Abstract Candidate adoptive T cell therapies (ACT), such as tumor infiltrating lymphocyte (TIL) treatments, have resulted in unprecedented and durable efficacy in clinical trials. Despite this, the manufacturing requires viable tumor resection, and TIL expansion conditions have the potential to promote T cell exhaustion. Moreover, a large proportion of patients do not respond to treatment, possibly due to exhaustion or to bystander T cells that are not tumor-specific present in the product candidates. In addition, we have shown that naturally occurring pro-tumor T cell responses to tumor-specific antigens we term Inhibigens™ are generated in nearly every subject with cancer; these T cells may be inadvertently expanded in the non-specific TIL manufacturing process. In animal models, Inhibigen-specific responses drive tumor hyperprogression. To avoid these pro-tumor T cells and improve upon ACT limitations, we are developing GEN-011, a neoantigen-targeted, peripheral T cell (NPT) therapy. GEN-011 is designed to contain primarily tumor-specific T cells with broad specificity and limited exhaustion, starting from easily accessible peripheral blood. Putatively beneficial neoantigen targets and deleterious pro-tumor Inhibigen targets are identified through measurement of cytokines in the assay supernatants of an in vitro ATLAS™ screen, in which each mutation identified in a patient's tumor is screened with the patient's own peripheral CD4+ and CD8+ T cells in a recall (overnight) assay, without algorithm prioritization. Next, the patient's peripheral T cells and monocyte-derived dendritic cells are incorporated into the PLANET™ manufacturing process where they are specifically stimulated with up to 30 ATLAS-verified neoantigens, avoiding Inhibigens, in a scalable, closed system. Development and engineering runs performed at scale show that the NPTs are up to 96% tumor-specific, with responses maintained for up to 89% of the intended neoantigen targets. They are non-exhausted effector and central memory T cells that express both proliferative and tissue homing markers. In addition to being highly polyfunctional, secreting multiple combinations of IFNγ, Granzyme B, TNFα, and MIP1α in response to specific neoantigens, they are also cytolytic in vitro and express memory-progenitor stem-like cell markers. The TITAN™ clinical trial evaluating GEN-011 NPTs is ongoing (NCT04596033). TiTAN is an open-label, multi-center Phase1/2a trial evaluating safety, tolerability, T cell persistence and proliferation, and clinical efficacy. The TiTAN clinical trial is testing two dosing regimens, a repeated lower dose regimen of GEN-011 without lymphodepletion and a single high dose administration of GEN-011 NPTs after lymphodepletion. Both groups will receive interleukin-2 after GEN-011 NPT dosing. By enriching healthy, broadly-specific neoantigen-targeted T cells and avoiding Inhibigens, the GEN-011 NPTs may represent an accessible and promising ACT for treating solid tumors. Citation Format: James Perry, Pranay D. Khare, Mercay Reuter, Daniel B. DeOliveira, Manish Jain, Colleen Winstead, Hubert Lam, Thomas Davis, Ray Stapleton, Jessica B. Flechtner. GEN-011: A neoantigen-targeted peripheral blood-derived T cell therapy that has broad neoantigen specificity and high T cell purity while avoiding pro-tumor T cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P005.

