Potentiation Of Antitumor Immunity Research Articles

Enhancing Cancer Immunotherapy: A Novel Approach through the Integration of Oncolytic Virotherapy with CAR-T and NK Cell Therapies

A two-pronged approach to fighting cancer, oncolytic virotherapy (OV) combines direct tumor cytotoxicity with potentiation of anti-tumor immunity. Combining OVs and cell therapies, such as CAR-T and NK cells, has been proposed as an enhanced therapeutic approach. Additionally, it has been demonstrated that adding oncolytic virotherapy to combination medicines increases their therapeutic potential. This article explains the mechanisms underlying oncolytic viruses, the beneficial interactions between them and immune cell therapies, as well as the drawbacks and difficulties of this strategy. We highlight the novel approach of combining CAR T/NK cell treatments and oncolytic virotherapy, highlighting current developments and speculating on the future trajectories of this frontier in cancer immunotherapy.

Next-Generation Antisense Oligonucleotide of TGF-β2 Enhances T Cell-Mediated Anticancer Efficacy of Anti-PD-1 Therapy in a Humanized Mouse Model of Immune-Excluded Melanoma.

Anti-programmed death-1 (PD-1) immunotherapy is one of the most promising therapeutic interventions for treating various tumors, including lung cancer, bladder cancer, and melanoma. However, only a subset of patients responds to anti-PD-1 therapy due to complicated immune regulation in tumors and the evolution of resistance. In the current study, we investigate the potential of a novel transforming growth factor-beta2 (TGF-β2) antisense oligonucleotide (ngTASO), as a combination therapy with an anti-PD-1 antibody in melanoma. This study was conducted in a melanoma-bearing human immune system mouse model that recapitulates immune-excluded phenotypes. We observed that the TGF-β2 blockade by ngTASO in combination with PD-1 inhibition downregulated the tumor intrinsic β-catenin, facilitated the infiltration of CD8+ cytotoxic lymphocytes (CTLs) in the tumor, and finally, enhanced the antitumor immune potentials and tumor growth delays. Blockade of TGF-β2 combined with PD-1 inhibition also resulted in downregulating the ratio of regulatory T cells to CTLs in the peripheral blood and tumor, resulting in increased granzyme B expression. In addition, co-treatment of ngTASO and anti-PD-1 augmented the PD-L1 expression in tumors, which is associated with an improved response to anti-PD-1 immunotherapy. These results indicate that the combination of ngTASO and anti-PD-1 exerts an enhanced T cell-mediated antitumor immune potential. Hence, co-inhibition of TGF-β2 and PD-1 is a potentially promising immunotherapeutic strategy for immune-excluded melanoma.

Near-Infrared-Enpowered Nanomotor-Mediated Targeted Chemotherapy and Mitochondrial Phototherapy to Boost Systematic Antitumor Immunity.

Phototherapy is an important strategy to inhibit tumor growth and activate antitumor immunity. However, the effect of photothermal/photodynamic therapy (PTT/PDT) is restricted by limited tumor penetration depth and unsatisfactory potentiation of antitumor immunity. Here, a near-infrared (NIR)-driven nanomotor is constructed with a mesoporous silicon nanoparticle (MSN) as the core, end-capped with Antheraea pernyi silk fibroin (ApSF) comprising arginine-glycine-aspartate (RGD) tripeptides. Upon NIR irradiation, the resulting ApSF-coated MSNs (DIMs) loading with photosensitizers (ICG derivatives, IDs) and chemotherapeutic drugs (doxorubicin, Dox) can efficiently penetrate into the internal tumor tissues and achieve effective phototherapy. Combined with chemotherapy, a triple-modal treatment (PTT, PDT, and chemotherapy) approach is developed to induce the immunogenic cell death of tumor cells and to accelerate the release of damage-associated molecular patterns. In vivo results suggest that DIMs can promote the maturation of dendritic cells and surge the number of infiltrated immune cells. Meanwhile, DIMs can polarize macrophages from M2 to M1 phenotypes and reduce the percentages of immunosuppressive Tregs, which reverse the immunosuppressive tumor microenvironment and activate systemic antitumor immunity. By achieving synergistic effects on the tumor inhibition and the antitumor immunity activation, DIMs show great promise as new nanoplatforms to treat metastatic breast cancer.

Salvia miltiorrhiza in Breast Cancer Treatment: A Review of Its Phytochemistry, Derivatives, Nanoparticles, and Potential Mechanisms.

