In vivo anti-melanoma efficacy of allo-restricted CTLs specific for melanoma expanded by artificial antigen-presenting cells

Cytotoxic CD8⁺ T cells are key effectors in the immunotherapy of malignant and viral diseases. However, autologous T cell responses to tumor antigens presented by self-MHC are usually weak and ineffective. Allo-restricted T cells represent a potent source of tumor-specific T cells for adoptive immunotherapy. This study reports in vivo anti-melanoma efficacy of the pTRP2-specific allo-restricted CTLs expanded from the BALB/c splenocytes by multiple stimulations with aAPCs made by coating H-2K^b-Ig/pTRP2 dimeric complexes, anti-CD28 antibody, 4-1BBL molecules and CD83 molecules to cell-sized latex beads. The induced allo-restricted CTLs exhibited specific lysis against RMA-S cells pulsed with the peptide pTRP2 and H-2K^b+ melanoma cells expressing TRP2, while a murine Lewis lung carcinoma cell line 3LL could not be recognized by the CTLs. The peptide-specific activity was inhibited by anti-H-2K^b monoclonal antibody Y3. Adoptive transfer of the allo-restricted CTLs specific for malignant melanoma expanded by the aAPCs can mediate effective anti-melanoma response in vivo. These results suggested that the specific allo-restricted CTLs expanded by aAPCs coated with an MHC-Ig/peptide complex, anti-CD28 antibody, 4-1BBL and CD83 could be a potential option of specific immunotherapy for patients with malignant melanoma. X.-l. Lu and X.-b. Jiang have contributed equally to this work. An erratum to this article can be found at     

A new tyrosinase epitope recognized in the HLA-B*4002 context by CTL from melanoma patients

Melanoma reactive CTL were obtained by stimulating PBL from a melanoma patient in remission since 1994 following adjuvant TIL immunotherapy, with the autologous melanoma cell line. They were cloned by limiting dilution. One CTL clone recognized melanoma cell lines expressing tyrosinase and the B*4002 molecule, either spontaneously or upon transfection. We demonstrated that this clone recognizes the tyrosinase-derived nonapeptide 316-324 (ADVEFCLSL) and the overlapping decapeptide 315–324 (SADVEFCLSL). We derived two distinct additional specific CTL clones from this same patient that were also reactive against B*4002 melanoma cell lines, suggesting a relative diversity of this specific repertoire in this patient. Stimulating PBMC derived from four additional B*4002 melanoma patients with the tyrosinase 316–324 nonapeptide induced the growth of specific cells for two of the patients, demonstrating the immunogenicity of this new epitope. Our data show that this nonapeptide is a new tool that could be used to generate melanoma-specific T cells for adoptive immunotherapy or serve as a peptide vaccine for HLA-B*4002 melanoma patients.     

Active specific immunotherapy with hapten-modified autologous melanoma cell vaccine

 We have developed a novel approach to cancer immunotherapy – an autologous whole-cell vaccine modified with the hapten dinitrophenyl (DNP). This approach elicits significant inflammatory responses in metastatic sites and some objective tumor responses. Post-surgical adjuvant immunotherapy with DNP-modified melanoma vaccine in a setting of micrometastatic disease produces significant survival prolongation in stage III melanoma patients. Histologically, the inflammatory responses of the tumor consist of infiltration by lymphocytes, the majority of which are CD8⁺, HLA-DR⁺ T cells. T cells from these lesions tend to have mRNA for interferon γ. T cell receptor analysis suggests that the tumor-infiltrating T cells are clonally expanded. DNP-modified vaccine also induces T cells in the peripheral blood, which respond to DNP-modified autologous cells in a hapten-specific, MHC-restricted manner. Moreover, a T cell line generated from these lymphocytes responded to only a single HPLC fraction of MHC-associated, DNP-modified tumor peptides. Since inflammatory responses in metastases were not consistently associated with dramatic tumor regression, we considered the possibility of immunosuppression at the tumor site. We found that mRNA for the anti-inflammatory cytokine, interleukin-10 (IL-10) is expressed in most metastatic melanoma tissues and subsequently demonstrated that IL-10 protein is produced by melanoma cells. Thus the efficacy of DNP vaccine could be further enhanced by inhibition of IL-10 production or binding. Finally, we expect these results obtained with melanoma to be applicable to other human cancers. Received: 6 August 1996 / Accepted: 20 September 1996     

