Methods of treatment using conditional superagonist CTL ligands for the promotion of tumor-specific CTL responses

What is described is a method of treatment of a patient with a tumor, comprising administering a cell responsive to a peptide comprising a tumor epitope, wherein the tumor epitope comprises an amino acid substitution in a tumor antigen. The tumor antigen is preferably selected from the group consisting of NYESO-I157-165, NYESO-II157-170, or MART-126-35, preferably SEQ ID NOS: 1-351, 361-376, and 392-401.

1. A method of treatment of a patient with a tumor, comprising administering a cell responsive to a peptide comprising a tumor epitope, wherein the tumor epitope comprises a sequence selected from the group consisting of SEQ ID NOS: 363-365 and 368-375.

2. The method of claim 1, wherein the tumor epitope comprises a sequence selected from the group consisting of SEQ ID NOS: 368-375.

3. The method of claim 2, wherein the tumor epitope comprises a sequence consisting of SEQ ID NOS: 372, 374, or 375.

4. The method of claim 1, wherein the tumor epitope comprises a sequence selected from the group consisting of SEQ ID NOS: 363-365.

5. The method of claim 4, wherein the tumor epitope comprises a sequence selected from the group consisting of SEQ ID NOS: 363 and 365.

6. The method of claim 1, wherein the tumor epitope comprises a multiplicity of sequences selected from the group consisting of SEQ ID NOS: 363-365 and 368-375.

7. The method of claim 6, wherein the tumor epitope comprises a multiplicity of sequences selected from the group consisting of SEQ ID NOS: 363-365.

8. The method of claim 7, wherein the tumor epitope comprises a sequence selected from the group consisting of SEQ ID NOS: 363 and 365.

9. The method of claim 6, wherein the tumor epitope comprises a multiplicity of sequences selected from the group consisting of SEQ ID NOS: 368-375.

10. The method of claim 9, wherein the tumor epitope comprises a sequence selected from the group consisting of SEQ ID NOS: 372, 374, and 375.

11. A method of treating a patient with a tumor, comprising administering a pharmaceutical composition to said patient, wherein said pharmaceutical composition comprising a peptide comprising a tumor epitope, wherein the tumor epitope comprises a sequence selected from the group consisting of SEQ ID NOS: 363-365 and 368-375.

12. The method of claim 11, wherein the tumor epitope comprises a sequence selected from the group consisting of SEQ ID NOS: 368-375.

13. The method of claim 12, wherein the tumor epitope comprises a sequence selected from the group consisting of SEQ ID NOS: 372, 374, and 375.

14. The method of claim 11, wherein the tumor epitope comprises a sequence selected from the group consisting of SEQ ID NOS: 363-365.

15. The method of claim 14, wherein the tumor epitope comprises a sequence selected from the group consisting of SEQ ID NOS: 363 and 365.

16. The method of claim 11, wherein the tumor epitope comprises a multiplicity of sequences selected from the group consisting of SEQ ID NOS: 363-365 and 368-375.

17. The method of claim 16, wherein the tumor epitope comprises a multiplicity of sequences selected from the group consisting of SEQ ID NOS: 363-365.

18. The method of claim 17, wherein the tumor epitope comprises a sequence selected from the group consisting of SEQ ID NOS: 363 and 365.

19. The compound of claim 16, wherein the tumor epitope comprises a multiplicity of sequences selected from the group consisting of SEQ ID NOS: 368-375.

20. The compound of claim 19, wherein the tumor epitope comprises a sequence selected from the group consisting of SEQ ID NOS: 372, 374, and 375.

This application is a continuation of U.S. patent application Ser. No. 15/098,274 filed Apr. 13, 2016, issued as U.S. Pat. No. 10,328,135, which is a continuation of U.S. patent application Ser. No. 13/696,303 filed on Jan. 8, 2013, issued as U.S. Pat. No. 9,314,516, which is a national phase application of PCT application no. US2011/035272 filed May 4, 2011, which claims benefit under claims benefit under 35 U.S.C. § 119(e) of Provisional U.S. patent application No. 61/331,260 filed May 4, 2010, the contents of which herein are incorporated by reference in their entirety.

This invention was made with government support under CA122904 awarded by the National Institutes of Health and National Cancer Institute. The government has certain rights to the invention.

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 11, 2014, is named 105003.000128_SL.txt and is 69,434 bytes in size.

What is described is a method of treating cancer by administering immune cells responsive to epitopes of tumor antigens, preferably NYESO-I_157-165, NYESO-II_157-170, or MART-1_26-35tumor epitopes.

