Search

Production of clinical-grade gammaretroviral vectors for ex vivo gene delivery requires a scalable process that can rapidly generate large amounts of vector supernatant, clear large numbers of residual packaging cells with minimal decreases in vector titer, and satisfy all current regulatory guidelines regarding product biosafety. To that end, we have developed a simplified method that is compliant with current good manufacturing practices for the production of clinical-grade gammaretroviral vectors in a clinical research environment. We validated a large-scale production platform utilizing 1,700-cm(2) expanded surface roller bottles and a "modified" step-filtration process consisting of a 40/150-μm dual-screen filter for aggregate removal followed by a Sepacell 500II leukocyte reduction filter for removal of residual packaging cells. This clarification process can clear at least 2 × 10(9) viable producer cells using a single filter set-up without any significant loss of titer post-filtration. This platform typically generates 18 liters of vector supernatant to support small-scale clinical trials, but can easily be scaled up to 70 liters during a single manufacturing run. To date, this platform has generated five clinical-grade gammaretroviral vector products, four of which are now being used in adoptive cell therapy clinical trials for the treatment of a variety of solid cancers.

Purpose: Carcinoembryonic antigen (CEA) is a tumor-associated protein expressed on a variety of adenocarcinomas. To develop an immunotherapy for patients with cancers that overexpress CEA, we isolated and genetically modified a T-cell receptors (TCRs) that specifically bound a CEA peptide on human cancer cells. Experimental Design: HLA-A2.1 transgenic mice were immunized with CEA:691-699. A CEA-reactive TCR was isolated from splenocytes of these mice and was genetically introduced into human peripheral blood lymphocytes via RNA electroporation or retroviral transduction. Amino acid substitutions were introduced throughout the complementarity determining regions (CDR1, CDR2, and CDR3) of both TCR α and β chains to improve recognition of CEA. Results: Murine lymphocytes bearing the CEA-reactive TCR specifically recognized peptide-loaded T2 cells and HLA-A2.1+ CEA+ human colon cancer cells. Both CD8+ and CD4+ human lymphocytes expressing the murine TCR specifically recognized peptide-loaded T2 cells. However, only gene-modified CD8+ lymphocytes specifically recognized HLA-A2.1+ CEA+ colon cancer cell lines, and tumor cell recognition was weak and variable. We identified two substitutions in the CDR3 of the α chain that significantly influenced tumor cell recognition by human peripheral blood lymphocytes. One substitution, T for S at position 112 (S112T), enhanced tumor cell recognition by CD8+ lymphocytes, and a second dually substituted receptor (S112T L110F) enhanced tumor cell recognition by CD4+ T cells. Conclusions: The modified CEA-reactive TCRs are good candidates for future gene therapy clinical trials and show the power of selected amino acid substitutions in the antigen-binding regions of the TCR to enhance desired reactivities.

Adoptive cell transfer (ACT) of tumor-reactive lymphocytes has been shown to be an effective treatment for cancer patients. Studies in murine models of ACT indicated that antitumor efficacy of adoptively transferred T cells is dependent on the differentiation status of the cells, with lymphocyte differentiation inversely correlated with in vivo antitumor effectiveness. T-cell in vitro development technologies provide a new opportunity to generate naive T cells for the purpose of ACT. In this study, we genetically modified human umbilical cord blood–derived hematopoietic stem cells (HSCs) to express tumor antigen-specific T-cell receptor (TCR) genes and generated T lymphocytes by coculture with a murine cell line expressing Notch-1 ligand, Delta-like-1 (OP9-DL1). Input HSCs were differentiated into T cells as evidenced by the expression of T-cell markers, such as CD7, CD1a, CD4, CD8, and CD3, and by detection of TCR excision circles. We found that such in vitro differentiated T cells expressed the TCR and showed HLA-A2–restricted, specific recognition and killing of tumor antigen peptide–pulsed antigen-presenting cells but manifested additional natural killer cell–like killing of tumor cell lines. The genetic manipulation of HSCs has broad implications for ACT of cancer. [Cancer Res 2007;67(6):2425–9]

Neoantigens unique to each patient's tumor can be recognized by autologous T cells through their T-cell receptor (TCR) but the low frequency and/or terminal differentiation of mutation-specific T cells in tumors can limit their utility as adoptive T-cell therapies. Transfer of TCR genes into younger T cells from peripheral blood with a high proliferative potential could obviate this problem. We generated a rapid, cost-effective strategy to genetically engineer cancer patient T cells with TCRs using the clinical Sleeping Beauty transposon/transposase system. Patient-specific TCRs reactive against HLA-A*0201-restriced neoantigens AHNAK(S2580F) or ERBB2(H473Y) or the HLA-DQB*0601-restricted neoantigen ERBB2IP(E805G) were assembled with murine constant chains and cloned into Sleeping Beauty transposons. Patient peripheral blood lymphocytes were coelectroporated with SB11 transposase and Sleeping Beauty transposon, and transposed T cells were enriched by sorting on murine TCRβ (mTCRβ) expression. Rapid expansion of mTCRβ(+) T cells with irradiated allogeneic peripheral blood lymphocytes feeders, OKT3, interleukin-2 (IL-2), IL-15, and IL-21 resulted in a preponderance of effector (CD27(-)CD45RA(-)) and less-differentiated (CD27(+)CD45RA(+)) T cells. Transposed T cells specifically mounted a polyfunctional response against cognate mutated neoantigens and tumor cell lines. Thus, Sleeping Beauty transposition of mutation-specific TCRs can facilitate the use of personalized T-cell therapy targeting unique neoantigens.