Lymphocytes cytotoxic to uveal and skin melanoma cells from peripheral blood of ocular melanoma patients

Summary To study antitumor immunity in patients with choroidal melanoma, T cells were generated from the peripheral blood of choroidal melanoma patients by mixed lymphocyte/tumor cell culture (MLTC). Because autologous tumors are generally unavailable, an allogeneic choroidal melanoma cell line, OCM-1, was used as the specific stimulus. Lymphocyte cultures from 27 patients were characterized by cell-surface phenotypes, patterns of reactivity towards cells of the melanocytic origin and T-cell-receptor gene usage. Antimelanoma reactivity was found in cell-sorter-purified CD4⁺ and CD8⁺ T cell subsets. To analyze this reactivity, sorter-purified CD4⁺ and CD8⁺ cells from a MLTC were cloned by limiting dilution in the presence of exogenous interleukin-2 and interleukin-4 as well as irradiated OCM-1. Under these conditions, CD4⁺ T cells did not proliferate, perhaps because of the absence of antigen-presenting cells. However, CD8⁺ grew vigorously and 29 cytolytic CD8⁺ T cell clones were isolated. On the basis of their pattern of lysis of OCM-1, a skin melanoma cell line M-7 and its autologous lymphoblastoid cell line LCL-7, the clones were categorized into three groups. Group 1, representing 52% of the clones, lysed all three target cells, and are alloreactive. However, since OCM-1 and M-7 did not share class I antigens, these clones recognized cross-reactive epitope(s) of the histocompatibility locus antigen (HLA) molecule. Group 2, constituting 28% of the clones, lysed both the ocular and skin melanoma cell lines but not LCL-7, and were apparently melanoma-specific. Unlike classical HLA-restricted cytolytic T lymphocytes, these T cells might mediate the lysis of melanoma cells via other ligands or a more degenerate type of HLA restriction. For the latter, the HLA-A2 and -A28 alleles would have to act interchangeably as the restriction element for shared melanoma-associated antigen(s). Group 3, representing only 10% of the T cell clones, was cytotoxic only to OCM-1, but not to M-7 or LCL-7. These clones may recognize antigens unique to ocular melanoma cells. Our data suggest that choroidal melanoma patients can recognize melanoma-associated antigens common to both ocular and cutaneous melanoma cells, and presumbly their autologous tumor. Thus, choroidal melanoma, like its skin counterpart, may be responsive to immunotherapeutic regimens such as active specific or adoptive cellular immunotherapy.This work is supported by National Institutes of Health research grants CA 36 233 and EY 9031, the Lucy Adams Memorial Fund and support from the Concern Foundation     

Incubation of antigen-sensitized T lymphocytes activated with bryostatin 1 + ionomycin in IL-7 + IL-15 increases yield of cells capable of inducing regression of melanoma metastases compared to culture in IL-2

Regression of established tumors can be induced by adoptive immunotherapy (AIT) with tumor draining lymph node (DLN) lymphocytes activated with bryostatin and ionomycin (B/I). We hypothesized that B/I-activated T cells cultured in IL-7 + IL-15 might proliferate and survive in culture better than cells cultured in IL-2, and that these cells would have equal or greater anti-tumor activity in vivo. Tumor antigen-sensitized DLN lymphocytes from either wild-type or T cell receptor transgenic mice were harvested, activated with B/I, and expanded in culture with either IL-2, IL-7 + IL-15 or a regimen of alternating cytokines. Cell yields, proliferation, apoptosis, phenotypes, and in vitro responses to tumor antigen were compared for cells grown in different cytokines. These T cells were also tested for anti-tumor activity against melanoma lung metastases established by prior i.v. injection of B16 melanoma cells. IL-7 + IL-15 or alternating cytokines resulted in much faster and prolonged proliferation and much less apopotosis of B/I-activated T cells than culturing the same cells in IL-2. This resulted in approximately tenfold greater yields of viable cells. Culture in IL-7 + IL-15 yielded higher proportions of CD8+ T cells and a higher proportion of cells with a central memory phenotype. Despite this, T cells grown in IL-7 + IL-15 had higher IFN-γ release responses to tumor antigen than cells grown in IL-2. Adoptive transfer of B/I-activated T cells grown in IL-7 + IL-15 or the alternating regimen had equal or greater efficacy on a “per-cell” basis against melanoma metastases. Activation of tumor antigen-sensitized T cells with B/I and culture in IL-7 + IL-15 is a promising modification of standard regimens for production of T cells for use in adoptive immunotherapy of cancer.     