Breast cancer is one of the most deadly malignancies in women worldwide. Salvia miltiorrhiza, a perennial plant that belongs to the genus Salvia, has long been used in the management of cardiovascular and cerebrovascular diseases. The main anti-breast cancer constituents in S. miltiorrhiza are liposoluble tanshinones including dihydrotanshinone I, tanshinone I, tanshinone IIA, and cryptotanshinone, and water-soluble phenolic acids represented by salvianolic acid A, salvianolic acid B, salvianolic acid C, and rosmarinic acid. These active components have potent efficacy on breast cancer in vitro and in vivo. The mechanisms mainly include induction of apoptosis, autophagy and cell cycle arrest, anti-metastasis, formation of cancer stem cells, and potentiation of antitumor immunity. This review summarized the main bioactive constituents of S. miltiorrhiza and their derivatives or nanoparticles that possess anti-breast cancer activity. Besides, the synergistic combination with other drugs and the underlying molecular mechanisms were also summarized to provide a reference for future research on S. miltiorrhiza for breast cancer treatment.

The Potentiation of Anti-Tumor Immunity by Tumor Abolition with Alpha Particles, Protons, or Carbon Ion Radiation and Its Enforcement by Combination with Immunoadjuvants or Inhibitors of Immune Suppressor Cells and Checkpoint Molecules.

The delivery of radiation therapy (RT) for cancer with intent to cure has been optimized and standardized over the last 80 years. Both preclinical and clinical work emphasized the observation that radiation destroys the tumor and exposes its components to the immune response in a mode that facilitates the induction of anti-tumor immunity or reinforces such a response. External beam photon radiation is the most prevalent in situ abolition treatment, and its use exposed the “abscopal effect”. Particle radiotherapy (PRT), which has been in various stages of research and development for 70 years, is today available for the treatment of patients in the form of alpha particles, proton, or carbon ion radiotherapy. Charged particle radiotherapy is based on the acceleration of charged species, such as protons or carbon-12, which deposit their energy in the treated tumor and have a higher relative biological effectiveness compared with photon radiation. In this review, we will bring evidence that alpha particles, proton, or carbon ion radiation can destroy tumors and activate specific anti-tumor immune responses. Radiation may also directly affect the distribution and function of immune cells such as T cells, regulatory T cells, and mononuclear phagocytes. Tumor abolition by radiation can trigger an immune response against the tumor. However, abolition alone rarely induces effective anti-tumor immunity resulting in systemic tumor rejection. Immunotherapy can complement abolition to reinforce the anti-tumor immunity to better eradicate residual local and metastatic tumor cells. Various methods and agents such as immunoadjuvants, suppressor cell inhibitors, or checkpoint inhibitors were used to manipulate the immune response in combination with radiation. This review deals with the manifestations of particle-mediated radiotherapy and its correlation with immunotherapy of cancer.

Targeting Checkpoint Receptors and Molecules for Therapeutic Modulation of Natural Killer Cells.

Among the most promising therapeutic modalities for cancer treatment is the blockade of immune checkpoint pathways, which are frequently co-opted by tumors as a major mechanism of immune escape. CTLA-4 and PD-1 are the representative examples, and their blockade by therapeutic antibodies leads to enhanced anti-tumor immunity with durable clinical responses, but only in a minority of patients. This has highlighted the need to identify and target additional immune checkpoints that can be exploited to further enhance immune responses to refractory cancers. These emerging targets include natural killer (NK) cell-directed checkpoint receptors (KIR and CD94/NKG2A) as well as the NK- and T cell-expressed checkpoints TIM-3, TIGIT, CD96, and LAG-3. Interestingly, the potentiation of anti-tumor immunity by checkpoint blockade relies not only on T cells but also on other components of the innate immune system, including NK cells. NK cells are innate lymphoid cells that efficiently kill tumor cells without MHC specificity, which is complementary to the MHC-restricted tumor lysis mediated by cytotoxic T cells. However, the role of these immune checkpoints in modulating the function of NK cells remains unclear and somewhat controversial. Unraveling the mechanisms by which these immune checkpoints function in NK cells and other immune cells will pave the way to developing new therapeutic strategies to optimize anti-tumor immunity while limiting cancer immune escape. Here, we focus on recent findings regarding the roles of immune checkpoints in regulating NK cell function and their potential application in cancer immunotherapy.

