BR112016021370B1 - METHODS FOR EX VIVO EXPANSION OF TUMOR INFILTRATING LYMPHOCYTES - Google Patents

Abstract

METHODS FOR EX VIVO EXPANSION OF TUMOR INFILTRATING LYMPHOCYTES AND FOR IDENTIFICATION OF AN AGENT FOR SUCH EXPANSION FOR USE IN ADOPTIVE CELL THERAPY, AS WELL AS USE OF A COMPOSITION COMPRISING EXPANDED LYMPHOCYTES. Compositions and methods for ex vivo expansion of tumor-infiltrating lymphocytes for use in adoptive cell therapy (ACT) are disclosed. Also described are compositions and methods for identifying an agent for ex vivo expansion of tumor infiltrating lymphocytes for use in ACT. Also disclosed are methods for treating cancer using the tumor-infiltrating lymphocytes expanded by the disclosed methods.

Description

CROSS-REFERENCE TO RELATED ORDERS

[001] This application claims the benefit of US Provisional Patent Application 61/955,970, filed on March 20, 2014, and US Provisional Patent Application 61/973,002 filed on March 31, 2014, which are incorporated herein by reference in your totality.

BACKGROUND

[002] Adoptive cell therapy (ACT) with tumor infiltrating lymphocytes (TIL) is a promising form of T cell-based immunotherapy. Preparation of TIL involves surgical resection and TIL expansion of melanoma tumors. In the face of adequate TIL expansion, patients undergo lymphodepletion chemotherapy, and TIL adoptive transfer followed by high doses of IL-2. The Department of Surgery at the National Cancer Institute pioneered this treatment for metastatic melanoma and reported a response rate of about 50% in treated patients, with ~20% of patients achieving durable complete responses (Rosenberg SA, et al. Clinical Cancer Research: An Official Bulletin of the American Association for Cancer Research. 2011 17(13): 4550-7). The interesting durability of responses to ACT is a hallmark of this treatment and appears to be superior to existing treatments for melanoma. ACT relies on T-cell infiltration of tumors before harvesting, successful ex vivo expansion of TIL, and potent antitumor effector function after transfer. Although TIL ACT is effective for melanoma, durable response rates need improvement. Shortening the expansion period for initial TIL growth and improving the tumor specificity of expanded TILs may increase response rates in patients treated with autologous TIL.

SUMMARY

[003] Compositions and methods for ex vivo expansion of tumor-infiltrating lymphocytes for use in adoptive cell therapy (ACT) are disclosed. In some embodiments, the methods involve culturing lymphocytes to produce expanded lymphocytes in a culture medium comprising a toll-like receptor (TLR) agonist, in an amount effective to improve specificity. expanded lymphocyte tumor. In some embodiments, the methods involve culturing lymphocytes to produce expanded lymphocytes in a culture medium comprising a stimulatory or peptidomimetic peptide. In some embodiments, the peptidomimetic is a peptoid or peptide-peptoid hybrid. In some embodiments, the peptoid or peptide-peptoid hybrid is stabilized by a hydrocarbon crosslink.

[004] Also disclosed are methods for treating cancer using tumor-infiltrating lymphocytes expanded by the disclosed methods. In some embodiments, the methods involve obtaining autologous tumor-infiltrating lymphocytes from the subject, culturing the lymphocytes in a culture medium comprising a toll-like receptor (TLR) agonist to produce expanded lymphocytes, treating the subject with myeloablative chemotherapy of lymphodepletion, and administering the expanded lymphocytes to the mammal.

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

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

[007] Also described are compositions and method for identifying an agent for ex vivo expansion of tumor infiltrating lymphocytes for use in ACT. The methods may involve contacting tumor-infiltrating lymphocytes with a candidate peptide or peptidomimetic from a peptide or peptidomimetic library for the ability to selectively bind to tumor-infiltrating lymphocytes. In some embodiments, the peptidomimetic is a peptoid or peptide-peptoid hybrid. In some embodiments, the peptoid or peptide-peptoid hybrid is stabilized by a hydrocarbon crosslink. The method may further involve screening for the effect of a binding peptide or peptidomimetic on the proliferation of tumor-infiltrating lymphocytes. In some embodiments, identification of a candidate peptide or peptidomimetic that enhances proliferation of tumor-infiltrating lymphocytes identifies an agent for ex vivo expansion of tumor-infiltrating lymphocytes for use in ACT. Agents identified by these methods can be used to expand tumor-infiltrating lymphocytes for use in ACT.

[008] 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.

DESCRIPTION OF FIGURES

[009] Figures 1A to 1D show examples of peptoid bodies.

[0010] Figure 2 shows a scheme for preparing a peptoid body comprising a linker that is attached to a resin and a second linker comprising a peptoid-peptoid sequence.

[0011] Figure 3 shows a scheme for preparing a peptoid body comprising a linker which is linked to a resin and a second linker which is a beta sheet binding promoter.

[0012] Figures 4A to 4C show examples of interlinked peptide-peptide hybrids.

[0013] Figure 5 shows an example of a positional library scan board.

[0014] Figure 6 shows an example of a reaction scheme to stabilize the backbone of a cyclic beta-staple-like peptoid-peptide hybrid using a meta-xylenyl group.

[0015] Figure 7 shows an example of a reaction scheme for stabilizing the cyclic beta-clip-like peptide-peptoid hybrid backbone by cross-linking and augmenting propargyl side chains that are on closer peptide side chains.

