Charles S. Carter

Use of tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. A preliminary report.

Lymphocytes extracted from freshly resected melanomas can be expanded in vitro and can often mediate specific lysis of autologous tumor cells but not allogeneic tumor or autologous normal cells. We treated 20 patients with metastatic melanoma by means of adoptive transfer of these tumor-infiltrating lymphocytes and interleukin-2, after the patients had received a single intravenous dose of cyclophosphamide. Objective regression of the cancer was observed in 9 of 15 patients (60 percent) who had not previously been treated with interleukin-2 and in 2 of 5 patients (40 percent) in whom previous therapy with interleukin-2 had failed. Regression of cancer occurred in the lungs, liver, bone, skin, and subcutaneous sites and lasted from 2 to more than 13 months. Toxic effects of interleukin-2 occurred, although the treatment course was short (five days); these side effects were reversible. It appears that in patients with metastatic melanoma, this experimental treatment regimen can produce higher response rates than those achieved with interleukin-2 administered alone or with lymphokine-activated killer cells. It is too early to determine whether this new form of immunotherapy can improve survival, but further trials seem warranted.

T Lymphocyte-Directed Gene Therapy for ADA− SCID: Initial Trial Results After 4 Years

In 1990, a clinical trial was started using retroviral-mediated transfer of the adenosine deaminase (ADA) gene into the T cells of two children with severe combined immunodeficiency (ADA− SCID). The number of blood T cells normalized as did many cellular and humoral immune responses. Gene treatment ended after 2 years, but integrated vector and ADA gene expression in T cells persisted. Although many components remain to be perfected, it is concluded here that gene therapy can be a safe and effective addition to treatment for some patients with this severe immunodeficiency disease.

Lymphopenia and interleukin-2 therapy alter homeostasis of CD4+CD25+ regulatory T cells

CD4+CD25+ regulatory T (Treg) cells have a crucial role in maintaining immune tolerance. Mice and humans born lacking Treg cells develop severe autoimmune disease, and depletion of Treg cells in lymphopenic mice induces autoimmunity. Interleukin (IL)-2 signaling is required for thymic development, peripheral expansion and suppressive activity of Treg cells. Animals lacking IL-2 die of autoimmunity, which is prevented by administration of IL-2–responsive Treg cells. In light of the emerging evidence that one of the primary physiologic roles of IL-2 is to generate and maintain Treg cells, the question arises as to the effects of IL-2 therapy on them. We monitored Treg cells during immune reconstitution in individuals with cancer who did or did not receive IL-2 therapy. CD4+CD25hi cells underwent homeostatic peripheral expansion during immune reconstitution, and in lymphopenic individuals receiving IL-2, the Treg cell compartment was markedly increased. Mouse studies showed that IL-2 therapy induced expansion of existent Treg cells in normal hosts, and IL-2–induced Treg cell expansion was further augmented by lymphopenia. On a per-cell basis, Treg cells generated by IL-2 therapy expressed similar levels of FOXP3 and had similar potency for suppression compared to Treg cells present in normal hosts. These studies suggest that IL-2 and lymphopenia are primary modulators of CD4+CD25+ Treg cell homeostasis.

Tumor localization of adoptively transferred indium-111 labeled tumor infiltrating lymphocytes in patients with metastatic melanoma.

Lymphoid cells infiltrating into human tumors can be expanded in vitro in medium containing interleukin-2 (IL-2). Adoptive transfer of these tumor-infiltrating lymphocytes (TIL) mediates potent antitumor effects in murine tumor models. Clinical trials to evaluate the efficacy of these cells in patients with advanced cancer are underway. We have investigated whether infused TIL labeled with indium 111 (111In) oxine can traffic and localize to metastatic deposits of tumor. Six patients with metastatic malignant melanoma who had multiple sites of subcutaneous, nodal, and/or visceral disease were the subjects of the study. The patients received cyclophosphamide 36 hours before receiving the intravenous (IV) infusion of TIL followed by IL-2 IV every eight hours. The distribution and localization of the TIL were evaluated using serial whole body gamma camera imaging, serial blood and urine samplings, and serial biopsies of tumor and normal tissue. 111In-labeled TIL localized to lung, liver, and spleen within two hours after the infusion of activity. Activity in the lung diminished within 24 hours. As early as 24 hours after injection of 111In-labeled TIL, localization of TIL to sites of metastatic deposits was demonstrated in all six patients using either imaging studies or biopsy specimens or both. 111In activity in tumor tissue biopsies ranged from three to 40 times greater than activity in normal tissue. A progressive increase in the radioactive counts at sites of tumor deposit was seen. This study shows that labeled TIL can localize preferentially to tumor, and provides information concerning the possible mechanism of the therapeutic effects of TIL.

T cell-depleted bone marrow transplantation and delayed T cell add- back to control acute GVHD and conserve a graft-versus-leukemia effect

Thirty-eight patients with hematological malignancies, received T cell-depleted marrow transplants (BMT) and cyclosporine to prevent acute graft-versus-host disease (aGVHD), followed by delayed add-back of donor lymphocytes to prevent leukemia relapse. In 26 patients scheduled for donor T cell add-back of 2 × 106 cells/kg on day 30 and 5 × 107 cells/kg on day 45 (schedule 1), the overall probability of grade ⩾II aGVHD developing was 31.5%, with a 15.5% probability of aGVHD occurring after T cell add-back. In 12 patients receiving 107 donor T cells/kg on day 30 (schedule 2), the probability of grade ⩾II aGVHD was 100%. The incidence of grade III–IV aGVHD was higher in schedule 2 than in schedule 1 (P = 0.02). Of 24 evaluable patients, 10 (46%) developed chronic GVHD which was limited in eight and extensive in two. Current disease-free survival for 18 patients at standard risk for relapse (chronic myeloid leukemia (CML) in chronic or accelerated phase, acute myeloid leukemia in remission) vs 20 patients with more advanced leukemia or multiple myeloma were respectively 72% vs 12% (P < 0.01) with a 29% vs 69% probability of relapse (P = 0.08). In 12 CML patients surviving more than 3 months, PCR analysis of the BCR/ABL transcript showed that minimal residual disease after T cell add-back was transient except in two patients who developed hematological relapse. Results indicate that the risk of acute GVHD is low following substantial T cell doses, transfused 45 days after transplant, using cyclosporine prophylaxis. Furthermore a graft-versus-leukemia effect was conserved.

