Blogs

Dietary fiber as a prebiotic for the gut microbiome

The microbiome, diet, and immune response: From observation to intervention
Jennifer McQuade, MD, MS, MA (The University of Texas MD Anderson Cancer Center) reviewed the recent data linking the microbiome with immune checkpoint inhibitors efficacy in patients with cancer. Within this context, Dr. McQuade described an ongoing study to identify factors that influence the content of the microbiome and could potentially influence response to immunotherapy. A previous dietary assessment of 438 patients with melanoma identified dietary fiber as prebiotic factor in establishing the gut microbiome. Sufficient fiber intake in patients receiving immunotherapy for melanoma was also associated with improved progression free survival. Preclinical studies with mice have shown that fiber deprivation shifts the gut microbiome of mice and that the tumors of mice on high-fiber diets exhibit a stronger response to immunotherapy when compared to the tumors of mice on fiber-deprived diet. This response was largely mediated by the microbiome, as an increased response to immunotherapy was not observed in germ-free mice on high-fiber diets. An ongoing controlled feeding study in patients with melanoma is investigating whether these effects are seen in humans. In a Phase I study, melanoma survivors (n = 10) received a diverse fixed diet rich in whole foods, fruits, and vegetables, providing up to 50 g of fiber per day for a six-week period. The primary endpoint of patient compliance was met, indicating that this approach is safe and tolerable. Patients whose fixed diet represented a larger increase in fiber intake exhibited larger shifts in their microbiome. Changes in circulating metabolome during the high-fiber diet were also observed; while some changes were due to the high-fiber diet, other changes were due to more general dietary changes, such as changes in fat and protein sources. Enrollment is ongoing for the Phase II study in which patients actively receiving immune checkpoint inhibitor therapy are randomly assigned to a group receiving high fiber diet vs. a group who will receive recommendations for healthy diet but will determine their individual food intake. Preliminary results suggest that a high fiber diet is associated with higher abundance of pro-response bacteria and improved response to immunotherapy and with significant shifts in the metabolome. Future directions for this study include combining observational and interventional profiling to better understand the relationship between diet, microbiome, metabolome, and immune interactions in order to design more specific probiotics for patients on immunotherapy. Behavioral changes in patient populations also need to be identified in order to make the dietary/metabolic changes sustainable.

Stimulation of anti-cancer immunity by the gut microbiota

Microbiota-specific T cells support tertiary lymphoid structure formation and anti-colorectal cancer immunity
Timothy Hand, PhD, (UPMC Children’s Hospital of Pittsburgh) described preclinical studies elucidating the role of epithelial-associated bacteria from gut microbiome in activating an immune response, and highlighting the role of CD4+ T cells and B cells in mediating this association. In a series of elegant preclinical experiments, in which the Helicobacter hepaticus (Hhep) bacteria were introduced in a mouse model of colorectal cancer (CRC), after formation of colorectal tumors, tumors in mice with Hhep colonization fewer and smaller: Importantly, Hhep+ mice exhibited long-term control of these tumors and improved survival. Single cell RNAseq of the colon cancer tissue from these mice also revealed a striking increase in cytotoxic lymphocytes in this group, compared to control Hhep- mice. In addition, Hhep colonization also lead to increased T cell and B cell infiltration of tumors, specifically T follicular helper cells, and the development of larger, more organized tertiary lymphoid structures (TLS). This finding is of particular interest given the growing body of evidence linking TLS with improved outcomes to immune checkpoint inhibition, and better prognosis in general across tumor types. The TLS are connected to tumors, draining the tumor and acting as a platform for immune cells. Mice defective in formation of T follicular helper cells that are colonized with Hhep do not form tertiary lymphoid structures and experience decreased tumor shrinkage, suggesting T follicular helper cells are necessary for this immune response. These findings are supported by work in humans, showing that high levels of CD4+ T follicular helper cells are associated with higher survival rates in patients with stage IV CRC. Additionally, CRC tumors from humans contain Campylobacter species similar to Hhep. These results suggest that some bacteria of the gut microbiota can activate anti-tumor immunity, suggesting a therapeutic approach for treating CRC.

Dual role of the immune system in metastasis

Immune regulation of dormancy
Mikala Egeblad, PhD (Cold Spring Harbor Laboratory) described factors that cause a disseminated dormant cancer cell at a secondary site to become part of an active metastasis. Dr. Egeblad started by reporting on previous works showing endotoxin-induced inflammation can awaken disseminated dormant cancer cells to form metastatic disease. Sustained inflammation involves several mediators within the tumor microenvironment. Among others, Dr. Egeblad group has identified that neutrophil extracellular traps (NETs) drive metastases development by promoting migration and invasion of cancer cells to blood vessels, extravasion of the cancer cell from the blood vessel, providing a metastatic niche for cancer cells, and suppressing anti-cancer immunity. Importantly, LPS is not only a NET inducer, but also increases the levels of stress response hormones. Against this evidence, another the aim of these studies was to determine whether stress hormones also contribute to awakening dormant disseminated cells to form metastases. In physical restraint mouse models, chronic stress induced quiescent cancer cells to form lung metastases as well as an increase in NET formation. Metastasis development was dependent on NET formation. Along with NETs presence, also T cells depletion was shown to awaken dormant cells and increase in metastases, providing additional evidence on the mechanisms leading to disseminated dormant cells awakening. Lastly, Dr. Egeblad described a model of quiescent, disseminated pancreatic adenocarcinoma, in which mice immunized against cancer cells had undetectable levels of metastatic lesions after injection with latent pancreatic cancer cells, while immunized mice exposed to stress coupled with T cell or NK cell suppression formed metastases in the liver. These results suggest a duality in the role of the immune system in metastases, in which T cells restrain metastasis, while myeloid-derived immune cells like neutrophils, when under stress, promote metastasis.

Review of tissue-based biomarkers in adjuvant and neoadjuvant settings

Predictive biomarkers for adjuvant immunotherapy

Janis Taube, MD, MSc, (Johns Hopkins University School of Medicine) discussed the current knowledge surrounding tissue-based biomarkers in the neoadjuvant and adjuvant immune checkpoint inhibition therapy. Key clinical trials on adjuvant immunotherapy and tissue-based biomarkers were reviewed. For trials addressing the efficacy of adjuvant immunotherapy in treating renal cell carcinoma, urothelial carcinoma, and non-small cell lung cancer (NSCLC), tumors with high PD-L1 expression levels largely drove the advantages observed in treatment groups receiving immunotherapy. This observation is consistent with what has been reported in patients with advanced NSCLC receiving PD-(L)1 inhibition within registrational clinical trials. Together, these data suggest that PD.-L1 negative tumors may not derive the same benefit from adjuvant immune checkpoint therapies, as patients with PD-L1 positive tumors do. With regards to melanoma, the stratification in overall survival was similar for PD-L1 positive tumors compared to PD-L1 negative tumors. The CheckMate 238 trial comparing adjuvant nivolumab versus ipilimumab in stage IIIB-C and stage IV melanoma was updated with four-year efficacy data and overall survival; nivolumab shows an advantage over ipilimumab for Relapse-Free Survival (RFS) and Distant Metastasis-Free Survival, but not for Overall Survival (OS), which could likely reflect the crossover of patients on ipilimumab switching to nivolumab. Of note, RFS was increased along with the increase of PD-L1 expression, again highlighting the strong influence of PD-L1 expression on clinical outcomes in with (neo)adjuvant immunotherapy. As many of these studies are relatively recent, RFS and DFS numbers have been provided, but for OS data are still in development. Dr. Taube also noted that studies of neoadjuvant immunotherapy have provided unique opportunities to study resected tumors to determine how immunotherapy affects expression of tissue-based biomarkers. Over 100 clinical trials of neoadjuvant immunotherapy are in process, and most of these trials use pathological responses such as Pathological Complete Response (pCR) or major pathological response (MPR) as primary endpoints. This change in endpoints allows patients responses to be measured in shorter periods of time (weeks/months) allowing tailored adjustments in therapy. It remains to be determined whether pCR can serve as a surrogate measurement for EFS for NSCLC, while pooled data from melanoma suggest this is the case. Pathologic responses to neoadjuvant treatment regimens can potentially inform choices of adjuvant therapy. Along with PD-L1 expression a wide spectrum of biomarkers of (neo)adjuvant immunotherapy efficacy is under investigation, including but not limited to TMB, NIF-gamma gene expression signatures, CD8+ T cell density, and B2M status. However, there is still a need to learn more about existing tissue-based biomarkers and to identify new tissue- and blood-based biomarkers to match patients to the most effective therapies.

Identifying blood-based biomarkers in the adjuvant setting

Peripheral blood ‘omics’ in the adjuvant setting

Tomas Kirchhoff, PhD (Perlmutter Cancer Center, NYU Grossman School of Medicine) described current efforts to identify blood-based predictive biomarkers for immune checkpoint inhibitors in the adjuvant setting. Current biomarkers for ICI efficacy such as circulating IL-6 levels, soluble PD-L1 or blood-based tumor mutational burden (TMB) have several limitations in predicting outcomes, and additional clinically available markers are needed to improve our prediction ability. Immunophenotyping analyses and systems biology approaches have been important in identifying transcriptional regulatory networks that govern T cell activation and differentiation pathways. Ongoing differential gene expression analyses have identified baseline differences in gene expression in peripheral CD8+ T cells between responders and non-responders to nivolumab and ipilimumab + nivolumab in metastatic melanoma. Learning from data generated in the metastatic setting, Dr. Kirchoff summarized how we can apply the technologies and approaches in the adjuvant setting. Overexpression one gene, currently called HMAX, has been associated with non-responders. The name of HMAX and data surrounding HMAX will be published soon. Single cell RNA sequencing of peripheral CD8+ T cells from relapsed vs non-relapsed patients treated with nivolumab and ipilimumab + nivolumab have also identified HMAX as being expressed at higher levels in patients who have experienced a relapse. Gene expression and transcription factor data have generated a predictive regulatory network of CD4/CD8 T cells, and in non-relapsed patients, HMAX is in a more isolated network of transcription factors compared to relapsed patients. These findings highlight the emerging importance of cellular, genomic, and transcriptomic studies of peripheral blood cells to discover new biomarkers of immunotherapy efficacy in metastatic and adjuvant settings. However, the heterogeneity of peripheral immune cells makes composite biomarker studies complex and additional studies are needed to further improve our ability to leverage these data to predict cancer relapse.

