Gp41 Peptide Research Articles

Association between clinical and disease characteristics and detectable or undetectable baseline ctDNA in patients with metastatic uveal melanoma.

9537 Background: Tebentafusp, a bispecific soluble TCR specific for a gp100 peptide, is licensed for the treatment of metastatic uveal melanoma (mUM) in HLA-A*02:01+ individuals. 82% of patients (pts) with mUM in the IMCgp100-102 (previously treated; 2L+) and 61% in IMCgp100-202 (frontline; 1L) studies, respectively, had detectable baseline ctDNA. There is a strong relationship between decrease in ctDNA by 9 weeks and overall survival (OS) [Carvajal 2022; Hassel & Piperno-Neumann 2023]. Patients with undetectable baseline ctDNA had improved survival despite having macroscopic disease. In this study, we report the characteristics of baseline ctDNA undetectable vs detectable disease. Methods: Disease characteristics of 202 1L (Study 202) and 117 2L+ (Study 102) pts who had baseline undetectable or detectable ctDNA (unDNA vs detDNA) were compared in a post-hoc unplanned analysis. ctDNA detection was analysed at baseline and up to week 9 on tebentafusp using targeted mPCR-NGS assay for mutations in 15 genes including GNAQ, GNA11, SF3B1, CYSLTR2, PLCB4 and EIF1AX. Largest lesion size groups (≤ 3 cm, 3-8 cm, > 8 cm) were defined by American Joint Committee on Cancer 7th Ed. Presence of oncogenic mutations in GNAQ, GNA11, SF3B1 and BAP1 were assessed by WES on metastatic tissue biopsies. Results: Baseline ctDNA levels increased in association with size of largest lesion regardless of number of prior therapies. However, there was an association between disease burden (defined by largest lesion size) and unDNA vs detDNA in both studies, with all pts with largest liver lesion > 8 cm having detDNA. In Study 202, % of the population with unDNA vs detDNA was 50% vs 50% for ≤ 3cm and 27% vs 73% for 3.1-8 cm. While OS was similar for unDNA & detDNA in pts with ≤ 3cm lesions, OS was longer for pts with unDNA vs detDNA who had baseline tumor size 3.1-8 cm in both studies (Table). Within this 3.1-8 cm group, pts with unDNA tended to have lower LDH, AST, and ALP. Initial analysis showed no difference in the frequency of GNAQ, GNA11, SF3B1 or BAP1 mutations between unDNA and detDNA groups. Further data on somatic mutation defined groups and RNAseq gene expression in unDNA and detDNA groups will be presented. Conclusions: Patients with unDNA were more likely to have smaller disease burden than those with detDNA; however, many pts with unDNA had a significant burden of metastatic disease. Baseline unDNA was associated with improved OS outcome vs detDNA in pts with 3.1-8 cm baseline metastatic deposits, but not in pts with ≤ 3cm disease. No association was seen between oncogenic background of GNAQ/GNA11/SF3B1/BAP1 and unDNA vs detDNA. Clinical trial information: NCT02570308 ; NCT03070392 . [Table: see text]

Use of baseline and serial ctDNA dynamics to predict outcomes in patients treated with first-line tebentafusp, including those who were and were not treated beyond progression.

9536 Background: Tebentafusp (tebe), a bispecific soluble T cell receptor specific for a gp100 peptide, is licensed for the treatment of metastatic uveal melanoma (mUM) in HLA-A*02:01+ individuals. As part of the pivotal, Phase 3 IMCgp100-202 study, 378 mUM pts were randomized to receive tebe or investigators choice. Approximately 60% of patients (pts) with metastatic UM in the IMCgp100-202 study had detectable baseline ctDNA. We have reported the strong relationship between change in ctDNA at 9 weeks (wks) and overall survival (OS). Additionally, radiographic treatment response is a poor predictor of who is benefitting from therapy. We hypothesized that ctDNA dynamics would help identify those patients (pts) who benefit from treatment beyond progression (TBP). Methods: Sera (n=202 pts) collected at baseline and wk 9 on tebe were analyzed for ctDNA using a targeted mPCR-NGS assay for 15 genes including GNAQ, GNA11, SF3B1, PLBC4, EIF1AX, and CYSLTR2. RECISTv1.1 was assessed by investigators; TBP was allowed per protocol. OS was analyzed in subsets of pts who were not TBP, and those who were TBP and treated with tebe for < or > 16 wks; the approximate time point where TBP pts would have had confirmatory imaging, although confirmatory scan data is not available. At baseline, pts were determined to be ctDNA detectable or undetectable. At wk 9, pts were determined to have cleared ctDNA, < or ≥ 68% (0.5 log-fold) reduction, < or ≥ 50% reduction. Data cut-off was July 2023. Results: Of 202 pts randomized to tebe and with ctDNA at baseline and wk 9, 126 (62%) received TBP (80 <16 wks, 46 ≥ 16 wks). Undetectable ctDNA at baseline was associated with improved OS regardless of TBP, while TBP was associated with improved OS in those with baseline detectable ctDNA (hazard ratio, HR, 0.59, 95% confidence interval, CI, 0.4-0.88). Subgroup analysis of those with baseline detectable ctDNA demonstrated improved OS in only those who received TBP for ≥ 16 wks (HR 0.33; 95% CI 0.21-0.51). Clearance of ctDNA at wk 9 was associated with better OS than in those who did not have clearance. TBP was associated with better OS in pts who did not have clearance (HR 0.48, 95% CI 0.30-0.79), but not in those with clearance (HR 0.87, 95% CI 0.43-1.78). Finally, TBP was associated with improved outcomes in pts with ≥50% and ≥68% reduction, compared to those without reduction, HR 0.63 (95% CI 0.4-0.97) and 0.62 (95% CI 0.39-0.99), respectively. Conclusions: Baseline and wk 9 ctDNA detection predicts OS in mUM pts treated with first line tebe. The utility of ctDNA predicting pts most likely to benefit from TBP is less clear, although TBP seems to be more beneficial in those with reduction of ctDNA at wk 9. Optimization of time points, assay, and integration of ctDNA data with radiomics are necessary to understand the true utility of ctDNA in helping select which pts should receive TBP. Clinical trial information: NCT03070392 .