Generation of human-melanoma-specific T lymphocyte clones defining novel cytolytic targets with panels of newly established melanoma cell lines

Melanoma is a cancer where the immune system is believed to play an important role in the control of malignant cell growth. To study the variability of the immune response in melanoma patients, we derived melanoma cell lines from several HLA-A2⁺ and HLA-A2^– patients. The melanoma cell lines studied were designated FM3, FM6, FM9, FM28, FM37, FM45, FM55_P, FM55_M1 and FM55_M2 and were established from eight metastatic tumors as well as from one primary tumor from a total of seven different patients. On the basis of the ability of tumor cells to induce specific cytotoxic T lymphocytes (CTL) from peripheral blood lymphocytes (PBL) in mixed lymphocyte/tumor culture with HLA-A2⁺ melanoma cells, the FM3 cell line was characterized as highly immunogenic. To investigate the expression of different melanoma-associated antigens recognized by CTL on different melanoma cell lines, we selected the cell line FM3 for restimulation and further T cell cloning experiments. The lytic activity of CTL clones with good proliferative activity was examined using a panel of HLA-A2⁺ and HLA-A2^– melanoma cell lines. None of the tested HLA-A2^– melanoma cell lines were susceptible to lysis by the CTL clones, whereas allogeneic HLA-A2⁺ melanoma cell lines were lysed only by a few CTL clones. On the basis of their reactivity with different melanoma cell lines, it was possible to divide the present CTL clones into at least four groups suggesting the recognition of at least four different antigens. Three of these target structures probably are different from already-described HLA-A2-restricted melanoma-associated antigens, because their expression in the different melanoma cell lines do not correlate with the recognition of melanoma cells by these CTL. The results first indicate that poorly immunogenic melanoma cells may express melanoma-associated antigens, and also suggest that, by using CTL clones obtained against different HLA-class-I-matched melanoma cells, it is possible to define such antigens.     

Cancer/testis antigens can be immunological targets in clonogenic CD133<Superscript>+</Superscript> melanoma cells

“Cancer stem cells” that resist conventional treatments may be a cause of therapeutic failure in melanoma. We report a subpopulation of clonogenic melanoma cells that are characterized by high prominin-1/CD133 expression in melanoma and melanoma cell lines. These cells have enhanced clonogenicity and self-renewal in vitro, and serve as a limited in vitro model for melanoma stem cells. In some cases clonogenic CD133⁺ melanoma cells show increased expression of some cancer/testis (CT) antigens. The expression of NY-ESO-1 in an HLA-A2 expressing cell line allowed CD133⁺ clonogenic melanoma cells to be targeted for killing in vitro by NY-ESO-1-specific CD8⁺ T-lymphocytes. Our in vitro findings raise the hypothesis that if melanoma stem cells express CT antigens in vivo that immune targeting of these antigens may be a viable clinical strategy for the adjuvant treatment of melanoma. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Melanomas with concordant loss of multiple melanocytic differentiation proteins: immune escape that may be overcome by targeting unique or undefined antigens

Melanoma-reactive HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) lines generated in vitro lyse autologous and HLA-matched allogeneic melanoma cells and recognize multiple shared peptide antigens from tyrosinase, MART-1, and Pmel17/gp100. However, a subset of melanomas fail to be lysed by these T cells. In the present report, four different HLA-A*0201⁺ melanoma cell lines not lysed by melanoma-reactive allogeneic CTL have been evaluated in detail. All four are deficient in expression of the melanocytic differentiation proteins (MDP) tyrosinase, Pmel17/gp100, gp75/trp-1, and MART-1/Melan-A. This concordant loss of multiple MDP explains their resistance to lysis by melanoma-reactive allogeneic CTL and confirms that a subset of melanomas may be resistant to tumor vaccines directed against multiple MDP-derived epitopes. All four melanoma lines expressed normal levels of HLA-A*0201, and all were susceptible to lysis by xenoreactive-peptide-dependent HLA-A*0201-specific CTL clones, indicating that none had identifiable defects in antigen-processing pathways. Despite the lack of shared MDP-derived antigens, one of these MDP-negative melanomas, DM331, stimulated an effective autologous CTL response in vitro, which was restricted to autologous tumor reactivity. MHC-associated peptides isolated by immunoaffinity chromatography from HLA-A1 and HLA-A2 molecules of DM331 tumor cells included at least three peptide epitopes recognized by DM331 CTL and restricted by HLA-A1 or by HLA-A*0201. Recognition of these CTL epitopes cannot be explained by defined, shared melanoma antigens; instead, unique or undefined antigens must be responsible for the autologous-cell-specific anti-melanoma response. These findings suggest that immunotherapy directed against shared melanoma antigens should be supplemented with immunotherapy directed against unique antigens or other undefined antigens, especially in patients whose tumors do not express MDP. Received: 31 October 1997 / Accepted: 4 August 1999     