Predicting, monitoring, and treating CRS caused by bispecific antibodies

CRS from bispecific antibody therapies; not your grandmother’s CAR
Martin Hutchings, MD, PhD (Copenhagen University Hospital) reported on the incidence and risk factors for cytokine release syndrome (CRS) caused by CD3/CD20 bispecific antibodies (e.g. glofitamab and epcoritamab) in patients with recurrent/refractory B cell lymphomas. While CRS is a dominant side effect of glofitamab, it should be noted that the overall rate of CRS is higher with anti-CD19 chimeric antigen receptor (CAR) T-cell therapy; approximately 70% of patients in the glofitamab dose-escalation studies developed CRS, with 23% of patients experiencing grade 2 CRS or higher after the step-up dose. While overall CRS rates were similar between fixed-dosing and step-up dosing cohorts step-up dosing reduced the frequency of high-grade CRS. CRS development in this patient population is predictable, and usually begins within the first 24 hours after glofitamab infusion. Whereas late events develop more slowly and are generally of grade 1, very early CRS events occurring within the first 10 hours are more severe. It is important to note that incidence of higher grade CRS events were dose-dependent. In predictive models of grade ≥2 Glofitamab-induced CRS development, eight variables were included in to generate a CRS risk score, and the most heavily weighted factors in the CRS risk score were disease stage and tumor burden, which are partially overlapping measures. Importantly, Induction of IL-6 and TNFalpha were also directly associated with CRS incidence and severity. Cytokine induction was observed prior to the onset of clinical CRS, with TNFalpha as an earlier and more sensitive predictor of CRS compared to IL-6. TNFalpha testing combined with CRS risk score generate a predictive model of CRS. 50/54 patients classified as “low risk” did not experience CRS, and 23/35 patients classified as “high risk” experienced grade ≥2 CRS. In addition to step-up dosing, pre-treatment of obinituzumab (anti-CD20), and subcutaneous administration (epcoritmab and mosunetuzumab) are other strategies to mitigate CRS caused by CD3/CD20 bispecific antibodies. No studies have shown that using IL-6 inhibitors like tocilizumab to treat CRS impairs the anti-cancer activity of bispecific antibodies; however, tumor T-cell infiltration and tumor regression are not affected by blocking IL6/IL-R interactionin preclinical studies. Ongoing studies are investigating the efficacy of JAK inhibition, mTOR inhibition, and Src/ICK inhibition in preventing CRS. Dynamics of CRS are variable between patients, and they are dependent on the tumor antigen and effector cells. Although risk scores, predictive models, and cytokine testing can be useful for monitoring patients at high risk bispecific-induced CRS, they should not replace monitoring of clinical signs and symptoms.

Using chimeric CD3 fusion receptors and bispecifics to increase the efficacy of chimeric antigen receptor cells

5513 - Chimeric CD3 fusion receptors expressed on iPSC-derived universal TCR-less CAR-T and -NK cells synergize with bispecific engagers to enhance antitumor activity and limit antigen escape

Eigen Peralta, PhD (Fate Therapeutics) described an approach to improve the efficacy of chimeric antigen receptor (CAR) T cells in treating solid tumors. The authors reported on a novel chimeric CD3 epsilon fusion receptor (3e-28-3e* CFR) with a unique transmembrane and endodomain which enables surface expression of CD3e on the surface of TCR-less T cells and NK cells. The engineered CFR engages bispecific T cell engagers (BiTEs), thus CFR+ effector cells can potentially target multiple tumor antigens. TRAC knockout (T-KO) Jurkat cells expressing surface CFR and co-cultured with EpCAM+ target cells exhibited increased TCR activation when EpCAM-BiTE was added to the culture or when EpCAM-BiTE was self-secreted by the CFR+ T-KO Jurkat cells. CFR+ iPSC-derived CAR-T (iT) cells exhibited 90% cytolysis against target cells expressing the primary (CAR-specific) antigen and, in the presence of soluble EpCAM-BiTE, 99% cytolysis of target cells expressing the secondary antigen that were negative for the primary antigen.  CFR+ CAR-iT cells self-secreting BiTEs exhibited 70% cytolysis against heterogenous tumors, compared to 5% cytolysis by CFR+ CAR-iT cells not expressing BiTEs. These data suggest that CD3 expression and BiTE engagement can be combined in CAR-iT cells, and engagement of CFR+ CAR+ cells with BiTEs have the potential to limit antigen escape and to produce durable anti-tumor responses.

Addressing persistence of CAR-T cells

Engineering T cells for persistence
Marcela V. Maus, MD, PhD (Massachusetts General Hospital) provided insights to engineering CAR-T cells for persistence.  Previous work suggests correlation between the persistence of anti CD19 CD8-based CAR T cells and durable remission. Interestingly, Dr. Maus noted that for anti CD19 CD28-based CARs, expansion is more important for response, and that prolonged persistence is not necessarily needed if CAR-T cells produce an early robust anti-tumor effect that eliminates the tumor. Rather persistence should be just long enough to eliminate the tumor. Another important question raised by Dr. Maus is whether persistence and signatures differ by product.  To address this question Maus group examined a cohort of 32 patients treated with tisa-cel (CD8-based) or axi-cel (CD28-based). Single cell RNA-seq of 105 pre- and post-treatment PBMCs and infusion product samples was performed. Importantly, the authors noted that CD8+ T cell expansion correlated with response to tisa-cel but not axi-cel, indicating that co-stimulatory domains of CAR affect persistence. Different approaches to achieving higher persistence in CAR-T or –NK cells were also discussed. Allogenic cord-blood derived CAR-NK cells expressing transgenic IL-15 cytokines exhibit persistence, and persistence was associated with response to treatment. Membrane bound IL-7 constructs and mutations in the CD3 ITAM domains or in CD28 costimulatory domains that alter cellular signaling can also increase CAR-T persistence and improve CAR anti-tumor activity. Approaches such as bispecific CAR-T cells, or approaches to block host cell-mediated cleavage of CARs also improve efficacy of CAR cells. Additional preclinical and clinical approaches are being taken to decrease the frequency of destruction of CAR cells by host T cells. Although many approaches are being taken to increase persistence of CAR cells, it is still unclear how long persistence should last.

New approaches in CAR-NK therapy

Engineered NK cells: The next frontier in cancer immunotherapy
Katayoun Rezvani, MD, PhD (UT MD Anderson Cancer Center) provided an overview on the development of chimeric antigen receptor (CAR) NK cells. Natural Killer (NK) cells, innate lymphoid cells, pose little to no risk of graft versus host disease (GVHD), and NK cell surfaces contain a wide array of activating and inhibitory antigen receptors. NK mediate a strong anti-tumor response. In addition to transmembrane, signaling, and extracellular domains, armored CARs for CAR-NK cells also contain a cyotokine gene to promote cellular persistence and a caspase-mediated suicide switch. In the dose escalation portion of a phase I trial of testing the efficacy of CAR-NK cells for treating CD19-positive lymphoid tumors, 7/11 patients exhibited a complete response, and no cytokine release syndrome (CRS), neurotoxicity, or GVHD was observed, suggesting that this approach is also safe and tolerable. The phase II trial has been completed, and results are eagerly awaited. In a dosing trial for FT596, a multi-antigen targeted, off the shelf, iPSC-derived CAR-NK cell therapy, the overall response rate is 72% (44% complete response). In an approach to induce a memory phenotype, NK cells were preactivated with IL-12, -15, and –18 and expanded ex vivo. The cells exhibit upregulated expression of genes related to JAK-STAT signaling and IFN-gamma response, indicating the presence of memory-like responses. When these cells are precomplexed with AFM13, a tetravalent bispecific CD30/CD16 immune cell engager, they exhibit a strong anti-tumor response in mice. Clinical trials of these AFM13-complexed CAR-like memory CB-NK cells for treating refractory/relapsed CD30+ malignancies are ongoing. Dr. Rezvani next discussed potential mechanisms by which relapse occur during CAR-NK therapy. Interestingly mouse models indicate that trogocytosis may mediate relapse after CAR-NK therapy- by downregulating expression of the target antigen on tumor cells, leading to lower tumor antigen density. In addition this process also causes NK cell exhaustion and fratricide, ultimately resulting in CAR T depletion. Dual CAR NK cells expressing an activating ROR1 CAR and an inhibitory CS1 CAR exhibit a strong anti-tumor response in mice, and prevents fratricide and exhaustion. With regards to the use of CAR-NK cells to treat solid tumors, recent studies have shown glioma stem cells express NK activating ligand and are susceptible to killing by healthy NK cells. Gliobastoma tumor-infiltrated NK cells are deficient in cell killing capacity, and blockade of TGF-beta restored cell killing activity. TGF-beta receptor knockout NK cells exhibit a strong anti-tumor response against GSCs in mice. A Phase I dose escalation study for NK cells containing deleted TGF-beta receptor is being planned. CD70, which is expressed on many hematologic malignancies and some solid carcinomas, is also being investigated as a new CAR target for hematologic malignancies and solid tumors. CD79 CAR-NK cells show anti-tumor activity in mouse models, and Phase I clinical trials for CAR70/IL-15 NK cells are being planned for hematologic malignancies and for solid tumors. CAR- and engineered NK cells show promise for treating specific cancers, and combinatorial strategies, such as multi-targeting CAR systems, combining CAR engineering with gene editing, or combining CAR-NK cells with other cancer therapies are important for the next generation of CAR-NK immunotherapy.

