Rejection Of Established Tumors Research Articles

Nanocomplex-based TP53 gene therapy promotes anti-tumor immunity through TP53- and STING-dependent mechanisms

ABSTRACTLoss or mutation of TP53 has been linked to alterations in anti-tumor immunity as well as dysregulation of cell cycle and apoptosis. We explored immunologic effects and mechanisms following restoration of wild-type human TP53 cDNA in murine oral cancer cells using the therapeutic nanocomplex scL-53. We demonstrated scL-53 induces dose-dependent expression of TP53 and induction of apoptosis and immunogenic cell death. We further demonstrated both TP53-dependent and independent induction of tumor cell immunogenicity through the use of blocking mAbs, nanocomplex loaded with DNA plasmid with or without TP53 cDNA, empty nanocomplex and siRNA knockdown techniques. TP53-independent immune modulation was observed following treatment with nanocomplex loaded with DNA plasmid lacking TP53 cDNA and abrogated in STING-deficient tumor cells, supporting the presence of a cytoplasmic DNA sensing, STING-dependent type-I IFN response. Cooperatively, TP53- and STING-dependent alterations sensitized tumor cells to CTL-mediated lysis, which was further enhanced following reversal of adaptive immune resistance with PD-1 mAb. In vivo, combination scL-53 and PD-1 mAb resulted in growth control or rejection of established tumors that was abrogated in mice depleted of CD8+ cells or in STING deficient mice. Cumulatively, this work demonstrates 1) a direct anti-tumor effects of functional TP53; 2) non-redundant TP53- and STING-dependent induction of tumor cell immunogenicity following scL-53 treatment; and 3) that adaptive immune resistance following scL-53 treatment can be reversed with PD-based immune checkpoint blockade, resulting in the rejection or control of syngeneic murine tumors. These data strongly support the clinical combination of scL-53 and immune checkpoint blockade.

Correction: TALEN-Mediated Inactivation of PD-1 in Tumor-Reactive Lymphocytes Promotes Intratumoral T-cell Persistence and Rejection of Established Tumors.

Correction: TALEN-Mediated Inactivation of PD-1 in Tumor-Reactive Lymphocytes Promotes Intratumoral T-cell Persistence and Rejection of Established Tumors.

Resistance to CTLA-4 checkpoint inhibition reversed through selective elimination of granulocytic myeloid cells.

PurposeLocal immunosuppression remains a critical problem that limits clinically meaningful response to checkpoint inhibition in patients with head and neck cancer. Here, we assessed the impact of MDSC elimination on responses to CTLA-4 checkpoint inhibition.Experimental DesignMurine syngeneic carcinoma immune infiltrates were characterized by flow cytometry. Granulocytic MDSCs (gMDSCs) were depleted and T-lymphocyte antigen-specific responses were measured. Tumor-bearing mice were treated with MDSC depletion and CTLA-4 checkpoint blockade. Immune signatures within the human HNSCC datasets from The Cancer Genome Atlas (TCGA) were analyzed and differentially expressed genes from sorted human peripheral MDSCs were examined.ResultsgMDSCs accumulated with tumor progression and correlated with depletion of effector immune cells. Selective depletion of gMDSC restored tumor and draining lymph node antigen-specific T-lymphocyte responses lost with tumor progression. A subset of T-cell inflamed tumors responded to CTLA-4 mAb alone, but the addition of gMDSC depletion induced CD8 T-lymphocyte-dependent rejection of established tumors in all treated mice that resulted in immunologic memory. MDSCs differentially expressed chemokine receptors. Analysis of the head and neck cancer TCGA cohort revealed high CTLA-4 and MDSC-related chemokine and an MDSC-rich gene expression profile with a T-cell inflamed phenotype in > 60% of patients. CXCR2 and CSF1R expression was validated on sorted peripheral blood MDSCs from HNSCC patients.ConclusionsMDSCs are a major contributor to local immunosuppression that limits responses to checkpoint inhibition in head and neck cancer. Limitation of MDSC recruitment or function represents a rational strategy to enhance responses to CTLA-4-based checkpoint inhibition in these patients.

Correction: TALEN-Mediated Inactivation of PD-1 in Tumor-Reactive Lymphocytes Promotes Intratumoral T-cell Persistence and Rejection of Established Tumors.

