Growth Of Established Tumors Research Articles

Coating Dormant Collagenase-Producing Bacteria with Metal-Anesthetic Networks for Precision Tumor Therapy.

Tumor malignancy highly depends on the stiffness of tumor matrix, which mainly consists of collagen. Despite the destruction of tumor matrix is conducive to tumor therapy, it causes the risk of tumor metastasis. Here, metal-anesthetic network-coated dormant collagenase-producing Clostridium is constructed to simultaneously destruct tumor matrix and inhibit tumor metastasis. By metal-phenolic complexation and π-π stacking interactions, a Fe3+-propofol network is formed on bacterial surface. Coated dormant Clostridium can selectively germinate and rapidly proliferate in tumor sites due to the ability of carried Fe3+ ions to promote bacterial multiplication. Intratumoral colonization of Clostridium produces sufficient collagenases to degrade tumor collagen mesh and the loaded propofol restrains tumor metastasis by inhibiting tumor cell migration and invasion. Meanwhile, the delivered Fe3+ ions are reduced to the Fe2+ form by intracellular glutathione, thereby inducing potent Fenton reaction to trigger lipid peroxidation and ultimate ferroptosis of tumor cells. In addition to a satisfactory safety, a single intratumoral injection of coated dormant Clostridium not only effectively retards the growth of established large primary tumors, but also significantly suppresses distal lung metastasis in two different orthotopic tumor models. This work proposes a strategy to develop advanced therapeutics for malignant tumor treatment and metastasis prevention.

DYRK1B blockade promotes tumoricidal macrophage activity in pancreatic cancer

ObjectiveHighly malignant pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant immunosuppressive and fibrotic tumour microenvironment (TME). Future therapeutic attempts will therefore demand the targeting of tumours and stromal compartments...

Synergistic Effects of Metal-Organic Nanoplatform and Guanine Quadruplex-Based CpG Oligodeoxynucleotides in Therapeutic Cancer Vaccines with Different Tumor Antigens.

Oligodeoxynucleotides (ODNs) containing unmethylated cytosine-phosphate-guanosine (CpG) motifs are readily recognized by Toll-like receptor 9 on immune cells, trigger an immunomodulatory cascade, induce a Th1 -biased immune milieu, and have great potential as an adjuvant in cancer vaccines. In this study, a green one-step synthesis process was adopted to prepare an amino-rich metal-organic nanoplatform (FN). The synthesized FN nanoplatform can simultaneously and effectively load model tumor antigens (OVA)/autologous tumor antigens (dLLC) and immunostimulatory CpG ODNs with an unmodified PD backbone and a guanine quadruplex structure to obtain various cancer vaccines. The FN nanoplatform and immunostimulatory CpG ODNs generate synergistic effects to enhance the immunogenicity of different antigens and inhibit the growth of established and distant tumors in both the murine E.G7-OVA lymphoma model and the murine Lewis lung carcinoma model. In the E.G7-OVA lymphoma model, vaccination efficiently increases the CD4+, CD8+, and tetramer+CD8+ T cell populations in the spleens. In the Lewis lung carcinoma model, vaccination efficiently increases the CD3+CD4+ and CD3+CD8+ T cell populations in the spleens and CD3+CD8+, CD3-CD8+, and CD11b+CD80+ cell populations in the tumors, suggesting the alteration of tumor microenvironments from cold to hot tumors.

A rapid strategy to develop personalized cancer nanovaccines for different immunogenic tumors.

e14670 Background: Therapeutic cancer vaccines aim to train the immune system and elicit antitumor immune responses, which are expected to fill the unmet medical need of immune checkpoint inhibitors. Despite tremendous efforts over the past decades, it remains challenging to prepare personalized cancer vaccines using a facile and rapid approach due to tumor diversity. CpG-ODN is a promising immunoadjuvant in cancer immunotherapy, and many clinical trials have been performed or are ongoing. However, commonly used phosphorothioate-modified CpG ODN easily induces protein aggregation and its long-term side effects are concerned. Here, we designed hollow large-pore mesoporous organosilica with sulfide bond to simultaneously load unmodified CpG-ODN with guanine-quadruplex (G4) structures and different tumor antigens to prepare personalized cancer vaccines in a simple, fast, and effective way. Methods: High-immunogenicE.G7-OVA lymphoma or low-immunogenic Lewis lung carcinoma (LLC) cells were subcutaneously inoculated into the left flanks to establish tumor-bearing mice. On days 4, 7, and 10 post tumor inoculation, cancer vaccines prepared using model antigen (OVA) or autologous tumor antigen (LLC tumor cell lysate) were subcutaneously injected into the right flanks of mice, respectively. Saline group, free tumor antigen group, free tumor antigen + CpG-ODN group, tumor antigen + organosilica group were used as controls. The size of tumors was monitored. Results: Cancer vaccines against E.G7-OVA showed high loading efficiencies of >95% for both CpG-ODN and OVA, triggered antitumor immune response with high percentages of CD4+, CD8+, tetramer+CD8+ T cells in splenocytes, and effectively inhibited the growth of established tumor, compared with free OVA, free OVA + CpG-ODN, free OVA + organosilica groups. Cancer vaccines against LLC exhibited high loading efficiencies of >95% and >80% for CpG-ODN and LLC tumor cell lysate, activated antitumor immune response with high percentages of CD4+ and CD8+ T cells in splenocytes and CD8+ T, NK and M1 cells in tumor sites, and effectively inhibited the growth of established tumor, compared with control groups. Conclusions: Codelivery of CpG-ODN and different tumor antigens using hollow large-pore mesoporous organosilica elicits antitumor immunity against both high-immunogenic and low-immunogenic tumors.

