Castro, Sarah A

Abstract Description This study dissects mechanisms of spontaneous tumor rejection leading to better understanding of successful immune responses to cancer. Cancers persist despite expression of tumor-specific neoantigens promoting ongoing anti-tumor immune responses. While some occult tumor cells likely are cleared by effective anti-tumor immune responses, the targeted antigens are not easily identifiable as those cells spontaneously disappear. Congenic mouse breast carcinoma models expressing a defined protein mimicking neoantigens were used to dissect the protective spontaneous immune response. BALB/c (MHC H-2d) mice challenged with BALB/c breast tumors expressing the rat-erbB2 oncoprotein succumb to their tumors despite demonstrated tumor immunogenicity, mimicking known patient responses to cancer. Meanwhile, H-2b positive BALB.B and H-2d/H-2b F1 hybrid mice spontaneously eliminate genetically matched tumors in an MHC-II dependent manner. CD4+ T-cells and B-cells or the rat-erbB2-specific antibodies they produce are crucial mediators of this protective response. Differences in the tumor microenvironments in rat-erbB2+ tumor-bearing BALB.B and BALB/c mice at the time of fate decision in the models reflect the differences between effective and ineffective anti-tumor immunity. Identifying the nature of the optimal MHC-II antigens, how they activate protective CD4+ T-cells, and the interactions that promote protective antibodies will better inform future cancer therapies. Funding Sources Bernie Fineman Research Fund Mayo Clinic Center for Biomedical Discovery Mayo Clinic Department of Immunology Mayo Clinic Graduate School of Biomedical Sciences NIH Traning Grant 5 R25 GM 55252 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Background Cancers persist despite expression of immunogenic neoantigens and ongoing antitumor immune responses. While some occult tumors likely are cleared by effective antitumor immune responses, the targeted antigens are not easily identifiable as those tumors spontaneously disappear. Methods We used mouse models with a defined antigenic protein mimicking tumor-specific neoantigens to address the nature of these spontaneous anti-tumor immune responses. Results BALB/c (H-2d ) mice challenged with BALB/c breast tumors expressing the rat-erbB2 oncoprotein succumb to their tumors despite ongoing immune responses targeting tumor-specific model antigens. Meanwhile, congenic BALB.B (H-2b ) and H-2d/H-2b F1 hybrid mice spontaneously eliminate genetically matched tumors in a major histocompatibility complex (MHC)-II dependent manner. Adoptive transfer and immune cell depletion strategies revealed CD4+ T cells and CD20+ B cells are crucial mediators of the protective response in H-2b mice. Furthermore, passive transfer of immune serum from mice rejecting their tumors confers resistance in tumor antigen-tolerant animals with an inversely proportional relationship between tumor outgrowth and the amount of rat-erbB2 specific antibody present in tumor-bearing mice. Introduction of the rat-erb2 ectodomain into other H-2b tumor models also promotes their spontaneous tumor rejection. Notably, the tumor microenvironments differ in rat-erbB2+ tumor-bearing BALB.B and BALB/c mice at the time of fate decision in the models reflecting the differences between effective and ineffective tumor immune responses. Conclusions We find that the effective antitumor immunity targeting neoantigens in these breast cancer models is determined by MHC-II-restricted presentation of optimal cancer-associated antigens. These responses are dependent on CD4+ T cells, B cells, and antigen-specific antibodies.

Abstract 20 to 30% of breast cancers overexpress ERRB2, encoding the human epidermal growth factor-2 receptor (HER2). Overexpression of HER2 is associated with poorer prognosis, particularly in metastatic disease. Although HER2 therapeutics exists many patients eventually develop uncontrolled tumor growth. The BALB/c-neuT mouse has been a popular model for investigating HER2-positive breast cancer. We have found that immune responsiveness and MHC genotype are important factors in spontaneous tumor development and tumor rejection in the neuT model. To assess the influence of immune response potential on tumor biology we introduced the skg ZAP-70 mutation or the H-2 b MHC haplotype into the neuT model. Spontaneous tumors appeared with decreased frequency and metastasis or increased frequency and metastasis, respectively, in comparison to the BALB/c model. H-2 b tumors were readily transplantable into the breast fat pads of syngeneic BALB.B-neuT hosts which developed systemic immune abnormalities very similar to those observed in BALB/c - neuT or BALB/c WT mice bearing neuT tumors. However, BALB.B WT hosts initially accepted the grafts, but subsequently rejected them after they were established. Adoptive transfer of splenocytes from BALB.B WT mice which had rejected tumor conferred resistance to the tumor grafts, as well as to spontaneous tumors developing in BALB.B neuT littermates. The strong H-2b-mediated antitumor response in WT mice indicates efficient antigen presentation, and presumably leads to strong tolerance in neuT+ animals. The importance of an intact T cell signaling and MHC genotype indicate a critical role for T cell-mediated immunity and tolerance in the biology of the spontaneously developing tumor and metastasis.