Cytotoxic T lymphocytes can directly kill malignant cells, which express and display specific antigenic peptides in the context of specific class I MHC molecules. These antigenic peptides, often referred to as CTL epitopes, are peptides of unique amino acid sequence, usually 9-11 amino acids in length. The tumor-associated antigenic peptide that is being targeted can be used as a peptide-based vaccine to promote the anti-tumor CTL response. However, when the target peptide is derived from non-mutated differentiation antigens as is often the case (e.g. melanosomal proteins), it can be insufficient to engender robust and sustained anti-tumor CTL responses. This is a result of immune tolerance mechanisms that generally suppress or eliminate high avidity auto-reactive T cells. As a result of these mechanisms, the vast majority of tumor-specific CTL, specifically those that recognize non-mutated tumor-associated antigens, are eliminated in the thymus and in the periphery. What remains is a low frequency of tumor-specific CTL, and/or CTL that bear low avidity T cell receptors for the cognate tumor antigen.

One way to activate and mobilize these rare and low avidity tumor-specific CTL is with the use of superagonist altered peptide ligands (APLs). These are mutant peptide ligands that deviate from the native peptide sequence by one or more amino acids, and which activate specific CTL clones more effectively than the native epitope. These alterations either allow the peptide to bind better to the restricting class I MHC molecule or interact more favorably with the TCR of a given tumor-specific CTL subset. Superagonist APLs demonstrate favorable responses in clinical studies.

One method to identify superagonist APLs involves comparing the amino acid sequence of the tumor-associated CTL epitope to the so-called consensus binding motif for the restricting class I MHC allotype. Where the tumor-associated epitope deviates from the consensus sequence, the appropriate amino acids can be substituted, allowing the peptide to bind better to the class I MHC molecule. This approach is limited because not all poorly stimulatory CTL epitopes deviate from the consensus motif. Another approach involves substituting one or more specific amino acids into every position of the epitope; e.g., alanine scanning. Another approach includes making every single amino acid substitution at one or two positions—positions either predicted to play a role in class I MHC secondary binding or to be directly involved in engaging the TCR. All of these approaches are severely limited in scope, and potentially overlook a large number of superagonist APLs. Utilization of APLs remains limited due to a lack of comprehensive methods for which to identify them.

One aspect of the description is a method of treatment of a patient with a tumor, comprising administering a cell responsive to a peptide comprising a tumor epitope, wherein the tumor epitope comprises an amino acid substitution in a tumor antigen, and wherein the tumor antigen is selected from the group consisting of SEQ ID NOS: 1-351, 361-376, and 392-401.

Preferably the tumor epitope comprises an amino acid substitution in a tumor antigen, wherein the tumor antigen is SEQ ID NO: 144 or 228, and wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOS: 362-365 and 368-376.

In one embodiment, the tumor antigen is SEQ ID NO: 144, and wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOS: 368-376, preferably comprising a sequence consisting of SEQ ID NOS: 372, 374, or 375.

In another embodiment, the tumor antigen is SEQ ID NO: 228, and the compound comprises a sequence selected from the group consisting of SEQ ID NOS: 362-365, preferably a sequence consisting of SEQ ID NOS: 362, 363 or 365.

In another embodiment, the tumor antigen comprises a multiplicity of sequences selected from the group consisting of SEQ ID NOS: 362-365 and 368-376. Preferably, the wherein the compound comprises a multiplicity of sequences selected from the group consisting of SEQ ID NOS: 362-365, more preferably a sequence consisting of SEQ ID NOS: 362, 363 or 365. In another embodiment the tumor antigen comprises a multiplicity of sequences selected from the group consisting of SEQ ID NOS: 368-376, preferably a sequence consisting of SEQ ID NOS: 372, 374, or 375.

Another aspect of the description is a method of treating a patient with a tumor, comprising administering a pharmaceutical composition to said patient, wherein said pharmaceutical composition comprising a peptide comprising a tumor epitope, wherein the tumor epitope comprises an amino acid substitution in a tumor antigen, and wherein the tumor antigen comprising a sequence selected from the group consisting of SEQ ID NOS: 1-351, 361-376, and 392-401.

Preferably, the tumor epitope comprises an amino acid substitution in a tumor antigen, wherein the tumor antigen is SEQ ID NO: 144 or 228, and wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOS: 362-365 and 368-376.

In one embodiment, the tumor antigen is SEQ ID NO: 144, and wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOS: 368-376, preferably a sequence consisting of SEQ ID NOS: 372, 374, or 375.

In another embodiment, the tumor antigen is SEQ ID NO: 228, and the compound comprises a sequence selected from the group consisting of SEQ ID NOS: 362-365, preferably a sequence consisting of SEQ ID NOS: 362, 363 or 365.

In another embodiment, the tumor antigen comprises a multiplicity of sequences selected from the group consisting of SEQ ID NOS: 362-365 and 368-376. Preferably, the tumor antigen comprises a multiplicity of sequences selected from the group consisting of SEQ ID NOS: 362-365, more preferably, a sequence consisting of SEQ ID NOS: 362, 363 or 365. Alternatively, the tumor antigen comprises a multiplicity of sequences selected from the group consisting of SEQ ID NOS: 368-376, preferably a sequence consisting of SEQ ID NOS: 372, 374, or 375.