[0016] Figure 8 shows an example of a reaction scheme to stabilize cyclic beta-grampo-like peptoid-peptide hybrid backbone using an azide and an alkyne.

[0017] Figure 9 shows an example of a reaction scheme to stabilize hybrid backbone peptoid-peptide-like hybrid cyclic beta clamp using ring-closing metathesis (RCM).

DETAILED DESCRIPTION

[0018] Adoptive Cell Transfer (ACT) is a very effective form of immunotherapy and involves the transfer of immune cells with anti-tumor activity into cancer patients. ACT is a treatment approach that involves the in vitro identification of lymphocytes with antitumor activity, the in vitro expansion of these cells to large numbers, and their infusion into the cancer-bearing host. The lymphocytes used for adoptive transfer can be derived from the stroma of resected tumors (tumor infiltrating lymphocytes or TILs). They may also be derived from or from blood, if they are genetically engineered to express anti-tumor T cell receptors (TCRs) or chimeric antigen receptors (CARs), enriched with cultures of mixed lymphocyte tumor cells (MLTCs), or cloned using autologous antigen from cells and tumors presenting derived peptides. ACT in which the lymphocytes originate from the cancer-bearing host to be infused is termed autologous ACT. US 2011/0052530 relates to a method for carrying out adoptive cell therapy to promote cancer regression, primarily for the treatment of patients suffering from metastatic melanoma, which is hereby incorporated by reference in its entirety to these methods.

[0019] Compositions and methods for ex vivo expansion of tumor infiltrating lymphocytes (TILs) for use in ACT are disclosed. In some embodiments, the methods involve culturing 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 lymphocytes. expanded. The TLR agonist is in some embodiments a ligand for a TLR selected from the group consisting of TLR1, TLR2, TLR3, TLR4, and TLR9. For example, the TLR agonist can be a linker selected from the group consisting of Pam3CSK4, Pam3CSK4, poly I:C, Ribomunyl, and CpG ODN.

[0020] In some embodiments, the methods involve culturing lymphocytes to produce expanded lymphocytes in a culture medium comprising a peptide or peptidomimetic in an amount effective to improve the tumor specificity of the expanded lymphocytes. In some embodiments, the peptidomimetic is a peptoid or peptide-peptoid hybrid. In some embodiments, the peptoid or peptide-peptoid hybrid is stabilized by a hydrocarbon crosslink.

[0021] The peptoid portion can provide resistance to proteolysis and the peptide portion of peptoid-peptide hybrids can provide the ability to target the secondary beta hairpin-like secondary structure. These two contributions may result in a hybrid that is a good drug candidate for therapies where proteolysis is usually a limitation of therapy. As described herein, these peptide-peptoid hybrids can also be used for ex vivo expansion of tumor infiltrating lymphocytes (TIL) for use in ACT.

[0022] Examples of peptide-peptoid hybrids are described in WO 2013/192628 by McLaughlin et al., which is incorporated herein by reference to the library of peptoid bodies described herein. The peptoid bodies in WO 2013/192628 are cyclic peptoid-peptide hybrids, which can provide a backbone library using a pairwise combinatorial approach. Peptoid-cyclic peptide hybrids can adopt a secondary beta hairpin-like structure. This cyclic beta hairpin-like design results from alternating peptide-peptoid subunits in two antiparallel beta-strands. For example, the disclosed peptide-peptoid hybrid may have the chemical structure, for example, shown in formula I: or a pharmaceutically acceptable salt or hydrate thereof, wherein R1-R6 are independently organic groups.

[0023] In some embodiments, the peptide-cyclic peptide hybrids have the chemical structure shown in formula II: or a pharmaceutically acceptable salt or hydrate thereof, wherein the R groups are independently organic groups, R' is an organic group, or an organic sealing group to a resin or other substrate, ex is 1 to 3.

[0024] In some embodiments, the peptide-cyclic peptide hybrids have the chemical structure shown in formula III: or a pharmaceutically acceptable salt or hydrate thereof, wherein the R groups are independently organic groups, R' is an organic group, or an organic sealing group to a resin or other substrate, ex is 1 to 3.

[0025] In some embodiments, the peptoid-cyclic peptide hybrids have the chemical structure shown in formula IV: or a pharmaceutically acceptable salt or hydrate thereof, wherein the R groups are independently organic groups, R' is an organic group, or an organic sealing group to a residue, wherein the R groups are, independently, organic groups; R' is an organic group, or an organic sealing group to a resin or other substrate, eg is 1 to 3.

[0026] In some embodiments, the peptide-cyclic peptide hybrids have the chemical structure shown in formula V: or a pharmaceutically acceptable salt or hydrate thereof, wherein the R groups are independently organic groups; R' is an organic group, or an organic sealing group to a resin or other substrate, eg is 1 to 3.

[0027] In some embodiments, the peptide-cyclic peptide hybrids have the chemical structure shown in formula VI: or a pharmaceutically acceptable salt or hydrate thereof, wherein the R groups are independently organic groups, R' is independently an organic group, or an organic sealing group to a resin or other substrate, ex is 1 to 3.\

[0028] In some embodiments, the peptoid-cyclic peptide hybrids have the chemical structure shown in formula VII: or a pharmaceutically acceptable salt or hydrate thereof, wherein the R groups are independently organic groups, R' is independently an organic group, or an organic sealing group to a resin or other substrate, ex is 1 to 3.