Proteomimetic Polymers Trigger Potent Antigen-Specific T Cell Responses to Limit Tumor Growth.

Elicitation of effective antitumor immunity following cancer vaccination requires the selective activation of distinct effector cell populations and pathways. Here we report a therapeutic approach for generating potent T cell responses using a modular vaccination platform technology capable of inducing directed immune activation, termed the Protein-like Polymer (PLP). PLPs demonstrate increased proteolytic resistance, high uptake by antigen-presenting cells (APCs), and enhanced payload-specific T cell responses. Key design parameters, namely payload linkage chemistry, degree of polymerization, and side chain composition, were varied to optimize vaccine formulations. Linking antigens to the polymer backbone using an intracellularly cleaved disulfide bond copolymerized with a diluent amount of oligo(ethylene glycol) (OEG) resulted in the highest payload-specific potentiation of antigen immunogenicity, enhancing dendritic cell (DC) activation and antigen-specific T cell responses. Vaccination with PLPs carrying either gp100, E7, or adpgk peptides significantly increased the survival of mice inoculated with B16F10, TC-1, or MC38 tumors, respectively, without the need for adjuvants. B16F10-bearing mice immunized with gp100-carrying PLPs showed increased antitumor CD8+ T cell immunity, suppressed tumor growth, and treatment synergy when paired with two distinct stimulator of interferon gene (STING) agonists. In a human papillomavirus-associated TC-1 model, combination therapy with PLP and 2'3'-cGAMP resulted in 40% of mice completely eliminating implanted tumors while also displaying curative protection from rechallenge, consistent with conferment of lasting immunological memory. Finally, PLPs can be stored long-term in a lyophilized state and are highly tunable, underscoring the unique properties of the platform for use as generalizable cancer vaccines.

Abstract 6745: Crafting hyaluronic acid-based nanoparticles for enhanced LN targeting as a potent priming tool in immunotherapy

Abstract Background: Cholesterol-conjugated hyaluronic acid, synthesized by grafting cholesterol moieties onto hyaluronic acid, spontaneously formed nano-sized hydrogel of approximately 30-100nm in water. This hyaluronic acid derivative (HA nanogel) has demonstrated the ability to encapsulate various modalities, including low- and medium-sized molecules, peptides, and proteins. Previous reports have highlighted its efficacy in solubilizing poorly water-soluble drugs and serving as a sustained-release matrix. In this study, we explore the potential of HA nanogels encapsulating antigen peptides with adjuvant as a cancer vaccine and a priming tool for combinational immunotherapy. Method: We prepared HA nanogels as a novel peptide carrier for cancer vaccines and attempted to encapsulate various peptides, including neoantigen (mERK2) peptides expressed in immune checkpoint inhibitor (ICI) therapy-resistant fibrosarcoma cell line CMS5a, gp-100 peptides expressed in metastatic melanoma cell line B16F10, and MAGEA4. Our experiments covered APC uptake, antigen-specific CTL induction, and anti-tumor studies to demonstrate the utility of HA nanogels in these applications. Results: HA nanogel efficiently formed stable complexes with mERK2, gp-100 and MAGEA4 long peptides, each with diameters of 30-100nm. The facile and simple vaccine formulation processes with HA nanogel achieved high drug content and high encapsulation efficiency of 90% or higher. Fluorescently labeled mERK2 long peptide encapsulated in HA nanogel vaccines were subcutaneously administered to BALB/c mice, and uptake analysis of mERK2 long peptides in draining lymph nodes (dLN) was performed using flow cytometry. In comparison to the group receiving mERK2 long peptide without nanogel encapsulation, the HA nanogel group exhibited higher uptake by macrophages and dendritic cells in the dLN. Administering HA nanogel vaccines containing mERK2 long peptides with CpG oligoDNA resulted in higher antigen-specific CD8+ T cells compared to the group receiving mERK2 long peptides without encapsulation. Strong anti-tumor effects were demonstrated in mice models with subcutaneously transplanted CMS5a and B16F10 cell lines. The enhanced efficacy through co-administration with ICI was also confirmed. Conclusion: HA nanogel vaccines efficiently deliver antigen peptides to macrophages and dendritic cells, inducing antigen-specific CTL responses, thereby demonstrating anti-tumor effects against cold tumors such as CMS5a and B16F10. HA nanogels have the potential not only as carriers for cancer vaccines but also as priming tools for combinational immunotherapy. Citation Format: Takashi Nakai, Fumiyasu Momose, Kohei Yabuuchi, Makiko Yamane, Tae Hayashi, Linan Wang, Yoshiyuki Nakagawa, Shogo Aso, Toru Katsumata, Tsuyoshi Shimoboji, Yoshihiro Miyahara. Crafting hyaluronic acid-based nanoparticles for enhanced LN targeting as a potent priming tool in immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6745.