Cellular therapy from a systems biology perspective

Expanding the cell-therapy arsenal: CAR-T versus CAR-NK in 2022
Yvonne Y. Chen, PhD (University of California Los Angeles) presented specific considerations when engineering chimeric antigen receptor (CAR) T cells and how these principles may apply to CAR-NK cells. Engineering the next generation of CARs requires a synthetic biology approach in which biological parts build up genetic circuits to be used in translational applications. This approach can help optimizing the design CAR constructs to make them more effective and potentially more safe. An example of this is represented by the generation of CD19/CD20 bi-specific CAR-T therapy. In an ongoing phase 1 dose escalation study with a 3+3 design of bispecific CAR-T cells that target CD19 and CD20 to treat non-Hodgkin lymphoma, 7 of 9 patients achieved complete response, and high efficacy was observed at the lowest and second dosing levels, and median PFS has not been reached. No neurotoxicity or cases of cytokine release syndrome greater than grade 1 has been observed. In order for CAR T cells to function in the tumor microenvironment, TGF-beta CAR has also been engineered to activate primary human T cells and produce cytokines in dose-dependent manner. Bispecific TGF-beta CAR-T cells respond to TGF-beta and out-perform single-target CAR-T cell anti-cancer activity in mouse models of glioblastoma. To improve specificity of cell therapy and reduce cross-reactivity, T cells are being engineered to recognize intracellular tumor antigens with a COVERT synthetic protein. Engineered T cells release COVERT into target cells expressing the primary antigen. If the target cell expresses a secondary antigen (i.e., oncoprotein) the COVERT protein activated and induces apoptosis. A candidate COVERT protein is engineered granzyme B in which SUMO inhibitory peptide is fused to N-terminus of the protein. SENP1 protease, which is expressed in some cancer cells, cleaves SUMO to activate granzyme B. Many biological parts of cellular therapy such as CAR signaling and granzyme B appear to be transferable from T cells to NK cells. Some cell-specific differences may require alterations, such granzyme B in which SUMO inhibitory peptide is fused to N-terminus of the protein; SENP1 protease, which is expressed in some cancer cells, cleaves SUMO to activate granzyme B. Many biological parts of cellular therapy such as CAR signaling and granzyme B appear to be transferable from T cells to NK cells. Some cell-specific differences may require alterations, such as signaling domains, cell-type specific promoters and effector proteins. More knowledge of downstream proteins in T cells and NK cells is needed to predict how engineered constructs would transfer between cell types and what alterations may need to be made.  For this reason, simpler constructs would be more likely to function between NK cells and T cells. Questions surrounding cell persistence and durability also need to be addressed.

Immunosuppressive effects of the Unfolded Protein Response and other environmental stressors

Regulation of anti-tumor immunity by endoplasmic reticulum stress
Paulo Rodriguez, PhD (Moffitt Cancer Center) discussed the effects of endoplasmic reticulum (ER) stress and other environmental stressors on immune cells in the tumor. Factors in the tumor microenvironment (TME), including accumulation of misfolded protein and exposure to cytokines trigger ER stress and the unfolded protein response (UPR) activation leading to immune cell dysfunction. As ER stress increases, signaling molecules IRE1, ATF6, and PERK induce UPR activation. During the UPR, cells reprogram metabolic and signaling pathways to adapt to the stresses of the TME. If ER stress is severe, constant signaling through these mediators may lead to cell death mediated by the CHOP transcription factor. In a series of preclinical experiments aimed at determining the mechanisms by which ER stress contribute to an immune-suppressive tumor microenvironment, Dr. Rodriguez group and others identified that tumor associated myeloid cells such as tumor-infiltrating myeloid-derived suppressor cells (MDSCs) undergo ER stress and UPR activation. Of note, these tumor-associated MDSCs undergoing high ER stress were found to be more proficient in inhibiting activation of T cells (4% activation) compared to MDSCs in the non-cancerous spleen (37%). Reactive oxygen species and cytokines IL-4 and IL-6 are also known to induce IRE1 and ATF6 signaling in macrophages and suppress T cell activation in the tumor. The evidence that ER stress responses affect also human tumor-MDSC, derive from multiplexed immunofluorescence studies that revealed that tumor-infiltrating MDSCs from patients with non-small cell lung cancer have higher levels of PERK activation compared to myeloid cells from non-cancerous lungs. In support of this, the elimination of PERK in myeloid cells significantly delayed tumor growth in mice, and MDSCs from PERK knockout mice showed impaired suppression of T cells. Tumor-associated MDSCs from PERK knockout mice showed a higher capacity to engulf and destroy cancer cells compared to tumor-associated MDSCs from mice expressing PERK, suggesting that targeting PERK reprograms MDSCs and boosts immunotherapy. Other studies have found similar results in macrophages and with other signaling molecules, including ARF6 and IRE1. ER stress has a dual effect on T cells: acute ER stress activates T cells, while persistent ER stress suppresses T cells and induces apoptosis. In humans, CD8+ TILs show high levels of CHOP activation, correlating with decreased overall survival. Elimination of CHOP in T cells promotes anti-tumor immunity in vitro, and CHOP knockout CD8+ T cells exhibit more anti-tumor activity than wild type CD8+ T cells in mice. Limitation of glucose also activates IRE1 and leads to T cell dysfunction, and UPR has also been associated with upregulation of genes that cause T cell exhaustion. This body of work underscores how UPR and other cellular stresses affect MDSC and T cell activity in the TME and suggest that inhibitors of UPR could enhance the efficiency of cancer immunotherapies.

Using monoclonal antibodies and IL-2 variant to expand populations of stem-like T cells

Differentiating tumor-specific PD-1+TCF-1+ stem-like resource CD8 T cells towards a distinct effector T cell population through PD-1 mediated delivery of IL-2v
Laura Codarri Deak, PhD (Roche, Zurich, Switzerland) reported on the use of a monoclonal antibody against PDL1 to deliver a variant of IL-2 (IL2v) to tumors to expand the population of stem-like T cells. While IL-2 alone has shown activity in patients with cancer (e.g. melanoma, renal cell carcinoma), only a fraction of them responds to this treatment. In addition, the use of IL-2 is limited by the relatively poor tolerability and by the induction of regulatory T-cells (Treg) expansion. Therefore, additional strategies are necessary to optimally leverage the potential of IL-2 based therapies. Because the presence of PD1+ stem-like T cells correlate with better prognosis, and stronger response to immune checkpoint inhibitor therapy, researchers have developed a PD1-IL2v antibody conjugate that blocks PD-1, while activating IL-2 signaling via IL-2v delivery. This drug induced expansion, differentiation, and migration of stem-like T cells to provide durable anti-tumor immunity. PD1-IL2v binds preferentially to conventional T cells over regulatory T cells in vitro, and PD1-IL2v provides superior survival benefit and tumor eradication in preclinical mouse models of pancreatic cancer compared to anti PD1 + IL-2 combination therapy. Closer investigation has determined that PD1-IL2v targets tumor-reactive tumor-infiltrating lymphocytes (TILs) and expands effector memory of stem-like T cells and stem-like CD8 TILs, while anti PD1 expands differentiated T cells. The unique subset of Cd8+ TILs expanded by PD1-IL2v were active effector cells with no signs of exhaustion. PD1-IL2v provides a survival benefit and better tumor eradication in mouse models of multiple cancers, including neuroendocrine pancreatic cancers and sarcomas– provides survival benefit, better tumor eradication.  To determine whether these benefits translate to humans, PD1-IL2v is currently being tested in Phase I clinical trials.

Lessons learned from studies in the neoadjuvant immunotherapy setting

Maximizing translational discoveries in neoadjuvant IO clinical trials
Kellie N. Smith, PhD (Johns Hopkins University) shared insights gained from recent neoadjuvant studies, errors made early in the studies, and actions to take to maximize the success from correlative studies. Correlative studies in the metastatic setting are challenging, as immunotherapy has become the standard of care for many metastatic diseases, and biopsies and resections are no longer part of the standard care regimen. Most biopsies are obtained after immunotherapy, and samples are often small, with low quality and do fully capture tumor heterogeneity. In the neoadjuvant immunotherapy setting, biospecimens are obtained before and during treatment and post-surgery, so larger samples are obtained, which are more heterogenous and suitable for integrated large-scale bioinformatics studies. Importantly, as part of the surgical plan draining lymph-node and healthy tissues are also resected, which can provide valuable information as well as negative control for multi-omics studies. Correlative analyses on neoadjuvant immunotherapy studies require preparedness and communication between multiple healthcare providers and researchers. Specimens need to be collected in a format compatible with proposed experiments, so researchers need to know which experimental questions will be answered prior to specimen acquisition. Factors such as funding, experience of personnel, opportunities to share samples, and potential trade-offs should affect decisions on prioritization of experiments and research endpoints. Careful planning and maximizing biospecimens will allow research teams to perform a wide range of integrated “-omics” studies that generate large amounts of data for many correlative studies. Integrated analyses before and during treatment can generate key biological findings that identify new immunotherapy targets, new biomarkers, and new approaches to personalized medicine.