Abstract PO2-17-12: Phase I Trial of alpha-lactalbumin vaccine in high risk operable triple negative breast cancer (TNBC) and patients at high genetic risk for TNBC

Abstract Background: Triple-negative breast cancer (TNBC) has a poor prognosis and may be associated with germline mutations. α-Lactalbumin (aLA) is expressed in lactating breasts but not at other times or in other tissues. Expression of aLA is found in 70% of TNBC (PMID: 27322324) so could be an immunologic target for TNBC based on the “retired protein hypothesis” (PMID: 31926646). In pre-clinical studies, vaccination with aLA provided protection from development of autochthonous tumors in transgenic murine models of breast cancer and inhibited growth of established 4T1 transplantable breast tumors in BALB/c mice (PMID: 20512124). Methods: To determine the safety and immunogenicity of aLA, patients with early stage TNBC are being entered in a Phase I trial of aLA with GMP-grade zymosan adjuvant in Montanide ISA 51 VG vehicle. Subjects receive 3 vaccinations given once every 2 weeks. Events of Common Terminology Criteria for Adverse Events (CTCAE) grade ≥ 2 are considered dose-limiting toxicities (DLTs). Results: CTCAE toxicity by dose level is summarized below. All DLTs were injection site reactions, with ulceration and need for incisional drainage representing the grade 3 events. Seven (7) of 10 patients assayed to date have met protocol specified definitions of an immune response based on ELISpot assays to determine frequencies of T cells producing IFNγ and IL-17 in response to recombinant aLA. Assays for an additional 6 patients will be available in September 2023. Conclusion: Dose level ≤ 2 appears to be the maximum tolerated dose. Based on immune response, additional intermediate dose levels may be studied. An additional cohort of patients receiving concurrent anti-PD1 treatment is being accrued. Accrual of patients with BRCA1/2 or PALB2 mutations planning to undergo prophylactic mastectomy is beginning in order to define the toxicity and immunologic effects in this group and to determine whether inflammatory changes from occult lactational foci will be produced. Funding Source: Department of Defense (W81XWH-17-1-0592 and W81XWH-17-1-0593) Table 1 Worst Toxicity by Dose Level Citation Format: Justin Johnson, Emily Rhoades, Holly Levengood, Halle Moore, Megan Kruse, Erin Roesch, Jame Abraham, Brenna Elliott, Rachel Swartz, Holly Pederson, Elena Haury, Azka Ali, Tiffany Onger, Andrew Sciallis, Zahraa Al-Hilli, Wei Wei, Thaddeus Stappenbeck, George Budd. Phase I Trial of alpha-lactalbumin vaccine in high risk operable triple negative breast cancer (TNBC) and patients at high genetic risk for TNBC [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-17-12.

Omental Preadipocytes Stimulate Matrix Remodeling and IGF Signaling to Support Ovarian Cancer Metastasis.

Ovarian cancer can metastasize to the omentum, which is associated with a complex tumor microenvironment. Omental stromal cells facilitate ovarian cancer colonization by secreting cytokines and growth factors. An improved understanding of the tumor-supportive functions of specific cell populations in the omentum could identify strategies to prevent and treat ovarian cancer metastasis. Here, we showed that omental preadipocytes enhance the tumor initiation capacity of ovarian cancer cells. Secreted factors from preadipocytes supported cancer cell viability during nutrient and isolation stress and enabled prolonged proliferation. Coculturing with preadipocytes led to the upregulation of genes involved in extracellular matrix (ECM) organization, cellular response to stress, and regulation of insulin-like growth factor (IGF) signaling in ovarian cancer cells. IGF1 induced ECM genes and increased alternative NF-κB signaling by activating RelB. Inhibiting the IGF1 receptor initially increased tumor omental adhesion but decreased the growth of established preadipocyte-induced subcutaneous tumors as well as established intraperitoneal tumors. Together, this study shows that omental preadipocytes support ovarian cancer progression, which has implications for targeting metastasis. Significance: Omental preadipocyte-mediated IGF1 signaling promotes ovarian cancer tumorigenesis and metastasis via extracellular matrix remodeling, revealing a role for preadipocytes in regulating ovarian cancer progression and highlighting potential therapeutic targets for metastatic disease.