Detection of lymphocytes that target tumor-specific mutant neoantigens—derived from products encoded by mutated genes in the tumor—is mostly limited to tumor-resident lymphocytes(1,2), but whether these lymphocytes often occur in the circulation is unclear. We recently reported that intratumoral expression of the programmed cell death 1 (PD-1) receptor can guide the identification of the patient-specific repertoire of tumor-reactive CD8(+) lymphocytes that reside in the tumor3. In view of these findings, we investigated whether PD-1 expression on peripheral blood lymphocytes could be used as a biomarker to detect T cells that target neoantigens. By using a high-throughput personalized screening approach, we identified neoantigen-specific lymphocytes in the peripheral blood of three of four melanoma patients. Despite their low frequency in the circulation, we found that CD8(+)PD-1(+), but not CD8(+)PD-1(−), cell populations had lymphocytes that targeted 3, 3 and 1 unique, patient-specific neoantigens, respectively. We show that neoantigen-specific T cells and gene-engineered lymphocytes expressing neoantigen-specific T cell receptors (TCRs) isolated from peripheral blood recognized autologous tumors. Notably, the tumor-antigen specificities and TCR repertoires of the circulating and tumor-infiltrating CD8(+)PD-1(+) cells appeared similar, implying that the circulating CD8(+)PD-1(+) lymphocytes could provide a window into the tumor-resident antitumor lymphocytes. Thus, expression of PD-1 identifies a diverse and patient-specific antitumor T cell response in peripheral blood, providing a novel noninvasive strategy to develop personalized therapies using neoantigen-reactive lymphocytes or TCRs to treat cancer.

Genetic modification of dendritic cells (DCs) with recombinant vectors encoding tumor antigens may aid in developing new immunotherapeutic treatments for patients with cancer. Here, we characterized antigen presentation by human DCs genetically modified with plasmid cDNAs, RNAs, adenoviruses, or retroviruses, encoding the melanoma antigen gp100 or the tumor-testis antigen NY-ESO-1. Monocyte-derived DCs were electroporated with cDNAs or RNAs, or transduced with adenoviruses. CD34+ hematopoietic stem cell-derived DCs were used for retroviral transduction. Genetically modified DCs were coincubated with CD8+ and CD4+ T cells that recognized major histocompatibility complex class I- and class II-restricted epitopes from gp100 and NY-ESO-1, and specific recognition was evaluated by interferongamma secretion. Cytokine release by both CD8+ and CD4+ T cells was consistently higher in response to DCs modified with adenoviruses than cDNAs or RNAs, and maturation of DCs after genetic modification did not consistently alter patterns of recognition. Also, retrovirally transduced DCs encoding gp100 were well recognized by both CD8+ and CD4+ T cells. These data suggest that DCs transduced with viral vectors may be more efficient than DCs transfected with cDNAs or RNAs for the induction of tumor reactive CD8+ and CD4+ T cells in vitro and in human vaccination trials.

Purpose: Telomerase is an attractive target antigen for cancer immunotherapies because it is expressed in >85% of human tumors but is rarely found in normal tissues. A HLA-A*0201-restricted T-cell epitope was previously identified within telomerase reverse transcriptase hTERT:540-548. This peptide was reported to induce CTL that recognized tumor cells and transfectants that endogenously expressed telomerase. Therefore, we initiated a clinical protocol to evaluate the therapeutic and immunological efficacy of this peptide.Experimental Design: Fourteen patients with metastatic cancers were vaccinated with hTERT:540-548 emulsified in incomplete Freund’s adjuvant.Results: In 7 patients, peripheral blood mononuclear cells collected after immunization recognized hTERT:540-548, whereas those collected before vaccination did not. However, none of these CTLs recognized tumors that endogenously expressed telomerase, and none of the patients had an objective clinical response. Several highly avid T-cell clones were generated that recognized T2 cells pulsed with ≤1 nm hTERT:540-548, but none of these recognized HLA-A*0201+ hTERT+ tumors or cells transduced with the human telomerase reverse transcriptase (hTERT) gene. Also, an antibody specific for hTERT:540-548/HLA-A*0201 complexes stained peptide-pulsed cells but not telomerase+ tumors.Conclusions: Our results are discordant with previous studies and those of a clinical trial that claimed peripheral blood mononuclear cells from patients vaccinated with peptide-pulsed dendritic cells lysed hTERT+ tumors. However, our findings are consistent with a previous study that demonstrated that the hTERT:540-548 peptide is cleaved in the proteasome. These results suggest that hTERT:540-548 is not presented on the surfaces of tumor cells in the context of HLA-A*0201 and will not be useful for the immunotherapy of patients with cancer.

