IL15/IL15Rα complex induces an anti-tumor immune response following radiation therapy only in the absence of Tregs and fails to induce expansion of progenitor TCF1+ CD8 T cells.

Background: This work seeks to understand whether IL15-incorporating treatments improve response to radiotherapy and uncover mechanistic rationale for overcoming resistance to IL15 agonism using novel therapeutic combinations. Experimental Design: Orthotopic tumor models of PDAC were used to determine response to treatment. IL15-/- and Rag1-/- mouse models were employed to determine dependence on IL15 and CTLs, respectively. Flow cytometry was used to assess immune cell frequency and activation state. Phospho-proteomic analyses were used to characterize intracellular signaling pathways. Results: We show that the combination of radiation therapy (RT) and an IL15/IL15Ra fusion complex (denoted IL15c) fails to confer anti-tumor efficacy; however, a CD8-driven anti-tumor immune response is elicited with the concurrent administration of an aCD25 Treg-depleting antibody. Using IL15-/- and Rag1-/- mice, we demonstrate that response to RT + IL15c + aCD25 is dependent on both IL15 and CTLs. Furthermore, despite an equivalent survival benefit following treatment with RT + IL15c + aCD25 and combination RT + PD1-IL2v, a novel immunocytokine with PD-1 and IL2Rßγ binding domains, CTL immunophenotyping and phospho-proteomic analysis of intracellular metabolites showed significant upregulation of activation and functionality in CD8 T cells treated with RT + PD1-IL2v. Finally, we show the immunostimulatory response to RT + PD1-IL2v is significantly diminished with a concurrent lack of TCF+ CD8 T cell generation in the absence of functional IL15 signaling. Conclusions: Our results are illustrative of a mechanism wherein unimpeded effector T cell activation through IL2Rß signaling and Treg inhibition are necessary in mediating an anti-tumor immune response.

Optimizing Early Clinical Investigations in Cancer Immunotherapy: The Translational Journey of RG6292, a Novel, Selective Treg-Depleting Antibody.

The multifaceted IL-2/IL-2R biology and its modulation by promising therapeutic agents are highly relevant topics in the cancer immunotherapy field. A novel CD25-Treg-depleting antibody (Vopikitug, RG6292) has been engineered to preserve IL-2 signaling on effector T cells to enhance effector activation and antitumor immunity, and is currently being evaluated in the clinic. The Entry into Human-enabling framework described here investigated the characteristics of RG6292, from in vitro quantification of CD25 and RG6292 pharmacology using human tissues to in vivo assessment of PK/PD/safety relationships in cynomolgus monkeys as non-human primate species (NHP). Fundamental knowledge on CD25 and Treg biology in healthy and diseased tissues across NHP and human highlighted the commonalities between these species in regard to the target biology and demonstrated the conservation of RG6292 properties between NHP and human. The integration of in vitro and in vivo PK/PD/safety data from these species enabled the identification of human relevant safety risks, the selection of the most appropriate safe starting dose and the projection of the pharmacologically-relevant dose range. The first clinical data obtained for RG6292 in patients verified the appropriateness of the described approaches as well as validated the full clinical relevance of the projected safety, PK, and PD profiles from animal to man. This work shows how the integration of mechanistic non-clinical data increases the predictive value for human, allowing efficient transition of drug candidates and optimizations of early clinical investigations.

CD25 targeting with the afucosylated human IgG1 antibody RG6292 eliminates regulatory T cells and CD25+ blasts in acute myeloid leukemia.

