Immuno-PET Monitoring of Lymphocytes Using the CD8-Specific Antibody REGN5054.

Assessment of immune-cell subsets within the tumor immune microenvironment is a powerful approach to better understand cancer immunotherapy responses. However, the use of biopsies to assess the tumor immune microenvironment poses challenges, including the potential for sampling error, restricted sampling over time, and inaccessibility of some tissues/organs, as well as the fact that single biopsy analyses do not reflect discordance across multiple intrapatient tumor lesions. Immuno-positron emission tomography (PET) presents a promising translational imaging approach to address the limitations and assess changes in the tumor microenvironment. We have developed 89Zr-DFO-REGN5054, a fully human CD8A-specific antibody conjugate, to assess CD8+ tumor-infiltrating lymphocytes (TIL) pre- and posttherapy. We used multiple assays, including in vitro T-cell activation, proliferation, and cytokine production, and in vivo viral clearance and CD8 receptor occupancy, to demonstrate that REGN5054 has minimal impact on T-cell activity. Preclinical immuno-PET studies demonstrated that 89Zr-DFO-REGN5054 specifically detected CD8+ T cells in lymphoid tissues of CD8-genetically humanized immunocompetent mice (VelociT mice) and discerned therapy-induced changes in CD8+ TILs in two models of response to a CD20xCD3 T-cell activating bispecific antibody (REGN1979, odronextamab). Toxicology studies in cynomolgus monkeys showed no overt toxicity, and immuno-PET imaging in cynomolgus monkeys demonstrated dose-dependent clearance and specific targeting to lymphoid tissues. This work supports the clinical investigation of 89Zr-DFO-REGN5054 to monitor T-cell responses in patients undergoing cancer immunotherapy.

A Biparatopic Antibody-Drug Conjugate to Treat MET-Expressing Cancers, Including Those that Are Unresponsive to MET Pathway Blockade.

Lung cancers harboring mesenchymal-to-epithelial transition factor (MET) genetic alterations, such as exon 14 skipping mutations or high-level gene amplification, respond well to MET-selective tyrosine kinase inhibitors (TKI). However, these agents benefit a relatively small group of patients (4%-5% of lung cancers), and acquired resistance limits response durability. An antibody-drug conjugate (ADC) targeting MET might enable effective treatment of MET-overexpressing tumors (approximately 25% of lung cancers) that do not respond to MET targeted therapies. Using a protease-cleavable linker, we conjugated a biparatopic METxMET antibody to a maytansinoid payload to generate a MET ADC (METxMET-M114). METxMET-M114 promotes substantial and durable tumor regression in xenografts with moderate to high MET expression, including models that exhibit innate or acquired resistance to MET blockers. Positron emission tomography (PET) studies show that tumor uptake of radiolabeled METxMET antibody correlates with MET expression levels and METxMET-M114 efficacy. In a cynomolgus monkey toxicology study, METxMET-M114 was well tolerated at a dose that provides circulating drug concentrations that are sufficient for maximal antitumor activity in mouse models. Our findings suggest that METxMET-M114, which takes advantage of the unique trafficking properties of our METxMET antibody, is a promising candidate for the treatment of MET-overexpressing tumors, with the potential to address some of the limitations faced by the MET function blockers currently in clinical use.

Generation of T-cell-redirecting bispecific antibodies with differentiated profiles of cytokine release and biodistribution by CD3 affinity tuning.

T-cell-redirecting bispecific antibodies have emerged as a new class of therapeutic agents designed to simultaneously bind to T cells via CD3 and to tumor cells via tumor-cell-specific antigens (TSA), inducing T-cell-mediated killing of tumor cells. The promising preclinical and clinical efficacy of TSAxCD3 antibodies is often accompanied by toxicities such as cytokine release syndrome due to T-cell activation. How the efficacy and toxicity profile of the TSAxCD3 bispecific antibodies depends on the binding affinity to CD3 remains unclear. Here, we evaluate bispecific antibodies that were engineered to have a range of CD3 affinities, while retaining the same binding affinity for the selected tumor antigen. These agents were tested for their ability to kill tumor cells in vitro, and their biodistribution, serum half-life, and anti-tumor activity in vivo. Remarkably, by altering the binding affinity for CD3 alone, we can generate bispecific antibodies that maintain potent killing of TSA + tumor cells but display differential patterns of cytokine release, pharmacokinetics, and biodistribution. Therefore, tuning CD3 affinity is a promising method to improve the therapeutic index of T-cell-engaging bispecific antibodies.