IMMU-52. LOW-INTENSITY PULSED ULTRASOUND CONDUCTS ANTIGEN-INDEPENDENT T CELL HOMING INTO THE BRAIN PARENCHYMA WITH THE INTACT BLOOD-BRAIN BARRIER

Abstract The blood-brain barrier (BBB) protects the brain parenchyma from excessive inflammation but also leads to poor immune cell homing, hampering the success of immunotherapies, such as ones for low-grade glioma, where the BBB is intact. Low-intensity pulsed ultrasound (LIPU) combined with the application of microbubbles allows the non-invasive, transient opening of the BBB and recent studies have observed immune cell infiltration into malignant gliomas following LIPU. While these data warrant mechanistic investigations on the effects of LIPU in preclinical settings, most mouse models of malignant glioma already display BBB disruption. Therefore, to study whether LIPU affects antigen-specific immune cell trafficking through the BBB, we created a novel immunocompetent mouse model of non-tumor bearing mice that display an intact BBB and express a minigene cassette encoding for four immunogenic peptides in glia cells under the glial fibrillary acidic protein (GFAP) promotor. These peptides are either presented by major histocompatibility complex (MHC) class I: human melanoma antigen glycoprotein (gp) 100 and lymphocytic choriomeningitis virus (LCMV)gp31) or class II: LCMV gp66 and tyrosinase-related protein 1. Minigene expression in astrocytes induced antigen-specific T cell proliferation and Interferon-γ release in vitro. Intravenous adoptive T cell transfer of naïve or respectively CD3/CD28-coactivated antigen-specific CD 8 T cells targeting gp100 peptide followed by immediate LIPU treatment induced T cell homing into the CNS parenchyma, and astrocyte-T cell interaction was confirmed by ex-vivo two-photon imaging. Furthermore, some animals that underwent immediate LIPU treatment after transfer of preactivated antigen-specific T cells developed neurological deficits such as ataxia, while no deficits were observed in those animals that underwent LIPU but did not receive T cells or received activated T cells but did not undergo LIFU treatment. In conclusion, LIPU might be a novel effective tool to support antigen-independent immune cell homing with the intact BBB.

HLA discordance between blood and tumor: A treatment conundrum?

e21577 Background: Tebentafusp is a bispecific gp100 peptide-HLA-directed CD3 T cell engager that is now FDA approved for HLA-A*02:01 patients with unresectable or metastatic uveal melanoma (UM). Tebentafusp requires the targeted gp100 peptide to be presented by the major histocompatibility complex Class I complex HLA-A*02:01 allele for optimal cell killing. HLA-A*02:01 has been reported to be carried by approximately 50% of the North American and Western European population. The clinical trials for tebentafusp all required central blood tests to determine HLA status. In March of 2021, Caris Life Sciences began the launch of HLA Genotype as a standard reporting element for patient tumors profiled by Caris. Somatic HLA mutations have been described in different tumor types and has strong implications for a possible mechanism of immune evasion. Tumor heterogeneity between tumors and within tumors may also impact HLA tumor testing. As more novel drugs that rely on HLA genotyping are developed, understanding the relationship between blood and tumor HLA testing has become crucial. Methods: A single institution retrospective analysis was performed of patients who had tumor HLA genotyping performed as part of their tumor profiling by Caris Life Sciences. HLA Genotyping by Caris was performed using Whole Exome Sequencing of the tumor. Of the patients who had tumor HLA genotyping results, patients who also had blood HLA typing were identified. The concordance rate of HLA-A status between blood testing and tumor testing was determined. The prevalence of HLA-A*02:01 in our patients was also determined (either blood or tumor). Results: Forty-seven patients with both tumor and blood HLA genotyping were identified (37 UM, 8 cutaneous melanoma, 1 mucosal melanoma, 1 renal cell carcinoma). The majority of the patients were Caucasian (n = 44), with only two Asian patients and one African patient. There were two UM patients whose tumor and blood HLA genotyping were discordant (concordance rate of 95.7%). One patient was found to be HLA-A*01:01, *026:01 by blood but tumor genotyping revealed HLA- A*01:01, A*02:01. The other patient was found to be HLA-A*02:01, A*01 by blood but tumor genotyping revealed HLA-A*03:01, A*33:03. The prevalence of HLA-A*02:01 in the entire group was 34%, while the prevalence of HLA-A*02:01 in the Caucasian patients was 36.4%. None of the non-Caucasian patients were HLA-A*02:01 by blood or tumor genotyping. Conclusions: Among our 47 patients with both tumor and blood HLA genotyping, we found two patients with discordant HLA results. Neither of the discordant UM patients had received tebentafusp yet, but it brings up a crucial question about how these patients may respond to a therapy that relies on the HLA-A*02:01 complex. Our study highlights the importance of continuing to use blood HLA genotyping as the standard test to determine eligibility for a treatment such as tebentafusp, as the concordance rate with tumor genotyping is high but not 100%.

Liposomal celecoxib combined with dendritic cell therapy enhances antitumor efficacy in melanoma.

Cancer vaccine efficacy is limited by the immunosuppressive nature of the tumor microenvironment created by inflammation, immune inhibitory factors, and regulatory T cells (Tregs). Inspired by the role of cyclooxygenase-2 (COX-2) in inflammation in the tumor site, we proposed that normalization of the tumor microenvironment by celecoxib as a COX-2 inhibitor might improve the efficacy of Dendritic Cell (DC) therapy in a melanoma model. In the present study, liposomal celecoxib (Lip-CLX) was combined with ex vivo generated DC vaccines pulsed with gp100 peptide (in liposomal and non-liposomal forms) for prophylactic and therapeutic evaluation in the B16F10 melanoma model. Tumor site analysis by flow cytometry demonstrated that intravenous administration of Lip-CLX at a dose of 1 mg/kg in four doses effectively normalized the tumor microenvironment by reducing Tregs and IL-10 production. Furthermore, in combination with DC vaccination (DC + Lip-peptide+Lip-CLX), it significantly increased tumor-infiltrating CD4+ and CD8+ T cells and secretion of IFN-γ. This combinatorial strategy produced an effective prophylactic and therapeutic antitumor response, which reduced tumor growth and prolonged the overall survival. In conclusion, our findings suggest that the liposomal celecoxib targets the inhibitory mechanisms of the tumor microenvironment and broadens the impact of DC therapy to improve the outcome of immunotherapy in solid tumors.

Chimeric Human Papillomavirus-16 Virus-like Particles Presenting P18I10 and T20 Peptides from HIV-1 Envelope Induce HPV16 and HIV-1-Specific Humoral and T Cell-Mediated Immunity in BALB/c Mice.

