Cancer therapy in mice using a pure population of CD8+ T cell specific to the AH1 tumor rejection antigen.

There is a growing interest in the use of patient-derived T cells for the treatment of various types of malignancies. The expansion of a polyclonal and polyspecific population of tumor-reactive T cells, with a subsequent infusion into the same donor patient, has been implemented, sometimes with positive results. It is not known, however, whether a set of T cells with a single antigen specificity may be sufficient for an effective therapy. To gain more insights in this matter, we used naturally occurring T cells recognizing a retroviral peptide (AH1), which is endogenous in many tumor cell lines of BALB/c origin and which serves as potent tumor rejection antigen. We were able to isolate and expand this rare population of T cells to numbers suitable for therapy experiments in mice (i.e., up to 30 × 106 cells/mouse). After the expansion process, T cells efficiently killed antigen-positive tumor cells in vitro and demonstrated tumor growth inhibition in two syngeneic murine models of cancer. However, AH1-specific T cells failed to induce complete regressions of established tumors. The incomplete activity was associated with a failure of injected T cells to survive in vivo, as only a very limited amount of T cells was found in tumor or secondary lymphoid organs 72 h after injection. These data suggest that future therapeutic strategies based on autologous T cells may require the potentiation of tumor-homing and survival properties of cancer-specific T cells.

A Novel Antibody-IL15 Fusion Protein Selectively Localizes to Tumors, Synergizes with TNF-based Immunocytokine, and Inhibits Metastasis.

IL15 is an immunostimulatory cytokine that holds promises for cancer therapy, but its performance (alone or as partner for fusion proteins) has often been limited by suboptimal accumulation in the tumor and very rapid clearance from circulation. Most recently, the Sushi Domain (SD, the shortest region of IL15 receptor α, capable of binding to IL15) has been fused to IL15-based anticancer products to increase its biological activity. Here, we describe two novel antibody fusion proteins (termed F8-F8-IL15 and F8-F8-SD-IL15), specific to the alternatively spliced EDA domain of fibronectin (a marker of tumor neoangiogenisis, expressed in the majority of solid and hematologic tumors, but absent in normal healthy tissues) and featuring the F8 antibody in single-chain diabody format (with a short linker between VH and VL, thus allowing the domains to pair with the complementary ones of another chain). Unlike previously described fusions of the F8 antibody with human IL15, F8-F8-IL15 and F8-F8-SD-IL15 exhibited a preferential uptake in solid tumors, as evidenced by quantitative biodistribution analysis with radioiodinated protein preparations. Both products were potently active in vivo against mouse metastatic colon carcinomas and in sarcoma lesion in combination with targeted TNF. The results may be of clinical significance, as F8-F8-IL15 and F8-F8-SD-IL15 are fully human proteins, which recognize the cognate tumor-associated antigen with identical affinity in mouse and man.

Antibody-mediated delivery of LIGHT to the tumor boosts natural killer cells and delays tumor progression.

LIGHT is a member of the tumor necrosis factor superfamily, which has been claimed to mediate anti-tumor activity on the basis of cancer cures observed in immunocompetent mice bearing transgenic LIGHT-expressing tumors. The preclinical development of a LIGHT-based therapeutic has been hindered by the lack of functional stability exhibited by this protein. Here, we describe the cloning, expression, and characterization of five antibody-LIGHT fusion proteins, directed against the alternatively spliced extra domain A of fibronectin, a conserved tumor-associated antigen. Among the five tested formats, only the sequential fusion of the F8 antibody in single-chain diabody format, followed by the LIGHT homotrimer expressed as a single polypeptide, yielded a protein (termed "F8-LIGHT") that was not prone to aggregation. A quantitative biodistribution analysis in tumor-bearing mice, using radio-iodinated protein preparations, confirmed that F8-LIGHT was able to preferentially accumulate at the tumor site, with a tumor-to-blood ratio of ca. five to one 24 hours after intravenous administration. Tumor therapy experiments, performed in two murine tumor models (CT26 and WEHI-164), featuring different levels of lymphocyte infiltration into the neoplastic mass, revealed that F8-LIGHT could significantly reduce tumor-cell growth and was more potent than a similar fusion protein (KSF-LIGHT), directed against hen egg lysozyme and serving as negative control of irrelevant specificity in the mouse. At a mechanistic level, the activity of F8-LIGHT was mainly due to an intratumoral expansion of natural killer cells, whereas there was no evidence of expansion of CD8 + T cells, neither in the tumor, nor in draining lymph nodes. Abbreviations: CTLA-4: Cytotoxic T-lymphocytes-associated protein 4; EGFR: Epidermal growth factor receptor; HVEM: Herpesvirus entry mediator; IFNγ: Interferon-gamma; LIGHT: Lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for binding to herpesvirus entry mediator, a receptor expressed on T lymphocytes; LTßR: Lymphotoxin beta receptor; NF-κB: Nuclear factor "kappa-light-chain-enhancer" of activated B cells; NK: Natural killer cells; PD-1: Programmed cell death protein 1; PD-L1: Programmed death-ligand 1; TNF: Tumor necrosis factor.