Mesothelin CAR-T cells expressing tumor-targeted immunocytokine IL-12 yield durable efficacy and fewer side effects

Chimeric antigen receptor (CAR)-modified T cell therapy has achieved remarkable efficacy in treating hematological malignancies, but it confronts many challenges in treating solid tumors, such as the immunosuppressive microenvironment of the solid tumors. These factors reduce the antitumor activity of CAR-T cells in clinical trials. Therefore, we used the immunocytokine interleukin-12 (IL-12) to enhance the efficacy of CAR-T cell therapy. In this study, we engineered CAR-IL12R54 T cells that targeted mesothelin (MSLN) and secreted a single-chain IL-12 fused to a scFv fragment R54 that recognized a different epitope on mesothelin. The evaluation of the anti-tumor activity of the CAR-IL12R54 T cells alone or in combination with anti-PD-1 antibody in vitro and in vivo was followed by the exploration of the functional mechanism by which the immunocytokine IL-12 enhanced the antitumor activity. CAR-IL12R54 T cells had potency to lyse mesothelin positive tumor cells in vitro. In vivo studies demonstrated that CAR-IL12R54 T cells were effective in controlling the growth of established tumors in a xenograft mouse model with fewer side effects than CAR-T cells that secreted naked IL-12. Furthermore, combination of PD-1 blockade antibody with CAR-IL12R54 T cells elicited durable anti-tumor responses. Mechanistic studies showed that IL12R54 enhanced Interferon-γ (IFN-γ) production and dampened the activity of regulatory T cells (Tregs). IL12R54 also upregulated CXCR6 expression in the T cells through the NF-κB pathway, which facilitated T cell infiltration and persistence in the tumor tissues. In summary, the studies provide a good therapeutic option for the clinical treatment of solid tumors.

A facile approach to preparing personalized cancer vaccines using iron-based metal organic framework.

Considering the diversity of tumors, it is of great significance to develop a simple, effective, and low-cost method to prepare personalized cancer vaccines. In this study, a facile one-pot synthetic route was developed to prepare cancer vaccines using model antigen or autologous tumor antigens based on the coordination interaction between Fe3+ ions and endogenous fumarate ligands. Herein, Fe-based metal organic framework can effectively encapsulate tumor antigens with high loading efficiency more than 80%, and act as both delivery system and adjuvants for tumor antigens. By adjusting the synthesis parameters, the obtained cancer vaccines are easily tailored from microscale rod-like morphology with lengths of about 0.8 μm (OVA-ML) to nanoscale morphology with sizes of about 50~80 nm (OVA-MS). When cocultured with antigen-presenting cells, nanoscale cancer vaccines more effectively enhance antigen uptake and Th1 cytokine secretion than microscale ones. Nanoscale cancer vaccines (OVA-MS, dLLC-MS) more effectively enhance lymph node targeting and cross-presentation of tumor antigens, mount antitumor immunity, and inhibit the growth of established tumor in tumor-bearing mice, compared with microscale cancer vaccines (OVA-ML, dLLC-ML) and free tumor antigens. Our work paves the ways for a facile, rapid, and low-cost preparation approach for personalized cancer vaccines.

Stereotactic ablative radiotherapy and FAPα-based cancer vaccine suppresses metastatic tumor growth in 4T1 mouse breast cancer

Background and purposeThis study tested the hypothesis that a novel combination of stereotactic ablation radiotherapy (SABR) and a cancer vaccine against fibroblast activation protein-alpha (FAPα) can suppress established tumor growth and impede potential metastasis. MethodsThe poorly immunogenic metastatic mouse mammary carcinoma 4T1 was used as a model. Mice were randomly assigned to five treatment groups: (1) untreated control, (2) FAPα-based cancer vaccine, (3) SABR, (4) SABR + pCDH (lentiviral control vector), (5) SABR + FAPα-based cancer vaccine (SABR/FAPα-Vax). FAPα-based cancer vaccine were administered subcutaneously every week for a total of three treatments. SABR was delivered to the primary tumor by 3 × 8 Gy after the first vaccination. ResultsConsistent with the poorly immunogenic nature of 4T1, tumor-bearing mice receiving FAPα-based cancer vaccine or SABR monotherapy showed a modest reduction in tumor volume and increased animal lifespan. In contrast, SABR/FAPα-Vax was well-tolerated, significantly reduced tumor burden, and increased survival compared to monotherapy. The increased survival correlated with inhibition of extracellular matrix (ECM) production, tumor vascularization and lymphangiogenesis. SABR/FAPα-Vax also resulted in an abscopal effect capable of eliminating lung metastases. SABR/FAPα-Vax recruited and activated CD8 + T cells to attack tumor cells and FAPα + stromal cells, and initiated suppressor cell reprogramming, including facilitating macrophage polarization toward an anti-tumor (M1) state, as well as depleting myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). ConclusionThese findings provide a novel therapeutic combination of radiation and FAPα-based cancer vaccine with promising results against poorly immunogenic metastatic cancer. This study may pave the way to overcome the therapeutic resistance caused by FAPα + CAFs.

Neoantigen-Based Nanovaccine In Combination with Immune Checkpoint Inhibitors Abolish Postsurgical Tumor Recurrence and Metastasis.