[0029] In some embodiments, the peptoid-cyclic peptide hybrids have the chemical structure shown in formula VIII:

[0030] or a pharmaceutically acceptable salt or hydrate thereof, wherein the R groups are independently organic groups, R' is independently an organic group, or an organic sealing group to a resin or other substrate, and x is 1 to 3.

[0031] In some embodiments, the peptide-cyclic peptide hybrids have the chemical structure shown in formula IX: or a pharmaceutically acceptable salt or hydrate thereof, wherein the R groups are independently organic groups, R' is an organic group, or an organic sealing group to a resin or other substrate, ex is 1 to 3.

[0032] In some embodiments, the peptide-cyclic peptide hybrids have the chemical structure shown in formula X: or a pharmaceutically acceptable salt or hydrate thereof, wherein the R groups are independently organic groups, R' is an organic group, or an organic sealing group to a resin or other substrate, ex is 1 to 3.

[0033] In some embodiments, the R groups of at least two adjacent peptoid-glycine sequences are 4-piperidinyl groups, for example, a compound of Formula II where x is 2:

[0034] The R groups of the peptoid-cyclic peptide hybrids of Formula I through XII above may be of almost any structure such that they do not contain a moiety that interrupts the complementary hydrogen bonding of the beta sheet structure within the peptoid-cyclic peptide hybrid . The R group can be equivalent to the side chains of amino acids, the non-amine portion of an amino acid, or modified non-amine portion of an amino acid. The R group can be a sugar, such as a monosaccharide or disaccharide, or a fatty acid, or modified variant thereof. The R group can be, but is not limited to, C1-C12alkyl, C1-C12hydroxyalkyl, C1-C12aminoalkyl, C1-C12 alkyl carboxylic acid, C2-C12alkyloxyalkyl, C2-C12alkenyl, C2-C12hydroxyalkenyl, C2-C12aminoalkenyl, C1- C12alkenyl carboxylic acid, C3-C14alkyloxyalkenyl, C6-C14 aryl, C6-C14hydroxyaryl, C6-C14aminoaryl, C6-C14 aryl carboxylic acid, C7-C15alkyloxyaryl, C4-C14heteroaryl, C4-C14hydroxyheteroaryl, C4-C14aminoheteroaryl, C4-C14heteroaryl carboxylic acid, C5-C15alkoxy-, C7-C15alkylaryl, C7-C15hydroxyalkylaryl, C7-C15aminoalkylaryl, C7-C15alkylaryl carboxylic acid, C8-C15alkoxyalkylaryl, or a chemically transformed product of any of these R groups, such as esters, thioesters, amides, thiols, or sulfonamides, in which alkyl groups may be linear, branched, multiply branched, cyclic or polycyclic. For example, R can be, a residue of a primary amine, which can be, but is not limited to, groups from the incorporation of 4-aminopiperidine, ethanolamine, allylamine, 1,4-diaminobutane, piperponylamine, 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-ethylsparagine, 3-aminoadipic acid, hydroxylysine, beta-alanine, allohydroxylysine propionic acid, 2-aminobutyric acid, 3-hydroxyproline, 4-aminobutyric acid, 4-hydroxyproline piperidic acid - nico, 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, nor-valine, 2,2'-diaminopimelic acid, norleucine, 2,3-diaminopropionic acid, ornithine, N-ethylglycine, or protected equivalents thereof, as an N-R peptoid unit in the peptoid-cyclic peptide hybrid.

[0035] In some embodiments, the peptoid-peptide hybrids can be produced on a solid support, such as a resin. Hybrids can then be cleaved from the supporting resin for use in the described methods. Figures 1 and 2 illustrate example Hydridspeptoid-peptide as resin-bound intermediates.

Example peptoid-peptide hybrids

[0036] Embodiment 1: A peptoid-peptide hybrid with a beta-clamp-like conformation, comprising a plurality of alternating peptoid-peptide sequences, each having at least one peptoid residue and one amino acid residue, wherein the -peptoid-peptide sequences form at least two antiparallel beta strands between a plurality of linkers, and wherein at least one linker is a beta sheet binding promoter.

[0037] Embodiment 2: The hybrid according to Embodiment 1, wherein at least one of the linker is the amino acid residue from the condensation of the linker precursor of the structure:

[0038] ; where θm quθ R' is an organic group, or an organic sealing group attached to a resin or other substrate and R" is H or a carboxylic acid protecting group.

[0039] Embodiment 3: The hybrid according to Embodiment 2, wherein R" is t-butyl, allyl, or benzyl.

[0040] Embodiment 4: The hybrid according to Embodiment 2, wherein the organic linking group attached to a resin or other substrate comprises a -NH(CH2)2- sealing group.

[0041] Embodiment 5: The hybrid according to Embodiment 1, wherein one of the linker is two peptoid residues.

[0042] Embodiment 6: The hybrid according to Embodiment 1, in which the peptide-cyclic peptide hybrid is: wherein the R groups are independently organic groups, R' is independently an organic group, or an organic sealing group to a resin or other substrate, and x is 1 to 3.

