] Example 1: Targeting TLRs to improve TIL expansion and activity Toll-like receptors (TLRs) are pattern recognition receptors that recognize a wide variety of microbial molecules. Binding of TLR ligands to TLRs expressed on macrophages and dendritic cells (DCs) leads to effective antigen presentation for T cell and host immune activation. In the tumor environment, macrophage and DC function is inhibited. As disclosed herein, this suppressed state can be reversed by administration of TLR ligands (Table 1). Exogenous TLR ligands added to TIL cultures can improve DC and macrophage function, leading to increased TIL expansion and improved tumor-specific immune responses.

[0077] [Table 1] To determine whether exogenous TLRs added to TIL cultures could increase TIL expansion and improve tumor selectivity, fresh melanoma tumors were cultured at 1–2 mm in medium supplemented with 6000 IU / ml IL-2. 2 Twelve fragments are cultured in IL-2 alone. An additional set of 12 fragments are treated with the following TLR ligands: Pam3CSK4 (1 μg / ml), a TLR1 / 2 ligand; The medium was replaced with fresh medium every 2-3 days. TILs were aliquoted into new wells when they reached confluence. After 10, 20, and 30 days of culture, the total number of tumor fragments that gave rise to TIL proliferation was recorded. In addition, cells were harvested and counted from each fragment. These cell counts were compared between the IL-2 control and TLR ligand-treated groups. Individual and pooled TILs from each fragment were co-cultured with autologous or HLA-matched and HLA-mismatched melanoma cells for 24 hours. Culture supernatants were harvested and IFN-γ was measured by standard ELISA. The goal of these experiments is to determine whether the addition of TLR ligands results in increased proliferation of fragment-derived TILs, increased proliferation of TILs, and / or increased tumor-specific activation of TILs. Example 2: Identification of peptoids for proliferation and activation of TILs In addition to TLR agonists, other costimulatory molecules can be expressed by TILs. A library of peptoids can be screened to identify compounds that enhance TIL proliferation and activation. A library of peptoids containing approximately 200,000 compounds is screened in a 384-well-like format. TIL cell lines are labeled with red quantum dots and screened to identify library hit compounds that selectively bind to red cells. Once several binding peptoids are identified, proliferation of TILs is tested in the presence of the peptoids. Peptoids that result in proliferation of TILs (stimulatory peptoids) are further tested in functional assays.

