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Circulating T cells from peripheral blood (PBL) can provide a rich and noninvasive source for antitumor T cells. By single-cell transcriptomic profiling of 36 neoantigen-specific T cell clones from 6 metastatic cancer patients, we report the transcriptional and cell surface signatures of antitumor PBL-derived CD8 + T cells (NeoTCR PBL ). Comparison of tumor-infiltrating lymphocyte (TIL)- and PBL-neoantigen-specific T cells revealed that NeoTCR PBL T cells are low in frequency and display less-dysfunctional memory phenotypes relative to their TIL counterparts. Analysis of 100 antitumor TCR clonotypes indicates that most NeoTCR PBL populations target the same neoantigens as TILs. However, NeoTCR PBL TCR repertoire is only partially shared with TIL. Prediction and testing of NeoTCR PBL signature-derived TCRs from PBL of 6 prospective patients demonstrate high enrichment of clonotypes targeting tumor mutations, a viral oncogene, and patient-derived tumor. Thus, the NeoTCR PBL signature provides an alternative source for identifying antitumor T cells from PBL of cancer patients, enabling immune monitoring and immunotherapies., Competing Interests: Declaration of interests R.Y., A.R.C., S.K., F.J.L. P.F.R., and S.A.R. are listed on a patent application (PCT/US2021/023225) that covers the use of NeoTCR(PBL) signature to identify antitumor TCRs., (Copyright © 2023. Published by Elsevier Inc.)

Efforts to apply adoptive cell transfer (ACT) immunotherapy to patients with common epithelial cancers have been stimulated by the demonstration that the majority of these patients contain lymphocytes reactive against the expressed products of their cancer mutations. Early efforts to specifically target these antigens have been promising., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023. Published by Elsevier Inc.)

Background: Adoptive transfer of tumor-infiltrating lymphocytes (TIL) fails to consistently elicit tumor rejection. Manipulation of intrinsic factors that inhibit T cell effector function and neoantigen recognition may therefore improve TIL therapy outcomes. We previously identified the cytokine-induced SH2 protein (CISH) as a key regulator of T cell functional avidity in mice. Here, we investigate the mechanistic role of CISH in regulating human T cell effector function in solid tumors and demonstrate that CRISPR/Cas9 disruption of CISH enhances TIL neoantigen recognition and response to checkpoint blockade., Methods: Single-cell gene expression profiling was used to identify a negative correlation between high CISH expression and TIL activation in patient-derived TIL. A GMP-compliant CRISPR/Cas9 gene editing process was developed to assess the impact of CISH disruption on the molecular and functional phenotype of human peripheral blood T cells and TIL. Tumor-specific T cells with disrupted Cish function were adoptively transferred into tumor-bearing mice and evaluated for efficacy with or without checkpoint blockade., Findings: CISH expression was associated with T cell dysfunction. CISH deletion using CRISPR/Cas9 resulted in hyper-activation and improved functional avidity against tumor-derived neoantigens without perturbing T cell maturation. Cish knockout resulted in increased susceptibility to checkpoint blockade in vivo., Conclusions: CISH negatively regulates human T cell effector function, and its genetic disruption offers a novel avenue to improve the therapeutic efficacy of adoptive TIL therapy., Funding: This study was funded by Intima Bioscience, U.S. and in part through the Intramural program CCR at the National Cancer Institute., Competing Interests: Declaration of interests M.C. is a co-founder of Intima Bioscience. B.R.W., S.A.R., and B.S.M. have received sponsored research support from Intima Bioscience. D.C.P., B.R.W., M.C., S.A.R., B.S.M., and N.P.R. have patents filed based on the findings described here., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: Metastatic breast cancer (mBrCa) is most often an incurable disease with only modest responses to available immunotherapies. This study investigates the immunogenicity of somatic mutations in breast cancer and explores the therapeutic efficacy in a pilot trial of mutation-reactive tumor-infiltrating lymphocytes (TILs) in patients with metastatic disease., Patients and Methods: Forty-two patients with mBrCa refractory to previous lines of treatment underwent surgical resection of a metastatic lesion(s), isolation of TIL cultures, identification of exomic nonsynonymous tumor mutations, and immunologic screening for neoantigen reactivity. Clinically eligible patients with appropriate reactivity were enrolled into one cohort of an ongoing phase II pilot trial of adoptive cell transfer of selected neoantigen-reactive TIL, with a short course of pembrolizumab (ClinicalTrials.gov identifier: NCT01174121)., Results: TILs were isolated and grown in culture from the resected lesions of all 42 patients with mBrCa, and a median number of 112 (range: 6-563) nonsynonymous mutations per patient were identified. Twenty-eight of 42 (67%) patients contained TIL that recognized at least one immunogenic somatic mutation (median: 3 neoantigens per patient, range: 1-11), and 13 patients demonstrated robust reactivity appropriate for adoptive transfer. Eight patients remained clinically eligible for treatment, and six patients were enrolled on a protocol of adoptive cell transfer of enriched neoantigen-specific TIL, in combination with pembrolizumab (≤ 4 doses). Objective tumor regression was noted in three patients, including one complete response (now ongoing over 5.5 years) and two partial responses (6 and 10 months)., Conclusion: Most patients with breast cancer generated a natural immune response targeting the expressed products of their cancer mutations. Adoptive transfer of TIL is a highly personalized experimental option for patients with mBrCa shown to be capable of mediating objective responses in this pilot trial and deserves further study., Competing Interests: Nikolaos ZacharakisPatents, Royalties, Other Intellectual Property: Provisional patents about TCRs filed and held by NIH (Inst) Sanghyun P. KimPatents, Royalties, Other Intellectual Property: I have filed a provisional patent that has no relationship to the current publication (Inst) Maria R. ParkhurstPatents, Royalties, Other Intellectual Property: Patent royalties Paul F. RobbinsPatents, Royalties, Other Intellectual Property: BioNTech US Inc—Royalty, Kite Pharma Inc—Royalty, Intellia Therapeutics Inc—Royalty, Intima Bioscience Inc—Royalty, Myst Therapeutics Inc—Royalty, Tailored Therapeutics Inc—Royalty, Ziopharm Oncology Inc—Royalty Richard M. SherryEmployment: Lyell Immunopharma James C. YangResearch Funding: Kite/Gilead (Inst)Patents, Royalties, Other Intellectual Property: Royalties on inventions may go to my institution (NCI), which may pass a portion (anonymously) to me Steven A. FeldmanConsulting or Advisory Role: Guidepoint Global, Kite, a Gilead Company, TCG Crossover Management LLCResearch Funding: Syncopation (Inst)Patents, Royalties, Other Intellectual Property: I have patents and resulting royalties from licensed patents from NCI/Surgery Branch, related to cellular immunotherapy Steven A. RosenbergResearch Funding: Kite, a Gilead Company (Inst), Iovance Biotherapeutics (Inst), ZIOPHARM Oncology (Inst)Patents, Royalties, Other Intellectual Property: Patents held by NIHNo other potential conflicts of interest were reported.