The notorious limitation of conventional surgical excision of primary tumor is the omission of residual and occult tumor cells, which often progress to recurrence and metastasis, leading to clinical treatment failure. The therapeutic vaccine is emerging as a promising candidate for dealing with the issue of postsurgical tumor residuals or nascent metastasis. Here, a flexible and modularized nanovaccine scaffold based on the SpyCatcher003-decorated shell (S) domain of norovirus (Nov) is employed to support the presentation of varied tumor neoantigens fused with SpyTag003. The prepared tumor neoantigen-based nanovaccines (Neo-NVs) are able to efficiently target to lymph nodes and engage with DCs in LNs, triggering strong antigen-specific T-cell immunity and significantly inhibiting the growth of established orthotopic 4T1 breast tumor in mice. Further, the combination of Neo-NVs and anti-PD-1 monoclonal antibody (mAb) produces significant inhibition on postsurgical tumor recurrence and metastasis and induces a long-lasting immune memory. In conclusion, the study provides a simple and reliable strategy for rapid preparing personalized neoantigens-based cancer vaccines and engaging checkpoint treatment to restore the capability of tumor immune surveillance and clearance in surgical patients.

Pegylated Liposomal Alendronate Biodistribution, Immune Modulation, and Tumor Growth Inhibition in a Murine Melanoma Model.

While tumor-associated macrophages (TAM) have pro-tumoral activity, the ablation of macrophages in cancer may be undesirable since they also have anti-tumoral functions, including T cell priming and activation against tumor antigens. Alendronate is a potent amino-bisphosphonate that modulates the function of macrophages in vitro, with potential as an immunotherapy if its low systemic bioavailability can be addressed. We repurposed alendronate in a non-leaky and long-circulating liposomal carrier similar to that of the clinically approved pegylated liposomal doxorubicin to facilitate rapid clinical translation. Here, we tested liposomal alendronate (PLA) as an immunotherapeutic agent for cancer in comparison with a standard of care immunotherapy, a PD-1 immune checkpoint inhibitor. We showed that the PLA induced bone marrow-derived murine non-activated macrophages and M2-macrophages to polarize towards an M1-functionality, as evidenced by gene expression, cytokine secretion, and lipidomic profiles. Free alendronate had negligible effects, indicating that liposome encapsulation is necessary for the modulation of macrophage activity. In vivo, the PLA showed significant accumulation in tumor and tumor-draining lymph nodes, sites of tumor immunosuppression that are targets of immunotherapy. The PLA remodeled the tumor microenvironment towards a less immunosuppressive milieu, as indicated by a decrease in TAM and helper T cells, and inhibited the growth of established tumors in the B16-OVA melanoma model. The improved bioavailability and the beneficial effects of PLA on macrophages suggest its potential application as immunotherapy that could synergize with T-cell-targeted therapies and chemotherapies to induce immunogenic cell death. PLA warrants further clinical development, and these clinical trials should incorporate tumor and blood biomarkers or immunophenotyping studies to verify the anti-immunosuppressive effect of PLA in humans.

A multi-targeted T-cell engagement of PD1-resistant colorectal cancer cells via delivery of glycopeptide conjugates.

e14571 Background: Glycan-based cancer immunotherapies are novel therapeutic strategies that use the pivotal role of aberrant glycosylation patterns in cancer progression. Targeting hybrid epitopes consisting of altered glycans and carrier peptides enhances the efficacy and specificity of the anti-tumor immune response. Here, we exploit ensembles of glycopeptide conjugates to steer a multi-targeted yet tumor-specific T-cell response, a technology we call GLAAD (glycopeptides with adjuvant and tumor-associated antigen delivery). By inducing T cells that recognize and eliminate tumor cells and promoting their antigenicity in a therapeutic setting, we show the potential of our GLAAD candidate GNU-101 to overcome the transient and heterogeneous responses frequently observed with traditional immunotherapies. Methods: Using the MC38 syngeneic mouse model of colorectal cancer (CRC) in a therapeutic setting, we tested whether GNU-101 reduces tumor burden when orally administered alone or combined with immune checkpoint inhibitors (ICI). On day 0, 20 C57/BL6J mice were injected subcutaneously with MC38 CRC cells in each flank. On day 6, mice were supplemented with 3% GNU-101 in the drinking water, while control mice received normal drinking water. On days 6, 9, 12, and 15, half of the mice in each group received an anti-PD1 antibody (aPD1), while the others received isotype control. On day 16, the mice were sacrificed. Results: In this therapeutic setting, a standalone therapy of GNU-101 successfully reduced tumor growth as demonstrated by a statistically significant reduction in both tumor volume and weight. As expected, the aPD1 therapy had only a non-significant effect on tumor growth. Remarkably, its efficacy was improved when combined with GNU-101, leading to a statistically significant reduction of >90% in tumor volume and weight. The analysis of the immune cell population in the tumor microenvironment revealed the activation of CD8 T cells and a reduction of immunosuppressive signals, which likely mediates the anti-tumor efficacy of GNU-101. This was obvious by a higher abundance of CD8 T cells producing IFNγ and secreting granzyme B & perforin and, conversely, lower amounts of Tim3+ CD8 T cells and regulatory T cells (FoxP3+ CD4 T cells) found in mice treated with GNU-101 vs control mice. Conclusions: We demonstrate the immunomodulatory and therapeutic potential of GNU-101 in a pre-clinical mouse study in which the growth of established tumors was significantly reduced, either as a standalone therapy or in combination with existing treatment. By specifically promoting anti-cancer CD8+ T cells while reducing immunosuppressive signals, this easy-to-administer glycopeptide has the potential to create the conditions for long-term cancer control and improve treatment outcomes in cancer that are frequently resistant to immunotherapy, such as many epithelial cancers.