Background: Acute Myeloid leukemia is a heterogeneous disease that requires novel targeted treatment options tailored to the patients' specific microenvironment and blast phenotype. Methods: We characterized bone marrow and/or blood samples of 37 AML patients and healthy donors by high dimensional flow cytometry and RNA sequencing using computational analysis. In addition, we performed ex vivo ADCC assays using allogeneic NK cells isolated from healthy donors and AML patient material to test the cytotoxic potential of CD25 Mab (also referred to as RG6292 and RO7296682) or isotype control antibody on regulatory T cells and CD25+ AML cells. Results: Bone marrow composition, in particular the abundance of regulatory T cells and CD25 expressing AML cells, correlated strongly with that of the blood in patients with time-matched samples. In addition, we observed a strong enrichment in the prevalence of CD25 expressing AML cells in patients bearing a FLT3-ITD mutation or treated with a hypomethylating agent in combination with venetoclax. We adopted a patient-centric approach to study AML clusters with CD25 expression and found it most highly expressed on immature phenotypes. Ex vivo treatment of primary AML patient samples with CD25 Mab, a human CD25 specific glycoengineered IgG1 antibody led to the specific killing of two different cell types, CD25+ AML cells and regulatory T cells, by allogeneic Natural Killer cells. Conclusion: The in-depth characterization of patient samples by proteomic and genomic analyses supported the identification of a patient population that may benefit most by harnessing CD25 Mab's dual mode of action. In this pre-selected patient population, CD25 Mab could lead to the specific depletion of regulatory T cells, in addition to leukemic stem cells and progenitor-like AML cells that are responsible for disease progression or relapse.

Selective targeting of IL2Rßγ combined with radiotherapy triggers CD8- and NK-mediated immunity, abrogating metastasis in HNSCC.

The implementation of cancer immunotherapies has seen limited clinical success in head and neck squamous cell carcinoma (HNSCC). Interleukin-2 (IL-2), which modulates the survival and functionality of lymphocytes, is an attractive target for new immunotherapies but one that is limited by presence of regulatory T cells (Tregs) expressing the high-affinity IL-2Rα. The bispecific immunocytokine PD1-IL2v preferentially delivers IL-2 signaling through IL-2Rßγ on PD-1-expressing cells. Selectively targeting the intermediate-affinity IL-2Rßγ can be leveraged to induce anti-tumor immune responses in effector T cells and natural killer (NK) cells while limiting the negative regulation of IL-2Rα activation on Tregs. Using radiation therapy (RT) in combination with PD1-IL2v improves local tumor control and survival, and controls metastatic spread in orthotopic HNSCC tumor models. PD1-IL2v drives systemic activation and expansion of circulating and tumor-infiltrating cytotoxic T cells and NK cells while limiting Treg-mediated immunosuppression. These data show that PD1-L2v induces durable systemic tumor control in HNSCC.

Simultaneous targeting of PD-1 and IL-2Rßγ with radiation therapy inhibits pancreatic cancer growth and metastasis.

In pancreatic ductal adenocarcinoma (PDAC) patients, we show that response to radiation therapy (RT) is characterized by increased IL-2Rß and IL-2Rγ along with decreased IL-2Rα expression. The bispecific PD1-IL2v is a PD-1-targeted IL-2 variant (IL-2v) immunocytokine with engineered IL-2 cis targeted to PD-1 and abolished IL-2Rα binding, which enhances tumor-antigen-specific T cell activation while reducing regulatory T cell (Treg) suppression. Using PD1-IL2v in orthotopic PDAC KPC-driven tumor models, we show marked improvement in local and metastatic survival, along with a profound increase in tumor-infiltrating CD8+ T cell subsets with a transcriptionally and metabolically active phenotype and preferential activation of antigen-specific CD8+ T cells. In combination with single-dose RT, PD1-IL2v treatment results in a robust, durable expansion of polyfunctional CD8+ T cells, T cell stemness, tumor-specific memory immune response, natural killer (NK) cell activation, and decreased Tregs. These data show that PD1-IL2v leads to profound local and distant response in PDAC.

Preclinical models for prediction of immunotherapy outcomes and immune evasion mechanisms in genetically heterogeneous multiple myeloma.