GD3-reactive antibodies can inhibit the lysis of autologous tumor cells by tumor-infiltrating lymphocytes

Summary GD3 is expressed in high concentrations on melanoma cells and may serve as a useful target antigen for mAb-mediated immunotherapy. Monoclonal antibodies (mAbs) against GD3 stimulate cell-mediated immune responses against tumor cells in vitro and this activity may contribute to antitumor effects in patients with melanoma treated with GD3-reactive mAbs. In the present study the effects of GD3-reactive mAbs on autologous tumor cell lysis by a human melanoma-derived tumor-infiltrating lymphocyte (TIL) population were examined. Unlike results reported for other GD3⁺ T cells isolated from melanoma patients, the tumor-specific lytic activity of the TIL line was inhibited by incubation with mAbs against GD3. Other melanoma-reactive mAbs, including those against GD2 and the high-molecular-weight melanoma-associated Ag, had no effect on the TIL lytic activity. Overall, these results indicate that mAbs against GD3 may have different effects on T cell/tumor cell interactions.     

Generation of specific anti-melanoma reactivity by stimulation of human tumor-infiltrating lymphocytes with MAGE-1 synthetic peptide

The MAGE-1 gene encodes a tumor-specific antigen, MZ2-E, which is recognized by cloned, specific cytolytic T cells (CTL) derived from the peripheral blood of a patient with melanoma. We have produced a MAGE-1-specific CTL line derived from the tumor-infiltrating lymphocytes (TIL) of a melanoma patient by weekly restimulation with autologous EBV-B cells pulsed with the synthetic HLA-A1-restricted MAGE-1 epitope nonapeptide EADPTGHSY. The 1277. A TIL line grew in long-term culture in low-dose interleukin-2 (IL-2) and IL-4, and exhibited antigen-specific, MHC-class-I-restricted lysis of HLA-A1-bearing MAGE-1⁺ cell lines. Cytolysis of target cells pulsed with the synthetic MAGE-1 decapeptide KEADPTGHSY was superior to that of cells pulsed with the immunodominant nonapeptide. Single amino-acid or even side-chain substitutions in the immunodominant nonamer abrogated cytolysis. 1277. A TIL specifically secreted tumor necrosis factor after co-incubation with HLA-A1-expressing MAGE-1⁺ cell lines or fresh tumor. These data suggest that tumor-antigen-specific, MHC-restricted CTL may be grown from TIL in the presence of synthetic epitope peptides and expanded for adoptive immunotherapy in melanoma patients.     

Melanoma-specific tumor-infiltrating lymphocytes but not circulating melanoma-specific T cells may predict survival in resected advanced-stage melanoma patients

Purpose: To study the effect of autologous tumor cell vaccinations on the presence and numbers of circulating CD8⁺ T cells specific for tumor-associated antigens (TAA) in metastatic melanoma patients. To investigate the correlation between the presence of tumor-infiltrating lymphocytes (TIL) and circulating TAA-specific CD8⁺ T cells before and after autologous tumor cell vaccination with overall survival. Experimental design: Twenty-five stage III and resected stage IV metastatic melanoma patients were adjuvantly treated with a series of intracutaneously injected autologous tumor cell vaccinations, of which the first two contained BCG as an immunostimulatory adjuvant. Tumor samples and blood samples obtained before and after vaccination of these patients were studied for the presence of TAA-specific T cells using HLA-tetramers and results were correlated with survival. Results: In 5 of 17 (29%) melanoma patients, circulating TAA-specific T cells were detectable prior to immunizations. No significant changes in the frequency and specificity were found during the treatment period in all patients. Presence of circulating TAA-specific T cells was not correlated with survival (log rank, P=0.215). Inside melanoma tissue, TAA-specific TIL could be detected in 75% of 16 available tumor samples. In case of detectable TAA-specific TIL, median survival was 22.5 months compared to median survival of 4.5 months in case of absence of TAA-specific T cells (log rank, P=0.0094). In none of the patients, TAA-specific T cells were found both in tumor tissue and blood at the same time. Conclusions: These data suggest that the presence of TAA-specific TILs forms a prognostic factor, predicting improved survival in advanced-stage melanoma patients. The absence of TAA-specific T cells in the circulation suggests that homing of the tumor-specific T cell population to the tumor site contributes to the effectiveness of antitumor immunity. J.B.A.G. Haanen and A. Baars contributed equally to this work.     