Tumor-specific T cells recognize patient-specific antigens more frequently than known oncoproteins

563 / 17 - Circulating tumor-specific T cells preferentially recognize patient-specific mutational neoantigens and infrequently recognize shared cancer driver mutations
Barbara Sennino, PhD (PACT Pharma) presented data from imPACT Isolation Technology (R), a T cell isolation platform that selectively captures cancer-specific CD8+ T cells from blood from cancer patients. This platform identifies non-synonymous mutations in genes, including cancer driver genes. Bioinformatic pipelines predicted HLA-binding neoantigens (neoAG), and a library of predicted neo-Ag peptide-HLA molecules was used to interrogate patient T cells. Paired T cell receptor (TCR) sequences were derived from single cell sorting of T cells. TCRs from antigen-experienced CD8+ T cells were validated by generating T cells expressing the edited neoTCRs. Neoantigen-specific T cells were isolated from peripheral blood samples of 243 patients with a variety of solid tumors. An average of 352 neoAg-HLA complexes were predicted per patient, and a total of 48,480 neoAG-HLA complexes were predicted, representing 7,711 unique mutations. 2% of the total neoAg-HLA complexes represented known oncogene driver mutations. Importantly, the author noted that the predicted peptide-HLA binding affinity for cancer driver mutation was significantly lower compared to patient specific neoAg. In detail, among a total of 216 functionally characterized TCRs, 94% recognized patient-specific neoAgs, and only 6% recognized known cancer driver proteins. Human T cells were engineered to express each TCR, and the engineered T cells became functionally active upon exposure to the matching antigen, regardless of the matching antigen being a neoAg or a known cancer driver protein. This study indicates that imPACT Isolation Technology can identify TCRs against patient-specific neoantigens and known oncogenic driver mutations. TCRs recognizing known oncogenic driver mutations were functionally similar to TCRs recognizing patient-specific neoantigens.

Targeting the tumor microenvironment with CAR T cells secreting T cell engaging molecules

569 / 4 - Mesothelin CAR T cells secreting FAP specific T cell engaging molecule (TEAM) target pancreatic cancer and its tumor microenvironment (TME)
Marc Wehrli, PhD, MD (Massachusetts General Hospital Cancer Center) reported on an effort targeting cancer-associated fibroblasts (CAFs) to make the tumor microenvironment more hospitable to chimeric antigen receptor (CAR) T cells that target the surface pancreatic tumor antigen mesothelin (meso CAR). A novel bicistronic lentiviral vector that encodes a mesothelin CAR and a secreted T cell engaging molecule (TEAM) that targets fibroblast activation protein (FAP), which is expressed by CAFs (CAR^TEAM), was constructed. In vitro assays show that TEAM secreted by CAR^TEAM cells interacts with and recruits bystander cytotoxic T cells against CAFs. Real-time cell analysis of co-culture assays with a pancreatic cancer cell line and CAFs expressing FAB show destruction of the pancreatic cancer cells by CAR^TEAM cells and cell death of CAFs through TEAM-mediated recruitment of T cells and CAR^TEAM cells. Cell-killing capabilities of the CAR^TEAM construct is superior to meso CAR-T cells and meso CAR-T cells secreting a nonspecific TEAM. In vivo experiments of subcutaneously injected tumor cells mixed with CAFs demonstrated that CAR^TEAM cells exhibit increased pancreatic tumor control activity compared to meso CAR-T cells and meso CAR-T cells secreting a nonspecific TEAM. These results provide promise for a new dual targeting strategy that uses TEAM-secreting T cells to target pancreatic cancer cells and the tumor microenvironment.

Isolation and expansion of reactive TILs from RCC tumors

575 / 10 - Ex vivo expansion of tumor infiltrating lymphocytes (TIL) from human renal cell carcinoma (RCC) tumors
Mohammed Alkhouli, PhD (H. Lee Moffitt Cancer Center & Research Institute) reported a feasibility study to expand tumor reactive tumor infiltrating lymphocytes (TILs) from surgically resected renal cell carcinomas (RCCs). Tumors were isolated from 44 patients and included different subtypes of renal tumors: 81.82% from clear cell RCC, 9.09% from papillary RCC, and 2.27% from chromophobe RCC. Tumor fragments were cultured with high does IL-2 for four weeks. TILs were grown from 39/44 (88.64%) of tumor samples, and two-thirds of TILs were tumor reactive. The proportion of isolated CD4+ T cells was significantly higher than the proportion of isolated CD8+ T cells. TIL expansion was also examined under hypoxic conditions, as hypoxia within the tumor microenvironments of RCC has previously been associated with resistance to immunotherapy and with poor prognosis. TIL yield in hypoxic conditions was lower than TIL yield in atmospheric conditions (20% O₂), suggesting that hypoxia negatively affects TIL production. TILs grown in hypoxic conditions exhibited a higher frequency of CD45RA- CCR7+ central memory T-cells compared to those grown under normal conditions. TILs were further expanded by a rapid expansion protocol (REP) of stimulation with anti-CD3 antibody in the presence of allogenic feeder cells under atmospheric conditions (20% O₂) and under hypoxic conditions (5% O₂). TILs that underwent REP were able to expand in hypoxic conditions and exhibited a higher frequency of CD69+ CD103+ memory T-cells (19%) compared to the starting T cell population (0.11%) and to TILs expanded at atmospheric conditions (0.17%). When cultured with autologous tumor cells in atmospheric and hypoxic conditions, TILs expanded in hypoxic conditions outperformed TILs expanded in atmospheric conditions with regards to cytokine release (TNF-alpha, IFN-gamma, and granzyme B), regardless of culture conditions. This study indicates that functional reactive TILs can be isolated and expanded from RCC tumors and adapting TILs to hypoxia can enhance their anti-tumor activity in normoxic and hypoxic settings.

Analysis of regulatory T cell clusters among patient populations

595 / 5 - Distinct tumor infiltrating Treg lineages are associated with response to anti-PD1 checkpoint blockade in NSCLC
Arbor G. Dykema (Johns Hopkins University) reported on the different populations of regulatory T cells (Tregs) in the tumor and tumor microenvironment of non-small cell lung cancer (NSCLC). T cells were isolated from tissue and peripheral blood samples from 15 patients treated with neoadjuvant nivolumab for non-small cell lung cancer (NSCLC) and from 10 patients untreated for NSCLC. Single-cell RNA sequencing- TCR-sequencing was performed on CD3+ cells, and 72,251 CD4+ FoxP3+ T regs were analyzed by refined clustering and pseudotime and differential gene analyses. 7 separate Treg clusters that reflect different functionalities in the tumor microenvironment were identified. Two separate Treg subsets represent an activated state that express members of the tumor necrosis factor receptor superfamily (e.g., OX40, 41BB, GITR) or a resting state. Tumors that are non-responsive to immune checkpoint blockade therapy are enriched for activated Tregs. Patients who respond to immune checkpoint inhibitor therapy tend to have less active Treg subsets, and RNA velocity analyses infer a shift from activated Tregs to a more resting population.  Untreated patients display the opposite Treg subsets, suggesting more Tregs are active and in a terminal differentiated state. In-depth analyses of tumor Tregs like this can identify specific biomarkers that when targeted, could inhibit a specific subset of Tregs, improving response to immune checkpoint blockade therapy and reducing incidence of adverse events.

Timing of CTLA-4 Ig therapy affects its therapeutic benefits

662 - Late CTLA-4 Ig treatment improves antitumor efficacy of immunotherapy
Stephen Mok, PhD (University of Texas MD Anderson Cancer Center) presented data on the effects of CTLA-4 Ig (abatacept), an inhibitor of T cell stimulation, on anti-tumor activity of immunotherapy. CTLA-4 Ig has been shown to reduce immune-related adverse events (irAEs), including cardiotoxicity, in mice receiving anti-CTLA-4 and anti-PD-1 combination immune checkpoint therapies, but its effect on the anti-tumor response had not been investigated. Mice were injected with CTLA-4 Ig during anti-CTLA-4 immunotherapy (early) or upon completion of immunotherapy (late). CTLA-4 Ig administered early in therapy compromised the anti-tumor response of immunotherapy, while late administration of CTLA4-Ig improved anti-tumor efficacy. Similar results were observed with anti-PD-1 immunotherapy and with combination anti-CTLA-4 and anti-PD-1 combination immunotherapy. In each of these cases, late CTLA-4 Ig resulted in improved anti-tumor efficacy of immune checkpoint inhibition. The improved anti-tumor response observed with CTLA-4 Ig was dependent on CD80/86. Importantly, Dr. Mok also noted that the frequency of CD4 and ICOS+ Foxp3+ regulatory T cells decreased in the CTLA-4 Ig late treatment group. Improved function of anti-CTLA-4 immunotherapy was also observed in mice depleted for regulatory T cells, suggesting that the benefit of late administration of CTLA-4 Ig is dependent on the depletion of regulatory T cells. These results suggest that CTLA-4 Ig has a differential role throughout treatment with immune checkpoint inhibitors and that combining checkpoint blockade with late CTLA-4 Ig treatment has potential therapeutic benefits.