[0078] The effect of stimulatory peptoids on T cell function is measured using a human T cell line (AS1). AS1 cells are activated human CD8+ T cells that show specificity for the 624 melanoma cell line but not for the HLA-mismatched 888 melanoma cell line. AS1 cells are cultured in 6000 IU / ml IL-2 in the presence of increasing doses of stimulatory peptoid. Cells are counted on days 3, 7, 10, 14, and 21 to determine the dose of the stimulatory peptoid that leads to increased proliferation of AS1 cells. IFN-γ, a cytokine secreted by activated T cells, is measured to determine whether culture with stimulatory peptoids leads to improved T cell function. AS1 cells are treated with the optimal dose of the stimulatory peptoid body determined above. AS1 cells alone and anti-4 cells were used as controls. 1BB antibody-treated AS1 cells are included. After 7 days, AS1 cells are harvested and co-cultured with 624 melanoma cells. As a negative control, AS1 cells are co-cultured with 888 melanoma cells. After 24 hours, supernatants are harvested and IFN-γ production is measured by ELISA. Comparisons are made between AS1 alone and AS1 treated with stimulatory peptoids. To further investigate the efficacy of stimulatory peptoids, T cells are harvested from tumors of 10 patients with metastatic melanoma participating in an ongoing IRB-approved clinical trial. Tumor fragments are cultured in medium containing 6000 IU / ml IL-2 to generate pools of T cells as previously described (Pilon-Thomas S,et.al J Immunother. 2012 35(8):615-20). Twelve fragments per condition are supplemented with irrelevant or stimulatory peptoids. Fragments treated with isotype IgG or anti-41BB antibody (10 μg / ml) are used as controls. After 21 days, T cells are harvested and counted. To measure activation, T cells are co-cultured with autologous or HLA-matched tumor cells. T cells alone and T cells co-cultured with HLA-mismatched tumor cells are included as negative controls. T cells cultured in the presence of CD3 / CD28 are included as positive controls. Supernatants are harvested after 24 hours and IFN-γ production is measured by ELISA. Additionally, T cell proliferation is measured on days 7, 14, and 21 to determine whether co-culture with stimulatory peptoids results in increased proliferation of tumor-infiltrating T cells. These studies determine whether treatment of T cells with anti-PD1 peptoid bodies improves proliferation and activation of anti-melanoma T cells. Example 3: Evaluation of TIL activity in a mouse model The main drawback of TIL studies is the inability to test TILs in an in vivo model. A mouse model to measure the efficacy of TILs is developed. NSG mice (B and T cell deficient mice, purchased from Jackson Laboratories) are implanted with primary patient melanoma tumors in the abdomen. When the tumors reach a diameter of 5 mm, 1 × 10 7 1000-fold expanded TILs are then transferred into the mouse. Tumor growth and survival are measured. This model is used to test whether TILs grown in standard IL-2 medium, medium containing TLR ligands, or medium containing stimulatory peptoids result in better tumor rejection in vivo. Example 4: Positional scanning with 69 different substituents In some embodiments, a molecular library was prepared that represents a mixture of 69 compounds defined by the general structure of FIG. 4A, with various substituents (listed in Table 2) at either the R1, R2, or R3 positions. Here, all remaining R1 and R2, or R1 and R3, or R2 and R3 positions represent each of the possible 69×69 combinations. This is equivalent to 69×69×3, or 14,283 different spots. This number is significantly less than if all possible combinations of 69×69×69, or 328,509 spots, had to be made, which would require 952.2 plates with 345 spots per plate. The positional scanning approach described above requires only 41.4 plates.

[0079] In this embodiment of the positional scanning method, only about 1 / 69 of pure material is displayed per spot. If a hit compound is found by screening, the hit compound is prepared again and its activity is verified. This method gives about 1.4% of the specific sequences displayed per well, and the preferred side chain can be determined by comparing with the hit compounds from other plates that display each side chain in each of the above three side chains individually.

[0080] [Table 2] TIFF2025041755000016.tif177170 Figure 5 is an example of a plate used for position scanning. Spots 1-69 will have the same R1 and each of the individual in all spots, 69 different side chains in R2, and a mixture of all 69 compounds displayed in the R3 position. Spots 70-138 will similarly have all R1 positions displaying side chain 2, then each of the 69 different side chains in R2, a mixture of the 69 different side chains displayed in the R3 position, and so on, covering all possible combinations. A specific R2 plate will have a mixture of 69 compounds displayed in the R1 position, then all of the 69 spots for R2 with the same side chain equal to compound 1, then each individual 69 compound for R3 similar to the R1 specific plate described above, then the remaining combinations, where the R1 position displays each of the 69 compounds, R2 displays a mixture of 69 compounds, and in the R3 position spots 1-69 all have the same side chain.

[0081] An example of the results of the above position screening is also shown in Figure 5. The first shaded spot is the best The plate has an R1 side chain, which is equal to compound 2, and R2 is equal to compound 26, with at least one of the 69 possible compounds at position R3. The next spot shows that compound 3 is available at R1, and R2 has compound 10, with at least one of the 69 possible compounds at position R3. The last spot on the plate shows that R1 is equal to compound 5, and for R2 is compound 55, with at least one of the 69 possible compounds at position R3. By analyzing the results of similar screens from plates with defined R2 and R3 positions, the exact sequence can be determined.