HSP70iQ435A to subdue autoimmunity and support anti-tumor responses

Developing immunosuppressive therapies for autoimmune diseases comes with a caveat that immunosuppression may promote the risk of developing other conditions or diseases. We have previously shown that biolistic delivery of an expression construct encoding inducible HSP70 (HSP70i) with one amino acid modification in the dendritic cell (DC) activating moiety 435–445 (HSP70i_Q435A) to mouse skin resulted in significant immunosuppressive activity of autoimmune vitiligo, associated with fewer tissue infiltrating T cells. To prepare HSP70i_Q435A as a potential therapeutic for autoimmune vitiligo, in this study we evaluated whether and how biolistic delivery of HSP70i_Q435A in mice affects anti-tumor responses. We found that HSP70i_Q435A in fact supports anti-tumor responses in melanoma-challenged C57BL/6 mice. Biolistic delivery of the HSP70i_Q435A-encoding construct to mice elicited significant anti-HSP70 titers, and anti-HSP70 IgG and IgM antibodies recognize surface-expressed and cytoplasmic HSP70i in human and mouse melanoma cells. A peptide scan revealed that the anti-HSP70 antibodies recognize a specific C-terminal motif within the HSP70i protein. The antibodies elicited surface CD107A expression among mouse NK cells, representative of antibody-mediated cellular cytotoxicity (ADCC), supporting the concept, that HSP70i_Q435A-encoding DNA elicits a humoral response to the stress protein expressed selectively on the surface of melanoma cells. Thus, besides limiting autoimmunity and inflammation, HSP70i_Q435A elicits humoral responses that limit tumor growth and may be used in conjunction with immune checkpoint inhibitors to not only control tumor but to also limit adverse events following tumor immunotherapy.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12192-021-01229-x.     

Antitumor effects of a novel monoclonal antibody with high binding affinity to ganglioside GD3

Ganglioside GD3, which is one of the major gangliosides expressed on the cell surface human tumors of neuroectodermal origin, has been studied as a target molecule for passive immunotherapy. We established ten kinds of anti-GD3 monoclonal antibodies (mAb) of the mouse IgG3 subclass by immunization with purified GD3 and melanoma cells. One of the established mAb, KM641, showed major reactivity with GD3 and minor reactivity with GQ1b out of 11 common gangliosides in an enzymelinked immunosorbent assay. Immunostaining of gangliosides, separated on thin-layer chromatography plates, using KM641 revealed that most of the melanoma cell lines contained immunoreactive GD3 and GD3-lactone at a high level, but only the adrenal gland and the urinary bladder out of 21 human normal tissues had immunoreactive GD3. In immunofluorescence, KM641 bound to a variety of living tumor cell lines especially melanoma cells, including some cell lines to which another anti-GD3 mAb R24, established previously, failed to bind. High-affinity binding of KM641 to a tumor cell line was quantified by Scatchard analysis (K _d = 1.9×10^–8 M). KM641 exerted tumor-killing activity in the presence of effector cells or complement against melanoma cells expressing GD3 at a high level. Not only natural killer cells but also polymorphonuclear cells were effective as the effector cells in antibody-dependent cellular cytotoxicity. Intravenous injection of KM641 markedly suppressed the tumor growth of a slightly positive cell line, C24.22 (7.2×10⁵ binding sites/cell), as well as a very GD3-positive cell line, G361 (1.9×10⁷ binding sites/cell), inoculated intradermally in nude mice. KM641, characterized by a high binding affinity for GD3, has the potential to be a useful agent for passive immunotherapy of human cancer.     