A common theme across multiple successful immunotherapies for cancer is the recognition of tumor-specific mutations (neoantigens) by T cells. The rapid discovery of such antigen responses could lead to improved therapies through the adoptive transfer of T cells engineered to express neoantigen-reactive T cell receptors (TCRs). Here, through CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) and TCR-seq of non-small cell lung cancer (NSCLC) tumor-infiltrating lymphocytes (TILs), we develop a neoantigen-reactive T cell signature based on clonotype frequency and CD39 protein and CXCL13 mRNA expression. Screening of TCRs selected by the signature allows us to identify neoantigen-reactive TCRs with a success rate of 45% for CD8 + and 66% for CD4 + T cells. Because of the small number of samples analyzed (4 patients), generalizability remains to be tested. However, this approach can enable the quick identification of neoantigen-reactive TCRs and expedite the engineering of personalized neoantigen-reactive T cells for therapy., Competing Interests: Declaration of interests The authors declare no competing interests. A non-provisional international patent has been filed based on the work described in this study., (Published by Elsevier Inc.)

The accurate identification of antitumor T cell receptors (TCRs) represents a major challenge for the engineering of cell-based cancer immunotherapies. By mapping 55 neoantigen-specific TCR clonotypes (NeoTCRs) from 10 metastatic human tumors to their single-cell transcriptomes, we identified signatures of CD8 + and CD4 + neoantigen-reactive tumor-infiltrating lymphocytes (TILs). Neoantigen-specific TILs exhibited tumor-specific expansion with dysfunctional phenotypes, distinct from blood-emigrant bystanders and regulatory TILs. Prospective prediction and testing of 73 NeoTCR signature-derived clonotypes demonstrated that half of the tested TCRs recognized tumor antigens or autologous tumors. NeoTCR signatures identified TCRs that target driver neoantigens and nonmutated viral or tumor-associated antigens, suggesting a common metastatic TIL exhaustion program. NeoTCR signatures delineate the landscape of TILs across metastatic tumors, enabling successful TCR prediction based purely on TIL transcriptomic states for use in cancer immunotherapy.