Preclinical PET imaging with the novel human antibody 89Zr-DFO-REGN3504 sensitively detects PD-L1 expression in tumors and normal tissues.

BACKGROUND: Programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) blocking antibodies including cemiplimab have generated profound clinical activity across diverse cancer types. Tumorous PD-L1 expression, as assessed by immunohistochemistry (IHC), is an accepted predictive marker of response to therapy in some cancers. However, expression is often dynamic and heterogeneous, and therefore not reliably captured by IHC from tumor biopsies or archival samples. Thus, there is significant need for accurate whole-body quantification of PD-L1 levels. METHODS: We radiolabeled the novel human anti-PD-L1 antibody REGN3504 with zirconium-89 (89Zr) using the chelator p-SCN-Bn-Deferoxamine to enable non-invasive immuno-positron emission tomography (immuno-PET) of PD-L1 expression. PET imaging assessed the localization of 89Zr-REGN3504 to multiple human tumor xenografts. Mice genetically humanized for PD-1 and PD-L1 were used to assess the biodistribution of 89Zr-REGN3504 to normal tissues and the estimated human radiation dosimetry of 89Zr-REGN3504 was also determined. Pharmacokinetics of REGN3504 was assessed in monkeys. RESULTS: Clear localization of 89Zr-REGN3504 to human tumor xenografts was observed via PET imaging and ex vivo biodistribution studies demonstrated high (fourfold to sixfold) tumor:blood ratios. 89Zr-REGN3504 specifically localized to spleen and lymph nodes in the PD-1/PD-L1 humanized mice. 89Zr-REGN3504 immuno-PET accurately detected a significant reduction in splenic PD-L1 positive cells following systemic treatment with clodronate liposomes. Radiation dosimetry suggested absorbed doses would be within guidelines for other 89Zr radiolabeled, clinically used antibodies. Pharmacokinetics of REGN3504 was linear. CONCLUSION: This work supports the clinical translation of 89Zr-REGN3504 immuno-PET for the assessment of PD-L1 expression. Future clinical studies will aim to investigate the utility of 89Zr-REGN3504 immuno-PET for predicting and monitoring response to anti-PD-1 therapy.

Antibody drug conjugates of cleavable amino-benzoyl-maytansinoids.

ADCs based on the natural product maytansine have been successfully employed clinically. In a previous report, ADCs based on hydrophilic non-cell permeable maytansinoids was presented. The authors in this report further explore the maytansine scaffold to develop tubulin inhibitors capable of cell permeation. The research resulted in amino-benzoyl-maytansinoid payloads that were further elaborated with linkers for conjugating to antibodies. This approach was applied to MUC16 tumor targeting antibodies for ovarian cancers. A positive control ADC was evaluated alongside the amino-benzoyl-maytansinoid ADC and the efficacy observed was equivalent while the isotype control ADCs had no effect.

A Mucin 16 bispecific T cell-engaging antibody for the treatment of ovarian cancer.

Advanced ovarian cancer is frequently treated with combination chemotherapy, but high recurrence rates show the need for therapies that can produce durable responses and extend overall survival. Bispecific antibodies that interact with tumor antigens on cancer cells and activating receptors on immune cells offer an innovative immunotherapy approach. Here, we describe a human bispecific antibody (REGN4018) that binds both Mucin 16 (MUC16), a glycoprotein that is highly expressed on ovarian cancer cells, and CD3, thus bridging MUC16-expressing cells with CD3+ T cells. REGN4018 induced T cell activation and killing of MUC16-expressing tumor cells in vitro. Binding and cytotoxicity of REGN4018 in vitro were minimally affected by high concentrations of CA-125, the shed form of MUC16, which is present in patients. In preclinical studies with human ovarian cancer cells and human T cells in immunodeficient mice, REGN4018 potently inhibited growth of intraperitoneal ovarian tumors. Moreover, in a genetically engineered immunocompetent mouse expressing human CD3 and human MUC16 [humanized target (HuT) mice], REGN4018 inhibited growth of murine tumors expressing human MUC16, and combination with an anti-PD-1 antibody enhanced this efficacy. Immuno-PET imaging demonstrated localization of REGN4018 in MUC16-expressing tumors and in T cell-rich organs such as the spleen and lymph nodes. Toxicology studies in cynomolgus monkeys showed minimal and transient increases in serum cytokines and C-reactive protein after REGN4018 administration, with no overt toxicity. Collectively, these data demonstrate potent antitumor activity and good tolerability of REGN4018, supporting clinical evaluation of REGN4018 in patients with MUC16-expressing advanced ovarian cancer.