Abstract 18: Evaluating the contribution of anti-myeloma immunity for the efficacy of oncolytic reovirus therapy

Abstract Introduction: The aim of this study was to examine the contribution of antitumor immunity for the efficacy of oncolytic reovirus therapy against multiple myeloma (MM). Oncolytic viruses (OV) have two main mechanisms of action; direct lytic killing and potentiation of antitumor immunity. The direct oncolytic potential of reovirus in MM has previously been demonstrated both in vitro and in vivo, but the importance of an enhanced immunologic antitumor response remains underinvestigated. Reovirus-induced antitumor immunity has been demonstrated in other types of cancer such as melanoma and chronic leukemia, and is of major importance for the efficacy of OV treatment. Thus, it was hypothesized that reovirus-induced antimyeloma immunity would contribute significantly to the efficacy of reovirus treatment for MM. Experimental Procedures: C57BL/KaLwRij mice were used in the 5TGM1 model system to establish MM in vivo. This model closely resembles human MM with induction of osteolytic bone disease and secretion of paraprotein. C57BL/KaLwRij mice have a fully functional immune system, comparable to C57BL/6 mice, and to our knowledge, this is the first immunocompetent model of MM for the study of reovirus efficacy. After establishment of MM in the bone by intravenous injection of bone-homing 5TGM1 cells, mice were treated with repeated injections of reovirus or PBS. Upon sacrifice, direct cytotoxicity and immune activation was examined using flow cytometry. All animal experiments were performed under an appropriate project license following approval by a local ethical review committee. In vivo findings were translated into human in vitro studies, using MM cell lines, healthy donor (HD) blood, and MM patient samples. Reovirus-induced Natural Killer (NK) cell activation and degranulation was examined using flow cytometry and priming of myeloma-specific T cells was performed using long-term priming cultures. Results: Tumor burden was reduced by reovirus treatment both in the bone marrow (BM) and spleen of tumor-bearing mice. The immune cell populations, including NK cells, CD4+ T cells, and CD8+ T cells, were restored to levels of tumor-naïve mice in the BM. NK cells were activated in the BM following reovirus treatment, which indicates the onset of an innate immune response. In the spleen, an increase in CD4+ T cells, in combination with CD8+ activation, was indicative of an early adaptive immune response. These results translate into human in vitro findings, using both HD and MM patient samples, with activation of NK cells in response to reovirus treatment and subsequent enhancement of NK cell degranulation and killing of MM target cells. Encouragingly, reovirus-activated NK cells were able to kill OPM2 cells, which are resistant to direct lytic killing. Preliminary human in vitro studies suggest that reovirus treatment can prime antimyeloma CD8+ T cells for the induction of a long-term protective response. Conclusions: Introducing a viral agent into the body requires a delicate immunologic balance to avoid neutralizing the virus by an antiviral response and simultaneously allowing the enhancement of antitumor immunity. The importance of enhanced antitumor immunity for OV therapy efficacy is becoming more widely recognized and antiviral immunity can in some circumstances contribute to tumor eradication. The findings in this study indicate that the antitumor immune response is also important in the MM setting, with activation of both innate and adaptive immune responses resulting in enhanced killing of MM cells, in particular those resistant to direct lytic killing. This suggests that both arms of OV therapy could play a role for MM eradication, including any minimal residual disease. Future work will explore whether the antimyeloma response can be further enhanced by combinatorial treatments, including current standard of care treatments. Citation Format: Louise M. E. Müller, Christopher Parrish, Gemma Migneco, Gina B. Scott, Matthew Holmes, Alan A. Melcher, Michelle A. Lawson, Gordon Cook, Fiona Errington-Mais. Evaluating the contribution of anti-myeloma immunity for the efficacy of oncolytic reovirus therapy [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 18.

Baseline tumor growth and immune control in laboratory mice are significantly influenced by subthermoneutral housing temperature

We show here that fundamental aspects of antitumor immunity in mice are significantly influenced by ambient housing temperature. Standard housing temperature for laboratory mice in research facilities is mandated to be between 20-26 °C; however, these subthermoneutral temperatures cause mild chronic cold stress, activating thermogenesis to maintain normal body temperature. When stress is alleviated by housing at thermoneutral ambient temperature (30-31 °C), we observe a striking reduction in tumor formation, growth rate and metastasis. This improved control of tumor growth is dependent upon the adaptive immune system. We observe significantly increased numbers of antigen-specific CD8(+) T lymphocytes and CD8(+) T cells with an activated phenotype in the tumor microenvironment at thermoneutrality. At the same time there is a significant reduction in numbers of immunosuppressive MDSCs and regulatory T lymphocytes. Notably, in temperature preference studies, tumor-bearing mice select a higher ambient temperature than non-tumor-bearing mice, suggesting that tumor-bearing mice experience a greater degree of cold-stress. Overall, our data raise the hypothesis that suppression of antitumor immunity is an outcome of cold stress-induced thermogenesis. Therefore, the common approach of studying immunity against tumors in mice housed only at standard room temperature may be limiting our understanding of the full potential of the antitumor immune response.