The clinical impact of soluble natural killer cell group 2-member D (NKG2D) receptor ligands on tumor tumorigenicity and anti-tumor immunity.

e14557 Background: NKG2D is an activating receptor expressed on all human natural killer (NK) cells and CD8 T-cells. It is widely accepted that human tumors shed soluble NKG2D ligands, such as soluble major histocompatibility complex-I chain-related molecule (sMIC), to evade immune activation. Harnessing the NKG2D/NKG2D-ligand axis has emerged as a promising avenue for immunotherapy. However, controversy exists over whether soluble NKG2D ligands are immunosuppressive or immunostimulatory. Methods: Cell lines for melanoma (B16F10), Lewis lung carcinoma (LLC1), and prostate cancer (TRAMP-C2, T-C2) were used as parental cell lines in this study. Derivative cell lines over-expressing the soluble human NKG2D ligand sMICB (B16F10-sMICB, TC2-sMICB and LLC-sMICB) were created. We inoculated these 3 syngeneic transplantable tumor models and their respective parental lines into mice and compared tumor incidence/growth over time. We also compared surface markers representative of cell stemness (SCA-1, CD166 and CD133) in mouse cancer cells between the sMICB-expressing tumor cells and their respective parental cell lines. We then characterized the tumor immune microenvironment by flow cytometry. Results: B16F10-sMICB mice demonstrated a significant delay in tumor development compared to control mice (23 vs. 6 days, p < 0.05). Over longer follow-up, rapid tumorigenesis still occurred in the experimental group, with a 50% tumor incidence by day 26. By contrast, the TRAMP-C2-sMICB mice demonstrated higher tumorigenic ability (p < 0.05). LLC-sMICB mice demonstrated no difference in tumorigenicity. Time to tumor onset was more strongly associated with expression of stemness-like markers rather than sMIC. Once tumors were established, they grew significantly more aggressively in the TRAMP-C2-sMICB, LLC-sMICB and B16F10-sMICB lines vs. their parental counterparts. sMICB expressing tumors contained significantly lower percentages of NK cells and CD8 T-cells in tumor infiltrates than their respective parental lines. NK cells and CD8 T Cells from sMICB-expressing tumors had an impaired capacity to produce interferon gamma. Conclusions: Expression of a soluble NKG2D ligand had minimal impact on tumor establishment but facilitated more aggressive growth of established tumors due to immune suppression. Soluble NKG2D ligands imprint negative anti-tumor immunity onto established tumors. Our data differentiated the impact of NKG2D ligands on tumor onset vs. tumor progression and reinforced the translational concept that soluble NKG2D ligands are strong therapeutic targets for future study.

Abstract 3035: Phase I trial of alpha-lactalbumin vaccine in high-risk operable triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) has a poor prognosis and may be associated with germline mutations. α-lactalbumin (aLA) is expressed in lactating breasts but not at other times or in other tissues. Expression of aLA is found in 70% of TNBC (PMID: 27322324) so could be an immunologic target for TNBC based on the “retired protein hypothesis” (PMID: 31926646). In pre-clinical studies vaccination with aLA inhibited growth of established breast tumors and provided protection from development of autochthonous tumors in transgenic murine models of breast cancer and against 4T1 transplantable breast cancer in BALB/c mice (PMID: 20512124). Methods: To determine the safety and immunogenicity of aLA, patients with early stage TNBC are being entered in a Phase I trial of aLA with GMP-grade zymosan adjuvant in Montanide ISA 51 VG vehicle. Subjects receive 3 vaccinations given once every 2 weeks. Events of Common Terminology Criteria for Adverse Events (CTCAE) grade ≥2 are considered dose-limiting toxicities (DLTs). Results: CTCAE toxicity by dose level is summarized below. All DLTs were injection site reactions, with ulceration and need for incisional drainage representing the grade 3 events. ELISpot assays to determine frequencies of T-cells producing IFN-γ and IL-17 in response to recombinant aLA and ELISA assays of antibody response to aLA will be available in December 2022. Conclusion: Dose level 2 appears to be the maximum tolerated dose. Accrual to dose levels 1 and 2 will be expanded to further define toxicity and immunologic effects. Accrual of patients with BRCA1 or PALB2 mutations planning to undergo prophylactic mastectomy is beginning in order to define the toxicity and immunologic effects in this group and to determine whether inflammatory changes from occult lactational foci will be produced. Funding Source: Department of Defense (W81XWH-17-1-0592 and W81XWH-17-1-0593) Worst Toxicity by Dose Level Dose Level aLAC (mcg) dose Zymosan (mcg) dose n Patients n Grade 0 n Grade 1 n Grade 2 n Grade 3 1 10 10 3 3 2 100 10 3 3 3 500 10 3 2 1 Original 2 100 100 1 1 Citation Format: George Thomas Budd, Justin M. Johnson, Emily Rhoades, Halle Moore, Holly Levengood, Megan Kruse, Erin Roesch, Jame Abraham, Brenna Elliott, Elena Haury, Rachel Swartz, Holly Pederson, Zahraa Al Hilli, Vincent Tuohy. Phase I trial of alpha-lactalbumin vaccine in high-risk operable triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3035.