The historical lack of preclinical models reflecting the genetic heterogeneity of multiple myeloma (MM) hampers the advance of therapeutic discoveries. To circumvent this limitation, we screened mice engineered to carry eight MM lesions (NF-κB, KRAS, MYC, TP53, BCL2, cyclin D1, MMSET/NSD2 and c-MAF) combinatorially activated in B lymphocytes following T cell-driven immunization. Fifteen genetically diverse models developed bone marrow (BM) tumors fulfilling MM pathogenesis. Integrative analyses of ∼500 mice and ∼1,000 patients revealed a common MAPK-MYC genetic pathway that accelerated time to progression from precursor states across genetically heterogeneous MM. MYC-dependent time to progression conditioned immune evasion mechanisms that remodeled the BM microenvironment differently. Rapid MYC-driven progressors exhibited a high number of activated/exhausted CD8+ T cells with reduced immunosuppressive regulatory T (Treg) cells, while late MYC acquisition in slow progressors was associated with lower CD8+ T cell infiltration and more abundant Treg cells. Single-cell transcriptomics and functional assays defined a high ratio of CD8+ T cells versus Treg cells as a predictor of response to immune checkpoint blockade (ICB). In clinical series, high CD8+ T/Treg cell ratios underlie early progression in untreated smoldering MM, and correlated with early relapse in newly diagnosed patients with MM under Len/Dex therapy. In ICB-refractory MM models, increasing CD8+ T cell cytotoxicity or depleting Treg cells reversed immunotherapy resistance and yielded prolonged MM control. Our experimental models enable the correlation of MM genetic and immunological traits with preclinical therapy responses, which may inform the next-generation immunotherapy trials.

Elective nodal irradiation mitigates local and systemic immunity generated by combination radiation and immunotherapy in head and neck tumors.

In the setting of conventional radiation therapy, even when combined with immunotherapy, head and neck cancer often recurs locally and regionally. Elective nodal irradiation (ENI) is commonly employed to decrease regional recurrence. Given our developing understanding that immune cells are radio-sensitive, and that T cell priming occurs in the draining lymph nodes (DLNs), we hypothesize that radiation therapy directed at the primary tumor only will increase the effectiveness of immunotherapies. We find that ENI increases local, distant, and metastatic tumor growth. Multi-compartmental analysis of the primary/distant tumor, the DLNs, and the blood shows that ENI decreases the immune response systemically. Additionally, we find that ENI decreases antigen-specific T cells and epitope spreading. Treating the primary tumor with radiation and immunotherapy, however, fails to reduce regional recurrence, but this is reversed by either concurrent sentinel lymph node resection or irradiation. Our data support using lymphatic sparing radiation therapy for head and neck cancer.

Fibroblast activation protein-targeted-4-1BB ligand agonist amplifies effector functions of intratumoral T cells in human cancer.

BACKGROUND: The costimulatory receptor 4-1BB (CD137, TNFRSF9) plays an important role in sustaining effective T cell immune responses and is investigated as target for cancer therapy. Systemic 4-1BB directed therapies elicit toxicity or low efficacy, which significantly hampered advancement of 4-1BB-based immunotherapy. Therefore, targeted delivery of 4-1BB agonist to the tumor side is needed for eliciting antitumor efficacy while avoiding systemic toxicity. METHODS: We analyzed the immunostimulatory properties of a fibroblast activation protein (FAP)-targeted 4-1BB agonist (FAP-4-1BBL) by assessing tumor-infiltrating lymphocytes' (TIL) activity from patients with non-small cell lung cancer and epithelial ovarian cancer. RESULTS: Combination treatment with FAP-4-1BBL and T cell receptor stimulation by either anti-CD3 or T cell bispecific antibodies significantly enhanced TIL activation and effector functions, including T cell proliferation, secretion of proinflammatory cytokines and cytotoxicity. Notably, costimulation with FAP-4-1BBL led to de novo secretion of interleukin (IL)-13. This was associated with cytokine-mediated tumor cell apoptosis, which was partially dependent on IL-13 alpha 1/2 receptors and STAT6 phosphorylation. CONCLUSIONS: Our study provides mechanistic insights into T cell stimulation induced by FAP-4-1BBL in primary human tumors and supports the investigation of FAP-4-1BBL compound in early clinical trials.