What is described herein is a method to screen for potential superagonist APLs of a clinically relevant tumor-associated antigen, including NY-ESO-1 and MART-1. Rather than screening a limited subset of possible agonists, this technique allows screening of every single amino acid mutant of tumor epitope in a rapid and cost-effective manner. This approach to identifying APLs is effective, given the difference even subtle amino acid substitutions have on specific T cell response. Since superagonist APL structure cannot be predicted, the method described generates candidate APLS by a comprehensive screening technique. Another aspect of unpredictability is that a given agonist APL may be more or less effective for different patients. While a given agonist APL might have a high stimulatory capacity for one patient it could be relatively ineffective for another patient. Apparently, different clones are being mobilized with different agonist peptides. This heightens the need for panels of superagonist APLs for use in a therapeutic setting.

Another aspect of unpredictability is that a given agonist APL may be more or less effective for different patients. While a given agonist APL might have a high stimulatory capacity for one patient it could be relatively ineffective for another patient. Apparently, different clones are being mobilized with different agonist peptides. This heightens the need for panels of superagonist APLs for use in a therapeutic setting.

Unique antigens result from point mutations in genes that are expressed ubiquitously. The mutation usually affects the coding region of the gene and is unique to the tumor of an individual patient or restricted to very few patients. Antigens that are strictly tumor-specific may play an important role in the natural anti-tumor immune response of individual patients. These are listed in Table 1.

These epitopes are characteristic of lung carcinoma, melanoma, chronic myeloid leukemia, colorectal carcinoma, gastric carcinoma, endometrial carcinoma, head and neck squamous cell carcinoma, lung squamous cell carcinoma, renal cell carcinoma, bladder tumor, non-small cell lung carcinoma, head and neck squamous cell carcinoma, pancreatic adenocarcinoma, sarcoma, promyelocytic leukemia, myeloid leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, breast cancer, and prostate carcinoma.

Each epitope is associated with a particular HLA haplotype, either a class I or class II MHC antigen, as shown in Tables 1-4.

Shared antigens are present on many independent tumors. One group corresponds to peptides encoded by “cancer-germline” genes that are expressed in many tumors but not in normal tissues. Some are listed in Table 2.

A second group of shared tumor antigens, named differentiation antigens, are also expressed in the normal tissue of origin of the malignancy. Antigens of this group are not tumor-specific, and their use as targets for cancer immunotherapy may result in autoimmunity towards the corresponding normal tissue. Autoimmune toxicity should not be an issue, however, in situations where the tissue expressing the antigen is dispensable or even resected by the surgeon in the course of cancer therapy, as would be the case for prostate specific antigen (PSA). These antigens are listed in Table 3.

Shared antigens of the third group are expressed in a wide variety of normal tissues and overexpressed in tumors. Because a minimal amount of peptide is required for CTL recognition, a low level of expression in normal tissues may mean that autoimmune damage is not incurred. However, this threshold is difficult to define, as is the normal level of expression of those genes for each cell type. A list of these is in Table 4.

After selecting the particular tumor specific epitope, random amino acid substitutions are introduced. Oligonucleotide sequences encoding the peptide epitope are designed and cloned in an appropriate vector. Mutagenesis can be done according to the skill of the ordinary worker at each amino acid position of the peptide. The mutant may have substitutions at 1, 2, 3, 4, 5, 6 or more positions, depending on the particular epitope.

The positional libraries are designed such that the codon of interest is totally randomized (NNN), resulting in a pool of oligonucleotides which contains every given codon sequence. This mutagenesis approach might be likened to a slot machine which contains three positions (a codon) and each position has the same 4 possibilities (A, C, G, or T). When pulled, there is a 1 in 64 chance of getting any combination of 3. If pulled 100 times there is a high probability that every sequence will be represented (80% certainty, according to a Poisson distribution). Here, the 100 pulls represent 100 bacterial colonies, each containing a different mutant agonist peptide-encoding oligonucleotide. When cloned and expressed, each amino acid should be represented in a library of 100, with 80% certainty, according to a Poisson distribution. A positional library can be generated for each position (amino acid) of the target peptide. The APL minigene constructs are fused to a 6×-histidine tag (SEQ ID NO:415) and can easily be separated from bacterial proteins on Co′-coated paramagnetic beads.

The mutagenized epitopes are preferentially expressed in cells as part of an expression vector, more preferentially as a fusion protein. The preferred host for the expression vector is bacterial, e.g., a strain of E. coli. Most preferred is an inducible expression system. A mutant library is generated using the expression vector in the host cell. Preferentially, the library is distributed in liquid culture, most preferentially in 96 well plates. The cells accumulate a recombinant protein comprising the sequence of the mutagenized epitopes.