[0043] Embodiment 7: The hybrid according to Embodiment 6, wherein R is independently C1-C12alkyl, C1-C12hydroxyalkyl, C1-C12aminoalkyl, C1-C12 alkyl carboxylic acid, C2-C12alkyloxyalkyl, C2-C12alkenyl, C2-C12hydroxyalkenyl, C2-C12aminoalkenyl, C1-C12alkenyl carboxylic acid, C3-C14alkyloxyalkenyl, C6-C14 aryl, C6-C14hydroxyaryl, C6-C14aminoaryl, C6-C14aryl carboxylic acid, C7-C15alkyloxyaryl, C4-C14heteroaryl, C4-C14hydroxyhetero aryl, C4 - C14aminoheteroaryl, C4-C14heteroaryl carboxylic acid, C5-C15alkoxy-, C7-C15alkylaryl, C7-C15hydroxyalkylaryl, C7-C15aminoalkylaryl, C7-C15alkylaryl carboxylic acid, C8-C15alkoxyalkylaryl, or any chemically transformed structure thereof.

[0044] Embodiment 8: The hybrid according to Embodiment 7, wherein the chemically transformed structure comprises an ester, thioester, thiol, amide or sulfonamide.

[0045] Embodiment 9: The hybrid according to Embodiment 6, wherein R is independently a residue of a primary amine: 4-aminopiperidine; ethanolamine; allylamine; 1,4-diaminobutane; piperponylamine; 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; beta-alanine; propionic acid allohydroxylysine; 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 equivalents thereof.

[0046] Embodiment 10: The hybrid according to Embodiment 6, wherein at least one R is a 4-aminopiperidine residue.

[0047] Embodiment 11: The hybrid according to Embodiment 1, wherein all amino acid residues are glycine residues.

[0048] Peptide-peptide hybrids can be further stabilized by cross-linking between amino acid side chains, and/or N-substitutions on glycines, and/or backbone cyclization. Such peptoids are referred to herein as "interlinked peptoids"; whereas, such peptoid-peptide hybrids are referred to herein as "interlinked peptoid-peptide hybrids".

[0049] Interlinking of peptoids or peptoid-peptide hybrids involves chair-side-chair linkages and/or backbone cyclization to stabilize the peptoids or peptoid-peptide hybrids. Thus, the present invention extends the peptide stabilization approach using stabilized side chain linkages to stabilizing peptoids or peptide-peptide hybrids.

[0050] For the purpose of the present invention, the term "side chain" includes an amino acid side chain as well as the N-atom-linked moiety of the N-substituted glycine.

[0051] Several possible side-chain-side-chain linkages (herein referred to as intramolecular cross-linking) can be designed. Intramolecular cross-linking between the two side chains of the peptoids or the peptoid-peptide hybrids of the present invention can be mediated through chemical reactions between the side chains, which can also involve additional chemicals.

[0052] For example, intramolecular cross-linking can be mediated through a chemical moiety which is not a part of the side chains and in which the side chains link to each other through the chemical moiety. An example of the chemical moiety that forms the intramolecular crosslink is depicted in Figures 6-9.

[0053] In some embodiments, the intramolecular cross-linking is established by the RCM approach (ring-closing metathesis) or according to a Click or Aileron reaction (eg, 3+2 copper-catalyzed cycloditation) described in a US Patent of Number 5,811,515, which is incorporated by reference in its entirety herein. An example of intramolecular cross-linking mediated via the RCM approach is depicted in Figure 9.

[0054] In another embodiment, the intramolecular cross-linking between the two side chains is established by the formation of a chemical bond, for example through a condensation reaction, between the functional groups present on the side chains. A condensation reaction is a chemical reaction in which two molecules or fractions (functional groups) combine through a chemical bond and the reaction involves the loss of one or more small molecules. Examples of condensation reaction between side chains that can be used in producing intramolecular cross-linking according to the present invention are well known to one skilled in the current art and such embodiments are within the scope of the present invention.

[0055] Additional examples of interlinked peptoids with some intramolecular crosslinks are shown in Figures 4A-4C.

[0056] Additional examples of peptide cross-linking are disclosed in United States Patent Numbers 8592377, 8324428, 8198405, 7786072, 7723469, 7192713, the contents of which are incorporated herein by reference in their entirety. One skilled in the current art can envision using various crosslinks described in these patent documents in preparing the crosslinked peptoids and the crosslinked peptoid-peptide hybrids according to the present invention and such embodiments are within the scope of the present invention.

[0057] Accordingly, the present invention provides an interlinked peptoid comprising a plurality of N-substituted glycines, wherein at least two of the N-substituted glycines are linked together by intramolecular cross-linking, and wherein the length and Intramolecular cross-linking geometry provides stability to the peptoid.

[0058] In some cases, the crosslinked peptide-peptoid hybrid may comprise a plurality of amino acids and the plurality of N-substituted glycines, wherein at least two residues of the plurality of amino acids and the plurality of N-substituted glycines are linked together others by an intramolecular cross-link, and where the length and geometry of the intramolecular cross-link provides stability to the peptide-peptide hybrid.

[0059] In one embodiment two N-substituted glycine residues or two amino acid residues are linked to each other by a cross-link. In another embodiment, an N-substituted glycine residue is cross-linked to an amino acid residue.

[0060] In another embodiment, the interlinked peptide-peptoid hybrid is a cyclic peptide-peptoid hybrid. A cyclic peptoid-peptide hybrid comprises a plurality of alternating peptide-peptide sequences, each having at least one peptoid residue and one amino acid residue, wherein the peptoid-peptide sequences form at least two antiparallel beta strands.

[0061] In some embodiments, the intramolecular crosslink is an all hydrocarbon crosslink.

[0062] In some embodiments, the peptoid or peptoid-peptide hybrid comprises more than one intramolecular crosslink, for example, two, three, or four intramolecular crosslinks.

