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SCLC is a highly aggressive tumor with a 5-year survival rate of less than 6%. A heterogeneous disease, SCLC is classified into four subtypes that include tumors with neuroendocrine and non-neuroendocrine features. Immune checkpoint blockade has been recently added for the frontline treatment of SCLC; however, this therapy has only led to modest clinical improvements. The lack of clinical benefit in a cancer type known to have a high tumor mutational burden has been attributed to poor T-cell infiltration and low expression of MHC-class I in most SCLC tumors. In an attempt to devise a more effective immunotherapeutic regimen, this study investigated an alternate approach on the basis of the use of the clinical-stage interleukin-15 superagonist, N-803.Preclinical models of SCLC spanning all molecular subtypes were used to evaluate the susceptibility of SCLC to natural killer (NK)-mediated lysis in vitro, including NK cells activated by N-803. Antitumor activity of N-803 was evaluated in vivo with a xenograft model of SCLC.In vitro and in vivo data revealed differences in susceptibility of SCLC subtypes to lysis by NK cells and that NK cells activated by N-803 effectively lyse SCLC tumor cells across all variant subtypes, regardless of their expression of MHC-class I.These findings highlight the potential of a novel immune-based intervention using a cytokine-based therapeutic option for the treatment of SCLC. We hypothesize that N-803 may provide benefit to most patients with SCLC, including those with immunologically cold tumors lacking MHC expression.

High affinity natural killer cells (haNKs) are a cell therapy product capable of mediating both direct and antibody-dependent cell-mediated cytotoxicity (ADCC). These cells may be particularly useful in tumors that escape T-cell anti-tumor immunity by harboring antigen processing and presentation defects. Here, we demonstrated that haNKs directly kill both HPV-positive and negative head and neck squamous cell carcinoma cells. Variable tumor cell sensitivity to haNK direct cytotoxicity did not correlated with MHC class I chain-related protein A or B (MICA or MICB) expression. Importantly, haNK killing was significantly enhanced via ADCC mediated by cetuximab or avelumab in cells with higher baseline EGFR or PD-L1 expression, respectively. The ability of IFNγ to induce tumor cell PD-L1 expression correlated with enhanced PD-L1-specific ADCC. IFNγ induced neither tumor cell EGFR expression nor EGFR-specific ADCC. Although a single dose of 8 Gy IR did not appear to directly enhance susceptibility to haNK killing alone, enhanced PD-L1- and EGFR-mediated ADCC after IR correlated with increased PD-L1 and EGFR expression in one of four models. This pre-clinical evidence supports the investigation of haNK cellular therapy in combination with ADCC-mediating mAbs, with or without IR, in the clinical trial setting for patients with advanced HNSCCs. Given the MHC-unrestricted nature of this treatment, it may represent an opportunity to treat patients with non-T-cell inflamed tumors.

PurposeDespite the strong interest in combining stereotactic ablative radiation therapy (SAR) with immunotherapy, limited data characterizing the systemic immune response after SAR are available. We hypothesized that the systemic immune responseto SAR would differ by irradiated site owing to inherent differences in the microenvironment of various organs.Methods and materialsPatients receiving SAR to any organ underwent prospective blood banking before and 1 to 2weeks after SAR. Peripheral blood mononuclear cells (PBMCs) and serum were isolated. PBMCs were stained with fluorophore-conjugated antibodies against T and natural killer (NK) cell markers. Cells were interrogated by flow cytometry, and the results were analyzed using FlowJo software. Serum cytokine and chemokine levels were measured using Luminex. We analyzed the changes from before to after therapy using paired t tests or 1-way analysis of variance (ANOVA) with Bonferroni's post-test.ResultsA total of 31 patients had evaluable PBMCs for flow cytometry and 37 had evaluable serum samples for Luminex analysis. The total number of NK cells andcytotoxic (CD56dimCD16+) NK cells decreased (P=.02) and T-cell immunoglobulin- and mucin domain-containing molecule-3-positive (TIM3+) NK cells increased (P=.04) after SAR to parenchymal sites (lung and liver) but not tobone or brain. The total memory CD4+ T cells, activated inducible co-stimulator-positive and CD25+CD4+ memory T cells, and activated CD25+CD8+ memory Tcells increased after SAR to parenchymal sites but not bone or brain. The circulating levels of tumor necrosis factor-α (P=.04) and multiple chemokines, including RANTES (P=.04), decreased after SAR to parenchymal sites but not bone or brain.ConclusionsOur data suggest SAR to parenchymal sites induces systemic immune changes, including a decrease in total and cytotoxic NK cells, an increase in TIM3+ NK cells, and an increase in activated memory CD4+ and CD8+ T cells. SAR to nonparenchymal sites did not induce these changes. By comparing the immune response after radiation to different organs, our data suggest SAR induces systemic immunologic changes that are dependent on the irradiated site.

Human papillomavirus (HPV) is associated with the etiology of cervical carcinoma, head and neck squamous cell carcinoma, and several other cancer types. Vaccines directed against HPV virus-like particles and coat proteins have been extremely successful in the prevention of cervical cancer through the activation of host HPV-specific antibody responses; however, HPV-associated cancers remain a major public health problem. The development of a therapeutic vaccine will require the generation of T-cell responses directed against early HPV proteins (E6/E7) expressed in HPV-infected tumor cells. Clinical studies using various vaccine platforms have demonstrated that both HPV-specific human T cells can be generated and patient benefit can be achieved. However, no HPV therapeutic vaccine has been approved by the Food and Drug Administration to date. One method of enhancing the potential efficacy of a therapeutic vaccine is the generation of agonist epitopes. We report the first description of enhancer cytotoxic T lymphocyte agonist epitopes for HPV E6 and E7. While the in silico algorithm revealed six epitopes with potentially improved binding to human leukocyte antigen–A2 allele (HLA-A2)–Class I, 5/6 demonstrated enhanced binding to HLA-Class I in cell-based assays and only 3/6 had a greater ability to activate HPV-specific T cells which could lyse tumor cells expressing native HPV, compared to their native epitope counterparts. These agonist epitopes have potential for use in a range of HPV therapeutic vaccine platforms and for use in HPV-specific adoptive T- or natural killer–cell platforms.