CD25-Treg-depleting antibodies preserving IL-2 signaling on effector T cells enhance effector activation and antitumor immunity.

Intratumoral regulatory T cell (Treg) abundance associates with diminished anti-tumor immunity and poor prognosis in human cancers. Recent work demonstrates that CD25, the high affinity receptor subunit for IL-2, is a selective target for Treg depletion in mouse and human malignancies; however, anti-human CD25 antibodies have failed to deliver clinical responses against solid tumors due to bystander IL-2 receptor signaling blockade on effector T cells, which limits their anti-tumor activity. Here we demonstrate potent single-agent activity of anti-CD25 antibodies optimized to deplete Tregs whilst preserving IL-2-STAT5 signaling on effector T cells, and demonstrate synergy with immune checkpoint blockade in vivo. Pre-clinical evaluation of an anti-human CD25 (RG6292) antibody with equivalent features demonstrates, in both non-human primates and humanized mouse models, efficient Treg depletion with no overt immune-related toxicities. Our data supports the clinical development of RG6292 and evaluation of novel combination therapies incorporating non-IL-2 blocking anti-CD25 antibodies in clinical studies.

The PET-Tracer 89Zr-Df-IAB22M2C Enables Monitoring of Intratumoral CD8 T-cell Infiltrates in Tumor-Bearing Humanized Mice after T-cell Bispecific Antibody Treatment.

CD8-expressing T cells are the main effector cells in cancer immunotherapy. Treatment-induced changes in intratumoral CD8+ T cells may represent a biomarker to identify patients responding to cancer immunotherapy. Here, we have used a 89Zr-radiolabeled human CD8-specific minibody (89Zr-Df-IAB22M2C) to monitor CD8+ T-cell tumor infiltrates by PET. The ability of this tracer to quantify CD8+ T-cell tumor infiltrates was evaluated in preclinical studies following single-agent treatment with FOLR1-T-cell bispecific (TCB) antibody and combination therapy of CEA-TCB (RG7802) and CEA-targeted 4-1BB agonist CEA-4-1BBL. In vitro cytotoxicity assays with peripheral blood mononuclear cells and CEA-expressing MKN-45 gastric or FOLR1-expressing HeLa cervical cancer cells confirmed noninterference of the anti-CD8-PET-tracer with the mode of action of CEA-TCB/CEA-4-1BBL and FOLR1-TCB at relevant doses. In vivo, the extent of tumor regression induced by combination treatment with CEA-TCB/CEA-4-1BBL in MKN-45 tumor-bearing humanized mice correlated with intratumoral CD8+ T-cell infiltration. This was detectable by 89Zr-IAB22M2C-PET and γ-counting. Similarly, single-agent treatment with FOLR1-TCB induced strong CD8+ T-cell infiltration in HeLa tumors, where 89Zr-Df-IAB22M2C again was able to detect CD8 tumor infiltrates. CD8-IHC confirmed the PET imaging results. Taken together, the anti-CD8-minibody 89Zr-Df-IAB22M2C revealed a high sensitivity for the detection of intratumoral CD8+ T-cell infiltrates upon either single or combination treatment with TCB antibody-based fusion proteins. These results provide further evidence that the anti-CD8 tracer, which is currently in clinical phase II, is a promising monitoring tool for intratumoral CD8+ T cells in patients treated with cancer immunotherapy. SIGNIFICANCE: Monitoring the pharmacodynamic activity of cancer immunotherapy with novel molecular imaging tools such as 89Zr-Df-IAB22M2C for PET imaging is of prime importance to identify patients responding early to cancer immunotherapy.

Therapeutic window of an EpCAM/CD3-specific BiTE antibody in mice is determined by a subpopulation of EpCAM-expressing lymphocytes that is absent in humans.