CD4+ T helper cells may play a critical role in the induction and maintenance of a therapeutic immune response to cancer. To evaluate the efficacy with which a recombinant tumor-associated protein can induce antigen-reactive CD4+ T cells, we stimulated peripheral blood lymphocytes from patients with melanoma in vitro with the purified melanoma antigen gp100 produced in Escherichia coli. In preliminary experiments, we observed that peripheral blood mononuclear cells could process and present known HLA-DRbeta1*0401 and HLA-DRbeta1*0701 restricted epitopes to gp100-reactive CD4+ T cell lines after being loaded exogenously with protein. Therefore, we used autologous protein-loaded peripheral blood mononuclear cells as antigen presenting cells. From four of nine patients who expressed both HLA-DRbeta1*0401 and HLA-DRbeta1*0701, we raised five gp100-reactive CD4+ T cell populations that secreted TH1 type cytokines in response to exogenously loaded protein as well as target cells that endogenously expressed gp100 and MHC class II molecules, including transfectants and melanoma cells. Four of the five cultures specifically recognized the known HLA-DRbeta1*0401 and HLA-DRbeta1*0701 restricted epitopes gp100:44-59 and gp100:170-190, respectively. The fifth culture, and 30 T cell clones derived from it, specifically recognized a new peptide, gp100:420-435, in the context of HLA-DRbeta1*0701. These results suggest that recombinant tumor-associated proteins may be clinically applicable for the generation of CD4+ T helper cells in active vaccination strategies or adoptive cellular immunotherapies.

The use of reverse immunology may be necessary to identify new tumor-associated antigens, particularly for cancers, against which tumor-reactive T cell populations have been difficult to establish. One approach has been to screen peptides derived from a candidate antigen with high major histocompatibility complex (MHC) binding affinities for the induction of tumor-reactive T lymphocytes in vitro. However, many candidate antigens that are overexpressed in tumors are nonmutated self-proteins, and unlike foreign or mutated proteins, immunodominant epitopes may not be expressed at high density on the surface of tumor cells. Therefore, to identify tumor-associated epitopes, it may be necessary to screen large panels of peptides with wide ranges of MHC binding affinities. The current methodology of stimulating peripheral blood lymphocytes (PBL) from donors expressing the MHC molecule of interest with individual peptides is impractical for screening such large panels. Therefore, we evaluated the use of mixtures of peptides with variable MHC binding affinities for the induction of tumor-reactive T lymphocytes with the melanoma antigens gp100 and an alternate isoform of tyrosinase-related protein 2 (TRP2-6b) as models. A mixture of 10 known human leukocyte antigen (HLA)-A*0201-restricted peptides from gp100 induced melanoma-reactive cytotoxic T lymphoycte (CTL) from multiple patients with metastatic melanoma. The majority of these T cell populations recognized the known immunodominant epitopes gp100:209-217 and gp100:280-288, even though the HLA-A*0201 binding affinities of these peptides were much lower than other peptides in the mixture. Similarly, melanoma-reactive CTL were generated with a mixture of HLA-A*0201-restricted peptides from TRP2-6b, and these responses were directed against the previously identified tumor-associated epitopes TRP2-6b:180-188, TRP2-6b:288-296 and TRP2-6b:403-411. These results suggest that the use of peptide mixtures may facilitate the identification of new tumor-associated antigens through the application of reverse immunology.

Multiple human cancer Ags have been identified, although little is known concerning which would be most effectively used in cancer immunotherapy. To gain insight into the selection of appropriate Ags, the immunologic reactivity of a patient who had a durable complete regression of melanoma metastases was measured. PBMCs were directly cloned using the monoclonal anti-CD3 Ab OKT3 and IL-2 without any bias introduced by previous culture. A lymphocyte clone recognized a previously unknown shared melanoma Ag that was identified as the BING-4 protein encoded in a gene-rich region of the extended class II MHC. The HLA-A2-restricted BING-4 immunodominant peptide was translated from a 10-aa-long alternative open reading frame. In vitro sensitization against this peptide generated lymphocytes reactive against HLA-A2+ melanomas. Real-time semiquantitative RT-PCR analysis revealed that 8 of 15 melanoma cell lines overexpressed BING-4, and this correlated with recognition by lymphocytes. Overexpression was not found in normal tissues or other tumor types. Thus, BING-4 represents another candidate Ag for possible use in the immunotherapy of patients with melanoma.

Meeting abstracts The adoptive transfer of tumor infiltrating lymphocytes (TIL) can mediate the regression of metastatic melanoma [[1][1]]. In addition, the adoptive transfer of lymphocytes expressing T cell receptors (TCRs) specifically reactive with antigens expressed on melanoma cells can

T Cells for Epithelial Tumors Malignant tumors harbor genetic alterations. Recently, adoptive T cell therapies have taken advantage of this: T cells specific for mutations in tumors are infused into patients to generate an antitumor immune response. Although therapeutic benefit has been seen for melanomas, effectiveness against more common epithelial tumors is unclear. Using whole-exome sequencing, Tran et al. (p. 641 ) identified tumor-infiltrating CD4+ T cells specific for a mutated antigen expressed by a tumor from a patient with metastatic cholangiocarcinoma. Infusion of this patient with an expanded-population, mutation-specific T cell resulted in tumor regression and stabilization of disease.