Synergistic potentiation of antitumor immunity with light-activated drug therapy and anti-CTLA4 antibody treatment.

e13034 Background: Talaporfin sodium (TS) is a light-activated, locally cytoreductive drug in Phase 3 clinical trials for several different cancers. It is activated intratumorally with a thin, flexible LED array. TS treatment has been shown in preclinical studies to activate CD8+-dependent anti-tumor immunity in a model of metastatic breast cancer. Anti-CTLA4 antibody is an immunotherapy that acts by suppressing negative regulation of immune effectors. It has recently completed a successful Phase 3 clinical trial in melanoma patients in which it was shown to prolong survival.TS and anti-CTLA4 antibody have great potential to synergistically reduce primary tumor burden and generate systemic anti-tumor immunity. Methods: This study examined combination treatment with these two therapies in a murine prostate cancer tumor model (C57BL/6 + TRAMP C2). A large, established tumor on the left side of each mouse was treated with light-activated TS (or light-only control). One day after treatment, mice were injected intradermally with 0.5 x 106 TRAMP C2 cells on the opposite flank. Anti-CTLA4 (or vehicle control) was given on days 1, 4, 7 and 10. Progression of the untreated tumor was measured. Results: Growth of untreated tumors was prevented or significantly delayed in 55% and 33% of combination-treated animals, respectively (n = 9). In addition, contralateral tumor growth was prevented in 1 of 9 animals given talaporfin sodium alone while another animal’s tumor showed growth delay. None of the untreated controls or anti-CTLA4 only animals showed a similar response. Individual combination-treated mice with delayed tumor growth often showed an atypical tumor growth curve in which progression either tapered off or began to reverse between 30 and 40 days post treatment. Conclusions: Reduction of tumor using local treatment with a light-activated drug could potentiate the anti-tumor immune response resulting from anti-CTLA4 treatment and prevent growth of secondary tumors in a preclinical prostate cancer model.

Synergistic potentiation of anti-tumor immunity with light-activated drug therapy and anti-CTLA4 antibody treatment.

27 Background: Talaporfin sodium (TS) is a light-activated, locally cytoreductive drug in phase III clinical trials for several different cancers. It is activated intratumorally with a thin, flexible LED array. TS treatment has been shown in preclinical studies to activate CD8+-dependent anti-tumor immunity in a model of metastatic breast cancer. Anti-CTLA4 antibody is an immunotherapy that acts by suppressing negative regulation of immune effectors. It has recently completed a successful phase III clinical trial in melanoma patients in which it was shown to prolong survival. TS and anti-CTLA4 antibody have great potential to synergistically reduce primary tumor burden and generate systemic anti-tumor immunity. Methods: This study examined combination treatment with these two therapies in a murine prostate cancer tumor model (C57BL/6 + TRAMP C2). A large, established tumor on the left side of each mouse was treated with light-activated TS (or light- only control). One day after treatment, mice were injected intradermally with 0.5 × 106 TRAMP C2 cells on the opposite flank. Anti-CTLA4 (or vehicle control) was given on days 1, 4, 7 and 10. Progression of the untreated tumor was measured. Results: Growth of untreated tumors was prevented or significantly delayed in 55% and 33% of combination-treated animals, respectively (n = 9). In addition, contralateral tumor growth was prevented in 1 of 9 animals given talaporfin sodium alone while another animal's tumor showed growth delay. None of the untreated controls or anti-CTLA4 only animals showed a similar response. Individual combination-treated mice with delayed tumor growth often showed an atypical tumor growth curve in which progression either tapered off or began to reverse between 30 and 40 days post treatment. Conclusions: Reduction of tumor using local treatment with a light-activated drug could potentiate the anti-tumor immune response resulting from anti-CTLA4 treatment and prevent growth of secondary tumors in a preclinical prostate cancer model. [Table: see text]

Potentiation of antitumor immunity by antibody-directed enzyme prodrug therapy.