Summary Background Therapeutic cancer vaccines have shown activity in metastatic castration-resistant prostate cancer (mCRPC), and methods are being assessed to enhance their efficacy. Ipilimumab is an antagonistic monoclonal antibody that binds cytotoxic T-lymphocyte-associated protein 4, an immunomodulatory molecule expressed by activated T cells, and to CD80 on antigen-presenting cells. We aimed to assess the safety and tolerability of ipilimumab in combination with a poxviral-based vaccine targeting prostate-specific antigen (PSA) and containing transgenes for T-cell co-stimulatory molecule expression, including CD80. Methods We did a phase 1 dose-escalation trial, with a subsequent expansion phase, to assess the safety and tolerability of escalating doses of ipilimumab in combination with a fixed dose of the PSA-Tricom vaccine. Patients with mCRPC received 2×10 8 plaque-forming units of recombinant vaccinia PSA-Tricom subcutaneously on day 1 of cycle 1, with subsequent monthly boosts of 1×10 9 plaque-forming units, starting on day 15. Intravenous ipilimumab was given monthly starting at day 15, in doses of 1, 3, 5, and 10 mg/kg. Our primary goal was to assess the safety of the combination. This study is registered with ClinicalTrials.gov, number NCT00113984. Findings We completed enrolment with 30 patients (24 of whom had not been previously treated with chemotherapy) and we did not identify any dose-limiting toxic effects. Grade 1 and 2 vaccination-site reactions were the most common toxic effects: three of 30 patients had grade 1 reactions and 26 had grade 2 reactions. 21 patients had grade 2 or greater immune-related adverse events. Grade 3 or 4 immune-related adverse events included diarrhoea or colitis in four patients and grade 3 rash (two patients), grade 3 raised aminotransferases (two patients), grade 3 endocrine immune-related adverse events (two patients), and grade 4 neutropenia (one patient). Only one of the six patients previously treated with chemotherapy had a PSA decline from baseline. Of the 24 patients who were chemotherapy-naive, 14 (58%) had PSA declines from baseline, of which six were greater than 50%. Interpretation The use of a vaccine targeting PSA that also enhances co-stimulation of the immune system did not seem to exacerbate the immune-related adverse events associated with ipilimumab. Randomised trials are needed to further assess clinical outcomes of the combination of ipilimumab and vaccine in mCRPC. Funding US National Institutes of Health.

We compared the effects of yeast-treated human dendritic cells (DCs) with CD40L-matured human DCs for the induction of effector cells and the number and functionality of CD4 + CD25 + CD127 − FoxP3 + regulatory T cells (Tregs). DCs were treated with yeast or CD40L and cocultured with isolated autologous CD4 + T cells. CD4 + CD25 + CD127 − T cells isolated from the coculture of CD4 + T cells plus yeast-treated DCs (yeast coculture) had a lower expression of FoxP3 and decreased suppressive function compared to CD4 + CD25 + CD127 − T cells isolated from the coculture of CD4 + T cells plus CD40L-treated DCs (CD40L coculture). Also, compared to the CD40L coculture, the yeast coculture showed increases in the ratio of CD4 + CD25 + activated T cells to Tregs and in the production of Th1-related cytokines (IL-2, TNF-α, IFN-γ) and IL-6. In addition, yeast-treated DCs used as antigen-presenting cells (APCs) incubated with the tumor antigen CEA enhanced the proliferation of CEA-specific CD4 + T cells compared to the use of CD40L-matured DCs used as APCs. This is the first study to report on the role of yeast-treated/matured human DCs in reducing Treg frequency and functionality and in enhancing effector to Treg ratios. These results provide an additional rationale for the use of yeast as a vector in cancer vaccines.

Tumor-associated antigens are weakly immunogenic. Human carcinoembryonic antigen (CEA) is overexpressed on a wide range of human carcinomas and represents an attractive target for cancer immunotherapy. This study analyzes the ability of a Saccharomyces cerevisiae vector containing the transgene encoding CEA (yeast-CEA) to activate human dendritic cells (DCs) and stimulate CEA-specific T-cell responses. We demonstrate for the first time that treatment with yeast-CEA can activate human DCs, resulting in increases in surface expression of CD80, CD83, CD54, CD58, and MHC class II, and increased production by DCs of IL-12p70, TNF-alpha, IFN-gamma, IL-8, IL-2, IL-13, IL-10, and IL-1beta. We also show that human DCs treated with yeast-CEA can activate CEA-specific T-cell lines and can act as antigen-presenting cells (APCs) to generate CEA-specific T-cell lines capable of lysing CEA(+) human tumor cells. Gene expression profiles of human DCs treated with yeast-CEA show increased expression of numerous genes involved in the production of chemokines and cytokines and their receptors, and genes related to antigen uptake, antigen presentation, and signal transduction.

The prevalence of obesity, an established risk factor for several chronic diseases, including cancer, has risen dramatically over the past 4 decades. Dietary change and/or increased physical activity are the most commonly recommended lifestyle-based strategies for preventing or reversing obesity. One of several physiological systems that may be enhanced by dietary change and exercise is the immune system. In this study, we examined the effects of energy restriction (ER; 30% reduction relative to control energy intake) and/or exercise (EX; voluntary wheel running) on systemic and mucosal immune function. Female C57BL/6 mice were randomized into 4 treatment conditions: 1) controls consumed ad libitum (AL); 2) AL with access to running wheels (AL + EX); 3) 30% ER; and 4) 30% ER with access to running wheels (ER + EX). Both ER and EX reduced spleen weight and the number of splenic T and B lymphocytes (P < 0.05). ER enhanced natural killer (NK) cell function, but reduced concanavalin A (Con A)-induced T-cell proliferation (P < 0.05). In contrast, EX enhanced Con A-induced proliferation and cytokine production from Peyer's patch cells (P < 0.05). These data suggest that ER and EX enhance some, but not all, components of the immune system and are likely working via different biological mechanisms to regulate NK and T-cell function.