Antibody drug conjugates of cleavable amino-alkyl and aryl maytansinoids.

Natural products have been used for many medicinal purposes for centuries. Antibody drug conjugates (ADCs) have utilized this rich source of small molecule therapeutics to produce several clinically useful treatments. ADCs based on the natural product maytansine have been successful clinically. The authors further the utility of the anti-cancer natural product maytansine by developing efficacious payloads and linker-payloads for conjugating to antibodies. The success of our approach was realized in the EGFRvIII targeting ADC EGFRvIII-16. The ADC was able to regress tumors in 2 tumor models (U251/EGFRvIII and MMT/EGFRvIII). When compared to a positive control ADC, the efficacy observed was similar or improved while the isotype control ADCs had no effect.

Preclinical Activity of the Novel Anti-Prolactin Receptor (PRLR) Antibody-Drug Conjugate REGN2878-DM1 in PRLR-Positive Breast Cancers.

The Prolactin Receptor (PRLR) is a type 1 cytokine receptor that is expressed in a subset of breast cancers and may contribute to its pathogenesis. It is relatively overexpressed in approximately 25% of human breast tumors while expressed at low levels in some normal human tissues including the mammary gland. We developed an anti-PRLR antibody-drug conjugate (ADC), to target PRLR-positive breast cancer. REGN2878-DM1 is comprised of a fully human high-affinity function-blocking anti-PRLR IgG1 antibody (REGN2878) conjugated via a noncleavable SMCC linker to the cytotoxic maytansine derivative DM1. Both unconjugated REGN2878 and conjugated REGN2878-DM1 block PRL-mediated activation in vitro and are rapidly internalized into lysosomes. REGN2878-DM1 induces potent cell-cycle arrest and cytotoxicity in PRLR-expressing tumor cell lines. In vivo, REGN2878-DM1 demonstrated significant antigen-specific antitumor activity against breast cancer xenograft models. In addition, REGN2878-DM1 showed additive activity when combined with the antiestrogen agent fulvestrant. These results illustrate promising antitumor activity against PRLR-positive breast cancer xenografts and support the evaluation of anti-PRLR ADCs as potential therapeutic agents in breast cancer. Mol Cancer Ther; 16(7); 1299-311. ©2017 AACR.

Oxygen pressures in the interstitial space of skeletal muscle and tumors in vivo.

A new Oxyphor (Oxyphor G3) has been used to selectively determine the oxygen pressure in interstitial (pericellular) spaces. Oxyphor G3 is a Pd-tetrabenzoporphyrin, encapsulated inside generation 2 poly-arylglycine (AG) dendrimer, and therefore is a true near infrared oxygen sensor, having a strong absorption band at 636nm and emission near 800nm. The periphery of the dendrimer is modified with oligoethylene glycol residues (Av. MW 350) to make the probe water soluble and biologically inert. Oxyphor G3 was injected along "tracks" in the tissue using a small needle (30gage or less) and remained in the pericellular space, allowing oxygen measurements for several hours with a single injection. The oxygen pressure distributions (histograms) were compared with those for Oxyphor G2 in the intravascular (blood plasma) space. In normal muscle, in the lower oxygen pressure region of the histograms (capillary bed) the oxygen pressure difference was small. At higher oxygen pressures in the histograms there were differences consistent with the presence of high flow vessels with oxygen pressures substantially above those of the surrounding interstitial space. In tumors, the oxygen pressures in the two spaces were similar but with large differences among tumors. In mice, anesthesia with ketamine plus xylazine markedly decreased oxygen pressures in the interstitial and intravascular spaces compared to awake or isoflurane anesthetized mice.

Hemodynamic responses to antivascular therapy and ionizing radiation assessed by diffuse optical spectroscopies.