CD4+ T cells kill HLA-class-II-antigen-positive melanoma cells presenting peptide in vitro

Purpose: Most melanoma cell lines express HLA class II antigens constitutively or can be induced to do so with interferon γ (IFNγ). We have previously demonstrated that peptide-specific CD4⁺ T cells proliferate in response to HLA-class-II-antigen-mediated peptide presentation by melanoma cells in vitro and produce interleukin-10 (IL-10) and (IFNγ). We asked whether the responding T cells kill the tumor cells and, if so, whether direct cell contact was required. Methods: Two HLA class II⁺ melanoma cell lines derived from metastases were co-cultured with a human CD4⁺ T cell clone specific for influenza hemagglutinin peptide (HA). T cells, melanoma, and HA were co-cultured for 48 h. Melanoma cells with and without HA and/or T cells served as controls. After 36 h, the medium was removed for cytokine analysis by enzyme-linked immunosorbent assay (ELISA). Twelve hours later non-adherent cells were washed away and the adherent melanoma cells were trypsinized and counted. Dual-chamber culture plates were used to determine whether cell contact and/or exposure to cytokine were required for tumor cell death. Results: Melanoma cell counts were over 80% lower in wells containing T cells than in wells with melanoma and peptide alone (P < 0.05). ELISA of supernatants revealed production of IFNγ and IL-10 by the responding T cells. Direct T cell contact with tumor cells was not required for tumor cell death, as melanoma cells were killed when they shared medium but had no contact with T cells responding to peptide presentation by HLA-class-II-antigen-positive melanoma cells in a separate chamber. Blocking antibody to IFNγ but not IL-10 prevented melanoma cell death at levels of cytokine similar to that present in co-culture assays. Conclusions: Peptide-specific CD4⁺ T cells kill melanoma cells in vitro when they recognize peptide presented by the tumor cell in the context of HLA class II antigen. Direct cell contact is not required, suggesting that it is a cytokine-mediated event. Immunotherapy, using primed CD4⁺ T cells and peptide, may be beneficial in patients whose tumors express HLA class II antigens or can be induced to do so with IFNγ. Received: 1 July 1999 / Accepted: 17 September 1999     

Generation of cytotoxic effector cells against human melanoma

Metastatic or tumor-draining lymph nodes from six of nine melanoma patients undergoing lymph node dissection for metastatic melanoma generated cytotoxic T cells against autologous melanoma when these lymph node cells were treated by in vitro sensitization and recombinant interleukin-2 (IL-2). During the initial lymphocyte culture (2–6 weeks), cross-reactivity with autologous tumor cells, K562 and Daudi cells was usually noted. Cold-target inhibition assay with K562 and Daudi showed K562/Daudi-associated antigens on melanoma cells. During the later phase of lymphocyte culture with repeated in vitro sensitization (over 6–10 weeks), cytotoxicity was noted against autologous and allogeneic melanoma cells but not against K562, Daudi cells or autologous fibroblasts. Repeated in vitro sensitization resulted in the selection of specific cytotoxic lymphocytes against melanoma. Cold-target inhibition assay with autologous and allogeneic melanoma cells revealed shared and individual antigens. Using blocking monoclonal antibodies, MHC-restricted killing was noted in the autologous system. Further, both the autologous and allogeneic systems could be mediated through adhesion molecules such as ICAM-1 and LFA-3 on melanoma cells and LFA-1 on T cells. This study suggests that a constellation of cytotoxic effector cells and melanoma-associated antigens may be pivotal in tumor killing. Thus, future adoptive immunotherapy should modulate and enhance this complex interaction.This work was supported by an Elsa, U. Pardee Foundation grant, the Arizona Chronic Disease Research Commission grant and partly by grant CA23074 from the National Institutes of Health, Bethesda, MD, 20892     

Focal adhesion kinase as an immunotherapeutic target

Background  Focal adhesion kinase (FAK) is a ubiquitously expressed non-receptor tyrosine kinase involved in cancer progression and metastasis that is found overexpressed in a large number of tumors such as breast, colon, prostate, melanoma, head and neck, lung and ovary. Thus, FAK could be an attractive tumor associated antigen (TAA) for developing immunotherapy against a broad type of malignancies. In this study, we determined whether predicted T cell epitopes from FAK would be able to induce anti-tumor immune cellular responses. Methods  To validate FAK as a TAA recognized by CD4 helper T lymphocytes (HTL), we have combined the use of predictive peptide/MHC class II binding algorithms with in vitro vaccination of CD4 T lymphocytes from healthy individuals and melanoma patients. Results  Two synthetic peptides, FAK_143–157 and FAK_{1,000–1,014}, induced HTL responses that directly recognized FAK-expressing tumor cells and autologous dendritic cells pulsed with FAK-expressing tumor cell lysates in an HLA class II-restricted manner. Moreover, since the FAK peptides were recognized by melanoma patient’s CD4 T cells, this is indicative that T cell precursors reactive with FAK already exist in peripheral blood of these patients. Conclusions  Our results provide evidence that FAK functions as a TAA and describe peptide epitopes that may be used for designing T cell-based immunotherapy for FAK-expressing cancers, which could be used in combination with newly developed FAK inhibitors.     