Cancer testis antigens, such as NY-ESO-1, are expressed in a variety of prevalent tumors and represent potential targets for T-cell receptor (TCR) gene therapy. DNA encoding a murine anti-NY-ESO-1 TCR gene (mTCR) was isolated from immunized HLA-A*0201 transgenic mice and inserted into a γ-retroviral vector. Two mTCR vectors were produced and used to transduce human PBL. Transduced cells were cocultured with tumor target cell lines and T2 cells pulsed with the NY-ESO-1 peptide, and assayed for cytokine release and cell lysis activity. The most active TCR construct was selected for production of a master cell bank for clinical use. mTCR-transduced PBL maintained TCR expression in short-term and long-term culture, ranging from 50% to 90% efficiency 7-11 days after stimulation and 46%-82% 10-20 days after restimulation. High levels of interferon-γ secretion were observed (1000-12000 pg/mL), in tumor coculture assays and recognition of peptide-pulsed cells was observed at 0.1 ng/mL, suggesting that the new mTCR had high avidity for antigen recognition. mTCR-transduced T cells also specifically lysed human tumor targets. In all assays, the mTCR was equivalent or better than the comparable human TCR. As the functional activity of TCR-transduced cells may be affected by the formation of mixed dimers, mTCRs, which are less likely to form mixed dimers with endogenous hTCRs, may be more effective in vivo. This new mTCR targeted to NY-ESO-1 represents a novel potential therapeutic option for adoptive cell-transfer therapy for a variety of malignancies.

Tyrosinase has many advantages as a target antigen for the immunotherapy of patients with melanoma because it is expressed in nearly all melanoma specimens with a high degree of cellular homogeneity, and its distribution in normal tissues is limited to melanocytes. To broaden our ability to direct cellular immune responses against this protein, we pursued an investigation to identify new shared human leukocyte antigen (HLA)-A2.1 restricted epitopes from tyrosinase. Peptides were synthesized that fit a permissive HLA-A2.1 binding motif and did not span common sites of polymorphism. The binding affinity of each peptide to HLA-A2.1 relative to a standard peptide with intermediate binding affinity was evaluated in a competitive inhibition assay. Twelve peptides were selected that had binding affinities within 80% of that of the standard peptide, and these were used to stimulate peripheral blood mononuclear cells (PBMC) in vitro from three HLA-A2.1+ patients with metastatic melanoma. Cytotoxic T lymphocytes that specifically recognized peptide-pulsed target cells as well as HLA-A2.1+ tyrosinase+ melanoma cells were raised from one patient with tyrosinase:8-17 (CLLWSFQTSA). To evaluate further the immunogenicity of this peptide, PBMC from 23 HLA-A2.1+ patients were stimulated in vitro with tyrosinase:8-17. Eleven bulk T-cell cultures demonstrated specific peptide recognition, and six of these also recognized HLA-A2.1+ tyrosinase+ melanoma cells. These data suggest that tyrosinase:8-17 may be clinically useful for the treatment of patients with melanoma.

NY-ESO-1 is an attractive candidate tumor antigen for the development of immunotherapy for a wide variety of cancers. It is expressed in multiple types of tumors, but its normal tissue distribution is predominantly limited to the testes and ovaries; furthermore, both humoral and cellular immune responses can be mounted against this protein. Three overlapping HLA-A2.1-restricted T-cell epitopes have been identified within NY-ESO-1. In this investigation, the authors evaluated the in vitro immunogenicity of these peptides. From 2 of 12 HLA-A2.1+ patients with metastatic melanoma, peptide-reactive cytotoxic T-lymphocytes were generated using either NY-ESO-1:157-167 or NY-ESO-1:157-165 but not NY-ESO-1:155-163. Because NY-ESO-1:157-165 is a 9 amino acid peptide completely contained within NY-ESO-1:157-167, it seemed likely that this peptide was the minimal determinant, and thus it was selected for continued study. An amino acid substitution of C to V was introduced into NY-ESO-1:157-165 at P9 to attempt to improve its immunogenicity by enhancing its binding affinity to HLA-A2.1 and increasing its stability in solution, because the C residue is readily oxidized, leading to dimerization of the peptide. From 5 of 20 HLA-A2.1+ patients with metastatic melanoma, NY-ESO-1:157-165(165V) stimulated cytotoxic T-lymphocytes in vitro, which recognized peptide-pulsed target cells and HLA-A2.1+ NY-ESO-1+ tumor cells, suggesting that this peptide may be clinically valuable for the treatment of patients with NY-ESO-1+ tumors.

To isolate melanoma Ags recognized by T cells, cDNA libraries made from melanoma cell lines were screened with four CTLs derived from tumor infiltrating lymphocytes (TIL) that were able to recognize melanoma cells in a HLA-A1, -A2, or -A3 restricted manner. Although cDNAs encoding the previously identified melanoma Ags, tyrosinase and gp100, were isolated, these TIL were found to recognize previously unidentified peptides. An HLA-A1-restricted CTL, TIL1388, was found to recognize a tyrosinase peptide (SSDYVIPIGTY), and an HLA-A3-restricted CTL, TIL1351, recognized a gp100 peptide (LIYRRRLMK). CTL clones isolated from the HLA-A2-restricted TIL1383 recognized a gp100 peptide (RLMKQDFSV). HLA-A2-restricted CTL, TIL1200, recognized a gp100 peptide (RLPRIFCSC). Replacement of either cysteine residue with α-amino butyric acid in the gp100 peptide, RLPRIFCSC, enhanced CTL recognition, suggesting that the peptide epitope naturally presented on the tumor cell surface may contain reduced cysteine residues. Oxidation of these cysteines might have occurred during the course of the synthesis or pulsing of the peptide in culture. These modifications may have important implications for the development of efficient peptide-based vaccines. These newly identified peptide epitopes can extend the ability to perform immunotherapy using synthetic peptides to a broader population of patients, especially those expressing HLA-A1 or HLA-A3 for whom only a few melanoma epitopes have previously been identified.