Purpose: Immunotherapies mediate the regression of human tumors through recognition of tumor antigens by immune cells that trigger an immune response. Mutations in the RAS oncogenes occur in about 30% of all patients with cancer. These mutations play an important role in both tumor establishment and survival and are commonly found in hotspots. Discovering T-cell receptors (TCR) that recognize shared mutated RAS antigens presented on MHC class I and class II molecules are thus promising reagents for "off-the-shelf" adoptive cell therapies (ACT) following insertion of the TCRs into lymphocytes., Experimental Design: In this ongoing work, we screened for RAS antigen recognition in tumor-infiltrating lymphocytes (TIL) or by in vitro stimulation of peripheral blood lymphocytes (PBL). TCRs recognizing mutated RAS were identified from the reactive T cells. The TCRs were then reconstructed and virally transduced into PBLs and tested., Results: Here, we detect and report multiple novel TCR sequences that recognize nonsynonymous mutant RAS hotspot mutations with high avidity and specificity and identify the specific class-I and -II MHC restriction elements involved in the recognition of mutant RAS., Conclusions: The TCR library directed against RAS hotspot mutations described here recognize RAS mutations found in about 45% of the Caucasian population and about 60% of the Asian population and represent promising reagents for "off-the-shelf" ACTs., (©2021 The Authors; Published by the American Association for Cancer Research.)

Tumor neoepitopes presented by major histocompatibility complex (MHC) class I are recognized by tumor-infiltrating lymphocytes (TIL) and are targeted by adoptive T-cell therapies. Identifying which mutant neoepitopes from tumor cells are capable of recognition by T cells can assist in the development of tumor-specific, cell-based therapies and can shed light on antitumor responses. Here, we generate a ranking algorithm for class I candidate neoepitopes by using next-generation sequencing data and a dataset of 185 neoepitopes that are recognized by HLA class I-restricted TIL from individuals with metastatic cancer. Random forest model analysis showed that the inclusion of multiple factors impacting epitope presentation and recognition increased output sensitivity and specificity compared to the use of predicted HLA binding alone. The ranking score output provides a set of class I candidate neoantigens that may serve as therapeutic targets and provides a tool to facilitate in vitro and in vivo studies aimed at the development of more effective immunotherapies., Competing Interests: Competing interests The authors declare no competing interests.

Engineered T cells expressing tumor-specific T-cell receptors (TCRs) are emerging as a mode of personalized cancer immunotherapy that requires identification of TCRs against the products of known driver mutations and novel mutations in a timely fashion. We present a nonviral and non-next-generation sequencing platform for rapid, and efficient neoantigen-specific TCR identification and evaluation that does not require the use of recombinant cloning techniques. The platform includes an innovative method of TCRα detection using Sanger sequencing, TCR pairings and the use of TCRα/β gene fragments for putative TCR evaluation. Using patients' samples, we validated and compared our new methods head-to-head with conventional approaches used for TCR discovery. Development of a unique demultiplexing method for identification of TCRα, adaptation of synthetic TCRs for gene transfer, and a reliable reporter system significantly shortens TCR discovery time over conventional methods and increases throughput to facilitate testing prospective personalized TCRs for adoptive cell therapy.

BACKGROUNDTherapeutic vaccinations against cancer have mainly targeted differentiation antigens, cancer-testis antigens, and overexpressed antigens and have thus far resulted in little clinical benefit. Studies conducted by multiple groups have demonstrated that T cells recognizing neoantigens are present in most cancers and offer a specific and highly immunogenic target for personalized vaccination.METHODSWe recently developed a process using tumor-infiltrating lymphocytes to identify the specific immunogenic mutations expressed in patients' tumors. Here, validated, defined neoantigens, predicted neoepitopes, and mutations of driver genes were concatenated into a single mRNA construct to vaccinate patients with metastatic gastrointestinal cancer.RESULTSThe vaccine was safe and elicited mutation-specific T cell responses against predicted neoepitopes not detected before vaccination. Furthermore, we were able to isolate and verify T cell receptors targeting KRASG12D mutation. We observed no objective clinical responses in the 4 patients treated in this trial.CONCLUSIONThis vaccine was safe, and potential future combination of such vaccines with checkpoint inhibitors or adoptive T cell therapy should be evaluated for possible clinical benefit in patients with common epithelial cancers.TRIAL REGISTRATIONPhase I/II protocol (NCT03480152) was approved by the IRB committee of the NIH and the FDA.FUNDINGCenter for Clinical Research, NCI, NIH.