Skin‐resident memory T cells as a potential new therapeutic target in vitiligo and melanoma

Tissue‐resident memory T (T_RM) cells are abundant in the memory T cell pool and remain resident in peripheral tissues, such as the skin, where they act as alarm sensors or cytotoxic killers. T_RM cells persist long after the pathogen is eliminated and can respond rapidly upon reinfection with the same antigen. When aberrantly activated, skin‐located T_RM cells have a profound role in various skin disorders, including vitiligo and melanoma. Autoreactive T_RM cells are present in human lesional vitiligo skin and mouse models of vitiligo, which suggests that targeting these cells could be effective as a durable treatment strategy for vitiligo. Furthermore, emerging evidence indicates that induction of melanoma‐reactive T_RM cells is needed to achieve effective protection against tumor growth. This review highlights seminal reports about skin‐resident T cells, focusing mainly on their role in the context of vitiligo and melanoma, as well as their potential as therapeutic targets in both diseases.     

Immunosensitization with a Bcl-2 small molecule inhibitor

Several tumor immunotherapy approaches result in a low percentage of durable responses in selected cancers. We hypothesized that the insensitivity of cancer cells to immunotherapy may be related to an anti-apoptotic cancer cell milieu, which could be pharmacologically reverted through the inhibition of antiapoptotic Bcl-2 family proteins in cancer cells. ABT-737, a small molecule inhibitor of the antiapoptotic proteins Bcl-2, Bcl-w and Bcl-x_L, was tested for the ability to increase antitumor immune responses in two tumor immunotherapy animal models. The addition of systemic therapy with ABT-737 to the immunization of BALB/c mice with tumor antigen peptide-pulsed dendritic cells (DC) resulted in a significant delay in CT26 murine colon carcinoma tumor growth and improvement in survival. However, the addition of ABT-737 to either a vaccine strategy involving priming with TRP-2 melanoma antigen peptide-pulsed DC and boosting with recombinant Listeria monocytogenes expressing the same melanoma antigen, or the adoptive transfer of TCR transgenic cells, did not result in superior antitumor activity against B16 murine melanoma. In vitro studies failed to demonstrate increased cytotoxic lytic activity when testing the combination of ABT-737 with lymphokine activated killer (LAK) cells, or the death receptor agonists Fas, TRAIL-ligand or TNF-alpha against the CT26 and B16 cell lines. In conclusion, the Bcl-2 inhibitor ABT-737 sensitized cancer cells to the antitumor effect of antigen-specific immunotherapy in a vaccine model for the CT26 colon carcinoma in vivo but not in two immunotherapy strategies against B16 melanoma.     

The antibody response against MART‐1 differs in patients with melanoma‐associated leucoderma and vitiligo

Patients with melanoma may develop skin depigmentation spontaneously or following therapy, referred to as melanoma‐associated leucoderma (MAL). As clinical presentation of MAL may precede primary/metastatic melanoma detection, recognition of MAL is important to prevent its misdiagnosis as vitiligo and the subsequent application of immunosuppressive treatment. To reveal the immunity involved in MAL development, we investigated the presence of antibody and T‐cell immune responses directed against the melanocyte‐differentiation‐antigens MART‐1 (Melan‐A), tyrosinase and gp100 in patients with MAL, as compared to patients with vitiligo. Autoantibodies to gp100 and tyrosinase were commonly found in both diseases. Interestingly, MART‐1 antibodies were only present in patients with MAL. Melanocyte antigen‐specific T cells were found in all patients, with relatively more specific T cells in patients with active vitiligo. Although MAL and vitiligo may appear clinically similar, our results indicate that the humoral immune responses against MART‐1 differ between these diseases, which can help to differentiate MAL from vitiligo.