Many tumor-associated antigens are nonmutated, poorly immunogenic tissue differentiation antigens. Their weak immunogenicity may be due to “self”-tolerance. To induce autoreactive T cells, we studied immune responses to gp100/pmel 17, an antigen naturally expressed by both normal melanocytes and melanoma cells. Although a recombinant vaccinia virus (rVV) encoding the mouse homologue of gp100 was nonimmunogenic, immunization of normal C57BL/6 mice with the rVV encoding the human gp100 elicited a specific CD8+ T cell response. These lymphocytes were cross-reactive with mgp100 in vitro and treated established B16 melanoma upon adoptive transfer. To understand the mechanism of the greater immunogenicity of the human version of gp100, we characterized a 9-amino acid (AA) epitope, restricted by H-2Db, that was recognized by the T cells. The ability to induce specific T cells with human but not mouse gp100 resulted from differences within the major histocompatibility complex (MHC) class I–restricted epitope and not from differences elsewhere in the molecule, as was evidenced by experiments in which mice were immunized with rVV containing minigenes encoding these epitopes. Although the human (hgp10025–33) and mouse (mgp10025–33) epitopes were homologous, differences in the three NH2-terminal AAs resulted in a 2-log increase in the ability of the human peptide to stabilize “empty” Db on RMA-S cells and a 3-log increase in its ability to trigger interferon γ release by T cells. Thus, the fortuitous existence of a peptide homologue with significantly greater avidity for MHC class I resulted in the generation of self-reactive T cells. High-affinity, altered peptide ligands might be useful in the rational design of recombinant and synthetic vaccines that target tissue differentiation antigens expressed by tumors.

The cloning of the genes encoding cancer antigens has opened new possibilities for the treatment of patients with cancer. In this study, immunodominant peptides from the gp100 melanoma-associated antigen were identified, and a synthetic peptide, designed to increase binding to HLA-A2 molecules, was used as a cancer vaccine to treat patients with metastatic melanoma. On the basis of immunologic assays, 91% of patients could be successfully immunized with this synthetic peptide, and 13 of 31 patients (42%) receiving the peptide vaccine plus IL-2 had objective cancer responses, and four additional patients had mixed or minor responses. Synthetic peptide vaccines based on the genes encoding cancer antigens hold promise for the development of novel cancer immunotherapies.

The interaction between infected leukocytes and mucosal epithelia is likely to play a critical role in the sexual transmission of certain diseases. It may be possible to prevent the transmission of intracellular pathogens by inhibiting leukocyte-epithelial interactions. Monoclonal antibodies (MAbs) against leukocyte adhesion receptors can provide specific and potent inhibition of leukocyte-epithelial interactions, but systemic delivery of such MAbs is not desirable; therefore, a new method for local delivery of anti-adhesion MAbs to an exposed mucosal surface was developed using polymeric controlled-release devices. To evaluate the prophylactic potential of MAbs, leukocyte adhesion to the apical surface of a monolayer of T84 cells was examined in the presence of MAbs against human leukocyte surface proteins. MAbs against the MAC-1 adhesion receptor inhibited neutrophil attachment to T84 cells by as much as 97%. MAbs against murine leukocyte receptors were produced from several hybridomas and incorporated into poly(ethylene-co-vinyl acetate) (EVAc) devices. During incubation in buffered saline, small polymer discs continuously released active MAbs for 10 days. After insertion into the vaginal canals of mice, these polymer disks produced high levels of anti-MAC-1 MAb for several days. Polymeric controlled-release devices provide a new technique for using MAbs against adhesion receptors as a means of long-term, localized sexually transmitted disease (STD) prophylaxis.

Tumor infiltrating lymphocytes (TILs) derived from tumor-bearing patients recognize tumor-associated antigens presented by major histocompatibility complex (MHC) class I molecules. The infusion of TIL586 along with interleukin (IL) 2 into an autologous patient with metastatic melanoma resulted in the objective regression of tumor. A gene encoding a tumor antigen recognized by TIL586 was recently isolated and shown to encode gp75. Here we report that an antigenic peptide, MSLQRQFLR, recognized by TIL586 was not derived from the normal gp75 protein. Instead, this nonamer peptide resulted from translation of an alternative open reading frame of the same gene. Thus, the gp75 gene encodes two completely different polypeptides, gp75 as an antigen recognized by immunoglobulin G antibodies in sera from a patient with cancer, and a 24-amino acid product as a tumor rejection antigen recognized by T cells. This represents the first demonstration that a human tumor rejection antigen can be generated from a normal cellular gene using an open reading frame other than that used to encode the normal protein. These findings revealed a novel mechanism for generating tumor antigens, which may be useful as vaccines to induce tumor-specific cell-mediated immunity against cancer.