Antibody-directed enzyme prodrug therapy (ADEPT) has displayed antitumor activity in animal models and clinical trials. We examined whether antitumor immunity is generated during ADEPT by employing an immunoenzyme composed of the monoclonal antibody (MAb) RH1 conjugated to beta-glucuronidase to target rat AS-30D hepatocellular carcinoma tumors. A glucuronide prodrug of p-hydroxyaniline mustard was used to treat malignant ascites after immunoenzyme localization at the cancer cells. ADEPT cured more than 96% of Sprague-Dawley rats bearing advanced malignant ascites, and all cured rats were protected from a lethal challenge of AS-30D cells. Immunization with radiation-killed AS-30D cells or AS-30D cells coated with immunoenzyme did not provide tumor protection. Likewise, ex vivo treatment of tumor cells by ADEPT before injection into rats did not protect against a tumor challenge. AS-30D and N1-S1 hepatocellular carcinoma cells but not unrelated syngeneic tumor cells were lysed by peritoneal exudate cells isolated from ADEPT-cured rats. Depletion of CD8(+) but not CD4(+) T cells or natural killer (NK) cells reduced the cytolytic activity of peritoneal lymphocytes. ADEPT did not cure tumor-bearing rats depleted of CD4(+) and CD8(+) T cells even though it was curative when given 7 days after tumor transplantation in rats with an intact immune system, indicating that ADEPT can synergize with host immunity to increase therapeutic efficacy. These results have important implications for the clinical application of ADEPT.

Some approaches to improve the therapeutic effectiveness of adoptive chemoimmunotherapy with spleen cells from melphalan-treated BALB/c mice bearing a large MOPC-315 tumor.

Spleen cells from BALB/c mice that are in the process of eradicating a large MOPC-315 tumor following low-dose L-PAM therapy (L-PAM TuB spleen cells) were previously shown to be capable of bringing about the complete regression of a large (15 to 20 mm) s.c. MOPC-315 tumor in a substantial percentage of T-cell-deficient (athymic nude) mice that had been treated with low-dose L-PAM (adoptive chemo-immunotherapy; ACIT). Here we show that aggressive depletion of CD4+T-cells by treatment both of spleen-cell donors and of recipients with anti-L3T4 monoclonal antibody (MAb) greatly improved the therapeutic effectiveness of L-PAM TuB spleen cells in ACIT. In the light of the apparent importance of cytotoxic T-lymphocytes (CTLs) for tumor eradication in low-dose L-PAM-treated MOPC-315-tumor bearers, it is interesting that treatment of L-PAM TuB spleen-cell donors with anti-L3T4 MAb was found to result in the generation of enhanced splenic anti-MOPC-315 cytotoxicity. Although most athymic nude mice in which the tumor had apparently completely regressed following ACIT remained tumor-free, approximately 1/3 of the mice relapsed. However, a substantial percentage of the relapsing mice were rescued by a low dose of L-PAM, which was not effective in causing tumor regression in athymic nude mice bearing a comparably large tumor if the mice had not been subjected previously to ACIT. Almost all athymic nude mice that had been "cured" of a large MOPC-315 tumor by ACIT but did not resist a subsequent MOPC-315 tumor challenge were rescued by low dose L-PAM. Thus, the therapeutic effectiveness of L-PAM TuB spleen cells in ACIT may be improved by aggressive depletion of CD4+ T-cells, suggesting that a low dose of L-PAM, which leads to the acquisition of potent splenic-tumor-eradicating immunity in BALB/c mice bearing a large MOPC-315 tumor, does not eliminate completely (or possibly not at all) the inhibitory activity of CD4+ T-cells. In addition, athymic nude mice that are not endowed with fully protective tumor-eradicating immunity following ACIT still have a substantial residual anti-tumor immune potential that can be exploited to bring about eradication of a large tumor burden following low-dose L-PAM therapy.

Suppressive effect of portal venous inoculation with tumor cells on the anti-tumor immune potential.