Engineering CARs for persistence and functionality in solid tumors

Multi-modular genetic engineering approaches to overcoming barriers in CAR T cell therapy for solid cancers
Martin Pule, MB, FRCPath (University College London Hospital) reported ongoing efforts to design and test novel anti-GD2 CAR T cells to treat neuroblastoma. The original CAR was bicistronic gamma retroviral vector and expressed an RQR8 safety switch, an anti-GD2 CAR, and a second-generation CD28 zeta endodomain. Because of the risk of neurotoxicity, a phase I dose escalation studies started with low doses and no lymphodepletion/conditioning, and slowly added conditioning and increased doses. 3 of 6 patients who received CAR-T cell doses of 10⁸ and 10⁹ cells/m² and lymphodepletion/conditioning with cyclophosphamide and fludarabine responded to treatment. One patient with high disease burden exhibited CRS and various adverse events associated with macrophage activation syndrome but no neurotoxicities. Reduced tumor burden was observed on Day 28, but disease regrowth was observed on Day 45. The other two patient who responded to treatment exhibited similar responses, suggesting GD2 CAR-T cell treatment of neuroblastoma is safe and produces an anti-tumor response, albeit transient. In order to achieve persistence and function in the neuroblastoma tumor microenvironment, a modular GD2 CAR-T cell (MCARGD2-T cell) was designed. In addition to the GD CAR and RQR8 safety switch, the construct includes a truncated SHP2 (dSHP2) to inactivate immune checkpoint receptors, a truncated TGF beta receptor (dnTBR2) so CAR-T cells are resistant to TGF beta-mediate inhibition, and a constitutively active cytokine signal (IL7 CCR) for persistence. T cells were co-transduced with two vectors, and the GD2 CAR and safety switch were included on both vectors. MCARGD2-T cells remained active in the presence of PDL1 and in the presence of TGF beta, suggesting dSHP2 inhibits immune checkpoint signaling and dnTBR2 is blocking signaling by TGF beta. MCARGD2-T cells proliferated in the absence of exogenous cytokine in an antigen-dependent manner and exhibited a lower level of turnover when co-cultured with tumor cells, compared to the first-generation GD2-CAR T-cells. Addition of each module to first-generation GD2 CAR-T cells shows an incremental benefit in anti-tumor activity and immunologic activity. Subjects are being recruited for Phase I trials of MCARGD2 T-cells to treat relapsed/refractory neuroblastoma. While clinical trials of the first-generation GD2 CAR-T cells showed promise, the preclinical results with MCARGD2-T cells suggest that in order for CAR T-cells to successfully treat solid tumors, CAR vectors will require a more complex, modular design to retain persistence and functionality in the tumor microenvironment.

Augmenting safety and potency of CAR T cells
Crystal Mackall, MD (Stanford University) reported novel approaches in using T cells to treat solid tumors. Trials investigating the use of GD2 CAR-T cells to treat diffuse midline gliomas show a significant pattern of tumor shrinkage, but the clinical benefit is not fully correlated with the depth of response. To achieve durable benefit repetitive dosing every 28 days is required, suggesting that treating solid tumors requires a longer period of immune pressure, compared to B cell lymphomas. The success seen with repetitive dosing is likely because CAR-T cells are prone to exhaustion and because easing signaling of CAR over time leads to restoration of T cell function. To address T cell exhaustion, the SNIP platform, which regulates CAR signaling within the CAR-T cell, was developed. In the SNIP platform, a hepatitis C protease-based module expressed in trans with a CAR containing a cleavage site. In baseline conditions, the protease is active, cleaving the CAR signaling domain and turning the CAR off. In presence of drug, (grazoprevir, an FDA-approved Hepatitis C antiviral), the protease inhibited, and CAR is not cleaved and produces signal. The SNIP platform has been optimized so there is no leaky CAR activity without the drug. CAR activity is present for approximately 21 hours after drug exposure. Originally developed as safety switch to turn off CAR expression at the onset of toxicity, but SNIP-CARs exhibit potent anti-tumor activity against solid tumors when the system is on. Compared to constitutively active CAR-T cells, tumor-infiltrating SNIP-CAR T cells expand better in mouse models and exhibit more stemness, less exhaustion (as measured by CD39 levels), greater functionality when challenged with antigen. SNIP-CAR T cells develop stem-like and cytotoxic characteristics while constitutive CAR-T cells develop cytotoxic characteristics. Reducing dosing of drug in preclinical models prevents toxicity, but still maintains conditions for tumor control. Pharmacokinetic cycling of the drug creates a transient resting period of CAR-T cells, which may prevent CAR-T cell exhaustion. Reducing CAR-T cell activity could also allow for a therapeutic window in which CAR-T cells specifically respond to cancerous tissues expression expressing the CAR antigen at high levels but not to non-cancerous tissues expressing low levels of CAR antigen.

In a separate attempt to improve CAR-T cell persistence and functionality, a genome-wide knockout CRISPR screen identified genes that restrict T cell fitness. The screen identified MED12, CCNC, and other components of the kinase module of the Mediator Complex Kinase, a transcription regulator in all cells. Deletion of components of the Mediator kinase module increased T cell fitness, while deletion of components of the core module decreased T cell fitness.  When Mediator regulates transcription, the kinase module dissociates from the Core Mediator, and the Core brings together transcriptional machinery. MED12-deficient T cells show increased IL2-dependent expansion. Antigen-induced cytokine production was also enhanced, measured by production of inflammation-associated cytokines and chemokines that are known to recruit T cells into the tumor. MED12-deficient T cells also show increased metabolic fitness and tumor control in mice, and they are enriched for effector memory. MED12-deficient T cells are more sensitive to IL-2 and express more copies of IL2 receptor alpha on their surface. Knock-down of other components of the mediator kinase module generates phenocopies of MED12-knockout T cells. These results point to a role for the Mediator transcription regulator in T cell biology, suggesting the Mediator Kinase Module regulates T cell fitness. These insights may have future applications in increasing the efficacy and persistence of CAR-T cells and other engineered cell therapies.

Addressing unanswered questions in the treatment of triple-negative breast cancer

Checkpoint inhibition for early-stage triple-negative breast cancer: Does sequencing and timing of administration matter?
Sara M. Tolaney, MD, MPH (Dana-Farber Cancer Institute) addressed unanswered questions regarding the standard of care for triple negative breast cancer (TNBC). The KEYNOTE-522 clinical trial randomized patients with stage II and III triple negative breast cancer to receive standard chemotherapy with carboplatin, taxanes and anthracycline with versus without pembrolizumab as preoperative therapy. After surgery patient who received preoperative immunotherapy continued pembrolizumabfor additional 9 cycles. In this study,  patients in the immunotherapy arm had a significantly higher rate of pathologic complete response and longer event free survival (84.5% vs 76.8% at 36 months). Based on these findings, pembrolizumab + chemotherapy is the standard of care for Stage 2 or 3 triple negative breast cancer in the neoadjuvant (pre-operative) setting. These gains in the treatment of breast cancer have raised new questions:
How can we select which patients need preoperative checkpoint inhibition? The KEYNOTE-522 study found that PD-L1 expression did not predict benefit to therapy. PD-L1 positivity is associated with better outcomes from either combination therapy or chemotherapy alone. Treatment benefits are also seen regardless of node status. More research is needed to identify biomarkers to predict which patients would benefit from immune checkpoint inhibitor therapy and which patients would get positive results from chemotherapy alone.
Does it matter if combination therapy is administered preoperatively vs adjuvantly? Dying tumors release tumor-specific antigens, so administering immune checkpoint inhibitors when the tumor is present may lead to an increase in memory T cells, a hypothesis supported by preclinical models. Multiple clinical trials are investigating the benefits of immune checkpoint inhibitors in the adjuvant setting. The IMpassion030 clinical trial is investigating whether adjuvant combination therapy provides clinical benefits over adjuvant chemotherapy alone. The SWOG S1418 trial is comparing pembrolizumab vs observation for TNBC patients with residual invasive disease, and the A-BRAVE Phase 3 trial compares adjuvant avelumab therapy vs observation in patients at high risk for relapse who completed surgery and chemotherapy. One caveat with these ongoing trials is that patients in these trials did not receive pre-operative immunotherapy and therefore these studies do not address  the question of whether to continue adjuvant immune checkpoint inhibitor therapy after patients who received preoperative pembrolizumab present with residual disease post-surgery.
Does choice of chemotherapy background matter? The TONIC Trial suggests anthracyclines and platinum are associated with greater activity. The NeoTRIP trials, which omitting anthracyclines, showed no improvement in pCR with the addition of atezolizumab to a platinum/taxane backbone. EFS data are not available, but early data suggest that anthracycline therapy is a factor to consider when determining benefit from chemotherapy. There are also questions surrounding the addition of platinum to anthracycline and taxane, but individual preoperative trials have produced varying results regarding EFS. Carboplatin does increase pCR but also toxicity; there is uncertainty whether it directly benefits EFS, but it is part of the current standard of care because it was a backbone therapy component in the KEYNOTE-522 study. More research is needed to determine whether the chemotherapy backbone can be de-escalated with the addition of checkpoint inhibition.
Does sequence of checkpoint and chemotherapy impact outcome? In the GEPARNUEVO trial, patients received durvalumab or placebo prior to checkpoint + chemotherapy; Patients who received durvalumab prior to combination therapy showed improved pCR rates and an increase in tumor infiltrating lymphocytes, suggesting that administering checkpoint inhibitor therapy prior to combination therapy does provide a therapeutic benefit.
Do patients need adjuvant checkpoint inhibition after surgery? Comparing outcomes from two independent clinical trials provide some insights to address this question. In the GEPARNUEVO trial, patients received checkpoint inhibition only in the preoperative setting; checkpoint inhibitors were not administered in the adjuvant setting. Patients in the KN522 trial received checkpoint inhibitors in the pre- and post-operative settings. The 3-year IDFS rates, as well as clinical outcomes among patients who achieved a pCR were similar, raising the question of whether postoperative immunotherapy is necessary.  The OptimICR-pCR trial, which is currently being planned, will directly address this question. Patients with pCR after preoperative chemotherapy + pembrolizumab will be randomized between two to receive post-operative pembrolizumab or a placebo.
Do all patients with residual TNBC need pembrolizumab in addition to chemotherapy? Based on results from the CREATE-X trial, the current standard of care is adjuvant capecitabine after neoadjuvant chemotherapy and surgery for patients with residual TNBC. Data from metastatic setting suggest that adjuvant combination checkpoint inhibitor therapy + chemotherapy is safe and without synergistic toxicities for patients without germline BRCA mutations, and therefore could be considered an options for patients with residual disease. For patients with germline BRCA mutations, the use of olaparib leads to improvements in EFS and OS, but questions remain whether to use olaparib, pembrolizumab, or a combination of the two. More randomized clinical trials are needed to completely address this question.