[0082] 69 compounds with similar reactivity are required in the SN2 reaction to produce secondary amines. Although large structural variability is possible, some possible side chains are shown in Table 2. Example 5: Stapling method to stabilize cyclic β-hairpin-like peptoid-peptide hybrid scaffolds There are several possible stapling methods that can be used to stabilize the cyclic β-hairpin-like peptoid-peptide hybrid backbone. Stapling two peptoid side chains is the easiest to achieve from a synthetic standpoint, pre-constructing the two peptoid side chains in close proximity to each other, which reinforces the overall backbone conformation that corresponds to the desired cyclic β-hairpin-like secondary structure. The easiest pair to staple is a propargylamine in the peptoid side chain to be stapled and react the two terminal alkynes with a bifunctional diazide that has a stable organic linker that spans the distance between the peptoid side chains. The organic linker requires three more atoms to span the distance. A meta-xylenyl group is shown in FIG. 6, but any combination of stable atoms can be used. In addition, the linkage need not be just a linker, as the group can also be used, for example, to optimize pharmacokinetic properties.

[0083] Alternatively, the same or different linkers can be used to staple the propargyl side chains on the more adjacent peptoid side chains as shown in Figure 7. Side chains from amino acid sides and adjacent peptoid side chains are also possible as described herein, but are not shown. A more comprehensive scheme using azides and alkynes is shown in Figure 8. RCM (ring-closing metathesis) reactions can also be used, as shown by the comprehensive example in Figure 9.

[0084] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which the publications are cited are specifically incorporated by reference.

[0085] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.< / plus>

Claims

1. 1. A method for ex vivo expansion of tumor-infiltrating lymphocytes for use in adoptive cell therapy (ACT), comprising culturing the tumor-infiltrating lymphocytes in a medium containing a Toll-like receptor (TLR) agonist in an amount effective to improve tumor specificity of the expanded lymphocytes to produce the expanded lymphocytes.

2. 1. A method of treating cancer in a subject, comprising: a) obtaining autologous tumor-infiltrating lymphocytes from said subject; b) culturing the lymphocytes in a medium containing a Toll-like receptor (TLR) agonist to produce expanded lymphocytes; c) treating said subject with non-myeloablative lymphodepleting chemotherapy; d) administering the expanded lymphocytes to the mammal; The method of treatment.

3. 3. The method of claim 1 or 2, wherein the TLR agonist is a ligand for a TLR selected from the group consisting of TLR1, TLR2, TLR3, TLR4, and TLR9.

4. The method of any one of claims 1 to 3, wherein the TLR agonist comprises a ligand selected from the group consisting of Pam3CSK4, Pam3CSK4, poly I:C, Ribomunyl, and CpG ODN.

5. The method of any one of claims 1 to 4, wherein the cancer is a solid tumor.

6. The method of any one of claims 1 to 5, wherein the cancer is selected from the group consisting of melanoma, ovarian cancer, breast cancer, and colon cancer.

7. The method of any one of claims 1 to 6, wherein the cancer is metastatic.

8. The method of any one of claims 1 to 7, wherein the cancer is recurrent.

9. The method of any one of claims 1 to 8, wherein the subject is a human.

10. 1. A method for identifying an agent for ex vivo expansion of tumor infiltrating lymphocytes for use in adoptive cell therapy (ACT), comprising: contacting tumor infiltrating lymphocytes with a candidate peptide or peptidomimetic from a library of peptides or peptidomimetics for the ability to selectively bind to said tumor infiltrating lymphocytes; screening the effect of the binding peptide or peptide mimetic on the proliferation of said tumor infiltrating lymphocytes; Including, The method, wherein identifying a candidate peptide or peptidomimetic that increases proliferation of the tumor infiltrating lymphocytes identifies an agent for ex vivo expansion of tumor infiltrating lymphocytes for use in ACT.

11. 1. A method for ex vivo expansion of tumor-infiltrating lymphocytes for use in adoptive cell therapy (ACT), comprising culturing the lymphocytes in a medium containing a peptide or peptidomimetic to produce expanded lymphocytes.

12. The method of claim 11, wherein the peptide or peptidomimetic is identified by the method of claim 9.

13. The method of any one of claims 10 to 12, wherein the peptidomimetic is a peptoid or a peptide-peptoid hybrid.

14. The method of claim 13, wherein the peptoid or peptide-peptoid hybrid is stabilized by a carbohydrate staple.

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