Three agonist antibodies in combination with high-dose IL-2 eradicate orthotopic kidney cancer in mice

<p>Abstract</p> <p>Background</p> <p>Combination immunotherapies can be effective against subcutaneous tumors in mice but the effect against orthotopic malignant disease is less well characterized. In particular, a combination of three agonist antibodies, termed Tri-mAb, consisting of anti-DR5, anti-CD40 and anti-CD137 has previously been demonstrated to eradicate a large proportion of subcutaneous renal cell carcinoma (Renca) tumors (75% long-term survival), but the effect against orthotopic disease is not known.</p> <p>Purpose</p> <p>To determine the relative response of orthotopic tumors, we inoculated Renca into the kidney followed by treatment with Tri-mAb.</p> <p>Results</p> <p>We found that orthotopic tumors responded much less to treatment (~13% survival), but a significant improvement in survival was achieved through the addition of IL-2 to the treatment regimen (55% survival). All three agonist antibodies and high dose IL-2, 100,000 IU for up to six doses, were required. CD8⁺T cells were also required for optimal anti-tumor responses. Coadministration of IL-2 led to enhanced T cell activity as demonstrated by an increased frequency of IFN-gamma-producing T cells in tumor-draining lymph nodes, which may have contributed to the observed improvement of therapy against kidney tumors.</p> <p>Implications</p> <p>Responses of subcutaneous tumors to immunotherapy do not necessarily reflect how orthotopic tumors respond. The use of combination immunotherapy stimulating multiple facets of immunity and including cytokine support for T cells can induce effective anti-tumor responses against orthotopic and metastatic tumors.</p

Anti-tumor activity of genetically redirected T cells against orthotopic kidney cancer in mice

In order to test the effectiveness of adoptive immunotherapy against renal cell carcinoma (RCC) in a mouse model relevant to human disease, we first generated two murine renal cell carcinoma cell lines expressing the human tumor associated antigen erbB2 by genetic modification of the Renca and SIRCC cell lines. When injected into the kidneys of mice, these cell lines rapidly formed a primary kidney tumor that metastasized to lung, liver and various peritoneal locations Tumor-specific mouse T cells were generated by genetic modification of splenic T cells in vitro. The transgene encoded a chimeric receptor (T-body) specific for erbB2, in which a single-chain (scFv) anti-erbB2 antibody was linked to T cell activation and costimulatory domains (scFv-erbB2-CD28-zeta), to enable tumor recognition in a major histocompatibility complex-independent manner. Gene modified T cells expressed the T-body on their surface, and could respond specifically against erbB2-expressing tumor cells as demonstrated by secretion of interferon-gamma. Adoptive transfer of T-body-expressing T cells could lead to inhibition of primary tumor and metastases. Systemic delivery of these gene modified T cells led to complete eradication of disease in a proportion of mice, and this was associated with peri-vascular tissue inflammation composed of activated lymphoid cells, with macrophages and lesser numbers of eosinophils and neutrophils. This study demonstrated, for the first time, the efficacy of genetically redirected T cells against orthotopic renal cell carcinoma in mice

Evidence that maturation of <i>P. chabaudi</i> in <i>Ank-1^MRI23420/+</i> RBCs is impaired.

<p>(A) Nuclear yellow staining of parasite DNA. (B) TUNEL staining of fragmented parasite DNA, and (C) merged TUNEL and nuclear yellow staining superimposed on brightfield showing infected RBC. (D) Percentage of double positive for nuclear yellow and TUNEL stained parasites in regards to overall parasitemia in RBCs of both cohorts. Blood was collected 6–8 hours (ring stage) and 18–20 hours after invasion (trophozoite stage). Error bars indicate SEM and statistical differences with a p-value <0.05 were indicated by (*).</p

Identification of the Ank-1MRI23420 allele.

<p>(A) Haplotypes of 2nd generation offspring. Narrowing down critical interval using microsatellites on chromosome 8. Grey boxes represent heterozygous and white boxes homozygous mice. The candidate interval for Ank-1MRI23420 was refined to 22–25 Mb on Chr. 8. (B) DNA sequence electropherograms showing the T to A transversion in exon 11 for wt, Ank-1MRI23420/+ and Ank-1MRI23420/MRI23420 mice. (C) Schematic view of the amino acid change. (D) Representation of the position of the mutation within the protein.</p

Impaired phenotype of <i>P. chabaudi</i> parasites in <i>Ank-1^MRI23420/+</i> RBCs.

<p>Giemsa stained blood smears showing the morphology of parasites in wt and <i>Ank-1^MRI23420/+</i> mice 6–8 hours (A-B) and 18–20 hours (C-D) post-invasion. Black arrows indicate condensed phenotype of intraerythrocytic parasites observed in heterozygous mutant mice.</p

Protein levels of ankyrin-1 analysis.

<p>(A) Immunoblot of erythrocytic ankyrin-1 in wt, Ank-1MRI23420/+, and Ank-1MRI23420/2340 mice with the N-terminal antibody ANK-1 p89. Quantification of the intensity of bands by densitometry for full-size ankyrin-1 (210 kDa) (B) and the truncated species (C) (calculated size 49 kDa). Error bars indicate SEM.</p

Identification of <i>Ank-1^MRI23420/+</i> and <i>Ank-1^{MRI23420/23420}</i> mice.

<p>Giemsa stained peripheral blood smears from (A) wt, (B) <i>Ank-1^MRI23420/+,</i> and (D) <i>Ank-1^{MRI23420/MRI23420}</i> mice. Black arrows indicate microcytic RBC in <i>Ank-1^MRI23420/+</i> and spherocytes in <i>Ank-1^{MRI23420/23420}</i>. (C) Jaundiced postnatal day 1 of <i>Ank-1^{MRI23420/MRI23420}</i> pup and an <i>Ank-1^MRI23420/+</i> control littermate.</p

Haematological parameters on wild-type and Ank-1^MRI23420/+ mice.

<p>Automated full blood analyses were obtained on 69 wt and 72 <i>Ank-1^MRI23420</i> mice at 7 weeks of age. Values are represented as mean value ±SEM. Statistical differences are indicated as (*) p-value <0.05 and (**) p-value<0.001. WBC indicates white blood cell count; RBC, red blood cell count; HBG, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RDW, red cell distribution width; PLT, platelet count; and %Retic, %Reticulocytosis.</p

Infection rate of <i>P. chabaudi</i> in <i>Ank-1^MRI23420/+</i> compared to wt RBCs is impaired.

<p>Uninfected RBCs from wt and <i>Ank-1^MRI23420/+</i> mice were labelled individually with different fluorescent dyes mixed at a 1∶1 ratio and injected into infected animals of both groups. 10 hours later tail blood was taken and analysed on flow cytometry gating for infected RBCs +ve for either dye (ATTO 633 & 495). Results were calculated as the mean of normal and reversible labelled RBCs from each group for each background separately. Error bars are presented as SEM and (*) indicate statistical differences with a p-value <0.0001.</p