Correction: TALEN-Mediated Inactivation of PD-1 in Tumor-Reactive Lymphocytes Promotes Intratumoral T-cell Persistence and Rejection of Established Tumors.

TALEN-Mediated Inactivation of PD-1 in Tumor-Reactive Lymphocytes Promotes Intratumoral T-cell Persistence and Rejection of Established Tumors.

Despite the promising efficacy of adoptive cell therapies (ACT) in melanoma, complete response rates remain relatively low and outcomes in other cancers are less impressive. The immunosuppressive nature of the tumor microenvironment and the expression of immune-inhibitory ligands, such as PD-L1/CD274 by the tumor and stroma are considered key factors limiting efficacy. The addition of checkpoint inhibitors (CPI) to ACT protocols bypasses some mechanisms of immunosuppression, but associated toxicities remain a significant concern. To overcome PD-L1-mediated immunosuppression and reduce CPI-associated toxicities, we used TALEN technology to render tumor-reactive T cells resistant to PD-1 signaling. Here, we demonstrate that inactivation of the PD-1 gene in melanoma-reactive CD8(+) T cells and in fibrosarcoma-reactive polyclonal T cells enhanced the persistence of PD-1 gene-modified T cells at the tumor site and increased tumor control. These results illustrate the feasibility and potency of approaches incorporating advanced gene-editing technologies into ACT protocols to silence immune checkpoints as a strategy to overcome locally active immune escape pathways. Cancer Res; 76(8); 2087-93. ©2016 AACR.

Abstract A088: Increase lymphocyte infiltration overcomes tumor resistance to checkpoint blockade

Abstract Spontaneous T cell-inflamed tumor microenvironment is associated with clinical responses to immunotherapies, including immune checkpoint blockades. However, majority of established tumors often inhibit peripheral T cells entering tumor tissues for tumor destruction and/or prevent effective T cell priming. Previous studies in our lab have shown that upregulation of the tumor necrosis factor superfamily member LIGHT (also known as TNFSF14) in peripheral tissues results in T cell activation and migration into non-lymphoid tissues and formation of lymphoid-like structures, which lead to rapid T cell-mediated tissue destruction. Furthermore, local expression of LIGHT by tumor cells may promote the formation of lymphoid-like structures for direct T-cell sequestration and activation, leading to tumor regression. Those data suggest LIGHT as an interesting candidate to create T cell-inflamed tumor environment. To better evaluate the therapeutic efficacy of human LIGHT protein in both murine and human systems, we engineered human LIGHT by random mutation using yeast-display system, and were able to generate a human LIGHT mutant which binds to both murine and human receptors while remains stable. To specifically target LIGHT to tumor tissues, an antibody-LIGHT fusion protein was generated. In different mouse models, we found that antibody-LIGHT induces a T cell inflamed tumor environment. This is correlated with an upregulation of inflammatory cytokines and adhesion molecules, and leads to the rejection of established tumors. More importantly, we observed that LIGHT-mediated T cell inflamed environment is critical for restoring the efficacy of checkpoint blockade therapy. When we blocked lymphocyte trafficking, a tumor previously responding to checkpoint blockade became resistant to therapy. This process can be reversed by treating with LIGHT. Our study indicates that targeting LIGHT may be an effective strategy for cancer immunotherapy, especially those who do not response to checkpoint blockade due to the lack of infiltration. Citation Format: Haidong Tang, Yang Wang, Yang-Xin Fu. Increase lymphocyte infiltration overcomes tumor resistance to checkpoint blockade. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A088.

Abstract B19: Tumor-specific structural rearrangements as potential targets for anticancer vaccines.

