Jeannette C. Oosterwijk-Wakka

Immune responses to transgene and retroviral vector in patients treated with ex vivo–engineered T cells

Adoptive transfer of immune effector cells that are gene modified by retroviral transduction to express tumor-specific receptors constitutes an attractive approach to treat cancer. In patients with metastatic renal cell carcinoma, we performed a study with autologous T cells genetically retargeted with a chimeric antibody receptor (CAR) directed toward carbonic anhydrase IX (CAIX), an antigen highly expressed in renal cell carcinoma. In the majority of patients, we observed distinct humoral and/or cellular anti-CAIX-CAR T-cell immune responses in combination with a limited peripheral persistence of transferred CAIX-CAR T cells in the majority of patients. Humoral immune responses were anti-idiotypic in nature and neutralized CAIX-CAR-mediated T-cell function. Cellular anti-CAIX-CAR immune responses were directed to the complementarity-determining and framework regions of the CAR variable domains. In addition, 2 patients developed immunity directed against presumed retroviral vector epitopes. Here, we document the novel feature that therapeutic cells, which were ex vivo engineered by means of transduction with a minimal γ-retroviral vector, do express immunogenic vector-encoded epitopes, which might compromise persistence of these cells. These observations may constitute a critical concern for clinical ex vivo γ-retroviral gene transduction in general and CAR-retargeted T-cell therapy in particular, and underscore the need to attenuate the immunogenicity of both transgene and vector.

Molecular cloning and immunogenicity of renal cell carcinoma-associated antigen G250

The molecular cloning of the cDNA and gene encoding the renal cell carcinoma (RCC)‐associated protein G250 is described. This protein is one of the best markers for clear cell RCC: all clear‐cell RCC express this protein, whereas no expression can be detected in normal kidney and most other normal tissue. Antibody studies have indicated that this molecule might serve as a therapeutic target. In view of the induction/up‐regulation of G250 antigen in RCC, its restricted tissue expression and its possible role in therapy, we set out to molecularly define the G250 antigen, which we identified as a transmembrane protein identical to the tumor‐associated antigen MN/CAIX. We determined, by FISH analysis, that the G250/MN/CAIX gene is located on chromosome 9p12‐13. In view of the relative immunogenicity of RCC, we investigated whether the G250 antigen can be recognized by TIL derived from RCC patients. The initial characterization of 18 different TIL cultures suggests that anti‐G250 reactivity is rare. Int. J. Cancer 85:865–870, 2000.

Vaccination of patients with metastatic renal cell carcinoma with autologous dendritic cells pulsed with autologous tumor antigens in combination with interleukin-2: a phase 1 study.

Dendritic cells (DC) have been recognized as the most potent antigen presenting cells (APC) of the immune system. We performed a phase 1 study in twelve patients with metastatic renal cell carcinoma (RCC) using autologous immature DC loaded with autologous tumorlysate (TuLy) as a vaccine based on our earlier in vitro observations that such DC can activate tumor-specific cytotoxic T-lymphocytes. The treatment was combined with low-dose interleukin (IL)-2, as this has shown benefit in DC-based therapies. Patients received three intradermal vaccinations at two weekly intervals, and, after each vaccination, IL-2 was administered for 5 consecutive days. In six patients, keyhole-limpet hemocyanin (KLH) was added to the DC culture for immunologic monitoring purposes. In general, DC phenotype was CD14low, CD86high, CD40high, CD80low, and CD83low. We noticed that the number of CD14+ cultured DC increased during treatment. Nevertheless, ovalbumin uptake remained high, underlining that these cells were still functional immature DC. The vaccine was able to elicit cellular anti-KLH responses, emphasizing the ability of the injected DC to mount an immunologic response. However, proliferative responses against TuLy were not detected, and humoral responses against TuLy or KLH were absent. Objective clinical responses were not observed, but extended stable disease was noted. The absence of cellular, humoral, or clinical antitumor responses suggests that the vaccination strategy with immature DC has little benefit for patients with advanced RCC. Nevertheless, this study shows the feasibility of a completely autologous DC and tissue culture methodology for the generation of TuLy pulsed DC.