Abstract 1813: Selective ITK blockade induces antitumor responses and enhances efficacy to immune checkpoint inhibitors in preclinical models

Abstract Interleukin 2 inducible T cell kinase (ITK) plays a role in both T cell receptor (TCR) signaling and T helper cell differentiation. ITK-/- mice exhibit defects in Th2 differentiation while retaining the ability to differentiate into Th1 cells that secrete IFNγ. We generated CPI-818, a covalent inhibitor of ITK (KD 2.5nM) with > 100 fold selectivity over resting lymphocyte kinase (RLK) and other TEC family kinases. This drug is now being evaluated as a single agent in an ongoing Phase 1 clinical trial in refractory T cell lymphoma where objective tumor responses have been observed. In the studies reported here, we have investigated the immunomodulatory effects of specific ITK blockade of human T cells with CPI-818 in vitro and in vivo in murine tumor models. Analysis of the cytokine profiles in human CD4 T cells differentiated under non-polarizing, Th1 or Th2 polarizing conditions showed that CPI-818 inhibited generation of Th2-associated cytokines whereas, production of Th1 cytokines such as IFNγ were largely unaffected. Suppression of IL-2-mediated proliferative response occurred only at high concentrations of CPI-818 (>1uM). The effect of CPI-818 on anti-tumor immunity was evaluated in both murine syngeneic CT26 colon cancer and EL4 T cell lymphoma models. In vivo treatment of animals with established tumors showed single-agent anti-tumor activity in both models. Combining CPI-818 with suboptimal doses of anti-PD1 and anti-CTLA4 synergistically inhibited the growth of established CT26 tumors, leading to complete tumor elimination in 100% treated animals. Furthermore, triple combination therapy elicited durable anti-tumor immune memory after animals were rechallenged with a new engraftment of CT26 cells. Enhancement of anti-tumor activity required CD8+ T cells as in vivo antibody-mediated depletion of CD8, but not CD4, abolished the efficacy of CPI-818 monotherapy. Consistent with that, we found increased tumor-infiltrating CD8+ T cells and increased ratio of CD8: Treg in the responding tumors of CPI-818 treated animals. Levels of several exhaustion makers were down-regulated by treatment with CPI-818, suggesting that inhibition of ITK by CPI-818 produces favorable changes in the tumor microenvironment. Our findings provide insights into the effects of selective ITK blockade on tumor immunity and its potential role in immunotherapy. Citation Format: Lih-Yun Hsu, Rahul D. Pawar, Dan Li, Poorva Ghosh, Kevin Nguyen, Carlene Williams, Yuqin Song, Ning Ding, Erik Verner, Richard A. Miller. Selective ITK blockade induces antitumor responses and enhances efficacy to immune checkpoint inhibitors in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1813.

Abstract 593: Bispecific antibody against TIGIT and PVRIG enhances antitumor immunity

Abstract T cell immunoreceptor with Ig and ITIM domains (TIGIT) and poliovirus receptor-related immunoglobulin domain-containing protein (PVRIG) are two immune checkpoint receptors. They compete with CD226 for binding to CD155 or CD112 and suppress the activity of NK cells and T cells. Here we developed a bispecific antibody KA-1874 targeting TIGIT with an anti-PVRIG scFv fused to the C-terminus. KA-1874 is a fully human IgG4 with high binding affinity to TIGIT (KD=0.08nM) and PVRIG (KD=0.17nM). KA-1874 blocks both bindings of PVRIG-mFc to PVRL2 and TIGIT-mFc to CD155. In cell-based luciferase reporter assays, KA-1874 blocks PVRIG and TIGIT-mediated inhibitory signaling and enhances CD3 signaling with EC50 of 1.1nM and 1.4nM respectively, while COM701 against PVRIG and EOS488 against TIGIT both with EC50 above 2nM. In a CMV recall assay, PBMC increases INF-γ secretion when cultured with KA-1874, suggesting KA-1874 enhances primary T cell function. Immunodeficient mice humanized with PBMC are transplanted with A374 tumor cells. Co-injection of KA-1874 and pembrolizumab, which against PD-1, suppress established tumor growth. In comparison, KA-1874 or pembrolizumab alone fails to inhibit tumor growth. The above results indicate KA-1874 is an excellent candidate therapeutical antibody for cancer immunotherapy. Citation Format: Jiabei Liang, Xiao Gao, Cong Zhang, Hui Zhao, Hao Peng, Feng Hao, Jinying Ning, Guojin Wu. Bispecific antibody against TIGIT and PVRIG enhances antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 593.