Background: Neoantigen (neoAg)-specific T cells can be found in the peripheral blood of patients with solid tumors, and the infiltration of fresh, peripherally derived T cells into tumors has been associated with successful outcomes after checkpoint blockade therapy. We previously described the development of our PLANET™ manufacturing process to create NPTs, based on the empirical identification of neoAgs using the ex vivo ATLAS™ bioassay, for adoptive transfer into patients with cancer. We have initiated the TiTAN™ clinical trial (NCT04596033) to evaluate this candidate immunotherapy, GEN-011, in subjects with checkpoint refractory solid tumors. Here we report the successful manufacturing of NPTs to support the ongoing trial. Methods: Apheresis, FFPE tumor and saliva samples were procured from subjects who consented to participate in the trial. Monocytes and T cells were isolated and cryopreserved. Patient-specific neoAgs against which their T cells were responsive were identified with ATLAS, and up to 30 neoAgs were prioritized for manufacture; pro-tumor Inhibigens™ were excluded. Cryopreserved peripheral blood monocytes and T cells were thawed, monocytes derived into dendritic cells, and then neoAg-specific T cells expanded in the fully closed PLANET manufacturing process. The NPTs were formulated and cryopreserved for administration to patients after release testing. Results: Subjects with five different solid tumor types contributed to these data: CSC, NSCLC, SCLC, SCCHN, and melanoma. A median of 2.1bn monocytes and 6bn T cells were cryopreserved from apheresis products (N=17). The median TMB was 1.8mut/Mb (range 0.01-36.6) with 473 somatic mutations (range 32-8893); the number of non-synonymous mutations ranged from 9 to 767. ATLAS screens identified a mean of 13±4 neoAgs and 11±3 Inhibigens, resulting in an average of 13 (range 2-30) unique neoAgs in each PLANET manufacturing process. Upon conclusion of manufacturing, the mean yield per patient was 1.6±0.3bn NPTs across runs (N=9) completed by the time of data cutoff. Characterization tests revealed the NPTs were consistently 80-90% CD8+ and 10-20% CD4+ T cells, of which 97% (range 64.9-99.8%) were effector memory and 1% (range 0.1-32.3%) central memory. The median purity at release was 98% with 82% viability. By functional assessments, the NPTs retained specificity for 91% (range 82-100%) of their intended neoAg targets (N=4). Administratable doses were successfully manufactured for 100% of patients to date. Conclusions: NPTs can routinely be manufactured in a GMP setting to treat patients with solid tumors. By expanding fresh, non-exhausted NeoAg-specific T cells with known tumor specificity from the periphery, GEN-011 has the potential to provide clinical benefits of TIL with greater accessibility and minimal irrelevant T cells. The TiTAN trial is ongoing. Citation Format: Harshal Zope, Rounak Nande, Manish Jain, Charley Hubbard, Louisa Dowal, James Foti, James Loizeaux, Crystal Cabral, Daniel B. DeOliveira, Guohan Yang, Mercay Reuter, Jessica Baker Flechtner, Raymond Stapleton. The PLANET manufacturing process reproducibly generates high-quality neoantigen-targeted peripheral T cells (NPTs) for adoptive T cell therapy in the TiTAN clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2745.

Adoptive T cell therapy using ex vivo expanded autologous tumor-infiltrating lymphocytes (TIL) has resulted in notable tumor regression across multiple solid tumor indications. Despite their success, TIL harvest requires invasive surgery and sterile isolation. In addition, extracted cells from an immunosuppressive tumor microenvironment (TME) often have impaired functions, low neoantigen-specificity and require high dose cytokine for expansion. All possible reasons for non-responders or post-treatment relapse. Generating T cell therapies specifically expanded against neoantigen targets and derived from peripheral blood lymphocytes may overcome these limitations. The ATLAS bioassay identifies patient-specific neoantigen targets of T cells, and Inhibigens™, tumor antigens that are detrimental to protective immune responses. Using PLANET™, a robust and scalable closed manufacturing process, T cells specific for up to 30 ATLAS-identified neoantigens (excluding Inhibigens) are expanded, providing considerable breadth of tumor recognition and the potential to limit tumor escape. Use of peripheral T cells leverages robust cells not suppressed by the TME and eliminates the need for invasive tumor resection. Our data show that NPTs are specific for up to 89% of targeted neoantigens, consist of non-exhausted effector and central memory cells, and express proliferative and tissue homing markers. NPTs are highly polyfunctional, secreting multiple combinations of IFNγ, Granzyme B, TNFα, and MIP1α in response to specific neoantigens. Evaluation of markers for memory-progenitor stem-like features in NPTs are being explored and will be discussed. The TITAN™ clinical trial evaluating GEN-011 NPTs is ongoing (NCT04596033).