While much progress has been made in the treatment of early-stage TNBC, more research is needed to identify biomarkers and other treatment and treatment sequences to best tailor treatments for individual patients.

Immunotherapy for resectable lung cancer: Clinical use and molecular predictors of benefit
Patrick Forde, MBBCh (Johns Hopkins University) provided updates on clinical trials investigating the efficacy of neoadjuvant immune checkpoint inhibitors in treating non-small cell lung cancer (NSCLC). In an initial pilot clinical trial of 20 patients who received neoadjuvant nivolumab prior to surgical resection, 50% of patients exhibited a major pathologic response, in both PD-L1 positive and negative cases. Of note, a high mutational burden was found to correlated with patient response to nivolumab. Interestingly, among 14 evaluable patients, the molecular response in ctDNA mirrored the pathologic response. In the NADIM trial, patients with Phase IIIA NSCLC received 3 cycles of nivolumab + chemotherapy in the neoadjuvant setting and post-operative nivolumab in this single-arm trial. The primary endpoint was PFS at 24 months. 46 patients enrolled, and the 24-month PFS was 77%. Notably, 74% (34/46) exhibited a major pathologic response (MPR), and 57% (26/46) exhibited a pathologic complete response. Other Phase III trials of neoadjuvant chemotherapy + PD-1/PD-L2 checkpoint blockade in NSLC are ongoing. In the CheckMate 816 trial, patients with resectable stage IB to IIIA NSCLC receive neoadjuvant (pre-surgery) nivolumab + platinum-double chemotherapy or platinum doublet chemotherapy followed by optional adjuvant chemotherapy and/or radiation therapy. Median EFS in patients who received neoadjuvant nivolumab + chemotherapy was 31.6 months, compared to 20.8 months for patients who received neoadjuvant chemotherapy. The hazard ratio for disease progression, disease recurrence, or death was 0.63, indicating that adding nivolumab to neoadjuvant chemotherapy reduced risk of recurrence/disease progression/death by 37%. Nivolumab + chemotherapy doubled EFS for patients with stage IIIA cancer and for patients with PD-L1 positive NSCLC, indicating disease stage and PD-L1 status as predictors of therapeutic benefit. Less, if any benefit, is seen in patients with stage IB or II disease or in patients with PD-L1 negative cancer. pCR rate for patients who received nivolumab + chemotherapy was 24%, compared to 2% for patients who received chemotherapy. Patients who exhibited pCR had a risk of recurrence/disease progression/death reduced by 87%, indicating that pCR may be a strong predictor of long-term outcomes. Clearance of ctDNA during neoadjuvant treatment showed a trend toward improved EFS, but it was not statistically significant. Dr. Forde also reviewed the the IMpower 010 trial which led to the approval of atezolizumab for treatment of NSCLC in the adjuvant setting. Also in this study, Adjuvant atezolizumab was associated with increased benefit particularly among patients with high PD-L1 expression (HR = 0.43 for TC >= 50%). In the phase III, randomized PEARL/KEYNOTE-091 trial, which compared adjuvant pembrolizumab versus placebo in patients with stage IB-III resected NSCLC, pembrolizumab significantly improved the disease-free survival in the entire patient population. However, in this study a high PD-L1 expression was not associated with outcomes. It is important to note that 0.7% of the population experienced treatment-related deaths, suggesting that more studies need to investigate prevention of toxicities and adverse effects. Although more studies are needed to investigate adjuvant treatment and to identify and verify biomarkers predictive of patient response, immunotherapy is now included as a standard of care for patients with resectable NSCLC, either as nivolumab + chemotherapy in the neoadjuvant setting or as chemotherapy followed by atezolizumab in the adjuvant setting.

Bolstering the microbiome during immunotherapy

Gut microbiome in immuno-oncology: From a biomarker to adjuvant therapeutic
Bertrand Routy, MD, PhD (University of Montreal) presented recent findings regarding the gut microbiota and its effects on patient response to immunotherapy. Studies of renal cell carcinoma and non-small cell lung cancer indicate that antibiotic treatment causes dysbiosis in the gut microbiota, and these changes are associated with a decreased response to immune checkpoint inhibitors across different tumor types. In a recent study investigating the use of charcoal adsorbent-based product (DAV132) to sequester antibiotic in the gut and to prevent microbiota destruction, patients were randomly divided in two groups (n=24, 12 per group), and one group received intravenous administration of ceftazidime/Avibactam (CZA) every eight hours for four days, and the treatment group received the same course of CZA along with oral administration of DAV132 every eight hours for one week. DAV132 did not affect the concentration of CZA in plasma, but it did reduce the concentration of CZA in feces. Gut microbiota diversity significantly decreased in patients who received CZA with no DAV132 early during treatment, but by Day 35, diversity had recovered to normal levels. Patients who received DAV132 exhibited no significant decrease in microbiota diversity throughout the treatment. The impact of DAV132 on immune checkpoint inhibitor therapy was tested in mouse cancer models. Germ-free mice received fecal microbial transplants (FMTs) from healthy volunteers in the DAV132 trials. Mice receiving FMTs from patients who did not receive DAV132 did not respond to anti-PD-1 therapy, while mice who received FMT with DAV132 responded to anti-PD-1, indicating DAV132 may ameliorate the negative effects of antibiotics on gut microbiota and on response to immune checkpoint inhibitors. Combination therapy of FMT + anti-PD1 therapy for treating metastatic melanoma was examined in a Canadian trial. 14 anti-PD1 naïve patients received FMT 7 days before administration of first anti-PD1 treatment. Safety was the primary objective: adverse events of grade 2 or 3 were observed in 4/14 patients. 3/14 patients exhibited a complete response, the objective response rate was 71.4% (10/14), and clinical benefit rate was 85.7% (12/14). FMTs from patients in the trial produced corresponding outcomes in mouse models: Mice receiving FMTs from patients who had responded to FMT + antiPD-1 therapy also responded to anti-PD1 therapy. The fruit Camu Camu (CC) is currently being investigated as a prebiotic that promotes diversity in the gut microbiome. Mouse models of fibrosarcoma and breast cancer show that CC extract has an additive effect to anti-PD1 activity. Catalagin polyphenol, a CC component that causes anti-PD1 efficacy, was tested in germ-free mice and in mice with intact gut microbiomes, and its effect was only observed in mice with intact gut microbiomes. Castalagin promoted infiltration of Cd8+ T lymphocytes to tumors and shifted both the gut microbiota composition toward more favorable species. Clinical trials investigating the effects of CC and anti-PD1 therapy for treating NSCLC and melanoma will be opening soon. The gut microbiome originally represented a biomarker to predict response to immune checkpoint inhibitors, but the studies presented lay the groundwork for using FMTs, antibiotic sequestration, and prebiotics as adjuvant therapy in combination with immunotherapy.

Factors that modulate myeloid cell participation in melanoma affect anti-tumor immunity

Germline and microbiome variants impact immunotherapy efficacy through modulation of myeloid cells
Thomas Gajewski, MD, PhD (University of Chicago) provided new insights to factors that influence the level of immune cell infiltration of a tumor, including cell-intrinsic oncogenic events, composition of gut microbiota, and polymorphisms in immune regulatory genes. Past studies have indicated beta-catenin expression as an important cell-intrinsic oncogenic effect. Tumors with active beta-catenin signaling fail to recruit Batf3-dendritic cells to the tumor and do not respond to anti-PD1 therapy. These results suggest dendritic cells contribute to an anti-tumor effect in the tumor microenvironment (TME), and more information is needed on how to recruit and activate dendritic cells in tumors that lack them. With regards to the gut microbiota, germ free mice received fecal microbiota transplants (FMTs) from human donors who responded to anti-PD-1 therapy (responders) and from human donors who did not respond (non-responders). Stable colonies of germ-free mice with donor gut microbiota have been generated and kept as separate lines. Mice have been bred, and the first and second generations of mice post-FMT maintain responder/non-responder phenotype, suggesting microbiota can be propagated generationally and provide a stable model system for investigation of differences in the gut microbiome. FMTs between responders and non-responder mice make non-responders responsive to anti-PD-1 therapy and vice versa, suggesting microbes of the gut can positively and negatively regulate response to immunotherapy. Single cell RNA seq of TILs in the gut did not identify many differences between responders vs non-responders. Analysis of macrophages indicated a shift to M2 phenotype in non-responders and a shift to an M1 phenotype in responders suggesting that composition of gut bacteria may induce immunophenotypic changes in myeloid cells to support anti-tumor immunity. Serum metabolites from responder mice vs. non-responder mice have also been examined, and specific metabolites have been identified. With regards to polymorphisms in immune regulatory genes, germline SNPs in the PKC delta gene have been associated with immune cell infiltration in metastatic melanoma. PKC delta knockout mice show enhanced response to anti-PD-L1 therapy and improved immune-mediated tumor control. Loss of function PKC delta variants are associated with greater immune cell infiltration of the tumor, and myeloid cells in the TME shift from M2 to M1 signature, suggesting role for PKC delta in controlling the shift from M2 to M1. Mutations in PKC delta are associated with the autoimmune disease lupus, and CTLA4 and PD1 have been identified as genes that were polymorphic and linked to autoimmunity, providing a new lens for therapeutic targets. Ongoing studies of tumor and host factors that modulate myeloid cell participation in the anti-tumor response will also provide new insights to therapeutic targets.