A Clinical Trial With Chimeric Monoclonal Antibody WX-G250 and Low Dose Interleukin-2 Pulsing Scheme for Advanced Renal Cell Carcinoma

PURPOSE WX-G250 is a chimeric monoclonal antibody that binds to carbonic anhydrase IX(G250/MN), which is present on greater than 95% of RCCs of the clear cell subtype. The suggested working mechanism of WX-G250 is by ADCC. Because the number of activated ADCC effector cells can be increased by a low dose interleukin-2 pulsing schedule, a multicenter study was initiated to investigate whether WX-G250 combined with LD-IL-2 could lead to an improved clinical outcome in patients with progressive RCC. MATERIALS AND METHODS A total of 35 patients with progressive clear cell RCC received weekly infusions of WX-G250 for 11 weeks combined with a daily LD-IL-2 regimen. Patients were monitored longitudinally for ADCC capacity. Radiological assessment of metastatic lesions was performed at week 16 and regularly until disease progression. RESULTS A durable clinical benefit was achieved in 8 of 35 patients (23%), including 3 with a partial response and 5 with stabilization at 24 weeks or greater. Mean survival was 22 months. In general treatment was well tolerated with little toxicity. The number of effector cells increased during treatment but lytic capacity per cell did not increase. ADCC and clinical outcome did not appear to correlate. CONCLUSIONS WX-G250 combined with LD-IL-2 in patients with metastatic RCC is safe and well tolerated. With a substantial clinical benefit and a median survival of 22 months in patients with metastatic RCC who have progressive disease at study entry combination therapy showed increased overall survival compared to WX-G250 monotherapy. Survival was at least similar to that of currently used cytokine regimens but with a favorable toxicity profile.

Two-step radio-immunotargeting of renal-cell carcinoma xenografts in nude mice with anti-renal-cell-carcinoma X anti-DTPA bispecific monoclonal antibodies

The specificity of antibodies offers unique opportunities to target tumors with radionuclides. However, due to the slow clearance of radiolabeled antibody, relatively high background is observed in non‐target organs. Pre‐targeting protocols using bispecific monoclonal antibodies (bsMAbs) and radiolabeled chelates may overcome this problem. We have evaluated the anti‐renal‐cell‐carcinoma (RCC) × anti‐DTPA bsMAb G250 x DTIn1 for 2‐step targeting of RCC tumors in nude mice. Tumor uptake of 111In‐DTPA was similar up to a 3‐day interval between bsMAb and 111In‐DTPA injections and decreased thereafter. The effect of G250 x DTIn1 protein dose was studied. High tumor uptake was seen at 1 to 4 μg, whereas at higher doses uptake decreased. Tumor was saturated with 15 μg bsMAb. At the saturating bsMAb dose the 111In‐DTPA amount was varied. High tumor uptake was observed at a 10‐fold molar excess 111In‐DTPA, whereas at higher excess uptake decreased. After priming with 15 μg bsMAb and targeting with a 10‐fold molar excess 111In‐DTPA, the biodistribution of 111In‐DTPA was studied for 1 to 48 hr after injection. Good tumor retention of 111In‐DTPA was observed, while the radiolabel cleared rapidly from the blood. Consequently, tumor‐to‐blood ratios increased with time to 500 at 24 hr after injection. In conclusion, RCC xenografts can be targeted efficiently using G250 x DTIn1 and 111In‐DTPA. However, this requires careful tuning of bsMAb protein dose and 111In‐DTPA dose. Using the optimal protein dose and 111In‐DTPA dose, high 111In‐DTPA tumor uptake and tumor‐to‐blood ratios can be obtained, thus providing good perspectives for diagnostic and therapeutic use in humans. Int. J. Cancer 75:74–80, 1998.© 1998 Wiley‐Liss, Inc.

Targeted therapy of renal cell carcinoma: synergistic activity of cG250-TNF and IFNg.

Immunotherapeutic targeting of G250/Carbonic anhydrase IX (CA‐IX) represents a promising strategy for treatment of renal cell carcinoma (RCC). The well characterized human‐mouse chimeric G250 (cG250) antibody has been shown in human studies to specifically enrich in CA‐IX positive tumors and was chosen as a carrier for site specific delivery of TNF in form of our IgG‐TNF‐fusion protein (cG250‐TNF) to RCC xenografts. Genetically engineered TNF constructs were designed as CH2/CH3 truncated cG250‐TNF fusion proteins and eucariotic expression was optimized under serum‐free conditions. In‐vitro characterization of cG250‐TNF comprised biochemical analysis and bioactivity assays, alone and in combination with Interferon‐γ (IFNγ). Biodistribution data on radiolabeled [125J] cG250‐TNF and antitumor activity of cG250‐TNF, alone and in combination with IFNγ, were measured on RCC xenografts in BALB/c nu/nu mice. Combined administration of cG250‐TNF and IFNγ caused synergistic biological effects that represent key mechanisms displaying antitumor responses. Biodistribution studies demonstrated specific accumulation and retention of cG250‐TNF at CA‐IX‐positive RCC resulting in growth inhibition of RCC and improved progression free survival and overall survival. Antitumor activity induced by targeted TNF‐based constructs could be enhanced by coadministration of low doses of nontargeted IFNγ without significant increase in side effects. Administration of cG250‐TNF and IFNγ resulted in significant synergistic tumoricidal activity. Considering the poor outcome of renal cancer patients with advanced disease, cG250‐TNF‐based immunotherapeutic approaches warrant clinical evaluation.