Sustained, local delivery of immunomodulatory cytokines is under investigation for its ability to enhance vaccine and anti-tumor responses both clinically and preclinically. This study evaluates the ability of chitosan, a biocompatible polysaccharide, to (1) control the dissemination of a cytokine, GM-CSF, and (2) enhance the immunoadjuvant properties of GM-CSF. While cytokines have previously been delivered in lipid-based adjuvants and other vehicles, these do not have the clinical safety profile or unique properties of chitosan. We found that chitosan solution maintained a measurable depot of recombinant GM-CSF (rGM-CSF) at a subcutaneous injection site for up to 9 days. In contrast, when delivered in a saline vehicle, rGM-CSF was undetectable in 12-24h. Furthermore, a single s.c. injection of 20 microg rGM-CSF in chitosan solution (chitosan/rGM-CSF(20 microg)) transiently expanded lymph nodes up to 4.6-fold and increased the number of MHC class II expressing cells and dendritic cells by 7.4-fold and 6.8-fold, respectively. These increases were significantly greater than those measured when rGM-CSF was administered in saline at the standard preclinical dose and schedule, i.e. 4 daily s.c. injections of 20 microg. Furthermore, lymph node cells from mice injected with chitosan/rGM-CSF(20 microg) induced greater allogeneic T cell proliferation, indicating enhanced antigen presenting capability, than lymph node cells from mice injected with rGM-CSF alone. Finally, in vaccination experiments, chitosan/rGM-CSF was superior to either chitosan or rGM-CSF alone in enhancing the induction of antigen-specific CD4(+) proliferation, peptide-specific CD8(+) pentamer staining and cytotoxic T cell lysis. Altogether, chitosan/rGM-CSF outperformed standard rGM-CSF administrations in dendritic cell recruitment, antigen presentation and vaccine enhancement. We conclude that chitosan solution is a promising delivery platform for the sustained, local delivery of rGM-CSF.

The development of safe, novel adjuvants is necessary to maximize the efficacy of new and/or available vaccines. Chitosan is a non-toxic, biocompatible, biodegradable, natural polysaccharide derived from the exoskeletons of crustaceans and insects. Chitosan's biodegradability, immunological activity and high viscosity make it an excellent candidate as a depot/adjuvant for parenteral vaccination. To this end, we explored chitosan solution as an adjuvant for subcutaneous vaccination of mice with a model protein antigen. We found that chitosan enhanced antigen-specific antibody titers over five-fold and antigen-specific splenic CD4+ proliferation over six-fold. Strong increases in antibody titers together with robust delayed-type hypersensitivity (DTH) responses revealed that chitosan induced both humoral and cell-mediated immune responses. When compared with traditional vaccine adjuvants, chitosan was equipotent to incomplete Freund's adjuvant (IFA) and superior to aluminum hydroxide. Mechanistic studies revealed that chitosan exhibited at least two characteristics that may allow it to function as an immune adjuvant. First, the viscous chitosan solution created an antigen depot. More specifically, less than 9% of a protein antigen, when delivered in saline, remained at the injection site after 8 h. However, more than 60% of a protein antigen delivered in chitosan remained at the injection site for 7 days. Second, chitosan induced a transient 67% cellular expansion in draining lymph nodes. The expansion peaked between 14 and 21 days after chitosan injection and diminished as the polysaccharide was degraded. These mechanistic studies, taken together with the enhancement of a vaccine response, demonstrate that chitosan is a promising and safe platform for parenteral vaccine delivery.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent immune stimulant when administered with different vaccines. Optimal use of GM-CSF resides in its ability to act locally to stimulate the proliferation and maturation of professional antigen-presenting cells (APCs) (i.e., Langerhans' cells) at the injection site. GM-CSF was engineered into a replication-incompetent recombinant avian (fowlpox) virus (rF-GM-CSF) and a single subcutaneous injection resulted in a sustained enrichment of activated dendritic cells within the regional draining lymph nodes. Those changes were attributed to local GM-CSF production at the injection site by rF-GM-CSF-infected cells. Studies were carried out in which mice were administered different types of beta-galactosidase (beta-gal)-based vaccines--whole protein, peptide, recombinant poxviruses--and GM-CSF was administered either as a single injection of rF-GM-CSF or four daily bolus injections of the recombinant protein. The use of rF-GM-CSF either improved the immune adjuvant effect, as observed for poxvirus-based vaccines, or was equivalent to rGM-CSF, as observed with the beta-gal protein vaccine. It is important to note that with either the replication-competent (vaccinia) or replication-incompetent (fowlpox) vaccines expressing LacZ, strong CTL responses directed against beta-gal were induced only when rF-GM-CSF was used as the immune adjuvant. Engineering GM-CSF into a recombinant fowlpox virus offers an excellent vehicle for the delivery of this cytokine as an immune adjuvant with specific vaccine platforms. In particular, delivery of GM-CSF via the rF-GM-CSF construct would be preferred over bolus injections of rGM-CSF when used as an immune adjuvant with whole protein or recombinant poxvirus-based vaccines. The study underscores the importance of defining the appropriate delivery form of an immune adjuvant, such as GM-CSF, relative to the immunization strategy to maximize the host immune responses against a specific antigen.

Human tumor-associated antigens (TAAs) are weak immunogens. One strategy for increasing the immunogenicity of TAAs is to generate altered peptide ligands. In the studies reported here, microarray technology has been used to compare gene expression profiles of a human T cell line that was derived from the peripheral blood of a cancer patient vaccinated with a carcinoembryonic antigen (CEA)-based vaccine. We compared the gene expression profiles of this CEA-specific CD8 T cell line when (a) stimulated with the native peptide used to derive this line vs. no peptide, and (b) stimulated with its TCR enhancer agonist epitope vs. no peptide. The results demonstrate that the effect on the T cell line, when stimulated with the agonist peptide, is not an enhanced quantitative expression of the same genes or gene sets induced by the native peptide, but is rather a nearly reciprocal upregulation of different gene sets. The gene for the chemokine lymphotactin, which was overexpressed only in T cells stimulated with the agonist peptide, stood out above all others. This finding was extended using other T cell lines, and another set of agonist and native peptides from another TAA. ELISPOT and ELISA assays for lymphotactin confirmed and extended these findings. These studies suggest a potential role for lymphotactin in the T-cell activation processes and subsequent anti-tumor events.