A novel platform to identify drivers of the tumor microenvironment

Investigating cancer determinants of tumor immune composition
Brian Brown, PhD (Icahn School of Medicine at Mount Sinai) described Perturb-map, a novel in vivo spatial functional genomics platform to identify genetic determinants of the immune composition of the tumor microenvironment and tumor immunity. CRISPR was used to knock out over 30 candidate genes in parallel in a mouse model of lung cancer, and tumor growth, histopathology, and immune composition were assessed. Knocking out positive regulators of IFN gamma signaling led to reduction of tumor burden. By contrast, knockout of Socs1, a suppressor of cytokine signaling, and of TGF beta Receptor 2 (Tgfbr2) caused the most tumor growth. While knockout of Socs1 increased tumor growth, it also associated with high CD4+ and CD8+ T cell infiltration, and these tumors were more responsive to anti-PD-L1 therapy. Knockout of Tgfbr2 also conferred a growth advantage, but T cells were excluded from the tumor and the tumor microenvironment (TME) converted to a fibro-mucinous state. Dr. Brown also noted that subclonal immune composition has specific spatial distribution. When a Socs1 knockout tumor is near a Tgfbr2 knockout tumor, the Tgfbr2 knockout tumor is devoid of T cells, though T cells are plentiful in Socs1 knockout tumor. Similar patterns of local immune exclusion have been observed also in human tumors. Spatial transcriptomics of lesions showed that TGF beta signaling, which drives immune exclusion, was upregulated in Tgfbr2 knockout tumors. Signal upregulation was primarily from the activation of tumor fibroblasts. A model has been developed in which knockout of Tgfbr2 increases the pool of free TGF beta in the TME. More TGF beta is available to bind to stromal cells in the TME, causing immune exclusion in the tumor. Results from this study indicate that Perturb-map platform is a new tool to perform functional genomics analyses at single-cell resolution. This study also provides insights to the effects of TGF beta signaling on stromal cells and anti-tumor immunity in the TME.

Progress in the development of a vaccine for pancreatic cancer

Intercepting pancreatic cancer development with oncogene-targeted immunotherapy
Elizabeth M. Jaffee, MD (Johns Hopkins University) presented recent work in the development of a pancreatic cancer vaccine. Based on the model of immune editing, the ideal time to administer a cancer vaccine to patients with cancer would be very early in the disease stage, during the elimination stage, when the first or second oncogene activates the immune system. The immune cells would be exposed to the vaccine antigen early, allowing time for the immune system to react and adapt by developing T cell memory and diversifying T cells. Mutations in the KRAS oncogene are commonly observed in pancreatic cancer, and mutated KRAS (mKRAS) is expressed early in the pre-malignant stage of pancreatic cancer development, making KRAS an ideal candidate antigen for vaccine development. Pathology specimens with low grade/high grade pancreatic intraepithelial neoplasia (PanIN) from the same patient show high levels of CD8 T cells infiltration in low grade PanIN more than high grade PanIN. Foxp3 is more present in in high grade PanIN. Trials of a KRAS vaccine + anti-CTLA4 + Treg depletion alters Tregs and monocytes and prevents PanIN progression to cancer 100% of the time. T cell memory is also increased, indicating potential for long-term protection. An early trial of an off-the-shelf mKRAS peptide vaccine has been conducted in patients in the adjuvant setting. The vaccine represents six overlapping most frequent KRAS mutations in pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC). Vaccine is administered with immune checkpoint inhibitors (ipilimumab and nivolumab) after surgery. 8 patients have been vaccinated so far, and samples from 7 have been monitored for T cells. Vaccination increased activated  and cytokine-expressing CD8+ and CD4+ T cells. The ratio of mKRAS CD4 to CD8 T cells is 1.8:1. Single cell analyses classify functional T cell states and TCR expansion. 19 clonotypes have been identified to contain mKRAS-specific TCRs. 136/28,497 TCRs cluster with known mKRAS-specific TCRs. Assays to validate cytotoxicity and specificity of cloned TCRs in vitro are ongoing. In a second ongoing trial, off the shelf mKRAS peptide vaccine has been administered to patients at high risk (germline mutation carriers with lesions) of developing pancreatic cancer. Patients are currently being enrolled for the pilot study to test safety and immune response, which will be measured by quantifying mKRAS specific T cells, functionally profiling mKRAS specific T cells and identifying mKRAS-specific TCRs. A third trial will identify vaccine efficacy in the preadjuvant setting: off the shelf mKRAS peptide vaccine for Intraductal Papillary Mucinous Neoplasms (IPMNs) at high risk for becoming pancreatic cancer, will be administered prior to surgical resection. The surgical specimen will be used to track trafficking mKRAS-specific T cells to lesion and to characterize inhibitory signals. Pancreatic cancer is difficult to treat, and the overall prognosis for most patients is poor. Results from these trials are promising, suggesting that a vaccine to prevent the disease in early stages may be attainable.

A new engineered T cell receptor increases antigen sensitivity

Emerging principles to overcome tumor antigen escape
Michel Sadelain, MD, PhD (Memorial Sloan Kettering Cancer Center) presented recent research surrounding antigen sensitivity of chimeric antigen receptors (CARs) and how it relates to tumor antigen escape. Although CAR-T cell treatment has shown great success in treating hematological malignancies, relapses still occur in a significant fraction of patients. Some relapses are associated with diminished expression of the target antigen. CAR sensitivity varies, and multiple studies have shown that CARs with CD28/CD3z signaling domains are more sensitive than CARs with 4-1BB/CD3z signaling domains. Mitigation of tumor escape can be achieved through multiple approaches. Some CAR-T cells target two antigens by expressing two different CARs on the cell. Chimeric costimulatory receptors (CCRs) recognize a second antigen and stimulate the CAR if the second antigen is present, but the primary (CAR-specific) antigen is at low levels. CAR cells expressing a modified CCR “If/Better” gate targets only one CAR-specific antigen, but the CAR is more effective if the second CCR-specific antigen is present. The If/Better gates increase CAR-T cell persistence, and T cells expressing the ADGRE2/CLEC12A IF,BETTER gate outperforms T cells expressing the ADGRE2 CAR in acute myeloid leukemia antigen escape models. A novel HLA-independent T cell receptor (HIT receptor) recognizes CAR target with greater sensitivity, similar to TCR. HIT receptors utilize CAR heavy and light chains on the CD3 T cell receptor, so the CAR is HLA independent and CD3 dependent. When placed under control of the TRAC promoter, HIT receptors display greater antigen sensitivity compared to CARs, and cause more cell lysis under low concentrations of the CAR antigen. Anti-CD19 HIT receptors will destroy leukemia cells that express less than 20 copies of CD19 per cell in vitro. TRAC-anti CD 19 HIT cells and TRAC-anti CD 22 HIT cells destroy MM1S multiple myeloma cells, which express low levels of CD19 and CD22 that are barely detectable. Anti-CD19 and anti-CD22 TRAC-CAR-T cells show no activity against the MM1s cell line. Quantitative mass spectrometry of peptides produced by TRAC-CAR-T cells in the presence of antigen indicate that HIT cells express more proteins involved in T cell activation and in actin machinery, suggesting that HIT cells are more effective at synapse formation with the target cell and at the aggregation of cytotoxic granules. Degranulation experiments indicate that HIT T cell kill faster than CAR T cells under low antigen density. HIT T cells show superior anti-cancer activity in vivo, exhibiting more cell killing of leukemias and multiple myeloma cells and increasing overall survival, compared to CAR T cells. All CARs have a limit to antigen sensitivity, and the development of the HIT increases options for treating malignancies with low antigen densities.

Engineering the tumor microenvironment to drive systemic immunity

Radiation-enhanced antigen presentation and immune priming
Chandan Guha, MBBS, PhD (Albert Einstein College of Medicine, Montefiore Medical Center) presented results from clinical trials investigating radiation-enhanced immune priming and antigen presentation. The first trial discussed investigated if combination of stereotactic body radiotherapy (SBRT) to a single lesion and dendritic cell (DC) growth factor CDX-301/Flt3L is safe and has clinical activity in patients with metastatic NSCLC. Ablative doses of SBRT were administered (34 Gy x 1 Fx, 18 Gy x 3 Fx or 10 Gy x 5 Fx). No dose-limiting toxicity was observed in the 26 subjects. 16/26 patients achieved PFS at the 4-month follow-up.  Abscopal effects occurred in metastases in bone, lungs, pleural, and lymph nodes. 9/29 patients demonstrated abscopal effect, as measured by partial metabolic response, without further treatment. PET Response at 2 months and disease multifocality were highly prognostic of patient outcomes. Increases in conventional DC1, CD5- DC2 and CD14- DC3 were observed 2 to 4 weeks post-treatment. Movements in populations of DC cells were observed in patients who achieved abscopal effects. Changes in DC prevalence and localization led to the development of a second trial (in progress), which compares the effectiveness of the combination of CDX-301/Flt3L, CD40 agonist antibody, and SBRT to standard therapy for treating metastatic NSCLC. Results from this trial underscore the potential role for radiation therapy as an in-situ tumor vaccine and immunomodulatory drug in the treatment of metastatic disease.