Abstract T cells expressing chimeric antigen receptors (CAR T cells) are manufactured outside the body using non-physiological activation and in vitro culture protocols. The procedure takes 3 weeks and costs approximately $450,000 to administer. We have devised a scheme using mouse models for manufacturing CAR T cells in just 3 days employing a natural, polyclonal anti-viral immune response inside a lymph node (LN) using off the shelf reagents that can be applied widely in populations. CAR T cells emerge from the LN within a week, invade solid tumor, and eliminate the targeted antigen-positive tumor cells. Polyclonal activation of T cells is achieved by modifying the antigen presenting properties of lymph node resident professional antigen presenting cells (APC) through viral expression of allogeneic major histocompatibility antigens. The ensuing polyclonal activation occurs in the context of virus activated APC within the normal aarchitecture of the LN and confers robust T cell activation signals directing the development of cytolytic T cells. During the course of T cell activation, the T cells are transduced in situ with retroviruses encoding selected CARs. This approach could be applied to any number of different CARs to target a variety of tumors. T cells activated in the lymph node are functionally distinct from those activated using standard CAR T manufacturing protocols. We anticipate that CAR T cells generated in situ to natural stimuli will exhibit more normal immune regulatory properties that will impact their therapeutic utility. Because our approach uses off the shelf reagents and is administered using standard clinical procedures, cost to patients should be lowered and access to therapy increased.

Abstract The BALB/c-neuT mouse has been a faithful model for investigating metastatic breast cancer. Spontaneous breast tumors from BALB/c-neuT mice are readily transplantable orthotopically into syngeneic or rat neuT negative BALB/c WT hosts facilitating controlled studies of developing breast cancers. When introduced into the breast ducts of BALB/c WT hosts, lung metastases form similarly to how they appear in mice with spontaneous tumors. Tumor grafts grow out with comparable kinetics and induce similar systemic immune abnormalities in the spleens and lymph nodes of both lines of mice as seen in mice with spontaneous tumors. Factors inducing these abnormal immune cell configurations are not yet known. Immunization of BALB/c WT mice against the rat Her2 antigen can induce both T cell and antibody mediated rejection of the transplanted tumors, but these approaches have not been equally successful in syngeneic, rat neuT+ positive hosts, indicating tolerance to rat Her2 is a significant barrier to the induction of immunity against these breast tumors. The MHC encodes class I and class II antigen presenting molecules which are known to be highly polymorphic in both structure and function. We introduced the H-2b MHC haplotype into the MMTV-NeuT model to assess the influence of a different set of antigen presenting molecules on tumor biology. In BALB.B-neuT mice spontaneous tumors appeared with frequencies and in numbers similar to those seen in the BALB/c model. In anything, lung metastases are more frequent and more aggressive in BALB.B-neuT mice. Again, the tumors were readily transplantable into the breast fat pads of syngeneic BALB.B-neuT hosts which developed systemic immune abnormalities very similar to those observed in BALB/c -neuT or BALB/c WT mice. However, BALB.B WT hosts initially accepted the grafts, but subsequently rejected them after they were established. Mice that were in the process of rejecting the tumors retained normal cellularity within their spleens and lymph nodes indicating the importance of an ongoing immune response and/or tumor rejection in shaping the microenvironment in secondary lymphoid organs. Adoptive transfer of spleen cells from BALB.B WT mice which had rejected established tumor conferred resistance to the tumor grafts as long as B cells were included (P<0.01), indicating active humoral immunity was important to tumor rejection. A mechanistic understanding or how antibody-mediated immunotherapy works in patients is incomplete. This new model provides an opportunity to address this question. The Her2 ecto-domain is known to be shed from tumor cells. We propose a model in which the ecto-domain of rat neuT is shed, but is insufficiently immunogenic in BALB/c (H-2d) hosts, but highly immunogenic in BALB.B (H-2b) mice. Consistent with this model is the prediction using the Immune Epitope and Data Base Resource of the presence of a potentially immunogenic IAb-restricted class II epitope within the ecto-domain of rat Her2 neuT oncogene, but no such epitope restricted by IAd or IEd was identified. This provides a compelling hypothesis explaining why grafted spontaneous tumors grow in BALB/c, but not in BALB.B WT mice. An important prediction from this model is that linkage of a potent class II epitope to the Erbb2-encoded ecto-domain may provide a basis for a potent vaccine against Her2 positive breast tumors which should be a strategy adaptable to any MHC genotype. It also will be of interest to determine whether the presence of an antibody reactive against established tumor restores the normal cellular composition of secondary lymphoid organs which have been perturbed by established breast tumors. Citation Format: Sarah Anne Castro, Alvaro Pena, Khashayarsha Khazaie, Larry R. Pease. An immunogenic mouse model using BALB.B-MMTV-neuT breast cancers reveals spontaneous protective humoral immunity [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-15.