In this study, the HIV-1 P18I10 CTL peptide derived from the V3 loop of HIV-1 gp120 and the T20 anti-fusion peptide of HIV-1 gp41 were inserted into the HPV16 L1 capsid protein to construct chimeric HPV:HIV (L1:P18I10 and L1:T20) VLPs by using the mammalian cell expression system. The HPV:HIV VLPs were purified by chromatography. We demonstrated that the insertion of P18I10 or T20 peptides into the DE loop of HPV16 L1 capsid proteins did not affect in vitro stability, self-assembly and morphology of chimeric HPV:HIV VLPs. Importantly, it did not interfere either with the HIV-1 antibody reactivity targeting sequential and conformational P18I10 and T20 peptides presented on chimeric HPV:HIV VLPs or with the induction of HPV16 L1-specific antibodies in vivo. We observed that chimeric L1:P18I10/L1:T20 VLPs vaccines could induce HPV16- but weak HIV-1-specific antibody responses and elicited HPV16- and HIV-1-specific T-cell responses in BALB/c mice. Moreover, could be a potential booster to increase HIV-specific cellular responses in the heterologous immunization after priming with rBCG.HIVA vaccine. This research work would contribute a step towards the development of the novel chimeric HPV:HIV VLP-based vaccine platform for controlling HPV16 and HIV-1 infection, which is urgently needed in developing and industrialized countries.

Highly mutated monoclonal antibody 3F2 targets a conformational and strain-restricted epitope in human immunodeficiency virus gp41 with significant antibody-dependent cell cytotoxicity.

Identifying epitope targets by studying the native antibody (Ab) response can identify potential novel vaccine constructs. Studies suggest that long-term non-progressor (LTNP) subjects have inherent immune mechanisms that help to control viremia and disease progression. To explore a role for antibodies (Abs) in LTNP progression, our lab has previously characterized a number of highly mutated Abs that target conformational epitopes of the human immunodeficiency virus (HIV) envelope protein from a single LTNP subject (10076). One Ab clone, 10076-Q3-2C6, had significant cross-clade Ab-dependent cell cytotoxicity. To assess if other LTNP subjects produced similar Abs, we expressed another highly mutated Ab from another subject; subject 10002, clone 10002-Q1-3F2 (variable heavy chain, 63.2% amino acid sequence identity to predicted germline). After expression with its native light chain, the recombinant Ab 3F2 bound to the trimeric envelope protein of HIV (trimer), as well as to the ectodomain of gp41. 3F2 binding to gp41 peptide libraries was consistent with non-linear epitope binding and showed possible overlap with the epitope of 2C6. Ab competition assays suggested that 3F2 may bind near the immunodominant epitope 1 loop region (ID1) of gp41. 2C6 blocked the binding of ID1-loop-binding Abs and 3F2 to the trimer, but 3F2 failed to block 2C6 binding. Together, these results suggest that 3F2 binds to a non-linear conformational epitope primarily localized between the epitope of 2C6 and the ID1. Since they are targeted by functional Abs, a more complete understanding of these ID1 and near-ID1 epitopes may be exploited in future immunization strategies.

P345: AN IMMUNE SCREEN IDENTIFIES 5-NONLOXYTRYPTAMINE AS A NOVEL ANTI-CANCER AGENT CAPABLE OF IMPROVING ANTI-TUMOR IMMUNITY IN COMBINATION WITH ANTI-PD1 THERAPY

Background: Exploiting the immune system, particularly CD8+ T cells to eliminate tumors has revolutionized the treatment landscape. However, despite the evident successes of CD8+ T cell-targeting immunotherapies such as anti-PD1 checkpoint inhibitors, many patients fail to respond especially those with poorly immunogenic tumors. Additional obstacles to successful immunotherapy responses include therapy-induced toxicity, lack of bio-markers of response, an immunosuppressive environment leading to T cell dysfunction and exhaustion and poor immune infiltration highlighting a need for novel combinatorial approaches to augment immunotherapeutic activity. Aims: We aimed to uncover novel therapeutic agents capable of augmenting the anti-tumoral responses of CD8+ T cells in ex vivo systems and in vivo syngenetic poorly immunogenic tumor models. Methods: To uncover novel potentiators of T cell anti-tumor immunity, we carried out an ex vivo pharmacological screen, using Lymphocytic choriomeningitis virus (LCMV)-primed splenic T cells that were combined tumor cells expressing the LCMV gp33 (B16) peptide CTL epitope and incubated with an NIH 770 compound library. Hits that increased tumor-cell killing when combined with LCMV-primed splenic T cells were further validated ex vivo. The key candidate was assessed in vivo in C57BL/6J syngeneic tumor models. Immunocompromised NSG mice as well as antibody depletion were used validate CD8+ T cell dependence. In vitro and in vivo immunomodulatory effects were deciphered using flow cytometry, immunoblot, CRISPR/Cas9 and histological analyses. Results: We identified 5-Nonyloxytryptamine (5-NL), a serotonin receptor (HTR) agonist, as increasing the ability of T cells to target tumor cells. In vitro, 5-NL induced apoptosis in melanoma and childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) at low micromolar levels. 5-NL delayed tumor growth in vivo and the phenotype was dependent on the hosts’ immune system, specifically CD8+ T cells as their depletion abrogated the phenotype (Figure 1A-B). 5-NL’s pro-immune effects were attributed to the upregulation of antigen presenting machinery in melanoma, BCP-ALL and other poorly immunogenic tumors with low MHC-I/HLAA-C expression (Figure 1C). Importantly, the upregulation of MHC-I/HLAA-C occurred without concomitant increases in PD-L1 expression and was linked to upregulation of cAMP Response Element-Binding Protein (CREB) in vitro and in vivo. As demonstrated through CRISPR/Cas9 HTR1D knockout, the immunomodulatory and pro-apoptotic effects of 5-NL occurred independently of receptor target signaling. 5-NL was successfully combined with an anti-PD1 antibody in vivo for maximal tumor growth inhibition. Figure 1: The serotonin agonist 5-Nonyloxytryptamine (5-NL) was identified as potentiating T cell mediated anti-tumor immunity. (A) C57BL/6J or (B) CD8 depleted C57BL/6J mice were subcutaneously injected with 5 x 105 gp33 expressing tumor cells. 7 days post-tumor injection, mice were randomized and treated daily 6.25 mg/kg of 5-NL or vehicle for five consecutive days and tumor volume was measured (n = 9-12). H2-Db (MHC-I, murine cell lines) and HLAA-C (human cells lines) were assessed by FACS analysis following treatment with 5-NL for 24 hours (n = 3-9). Error bars in all experiments indicate SEM; *P < 0.05 as determined by a Student´s t-test (unpaired, 2 tailed). Image:Summary/Conclusion: This study demonstrates novel therapeutic opportunities for augmenting immune responses in poorly immunogenic tumors and increasing their responsiveness to immunotherapies.