The recombinant protein is released and separated from the host cells. This can be done by lysing the cells to release the recombinant protein. Preferentially, the mutagenized epitope is separated from other cellular proteins by adding protein binding magnetic beads (e.g. 6×-histidine (SEQ ID NO:415) specific magnetic beads) to cell lysates.

Initial screens can be done by combining beads containing recombinant mutagenized epitopes with dendritic cells and epitope-specific T cells and assaying for the production appropriate cytokines, including, but not limited to, interferon γ, interleukin-4, interleukin-10, and granulocyte macrophage colony-stimulating factor. That is, APLs are screened for the ability to activate epitope-specific T cell clones following cross-presentation of the bead-bound ligand on class I or class II MHC molecules by dendritic cells (DC).

Attempts by others to measure the functional avidity of tumor epitope-specific CTL generated via unmodified peptide with CTL generated via the analogs, have been hampered by the inability to generate CD8⁺/MART-1_26-35-tetramer positive T cell populations using a peptide having the natural amino acid sequence of the epitope. Using the methods described herein, the superagonist APLs elicit different antigen-specific CTL responses from patient to patient, and that the CTL populations generated by APL stimulation are capable of effectively killing tumors. Thus, these agonist APLs might be considered “conditional” superagonist ligands. Using unique tumor epitope-specific CTL clones in the initial screen that other potential superagonist peptides can be identified. Panels of potential tumor-associated superagonist peptides may be assembled, to ensure that one or more APLs are effective at generating potent anti-tumor CTL responses from a given patient.

To determine how well the identified agonist APLs could prospectively generate tumor epitope-specific CTL populations from peripheral blood mononuclear cell (PBMC) of tumor patients, the APLs were used to stimulate different patient PBMC samples under standard in vitro conditions. Preferentially, cultures of PBMC are treated with the mutagenized epitope and incubated for at least one week. CTLs can readily be measured using ordinary methods. For example, cells can be stained with FITC-labeled anti-CD8 antibodies and APC-labeled HLA-matched complexes and analyzed by flow cytometry.

The ability of an APL to generate CD4 T cells from PBMC of tumor patients is also a measure of the efficacy of the mutagenized epitope.

It may be necessary to probe a panel of APLs since the ability of a single APL to stimulate cells of every patient having the specific tumor cannot be assumed at the outset of measurements.

One aspect of the utility of the APLs lies in their ability to stimulate T cells of a cancer patient ex vivo or in vivo. The stimulated T cells are effector and regulator CD4.sup.+cells, including Th1, Th2, Th9 and/or Th17 cells. The stimulation can involve use of the APLs as purified peptides, or as intracellular products of APL minigenes. APL minigenes may also be expressed as a string of beads, i.e., multiple CTL genes within the same expression vector, or as part of a T helper protein as described in Fomsgaard et al., 1999 Vaccine 18:681-91; Ann et al., 1997 J Virol 1192-302; Toes et al., 1997 Proc Natl Acad Sci 94:14660-65; Gao et al., 2006 Vaccine 24:5491-97, hereby incorporated by reference in their entirety.

The potential use for these novel antigenic peptides includes their use in anti-tumor vaccine studies; use in adoptive immunotherapy to generate a wider array of anti-tumor CD4.sup.+ T cell clonotypes; the ability to alter the phenotype of T regulatory cells in order to more effectively activate anti-tumor CD4.sup.+ T cells.

Oligonucleotides were designed to have a complimentary 5′ KpnI site and a complimentary 3′ PstI site. The sequences of the saturation mutagenesis sense strands of the MART-1.sub.26-35 positional oligonucleotides are shown in Table 5 (each sense strand has a corresponding mutant antisense strand):

NNN represents totally randomized codons, any one of sixty-four codons. In a given positional library consisting of 100 mutant oligonucleotide pairings, each codon has high likelihood of being represented.

Variant polypeptide sequences are listed in Table 6.

Similarly, nucleotides encoding variant sequences of NY-ESO-1_157-170(SEQ ID NO:144) were synthesized that encoded the following sequences (Table 7).

Synthetic polypeptides having these sequences were suspended in DMSO.

The saturation mutagenesis oligonucleotides were cloned into the expression vector pQE40 (Qiagen). The plasmids were transformed into E. coli (M15 pREP). Mini-gene products were expressed as fusion proteins containing 6×-histidine tags (SEQ ID NO:415). Following recombinant protein induction, bacteria were lysed with 8M Urea, pH 8.0. Lysate was harvested and applied to Mg²⁺ coated paramagnetic beads (Talon beads, Dynal), which bind specifically to 6×-histidine (SEQ ID NO:415).

For saturation mutagenesis libraries, bacterial clones were cultured individually in wells of 96-well plates.