An engineered 4-1BBL fusion protein with "activity on demand".

Engineered cytokines are gaining importance in cancer therapy, but these products are often limited by toxicity, especially at early time points after intravenous administration. 4-1BB is a member of the tumor necrosis factor receptor superfamily, which has been considered as a target for therapeutic strategies with agonistic antibodies or using its cognate cytokine ligand, 4-1BBL. Here we describe the engineering of an antibody fusion protein, termed F8-4-1BBL, that does not exhibit cytokine activity in solution but regains biological activity on antigen binding. F8-4-1BBL bound specifically to its cognate antigen, the alternatively spliced EDA domain of fibronectin, and selectively localized to tumors in vivo, as evidenced by quantitative biodistribution experiments. The product promoted a potent antitumor activity in various mouse models of cancer without apparent toxicity at the doses used. F8-4-1BBL represents a prototype for antibody-cytokine fusion proteins, which conditionally display "activity on demand" properties at the site of disease on antigen binding and reduce toxicity to normal tissues.

Targeting an engineered cytokine with interleukin-2 and interleukin-15 activity to the neovasculature of solid tumors.

There is a growing interest in the antibody-based delivery of cytokines to the tumor environment as a means to boost the anti-cancer activity of tumor-resident T cells and NK cells. Here, we describe the expression and characterization of fusion proteins, featuring the L19 antibody (specific to the alternatively-spliced EDB domain of fibronectin) and an engineered cytokine with interleukin-2 and interleukin-15 properties. The cytokine moiety was fused either at the N-terminal or at the C-terminal extremity and both fusion proteins showed a selective tumor accumulation in a quantitative biodistribution experiment. The N-terminal fusion inhibited tumor growth in immunocompetent mice bearing F9 carcinomas or WEHI-164 sarcomas when used as single agent. The anticancer activity was compared to the one of the same cytokine payload used as recombinant protein or fused to an anti-hen egg lysozyme antibody, serving as negative control of irrelevant specificity in the mouse. These results indicate that the antibody-based delivery of engineered cytokines to the tumor neovasculature may mediate a potent anticancer activity.

A novel format for recombinant antibody-interleukin-2 fusion proteins exhibits superior tumor-targeting properties in vivo.

The targeted delivery of interleukin-2 to the tumor is gaining attention as an avenue to potentiate the action of T and NK cells at the site of disease. We have previously described the fusion of the L19 antibody, specific to the EDB domain of fibronectin, with human interleukin-2, using a non-covalent homodimeric diabody format. Here, we describe four novel formats for the L19-IL2 fusion, featuring different arrangements of antibody and IL2. A comparative quantitative biodistribution analysis in tumor-bearing mice using radioiodinated proteins revealed that the novel format (L19L19-IL2, with the antibody in single-chain diabody format) exhibited the best biodistribution results. In vitro assays on peripheral blood mononuclear cells showed a decrease activation of regulatory T cells when single IL2 domain was used. In vivo, both L19-IL2 and L19L19-IL2 inhibited tumor growth in immunocompetent mouse models of cancer. T-cell analysis revealed similar levels of CD4+ and FoxP3+ cells, with an expansion of the CD8+ T cell in mice treated with L19-IL2 and L19L19-IL2. The percentage of CD4+ regulatory T cells was markedly decreased with L19L19-IL2 combined with a mouse-specific PD-1 blocker. Collectively, these data indicate that the new L19L19-IL2 format exhibits favorable tumor-homing properties and mediates a potent anti-cancer activity in vivo.

Immunotherapy of CT26 murine tumors is characterized by an oligoclonal response of tissue-resident memory T cells against the AH1 rejection antigen.