The ELISPOT assay is used to detect T-cell responses in patients enrolled in vaccine clinical trials; it is a relatively sensitive assay that can be performed without in vitro stimulation (IVS) of PBMC. However, this assay may be limited in some cases because of a weak signal 1 (as is the case for tumor-associated antigens [TAA]), or by a limited number of PBMC available from patients. The objective of this study is to enhance the sensitivity of the ELISPOT by increasing the level of signal 2, in this case, B7-1 expression on antigen-presenting cells (APC), allowing for a more sensitive detection of antigen-specific T-cell precursors. C1RA2 cells were used as APC and were uninfected, or infected with either recombinant avipox (fowlpox)-B7-1 (rF-B7-1) or control fowlpox wild-type (FP-WT) vector. The expressions of B7-1, MHC Class II, as well as HLA-A2 on the infected cells were analyzed by flow cytometry. A nearly threefold increase in B7-1 mean fluorescence intensity (MFI) occurred in the rF-B7-1-infected C1RA2 cells with no changes in MHC Class II or HLA-A2 expression. Various PBMC/APC ratios were used to further analyze the use of rF-B7-1-infected C1RA2 cells as APC in the ELISPOT assay. Fewer APC were required to activate PBMC when C1RA2 infected with rF-B7-1 were used as APC. Furthermore, using a PBMC/APC ratio of 1:1, similar detection was achieved using fewer PBMC. In addition, we demonstrated that the reactivity can be blocked by adding anti-B7-1 antibody. We performed the assay using APC on five normal healthy donors. All five donors showed substantial increases in PF to the Flu matrix peptide (Flu peptide) using the rF-B7-1-infected C1RA2 cells. Finally, we evaluated five cancer patients who received a carcinoembryonic antigen (CEA) vaccine-based therapy. Increases in CEA peptide precursors were noted in all five patients using the B7-1-infected APC. In conclusion, we have demonstrated the ability to enhance the sensitivity of the ELISPOT assay through infection of C1RA2 with rF-B7-1.

The role of OX40L on the activation of T cells was investigated using poxvirus vectors expressing OX40L alone or in combination with three other T-cell costimulatory molecules: B7-1, ICAM-1, and LFA-3. Poxvirus vector-infected cells were used to stimulate naive or activated CD4 + and CD8 + T cells. These studies demonstrate that (a) OX40L plays a role in sustaining the long-term proliferation of CD8 + T cells in addition to the known effect on CD4 + T cells following activation, (b) OX40L enhances the production of Th1 cytokines (IL-2, IFN-γ, and TNF-α) from both CD4 + and CD8 + while no change in IL-4 expression was observed, and (c) the anti-apoptotic effect of OX40L on T cells is likely the result of sustained expression of anti-apoptotic genes while genes involved in apoptosis are inhibited. In addition, these are the first studies to demonstrate that the combined use of a vector driving the expression of OX40L with three other costimulatory molecules (B7-1, ICAM-1, and LFA-3) both enhances initial activation and then further potentiates sustained activation of naive and effector T cells.

It has now been demonstrated in both experimental models and recent clinical trials that certain "self" antigens, which are functionally non-immunogenic in the host, can become immunogenic if presented to the immune system in a certain way. Here, we describe recombinant vaccines and vaccine strategies that have been developed to induce and potentiate T-cell responses of the host to such self-antigens. These strategies include: (a) the use of recombinant poxvirus vectors in which the tumor-associated antigen (TAA) is inserted as a transgene. Recombinant vaccinia vaccines and recombinant avipox (replication-defective) vaccines have been employed to break tolerance to a self-antigen; (b) the use of diversified prime and boost strategies using different vaccines; and (c) the insertion of multiple T-cell co-stimulatory molecules into recombinant poxvirus vectors, along with the TAA gene, to enhance T-cell immune responses to the TAA and induce anti-tumor immunity.

The studies reported herein present the first in vitro and in vivo comparison of radioimmunoconjugates (RIC) radiolabeled with 177Lu using the acyclic CHX-A"-DTPA ligand and the macrocyclic ligands, C-DOTA and PA-DOTA. The in vivo studies include pharmacokinetics and biodistribution of the formed 177Lu-labeled immunoconjugates in a tumor bearing murine model with engineered monoclonal antibody HuCC49DeltaCH2. The in vitro analysis indicated that the CHX-A" RIC was superior with respect to immunoreactivity, radiolabeling with 177Lu, and specific activity. The in vivo pharmacokinetic data by itself indicated that the Lu(III)-PA-DOTA complex may not be as stable as Lu(III) complexes with CHX-A" or C-DOTA. All three RICs demonstrated tumor targeting of human colon carcinoma xenografts in athymic mice. In these biodistribution studies, there appears to be no overall pattern or trend of one RIC over the other two. Based on these in vitro and in vivo studies, the CHX-A" DTPA ligand should be considered a suitable bifunctional chelate for the radiolabeling of monoclonal antibodies with 177Lu for radioimmunotherapy applications.

Activation of T cells requires at least two signals: signal 1, via the T-cell receptor, and signal 2, in which a costimulatory molecule on the antigen presenting cell (APC) interacts with a ligand on the T cell. Dendritic cells (DCs) are the most potent APCs in part due to their expression of costimulatory molecules. DCs, however, constitute only a minor percentage of APCs in the body, and the in vitro preparation of DCs is both costly and time consuming. The studies reported here demonstrate that one can utilize other APCs, such as bone marrow progenitor cells (BMPCs) and make them markedly more effective as APCs; this was accomplished by their infection with recombinant poxviruses (either the replication-defective avipox or vaccinia), which contain transgenes for a triad of costimulatory molecules (B7-1, ICAM-1 and LFA-3, designated TRICOM). APCs infected with TRICOM vectors are shown to significantly enhance the activation of both naive and effector CD4(+) and CD8(+) T-cell populations. The use of TRICOM vectors in vaccine strategies is discussed.