Adoptive transfer of T-cell receptor (TCR) gene engineered T-cells targeting tumor-associated antigens can mediate cancer regression. Accumulating evidence suggests that the clinical success of many immunotherapies is mediated by T-cells targeting mutated neoantigens unique to the patient. We hypothesized that the most frequent TCR clonotypes infiltrating the tumor were reactive against tumor antigens. We thus developed a multi-step strategy that involved CDR3-TCRB deep sequencing of tumor infiltrating lymphocytes and matching of TCRA-TCRB pairs by pairSEQ and single cell RT-PCR. CDR3 TCRA-TCRB pairs identified with this approach, were then reconstructed to full length based on IMGT database [http://www.imgt.org], synthetized and cloned into non-viral and retroviral expression vectors. T-cells were gene-engineered with these TCRs and tested against tumor cell lines and antigen presenting cells expressing tandem minigenes encoding mutated tumor neoantigens and/or pulsed with corresponding mutated peptides. Analysis of 12 fresh metastatic melanomas revealed that in 11 samples, up to 5 tumor-reactive TCRs were present in the 5 most frequently occurring clonotypes including reactivity against neoantigens. These data demonstrate the feasibility of developing a rapid approach for the identification of tumor-reactive TCRs that can be used for personalized TCR-gene therapy.

Autologous T lymphocytes genetically engineered to express a murine T cell receptor (TCR) against human carcinoembryonic antigen (CEA) were administered to three patients with metastatic colorectal cancer refractory to standard treatments. All patients experienced profound decreases in serum CEA levels (74–99%), and one patient had an objective regression of cancer metastatic to the lung and liver. However, a severe transient inflammatory colitis that represented a dose limiting toxicity was induced in all three patients. This report represents the first example of objective regression of metastatic colorectal cancer mediated by adoptive T cell transfer and illustrates the successful use of a TCR, raised in human leukocyte antigen (HLA) transgenic mice, against a human tumor associated antigen. It also emphasizes the destructive power of small numbers of highly avid T cells and the limitations of using CEA as a target for cancer immunotherapy.

Adoptive immunotherapy using TCR-engineered PBLs against melanocyte differentiation Ags mediates objective tumor regression but is associated with on-target toxicity. To avoid toxicity to normal tissues, we targeted cancer testis Ag (CTA) MAGE-A3, which is widely expressed in a range of epithelial malignancies but is not expressed in most normal tissues. To generate high-avidity TCRs against MAGE-A3, we employed a transgenic mouse model that expresses the human HLA-A*0201 molecule. Mice were immunized with two HLA-A*0201–restricted peptides of MAGE-A3: 112–120 (KVAELVHFL) or MAGE-A3: 271–279 (FLWGPRALV), and T cell clones were generated. MAGE-A3–specific TCR α- and β-chains were isolated and cloned into a retroviral vector. Expression of both TCRs in human PBLs demonstrated Ag-specific reactivity against a range of melanoma and nonmelanoma tumor cells. The TCR against MAGE-A3: 112–120 was selected for further development based on superior reactivity against tumor target cells. Interestingly, peptide epitopes from MAGE-A3 and MAGE-A12 (and to a lesser extent, peptides from MAGE-A2 and MAGE-A6) were recognized by PBLs engineered to express this TCR. To further improve TCR function, single amino acid variants of the CDR3 α-chain were generated. Substitution of alanine to threonine at position 118 of the α-chain in the CDR3 region of the TCR improved its functional avidity in CD4 and CD8 cells. On the basis of these results, a clinical trial is planned in which patients bearing a variety of tumor histologies will receive autologous PBLs that have been transduced with this optimized anti–MAGE-A3 TCR.

The adoptive transfer of autologous tumor infiltrating lymphocytes (TIL) can mediate long term tumor regression in some patients with metastatic melanoma. Recent observations suggest that autologous melanoma TIL administered to patients in adoptive T cell therapy (ACT) protocols may frequently recognize multiple tumor-specific somatic mutations, findings that have been facilitated by advances in whole exome sequencing and RNAseq methods. In an attempt to evaluate the antigenic diversity of TIL and gain some insights into the potential association between the recognition of mutated antigens and clinical responses to TIL therapy, we analyzed between 7 and 30 individual cultures derived from resected melanoma tumor fragments and pooled populations of administered TIL from each of 5 patients for their ability to recognize mutated antigens expressed by patients’ autologous tumors. Two of the patients who were evaluated exhibited durable complete tumor regressions, 1 exhibited a partial response, and 2 were non-responders to ACT. Screening assays were carried out by evaluating the interferon gamma release stimulated by the co-culture of autologous patient TIL with autologous dendritic cells or EBV transformed B cells transfected with mini-genes encoding mutated antigens identified by exomic sequencing of patient tumors. Using this approach, we identified 10, 3, and 2 mutated antigens targeted by TIL from the 3 patients who responded to ACT. The TIL that were administered to 1 of the non-responders appeared to recognize at least 4 mutated antigens, whereas TIL administered to the second non-responder failed to recognize any of the mutated minigenes that were tested. Immuno-dominant mutated antigens that were recognized by the majority of the evaluated TIL fragment cultures, as well as the bulk population of infusion TIL, could be identified in each of the 4 patients in this study for whom mutated antigen targets could be identified. In addition, 1 or more sub-dominant mutated antigens that were recognized by one or a relatively small percentage of the screened TIL fragment cultures were identified from each of these same 4 patients. Future studies will be directed at developing methods for enriching T cells reactive with mutated epitopes from TIL or peripheral blood in an attempt to enhance the effectiveness of ACT for the treatment of patients with metastatic melanoma and that will hopefully lead to the development of effective therapies for the treatment of patients with other malignancies. Note: This abstract was not presented at the meeting. Citation Format: Jessica S. Crystal, Todd Prickett, Yong-Chen Lu, Jared J. Gartner, Maria Parkhurst, Alena Gros, Yong F. Li, Mona El-Gamil, Steven A. Rosenberg, Paul F. Robbins. Diversity of tumor infiltrating lymphocyte recognition of diverse mutated antigens in cutaneous melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-221. doi:10.1158/1538-7445.AM2015-LB-221