Supplementary Table from An Empirical Antigen Selection Method Identifies Neoantigens That Either Elicit Broad Antitumor T-cell Responses or Drive Tumor Growth

GEN-009-101 is an ongoing phase I/IIa study of a personalized neoantigen vaccine containing targets selected by ATLAS™, a bioassay using ex vivo screening of autologous T cells to identify stimulatory peptides and “Inhibigens™”, peptides that suppress immunity and could accelerate tumor progression. Previous data showed 99% of selected peptides generated immune responses. After next-generation tumor sequencing and ATLAS testing, GEN-009 is formulated with up to 20 stimulatory peptides and poly-ICLC as adjuvant. Eligible patients with advanced cancer received standard-of-care (SOC) PD-1 CPI +/- chemotherapy during vaccine manufacturing and then received 5 vaccine doses over 6 months along with continuation of PD-1 CPI. Patients who progressed prior to vaccination were allowed to receive alternate therapy followed by GEN-009 in combination with an appropriate salvage regimen. The contributions from GEN-009 are to be assessed using each patient as their own control based upon change in tumor volume pre- versus post-vaccination. Between 4 and 99 neoantigens were identified in 18 patients with assorted histologies. To date 8 patients were vaccinated with GEN-009: 6 in conjunction with SOC PD-1 CPI and 2 with refractory disease received a salvage regimen. 24 vaccine doses have been given. AEs related to GEN-009 include grade 1 injection site reactions in 4 patients, and no DLTs. The ATLAS profile of stimulatory and inhibitory peptides evolved during PD-1 therapy to reflect a greater number of stimulatory targets and fewer inhibigens, suggesting a novel mechanism for CPI. Fluorospot results showed 69% of peptides generated a CD8 response, 85% a CD4 response and that 38% of the peptides did both. Up to 18 enrolled patients will provide insight into the treatment effects. Vaccination with GEN-009 in combination with PD-1 based therapy for patients with advanced cancer is feasible with little additive toxicity to date. Preliminary data suggests an expansion of stimulatory targets with epitope spreading in the presence of CPI. While early data are promising, the antitumor effect of GEN-009 will need to be evaluated with greater patient numbers.

Background: Tumor-specific neoantigens provide personalized targets for immunotherapy.Vaccines against epitopes predicted by in silico approaches very rarely induce CD4 þ and CD8 þ ex vivo T cell responses regardless of formulation.ATLAS selects neoantigens for vaccine inclusion using ex vivo screening of all patient-specific mutations to identify pre-existing CD4 þ or CD8 þ T cell responses and to exclude inhibitory peptides that suppress immunity and accelerate tumor progression.Preliminary data suggest that the inhibitory peptide profile may predict tumor response to immunotherapy.Methods: GEN-009-101 is a phase I/IIa study testing safety, immunogenicity and clinical activity in immune responsive tumors.After next-generation tumor sequencing and ATLAS testing of autologous leukocytes, up to 20 stimulatory synthetic long peptides adjuvanted with poly-ICLC make each personalized vaccine.The immunogenicity pilot has enrolled 9 patients in remission to receive GEN-009 monotherapy.Results: 4 patients have participated to the primary immunogenicity readout at day 50 (some data pending).The 17 doses given across patients have induced only mild local discomfort and no DLT.ATLAS results show high interpatient variability.Vaccination has generated immune responses against 94% of administered peptides, and both CD8 þ and CD4 þ responses in a 44 hr ex vivo fluorospot assay.Ten-day in vitro stimulation (IVS) assays result in broader immune responses.Conclusions: GEN-009 is a neoantigen vaccine that identifies tumor specific immune targets from the individual patient's repertoire.Immunogenicity data show that ATLAS can, with very high frequency, identify relevant neoantigens and exclude suppressive peptides.Clinical vaccination with PD-1 blockade is in process.Clinical trial identification: NCT03633110, issued 6-Aug-2018.