Mice bearing CT26 tumors can be cured by administration of L19-mIL12 or F8-mTNF, two antibody fusion proteins which selectively deliver their cytokine payload to the tumor. In both settings, cancer cures crucially depended on CD8+ T cells and the AH1 peptide (derived from the gp70 protein of the murine leukemia virus) acted as the main tumor-rejection antigen, with ∼50% of CD8+ T cells in the neoplastic mass being AH1-specific after therapy. In order to characterize the clonality of the T cell response, its phenotype, and activation status, we isolated CD8+ T cells from tumors and secondary lymphoid organs and submitted them to T cell receptor (TCR) and total mRNA sequencing. We found an extremely diverse repertoire of more than 40 000 unique TCR sequences, but the ten most abundant TCRs accounted for >60% of CD8+ T-cell clones in the tumor. AH1-specific TCRs were consistently found among the most abundant sequences. AH1-specific T cells in the tumor had a tissue-resident memory phenotype. Treatment with L19-mIL12 led to overexpression of IL-12 receptor and of markers of cell activation and proliferation. These data suggest that the antitumor response driven by antibody-cytokine fusions proceeds through an oligoclonal expansion and activation of tumor-infiltrating CD8+ T cells.

The antibody-based delivery of interleukin-12 to solid tumors boosts NK and CD8+ T cell activity and synergizes with immune checkpoint inhibitors.

We describe the cloning and characterization of a novel fusion protein (termed L19-mIL12), consisting of murine interleukin-12 in single-chain format, sequentially fused to the L19 antibody in tandem diabody format. The fusion protein bound avidly to the cognate antigen (the alternatively spliced EDB domain of fibronectin), retained the activity of the parental cytokine and was able to selectively localize to murine tumors in vivo, as shown by quantitative biodistribution analysis. L19-mIL12 exhibited a potent antitumor activity in immunocompetent mice bearing CT26 carcinomas and WEHI-164 sarcomas, which could be boosted by combination with checkpoint blockade, leading to durable cancer eradication. L19-mIL12 also inhibited tumor growth in mice with Lewis lung carcinoma (LLC), but in this case, cancer cures could not be obtained, both in monotherapy and in combination. A microscopic analysis and a depletion experiment of tumor-infiltrating leukocytes illustrated the contribution of NK cells and CD8+ T cells for the anticancer activity observed in both tumor models. Upon L19-mIL12 treatment, the density of regulatory T cells (Tregs) was strongly increased in LLC, but not in CT26 tumors. A FACS analysis also revealed that the majority of CD8+ T cells in CT26 tumors were specific to the retroviral AH1 antigen.

Antibody-Based Delivery of Cytokine Payloads to Carbonic Anhydrase IX Leads to Cancer Cures in Immunocompetent Tumor-Bearing Mice.

Antibody-cytokine fusion proteins can have the potential to increase the density and activity of subsets of leukocytes within the tumor mass. Here, we describe the design, production, and characterization of four novel antibody-cytokine fusion proteins directed against human carbonic anhydrase IX, a highly validated marker of hypoxia that is overexpressed in clear cell renal cell carcinoma and other malignancies. As immunomodulatory payloads we used TNF, IL2, IFNα2 (corresponding to products that are in clinical use), and IL12 (as this cytokine potently activates T cells and NK cells). Therapy experiments were performed in BALB/c mice, bearing CT26 tumors transfected with human carbonic anhydrase IX, in order to assess the performance of the fusion proteins in an immunocompetent setting. The biopharmaceuticals featuring TNF, IL2, or IL12 as payloads cured all mice in their therapy groups, whereas only a subset of mice was cured by the antibody-based delivery of IFNα2. Although the antibody fusion with TNF mediated a rapid hemorrhagic necrosis of the tumor mass, a slower regression of the neoplastic lesions (which continued after the last injection) was observed with the other fusion proteins, and treated mice acquired protective anticancer immunity. A high proportion of tumor-infiltrating CD8+ T cells was specific to the retroviral antigen AH1; however, the LGPGREYRAL peptide derived from human carbonic anhydrase IX was also present on tumor cells. The results described herein provide a rationale for the clinical use of fully human antibody-cytokine fusions specific to carbonic anhydrase IX.

Antibody-based Delivery of TNF to the Tumor Neovasculature Potentiates the Therapeutic Activity of a Peptide Anticancer Vaccine.