One of the major obstacles in the successful clinical application of monoclonal antibodies has been the development of host immune responses to murine Ig constant and variable regions. While the CDR grafting of MAbs may alleviate many of these problems, the potential remains that one or more murine CDRs on the human Ig backbone of a “humanized” MAb may still be immunogenic. Studies were undertaken employing a MAb of potential clinical utility, CC49, to define those CDRs that are essential for antigen binding and those that may be immunogenic in humans. We previously developed a humanized CC49 (HuCC49) by grafting the MAb CC49 hypervariable regions onto frameworks of human MAbs. To identify those CDRs essential for binding, a panel of variant HuCC49 MAbs was generated here by systematically replacing each of the murine CDRs with their human counterparts. The relative affinity constant of each variant was determined. Serum from a patient who received murine CC49 was used to determine the potential immunogenicity of each CDR in humans. The serum was shown to react with the anti-CC49 variable region. Results showed that patients’ anti-idiotypic responses are directed mainly against LCDR3 and moderately against LCDR1 and HCDR2. These studies demonstrate for the first time that variants containing individual CDR substitutions of a humanized MAb can be constructed, and each CDR can be defined for the two most important properties for potential clinical utility: antigen binding and immunogenicity.

Many human cancers have been shown to contain activated forms of the Ras proto-oncogene. Mutations comprising amino acid changes at codons 12, 13 and 61 therefore represent unique targets for cancer immunotherapy. Recombinant Vaccinia viruses encoding point mutated Ras oncogenes have raised issues concerning the safety and transforming ability of these recombinant vaccines. Vaccinia virus, a representative of the orthopox virus genus, is a large DNA virus that is cytopathogenic and that replicates in the cytoplasm of the infected cell. However, it remains unclear whether orthopox viruses are capable of genetic interactions with infected cells. Our studies show that DNA isolated from cells infected with a recombinant Vaccinia virus expressing mutated Ras constituted a poor reagent for transfection into NIH3T3 cells for transformation analysis. Stable integration of a recombinant Vaccinia virus expressing mutant Ras DNA was not detected in recipient cells. This study also demonstrates that the crossover plasmids used to generate the recombinant virus where the activated Ras gene is under the control of a Vaccinia virus early promoter had low but detectable transforming efficiency in the NIH3T3 transformation assay. Analysis of the transfected cells indicated that Ras transcription was initiated upstream of the Vaccinia virus promoter. The introduction of wobble mutations as well as the truncation of the Ras protein removed the transforming capabilities of the crossover vector. This study demonstrates the potential problems and solutions in the use of point mutated oncogenes in live vectors for cancer vaccine development.

Adoptive T-cell transfer has been shown to be a potentially effective strategy for cellular immunotherapy in some murine models of disease. However, several issues remain unresolved regarding some of the basic features involved in effective adoptive transfer, such as the influence of specific peptide antigen (Ag) boost after T-cell transfer, the addition of IL-2 post-T-cell transfer, the trafficking of transferred T cells to lymphoid and nonlymphoid tissues, and the functional stability of recoverable CD4 + and CD8 + T cells. We investigated several of these parameters, particularly as they relate to the persistence and maintenance of effector functions of murine CD4 + and/or CD8 + T lymphocytes after adoptive cellular transfer into partially γ-irradiated syngeneic hosts. Our laboratory previously identified murine (H-2 d ) immunogenic CD4 + and CD8 + T-cell peptide epitopes reflecting codon 12 ras mutations as tumor-specific Ag. Therefore, the model system chosen here employed epitope-specific MHC class II-restricted CD4 + T cells and MHC class I-restricted CD8 + T cells produced from previously immunized BALB/c mice. Between 2 and 7 days after T-cell transfer, recipient mice received various combinations of peptide boosts and/or IL-2 treatments. At different times after the T-cell transfer, spleen and lung tissues were analyzed phenotypically to monitor the persistence of the immune T cells and functionally (via proliferation or cytotoxicity assays) to assess the maintenance of peptide specificity. The results showed that immune donor T lymphocytes (uncultured immune T cells or cloned T cells) were recoverable from the spleens and lungs of recipient mice after transfer. The recovery of Ag-specific T-cell responses was greatest from recipient mice that received peptide boosts and IL-2 treatment. However, mice that received a peptide boost without IL-2 treatment responded nearly as well, which suggested that including a peptide boost after T-cell transfer was more obligatory than exogenous IL-2 treatment to sustain adoptively transferred T cells in vivo. Ag-specific T-cell responses were weak in mice that either received IL-2 alone or did not receive the cognate peptide boost after T-cell transfer. The T-cell clones were also monitored by flow cytometry or RT-PCR based on expression of the T-cell receptor Vβ-chain, which was previously characterized. Ag-specific T cells were recovered from both spleens and lungs of recipient mice, demonstrating that the T-cell clones could localize to both lymphoid and nonlymphoid tissues. This study demonstrates that both uncultured and in vitro -cloned T lymphocytes can migrate to lymphoid tissues and nonlymphoid (e.g., lung) tissues in recipient hosts and that their functional activities can be maintained at these sites after transfer, if they are exposed to peptide Ag in vivo.

Phosphorylation sites for casein kinase I were introduced into chimeric monoclonal antibody CC49 (MAb-chCC49) by inserting a synthetic fragment (CK1) encoding two casein kinase I phosphorylation sites into an expression vector. The phosphorylation sites were created by incorporating the predicted consensus sequences for phosphorylation by the casein kinase I at the carboxyl terminus of the heavy-chain constant region of the MAb-chCC49. The resultant modified MAb-chCC49 (MAb-chCC49CK1) was expressed and purified. The MAb-chCC49CK1 protein can be phosphorylated by the casein kinase I with [gamma-32P]ATP to high radiospecific activity. The 32P-labeled MAb-chCC49CK1 protein binds to cells expressing TAG-72 antigens. The introduction of phosphorylation sites into MAb provides new reagents for the diagnosis and treatment of cancer. This demonstrates that, as was described for the cAMP-dependent protein kinase site, the casein kinase I recognition site can also be used to introduce phosphorylation sites into proteins.