Therapeutic efficacy of plant-produced Nivolumab in transgenic C57BL/6-hPD-1 mouse implanted with MC38 colon cancer

The therapeutic blockade of inhibitory PD-1 signaling has emerged as an effective approach for cancer immunotherapy. Nivolumab (Opdivo®), a monoclonal antibody (mAb) targeting the PD-1 immune checkpoint, is approved for treatment of several cancer indications. It functions by blocking the PD-1-mediated T-cell inhibition thus reinstating anticancer immune responses. Tremendous advances in plant biotechnology offer an alternative and economical strategy to produce therapeutic mAbs for immune-based therapies. In this study, recombinant anti-PD-1 Nivolumab was produced in Nicotiana benthamiana and the plant-produced anti-PD-1 mAb was exploited for cancer treatment in syngeneic mice model C57BL/6 mice that were used to test the antitumor efficacy of plant produced Nivolumab, along with commercial Opdivo®. C57BL/6 syngeneic mice treated with plant produced anti-PD-1 mAb exhibited reduction in the growth of established MC38 tumors. The plant produced Nivolumab treatment showed 82.9% antitumor effect in decreasing the tumor volume along with 50% tumor-free mice, whereas Opdivo® showed 90.26% reduction in volume without any tumor-free mice. Finally, plant-derived anti-PD-1 therapy was also well tolerated in tumor-bearing mice that correlated with no significant body weight changes. Overall, our plant-produced Nivolumab elicits significant inhibition of tumor growth in vivo and provides a proof-of-concept for the production of immunotherapy targeting PD-1.

A "trained immunity" inducer-adjuvanted nanovaccine reverses the growth of established tumors in mice

Innate immune cells are critical in antitumor immune surveillance and the development of antitumor adaptive cellular immunity. Trained innate immune cells demonstrate immune memory-like characteristics, producing more vigorous immune responses to secondary homologous or heterologous stimuli. This study aimed to investigate whether inducing trained immunity is beneficial when using a tumor vaccine to promote antitumor adaptive immune responses. A biphasic delivery system was developed with the trained immunity inducer Muramyl Dipeptide (MDP) and specific tumor antigen human papillomavirus (HPV) E7 peptide encapsulated by poly(lactide-co-glycolide)-acid(PLGA) nanoparticles (NPs), and the NPs along with another trained immunity agonist, β-glucan, were further embedded in a sodium alginate hydrogel. The nanovaccine formulation demonstrated a depot effect for E7 at the injection site and targeted delivery to the lymph nodes and dendritic cells (DCs). The antigen uptake and maturation of DCs were significantly promoted. A trained immunity phenotype, characterized by increased production of IL-1β, IL-6, and TNF-α, was induced in vitro and in vivo in response to secondary homologous or heterologous stimulation. Furthermore, prior innate immune training enhanced the antigen-specific INF-γ-expressing immune cell response elicited by subsequent stimulation with the nanovaccine. Immunization with the nanovaccine completely inhibited the growth of TC-1 tumors and even abolished established tumors in mice. Mechanistically, the inclusion of β-glucan and MDP significantly enhanced the responses of tumor-specific effector adaptive immune cells. The results strongly suggest that the controlled release and targeted delivery of an antigen and trained immunity inducers with an NP/hydrogel biphasic system can elicit robust adaptive immunity, which provides a promising tumor vaccination strategy.Graphical

Abstract OT2-10-02: Phase I trial of an alpha-lactalbumin vaccine in patients with operable triple-negative breast cancer (TNBC)