PURPOSE: There is a growing interest in the use of tumor antigens for therapeutic vaccination strategies. Unfortunately, in most cases, the use of peptide vaccines in patients does not mediate shrinkage of solid tumor masses.Experimental Design: Here, we studied the opportunity to boost peptide vaccination with F8-TNF, an antibody fusion protein that selectively delivers TNF to the tumor extracellular matrix. AH1, a model antigen to investigate CD8+ T-cell immunity in BALB/c mice, was used as vaccine. RESULTS: Peptide antigens alone exhibited only a modest tumor growth inhibition. However, anticancer activity could be substantially increased by combination with F8-TNF. Analysis of T cells in tumors and in draining lymph nodes revealed a dramatic expansion of AH1-specific CD8+ T cells, which were strongly positive for PD-1, LAG-3, and TIM-3. The synergistic anticancer activity, observed in the combined use of peptide vaccination and F8-TNF, was largely due to the ability of the fusion protein to induce a rapid hemorrhagic necrosis in the tumor mass, thus leaving few residual tumor cells. While the cell surface phenotype of tumor-infiltrating CD8+ T cells did not substantially change upon treatment, the proportion of AH1-specific T cells was strongly increased in the combination therapy group, reaching more than 50% of the CD8+ T cells within the tumor mass. CONCLUSIONS: Because both peptide vaccination strategies and tumor-homing TNF fusion proteins are currently being studied in clinical trials, our study provides a rationale for the combination of these 2 regimens for the treatment of patients with cancer.

Targeted Delivery of TNF Potentiates the Antibody-Dependent Cell-Mediated Cytotoxicity of an Anti-Melanoma Immunoglobulin.

The recombinant murine IgG2a antibody TA99, directed against a melanoma antigen, was used to study combination modalities that potentiate antibody-dependent cell cytotoxicity. As previously reported, IgG2a(TA99) was extremely efficacious in preventing the growth of B16 lung metastases. However, the same antibody mediated only minimal tumor growth retardation when used to treat established neoplastic masses. The therapeutic activity of IgG2a(TA99) could be substantially enhanced by co-administration with an antibody-cytokine fusion (TA99-murine tumor necrosis factor [mTNF]), consisting of the TA99 antibody in single-chain variable fragment format fused to murine TNF. This fusion protein efficiently killed endothelial cells in vitro and displayed only minimal activity against B16 melanoma cells. In vivo, TA99-mTNF boosted the influx of natural killer cells and macrophages into B16 melanoma lesions. Therapy studies with two different administration schedules showed that the combination of TA99-mTNF and IgG2a(TA99) was superior to the individual products used as single agents. The combination treatment converted most of the tumor mass into a necrotic lesion, but a vital tumor rim eventually regrew, even when dacarbazine was included in the therapeutic regimen. The treatment modality described in this article may be applicable to the treatment of melanoma patients, given the specificity of the gp75 antigen and its conservation across species.

Non-internalizing antibody-drug conjugates display potent anti-cancer activity upon proteolytic release of monomethyl auristatin E in the subendothelial extracellular matrix.

Antibody-drug conjugates (ADCs) represent a promising class of biopharmaceuticals with the potential to localize at the tumor site and improve the therapeutic index of cytotoxic drugs. While it is generally believed that ADCs need to be internalized into tumor cells in order to display optimal therapeutic activity, it has recently been shown that non-internalizing antibodies can efficiently liberate disulfide-linked drugs at the extracellular tumor site, leading to potent anti-cancer activity in preclinical animal models. Here, we show that engineered variants of the F16 antibody, specific to a splice isoform of tenascin-C, selectively localize to the subendothelial tumor extracellular matrix in three mouse models of human cancer (U87, A431, MDA-MB-231). A site-specific coupling of F16 in IgG format with a monomethyl auristatin E (MMAE) derivative, featuring a valine-citrulline dipeptide linker equipped with a self-immolative spacer, yielded an ADC product, which cured tumor-bearing mice at a dose of 7 mg/Kg. The observation of an efficient extracellular proteolytic cleavage of the valine-citrulline linker was surprising, as it has generally been assumed that this peptidic structure would be selectively cleaved by cathepsin B in intracellular compartments. The products described in this article may be useful for the treatment of human malignancies, as their cognate antigen is strongly expressed in the majority of human solid tumors, lymphomas and aggressive leukemias, while being virtually undetectable in most normal adult tissues.