Abstract A hallmark of cancer is the accumulation of mutations that allow cells to proliferate uncontrollably. Every tumor has a unique set of somatic mutations, which can be identified with next generation sequencing. In theory, CD8+ T cells can recognize mutated proteins present in human cancers if the mutations are presented as peptides bound to MHC class I molecules. We hypothesize that personalized, tumor-specific peptide vaccines will activate CD8+ T cells and induce tumor regression. To test this concept, we subjected four mouse mammary tumors to RNA sequencing using the Illumina sequencing platform. We identified 14 somatic point mutations and one fusion transcript unique to these tumors. In addition, we have optimized a vaccination strategy involving long peptides and the adjuvant poly (I:C) that results in massive proliferation of antigen-specific CD8+ T cells. When we used this optimized vaccination protocol to target tumor specific mutations, a strong T cell response was elicited towards the fusion protein, but only weak responses or no responses were elicited towards the point mutations. Currently, we are vaccinating mice with immunogenic mutated-peptides, and assessing whether the T cell response elicited by the vaccines causes regression or rejection of established tumors that harbor the same mutations. As the cost of sequencing the human genome continues to decrease, personalized vaccines that target tumor-specific mutations may become a treatment strategy that is clinically feasible. Citation Format: Spencer David Martin, Darin A. Wick, John R. Webb, Robert A. Holt, Brad H. Nelson. Tumor-specific structural rearrangements as potential targets for anticancer vaccines. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B19.

IL-15 augments antitumoral activity of an ErbB2/HER2 cancer vaccine targeted to professional antigen-presenting cells.

Targeted delivery of tumor-associated antigens to professional antigen-presenting cells (APC) is being explored as a strategy to enhance the antitumoral activity of cancer vaccines. Here, we generated a cell-based system for continuous in vivo production of a CTLA-4-ErbB2 fusion protein as a therapeutic vaccine. The chimeric CTLA-4-ErbB2 molecule contains the extracellular domain of CTLA-4 for specific targeting to costimulatory B7 molecules on the surface of APC, genetically fused to residues 1-222 of human ErbB2 (HER2) as an antigenic determinant. In wild-type BALB/c mice, inoculation of syngeneic epithelial cells continuously secreting the CTLA-4-ErbB2 fusion vaccine in the vicinity of subcutaneously growing ErbB2-expressing renal cell carcinomas resulted in the rejection of established tumors, accompanied by the induction of ErbB2-specific antibodies and cytotoxic T cells. In contrast, treatment with CTLA-4-ErbB2 vaccine-secreting producer cells alone was insufficient to induce tumor rejection in ErbB2-transgenic WAP-Her-2 F1 mice, which are characterized by pronounced immunological tolerance to the human self-antigen. When CTLA-4-ErbB2 producer cells were modified to additionally secrete interleukin (IL)-15, antigen-specific antitumoral activity of the vaccine in WAP-Her-2 F1 mice was restored, documented by an increase in survival, and marked inhibition of the growth of established ErbB2-expressing, but not antigen-negative tumors. Our results demonstrate that continuous in vivo expression of an APC-targeted ErbB2 fusion protein results in antigen-specific immune responses and antitumoral activity in tumor-bearing hosts, which is augmented by the pleiotropic cytokine IL-15. This provides a rationale for further development of this approach for specific cancer immunotherapy.

Morphologic Evidence of Anti-Tumor Specificity of T Cells Activated by Denritic Cells Derived from Peripheral Blood Mononuclear Cells of Thyroid Cancer Patients

Recent studies suggest that immunization with autologous dendritic cells (DCs) results in protective immunity and rejection of established tumors in various human malignancies. The purpose of this study is to determine whether DCs are generated from peripheral blood mononuclear cells (PBMNs) by using cytokines such as F1t-3 ligand (FL), granulocyte macrophage-colony stimulating factor (GM-CSF), IL-4, and TNF-α, and whether cytotoxic T cells activated against the thyroid cancer tissues by the DCs. Peripheral blood was obtained from 2 patients with thyroid cancer. DCs were established from PBMNs by culturing in the presence of FL, GM-CSF, IL-4, and TNF-α for 14 days. At day 14, the differentiated DCs was analyzed morphologically. The immunophenotypic features of DCs such as CDla, CD83, and CD86 were analyzed by immunofluorelescence microscopy. At day 18, DCs and T cells were incubated with thyroid cancer tissues or normal thyroid tissues for additional 4 days, respectively. DCs generated from the PBMNs showed the typical morphology of DCs. Activated cytotoxic T lymphocytes (CTLs) were observed also. DCs and the CTLs were attached to the cancer tissues on scanning electron microscope. The DCs activated the CTLs, which able to specifically attack the thyroid cancer. This study provides morphologic evidence that the coculture of T cells/cancer tissues activated the T cells and differentiated CTLs. The CTLs tightly adhered to cancer tissues and lysed cancer tissues vigorously. Therefore DCs could be used as potential vaccines in the immunotherapy.