Abstract CT166: Injection site reactions correlate to delayed type hypersensitivity tests and suggest that GP2 reverses immune suppression of trastuzumab-treated HER2 positive patients in a phase IIb study evaluating HER2/neu peptide GP2 (GLSI-100) vs. GM-CSF after adjuvant trastuzumab in HER2 positive women with breast cancer

Abstract The authors did not submit an updated abstract. The original abstract should be considered final. Citation Format: Snehal S. Patel, David B. McWilliams, Mira S. Patel, Jaye Thompson, F. Joseph Daugherty. Injection site reactions correlate to delayed type hypersensitivity tests and suggest that GP2 reverses immune suppression of trastuzumab-treated HER2 positive patients in a phase IIb study evaluating HER2/neu peptide GP2 (GLSI-100) vs. GM-CSF after adjuvant trastuzumab in HER2 positive women with breast cancer [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 CT166.

Evaluation of booster injections in maintaining peak immunity in a phase IIb study evaluating HER2/neu peptide GP2 (GLSI-100) versus GM-CSF alone after adjuvant trastuzumab in women with HER2-positive breast cancer.

LBA550 Background: Injection site reactions (ISR) of booster injections in the randomized, active-controlled, single-blinded, multicenter Phase IIb trial of GLSI-100 (GP2+GM-CSF) administered in the adjuvant setting to node-positive and high-risk node-negative breast cancer patients with tumors expressing HER2 have been analyzed. It was demonstrated that GLSI-100 safely elicited a potent immune response versus placebo, along with data showing ISRs correlate to immune response data such as Delayed Type Hypersensitivity (DTH). Methods: Patients were randomized and received GLSI-100 (500 mcg GP2: 125 mcg GM-CSF) or control (GM-CSF) via 6 intradermal injections every 3-4 weeks as part of the Primary Immunization Series (PIS) for 6 months and 4 booster injections every 6 months thereafter. Injection site reactions were assessed 48-72 hours after the injection. Booster injections were introduced mid-trial so not all patients were dosed with boosters. The largest perpendicular diameters of induration were measured, and the orthogonal mean was calculated to quantify ISRs, providing potential assessments of peak and nadir immunity during the PIS and booster phases of treatment. Results: The study enrolled 180 patients who were HER2 3+ positive and low HER2 expressors (1-2+). After 5 years of follow-up, the Kaplan-Meier estimated 5-year DFS rate in the 46 HER2 3+ patients treated with GLSI-100, who were in the efficacy population, was 100% versus 89.4% (95% CI:76.2, 95.5%) in the 50 patients treated with GM-CSF (p = 0.0338). A patient is in the efficacy population if they were treated, followed, and remained disease free over the first 6 months. Boosters were administered to 53 HER2 3+ patients (54%). The median peak ISR during the PIS in GLSI-100 treated patients was 92.1 mm versus 60.5 mm in the GM-CSF only treated control patients. Peak ISR in GLSI-100 treated patients occurred throughout the 10-dose dosing period, including during boosters. During the 6 dose PIS, 84% of the peak ISRs occurred between doses 3 through 6. Each booster is administered 6 months after the last injection, thus the booster ISR is likely measuring the nadir immune response. When comparing the peak ISR during PIS to the boosters, the nadir ISRs of the boosters were approximately 20 mm smaller. Conclusions: Administering GLSI-100 boosters at 6 month intervals to patients produced a consistent nadir ISR approximately 20 mm lower than the maximum PIS ISR of 92.1 mm which is still larger than the maximum ISR in GM-CSF only patients of 60.5 mm. A patient’s immune response a month after booster dosing would theoretically be the peak ISR, which will be measured in future trials by measuring T-cell response and DTH one month after booster injections, further helping to evaluate booster strategies to sustain peak immunity over longer periods of time. Clinical trial information: 00524277.

Baseline GP2 immune response as an independent prognostic factor in a phase IIb study evaluating HER2/neu peptide GP2 (GLSI-100) versus. GM-CSF alone after adjuvant trastuzumab in HER2-positive women with breast cancer.