Alterations in expression of cadherin-6 and E-cadherin during kidney development and in renal cell carcinoma

Objectives: Cell–cell adhesion mediated by cadherins is tight and stable and preserves tissue integrity. However, during tissue remodeling, e.g., development, adhesion may be modified for morphogenic movement. Similarly, during carcinogenesis, cell–cell adhesion might alter leading to a more aggressive phenotype. Here we describe cadherin expression patterns in developing, adult, and neoplastic kidney.Methods: Fetal kidneys were obtained from voluntary terminations of pregnancy and 43 renal cell carcinomas (RCC) and normal kidneys were obtained at nephrectomy. Frozen sections of these specimens were stained immunohistochemically using antibodies against E–cadherin (ECD), cadherin–6 (CAD6) and α–catenin (α–cat).Results: CAD6 was expressed in lower and middle limbs of the S–shaped bodies, structures that will develop into renal proximal tubules, which also express CAD6. Similarly, ECD was expressed in the upper limb of S–shaped bodies, structures which will develop into distal and collecting tubules which also express ECD. Twenty–four out of 43 RCC (55.8%) displayed a CAD6 (+)/ECD (–)/α–cat (+) phenotype. The other RCC had a CAD6 (+)/ECD (+)/α–cat (+) phenotype (10/43, 23.2%), CAD6 (–)/ECD (+)/α–cat (+) phenotype (3/43, 7.0%) or CAD6 (–)/ECD (–)/α–cat (+) phenotype (6/43, 14.0%), respectively. On the other hand, normal, heterogeneous, or absent expression of CAD6 was seen in 19, 15, and 9 tumors, whereas in 11, 2, and 30 tumors, respectively, ECD expression was seen. Survival curves showed that abnormal CAD6 expression correlated with a poor prognosis rather than abnormal ECD expression.Conclusions: The combination of cadherin expression appeared to be fixed relatively early during kidney organogenesis. Since almost all RCC originate from proximal tubular epithelial cells (CAD6 (+)/ECD (–)/α–cat (+)), only 55.8% of RCC retained the original cadherin phenotype. Alterations in expression of these molecules may be a reflection of the degree of dedifferentiation from the primary organ. In addition, scoring of expression patterns including heterogeneous expression could be a useful tool to estimate the malignancy potential of the tumor.

The effect of antibody protein dose of anti-renal cell carcinoma monoclonal antibodies in nude mice with renal cell carcinoma xenografts†

Antibodies preferentially can direct radionuclides to solid tumors. However, antibody uptake in tumors is often highly heterogeneous. This heterogeneity may be overcome by increasing antibody protein dose.

Application of Monoclonal Antibody G250 Recognizing Carbonic Anhydrase IX in Renal Cell Carcinoma

Monoclonal antibody G250 (mAbG250) recognizes a determinant on carbonic anhydrase IX (CAIX). CAIX is expressed by virtually all renal cell carcinomas of the clear cell type (ccRCC), but expression in normal tissues is restricted. The homogeneous CAIX expression in ccRCC and excellent targeting capability of mAbG250 in animal models led to the initiation of the clinical evaluation of mAbG250 in (metastatic) RCC (mRCC) patients. Clinical studies confirmed the outstanding targeting ability of mAbG250 and cG250 PET imaging, as diagnostic modality holds great promise for the future, both in detecting localized and advanced disease. Confirmation of the results obtained in the non-randomized clinical trials with unmodified cG250 is needed to substantiate the value of cG250 treatment in mRCC. cG250-Based radio immuno-therapy (RIT) holds promise for treatment of patients with small-volume disease, and adjuvant treatment with unmodified cG250 may be of value in selected cases. In the upcoming years, ongoing clinical trials should provide evidence for these assumptions. Lastly, whether cG250-based RIT can be combined with tyrosine kinase inhibitors, which constitutes the current standard treatment for mRCC, needs to be established.

Effect of tyrosine kinase inhibitor treatment of renal cell carcinoma on the accumulation of carbonic anhydrase IX‐specific chimeric monoclonal antibody cG250

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