Identification and isolation of mutation-specific T-cells is a major obstacle to the development of effective cancer immunotherapies. The tumor is a natural sink for neo-antigen specific cells, but these are far less frequent in peripheral blood, a more accessible and abundant source of T cells. Previous studies have revealed that PD-1 expression in the tumor microenvironment can identify the patient-specific repertoire of tumor and mutation-specific cells infiltrating human tumors. Here, we evaluated the potential utility of PD-1 expression on circulating lymphocytes to identify and enrich for mutation-specific cells. Selection of CD8+PD-1+ lymphocytes circulating in peripheral blood, but not the bulk or CD8+PD-1- cells, led to the direct enrichment of mutation-specific cells in three melanoma samples tested, with at least 1, 3 and 5 unique patient-specific neo-antigens recognized, respectively. The PD-1+ derived cells also displayed autologous tumor recognition. Furthermore, CD8+PD-1+ and PD-1hi cells isolated and expanded from the blood from a patient with colorectal cancer led to the identification of mutation-specific cells targeting two tumor neo-antigens. These findings provide a novel strategy to harness naturally-occuring mutation-specific T cells present in peripheral blood for the development of personalized T -cell based therapies to treat cancer.

The adoptive transfer of human tumor-reactive T lymphocytes into autologous patients can mediate the regression of metastatic melanoma. Here, the in vitro generation of melanoma-reactive T lymphocytes was compared using 3 common gamma-chain cytokines, interleukin (IL)-2, IL-7, and IL-15, alone or in combination. The proliferation, function, and phenotype were evaluated for tumor-reactive T cells derived from peripheral blood mononuclear cells (PBMCs) from patients previously immunized with the melanoma-associated peptide gp100:209-217(210M) and PBMCs transduced with a retrovirus encoding the alpha and beta chains of a gp100-reactive T-cell receptor (TCR). IL-7 alone did not induce significant proliferation of any tumor-reactive T-cell population, whereas IL-2 and IL-15 induced significant proliferation of tumor-reactive T lymphocytes from both sources. Cells cultured in the presence of IL-2 or IL-15 secreted comparable amounts of interferon-gamma and IL-2 in response to melanoma cells in vitro and were phenotypically similar in terms of costimulatory molecules (CD27 and CD28), cytokine receptors (CD25, CD122, and CD127), and a lymphoid homing molecule (CD62L). In addition, the proliferation, function, and phenotype of T cells cultured with combinations of IL-2, IL-7, and IL-15 were similar to those grown with IL-2 alone. The effects of these cytokines on TCR stimulation of CD45RA+ naive cells derived from adult patients and from human umbilical cord blood were also compared. Similar to the data with activated tumor-reactive T lymphocytes, IL-7 alone did not support significant proliferation of naive T cells after TCR stimulation with anti-CD3, although IL-2 and IL-15 induced comparable proliferation of T lymphocytes with similar phenotypic attributes.

Hybrid cells generated by fusing dendritic cells with tumor cells (DC-TC) are currently being evaluated as cancer vaccines in preclinical models and human immunization trials. In this study, we evaluated the production of human DC-TC hybrids using an electrofusion protocol previously defined for murine cells. Human DCs were electrically fused with allogeneic melanoma cells (888mel) and were subsequently analyzed for coexpression of unique DC and TC markers using FACS and fluorescence microscopy. Dually fluorescent cells were clearly observed using both techniques after staining with Abs against distinct surface molecules suggesting that true cell fusion had occurred. We also evaluated the ability of human DC-TC hybrids to present tumor-associated epitopes in the context of both MHC class I and class II molecules. Allogeneic DCs expressing HLA-A*0201, HLA-DRβ1*0401, and HLA-DRβ1*0701 were fused with 888mel cells that do not express any of these MHC molecules, but do express multiple melanoma-associated Ags. DC-888mel hybrids efficiently presented HLA-A*0201-restricted epitopes from the melanoma Ags MART-1, gp100, tyrosinase, and tyrosinase-related protein 2 as evaluated by specific cytokine secretion from six distinct CTL lines. In contrast, DCs could not cross-present MHC class I-restricted epitopes after exogenously loading with gp100 protein. DC-888mel hybrids also presented HLA-DRβ1*0401- and HLA-DRβ1*0701-restricted peptides from gp100 to CD4+ T cell populations. Therefore, fusions of DCs and tumor cells express both MHC class I- and class II-restricted tumor-associated epitopes and may be useful for the induction of tumor-reactive CD8+ and CD4+ T cells in vitro and in human vaccination trials.