Tumor rejection by local interferon gamma induction in established tumors is associated with blood vessel destruction and necrosis

It has been shown that injecting a suspension of IFN-γ-secreting tumor cells results in their rejection. This effect has been attributed to IFN-γ preventing tumor stroma formation but not to a direct effect on the cancer cells. However, it is not known, which influence IFN-γ has on tumors with an established stroma. To address this question, the plasmacytoma cell line J558L was transduced with a vector allowing doxycycline-inducible IFN-γ gene expression. After the injection of the tumor cells into mice, IFN-γ was induced at different time points. Tumors did not grow when inducing IFN-γ immediately after tumor cell inoculation, while approximately half of the tumors were rejected when IFN-γ was induced in early established tumors within 2 weeks. Induction of IFN-γ 2-3 weeks after tumor cell inoculation was less efficient (0-17% rejection). IFN-γ induction in established tumors led to a reduction of CD146(+) endothelial cells and massive necrosis. Together, we show that vascularized tumors can be rejected by local IFN-γ expression, but that rejection of established tumors was less efficient over time. This suggests that transplanted tumors became less susceptible to local IFN-γ treatment the better they are established.

Established B16 tumors are rejected following treatment with GM-CSF-secreting tumor cell immunotherapy in combination with anti-4-1BB mAb

Immunization with irradiated tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates potent, specific and long lasting anti-tumor immunity in clinical and preclinical settings. Efforts to further increase immunotherapy efficacy with immune-modulatory agents are under evaluation. Based on the immune-modulatory properties of 4-1BB (CD137), it has been postulated that agonistic 4-1BB antibodies may add additional anti-tumor efficacy to GM-CSF-secreting tumor cell immunotherapy. The combination of GM-CSF-secreting tumor cell immunotherapy and anti-4-1BB monoclonal antibody (mAb) treatment resulted in rejection of established tumors in the B16 melanoma model. These anti-tumor effects correlated with persistent tumor-specific CD8 + T cell responses. In addition, early tumor infiltration of functional CD8 + T cells and a greater expansion of antigen-specific memory T cells were found in mice treated with the combination therapy. In summary, an agonistic anti-4-1BB mAb combined with GM-CSF-secreting tumor cell immunotherapy may provide a novel and potent treatment strategy for patients with cancer.

A phase II study of the anti-CTLA4 monoclonal antibody (mAb), CP-675,206, in patients with refractory metastatic adenocarcinoma of the colon or rectum

3035 Background: The antitumor activity of antibodies to cytotoxic T lymphocyte-associated antigen 4 (CTLA4) has been demonstrated in a variety of murine tumor models, including rejection of established tumors and secondary exposure to tumor cells. This suggests that blockade of the inhibitory effects of CTLA4 can promote effective antitumor immune responses. CP-675,206 has also been shown to induce durable tumor responses in patients (pts) with metastatic melanoma in phase 1 and phase 2 clinical studies. The purpose of this study was to assess safety and efficacy of CTLA4 blockade with the fully human mAb CP-675,206 as single-agent therapy in pts with relapsed/ refractory colorectal cancer. Methods: A single-arm, multicenter, phase II trial of CP-675,206 was conducted in pts with measurable adenocarcinoma of the colon or rectum failing standard treatments and with an ECOG performance status of 0 or 1. Patients received 15 mg/kg Q90 days via IV infusion until disease progression. The primary objective was response rate by RECIST criteria. Secondary objectives included safety, duration of response, progression-free survival, and overall survival. Results: A total of 47 pts who received a median of 4 previous therapies (range, 1 to 9) were treated, and 46 experienced disease progression or death because of disease before reaching the planned second dose at 3 months. Grade 3 or 4 adverse events attributed to study drug were limited to diarrhea (n = 3, 6.4%) and idiopathic thrombocytopenia purpura (n = 1, 2.1%). Four pts (8.5%) had grade 2 diarrhea. Four pts received steroids and 2 received infliximab. One patient was removed for toxicity (diarrhea in the setting of what appeared to be treatment-related ulcerative colitis that was responsive to steroids). One patient (2%; 95% CI = 0%, 11%) had a stable ovarian mass and a substantial regression in an adrenal mass. This patient is continuing on study and has received a second dose. Conclusions: In heavily pretreated pts with colorectal cancer and good performance status, CP- 675,206 was tolerable. However, in this setting, CP-675,206 at 15 mg/kg did not demonstrate substantial single-agent activity. No significant financial relationships to disclose.