[0063] In some embodiments, the cross-links are between two or more amino acid residues or N-substituted glycine residues located on the same side of a beta sheet, thereby providing stability to the peptoid or peptoid-peptide hybrid. In one embodiment, the intramolecular cross-links are between two or more amino acid residues, or N-substituted glycine residues located within residues of a beta sheet, thereby providing stability to the peptoid or peptoid-peptide hybrid.

[0064] In some embodiments, the side chain may be selected from cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkylene; cyclic or acyclic alkenylene, branched or unbranched, substituted or unsubstituted; cyclic or acyclic alkynylene, branched or unbranched, substituted or unsubstituted; cyclic or acyclic heteroalkylene, branched or unbranched, substituted or unsubstituted; cyclic or acyclic heteroalkenylene, branched or unbranched, substituted or unsubstituted; heteroalkynylene, cyclic or acyclic, branched or unbranched, substituted or unsubstituted; substituted or unsubstituted arylene; substituted or unsubstituted heteroarylene; or substituted or unsubstituted acylene.

[0065] Additional examples of side chains and crosslinks that may be applied in the present invention are disclosed, for example, in US Patent Number 8,592,377 at column 37, line 26 through column 43, line 14; US Patent Number 8,198,405, column 3, row 54 through column 10, row 2 and column 25, row 14 through column 26, row 21; US Patent Number 7,786,072, column 5, row 44 to column 9, row 43 and column 11, row 16 through column 12, row 8; US Patent Number 7,723,469, column 5, row 30 through column 9, row 12 and column 24, row 60 through column 26, row 3; and US Patent Number 7,192,713, column 4, row 26 to column 9, row 45 and column 11, row 23 through column 12, row 18.

[0066] The cross-linked peptoids, peptide-peptide hybrids, and cross-linked cyclic peptide-peptoid hybrids of the present invention may also optionally further contain substitutions in the amino acid side chains and/or the N-substituted glycine residues, wherein the substitutions on the side chains additionally stabilize peptoids, peptoid-peptide hybrids, and cyclic peptoid-peptide hybrids. Non-limiting examples of various substitutions that can be used in the present invention are provided in Table 2.

[0067] Production of Tumor Infiltrating Lymphocyte (TIL) is a two-step process: 1) the pre-REP (Rapid Expansion) phase, where cells are cultured in standard laboratory media such as RPMI and treated TILs with reagents, such as irradiated feeder cells, and anti-CD3 antibodies to achieve the desired effect; and 2) the REP phase where the TIL is expanded into a large enough amount of culture to treat the patients. The REP phase requires cGMP grade reagents and 30-40L of culture medium. However, the pre-REP phase can utilize laboratory-grade reagents (under the assumption that the laboratory-grade reagents dilute out during the REP phase), making it easier to incorporate alternative strategies to improve production. TIL. Therefore, in some embodiments, the disclosed TLR agonist and/or peptide or peptidomimetic can be included in the culture medium during the pre-REP phase.

[0068] 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. they are TILs, and are isolated from the mammal to be treated, i.e. autologous transfer.

[0069] Autologous ACTs as described herein may also be performed by (i) culturing autologous expanded lymphocytes to produce expanded lymphocytes; (ii) administering non-myeloablative lymphodepleting chemotherapy to the mammal; and (iii) after administering non-myeloablative lymphodepleting chemotherapy, administering the expanded lymphocytes to the mammal. Autologous TILs can be obtained from the stroma of resected tumors. Tumor samples are obtained from patients and a cell suspension is obtained. The cell suspension may be obtained in any suitable way, for example, mechanically (tumor disruption using, for example, a gentleMACS(TM) Dissociator, MiltenyiBiotec, Auburn, Calif.) or enzymatically (for example, collagenase or DNase).

[0070] Expansion of lymphocytes, including tumor-infiltrating lymphocytes such as T cells can be achieved by any of a number of methods as are known in the art. For example, T cells can be rapidly expanded through non-specific T cell receptor stimulation in the presence of feeding lymphocytes and interleukin-2 (IL-2), IL-7, IL-15, IL-21, or their combi-nations. Non-specific T cell receptor stimulation can, for example, include about 30 ng/ml OKT3, a mouse anti-CD3 monoclonal antibody (available from Ortho-McNeil(R), Raritan, NJ or MiltenyiBiotec, Bergisch Gladbach, Germany). Alternatively, T cells can be rapidly expanded by stimulating peripheral blood mononuclear cells (PBMC) in vitro with one or more antigens (including antigenic portions thereof, such as the epitope(s), or a cell from the cancer, which may optionally be expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2), binding peptide, e.g., approximately 0.3 µM MART-1: 26-35 (27 L) or gp100: 209-217 (210M)), in the presence of a T-cell growth factor, such as about 200-400 Ill/ml, such as 300 IU/ml IL-2 or IL-15, IL-2 being preferred. In vitro induced T cells are rapidly expanded by restimulation with the same HLA-A2-pulsed cancer antigen(s) that express the antigen-presenting cells. Alternatively, the T cells can be restimulated with irradiated autologous lymphocytes, or with irradiated HLA-A2+ and IL-2 allogeneic lymphocytes, for example.