Melanoma cell lines A375 and MeI 526, CTL clones and the TAP-deficient cell line T2 were maintained in RPMI 1640, containing 25 mM HEPES, 2 mM L-glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin and 10% human serum from normal donors. Dendritic cells were prepared from adherent monocytes, isolated from the PBMC of HLA-A2⁺ healthy donors. IL-4 (500 U/mL; R&D Systems, Minneapolis, Minn.) and GM-CSF (800 U/mL; Amgen, Thousand Oaks, Calif.) were added to the monocytes to promote their differentiation into dendritic cells. MART-1_26-35-specific CTL clones were generated as described by Li et al., 2005. J Immunol 175:2261-69, hereby incorporated by reference in its entirety. PBMC used in this study were obtained from HLA-A2⁺ melanoma patients.

Saturation Mutagenesis APL Screen

Following the isolation of the recombinant mini-gene APL products on Talon beads, the bead-bound products were “fed” to 100,000 immature dendritic cells. Following a 4-hour incubation at 37° C., 100,000 MART-1_26-35-specific CTL clones were added to DC/bead preparations. Following a 12-hour incubation at 37° C., the supernatant was harvested and assayed for the concentration of IFN-γ induced by the APL clones. Anti-IFN-γ antibodies (Endogen) used in the sandwich ELISA were used at 1 μg/ml in PBS/0.1% BSA.

Variant MART-1_26-35agonist peptides identified using mutagenesis APL screening and their corresponding DNA sequences are shown in Table 8.

In Vitro PBMC Stimulations with Analog Peptides and Tetramer Staining

On day 0, monocyte-derived dendritic cells were pulsed with 1 μM of each MART-1_26-35analog peptide for 2 hours at 37° C. The DCs were washed and added to 500,000 HLA-A2⁺ PBMC from melanoma patients at a 1:20 ratio in 24-well plates. On day 2, 12.5 U/ml of IL-2, 5 ng/ml IL-7, 1 ng/ml IL-15, and 10 ng/ml of IL-21 were added to each culture. Cytokines were replenished every 2-3 days for 1-week. Following the 1-week primary stimulation, cultures were re-stimulated with 1×10⁶irradiated monocytes pulsed with 10 μM of the peptide used in the primary stimulation. IL-2, IL-7 and IL-15 were added to secondary stimulations on day 2. Cytokines were replenished every 2-3 days. 500,000 cells from each culture were stained with APC-labeled anti-CD8 antibody (Caltag Lab, Burlingame, Calif.) and PE-labeled MART-1_26-35HLA-A2.1 tetramers. Stained cells were analyzed using FACSCALIBUR™ flow cytometer and CELLQUEST™ (BD PharMingen) and analyzed using FlowJo software v8.5 (Tree Star, San Carlos, Calif.). Cells were stained with tetramers in 25 μl of 2% FCS/BSA for 1 hour at room temperature, followed by anti-CD8 antibody for 15 minutes at 4° C.

Generation of MART-1_26-35Polyclonal Cell Lines

Following in vitro peptide stimulation of HLA-A2⁺ PBMC from melanoma patients MelPt-B, MelPt-C, MelPt-D, MelPt-F and a healthy donor (Healthy-1) MART-1_26-35tetramer and CD8 positive cells were sorted and isolated on BD FACSaria. Isolated cells were replicated using 30 ng/ml anti-CD3 antibody (OKT3) and IL-2 at 50 U/ml in the presence of irradiated feeder PBMC and LCL for 2 weeks. IL-2 was replenished every 2-3 days. Following the stimulation, cultures were stained for the generation of MART-1_26-35tetramer and CD8 positive cell populations. The polyclonal cell lines were tested for lytic activity and TCR VP usage (MelPt-C only), as described in Example 6.

In Vitro Cytotoxicity Assay

Target cells were labeled with 100 μCi of⁵¹Cr and co-cultured with effector cells for 4 hours at 37° C. plus 5% CO₂. Targets were melanoma cell lines A375 (HLA-A2⁺/NY-ESO-1⁺) and Mel 526 (HLA-A2⁺/MART-1+), and T2 cells pulsed with 1 μM of MART-1_26-35(positive control) or NY-ESO-1₁₅₇-165 (negative control). Effector cells were MART-1_26-35-tetramer positive polyclonal cell lines generated with either A27L, E26S, or L33M peptides (SEQ ID NOS:362, 364, and 365, respectively). Assays were performed in triplicate at a 50:1, 25:1 or 12.5:1 effector to target ratio. Released⁵¹Cr was measured with a gamma scintillation counter and percent specific lysis was determined by using the formula: percent specific release=(experimental release-spontaneous release)/(maximum release-spontaneous release).

TCR Spectratype Analysis

TCR VP spectratype analysis was carried out by the Immune Monitoring Laboratory at Fred Hutchinson Cancer Research Center. Briefly, cDNA was generated from 1×10⁶MART-1_26-35tetramer staining polyclonal cell lines. Multiplex Vβ PCR primers were then used to amplify the variable regions of the complementarity-determining region 3 (CDR3) of the TCR β chain. Sequence analysis to determine the VP usage of the TCRs was conducted with GenScan.