Purpose: The purpose of this study was to evaluate antigen experienced T cells in peripheral blood lymphocytes (PBL) for responses to p53 neoantigens., Experimental Design: PBLs from patients with a mutated TP53 tumor were sorted for antigen-experienced T cells and in vitro stimulation (IVS) was performed with p53 neoantigens. The IVS cultures were stimulated with antigen-presenting cells expressing p53 neoantigens, enriched for 41BB/OX40 and grown with rapid expansion protocol., Results: T-cell responses were not observed in the PBLs of 4 patients who did not have tumor-infiltrating lymphocyte (TIL) responses to mutated TP53 . In contrast, 5 patients with TIL responses to mutated TP53 also had similar T-cell responses in their PBLs, indicating that the PBLs and TILs were congruent in p53 neoantigen reactivity. CD4 + and CD8 + T cells were specific for p53 R175H , p53 Y220C , or p53 R248W neoantigens, including a 78% reactive T-cell culture against p53 R175H and HLA-A*02:01. Tracking TCRB clonotypes (clonality, top ranked, and TP53 mutation-specific) supported the enrichment of p53 neoantigen-reactive T cells from PBLs. The same T-cell receptor (TCR) from the TIL was found in the IVS cultures in three cases and multiple unique TCRs were found in another patient. TP53 mutation-specific T cells also recognized tumor cell lines bearing the appropriate human leukocyte antigen restriction element and TP53 mutation, indicating these T cells could recognize processed and presented p53 neoantigens., Conclusions: PBL was a noninvasive source of T cells targeting TP53 mutations for cell therapy and can provide a window into intratumoral p53 neoantigen immune responses. See related commentary by Olivera et al., p. 1203 ., (©2020 American Association for Cancer Research.)

Tumor-resident lymphocytes can mount a response against neoantigens expressed in microsatellite-stable gastrointestinal (GI) cancers, and adoptive transfer of neoantigen-specific lymphocytes has demonstrated antitumor activity in selected patients. However, whether peripheral blood could be used as an alternative minimally invasive source to identify lymphocytes targeting neoantigens in patients with GI cancer with relatively low mutation burden is unclear. We used a personalized high-throughput screening strategy to investigate whether PD-1 expression in peripheral blood could be used to identify CD8+ or CD4+ lymphocytes recognizing neoantigens identified by whole-exome sequencing in 7 patients with GI cancer. We found that neoantigen-specific lymphocytes were preferentially enriched in the CD8+PD-1+/hi or CD4+PD-1+/hi subsets, but not in the corresponding bulk or PD-1- fractions. In 6 of 7 individuals analyzed we identified circulating CD8+ and CD4+ lymphocytes targeting 6 and 4 neoantigens, respectively. Moreover, neoantigen-reactive T cells and a T cell receptor (TCR) isolated from the CD8+PD-1+ subsets recognized autologous tumor, albeit at reduced levels, in 2 patients with available cell lines. These data demonstrate the existence of circulating T cells targeting neoantigens in GI cancer patients and provide an approach to generate enriched populations of personalized neoantigen-specific lymphocytes and isolate TCRs that could be exploited therapeutically to treat cancer.

Immunotherapies can mediate regression of human tumors with high mutation rates, but responses are rarely observed in patients with common epithelial cancers. This raises the question of whether patients with these common cancers harbor T lymphocytes that recognize mutant proteins expressed by autologous tumors that may represent ideal targets for immunotherapy. Using high-throughput immunologic screening of mutant gene products identified via whole-exome sequencing, we identified neoantigen-reactive tumor-infiltrating lymphocytes (TIL) from 62 of 75 (83%) patients with common gastrointestinal cancers. In total, 124 neoantigen-reactive TIL populations were identified, and all but one of the neoantigenic determinants were unique. The results of in vitro T-cell recognition assays demonstrated that 1.6% of the gene products encoded by somatic nonsynonymous mutations were immunogenic. These findings demonstrate that the majority of common epithelial cancers elicit immune recognition and open possibilities for cell-based immunotherapies for patients bearing these cancers. SIGNIFICANCE: TILs cultured from 62 of 75 (83%) patients with gastrointestinal cancers recognized neoantigens encoded by 1.6% of somatic mutations expressed by autologous tumor cells, and 99% of the neoantigenic determinants appeared to be unique and not shared between patients. This article is highlighted in the In This Issue feature, p. 983 ., (©2019 American Association for Cancer Research.)

Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TILs) targeting neoantigens can mediate tumor regression in selected patients with metastatic epithelial cancer. However, effectively identifying and harnessing neoantigen-reactive T cells for patient treatment remains a challenge and it is unknown whether current methods to detect neoantigen-reactive T cells are missing potentially clinically relevant neoantigen reactivities. We thus investigated whether the detection of neoantigen-reactive TILs could be enhanced by enriching T cells that express PD-1 and/or T cell activation markers followed by microwell culturing to avoid overgrowth of nonreactive T cells. In 6 patients with metastatic epithelial cancer, this method led to the detection of CD4+ and CD8+ T cells targeting 18 and 1 neoantigens, respectively, compared with 6 and 2 neoantigens recognized by CD4+ and CD8+ T cells, respectively, when using our standard TIL fragment screening approach. In 2 patients, no recognition of mutated peptides was observed using our conventional screen, while our high-throughput approach led to the identification of 5 neoantigen-reactive T cell receptors (TCRs) against 5 different mutations from one patient and a highly potent MHC class II-restricted KRASG12V-reactive TCR from a second patient. In addition, in a metastatic tumor sample from a patient with serous ovarian cancer, we isolated 3 MHC class II-restricted TCRs targeting the TP53G245S hot-spot mutation. In conclusion, this approach provides a highly sensitive platform to isolate clinically relevant neoantigen-reactive T cells or their TCRs for cancer treatment.

Adoptive transfer of T cells with engineered T-cell receptor (TCR) genes that target tumor-specific 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. To test this hypothesis, we developed a multistep strategy that involved TCRB deep sequencing of the CD8(+)PD-1(+) T-cell subset, matching of TCRA-TCRB pairs by pairSEQ and single-cell RT-PCR, followed by testing of the TCRs for tumor-antigen specificity. 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, which included reactivity against neoantigens. These data show the feasibility of developing a rapid, personalized TCR-gene therapy approach that targets the unique set of antigens presented by the autologous tumor without the need to identify their immunologic reactivity. Cancer Immunol Res; 4(9); 734-43. ©2016 AACR., (©2016 American Association for Cancer Research.)

Immunotherapy treatment of patients with metastatic cancer has assumed a prominent role in the clinic. Durable complete response rates of 20% to 25% are achieved in patients with metastatic melanoma following adoptive cell transfer of T cells derived from metastatic lesions, responses that appear in some patients to be mediated by T cells that predominantly recognize mutated antigens. Here, we provide a detailed analysis of the reactivity of T cells administered to a patient with metastatic melanoma who exhibited a complete response for over 3 years after treatment. Over 4,000 nonsynonymous somatic mutations were identified by whole-exome sequence analysis of the patient's autologous normal and tumor cell DNA. Autologous B cells transfected with 720 mutated minigenes corresponding to the most highly expressed tumor cell transcripts were then analyzed for their ability to stimulate the administered T cells. Autologous tumor-infiltrating lymphocytes recognized 10 distinct mutated gene products, but not the corresponding wild-type products, each of which was recognized in the context of one of three different MHC class I restriction elements expressed by the patient. Detailed clonal analysis revealed that 9 of the top 20 most prevalent clones present in the infused T cells, comprising approximately 24% of the total cells, recognized mutated antigens. Thus, we have identified and enriched mutation-reactive T cells and suggest that such analyses may lead to the development of more effective therapies for the treatment of patients with metastatic cancer. Cancer Immunol Res; 4(8); 669-78. ©2016 AACR., (©2016 American Association for Cancer Research.)

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, 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 tumor. 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.

Adoptively transferred tumor-infiltrating T lymphocytes (TILs) that mediate complete regression of metastatic melanoma have been shown to recognize mutated epitopes expressed by autologous tumors. Here, in an attempt to develop a strategy for facilitating the isolation, expansion, and study of mutated antigen-specific T cells, we performed whole-exome sequencing on matched tumor and normal DNA isolated from 8 patients with metastatic melanoma. Candidate mutated epitopes were identified using a peptide-MHC-binding algorithm, and these epitopes were synthesized and used to generate panels of MHC tetramers that were evaluated for binding to tumor digests and cultured TILs used for the treatment of patients. This strategy resulted in the identification of 9 mutated epitopes from 5 of the 8 patients tested. Cells reactive with 8 of the 9 epitopes could be isolated from autologous peripheral blood, where they were detected at frequencies that were estimated to range between 0.4% and 0.002%. To the best of our knowledge, this represents the first demonstration of the successful isolation of mutation-reactive T cells from patients' peripheral blood prior to immune therapy, potentially providing the basis for designing personalized immunotherapies to treat patients with advanced cancer.