A cytolytic T-lymphocyte (CTL) response has been generated to a protein antigen via oral administration in an experimental murine system. This was achieved by the formulation of the test antigen ovalbumin (OVA) in a pH-sensitive microsphere particle (microbead) with an acrylic-based coating to avoid its degradation by gastric enzymes. Comparative studies of orally administered enterocoated microbead-OVA particles, versus the more traditional formulation of OVA in adjuvant (DETOX-PC) given subcutaneously as immunogen, were conducted; both elicited comparable responses in the generation of antigen-specific lymphoproliferative and CTL responses. CTL lines generated via oral administration of antigen were shown to be CD3(+), CD4(-), and CD8(+). CTL lysis of OVA peptide-pulsed targets was shown to be inhibited by anti-CD8 antibody. Whereas oral administration of pH-sensitive enterocoated microbeads containing proteins has previously been shown to elicit antibody and lymphoproliferative T-cell responses to protein antigens, this is the first demonstration of the generation of an antigen-specific cytolytic T-cell response via oral administration of a protein immunogen formulated in such microbeads.

Point mutations in the ras proto-oncogenes, notably at codon 12, are found in high frequency of human malignancies and, thus, may be appropriate targets for the induction of tumor-specific T cell responses in cancer immunotherapy. In this study, we examined the mutant ras protein sequence reflecting the substitution of Gly to Val at position 12 as a putative point-mutated determinant for potential induction of an HLA-A2-reactive, CD8+ cytotoxic T lymphocyte (CTL) response. We identified the ras 4-12(Val12) sequence as a minimal 9-mer peptide, which displayed specific binding to HLA-A2 by T2 bioassays. Peptide binding to HLA-A2 on T2 cells was weak and required coincubation with exogenous beta(2)-microglobulin to facilitate and enhance complex formation. In contrast, the wild-type ras 4-12(Gly12) peptide failed to bind to HLA-A2 even in the presence of beta(2)-microglobulin, consistent with the hypothesis that the point mutation creates a C-terminus anchor residue. A CD8+ CTL line against the ras 4-12(Val12) peptide was derived in vitro from a normal HLA-A2+ donor using a model culture system consisting of T2 cells as antigen presenting cells pulsed with exogenous mutant ras peptide and beta(2)-microglobulin plus cytokines (interleukin-2 and 12). Functional characterization of CD8+ CTL line revealed (1) peptide-specific and HLA-A2-restricted cytotoxicity against a panel of peptide-pulsed targets; (2) no specific lysis using the normal ras peptide sequence; (3) half-maximal lysis with exogenous peptide of approximately 0.3 microM; (4) lysis of HLA-A2+ B cell lines infected with a recombinant vaccinia virus construct encoding the point-mutated human K-ras gene; and (5) specific lysis of the HLA-A2+ SW480 colon carcinoma cell line expressing the naturally occurring K-ras Val12 mutation. Maximal lysis of SW480 cells occurred following interferon (IFN)-gamma pretreatment, which correlated with enhanced HLA-A2 and ICAM-1 (CD54) expression. Specificity of lysis was revealed by the absence of lysis against a HLA-A2+ melanoma cell line (+/- IFN-gamma), which lacked the mutant Val12 mutation, and the inability of an irrelevant CD8+ CTL line to lyse SW480 (+/- IFN-gamma) unless the appropriate exogenous peptide was added. These findings demonstrated that tumor cells may endogenously process and express mutant ras epitopes, such as the 4-12(Val12) sequence, albeit in limiting amounts that may be potentiated by IFN-gamma treatment. These data support the biological relevance of this sequence and, thus, may have important implications for the generation of ras oncogene-specific CTL responses in clinical situations.

Twenty-seven ovarian cancer patients who failed chemotherapy entered a phase I/II trial of intraperitoneal 177Lu-CC49 antibody.Patients had disease confined to the abdominal cavity +/- retroperitoneal lymph nodes, adequate organ function, and no previous radiation.The most common side effects were delayed, transient arthralgia (10/27) and marrow suppression with 1.665 GBq/m2 (45 mCi/m2), which was considered the maximum tolerated dose. One of thirteen patients with gross disease had50% tumor reduction after therapy, whereas most others with gross disease progressed (one went off study with stable disease at 11 weeks). Seven of nine patients with1-cm nodules progressed inor =21 months, and two of nine remain without evidence of disease at 4 to 5 months. Of patients with microscopic or occult disease, one relapsed at 10 months and four of five remain without evidence of disease at6 to 35 months.Marrow suppression was the dose-limiting toxic effect of intraperitoneal immunotherapy with 177Lu-CC49. Antitumor effects were noted against chemotherapy-resistant ovarian cancer, even at lower dose levels, and resulted in prolonged disease-free survival of most patients with microscopic disease. This form of treatment deserves further study.

A recent conference focused on the advances, challenges and prospects of radioimmunodetection (RAID) and radioimmunotherapy (RAIT) of cancer. Emphasis was given to the underlying sciences of radio- chemistry, as well as its clinical and experimental applications.