Abstract Background: Triple-negative breast cancer (TNBC) is the subtype of breast cancer with the worst prognosis and is the subtype most often associated with germline mutations of BRCA1 as well as other genes. Alpha-lactalbumin (aLA) is a milk protein expressed in lactating breasts but not at other times or in other normal tissues. Expression of aLA is found in approximately 70% of TNBC (Cancers PMID: 27322324) so is an attractive immunologic target for TNBC based on the “retired protein hypothesis” (Semin Immunol PMID: 31926646). Pre-clinical studies have shown that vaccination with aLA inhibits the growth of established breast tumors and provides potent protection from development of autochthonous tumors in transgenic murine models of breast cancer and against 4T1 transplantable breast cancer in BALB/c mice (Nat Med PMID: 20512124). Specific Aims: We are conducting a Phase I trial of vaccination with an aLA vaccine in patients with early stage TNBC to demonstrate the safety of this approach and to document the ability to produce a relevant immunologic response to aLA. Trial Design: Patients are being entered into a Phase I trial of alpha-lactalbumin with GMP-grade zymosan adjuvant in Montanide ISA 51 VG vehicle. Subjects receive a total of 3 vaccinations administered once every 2 weeks. Toxic events of Common Terminology Criteria for Adverse Events (CTCAE) grade 2 or greater are considered dose-limiting. Dose levels being tested in the current protocol are alpha-lactalbumin/zymosan 0.01/0.01, 0.1/0.01, 0.5/0.01, 0.5/0.03, and 0.5 mg/0.06 mg. Patients are being monitored for toxicity until 84 days after the first vaccination or resolution of toxicity, whichever is later. Blood is being drawn prior to therapy and 14, 28, and 56 days after the first vaccination to assess cellular response using enzyme-linked immunosorbent spot (ELISpot) assays of interferon-gamma and interleukin-17 production in response to aLA. Humoral response to aLA vaccination is being assessed by enzyme-linked immunosorbent assay (ELISA). Eligibility Criteria: Patients with ER-negative, PR-negative, HER2-negative breast cancer of pathologic stage II-III or who had residual disease after standard pre-operative systemic therapy. Participants must be within 3 years of initiation of treatment and have no evidence of recurrence. Dose Selection: Doses are being escalated using a 3+3 trial design to determine the maximum tolerated dose, the lowest immunologic dose and the optimal immunologic dose Present Accrual and Target Accrual: Dependent on toxicity, 18-30 patients will be treated. Future Plans and Contact Information: After identification of the maximum tolerated dose and expansion of the dose levels associated with effective tumor immunity we plan to enroll 2 additional cohorts of patients: 1) patients who have completed pre-operative chemo-immunotherapy and are receiving pembrolizumab as standard of care therapy, and 2) patients without cancer planning to undergo prophylactic bilateral mastectomy. Interested parties may contact Dr. Budd at buddg@ccf.org. Funding Source: Department of Defense (W81XWH-17-1-0592 and W81XWH-17-1-0593) Citation Format: George Budd, Justin M. Johnson, Emily Rhoades, Halle Moore, Megan L. Kruse, Erin Roesch, Jame Abraham, Brenna Elliott, Elena Haury, Vincent K. Tuohy. Phase I trial of an alpha-lactalbumin vaccine in patients with operable triple-negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT2-10-02.

Abstract PD5-09: PD5-09 Oncogenic Pathways of Tumor Cells Signal to Stem-ness in TNBC: Relationship between Stem Cell Markers and PI3K-DDR Pathway Targeted Drugs

Abstract Background: The interest in cancer stem cells in TNBC is encouraged by the finding that CD44+/CD24- and ALDH1+ breast cancer stem cells (CSC) are enriched in TNBC and contribute to chemotherapy resistance and aggressive tumor progression (O’Conor et al., 2018; Ma et al. 2014). CD44+ CD24- phenotype can be utilized to determine TNBC patients’ prognosis (Shadbad et al., 2021; Wang et al., 2017). CD44 has also been known for its tumor-intrinsic function in TNBC (Kong et al., Cancer Res. 2020). We have reported a functional correlation between CD44+/CD24- cancer stem cells and 3D clonogenic growth in testing rational combinations of PI3K pathway inhibitors and Wnt-beta-catenin pathway inhibitors in TNBC (Aske et al., 2016; 2017). We also reported that doubling down on the PI3K pathway enhances the anti-tumor efficacy of PARP inhibitor in TNBC (De et al., 2014). Hypothesis: We hypothesized that the anti-tumor effect of PI3K pathway targeted drugs in combination with PARP inhibitor will regulate the expression of CSC in TNBC. Methods: To test our hypothesis, we examined the expression of stem cell markers CD44, CD44v6, and CD24 in MDA-MB-231, SUM149, and MDA-MB-468 TNBC cells in vitro and in vivo. IHC for CD44 and CD44v6 was standardized using tumor controls and identified in (1) BC TMAs, (2) TNBC cell lines, and (3) patients from our Avera cohort. FFPE sections of tumors from athymic mice (bearing xenograft tumors) treated with a combination of Apitolisib with Veliparib plus carboplatin were used to test stem cell markers by IHC. Staining was evaluated independently by a pathologist who was unaware of the study design. Mechanism-based in vitro studies were conducted to understand the mode of action of the drugs using flow cytometry and qRT-PCR. Results: CD44 was identified primarily in the membrane and to a lesser extent in the cytosol of tumor cells of BC patients (TMA and Avera cohort), TNBC cell lines, and xenograft-tumors. Apitolisib, in combination with Veliparib plus carboplatin, blocked the growth of established xenograft tumors by 80-90%, with a concomitant decrease in Ki67 levels. Membrane expression of CD44 inversely correlated to tumor sizes which significantly reduced in response to drug combinations. The relationship between the expression of CD44 and CD44v6 is being worked out, which will be presented at the meeting. Significance: Our findings showed that the anti-tumor effect of Veliparib plus carboplatin and Apitolisib effectively blocked the specific CD44v6 CSC population in TNBC xenograft tumors. Our results demonstrate the integral role of the PI3K-mTOR and the DDR pathways in the control of stem-ness orchestrating anti-tumor actions of PARP inhibitor and mTOR-PI3K dual inhibitors in TNBC. Citation Format: Nandini Dey, Jennifer Aske, Xiaoqian Lin, Adam Dale, Yuliang Sun, Pradip De. PD5-09 Oncogenic Pathways of Tumor Cells Signal to Stem-ness in TNBC: Relationship between Stem Cell Markers and PI3K-DDR Pathway Targeted Drugs [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD5-09.