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Graft-versus-host disease (GVHD) is a major complication following hematopoietic cell transplantation. Innate immunity plays a major role in the development of GVHD both by aiding allogeneic T cell responses and by damaging target organs directly. MyD88 is an adaptor protein for the majority of toll-like receptors. The complement system is a central component of the innate immune response. We studied the role of these two pathways in the development of GVHD using the C57BL/6 into BALB/c model. Lethally irradiated recipients of MyD88-/- T cells survived slightly longer than recipients of wild type cells (median survival time: 48 vs. 32 days, P<0.05). Similarly, treatment with an anti-C5 antibody (clone BB5.1, 1 mg/dose, i.p., three times a week for 4 weeks), that blocks all three pathways of complement activation and prevents cell lysis, also moderately prolonged the survival of the mice with GVHD compared with the isotype control group (median survival time: 43 vs. 32 days). Treatment of MyD88-/- T cell recipients with anti-C5 antibody protected 80% of the animals from lethal GVHD at 100 days post transplantation, while only 20% in the MyD88-/- T cell recipients treated with isotype antibody group and 30% in the wildtype T cell recipients treated with anti-C5 antibody group survived (Table, P<0.01, compared with other groups). All MyD88-/- T cell recipients treated with anti-C5 antibody developed GVHD. However, the disease was less severe in these animals than those in the recipients of a single pathway blockade as measured by body weight and other clinical signs of GVHD. The anti-C5 antibody did not seem to prevent GVHD through inhibiting T cell activation and expansion because it did not inhibit mixed lymphocyte reactions. Levels of multiple pro-inflammatory cytokines were lower in the MyD88-/- T cell recipients treated with anti-C5 antibody compared with those in the control groups. These data clearly demonstrate that, even though blockade of MyD88 or complement pathway alone may prevent GVHD, GVHD can be better controlled by blocking both MyD88 and complement pathways.Tabled 1Blockade of two independent innate immunity pathways synergistically prevent lethal graft-versus-host diseaseGroupsnSurvival at day +50Survival at day +100TCD BM alone10100%100%Wildtype + isotype1020%10%Wildtype + anti-C51040%30%MyD88-/- + isotype1050%20%MyD88-/- + anti-C51090%80%P<0.01, MyD88-/- + anti-C5 vs. other groups. A representative experiment of three is shown. Open table in a new tab P<0.01, MyD88-/- + anti-C5 vs. other groups. A representative experiment of three is shown.

Human melanocortin 4 receptor (hMC4R) mutations with in vitro functional effects are responsible for 0.5-2.5% of severe obesity. Designing ligands that are able to counteract this in vitro-associated molecular defect is crucial to develop specific anti-obesity drugs in these genetically associated cases. We analyzed the in vitro effect of two novel melanocortin agonists, IRC-022493 and IRC-022511, on typical hMC4R mutations chosen based on the nature of their functional alterations, i.e. intracytoplasmic retention and/or reduced basal activity and/or reduced α-MSH potency. We assessed the in vitro ability of IRC-022493 and IRC-022511 to bind and activate hMC4R mutants. These mutations were found earlier in 11 obese French patients (median age (range) was 17.6 years (5.7-48.0) and body mass index (BMI)-Z-score 4.2 s.d. (1.5-5.5). The MC4R agonists were responsible for a significant activation of mutated hMC4R depending on the functional characteristics of the mutations. Both agonists were able to activate mutated hMC4R with decreased α-MSH potency, associated with or without decreased basal activity, to the same extent than α-MSH in wild-type MC4R. This result suggests that those mutations would be the best targets for the MC4R agonists among MC4R mutation-bearing obese patients. No specific clinical phenotype was associated with the differential response to pharmacological agonists. We identified two novel melanocortin agonists that were able in vitro to efficiently activate mutated hMC4R with impaired endogenous agonist functional response. These results stimulate interest in the development of these drugs for hMC4R mutations-associated obesity.