Diffuse optical methods were used to monitor two different therapies in K1735 malignant mouse melanoma tumor models: anti-vascular therapy and radiation therapy. Anti-vascular therapy induced acute variation in hemodynamic parameters within an hour, and radiation therapy induced longitudinal changes within 2 weeks. During anti-vascular therapy, the drug Combretastatin A-4 3-O-Phosphate (CA4P, 2.5 mg/200 mul PBS/mouse) significantly decreased tissue blood flow (65%) and blood oxygenation (38%) one hour after injection. In the longitudinal study, single-fraction ionizing radiation (12 Gy x 1) induced significant reduction of tissue blood flow (36%) and blood oxygenation (24%) 14 days after radiation. The results correlated well with contrast enhanced ultrasound, tumor histology, and a nitroimidazole hypoxia marker (EF5). The research provides further evidence that noninvasive diffuse optical spectroscopies can be useful tools for monitoring cancer therapy in vivo.

Inhibition of tumor endothelial ERK activation, angiogenesis, and tumor growth by sorafenib (BAY43-9006).

Activation of the Raf-MEK-ERK signal transduction pathway in endothelial cells is required for angiogenesis. Raf is the kinase most efficiently inhibited by the multikinase inhibitor sorafenib, which has shown activity against certain human cancers in clinical trials. To understand the mechanisms underlying this activity, we studied how it controlled growth of K1735 murine melanomas. Therapy caused massive regional tumor cell death accompanied by severe tumor hypoxia, decreased microvessel density, increased percentage of pericyte-covered vessels, and increased caliber and decreased arborization of vessels. These signs of K1735 angiogenesis inhibition, along with its ability to inhibit Matrigel neovascularization, showed that sorafenib is an effective anti-angiogenic agent. Extracellular signal-regulated kinase (ERK) activation in tumor endothelial cells, revealed by immunostaining for phospho-ERK and CD34, was inhibited, whereas AKT activation, revealed by phospho-AKT immunostaining, was not inhibited in K1735 and two other tumor types treated with sorafenib. Treatment decreased endothelial but not tumor cell proliferation and increased both endothelial cell and tumor cell apoptosis. These data indicate that sorafenib's anti-tumor efficacy may be primarily attributable to angiogenesis inhibition resulting from its inhibition of Raf-MEK-ERK signaling in endothelial cells. Assessing endothelial cell ERK activation in tumor bio-psies may provide mechanistic insights into and allow monitoring of sorafenib's activity in patients in clinical trials.

Oxygen pressures in the interstitial space and their relationship to those in the blood plasma in resting skeletal muscle.

This study compared oxygen pressures (Po(2)), measured by oxygen-dependent quenching of phosphorescence, in the intravascular (blood plasma) space in the muscle with those in the interstitial (pericellular) space. Our hypothesis was that the capillary wall would not significantly impede oxygen diffusion from the blood plasma to the pericellular space. A new near-infrared oxygen sensitive probe, Oxyphor G3, was used to obtain oxygen distributions in the interstitial space. Oxyphor G3 is a Pd-tetrabenzoporphyrin encapsulated inside generation 2 poly-arylglycine (AG) dendrimer. The periphery of the dendrimer is modified with oligoethylene glycol residues (average molecular weight 350) to make the probe water soluble and biologically inert. Oxyphor G3 was injected into thigh muscle using a 30-gauge needle. Histograms of the Po(2) in the interstitial space were measured in awake and anesthetized animals and compared with those for Oxyphor G2 in the intravascular (blood plasma) space. For awake mice, the lowest 10% of Po(2) values for the interstitial and intravascular spaces (believed to represent capillary bed) were not significantly different [23.8 (SD 4.5) and 25 Torr (SD 4.3), respectively], whereas, in isoflurane-anesthetized mice, there was a small but significant (P = 0.01) difference [20.4 (SD 6.3) and 27.9 Torr (SD 3.5), respectively]. The peak values for the histograms for the interstitial space in awake and isoflurane-anesthetized mice were 40.8 (SD 7.5) and 36.9 Torr (SD 8.3), respectively, whereas those for the intravascular space were 52.2 (SD 4.9) and 55.9 Torr (SD 8.4), respectively, showing no significant difference due to isoflurane anesthesia. The histograms for the intravascular space were significantly wider, with more contribution at higher Po(2) values. A different anesthetic, ketamine plus xylazine injected intraperitoneally, caused a marked decrease in the tissue Po(2) values in both spaces, with the time course and extent of the decrease dependent on the time after injection and variable among mice. It was, therefore, not further used.