MuS110 is a BiTE antibody bispecific for murine EpCAM (CD326) and murine CD3. A recent study has shown that microS110 has significant anti tumor activity at well-tolerated doses as low as 5 microg/kg in orthotopic breast and lung cancer models (Amann et al. in Cancer Res 68:143-151, 2008). Here, we have explored the safety profile of microS110 at higher doses. Escalation to 50 microg/kg microS110 caused in mice transient loss of body weight, and transient piloerection, hypomotility, hypothermia and diarrhoea. These clinical signs coincided with serum peaks of TNF-alpha, IL-6, IL-2, IFN-gamma and IL-4, and an increase of surface markers for T cell activation. Because activation of T cells in response to BiTE antibodies is typically dependent on target cells, we analyzed mouse blood for the presence of EpCAM(+) cells. Various mouse strains presented with a subpopulation of 2-3% EpCAM(+) blood cells, mostly B and T lymphocytes, which was not detected in human blood samples. In vitro experiments in which the number of EpCAM(+) cells in blood samples was either reduced or increased suggested that both T cell activation and cytokine release in response to microS110 was dependent on the number of target-expressing cells. In support for a role of EpCAM(+) lymphocytes in the observed side effects, reduction of EpCAM(+) blood cells in mice via a low-dose pre treatment with microS110 dramatically increased the tolerability of animals up to at least 500 microg/kg of the BiTE antibody. This high tolerability to microS110 occurred in the presence of non-compromised T cells. No damage to EpCAM(+) epithelial tissues was evident from histopathological examination of animals daily injected with 100 microg/kg microS110 for 28 days. In summary, these observations suggest that side effects of microS110 in mice were largely caused by an acute T cell activation that was triggered by a subpopulation of EpCAM(+) lymphocytes. Because humans have extremely low numbers of EpCAM(+) cells in blood, this toxicity of an EpCAM-specific BiTE may be specific for mice.

Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy.

Endogenous costimulatory molecules on T cells such as 4-1BB (CD137) can be leveraged for cancer immunotherapy. Systemic administration of agonistic anti-4-1BB antibodies, although effective preclinically, has not advanced to phase 3 trials because they have been hampered by both dependency on Fcγ receptor-mediated hyperclustering and hepatotoxicity. To overcome these issues, we engineered proteins simultaneously targeting 4-1BB and a tumor stroma or tumor antigen: FAP-4-1BBL (RG7826) and CD19-4-1BBL. In the presence of a T cell receptor signal, they provide potent T cell costimulation strictly dependent on tumor antigen-mediated hyperclustering without systemic activation by FcγR binding. We could show targeting of FAP-4-1BBL to FAP-expressing tumor stroma and lymph nodes in a colorectal cancer-bearing rhesus monkey. Combination of FAP-4-1BBL with tumor antigen-targeted T cell bispecific (TCB) molecules in human tumor samples led to increased IFN-γ and granzyme B secretion. Further, combination of FAP- or CD19-4-1BBL with CEA-TCB (RG7802) or CD20-TCB (RG6026), respectively, resulted in tumor remission in mouse models, accompanied by intratumoral accumulation of activated effector CD8+ T cells. FAP- and CD19-4-1BBL thus represent an off-the-shelf combination immunotherapy without requiring genetic modification of effector cells for the treatment of solid and hematological malignancies.

CEA/CD3 bispecific antibody MEDI-565/AMG 211 activation of T cells and subsequent killing of human tumors is independent of mutations commonly found in colorectal adenocarcinomas.