Rapamycin (RAPA) is an immunosuppressive drug that prevents and treats graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplant (HCT). One possible mechanism for its efficacy is induction of tolerance, through increased number or enhanced survival of regulatory T cells. In our experiments, B10.D2 BM and splenocytes were injected into lethally irradiated BALB/cJ recipients. The mice received i.p. injections of either RAPA or vehicle control on days 1–28. There was a significant survival advantage in RAPA-treated mice. Evaluation of the skin biopsies showed a dense cellular infiltrate in RAPA-treated mice. Further characterization of these cells revealed a higher percentage of regulatory T cells characterized by FoxP3-positive cells in high-dose RAPA-treated mice as compared with controls on day 30. This effect appears to be dose dependent. When peripheral blood analysis for FoxP3-positive cells was performed, there was no significant difference observed in the RAPA-treated mice as compared with control mice. These data show a novel mechanism of rapamycin in GVHD, accumulation of regulatory T cells in the GVHD target tissue: the skin.

Several groups have independently demonstrated that memory T cells do not induce graft-versus-host disease in several different animal models. To test whether the same concept applies to humans, we compared the ability of memory T cells to respond to alloantigens with that of naive T cells. Purified T cells were first obtained from peripheral blood from healthy donors and then separated into memory and naive T cell subsets based on the expression of CD45RA (memory: CD45RA-, naive: CD45RA+). Memory T cells were then tested for their ability to respond to alloantigens by using several standard in vitro assays. In contrast to the mouse data, memory T cells proliferated equally well as naive T cells did in mixed lymphocyte culture. Despite the similar proliferative responses against alloantigens as those mediated by naive T cells, these same memory T cells failed to kill the allogeneic targets. Limiting dilution assay demonstrated that the frequency of allospecific cytotoxicity T cells was 6–68.5 fold less in memory T cells than that in naive T cells. Interleukin 2 was unable to restore the cytotoxicity against alloantigens. The results from skin explant assay further suggested that, in contrast to naive T cells, memory T cells might not be able to induce graft-versus-host disease. These data suggest that human memory T cells contain less alloantigen-specific cytotoxicity T cells and may not cause graft-versus-host disease upon in vivo transfer. Since clinical grade antibody is now available, this approach will soon be tested in humans for the prevention of graft-versus-host disease.

In previous work, we fractionated CD34+ umbilical cord blood (UCB) progenitors to purify cells that express high levels of aldehyde dehydrogenase (ALDH). The ALDHbr CD34+ cells were enriched with progenitors that engraft NOD/SCID mice in both short-term (6–8 week) and long-term (>18 week) transplants. In contrast, ALDHneg CD34+ progenitors were not a significant source of NOD/SCID repopulating cells. These data strongly imply that transplantable human progenitors express ALDH; however, one shortcoming for that work remains that human hematopoietic development in NOD/SCID mice is limited to the myeloid and B-lymphoid lineages. Because the pace of T cell engraftment is a critical clinical concern, we have adopted the use of the NOD/SCID-IL2Rγnull mouse xenograft transplant model. In the current study, 20 mice were transplanted with lineage-depleted ALDHbr CD34+ progenitors at doses that ranged from 3,000 to 30,000 cells. In control studies, similar doses of ALDHneg CD34+ cells did not provide long-term engraftment. So that we might characterize the behavior of individual UCB, each transplant graft was derived from a single UCB. In addition, each UCB was assayed in multiple different mice. Twelve of 13 mice that survived until 21 weeks post-transplant demonstrated multiple-lineage human hematopoietic engraftment. Human T, B, NK and myeloid cells were detected in the bone marrow and spleen. In addition, in 9 mice human T cells were detected in the thymus. Furthermore, 7 mice had sufficient thymus tissue to measure T cell Receptor Excision Circles (TREC). By that assay, 3 of those mice demonstrated active human T cell rearrangements. As anticipated, the level of engraftment to all tissues was cell dose-dependent. One strength for this xenograft model was that engraftment could be monitored over time within the peripheral blood. Human B cells engrafted within 7 weeks post-transplant and were detected in 17 of 19 mice (≥ 5 B cells/μL). At 16 weeks post-transplant, 15 of 18 mice maintained detectable B cells. In contrast, human T cells emerged later, beginning at 13 weeks post-transplant. However, even by 16 weeks post-transplant, T cells were only detectable in the peripheral blood of 6 of 18 mice (≥ 5 T cells/μL). These studies provided evidence that ALDHbr CD34+ cells establish both short- and long-term hematopoiesis in NOD/SCID-IL2Rγnull mice. The emergence of T cells was the most stringent test for long-term engraftment.