e12519 Background: Delayed type hypersensitivity (DTH) skin tests to GP2 were conducted in the randomized, active-controlled, single-blinded, multicenter Phase IIb trial investigating GLSI-100 (GP2+GM-CSF) administered in the adjuvant setting to node-positive and high-risk node-negative breast cancer patients with tumors expressing HER2. Previously it has been reported that subjects with baseline response to GP2 by DTH were more likely to experience a recurrence sooner than those without a positive baseline response. It was of interest to determine if this response was independent of other known prognostic factors. Methods: Patients were randomized and received GLSI-100 (500 mcg GP2: 125 mcg GM-CSF) or control (GM-CSF only) via 6 intradermal injections every 3-4 weeks as part of the Primary Immunization Series (PIS) for 6 months and 4 booster injections every 6 months thereafter. GP2 DTH skin tests were placed at baseline and after the 6th dose. After 48-72 hours, the largest perpendicular diameters of induration were measured and the orthogonal mean was calculated. Induration of over 5mm was considered a positive response. The relationship between prognostic factors such as demographic and disease characteristics and the baseline DTH response were investigated by logistic regression. Results: The study enrolled 180 patients who were HER2 3+ positive and low HER2 expressors (1-2+). After 5 years of follow-up, the Kaplan-Meier estimated 5-year DFS rate in the 46 HER2 3+ patients treated with GLSI-100, if completing the PIS, was 100% versus 89.4% (95% CI:76.2, 95.5%) in the 50 patients treated with GM-CSF (p = 0.0338). Just under 23% of subjects experienced an immune response to GP2 by DTH at baseline. The incidence was similar in patients who were HER2/neu positive and those considered low expressors and was equally distributed across GLSI-100 and placebo-treated subjects. Known prognostic factors such as hormone receptor status, stage at presentation, node status, etc. were evenly distributed across subjects that experienced a baseline DTH response to GP2 and those who did not. Each prognostic factor was investigated in a logistic model to determine if it predicted baseline DTH immune status. The prognostic factors did not predict baseline DTH immune response in the logistic models. There appears to be no correlation between baseline immune response to GP2 and known prognostic factors. Conclusions: Baseline GP2 immune response as measured by the delayed-type hypersensitivity test may be an independent prognostic factor for recurrence. Knowledge of this GP2 immune response may identify a patient with increased risk of rapid recurrence. Clinical trial information: 00524277.

Abstract P2-13-29: Analysis of GP2 immune response and relationship to recurrence in a prospective, randomized, placebo-controlled, single-blinded, multicenter, phase IIb study evaluating the reduction of recurrences using HER2/neu peptide GP2 (GLSI-100) vs. GM-CSF alone after adjuvant trastuzumab in HER2 positive women with operable breast cancer

Abstract Background: Delayed type hypersensitivity (DTH) skin tests in the randomized, active-controlled, single-blinded, multicenter Phase IIb trial investigating GLSI-100 (GP2+GM-CSF) administered in the adjuvant setting to node-positive and high-risk node-negative breast cancer patients with tumors expressing any degree of HER2 (immuno-histochemistry [IHC] 1-3+) (NCT00524277) have been analyzed. The trial enrolled HLA-A*02 patients randomized to receive GLSI-100 versus GM-CSF alone. The trial's primary objective was to determine if treatment with GLSI-100, a HER2-derived peptide, reduces recurrence rates. Analyses for this trial showing GLSI-100 to be efficacious, safe and immunogenic have been previously reported by Patel et al. and Mittendorf et al.. Methods: Consented patients were randomized and scheduled to receive GLSI-100 (500 mcg GP2: 125 mcg GM-CSF) or control (GM-CSF only) via 6 intradermal injections every 3-4 weeks as part of the Primary Immunization Series (PIS) for the first 6 months and 4 booster intradermal injections every 6 months thereafter. Boosters were introduced during the trial, thus some patients did not receive all 4 boosters. DTH skin tests were assessed at baseline and after the 6th dose with the orthogonal mean of each skin reaction measured 48-72 hours after injection using the sensitive ballpoint-pen method.. Results: The study enrolled 180 patients across 16 clinical sites with both HER2 3+ positive and low HER2 expressors (1-2+). After 5 years of follow-up, the Kaplan-Meier estimated 5-year DFS rate in the 46 HER2 3+ patients treated with GLSI-100, if the patient completed the PIS, was 100% versus 89.4% (95% CI:76.2, 95.5%) in the 50 placebo patients treated with GM-CSF (p = 0.0338). GLSI-100 was shown to be well tolerated with no SAEs deemed related to study medication and elicited a potent immune response measured by local skin tests and immunological assays. Injection site reactions were common, occurring in almost 100% of patients treated with either GLSI-100 or GM-CSF alone. Previous publications have reported the increase in DTH response reported among patients after treatment with GLSI-100. However, it was of interest to understand the positive DTH responses to GP2 noted at baseline. 22.8% of patients reacted to GP2 at baseline with induration of 5mm or greater. In the subgroup of patients who later experienced a breast cancer recurrence, 36.4% (8/22) had such a baseline response. Analysis of the time to recurrence among those recurring found that the median time to recurrence was 0.6 years for those with a baseline response while those that did not have a positive baseline DTH response to GP2 took 1.2 years to recur.. Conclusions: This study demonstrated that GLSI-100 safely elicited a potent immune response as evidenced by increased DTH skin responses with treatment paired with improved disease-free survival. It is theorized that a positive baseline DTH skin test to GP2 may be evidence of an existing immune response to GP2 associated with residual disease, impending recurrence, or prior treatments. Further studies assessing if GP2 immune response is an important prognosticator of cancer disease state or recurrence are planned. Citation Format: Snehal S Patel, David B McWilliams, Mira S Patel, Christine T Fischette, Jaye Thompson, F. Joseph Daugherty. Analysis of GP2 immune response and relationship to recurrence in a prospective, randomized, placebo-controlled, single-blinded, multicenter, phase IIb study evaluating the reduction of recurrences using HER2/neu peptide GP2 (GLSI-100) vs. GM-CSF alone after adjuvant trastuzumab in HER2 positive women with operable breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-29.

Anti-CD40 Antibody Fused to CD40 Ligand Is a Superagonist Platform for Adjuvant Intrinsic DC-Targeting Vaccines.