IL-4 affects dendritic cell signaling and remodels myeloid composition of tumors

3611 - Determining the regulation and role of mregDC in tumor immunity
Raphael Mattiuz, PhD (Icahn School of Medicine at Mount Sinai) reported new insights on the properties of dendritic cells (DCs) required to promote T cell anti-cancer activity. A new, conserved DC gene expression profile has been identified, called mature DC enriched in regulatory molecules (mregDCs). The mregDC gene expression profile includes maturation genes associated with T cell stimulation and migration to lymph nodes and genes associated with immunoregulation. MregDCs, are present in multiple cancer types (lung, liver, colon) and induced in DC1 and DC2 upon tumor antigen uptake, suggesting that mregDC identifies antigen-charged DCs that migrate to lymphoid structures to modulate tumor-specific T cell immune responses. IL-4 partially regulates the mregDC program. IL-4 blockade reduces growth of lung tumors and lung metastasis and increases levels of CD40 and IL-12, suggesting IL-4 blockade stimulates DC maturation. Deletion of IL-4 receptor A (IL-4RA) in dendritic cells does not affect tumor growth, but IL-4RA deletion in monocytes and monocyte progenitor cells does restrict tumor growth. IL-4RA deletion in DC cells does increase expression of IFN gamma receptor 1 (IFNGR1) on the DC cell surface, which is important for DC maturation. In mouse models of cancer, deletion of IFNGR1 in DCs decreases IL-12 tumor production in the tumor. Activated T cells are not present in tumors, but they are present in lymph nodes, suggesting IFN gamma controls IL-12 production and signaling in the tumor, and IL-12 activates T cells from lymph nodes to tumor sites. This study produces a model in which IFNGR signaling in mregDC cells promotes activation of cytotoxic T lymphocytes and promotes T helper cell differentiation to prime CD8+ T cells in the tumor. IL-4, produced by monocytes in the lymph nodes, has an inhibitory effect on mregDCs, IFN gamma signaling and IL-12 production. Spatial imaging has identified mregDCs in proximity to CD4+, CD8+ and PD-L1+ cells in lung tumors providing further support for this model. These data suggest that blocking IL-4R signaling reprograms myeloid compartmentalization in tumors, and clinical trials investigating the efficacy of combination IL-4R/PD-1 blockade for NSCLC is in development.

Identifying tumor- and T cell-intrinsic factors that determine cancer vaccine efficacy

Beyond tumor antigen immunogenicity: Determinants of protective immunity induced by therapeutic cancer vaccines
Lélia Delamarre, PhD (Genentech) presented new insights on the development of cancer vaccines. Over the last years novel machine learning models have improved neoantigen prediction, and data from large scale immunopeptidomic studies have contributed to the development of these more precise algorithms. In addition to the identification of putative neoantigens, Dr. Delamarre highlighted the importance of determining predicted neoantigens immunogenicity. To address this question,  in a recent study MC38 cell line and mouse models of colon adenocarcinoma were used to identify factors associated with effective neoantigen-specific T cell responses after vaccination. Two RNA-LPX vaccines (Deca-1 and Deca-2), each encoding 10 MC38 neoantigens, were administered to mice. Machine learning models predicted the vaccines to elicit immune responses of similar magnitudes. Both vaccines elicited T cell responses, and prophylactic vaccination with either vaccine prevented establishment of MC38 tumors. Vaccination with Deca-2 was protective in therapeutic settings, but vaccination with Deca-1 did not reverse growth of established MC38 tumors. Vaccine-induced CD8 T cells migrated to and infiltrated the tumor at comparable levels, but the phenotypes of vaccine-induced T cells were different between the two vaccines. Similar T cell receptor clones were observed between spleen and tumor for both Decatope 1 and Decatope 2. Single cell RNAseq analyses showed that vaccine-induced T cells from healthy vaccinated mice exhibited different gene expression profiles from vaccine-induced T cells from vaccinated tumor-bearing mice, suggesting that the phenotype of vaccine-induced T cells is differentially modulated by the tumor. Vaccine-induced T cells from Deca-1 vaccination exhibited a gene expression profile of reduced functionality at the tumor and in the spleen, while vaccine-induced T cells from Deca-2 exhibited a stronger cytotoxic/differentiated gene expression profile. Gene expression changed as vaccine-induced T cells from Deca-2 migrated between spleen and tumor, but these changes were not as prevalent in vaccine-induced T cells from Deca-1. While machine learning and large datasets have improved the process of generating immunogenic cancer vaccines, additional features could be incorporated to improve neoantigen prediction. Results from this study suggest that tumor-intrinsic factors such as tumor immunogenicity/neoantigen presentation and T cell-intrinsic factors also determine vaccine-induced T cell differentiation in the host.

Extended-half-life bispecific T cell engagers to treat hematological malignancies and solid tumors

BiTE / CD3 bispecific experience in solid tumors: Cinical updates and translational lessons learned
Dirk Nagorsen, MD, PhD (Amgen, Inc.) presented updates regarding the development and clinical trials of CD3 bispecific T cell engagers (BiTEs). Blinatumomab (CD19-targeting) was the first BiTE was approved by the FDA for treating relapsed/refractory acute lymphocytic leukemia, having shown to improve survival compared to chemotherapy. Half Life Extended (HLE) BitES have also been developed, and BCMA-targeting HLE BiTE produced a response in patients with multiple myeloma. Multiple Phase 1 clinical trials for BiTES targeting solid tumors have been initiated. Acapatamab/AMG 160 (PSMA HLE BiTE) targets prostate specific membrane antigen (PSMA), which is expressed at high levels on the cell surface of nearly all prostate cancers, including metastatic cancers.  PSMA expression in non-cancerous tissue expression is low, mostly cytoplasmic. Acapatamab was administered to patients with metastatic castration-resistant prostate cancer, by step dosing. Over 50% of patients have reductions in prostate specific antigen (PSA), and 34.3% exhibited PSA50 responses. 3 of 15 evaluable patients have RECIST responses, and 3 of 13 evaluable patients have CTC0 responses. Cytokine release syndrome (CRS) occurred in 39 of 43 patients (90.7%), with 11 (25.6%) experiencing grade 3 or higher. No treatment was discontinued due to CRS. A second HLE BiTE, Tarlatamab, targets DLL3, which is overexpressed on the surface of small cell lung cancer (SCLC) cells and other neuroendocrine tumors. DLL3 is minimally expressed or absent on non-cancerous tissue. 66 patients with relapsed/refractory SCLC participated in a phase I the dose-escalation trial, and patients had received a median of 2 prior lines of treatment. Partial responses were confirmed in 13/64 patients (20%), and 7/13 are still on treatment. The disease control rate is 47%. CRS was the most common adverse event, occurring in 29/66 (44%) patients; most cases were Grade 1, and one case 1/66 (2%) was grade 3. Early cytokine release was associated with development on CRS. All cases of CRS developed during treatment cycle 1, as CRS normally occurs early in BiTE therapy. Phase 1b trials testing tarlatamab for neuroendocrine prostate have been initiated, but no data are available. The efficacy proof-of-principle have been achieved for BiTES, and ongoing investigations of half-life-extended BiTES for treatment of hematological malignancies and solid tumors are promising. Additional efforts are being made to identify new BiTE targets and to mitigate CRS so treatment can be applied to additional malignancies.

Using the next generation of CARs and BiTEs to treat urothelial cancers

Clinical development of new targets for solid tumor immunotherapy, CAR T and BiTE
Tanya B. Dorff, MD (City of Hope) presented recent progress in the development of chimeric antigen receptor (CAR) T cells and bispecific T cell engagers (BiTEs) for treatment of urothelial cancers. Phase 1 trial of CAR-T cells targeting Prostate Stem Cell Antigen (PSCA) for treatment of metastatic castration-resistant prostate cancer (mCRPC) produced promising results. Clinically meaningful radiographic and PSA responses occurred in the majority of patients. Dose limiting toxicity was the development of cystitis. Early patient responses indicated that lymphodepletion with cycolphosphamide is necessary for CAR T efficacy, in agreement with previous preclinical results. In addition to destroying T cells, cycolphosphamide creates a more immunologically “warm” tumor microenvironment through increasing the numbers of dendritic cells and reducing the numbers of M2 macrophages in the tumor. Preconditioning with cycolphosphamide to improve the efficacy of CAR-T cells may apply to cellular therapy for all solid tumors. CAR-T cells targeting Prostate Specific Membrane Antigen (PSMA; POSEIDA P-PSMA-101) have also produced clinical and radiological responses. Tumors of responders exhibit T cell infiltration, and remission has been ongoing for seven to eight months. Some patients who exhibited an early PSA response later presented with progressive disease, highlighting the need for efforts to increase  the durability of treatment. Trials of BiTE XmAb antibody targeting six transmembrane epithelial antigen of the prostate (STEAP-1) and of CAR-T cells targeting Human Kallikrein 2 (KLK2) are ongoing. Toxicities associated with these treatments are usually on-target/off-tumor, when the CAR T or BiTE recognizes the target protein in non-cancerous tissues. Lastly, Dr. Dorff underscored that prostate cancers have a unique property of shifting from a hormonally driven tumor to a neuroendocrine tumor and that gene expression studies are ongoing to identify target antigens for CARs and BiTEs that could target both types of tumors. Creating dual targeting constructs is also an important focus for prostate cancer tumors and other solid tumors to improve the effectiveness of the treatment in the heterogeneous tumor environment.