Limited evidence exists that humans mount a mutation-specific T cell response to epithelial cancers. We used a whole-exomic-sequencing-based approach to demonstrate that tumor-infiltrating lymphocytes (TIL) from a patient with metastatic cholangiocarcinoma contained CD4+ T helper 1 (T(H)1) cells recognizing a mutation in erbb2 interacting protein (ERBB2IP) expressed by the cancer. After adoptive transfer of TIL containing about 25% mutation-specific polyfunctional T(H)1 cells, the patient achieved a decrease in target lesions with prolonged stabilization of disease. Upon disease progression, the patient was retreated with a >95% pure population of mutation-reactive T(H)1 cells and again experienced tumor regression. These results provide evidence that a CD4+ T cell response against a mutated antigen can be harnessed to mediate regression of a metastatic epithelial cancer.

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.

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.

Purpose: Adoptive transfer of tumor-infiltrating lymphocytes (TIL) can mediate regression of metastatic melanoma. However, many patients with cancer are ineligible for such treatment because their TIL do not expand sufficiently or because their tumors have lost expression of antigens and/or MHC molecules. Natural killer (NK) cells are large granular lymphocytes that lyse tumor cells in a non-MHC-restricted manner. Therefore, we initiated in a clinical trial to evaluate the efficacy of adoptively transferred autologous NK cells to treat patients with cancers who were ineligible for treatment with TIL., Experimental Design: Patients with metastatic melanoma or renal cell carcinoma were treated with adoptively transferred in vitro activated autologous NK cells after the patients received a lymphodepleting but nonmyeloablative chemotherapy regimen. Clinical responses and persistence of the adoptively transferred cells were evaluated., Results: Eight patients were treated with an average of 4.7 × 10(10) (± 2.1 × 10(10)) NK cells. The infused cells exhibited high levels of lytic activity in vitro. Although no clinical responses were observed, the adoptively transferred NK cells seemed to persist in the peripheral circulation of patients for at least one week posttransfer and, in some patients, for several months. However, the persistent NK cells in the circulation expressed significantly lower levels of the key activating receptor NKG2D and could not lyse tumor cell targets in vitro unless reactivated with IL-2., Conclusions: The persistent NK cells could mediate antibody-dependent cell-mediated cytotoxicity without cytokine reactivation in vitro, which suggests that coupling adoptive NK cell transfer with monoclonal antibody administration deserves evaluation., (©2011 AACR)

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.

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 alpha and beta 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 alpha 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.

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

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.

Immunotherapy with fusion of dendritic cells (DCs) and tumour cells potentially confers the advantages of DC antigen-presenting functionality and a continuous source of unaltered tumour antigens. However, fusion using chemical or viral fusogens has been inefficient. We have recently developed a high throughput electrofusion technique with which very efficient fusion rates (15-54%) were observed in over 300 experiments, using a variety of murine and human tumour cell lines. The fused cells display a mature DC phenotype and express tumour-associated antigens. In two pre-clinical animal models (B16 melanoma transduced with the LacZ gene and the MCA 205 fibrosarcoma), a single vaccination of mice bearing tumours established in the lung, brain and skin resulted in tumour regression and prolongation of life. However, therapeutic efficacy required the administration of adjuvants such as IL-12 and OX-40R mAbs. Effective immunotherapy also required the delivery of fusion cells directly into lymphoid organs (spleen or lymph nodes). Using five defined human T cell lines derived from melanoma patients, allogeneic DCs of HLA-A2, HLA-DR4 and HLA-DR7 haplotypes fused with MART-1, gp100, tyrosinase and TRP-2 expressing 888 mel melanoma cells were analysed for their ability to stimulate specific cytokine (IFN-gamma and GM-CSF) secretion. DC-888 mel hybrids presented all tumour-associated epitopes to both CD4 and CD8 T cell lines in the context of MHC class II and I molecules, respectively. The therapeutic efficacy of a DC-tumour fusion vaccine is now being evaluated for the treatment of metastatic melanoma.

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-DR beta 1*0401, and HLA-DR beta 1*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-DR beta 1*0401- and HLA-DR beta 1*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.