Adoptive Immunotherapy Combined With A Dendritic Cell-Based Vaccine Results in the Rejection of Established Tumor in A Murine Melanoma Model

s: Abstracts for the 20th Annual Scientific Meeting of the International Society for Biological Therapy of Cancer (Primary Authors are Italicized): CELLULAR THERAPIES FOR SOLID AND LIQUID TUMORS

Dendritic Cell Vaccination: Potential Immunotherapy for Adrenocortical Carcinoma

The ineffectiveness of conventional therapies in adrenocortical carcinoma (ACC) emphasize the need to search for novel treatment protocols. Diverse studies suggest that immunization with autologous dendritic cells (DCs) pulsed with tumor antigen result in protective immunity and rejection of established tumors. The DC vaccination outcomes mainly depend on 1) the generation and activation of DCs, 2) the number of DCs applied and 3) the (tumor) antigens used for immunization. Several protocols have been generated to obtain large numbers of activated human DCs either from CD34+ bone-marrow cells or from peripheral blood monocytes by culturing in media supplemented with GM-CSF and IL-4, followed by stimulation with TNF-α or monocyte-conditioned medium. Using the first protocol, we were able to induce a Th1 immune response (shown by an increased interferon-γ secretion) towards the polypeptide hormone calcitonin in 5 of 7 patients with metastasized medullary thyroid carcinoma. Immunohistochemical analyses of delayed-type hypersensitivity skin reactions revealed an antigen-specific perivascular and epidermal infiltration with CD4+ T-helper (Th) cells and CD8+ cytotoxic T cells. One of these patients showed a temporary clinical effect with a decrease in liver and lung metastases. A more recent study with an improved DC generation protocol with GM-CSF (1000 U/ml) and IFNalpha (1000 U/ml) following leukapheresis resulted in the induction of a Th1 immunity (IFN-γ intracytoplasmic cytokine staining) and cytotoxic CTLs (granzyme ELISpot assay) in 2 of 5 patients each (follow-up: 2–5 months). Our data indicate that vaccination with polypeptide hormone-pulsed DC result in the induction of a cellular immune response in patients with endocrine carcinomas, leading to clinical response in some. This approach may represent the basis for the development of a new therapeutic strategy in ACC as well. We have now started a DC-based vaccination trial in two patients with ACC (follow-up: ½ and 2 months each). Further follow-up of these and other patients enrolled into the study will answer the question of whether this strategy may represent a new therapy approach in ACC.

Indoleamine 2,3-dioxygenase contributes to tumor cell evasion of T cell-mediated rejection.

The priming of an appropriate anti-tumor T cell response rarely results in the rejection of established tumors. The characteristics of tumors that allow them to evade a T cell-mediated rejection are unknown for many tumors. We report on evidence that the expression of the immunosuppressive enzyme, indoleamine 2,3-dioxygenase (IDO) by mononuclear cells that invade tumors and tumor-draining lymph nodes, is 1 mechanism that may account for this observation. Lewis lung carcinoma (LLC) cells stimulated a more robust allogeneic T cell response in vitro in the presence of a competitive inhibitor of IDO, 1-methyl tryptophan. When administered in vivo this inhibitor also resulted in delayed LLC tumor growth in syngeneic mice. Our study provides evidence for a novel mechanism whereby tumors evade rejection by the immune system, and suggests the possibility that inhibiting IDO may be developed as an anti-cancer immunotherapeutic strategy.

Immunotherapy for medullary thyroid carcinoma by dendritic cell vaccination.