[0071] In some embodiments, non-myeloablative lymphodepletion chemotherapy is administered to the mammal prior to administration of tumor-infiltrating expanded lymphocytes to the mammal. The goal of lymphodepletion is to make room for infusion lymphocytes, in particular by eliminating regulatory T cells and other non-specific T cells that compete for homeostatic cytokines. Non-myeloablative lymphodepletion chemotherapy may be any type of suitable therapy which may be administered by any suitable route known to a person skilled in the art. Non-myeloablative lymphodepleting chemotherapy may comprise, for example, the administration of cyclophosphamide and fludarabine, particularly if the cancer is melanoma, which may be metastatic. A preferred route of administration for cyclophosphamide and fludarabine is intravenously. Likewise, any suitable dose of cyclophosphamide and fludarabine can be administered. Preferably, about 4080 mg/kg, such as about 60 mg/kg of cyclophosphamide is administered for about two days after which about 15-35 mg/m2, such as about 25 mg/m2, fludarabine it is given for about five days, particularly if the cancer is melanoma.

[0072] Tumor-specific reactivity of the expanded TILs can be tested by any method known in the art, for example, by measuring cytokine release (eg interferon-gamma) after co-culture with tumor cells. In one embodiment, the autologous ACT method comprises enriching the culture's TIL for CD8+ T cells prior to rapid expansion of the cells. After culturing the TILs in IL-2, the T cells are depleted of CD4+ cells and enriched for CD8+ cells, using, for example, a CD8 microbead separation (e.g., using a CliniMACS<plus>CDs microsphere system (Miltenyi Biotec )). In one embodiment of the method, a T cell growth factor that promotes the growth and activation of autologous T cells is administered to the mammal either concomitantly with the autologous T cells or subsequently with the autologous T cells. The T cell growth factor can be any appropriate growth factor that promotes the growth 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 can 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 IL2. IL-12 is a preferential T-cell growth factor.

[0073] Preferably, expanded lymphocytes produced by these methods are administered as an intra-arterial or intravenous infusion, which preferably lasts from about 30 to about 60 minutes. Other examples of routes of administration include the intraperitoneal, intrathecal and intralymphatic routes. Likewise, any suitable dose of lymphocytes can be administered. In one embodiment, about 1 x 1010 lymphocytes to about 15 x 1010 lymphocytes are administered.

[0074] The cancer treated by the disclosed compositions and methods can be any type of cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal or anorectal cancer, eye cancer, cancer of biliary cholestasis, cancer of the joints, cancer of the neck, gallbladder or pleura, cancer of the nose, cancer of the nasal cavity, middle ear, vulva, chronic lymphocytic leukemia, myeloid cancer chronic, cervical cancer, glioma, Hodgkin Lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non- Hodgkin cancer, ovarian cancer, peritoneum, omentum and mesentery cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, cancer, soft tissue cancer, testicular skin cancer, thyroid cancer, urethral cancer, bladder cancer and cancer of the digestive system, such as, for example, esophageal cancer, gastric cancer, pancreatic cancer, stomach cancer, bowel cancer, gastrointestinal carcinoid tumor, oral cavity cancer, colorectal cancer and gallbladder cancer.

[0075] The cancer can be a recurrent cancer. Preferably, the cancer is a solid cancer. Preferably, the cancer is melanoma, ovarian, breast and colorectal cancer, even more preferred is melanoma, in particular metastatic melanoma.

Definitions

[0076] The term "subject" refers to any subject who is the subject of administration or treatment. The subject may be a vertebrate, for example a mammal. Thus, the subject may be a human or veterinary patient. The term "patient" refers to an individual under treatment by a clinician, e.g., a physician.

[0077] The term "therapeutically effective" refers to the amount of the composition used is an amount sufficient to ameliorate one or more symptoms or causes of a disease or disorder. Such an improvement only requires a reduction or alteration, not necessarily elimination.

[0078] The term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, i.e., treatment directed specifically towards the amelioration of a disease, pathological condition or disorder, and also includes causal treatment, i.e., treatment aimed at removing the cause of the associated disease, pathological condition or disorder. . In addition, this term includes palliative care, that is, treatment designed to relieve symptoms rather than cure the disease, pathological condition or disorder; preventive treatment, that is, treatment aimed at minimizing or partially or completely inhibiting the development of the disease, pathological condition or associated disorder; and supportive treatment, that is, the treatment used to complement another specific therapy aimed at improving the disease, pathological condition or associated disorder.

[0079] The terms "peptide", "protein" and "polypeptide" are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of an amino acid with the alpha-amino group from another.

[0080] As used herein, "peptidomimetic" means a mimetic chain of a peptide that includes some alteration of normal peptide chemistry. Peptidomimetics typically improve some properties of the parent peptide, such as increased stability, increased efficacy, improved delivery, increased half-life, etc. Methods of producing peptidomimetics based on a known polypeptide sequence are described, for example, in US Patent Number 5,631,280; 5,612,895; and 5,579,250. Use of peptidomimetics may involve the incorporation of a non-amino acid residue with non-amide linkages at a given position. One embodiment of the present invention is a peptidomimetic, where the compound has a bond, peptide backbone or amino acid component replaced by a suitable mimic. Some non-limiting examples of unnatural amino acids that may be suitable amino acid mimics include β-alanine, butyric acid-L-α amino, butyric acid-L-Y amino, isobutyric acid-L-α amino, L-ε-amino caproic acid , 7-amino heptanoic 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.

[0081] The term "peptoid" refers to a class of peptidomimetics whose side chains are added to the nitrogen atom of the peptide backbone rather than to the α-carbons (as they are in amino acids).