CD40 is a potent activating receptor expressed on antigen-presenting cells (APCs) of the immune system. CD40 regulates many aspects of B and T cell immunity via interaction with CD40L expressed on activated T cells. Targeting antigens to CD40 via agonistic anti-CD40 antibody fusions promotes both humoral and cellular immunity, but current anti-CD40 antibody-antigen vaccine prototypes require co-adjuvant administration for significant in vivo efficacy. This may be a consequence of dulling of anti-CD40 agonist activity via antigen fusion. We previously demonstrated that direct fusion of CD40L to anti-CD40 antibodies confers superagonist properties. Here we show that anti-CD40-CD40L-antigen fusion constructs retain strong agonist activity, particularly for activation of dendritic cells (DCs). Therefore, we tested anti-CD40-CD40L antibody fused to antigens for eliciting immune responses in vitro and in vivo. In PBMC cultures from HIV-1-infected donors, anti-CD40-CD40L fused to HIV-1 antigens preferentially expanded HIV-1-specific CD8+ T cells versus CD4+ T cells compared to analogous anti-CD40-antigen constructs. In normal donors, anti-CD40-CD40L-mediated delivery of Influenza M1 protein elicited M1-specific T cell expansion at lower doses compared to anti-CD40-mediated delivery. Also, on human myeloid-derived dendritic cells, anti-CD40-CD40L-melanoma gp100 peptide induced more sustained Class I antigen presentation compared to anti-CD40-gp100 peptide. In human CD40 transgenic mice, anti-CD40-CD40L-HIV-1 gp140 administered without adjuvant elicited superior antibody responses compared to anti-CD40-gp140 antigen without fused CD40L. In human CD40 mice, compared to the anti-CD40 vehicle, anti-CD40-CD40L delivery of Eα 52-68 peptide elicited proliferating of TCR I-Eα 52-68 CD4+ T cells producing cytokine IFNγ. Also, compared to controls, only anti-CD40-CD40L-Cyclin D1 vaccination of human CD40 mice reduced implanted EO771.LMB breast tumor cell growth. These data demonstrate that human CD40-CD40L antibody fused to antigens maintains highly agonistic activity and generates immune responses distinct from existing low agonist anti-CD40 targeting formats. These advantages were in vitro skewing responses towards CD8+ T cells, increased efficacy at low doses, and longevity of MHC Class I peptide display; and in mouse models, a more robust humoral response, more activated CD4+ T cells, and control of tumor growth. Thus, the anti-CD40-CD40L format offers an alternate DC-targeting platform with unique properties, including intrinsic adjuvant activity.

Abstract IAP0301: Bispecific T cell engagers (TCEs) for treatment of solid tumors: Challenges and opportunities

Abstract T cell-engaging bispecific antibody constructs (TCEs) have the potential to engage all cytotoxic T cells for redirected cancer cell lysis, including CD8, CD4, gamma/delta, NKT and regulatory T cells. TCEs transiently connect T cells with target cells leading to formation of a cytolytic synapse. Once activated by TCEs, T cells can adopt a serial lysis mode and increase troop size by their local proliferation. In numerous animal models, TCEs can eradicate large tumors as single-agent therapy. TCEs show a remarkable single-agent activity in hematological diseases, including ALL, NHL (DLBCL and FL) and AML. A CD19/CD3-bispecific TCE called blinatumomab (Blincyto) has been approved in 2014, and six other TCEs targeting CD20, BCMA and CD123 are in late-stage development. With complete response rates &amp;gt;50% and durability of responses, TCEs are in par with autologous CD19 CAR-T cells in NHL but are easier to manufacture and allow for a faster treatment start. Treatment of solid tumor indications with TCEs is not as successful as treatment of blood-borne cancers. This may be due to issues with tumor penetration, the hostile tumor microenvironment and the scarcity of T cells in cold tumors. A variety of TCEs explore their single-agent activity against solid tumors by targeting surface antigen like PSMA, GPC3, GD2, or HER-2. Enhancements in the form of combination therapy with checkpoint inhibitors, or 4-1BB and CD28 agonists are being explored as ways of augmenting solid tumor activity of TCEs. The first TCE showing robust anti-tumor activity in a solid tumor indication is targeting a gp100 peptide/MHC (developed by Immunocore). It is made from a soluble TCR fragment of very high affinity for the gp100 peptide HLA*0201 complex fused to an anti-CD3 scFv fragment. Although only few partial responses were observed in uveal melanoma patients, the single-agent treatment showed a very robust improvement in overall survival with a hazard ratio of 0.51 compared to investigators’ choice. This data will inspire the development of more TCEs that are specific for peptide/MHC complexes. This class of targets can also be addresses by TCR-mimetic antibodies. TCEs using such antibodies showed high anti-tumor activity in mouse models against MAGE-A4, p53 mutant or tyrosinase peptide/MHC complexes, all targets that are otherwise not amenable to classical TCEs. Another emerging opportunity for treating cancers are conditional TCEs. These are infused as inactive precursors. Inside tumors, the locally produced proteases convert the precursors into highly active TCEs. One example is the so called ProTriTACs, where a half-life extending albumin-binding domain masks the CD3-binding domain. The mask confers a 500-fold reduction in the cytolytic activity of the TCE. Inside tumors, the mask is cleaved off and a TCE is activated that has a very short serum half-life once it is released from the tumor. Conditional TCEs promise not only a wider therapeutic index but can also leverage as targets tumor-associated antigens that are widely expressed on tumors but also on healthy normal tissues, such as EGFR or EpCAM/TROP1. On-target toxicity of TCEs relates to the quality of target antigens. Finding more tumor-specific surface antigens, appropriate pMHC targets (e.g., mutant oncogene epitopes) and conditional T cell engagers are ways to address this challenge. Off-target toxicity of TCEs relates to T cell activation because of redirected target cell lysis. In the worst case, a cytokine release syndrome is induced. In the best case, a low-level cytokine release is contributing to the efficacy of TCEs. CRS can be managed by steroids, step dosing, anti-IL6 antibody and/or dasatinib. Another promising means is subcutaneous administration of TCEs. A dozen TCE programs currently explore this route, which allows reduction of Cmax and of the initial cytokine release. TCEs will soon become a major weapon in the arsenal of cancer therapy. They will not only work in blood-borne cancers but will soon conquer select solid tumor indications. Peptide/MHC-targeting and conditional TCEs are very promising approaches to achieve solid tumor activity of TCEs. Citation Format: Patrick A. Baeuerle. Bispecific T cell engagers (TCEs) for treatment of solid tumors: Challenges and opportunities [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr IAP0301.