When administered as single agents, both interferon gamma (IFN-gamma) and interleukin 6 (IL-6) significantly increase carcinoembryonic antigen (CEA) and HLA class I antigen expression on the surface of human colorectal tumour cells. Studies were carried out to determine whether by combining those cytokines a synergistic enhancement of CEA and HLA expression could result. The findings revealed that the administration of 20 units IFN-gamma along with 1.7 ng IL-6, concentrations of each cytokine that individually induced minimal antigenic changes, together synergistically increased CEA and HLA class I as well as induced qualitative changes in HLA expression on WiDr human colon carcinoma cells. The magnitude of the synergistic increases in CEA and HLA class I expression were reminiscent of the level of antigen augmentation observed when administering 20- to 100-fold higher amounts of each cytokine as a single agent. Also, the addition of IL-6 potentiated the IFN-gamma induction of HLA class II expression. The combined administration of IL-6 potentiated the IFN-gamma did not have any additive or synergistic effects on the growth suppression of those tumour cells. Interestingly, utilization of specific neutralizing antibodies for type I interferons abrogated the increases of CEA and HLA expression seen with IL-6 treatment alone or in combination with IFN-gamma. Moreover, reverse transcriptase/polymerase chain reaction analyses revealed a constitutive expression as well as a temporal increase of IFN-beta mRNA transcripts in colon tumour cells treated with IL-6. Therefore, the findings provide indirect evidence that IFN-beta production seems to play a critical role in the ability of IL-6 to upregulate antigen expression alone or in combination with IFN-gamma. These findings provide insight into cytokine combinations that synergistically upregulate tumour-associated and normal HLA antigen expression on the surface of human tumour cells. Those results provide the rationale for the combined use of such cytokines to heighten tumour cell recognition in monoclonal antibody- or cell-mediated-based immunotherapeutic approaches.

Biodistribution of five different backbone-substituted derivatives of SCN-Bz-DTPA (1B4M-DTPA, 1M3B-DTPA, 1B3M-DTPA, GEM-DTPA and 2B-DTPA) linked to MAb B72.3 were compared to that of the parent molecule after labeling with 111indium. Athymic mice, bearing human colon carcinoma xenografts (LS-174T) were injected i.v. to determine the biodistribution of the MAb chelate conjugates. Three of the MAb metal chelate conjugates (1B4M-DTPA, 1M3B-DTPA, and 1B3M-DTPA), labeled with 111In showed efficient and stable tumor localization as well as a slower blood clearance rate than SCN-Bz-DTPA, GEM-DTPA or 2B-DTPA MAb chelate conjugates. Major differences were also seen in normal organ uptake, especially liver and spleen. Tumor-to-liver ratios rose as a function of time for 1B4M-DTPA, 1M3B-DTPA and 1B3M-DTPA MAb chelate conjugates with virtually no accumulation of the radiometal into this organ, as revealed by no increase in the liver-to-blood values. Small accretion in normal liver was noted for SCN-Bz-DTPA, GEM-DTPA or 2B-DTPA MAb chelate conjugates. The results reviewed here, and described previously (Roselli et al., 1991) demonstrate that the use in vivo of backbone-substituted forms of the SCN-Bz-DTPA, such as 1B4M-DTPA, 1M3B-DTPA, and 1B3M-DTPA bound to MAbs, can reduce uptake of indium to normal organs while maximizing the dose to tumor.

Great progress in imaging and therapy with radioimmunoconjugates was discussed at the fourth conference on radioimmunodetection and radioimmunotherapy of cancer. ∗ Monoclonal antibody-based cancer imaging agents are awaiting marketing approval and newer, potentially improved forms are in development. Tumor responses to radioimmunoconjugates are also being reported in increasing numbers, this is especially so with radiosensitive tumors and in regional or direct applications.

The expression of mouse mammary tumor virus (MMTV) gene products was examined in several different murine mammary tissues which showed no production of complete extracellular MMTV B-particles. Analyses by specific competition radioimmunoassay demonstrated that these mammary tissues contained large amounts of protein immunologically related to, or identical to, the 28,000-dalton major nonglycosylated MMTV core protein (p28), but no immunologically detectable 52,000-dalton MMTV envelope glycoprotein (gp52). Such noncoordinate MMTV genome expression was found in primary and transplanted mammary tumors, and in preneoplastic mammary tissues of BALB/c mice, as well as in normal mammary tissue of Swiss albino mice. These mammary tissues exemplify a naturally occurring animal model system for studying noncoordinate expression of MMTV gene products, and its possible significance for MMTV-mediated oncogenesis.

The major structural proteins of three type-D retroviruses are compared using sodium dodecyl sulfate polyacrylamide slab and tube gel electrophoresis and competitive radioimmunoassays. We report here that the virion-associated polypeptides of the endogenous virus of the langur (LV) and the nongerm line-transmitted Mason-Pfizer monkey virus (MPMV) of the rhesus have indistinguishable electrophoretic mobilities in polyacrylamide gels. Homologous radioimmunoassays (RIAs) for the major 27,000-dalton structural protein, (p27) of LV and for the p27 of MPMV also cannot distinguish these two viruses. LV and MPMV can be differentiated, however, by using either of the RlAs developed for their 10,000- to 12,000-dalton proteins. The major structural protein of the type-D retrovirus of New World squirrel monkeys (SMRV) is more immunoreactive with the endogenous virus of the Old World langur monkey than it is with MPMV. An interspecies RIA has also been developed using antiserum to LV to precipitate 125 I-labeled MPMV p27. This assay is more effective than either of the homologous LV or MPMV RIAs for the recognition of SMRV. These studies further define the structural and immunologic relationships among the type-D retroviruses of primates.

We have previously reported a high degree of selective binding of monoclonal antibody (MAb) B72.3 to a wide range of carcinoma versus normal tissues. While the diagnostic targeting of a primary or metastatic carcinoma lesion with a MAb is an end unto itself, it should also be considered as a first step toward the use of the MAb in tumor therapy. As we proceed with the use of MAbs in therapeutic applications, we are concomitantly characterizing the MAbs and associated tumor antigens to better understand their composition and nature ultimately in order to be able to manipulate their interaction. In this article we will discuss the use of MAbs in several aspects of clinical oncology, including tumor targeting clinical studies using MAb B72.3. These studies also include the regulation of tumor antigenic expression using preclinical models. We will present data on the use of a biological response modifier, recombinant human leukocyte (alpha) interferon (rHu-IFN-α), to amplify antigenic expression in carcinoma cells. We will discuss the use of MAb B72.3 in the localization of metastatic lesions in colorectal cancer patients, the findings of simultaneously administered B72.3 (i.v. and i.p.), and the pharmacokinetics associated with MAb administration. Finally, we will discuss the potential clinical uses of genetically engineered recombinant/chimeric MAbs, MAbs conjugated to toxins, drugs and/or radionuclides, MAb combinations, and second generation MAbs of predefined specificity.