The effect of portal venous (pv) administration of syngeneic tumor cells on the potential to generate anti-tumor immune resistance was examined. C3H/He mice were inoculated with syngeneic X5563 tumor cells via the pv or intravenous (iv) route. A single pv administration of 10,000 R X-irradiated X5563 cells into C3H/He mice not only failed to induce anti-X5563 immunity but also abolished the capability of the mice to develop anti-X5563 in vitro cytotoxic and in vivo protective immunity as induced by intradermal (id) inoculation of viable X5563 cells followed by the surgical resection of the tumor (immunization procedure). Such immunosuppression was also obtained by pv inoculation of tumor cells after the above immunization procedure. The immunosuppression induced by the pv injection of tumor cells before or after the immunization procedure was tumor-specific, since the pv sensitizing regimen using X5563 tumor cells did not interfere with the generation of immunity against another syngeneic tumor, MH134. More importantly, the pv route-induced suppression contrasted with the lack of inhibiting effect of iv inoculation with tumor cells on the induction of the tumor immunity. These results indicate that the host's tumor-specific immune capability is markedly suppressed in an anti-tumor sensitizing stage-independent manner when tumor cells enter the pv circulation.

Melphalan-induced enhancement of tumor cell immunostimulatory capacity as a mechanism for the appearance of potent antitumor immunity in the spleen of mice bearing a large metastatic MOPC-315 tumor.

Exposure of MOPC-315 cells from the primary tumor nodule to a low concentration (0.5 nmol/ml) of melphalan (L-phenylalanine mustard; L-PAM) rendered the tumor cells capable of bringing about the generation of a potent primary antitumor cytotoxic response. Accordingly, the level of antitumor cytotoxicity generated by normal spleen cells immunized in vitro with L-PAm-treated tumor cells was at least five-fold greater than the level generated in response to untreated tumor cells. The marked superiority of L-PAM-treated tumor cells over untreated tumor cells in bringing about the generation of antitumor cytotoxicity was evident over a wide range of responder to stimulator cell ratios. The higher level of antitumor cytotoxicity exhibited by normal spleen cells immunized with L-PAM-treated tumor cells as compared with untreated tumor cells was not merely the result of direct drug-mediated tumoricidal activity, thereby reducing the number of tumor cells present which can act as cold target cell inhibitors during the 51Cr release assay. This is apparent from the observation that the level of antitumor cytotoxicity generated in response to a given percentage of stimulator tumor cells pretreated with 0.5 nmol L-PAM/ml, a drug concentration associated with retention of 60% tumor cell proliferative capacity, is substantially greater than that generated in response to less than half that percentage of untreated stimulator tumor cells. Moreover, stimulator tumor cells exposed to a fully antiproliferative concentration of L-PAM brought about the generation of a higher level of antitumor cytotoxicity than stimulator tumor cells exposed to mitomycin C at a concentration which inhibited the proliferation of the tumor cells to the same extent as the L-PAM. A low concentration of L-PAM which was effective in rendering isolated tumor cells from the primary tumor nodule capable of bringing about the generation of antitumor cytotoxicity was also effective in inducing the appearance of potent antitumor immune potential in tumor bearer splenic cells containing metastatic tumor cells.

Cyclophosphamide-mediated enhancement of antitumor immune potential of immunosuppressed spleen cells from mice bearing a large MOPC-315 tumor

We have utilized 4-hydroperoxycyclophosphamide (4-HP-CY) as a probe for the immunomodulatory activity of the metabolites of cyclophosphamide (CY) since 4-HP-CY hydrolyzes spontaneously in aqueous solution to the same metabolites as those formed after in vivo conversion of CY by microsomal enzymes. Exposure of immunosuppressed MOPC-315 tumor bearer spleens to a low concentration of 4-HP-CY (0.1–3.0 μm) resulted in augmented antitumor immune potential. The level of antitumor immune potential exhibited by 4-HP-CY-treated tumor bearer spleen cells was not further augmented but was actually reduced by depletion of glass-adherent cells, a procedure which is effective in removing the cells known to have immunosuppressive activity (i.e. metastatic tumor cells and macrophages) from the spleen of untreated MOPC-315 tumor-bearing mice. In fact, 4-HP-CY was superior to depletion of glass-adherent cells in augmenting the antitumor immune potential of immunosuppressed tumor bearer spleen cells. When cells from the primary tumor nodule were incubated with a low concentration of 4-HP-CY which only marginally inhibited their proliferation, the drug completely abolished the suppressive activity of the cells for the in vitvo generation of antitumor cytotoxicity by normal spleen cells. Moreover, a high level of antitumor cytotoxicity developed when normal spleen cells were cultured vitro with 4-HP-CY-treated tumor cells at a wide range of ratios of spleen cells to tumor cells. Thus, in the MOPC-315 tumor model, metabolites of CY eliminate the inhibitory effectiveness of splenic suppressor cells and induce the appearance of immunopotentiating activity. The results obtained with 4-HP-CY in vitro provide support for the hypothesis that low-dose CY therapy of mice bearing a large MOPC-315 tumor leads to the appearance of augmented antitumor immune potential in their hitherto immunosuppressed spleen cells as a result of the in situ immunomodulatory effect of the drug on cells in the spleen.