Mart-1_26-35Specific CTL Clones can Detect Enhanced CTL Epitopes as Reflected by IFN-γ Expression

To identify superagonist APLs in this study, we utilize a novel genetic system. This system employs saturation mutagenesis of agonist peptide-encoding oligonucleotides, which when expressed in E. coli will contain position specific single amino acid substitutions. The positional libraries are designed such that the codon of interest is totally randomized (NNN), resulting in a pool of oligonucleotides which contains every given codon sequence. This mutagenesis approach might be likened to a slot machine which contains three positions (a codon) and each position has the same 4 possibilities (A, C, G or T). When pulled, there is a 1 in 64 chance of getting any combination of 3. If pulled 100 times there is a high probability that every sequence will be represented (80% certainty, according to a Poisson distribution). Here, the 100 pulls represent 100 bacterial colonies, each containing a different mutant agonist peptide-encoding oligonucleotide. When cloned and expressed, each amino acid should be represented in a library of 100, with 80% certainty, according to a Poisson distribution. A positional library can be generated for each position (amino acid) of the target peptide. The APL min-gene constructs are fused to a 6×-histidine tag (SEQ ID NO:415) and can easily be separated from bacterial proteins on Co²⁺-coated paramagnetic beads. APLs are screened for the ability to activate epitope-specific CTL clones following cross-presentation of the bead-bound ligand on class I MHC molecules by immature dendritic cells (DC).

To validate this system and to verify that it was sensitive enough to detect our model tumor-associated HLA-A2 restricted antigenic peptide, MART-1_26-35, as well as an APL superagonist epitope of MART-1_26-35, called MART-1_26-35A27L (henceforward referred to as A27L (SEQ ID NO:362)), oligonucleotides encoding the appropriate peptide sequences were cloned, expressed and assayed for the ability to activate antigen specific CTL clones as described in materials and methods. The CTL clone used in this assay, called M26-H1, is specific for MART-1_26-35, and expresses IFN-γ in response to HLA-A2/MART-1_26-35complexes. Here, the IFN-γ response elicited by the recombinant unmodified MART-1_26-35cross-presented construct is significantly higher than that elicited by the HLA-A2 restricted negative control, NYESO-1_157-165(FIG. 1). Further, the IFN-γ response elicited by the recombinant superagonist APL, A27L, was more than 2-fold higher than that elicited by the recombinant wild type construct. Yet, the activation of M26-H1 by the unmodified MART-1_26-35construct was clearly distinguishable from that elicited by the HLA-A2 restricted negative control construct, NYESO1_157-165. These results suggest that the HLA-A2 cross-presented recombinant ligands are sufficient to elicit detectable antigen-specific responses from CTL clones, and also that superagonist APLs can be distinguished based on an increase in IFN-γ expression, relative to the wild type CTL ligand.

Saturation Mutagenesis can Effectively Generate Random Amino Acids in the Parental Antigenic Peptide from which Enhanced Agonist APLs can be Identified

The saturation mutagenesis APL library screen depends on 200 μl bacterial expression cultures in 96-well plates. FIG. 1 shows that cross-presented recombinant ligands can be detected by antigen-specific CTL. However, in that experiment recombinant proteins were produced at high concentrations in 5 ml cultures. To determine whether the recombinant protein produced in these significantly smaller cultures would be sufficient to reflect detectable and varying degrees of activation, a position 2 (P2) library of MART-1_26-35(EXAGIGILTV (SEQ ID NO:416)) was constructed. By screening this library, in addition to determining if 200 μl cultures produce sufficient concentrations of recombinant protein previously identified superagonist APLs, including A27L could be identified from among 88 unique mutant APL clones. The P2 library screen (FIG. 2), using the CTL clone M26-H1, clearly shows that the wild type recombinant ligand MART-1_26-35elicits significantly more IFN-γ than the negative control. Furthermore, the APL clones from the library that contained leucine residues at P2 (A27L), elicited significantly more IFN-γ expression in comparison to the wild type ligand. Amino acid content was determined from replicated glycerol stock of the P2 bacterial library. Interestingly, APL clones containing methionine residues at P2 also elicited greater IFN-γ expression than wild type MART-1_26-35, although not as great as that elicited by the leucine containing APLs, A27L. Like A27L, A27M is a superagonist APL of MART-1_26-35. Thus, 200 μl bacterial cultures produce sufficient concentrations of the recombinant ligands to be detected in this screen. Also, superagonist APLs can be identified in a library of at least 88 unique APL clones.

Putative Enhanced CTL Epitopes of Mart-126-35A27L are Identified in APL Library Screens

On the basis of previous results demonstrating that superagonist APLs can be uncovered using the saturation mutagenesis screen, remaining positional libraries of MART-1_26-35, (with the exception of P10, which already contains an anchor residue that conforms to the HLA-A2 C-terminal consensus binding motif) were screened using similar methods. Because a potent superagonist APL of MART-1_26-35has already been identified in A27L, A27L was used as the basis for a mutational strategy. That is, leucine in position 2 was constant, while other positions were mutated independently. This would allow superagonist APLs to be identified that are more effective than A27L.