[0082] The term "lymphocyte tumor infiltration" or "TIL" refers to white blood cells, which have left the bloodstream and migrated to a tumor.

[0083] The term "regression" does not necessarily imply 100% or complete regression. Rather, there are varying degrees of regression, which one skilled in the art recognizes as having a potential therapeutic benefit or effect. The term also encompasses delaying the onset of the disease, or a symptom or condition thereof.

[0084] The term "crosslink" or "hydrocarbon crosslink" as used herein refers to the use of a hydrocarbon to stabilize the secondary structure of a synthetic peptide or peptidomimetic.

[0085] A number of embodiments of the invention have been described. However, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. In this sense, other modalities are within the scope of the following claims.

EXAMPLES Example 1: Targeting TLRs to improve TIL expansion and activity

[0086] 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 activation and host immunity. In the tumor environment, the function of macrophages and DCs is inhibited. As described herein, this repressed state can be reversed by administration of TLR ligands (Table 1). Exogenous TLR ligands added to TIL cultures can enhance DC and macrophage function, resulting in increased TIL expansion and enhanced tumor-specific immune responses. Table 1. TLR Receptors and Ligands

[0087] To determine whether exogenous TLR ligands added to TIL cultures are able to increase TIL expansion and improve tumor specificity, fresh melanoma tumors are cut into 1-2 mm 2 fragments in medium with su -supplement of 6000 IU/ml of IL-2. Twelve fragments are grown on IL-2 alone. Additional groups of 12 fragments are treated with the following TLR linkers: TLR1/2 Pam3CSK4 linker (1 µg/ml), TLR3 poly(I:C) linker (12.5 µg/ml), TLR4 Ribomunyl linker, a clinical grade bacterial extract (1 µg/ml), TLR9 CpG ODN2006 ligand (10 µg/ml). The culture medium is replaced with fresh medium every 2-3 days. TILs are split into new wells when they reach confluence. After 10, 20, and 30 days of culture, the total number of tumor fragments resulting in TIL growth are recorded. Furthermore, cells are collected from each fragment and counted. Cell numbers are compared between the IL-2 control group and the TLR ligand treated group. Individual pools of TIL from each fragment are co-cultured with autologous HLA-matched and non-matched melanoma cells for 24 hours. Culture supernatants are collected and IFN-gamma is measured by conventional ELISA. The purpose of these experiments is to determine whether the addition of TLR ligands results in increased TIL growth of fragments, increased TILA proliferation, and/or increased tumor-specific TIL activation.

Example 2: Identification of Peptoids for TIL proliferation and activation

[0088] In addition to TLR agonists, other costimulatory molecules can be expressed by TIL. A peptoid library can be screened to identify compounds that lead to better TIL proliferation and activation. A peptoid library containing approximately 200,000 compounds is scanned in a similar 384-well format. TIL cell lines are stained with red quantum dots and scanned to identify library hits that selectively bind to red cells. Once several binding peptoids are identified, TIL proliferation is examined in the presence of the peptoids. Peptoids that lead to TIL proliferation (stimulatory peptoids) are further examined in a functional assay.

[0089] Using a human T cell line (AS1), the stimulatory effects of peptoids are measured as a function of T cells. AS1 cells are activated CD8 + human T cells that demonstrate specificity against the 624 melanoma cell line , but not the HLA 888 mismatched melanoma cell line. AS1 cells are cultured in 6000 IU/ml of IL-2 in the presence of increasing doses of stimulatory peptoids. Cells are counted on days 3, 7, 10, 14, and 21 to determine the dose of stimulatory peptoids that leads to increased AS1 cell proliferation. To determine whether culture with stimulatory peptoids leads to increased T cell function, IFN-gamma, a cytokine secreted by activated T cells, is measured. AS1 cells are treated with the optimal dose of stimulatory peptoid bodies as determined above. Controls include AS1 cells alone and AS1 cells treated with anti-41BB antibody. After 7 days, AS1 cells are harvested and eco-cultured with 624 melanoma cells. As a negative control, AS1 cells are co-cultured with 888 melanoma cells. After 24 hours, supernatants are collected and IFN-gamma production is measured by ELISA. Comparisons are made between AS1 alone and AS1 treated with stimulant peptoids. To further explore the effectiveness of the stimulant peptoids, T cells are harvested from the tumors of 10 patients with metastatic melanoma enrolled in IRB approved clinical trials. Tumor fragments are cultured in media 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 ). Irrelevant or stimulatory peptides are added to 12 fragments per condition. 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-matched tumor cells are included as negative controls. T cells grown in the presence of CD3/CD28 cells are included as a positive control. After 24 hours, supernatants are collected and IFN-gamma production is measured by ELISA. In addition, T cell proliferation is measured on days 7, 14, and 21 to determine whether co-culture with stimulatory peptoids leads to increased proliferation of T cells that infiltrate the tumor. 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 murine model

[0090] A major disadvantage of TIL studies is the inability to test TIL in an in vivo model. A murine model is developed to measure the effectiveness of TIL. Using NSG mice (B and T-cell-depleted mice, purchased from Jackson Laboratories), melanoma tumors from primary patients are implanted into the flank. When the tumor reaches 5 mm in diameter, expanded 1x107 TIL are transferred from pooled patient samples. Tumor growth and survival are measured. Using this model, it is examined whether TIL grown in standard IL-2 medium, with TLR ligands, or with stimulatory peptoids lead to better in vivo tumor rejection.