Melphalan-mediated potentiation of antitumor immune responsiveness of immunosuppressed spleen cells from mice bearing a large MOPC-315 tumor.

Administration of a low dose of L-PAM (0.75 mg/kg) to mice bearing a large SC MOPC-315 tumor and extensive metastases led to the development of augmented antitumor immune potential in their hitherto immunosuppressed spleen cells. Such drug-induced potentiation of antitumor immune responsiveness appeared by day 2 after chemotherapy, and it could not be further enhanced but was actually reduced by depletion of glass-adherent cells, a procedure which is effective in depleting the cells known to have inhibitory activity (i.e., macrophages and metastatic tumor cells). To establish that L-PAM can lead to selective in situ abrogation of the inhibitory effectiveness of the splenic macrophages and metastatic tumor cells, we demonstrated that incubation of immunosuppressed tumor-bearer spleen cells with a low concentration of L-PAM in vitro also resulted in augmented antitumor immune potential that could not be further augmented by depletion of glass-adherent cells. L-PAM-mediated enhancement of the antitumor immune potential of immunosuppressed tumor bearer spleen cells was due at least in part to the effects of the drug on the splenic metastatic tumor cells. Isolated tumor cells treated with a low concentration of L-PAM were not only devoid of inhibitory activity for the primary in vitro antitumor immune response by normal spleen cells, but actually manifested a strong immunostimulatory capacity. Thus, L-PAM given at a low dose enhances the development of potent antitumor immunity which brings about the eradication of a large tumorigenic load that remains after the drug has been cleared from the circulation.

Suppression of antitumor immunity by macrophages in spleens of mice bearing a large MOPC-315 tumor.

We had shown previously that progression of MOPC-315 plasmacytoma growth is associated with an increase in the percentage of macrophages in the spleen as well as a decrease in the ability of tumor-bearer spleen cells to mount an antitumor cytotoxic response upon in vitro immunization. Here we provide evidence that macrophages in the MOPC-315 tumor-bearer spleen are responsible at least in part for the suppression of the generation of antitumor cytotoxicity. Accordingly, removal of most macrophages by depletion of phagocytic cells or Sephadex G-10-adherent cells from spleens of mice bearing a large tumor resulted in augmented antitumor immune potential. Also, Sephadex G-10-adherent spleen cells from tumor-bearing (but not normal) mice drastically suppressed the in vitro generation of antitumor cytotoxicity by normal spleen cells. The suppressive activity of these adherent cells did not reside in contaminating suppressor T cells, since it was not reduced by treatment with monoclonal anti-Thy 1.2 antibody plus complement. The Sephadex G-10-adherent cell population from the tumor-bearer spleen suppressed the in vitro generation of antitumor cytotoxicity against autochthonous tumor cells but not against allogeneic EL4 tumor cells, and hence the suppression was apparently specific. The suppressive activity of the Sephadex G-10-adherent cell population from tumor-bearer spleens was overcome by treatment of the tumor-bearing mice with a low curative dose of cyclophosphamide. This immunomodulatory effect of a low dose of the drug in overcoming the suppression mediated by the Sephadex G-10-adherent cell population enables the effector arm of the immune system of tumor-bearing mice to cooperate effectively with the drug's tumoricidal activity in tumor eradication.

Comparison of the Potentiation of Specific Tumor Immunity in Mice by Corynebacterium parvum or BCG 2

Two independent studies have compared killed C. parvum (CP) vaccine with viable BCG (Pasteur) and BCG (Glaxo) vaccines, respectively, for potentiation of antitumor immunity when injected with irradiated tumor cells into B6D2F1 AND CBA-T6T6 mice. Both studies concurred that, for a given number of irradiated tumor cells, larger amounts (dry weight equivalent) of BCG than CP were required to produce similar degrees of tumor immunity. Evidence also showed that stronger and more protracted immunity was mediated by CP than by BCG.