Ionizing radiation inhibits tumor neovascularization by inducing ineffective angiogenesis.

The vascular effects of ionizing radiation were examined in K1735 murine melanoma tumors. Single-fraction and fractionated radiation virtually arrested growth of these tumors for about a week, after which they resumed more rapid growth. Tumor microvessel density (MVD) and blood perfusion was unchanged seven days after radiation but decreased at later time points after irradiation, when they had grown 10-fold or more. Together with the finding of severe tumor hypoxia and VEGF induction in the latter tumors, the evidence pointed to vascular insufficiency and inhibited neovascularization in tumors that had grown substantially after radiation. Endothelial cell (EC) death detected by TUNEL staining only transiently increased the day following radiation, whereas EC proliferation detected by Ki-67 staining was increased in irradiated tumors that had grown substantially. The fact that increased EC proliferative activity produced fewer vessels suggests that angiogenesis is defective or ineffective after radiation. These results complement recent genetic evidence that EC damage from radiation plays a major role in tissue damage and antitumor efficacy to highlight the importance of EC and vasculature in radiation response. Our studies further show that radiation impact on tumor vasculature extends beyond near-term induction of EC death to more prolonged effects on their ability to support angiogenesis.

Selective cytokine inhibitory drugs with enhanced antiangiogenic activity control tumor growth through vascular inhibition.

Selective cytokine inhibitory drugs (SelCIDs) are a novel class of phosphodiesterase 4 inhibitors discovered during a thalidomide analog discovery program. These analogs were evaluated for their ability to inhibit tumor angiogenesis, vascularity, and growth. Two analogs (CC-7034 and CC-9088) were identified that had enhanced antiangiogenic activity in Matrigel assays compared with parental thalidomide. These analogs also inhibited the growth of established K1735 and RENCA murine tumors. Tumors whose growth was suppressed by SelCID treatment exhibited decreased vessel density together with increased tumor cell hypoxia and death. The decrease in vascularity produced by SelCID treatment is attributed to a selective loss of vessels devoid of pericyte coverage, suggesting that these agents target immature tumor vessels. That tumor cell death was localized to relatively avascular or hypoxic areas, coupled with the fact that none of the analogs was cytotoxic in vitro against the tumor cells, demonstrates that these analogs are novel antivascular agents with potent antitumor activity.

Tumor vessel development and maturation impose limits on the effectiveness of anti-vascular therapy.

The effect of anti-vascular agents on the growth of experimental tumors is well studied. Their impact on tumor vasculature, the primary therapeutic target of these agents, is not as well characterized, even though this primarily determines treatment outcome. Hypothesizing that the response of vessels to therapy is influenced by their stage of maturation, we studied vascular development and the vascular effects of therapy in several transplanted murine tumor models. Based on size, perfusion, endothelial cell (EC) proliferation, and the presence of pericytes, tumor vessels segregated into three categories. Least mature were highly proliferative, nonperfused EC sprouts emanating from functional vessels. Intermediate were small, perfused vessels which, like the angiogenic sprouts, were not covered by pericytes. Most mature were larger vessels, which were predominantly pericyte-covered with quiescent ECs and few associated sprouts. Thus, a developmental order, similar to that described during physiological neovascularization, was evident among vessels in growing tumors. This order markedly influenced tumor vessel response to anti-vascular therapy with recombinant interleukin-12. Therapy reduced tumor vessel density, which was attributable to a decrease in angiogenic sprouts and induction of EC apoptosis in pericyte-negative vessels. Although the great majority of vessels in growing tumors lacked pericyte coverage, selective loss of less mature vessels with therapy significantly increased the fraction of pericyte-positive vessels after therapy. These data indicate that the therapeutic susceptibility of tumor vasculature to recombinant murine IL-12 and, potentially, other anti-vascular agents is limited by its level of maturation. An implication is that tumor susceptibility is similarly limited, making pericyte coverage of tumor vasculature a potential indicator of tumor responsiveness.