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.

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.

It is generally considered that MHC class I-restricted antigens are recognized by CD8+ T cells, whereas MHC class II-restricted antigens are recognized by CD4+ T cells. In the present study, we report an MHC class I-restricted CD4+ T cell isolated from the tumor infiltrating lymphocytes (TILs) of a patient with metastatic melanoma. TIL 1383 I recognized HLA-A2+ melanoma cell lines but not autologous transformed B cells or fibroblasts. The antigen recognized by TIL 1383 I was tyrosinase, and the epitope was the 368-376 peptide. Antibody blocking assays confirmed that TIL 1383 I was MHC class I restricted, and the CD4 and CD8 coreceptors did not contribute significantly to antigen recognition. TIL 1383 I was weakly cytolytic and secreted cytokines in a pattern consistent with it being a Th1 cell. The avidity of TIL 1383 I for peptide pulsed targets is 10-100-fold lower than most melanoma-reactive CD8+ T cell clones. These CD4+ T cells may represent a relatively rare population of T cells that express a T-cell receptor capable of cross-reacting with an MHC class I/peptide complex with sufficient affinity to allow triggering in the absence of the CD4 coreceptor.

To study the induction of anti-"self" CD8+ T-cell reactivity against the tumor antigen gp100, we used a mouse transgenic for a chimeric HLA-A*0201/H-2 Kb molecule (A2/Kb). We immunized the mice with a recombinant vaccinia virus encoding a form of gp100 that had been modified at position 210 (from a threonine to a methionine) to increase epitope binding to the restricting class I molecule. Immunogens containing the "anchor-fixed" modification elicited anti-self CD8+ T cells specific for the wild-type gp100(209-217) peptide pulsed onto target cells. More important, these cells specifically recognized the naturally presented epitope on the surface of an A2/Kb-expressing murine melanoma, B16. These data indicate that anchor-fixing epitopes could enhance the function of recombinant virus-based immunogens.

Mac-1 (CD11b/CD18) is known to be involved in neutrophil (PMN) adhesion to endothelial cells and extracellular matrix. Although antibodies to CD 18 are being tested for therapy in humans, their role in PMN migration through the extracellular matrix is unknown. We used direct visualization to quantify PMN motility through reconstituted, three-dimensional gels of collagen type I. Gels were prepared with different concentrations of collagen (ranging from 0.1 to 1.0 mg/mL) and PMN migration was examined in the presence and absence of antibodies to CD18 (anti-CD18), with and without stimulation by N-formyl peptides. In low-concentration gels (<0.6 mg/mL), anti-CD18 had a significant influence on PMN migration, increasing motility in unstimulated PMN by 90% at 0.3 mg/mL collagen, and decreasing motility in N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN by 70% at 0.4 mg/mL collagen. But antiCD18 had no effect on the rate of cell migration through high-concentration collagen gels (>0.6 mg/mL). PMN migration through collagen gels is CD18-dependent but only under conditions of high hydration, suggesting that CD18-mediated effects (e.g., adhesion to gel fibers) are only important when the fiber density is relatively low. Anti-CD18 inhibited, but did not eliminate, the adhesion of fMLP-stimulated PMN to the surface of collagen gels, suggesting that cells use multiple mechanisms for gaining traction within the gel. Because of the multiple modes of interaction between motile cells and the deformable fiber matrix, blockade of one component, such as CD18, can enhance the rate of cell migration under one set of conditions, and inhibit under another.

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 alpha-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.

Tyrosinase-related protein 2 (TRP2) is a melanosomal enzyme expressed in most mammalian melanocytes and melanomas. This protein has been identified as a melanoma antigen recognized by tumor reactive CTLs derived from tumor infiltrating lymphocytes in the context of HLA-A31 and HLA-A33. The frequencies of these HLA-A alleles among melanoma patients in the United States is low (approximately 6% for HLA-A31 and approximately 2% for HLA-A33) compared with that of HLA-A*0201 (approximately 46%). Therefore, to extend significantly the use of TRP2-based immunotherapies for the treatment of patients with melanoma, we searched for new HLA-A*0201-restricted epitopes from this protein by screening TRP2-derived peptides for the induction of melanoma-reactive CTL. Fifty-one peptides were selected from TRP2 based on a permissive HLA-A*0201 binding motif, and the 21 peptides with the highest experimentally determined binding affinities were used to stimulate peripheral blood lymphocytes from HLA-A*0201+ melanoma patients in vitro. One peptide, TRP2(180-188) (SVYDFFVWL), induced CTLs from three of four patients that specifically recognized peptide-pulsed T2 cells, COS-7 cells expressing HLA-A*0201 and TRP2, and HLA-A2+ TRP2+ melanomas. TRP2(180-188) is identical to a previously identified TRP2 epitope recognized by murine melanoma-reactive CTLs in the context of H-2Kb. These results suggest that TRP2 may be useful for the development of murine tumor immunotherapy models and for the treatment of melanoma patients who are diverse in HLA expression.