The APL libraries were screened with two different high avidity MART-1_26-35-specific CTL clones. A high avidity TCR is defined as having the ability to recognize tumor cells that express both MART-1 and HLA-A2 class I molecules. The vast majority of the MART-1_26-35derivative mutant peptide clones screened from each of the positional libraries were not as effective as A27L at activating the MART-1_26-35-specific CTL clone (FIG. 3A and FIG. 3B). However, several clones from the P1, P3 and P8 libraries appeared to work similarly as well as the A27L recombinant construct. The initial screen was conducted by screening two unique APL library clones simultaneously in a single well. While this approach allows twice as many APL clones to be screened, the potency of any agonist APL in the pool is potentially underestimated in the initial screen.

Agonist candidates were selected and re-screened based on their ability to elicit more or comparable levels of IFN-γ from M26-H1 in the initial screen (FIG. 3B). When tested independently, both of the clones from the P3 libraries elicited less IFN-γ expression from the MART-1_26-35-specific CTL clone, relative to A27L. When re-screened independently, it was apparent that only one of the two mutant peptide clones from the P1 and P8 wells was responsible for the increased IFN-γ expression. The DNA encoding these putative MART-1_26-35agonist peptides was prepared from the duplicated bacterial glycerol stocks. The enhancing mutations for the P1 putative agonists contained either glycine (E26G) (SEQ ID NO:363) or serine (E26S) (SEQ ID NO:364) residues at P1 instead of the naturally occurring glutamate residue. The P8 putative agonist contained a methionine residue (L33M) (SEQ ID NO:365) at position 8 rather than the naturally occurring leucine residue. No additional putative agonists were identified from the library screens using the second CTL clone, M26-H2.

MART-1_26-35Agonist Peptides Display a Differential Capacity to Activate Different MART-1_26-35-Specific CTL Clones

To analyze the putative superagonist APLs on a molar basis, individual peptides were synthesized at greater than 90% purity. To determine whether these APLs would be similarly recognized by unique MART-1_26-35-specific CTL clones, the APLs were tested against four clones bearing unique T cell receptors (TCR). These included two high avidity CTL clones (M26-H1 and M26-H2) and two low avidity CTL clones (M26-L1 and M26-L2) (FIG. 4). Low-avidity TCR is here defined as having the ability to respond HLA-A2 positive peptide-pulsed target cells but not to cells displaying naturally processed and presented determinants from HLA-A2/MART-1 positive tumors. Low-avidity T cells have the potential to mediate antigen-specific cell and tissue destruction.

FIG. 4 panel A shows that each of the newly identified agonist peptides is similarly effective in activating M26-H1—the high-avidity CTL clone used in the initial screen (FIG. 3A and FIG. 3B) as compared to MART-1_26-35superagonist peptide, A27L. A similar pattern of activation was found when the identified agonist peptides are used to stimulate the CTL clone M26-H2. In contrast to the above results, the low-avidity MART-1_26-35-specific CTL clones yielded widely divergent results in response to different agonist peptides. For example, while the CTL clone M26-L1 recognizes the peptide E26S more than 100-fold better than A27L (based on half-maximal activation), the CTL clone M26-L2 recognizes A27L better than it does E26S. Similarly, while L33M is scarcely recognized by the CTL clone M26-L1, it is the most effective agonist for activating M26-L2. Thus, these analogs might be considered “conditional” agonists, as they do not elicit generalized patterns of activation among unique antigen-specific clonotypes.

MART-1_26-35APLs Demonstrate Patient-Specific Enhanced Generation of MART-1_26-35CTL Populations from the PBMC of Melanoma Patient Donors

To determine how well the identified agonist APLs could prospectively generate MART-1_26-35-specific CTL populations from melanoma patient peripheral blood mononuclear cell (PBMC) preparations, the APLs were used to stimulate eight different patient PBMC samples under standard in vitro conditions (Table 9).

These results show that MART-1_26-35APLs exhibit differential capacities to generate MART-1_26-35-specific CTL populations from the PBMC of different melanoma patient donors. APLs were used to stimulate PBMC cultures in vitro. Following a one-week secondary stimulation cells were stained with FITC-labeled anti-CD8 antibodies and APC-labeled HLA-A2/MART-1_26-35tetramers and analyzed by flow cytometry. Values are given as percent tetramer positive relative to a negative control. The fold difference relative to A27L is indicated in parentheses. Differences of more than two-fold are indicated in bold.