Individual or combinations of somatic mutations found in genes from colorectal cancers can redirect the effects of chemotherapy and targeted agents on cancer cell survival and, consequently, on clinical outcome. Novel therapeutics with mechanisms of action that are independent of mutational status would therefore fulfill a current unmet clinical need. Here the CEA and CD3 bispecific single-chain antibody MEDI-565 (also known as MT111 and AMG 211) was evaluated for its ability to activate T cells both in vitro and in vivo and to kill human tumor cell lines harboring various somatic mutations commonly found in colorectal cancers. MEDI-565 specifically bound to normal and malignant tissues in a CEA-specific manner, and only killed CEA positive cells. The BiTE® antibody construct mediated T cell-directed killing of CEA positive tumor cells within 6 hours, at low effector-to-target ratios which were independent of high concentrations of soluble CEA. The potency of in vitro lysis was dependent on CEA antigen density but independent of the mutational status in cancer cell lines. Importantly, individual or combinations of mutated KRAS and BRAF oncogenes, activating PI3KCA mutations, loss of PTEN expression, and loss-of-function mutations in TP53 did not reduce the activity in vitro. MEDI-565 also prevented growth of human xenograft tumors which harbored various mutations. These findings suggest that MEDI-565 represents a potential treatment option for patients with CEA positive tumors of diverse origin, including those with individual or combinations of somatic mutations that may be less responsive to chemotherapy and other targeted agents.

Antitumor activity of an EpCAM/CD3-bispecific BiTE antibody during long-term treatment of mice in the absence of T-cell anergy and sustained cytokine release.

muS110 is a BiTE antibody bispecific for murine EpCAM (CD326) and murine CD3. MT110, its human-specific analog, is in a clinical phase 1 trial for treatment of patients with adenocarcinoma of the lung or gastrointestinal tract. Recent studies have shown a therapeutic window for muS110, have explored single-dose toxicity of muS110, and have found that a 1-week low-dose treatment dramatically increased the tolerability of mice to very high doses of muS110 (Cancer Immunol. Immunother. 2009;58:95-109). Here we analyzed the impact of long-term, high-dose treatment of mice with muS110 on antitumor activity and functionality of T cells. After an initial self-limiting cytokine release, the 1-week adaptation period effectively blunted further cytokine production in response to a subsequent high-dose treatment with muS110. The much-increased tolerability of mice adapted to muS110 was not because of anergy of T cells. T cells isolated from chronically muS110-treated mice fully retained their cytotoxic potential, proliferative capacity, and responsiveness to stimulation by either muS110 or anti-CD3/anti-CD28/interleukin-2 when compared with T cells from control mice. Unimpaired T-cell performance was also evident from the effective prevention of orthotopic 4T1 breast tumor outgrowth in mice treated long term with escalating doses of muS110. Finally, we show that muS110 and MT110 recognize orthologous epitopes on mouse and human EpCAM proteins, suggesting that the target-related safety profile of muS110 in mice may be predictive for MT110 in humans.

Therapeutic window of MuS110, a single-chain antibody construct bispecific for murine EpCAM and murine CD3.

EpCAM (CD326) is one of the most frequently and highly expressed tumor-associated antigens known and recently has also been found on cancer stem cells derived from human breast, colon, prostate, and pancreas tumors. However, like many other tumor-associated antigens used for antibody-based immunotherapeutic approaches, EpCAM is expressed on normal tissues including epithelia of pancreas, colon, lung, bile ducts, and breast. To assess the therapeutic window of an EpCAM/CD3-bispecific single-chain antibody construct of the bispecific T-cell engager (BiTE) class, we constructed murine surrogate of MT110 (muS110) from single-chain antibodies specific for murine EpCAM and CD3 antigens. Immunhistochemical analysis showed that, with minor differences, the expression of EpCAM protein on a large variety of tissues from man and mouse was similar with respect to distribution and level. MuS110 exhibited significant antitumor activity at as low as 5 microg/kg in both syngeneic 4T1 orthotopic breast cancer and CT-26 lung cancer mouse models. Dosing of muS110 for several weeks up to 400 microg/kg by intraanimal dose escalation was still tolerated, indicating existence of a significant therapeutic window for an EpCAM-specific BiTE antibody in mice. MuS110 was found to have similar in vitro characteristics and in vivo antitumor activity as MT110, a human EpCAM/human CD3-bispecific BiTE antibody that currently is in formal preclinical development.