Example 4: Position scan with 69 different substituents

[0091] In some embodiments, a molecular library exhibiting a mixture of 69 compounds defined by the general structure in Figure 4A having various substituents (listed in Table 2) at either the R1, or R2, R3 positions is prepared where all other positions R1 and R2, Either R1 and R3 or R2 and R3 display each of the possible 69 x 69 combinations. This is equal to 69 x 69 x 3, ie. 14,283 different stitches. This number is much smaller than having to do every possible combination of 69 x 69 x 69, ie. 328,509 stitches which require 952.2 boards with 345 stitches per board. The positional scanning approach requires only 41.4 plates.

[0092] In this embodiment of the positional scanning method, only about 1/69° of a pure substance is displayed per point. When a scan finds a hit, the hit is re-prepared to check its activity. This approach gives ~1.4% of a particular sequence represented per well and by comparing hits from other plates that discretely show the side chains on each of the three side chains, the preferred side chains can be determined. Table 2. Examples of substituents in positions R1, R2, or R3:

[0093] Figure 5 is an example of a board used for positional scanning. Points 1-69 would have identical R1 at all points and each of the 69 individual and different side chains at R2 and a mixture of all 69 compounds shown at position R3. Points 70138 would similarly have all R1 positions displaying sidechain 2 and then each of the 69 different sidechains at position R2 and a mixture of 69 different sidechains displaying at position R3 and so on to cover all possible combinations. Specific R2 plates would have a mixture of 69 compounds shown at the R1 position, then all 69 spots with R2 having the same side chain as compound 1, and then each of the 69 individual compounds for the analogous R3 to the specific R1 plates described above, then the remaining combination where the R1 position will display each of the 69, the R2 will display the mixture of the 69 compounds, and points 1-69 will all have the same side chain at the R3 position.

[0094] An example of the positional scan results is also shown in Figure 5. The first shaded point has the best side chains of R1 with the side chain equal to compound 2 and R2 equals compound 26, and at least one of the 69 possible compounds in the R3 position. The next point shows that compound 3 is active at R1 and R2 has compound 10 and at least one of the 69 compounds at position R3. The last dot on the plate shows that R1 equals compound 5 and compound 55 for R2 and at least one of the 69 compounds at position R3. By analyzing analogous scan results from the defined R2 and R3 position plates the exact sequence can be determined.

[0095] The 69 compounds with similar reactivity in the SN2 reaction to make the secondary amines are needed. Huge structural variability is possible, but some possible side chains are shown in Table 2.

Example 5: Interlinking Methods to Stabilize a Beta Cyclic Clamp-Like Peptoid-Peptide Hybrid Skeleton

[0096] There are several viable interlinking methods that can be used to stabilize the beta cyclic hairpin-like peptoid-peptide hybrid backbone. Interlinking two peptoid side chains is the easiest to accomplish from a synthetic point of view and will pre-arrange those two peptoid side chains to be close together, and that proximity forces conformations with the overall backbone. that are compatible with the desired beta cyclic hairpin-like secondary structure. The easiest pairs to cross-link are propargyl amines on peptoid side chains to be cross-linked and react with these two alkyne termini with a bifunctional diazide with a stable organic linker spanning the distance between these peptoid side chains. The organic ligand needs 3 more atoms to cover that distance. The meta-xylenyl group is shown in Figure 6; however, any stable combination of atoms can be used. Furthermore, the chain does not have to be just a linker, as the group can also be used to optimize pharmacokinetic properties, for example.

[0097] Alternatively, the same or a different linker can be used to interconnect propargyl side chains that are on closer peptoid side chains, as shown in Figure 7. As described herein, a side chain of an amino acid side chain and a near peptoid side chain are also possible, but not shown. A more generic scheme using an azide and an alkyne is shown in Figure 8. The ring closing metathesis (RCM) reaction can also be used, as shown by the generic example in Figure 9.

[0098] Unless otherwise defined below, all technical and scientific terms used in this document have the same meaning as commonly understood by a person skilled in the art to which this invention belongs. Publications cited in this document and the materials cited are specifically incorporated by reference.

[0099] Those skilled in the art will recognize, or will be able to verify using no more than routine experimentation, and many equivalents to specific embodiments of the invention described herein. Such equivalents are intended to be covered by the following claims.

Claims (8)

1. Method for ex vivo expansion of tumor infiltrating lymphocytes for use in adoptive cell therapy (ACT) for cancer, comprising culturing the lymphocytes to produce expanded lymphocytes in a culture medium comprising a receptor-type agonist toll receptor (TLR) in an amount effective to improve the tumor specificity of the expanded lymphocytes. 2. Method according to claim 1, characterized in that the TLR agonist is a ligand for a TLR selected from the group consisting of TLR1, TLR2, TLR3, TLR4, and TLR9. 3. Method according to claim 1, characterized in that the TLR agonist comprises a ligand selected from the group consisting of Pam3CSK4, poly I:C, Ribomunil, and CpG ODN. 4. Method according to any one of claims 1 to 3, characterized in that the cancer is a solid tumor. 5. Method or use according to any one of claims 1 to 4, characterized in that the cancer is selected from the group consisting of melanoma, ovarian cancer, breast cancer, and colorectal cancer. 6. Method according to any one of claims 1 to 5, characterized in that the cancer is metastatic. 7. Method according to any one of claims 1 to 6, characterized in that the cancer recurs. 8. Method according to any one of claims 1 to 7, characterized in that the subject is a human.