One week following the second in vitro stimulation, cultures were stained with the wild-type MART-1_26-35/HLA-A2 tetramer. Similar to the observations made using different MART-1_26-35-specific CTL clones, none of the peptide ligands were universally effective in generating MART-1_26-35-specific CTL populations from all patient PBMC samples (FIG. 5). Any given APL was more or less effective in generating antigen-specific CTL from any given patient PBMC sample. For example, while the agonist peptide E26S is the least effective at generating MART-1_26-35-specific CD8 positive populations from the PBMC of MelPt-C (3-fold <A27L), it is the most effective APL for generating such T cell populations from MelPt-D (5-fold >A27L). Similarly, whereas the agonist peptide L33M is 14-fold more effective than A27L in generating of MART-1_26-35-specific CD8 positive populations from the PBMC of MelPt-E, it is 14-fold less effective than A27L in generating MART-1_26-35-specific CD8 populations from the PBMC of MelPt-G. These findings demonstrate that any one CTL ligand may not be effective at generating antigen-specific CTL populations from the PBMC of any given patient; and suggest the importance of establishing a panel of potential superagonist APLs.

CD8 Positive MART-1_26-35-Specific Polyclonal Cell Lines Generated with the Identified MART-1_26-35Agonist APLs can kill HLA-A2⁺ Tumors Expressing Endogenous MART-1

The use of altered peptide ligands poses the risk of generating antigen-specific T cells which display relatively low anti-tumor functional avidity. To determine whether the MART-1_26-35-specific CTL that were generated with these novel MART-1_26-35agonist peptides were of sufficient functional avidity to kill HLA-A2/MART-1 positive tumor targets, polyclonal lines of CD8 positive MART-1_26-35tetramer-staining cells were established from the PBMC of MelPt-B, MelPt-C, MelPt-D, MelPt-F or a healthy donor (Healthy 1), stimulated with either A27L, E26S or L33M agonist peptides (SEQ ID NOS:362, 364, and 365, respectively). These cell lines were screened for reactivity to unmodified MART-1_26-35peptide pulsed HLA-A2 positive targets and to HLA-A2/MART-1 positive tumor targets at varying effector to target ratios in a standard chromium release assay (Table 10).

The results illustrate that the CTL populations that were generated from each PBMC source with either of the altered peptide ligands can kill targets that display wild-type MART-1_26-35in the context of HLA-A2 and recognize the epitope with sufficient affinity to kill tumors expressing MART-1.

To determine whether unique or shared MART-1_26-35-specific CTL clonotypes were generated with each of the peptide ligands (A27L, E26S and L33M), spectratype analysis was performed on CTL lines derived from MelPt-C PBMC to determine their VP TCR usage. Results showed that the agonist peptides A27L, E26S and L33M generated CTL populations that primarily (>90%) utilized TCR Vβ24, Vβ8 and Vβ3, respectively. This suggests that the different analog peptides preferentially generate specific TCR utilizing CTL subsets. Taken together, these results demonstrate the ability of the identified APLs to elicit MART-1_26-35-specific CTL responses that are capable of directly killing MART-1 expressing tumors and suggest that unique MART-1_26-35-specific TCR subpopulations are being preferentially generated by the different MART-1_26-35analog peptides.

NY-ESO APLs

The methods of Examples 2-8 were used to generate enhanced agonist APLs. Results of a library screen are shown in FIG. 6. Clones showing activity were sequenced. Variant sequences with the most activity correspond to amino acid sequences of SEQ ID NOS:368-376.

Using the methods of Example 11 to analyze the putative superagonist APLs on a molar basis, individual peptides were synthesized at greater than 90% purity. To determine whether these APLs would be similarly recognized by unique NY-ESO-II-specific CTL clones, the APLs were tested against ten clones bearing unique TCR. FIG. 7 shows that each of the newly identified agonist peptides is similarly effective in activating CTL clones used in the initial screen in comparison to wild-type NY-ESO-II_157-170superagonist peptide and that different patterns of stimulation are obtained with different CTL clones. Specific CTL clones yielded widely divergent results in response to different agonist peptides. Similar to results obtained with MART superagonist peptides, these NY-ESO-II analogs might be considered “conditional” agonists, as they do not elicit generalized patterns of activation among unique antigen-specific clonotypes.

The NY-ESO-1_157-165C165V APL SEQ ID NO:376 was compared to wild-type NY-ESO-I_157-165SEQ ID NO:366 in effectively producing CTL from PBMC. FIG. 7 shows that the variant peptide had a higher avidity than the wild type sequence to a CD-8⁺ population.

FIG. 8A and FIG. 8B show the ability of several NY-ESO-1APL to stimulate CD-4⁺ fractions and PBMC, respectively. Results showed that NY-ESO-1APLs I162Q, Q164S, and F170W (SEQ ID NOS:372, 374, and 375, respectively) were the most effective in stimulating CD-4+ cells.

NY-ESO-1_157-170agonist peptides identified using mutagenesis APL screen and their corresponding DNA sequences are shown in the following Table 11.