SUMMARY: Tyrosinase has many advantages as a target antigen for the immunotherapy of patients with melanoma because it is expressed in nearly all melanoma specimens with a high degree of cellular homogeneity, and its distribution in normal tissues is limited to melanocytes. To broaden our ability to direct cellular immune responses against this protein, we pursued an investigation to identify new shared human leukocyte antigen (HLA)-A2.1 restricted epitopes from tyrosinase. Peptides were synthesized that fit a permissive HLA-A2.1 binding motif and did not span common sites of polymorphism. The binding affinity of each peptide to HLA-A2.1 relative to a standard peptide with intermediate binding affinity was evaluated in a competitive inhibition assay. Twelve peptides were selected that had binding affinities within 80% of that of the standard peptide, and these were used to stimulate peripheral blood mononuclear cells (PBMC) in vitro from three HLA-A2.1+ patients with metastatic melanoma. Cytotoxic T lymphocytes that specifically recognized peptide-pulsed target cells as well as HLA-A2.1+ tyrosinase+ melanoma cells were raised from one patient with tyrosinase:8-17 (CLLWSFQTSA). To evaluate further the immunogenicity of this peptide, PBMC from 23 HLA-A2.1+ patients were stimulated in vitro with tyrosinase:8-17. Eleven bulk T-cell cultures demonstrated specific peptide recognition, and six of these also recognized HLA-A2.1+ tyrosinase+ melanoma cells. These data suggest that tyrosinase:8-17 may be clinically useful for the treatment of patients with melanoma.

Summary Clinical observations in the interleukin (IL) 2-based immunotherapies suggest that T cells play a central role in the rejection of melanoma. Using cDNA expression cloning, we have isolated genes encoding melanoma antigens recognized by tumor-infiltrating T lymphocytes. These antigens are categorized as (a) melanocyte-specific melanosomal proteins (MART-1/melan A, gp100, tyrosinase, TRP-1, and TRP-2), (b) tumor-specific mutated proteins (β-catenin), and (c) others (p15). A variety of mechanisms has been identified for the generation of T cell epitopes on tumor cells. Some of the HLA-A2 binding epitopes from the melanosomal antigens appear to be subdominant self-determinants with relatively low major histocompatibility complex binding affinity. The effectiveness of adoptive transfer into patients of cytotoxic T lymphocytes recognizing the melanosomal antigens, the significant correlation between vitiligo development and clinical response in patients receiving IL-2-based immunotherapies, and the sporadic tumor regressions observed in some patients following immunization with the MART-1 or gp100 peptides in incomplete Freund's adjuvant or recombinant viruses expressing the MART-1 antigen suggest that these epitopes may represent tumor rejection antigens. Phase I immunization trials using peptides or recombinant viruses containing genes encoding the melanosomal antigens MART-1 or gp100, with or without co-administration of cytokines such as IL-2, IL-12, or granulocyte-macrophage colony-stimulating factor, are being conducted in the Surgery Branch of the National Cancer Institute. These studies may demonstrate the feasibility of using melanosomal proteins for the immunotherapy of patients with melanoma.

Tyrosinase was the first melanoma-associated antigen shown to be recognized by CD4+ T cells. In this study, we have identified two HLA-DRB1*0401-restricted peptides recognized by these T cells: Ty 56-70 and Ty 448-462. As with many of the MHC class I-restricted melanoma epitopes, both are nonmutated self peptides that have intermediate and weak MHC binding affinities, respectively. Mutated and truncated versions of these peptides were used to define their MHC binding anchor residues. Anchor residues were then modified to derive peptides with increased MHC binding affinities and T cell stimulatory properties. Ty 56-70 and Ty 448-462 enhance the list of immunogenic HLA-A2-, A24-, and B44-restricted tyrosinase peptides already described. Thus, tyrosinase provides a model for anti-melanoma vaccines in which a single molecule can generate multivalent immunization incorporating both CD4+ and CD8+ T cell responses.

Background To simplify clinical scale lymphocyte expansions, we investigated the use of the WAVE®, a closed system bioreactor that utilizes active perfusion to generate high cell numbers in minimal volumes. Methods We have developed an optimized rapid expansion protocol for the WAVE bioreactor that produces clinically relevant numbers of cells for our adoptive cell transfer clinical protocols. Results TIL and genetically modified PBL were rapidly expanded to clinically relevant scales in both static bags and the WAVE bioreactor. Both bioreactors produced comparable numbers of cells; however the cultures generated in the WAVE bioreactor had a higher percentage of CD4+ cells and had a less activated phenotype. Conclusions The WAVE bioreactor simplifies the process of rapidly expanding tumor reactive lymphocytes under GMP conditions, and provides an alternate approach to cell generation for ACT protocols.

The adoptive cell transfer (ACT) of autologous tumor infiltrating lymphocytes (TIL) can mediate durable, complete tumor regression in approximately 20% of patients with metastatic melanoma. Recent observations suggest that autologous melanoma TIL administered to multiple patients in ACT protocols can recognize 1 or more tumor-specific somatic mutations, findings facilitated by recent advances in whole exome sequencing and RNA-seq methods.

Meeting abstracts T cells targeting unique somatic mutations appear to play an important role in the antitumor responses observed following T cell transfer, and isolation of mutation-specific lymphocytes and T-cell receptors has become a major obstacle to the development of more effective

Meeting abstracts T cell-based immunotherapy shows promise for the successful treatment of advanced cancer. Indeed, adoptively transferred tumor infiltrating T lymphocytes (TIL) that mediated complete regression of metastatic melanoma have been shown to recognize neoantigens/mutated epitopes

Meeting abstracts The adoptive transfer of tumor infiltrating lymphocytes (TIL) can mediate the regression of metastatic melanoma. In addition, the adoptive transfer of lymphocytes genetically modified to express tumor reactive T cell receptors (TCRs) can mediate tumor regression. Many T cells from