A novel mouse mammary tumor virus (MMTV) has been isolated from mice of a subline of the BALB/cCrl Med mouse strain designated BALB/cV. Whereas breeding females of the parent BALB/cCrl Med colony have a mammary tumor incidence of 1%, 47% of the breeding females of the BALB/cV subline develop mammary tumors before 10 months of age. Foster nursing experiments demonstrated this virus, termed MMTV (BALB/c), was transmitted only by milk. The novel MMTV isolate was shown to be immunologically related to, but distinct, from the MMTV variants of C3H, GR, and RIII mice by a series of competition radioimmunoassays for the MMTV 28,000-dalton major core protein (p28), and the 52,000 (gp52)- and 36,000-dalton (gp36) major envelope glycoproteins. Monoclonal antibodies directed against MMTV gp36 were also used to clearly distinguish MMTV(BALB/c) from MMTV(C3H), MMTV(RIII), MMTV(GR), MMTV(C3HfC57BL), and MMTV(A). MMTV-specific proviral DNA content of mammary adenocarcinomas arising in the BALB/cV subline was examined with restriction endonucleases and the Southern blot technique, and compared to the MMTV proviral DNA content of BALB/cAnDe mammary tumors. The virus arising from these latter tumors has been termed MMTV(O). Analysis of Eco RI digests of high-molecular-weight DNA from both types of mammary tumors demonstrated additional MMTV-related proviral sequences when compared to the DNA of normal BALB/c tissues. The patterns generated with the restriction endonuclease Sac I distinguished the additional MMTV-specific proviral information in the mammary tumors of the BALB/cV mice from the proviral information in tissues containing the GR, C3H, or RIII MMTVs, as well as from the proviral information in the BALB/cAnDe mammary tumors. These immunological and molecular studies thus define MMTV(BALB/c) as a novel MMTV variant.

Monoclonal antibody B72.3 has been shown to be reactive with a high-molecular-weight glycoprotein complex termed TAG (tumor-associated glycoprotein)-72. By the avidin-biotin immunoperoxidase method, fine needle aspirates and corresponding surgically excised tumor tissues from both malignant and benign tissues were analyzed for TAG-72 expression. Staining (range, 1 to 100 per cent of tumor cells) with monoclonal antibody B72.3 was observed in needle aspirates from 18 of 18 adenocarcinomas and adenosquamous carcinomas of the lung, 17 of 21 adenocarcinomas of the breast, and six of six adenocarcinomas of the colon, as well as adenocarcinomas from other body sites. In contrast, small cell carcinomas of the lung, malignant melanomas, lymphomas, and sarcomas did not stain with the antibody. benign lesions from the breast, lung, pancreas, parotid, and thyroid also failed to stain. In 66 patients, tumor-bearing tissue had also been resected and was available for comparative examination with monoclonal antibody B72.3. In 62 of these 66 patients, the staining patterns in the aspirates were found to be predictive of the patterns of antibody reactivity in the comparable surgically resected tissues. From these studies it is concluded that monoclonal antibody B72.3 defines a tumor-associated antigen that is expressed in neoplastic cells but not in benign cells and is most selectively expressed in adenocarcinomas. This monoclonal antibody may be used as a novel adjunct for the diagnosis of carcinoma in fine needle aspiration biopsy specimens.

Protein products of the ras family of oncogenes were immunoprecipitated by an anti-p21 monoclonal antibody, separated by two-dimensional gel electrophoresis and subsequently detected by western immunoblot analysis using the same anti-p21 monoclonal antibody as a probe. Using this method, a 21 kDa oncogene protein (p21) was detected and characterized in cell lines containing Harvey (Ha), Kirsten (Ki), neuroblastoma (N), or cellular (proto) ras genes. The ras gene products from all cell types occurred with multiple forms differing in size, charge or in both parameters. Transforming ras oncogene proteins occurred in easily identifiable groups that were different from each other in molecular weight and charge, were distinctive for each ras gene type and were different from cellular ras equivalents. Similar, but not identical, family groups occurred in different cell types containing the same oncogene. The reproducible occurrence of unpredicted p21 forms suggests that previously unreported post-translational processing steps may be associated with the synthesis of certain oncogene products. This immunoprecipitation/two-dimensional gel/western blot technique is a simple method for the identification and characterization of p21 gene products.

131I-labeled monoclonal antibodies were used in therapeutic trials in two potentially useful clinical situations: disseminated melanoma (intravenously administered Fab fragments; 21 patients) and disseminated peritoneal adenocarcinomatosis (intraperitoneal injection of IgG; 5 patients). Acute toxicity observed is consistent with mild bone marrow suppression of acute radiation syndrome and the observed toxicity is dose related in a manner that conforms to the expected human response to total body irradiation. For single doses of both i.p. administered and intravenously administered 131I-labeled anti-tumor antibodies, 100 rad to red marrow, calculated by the absorbed dose fraction method (MIRD), appeared to be a threshold below which significant acute toxicity was unlikely.

The immunoreactivity of two monoclonal antibodies, CO17-1A (recognizing 17-1A antigen) and B72.3 (recognizing TAG-72), was examined in pancreatic tissues from individuals without pancreatic disease and from those with benign and malignant pancreatic disease. 17-1A antigen was found in all cells in the normal tissue, whereas TAG-72 was present in the duct cells in only one case. Both antigens were present in benign diseases; however, in some cells and in some conditions, TAG-72 was significantly less common (P less than .01 to .001) than 17-1A antigen. In pancreatic cancer, 17-1A antigen was present in 87% of cases and TAG-72 antigen was present in 92% of cases. The results indicate that B72.3 is more specific to pancreatic cancer than C017-1A.