Several different groups have independently demonstrated that non-allospecific memory T cells do not induce GVHD. The data related to the ability of allospecific memory T cells to induce GVHD have not been conclusive. In order to study this question more definitely, we have developed a novel GVHD model using TEa TCR transgenic mice as donors. TEa CD4+ TCR transgenic mice (C57BL/6 background) express a TCR that recognizes the peptide ASFEAQGLANIAVDKA in the context of I-Ab. This peptide corresponds to positions 52–68 from the alpha-chain of I-E class II molecules and is expressed in all APCs from H-2b/I-E+ strains such as CB6F1 mice. Titration experiment demonstrated that as few as 1 × 105 TEa cells were able to induce lethal GVHD in lethally irradiated CB6F1 recipients, making it an ideal model to study the ability of allospecific memory T cells to induce GVHD. Because sufficient numbers of allospecific memory phenotype T cells could not be obtained from the TEa mice or C57BL/6 CD45.1 mice containing TEa cells after priming, we chose the Rag1-/- model. TEa cells were first parked in Rag1-/- mice and then were immunized with irradiated CB6F1 cells. Eight weeks later, TEa cells were harvested from the spleens and effector memory T cells were obtained after depletion of CD62L+ cells. Many TEa cells that were parked in the Rag1-/- mice but were not immunized with alloantigens also obtained the effector memory T cell phenotype. These cells were termed as “unprimed TEM” while those from primed animals were termed as “primed TEM”. Both primed and unprimed effector memory TEa cells were able to respond to alloantigens in vitro. However, neither primed nor unprimed effector memory TEa cells was able to induce lethal GVHD in vivo and all animals in the effector memory T cell groups survived more than 100 days post transplantation. In contrast, all naive T cell recipients developed lethal GVHD and died within 35 days after transplantation. These data demonstrate that, similar to non-allospecific memory T cells, allospecific effector memory T cells also have decreased ability to induce GVHD when compared with naive T cells. This is an excellent model for us to study the unique immune response mediated by allospecific memory T cells in GVHD.

Hematopoietic and immune systems are sensitive to ionizing irradiation and the recovery is slow. Very few effective therapeutic agents are currently available for this condition. In this study, we investigated the effects of growth hormone on hematopoietic and immune recovery post irradiation and its ability to protect against lethal irradiation. The studies were performed using BALB/c mice. Recombinant human growth hormone (rhGH) was given at the dose of 20 μg/dose, i.p. or i.v., once a day, starting within one hour after irradiation. The hematologic and immune recovery was monitored weekly post irradiation. In a sublethally irradiated (5 Gy) model, treatment with rhGH for 30 days significantly accelerated the recovery of platelets in peripheral blood (59087±13860 vs. 42825±7425 on day +8, P=0.01). Similar trends were also observed in total white blood cells and all lymphocyte subsets tested (T, B, NK cells). Similar results were obtained when higher dose of radiation dose (7.5 Gy) was used. These data suggest that growth hormone can promote hematopoietic and immune recovery after irradiation. We next tested whether growth hormone can rescue animals from lethal irradiation. BALB/c mice were irradiated with 7.5 Gy and treated with rhGH for 35 days. As demonstrated in the table, 13 out of 20 mice in the growth hormone treated group survived more than 60 days after irradiation, whereas only 3 out of 20 mice survived more than 25 days in the saline control group. The radioprotective effect was still observed when higher dose of radiation (8.5 Gy) was used. These findings demonstrate that growth hormone has significant radioprotective effects even when given after total body irradiation.Tabled 1Growth hormone protects against lethal irradiationGroupsnSurvivalday +25day +60Saline control203/203/20Growth hormone2016/20⁎P<0.0113/20⁎P<0.01 P<0.01 Open table in a new tab