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 gamma 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.

Recognition of the melanoma Ag gp100 by tumor-infiltrating lymphocytes (TIL) in vitro has been correlated with tumor regression in patients with metastatic melanoma treated with the adoptive transfer of TIL plus IL-2. Three common gp100 epitopes have been identified that are recognized in the context of HLA-A2 by TIL from different patients: G9154 (KTWGQYWQV), G9209 (ITDQVPFSV), and G9280 (YLEPGPVTA). Upon stimulation with these peptides, melanoma-reactive CTL could be induced in vitro from PBL of some HLA-A2+ melanoma patients. However, numerous restimulations were required, and specific reactivity could not be generated in many patients. Therefore, to enhance the immunogenicity of gp100 peptides, amino acid substitutions were introduced into G9154, G9209, and G9280 at HLA-A*0201-binding anchor positions, but not at TCR contact residues, to increase peptide class I MHC-binding affinity. Several modified gp100 peptides bound with greater affinity to HLA-A*0201 than unmodified peptides and were recognized by TIL specific for the natural epitopes. These peptides were used to sensitize PBL from HLA-A2+ melanoma patients in vitro using peptide-pulsed autologous PBMC as stimulators. After five weekly restimulations with either the native G9209 or G9280 peptide, melanoma-reactive CTL could only be induced from two of seven patients. However, amino acid substitutions in these peptides enabled the induction of melanoma-reactive CTL from all seven patients. These results suggest that modified gp100 peptides may be more immunogenic than the native epitopes, and may be useful in immunotherapy protocols for patients with melanoma.

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.

The migration of leukocytes through mucus must be an essential factor in the mucosal immune response, particularly during mucosal invasion by pathogens. We used two methods to evaluate the ability of neutrophils to migrate through human midcycle cervical mucus: (i) direct visualization of cell migration within freshly collected mucus samples and (ii) quantification of cell penetration through a layer of mucus overlying a monolayer of cultured intestinal epithelial cells from the T84 cell line. Neutrophils migrated rapidly through human midcycle cervical mucus: the random motility coefficient (mu) ranged from 1.2 to 5.6 x 10(-8) cm2/sec in different mucus samples. The rate and pattern of neutrophil migration in cervical mucus were identical to those in a structurally similar collagen gel (0.4 mg/ml). Therefore, we also quantitatively examined the migratory behavior of human neutrophils, monocytes, and lymphocytes from peripheral blood, as well as leukocytes isolated from semen, in collagen gels as a model of cell migration in cervical mucus. In the absence of stimulation, mu was approximately 10(-8) cm2/s for neutrophils and lymphocytes; monocytes and seminal leukocytes were immotile. In all cases, mu increased with the addition of FMLP. Furthermore, neutrophils were able to penetrate layers of mucus overlying monolayers of T84 cells when the gel thickness was less than approximately 500 microns. These results suggest that leukocytes migrate effectively through mucus, a process which may be related to their ability to function in the mucosal immune system or to serve as vectors for the translocation of infectious pathogens like HIV.

Leukocytes must migrate through tissues to fulfill their role in the immune response, but direct methods for observing and quantifying cell motility have mostly been limited to migration on two-dimensional surfaces. We have now developed methods for examining neutrophil movement in a three-dimensional gel containing 0.1 to 0.7 mg/ml rat tail tendon collagen. Neutrophil-populated collagen gels were formed within flat glass capillary tubes, permitting direct observation with light microscopy. By following the tracks of individual cells over a 13.5-min observation period and comparing them to a stochastic model of cell movement, we quantified cell speed within a given gel by estimating a random motility coefficient (mu) and persistence time (P). The random motility coefficient changed significantly with collagen concentration in the gel, varying from 1.6 to 13.3 x 10(-9) cm2/s, with the maximum occurring at a collagen gel concentration of 0.3 mg/ml. The methods described may be useful for studying tissue dynamics and for evaluating the mechanism of cell movement in three-dimensional gels of extracellular matrix (ECM) molecules.