T cell engagers in solid tumors kick the door down

Tebentafusp, a Tcell-redirecting, bispecific-antibody-targeting, HLA∗02:01-restricted gp100 peptide and CD3, induces Tcells to kill glycoprotein 100 (gp100)-expressing tumor cells. Tebentafusp is the first Tcell engager to show a statistically significant overall survival improvement for the treatment of patients with solid tumors, and this has been reported in the New England Journal of Medicine.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

CD169 Defines Activated CD14+ Monocytes With Enhanced CD8+ T Cell Activation Capacity.

Monocytes are antigen-presenting cells (APCs) that play diverse roles in promoting or regulating inflammatory responses, but their role in T cell stimulation is not well defined. In inflammatory conditions, monocytes frequently show increased expression of CD169/Siglec-1, a type-I interferon (IFN-I)-regulated protein. However, little is known about the phenotype and function of these CD169+ monocytes. Here, we have investigated the phenotype of human CD169+ monocytes in different diseases, their capacity to activate CD8+ T cells, and the potential for a targeted-vaccination approach. Using spectral flow cytometry, we detected CD169 expression by CD14+ CD16- classical and CD14+ CD16+ intermediate monocytes and unbiased analysis showed that they were distinct from dendritic cells, including the recently described CD14-expressing DC3. CD169+ monocytes expressed higher levels of co-stimulatory and HLA molecules, suggesting an increased activation state. IFNα treatment highly upregulated CD169 expression on CD14+ monocytes and boosted their capacity to cross-present antigen to CD8+ T cells. Furthermore, we observed CD169+ monocytes in virally-infected patients, including in the blood and bronchoalveolar lavage fluid of COVID-19 patients, as well as in the blood of patients with different types of cancers. Finally, we evaluated two CD169-targeting nanovaccine platforms, antibody-based and liposome-based, and we showed that CD169+ monocytes efficiently presented tumor-associated peptides gp100 and WT1 to antigen-specific CD8+ T cells. In conclusion, our data indicate that CD169+ monocytes are activated monocytes with enhanced CD8+ T cell stimulatory capacity and that they emerge as an interesting target in nanovaccine strategies, because of their presence in health and different diseases.

Abstract CT256: A prospective, randomized, multicenter, double-blinded, placebo-controlled phase III trial of the HER2/neu peptide GP2 + GM-CSF versus bacteriostatic saline/WFI placebo as adjuvant therapy after any trastuzumab-based therapy in HER2-positive women with operable breast cancer

Abstract Background: GP2 is a biologic nine amino acid peptide of the HER2/neu protein delivered in combination with an FDA-approved immunoadjuvant Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF, Sargramostim, Leukine) that stimulates an immune response targeting HER2/neu expressing cancers. In a prospective, randomized, single-blinded, placebo-controlled, multicenter Phase IIb clinical trial completed in 2018, no recurrences were observed in the HER2/neu positive adjuvant setting after median 5 years of follow-up, if the HLA 2+ patient received the 6 primary intradermal injections over the first 6 months (p = 0.0338) in a pre-specified subgroup analysis. Furthermore, the GP2 immunotherapy elicited a potent immune response measured by local skin tests and immunological assays. Of the 138 patients that have been treated with GP2 to date over 4 clinical trials, GP2 treatment was well tolerated and no serious adverse events were observed related to the GP2 immunotherapy. This Phase III trial aims to reproduce the Phase IIb study and will explore the use of GP2 + GM-CSF as adjuvant therapy to prevent the recurrence of breast cancer in HER2/neu positive and HLA 2+ patients, post-surgery and following the first year treatment with any trastuzumab-based therapy. Trial Design: This Phase III trial is a prospective, randomized, double-blinded, multi-center study. After 1 year of trastuzumab-based therapy or an approved biosimilar, treatment with GP2 + GM-CSF or placebo (Bacteriostatic Saline/WFI ) will be administered intradermally for the 6 primary immunization series over the first 6 months and 5 subsequent boosters over the next 2.5 years for a total of 11 injections over 3 years of treatment. The participant duration of the trial will be 3 years treatment plus 2 years follow-up for a total of 5 years following the first year treatment with trastuzumab-based therapy or approved biosimilar. An interim analysis is planned and patients will be stratified based on prior and current treatments, among other factors. Eligibility Criteria: The majority of breast cancer patients will be HER2/neu positive and HLA 2+, disease-free, conventionally treated node-positive, post breast tumor removal surgery and following the first year treatment with trastuzumab-based therapy. Trial Objectives:To determine if GP2 therapy reduces recurrence in HER2/neu positive breast cancer patients. To monitor the in vitro and in vivo immunologic responses to GP2 therapy and correlate these responses with the clinical outcomes.To monitor for any unexpected adverse events and toxicities related to GP2 therapy. Accrual: The target enrollment is up to approximately 500 patients. Contact information: snehal.patel@greenwichlifesciences.com Funding: This trial is supported by Greenwich LifeSciences. Citation Format: Snehal S. Patel, David B. McWilliams, Christine T. Fischette, Jaye Thompson, F Joseph Daugherty, C Kent Osborne, Mothaffar F. Rimawi. A prospective, randomized, multicenter, double-blinded, placebo-controlled phase III trial of the HER2/neu peptide GP2 + GM-CSF versus bacteriostatic saline/WFI placebo as adjuvant therapy after any trastuzumab-based therapy in HER2-positive women with operable breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT256.