Recent studies suggest that immunization with autologeous dendritic cells (DCs) pulsed with tumor antigen result in protective immunity and rejection of established tumors in various human malignancies. The objective of this study was to develop a DC vaccination therapy in patients with metastasized medullary thyroid carcinoma (MTC). Mature DCs were generated from peripheral blood monocytes in the presence of granulocyte macrophage colony-stimulating factor, IL-4, and TNFalpha. After loading with calcitonin and carcinoembryonic antigen (CEA) peptide, 2-5 x 10(6) DCs were repeatedly delivered by sc injections. During follow-up (mean, 13.1 months) all patients developed a strong delayed-type hypersensitivity skin reaction caused by perivascular and epidermal infiltration with CD4+ memory T cells and CD8+ cytotoxic T cells. Clinical responses with a decrease of serum calcitonin and CEA were initially documented in three of seven patients. One of these patients had a complete regression of detectable liver metastases and a significant reduction of pulmonary lesions. T-cell response in this patient revealed a calcitonin- and CEA-specific immunreactivity. Our data indicate that vaccination with calcitonin and/or CEA peptide-pulsed DC results in the induction of a cellular, antigen-specific immune response in patients with MTC, leading to clinical response in some patients. Our approach may represent the basis for the development of new therapeutic strategies not only in MTC but also in other endocrine malignancies.

Cloning and characterization of the genes encoding the murine homologues of the human melanoma antigens MART1 and gp100.

The recent identification of genes encoding melanoma-associated antigens has opened new possibilities for the development of cancer vaccines designed to cause the rejection of established tumors. To develop a syngeneic animal model for evaluating antigen-specific vaccines in cancer therapy, the murine homologues of the human melanoma antigens MART1 and gp100, which were specifically recognized by tumor-infiltrating lymphocytes from patients with melanoma, were cloned and sequenced from a murine B16 melanoma cDNA library. The open reading frames of murine MART1 and gp100 encode proteins of 113- and 626-amino acids with 68.8 and 77% identity to the respective human proteins. Comparison of the DNA sequences of the murine MART1 genes, derived from normal melanocytes, the immortalized nontumorgenic melanocyte line Melan-a and the B16 melanoma, showed all to be identical. Northern and Western blot analyses confirmed that both genes encoded products that were melanocyte lineage proteins. Mice immunized with murine MART1 or gp100 using recombinant vaccinia virus failed to produce any detectable T-cell responses or protective immunity against B16 melanoma. In contrast, immunization of mice with human gp100 using recombinant adenoviruses elicited T cells specific for hgp100, but these T cells also cross reacted with B16 tumor in vitro and induced significant but weak protection against B16 challenge. Immunization with human and mouse gp100 together [adenovirus type 2 (Ad2)-hgp100 plus recombinant vaccinia virus (rVV)-mgp100], or immunization with human gp100 (Ad2-hgp100) and boosting with heterologous vector (rVV-hgp100 or rVV-mgp100) or homologous vector (Ad2-hgp100), did not significantly enhance the protective response against B16 melanoma. These results may suggest that immunization with heterologous tumor antigen, rather than self, may be more effective as an immunotherapeutic reagent in designing antigen-specific cancer vaccines.

Antitumor Activity of a <italic>Streptococcus pyogenes</italic> Preparation (OK-432). I. Sequential Effector Mechanisms Following a Single OK-432 Injection in F344 Rats Leading to the Rejection of Syngeneic MADB 106 Tumor Cells<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

The effector mechanisms evoked in tumor-bearing rats following a single injection of the avirulent Su strain of type 3, group A Streptococcus pyogenes (OK-432) were sequentially examined. F344 rats challenged ip with a lethal dose of the syngeneic MADB106 mammary carcinoma could survive more than 100 days when given 50 mg OK-432/kg ip 1 day after tumor challenge. When the responsible effector mechanisms were examined in this therapeutic model, two distinct effector phases distinguished by the number of tumor cells were evident. Phase I, 1-6 days following OK-432 injection, resulted in a sharp decrease in tumor cell numbers and was related to the direct antitumor cytotoxicity of OK-432 and was coincident with an increase in the number of polymorphonuclear neutrophils. However, by day 6 a sharp increase in tumor cell numbers was again observed. Subsequently, a second phase of tumor cell destruction was observed 7-20 days following OK-432 injection and was reflected in a strong lymphocyte-mediated cytotoxicity response as well as the production of complement-dependent cytotoxic antibody against the MADB106 tumor cells. Further, the adoptive transfer of either peritoneal exudate cells or sera from the phase II animals revealed that both factors may be responsible for the antitumor activity observed in this therapeutic model. In conclusion, this study has demonstrated that the antitumor effects seen with OK-432 are due to a combination of sequential effector mechanisms leading to the eventual rejection of established tumor.