Engraftment of Hematopoietic Progenitor Cells Transduced with the Fanconi Anemia Group C Gene (FANCC)

Fanconi anemia (FA) is an autosomal recessive disorder that leads to aplastic anemia. Mutations in the FANCC gene account for 10-15% of cases. FA cells are abnormally sensitive to DNA-damaging agents such as mitomycin C (MMC). Transfection of normal FANCC into mutant cells corrects this hypersensitivity and improves their viability in vitro. Four FA patients, representing the three major FANCC mutation subgroups, were entered into a clinical trial of gene transduction aimed at correction of the hematopoietic defect. Three patients received three or four cycles of gene transfer, each consisting of one or two infusions of autologous hematopoietic progenitor cells that had been transduced ex vivo with a retroviral vector carrying the normal FANCC gene. Prior to infusion, the FANCC transgene was demonstrated in transduced CD34-enriched progenitor cells. After infusion, FANCC was also present transiently in peripheral blood (PB) and bone marrow (BM) cells. Function of the normal FANCC transgene was suggested by a marked increase in hematopoietic colonies measured by in vitro cultures, including colonies grown in the presence of MMC, after successive gene therapy cycles in all patients. Transient improvement in BM cellularity coincided with this expansion of hematopoietic progenitors. A fourth patient, who received a single infusion of transduced CD34-enriched BM cells, was given radiation therapy for a concurrent gynecologic malignancy. The FANCC transgene was detected in her PB and BM cells only after recovery from radiation-induced aplasia, suggesting that FANCC gene transduction confers a selective engraftment advantage. These experiments highlight both the potential and difficulties in applying gene therapy to FA.

Allogeneic Lymphocytes Induce Tumor Regression of Advanced Metastatic Breast Cancer

PURPOSE Allogeneic T lymphocytes can induce regression of metastatic breast cancer through an immune-mediated graft-versus-tumor (GVT) effect in murine models. To determine if a clinical GVT effect exists against metastatic breast cancer, allogeneic lymphocytes were used as adoptive cellular therapy after a reduced-intensity chemotherapy conditioning regimen and allogeneic hematopoietic stem-cell transplantation (HSCT) from human leukocyte antigen-matched siblings. PATIENTS AND METHODS Sixteen patients with metastatic breast cancer that had progressed after treatment with anthracyclines, taxanes, hormonal agents, and trastuzumab, received allogeneic HSCT. The reduced-intensity transplant conditioning regimen consisted of cyclophosphamide and fludarabine. To distinguish an immunological GVT effect from any antitumor effect of cytotoxic chemotherapy in the transplant-conditioning regimen, allogeneic T lymphocytes were removed from the stem-cell graft and were subsequently administered late postallogeneic HSCT. Allogeneic lymphocytes containing 1 x 10(6), 5 x 10(6), and 10 x 10(6) CD3(+) cells/kg were infused on days +42, +70, and +98 post-allogeneic HSCT, respectively. RESULTS Objective tumor regressions occurred after day +28 post-allogeneic HSCT in six patients and were attributed to allogeneic lymphocyte infusions. Two of these responding patients had disease progression post-allogeneic HSCT before subsequent tumor regression. Tumor regressions occurred concomitantly with the establishment of complete donor T-lymphoid engraftment, were associated with the development of graft-versus-host disease (GVHD), and were abrogated by subsequent systemic immunosuppression for GVHD. CONCLUSION Allogeneic lymphocytes can induce regression of advanced metastatic breast cancer. These results indicate that an immunological GVT effect from allogeneic lymphocytes exists against metastatic breast cancer and provide rationale for further development of allogeneic cellular therapy for this largely incurable disease.

Dendritic cells signal T cells in the absence of exogenous antigen.

Interactions with self–major histocompatibility complex molecules on dendritic cells (DCs) are important for the survival of mature CD4+ T cells. We have followed the DC-mediated signal from the T cell surface to the nucleus and identified a pattern of activation that correlates with increased in vitro survival. This response is induced exclusively by DCs and is likely associated with a modulation of the T cell activation threshold. We have also found that DC-mediated activation results in antigen-independent cytokine gene expression, which points to a new role for DCs in shaping the cytokine milieu. Such antigen-independent activation of T cells may play a role in protective immunity, but may also induce and perpetuate autoimmune states such as multiple sclerosis.

Transplant dose of CD34(+) and CD3(+) cells predicts outcome in patients with haematological malignancies undergoing T cell-depleted peripheral blood stem cell transplants with delayed donor lymphocyte add-back.

We sought to optimize and standardize stem cell and lymphocyte doses of T cell‐depleted peripheral blood stem cell transplants (T‐PBSCT), using delayed add‐back of donor T cells (DLI) to prevent relapse and enhance donor immune recovery. Fifty‐one patients with haematological malignancies received a T‐PBSCT from an HLA‐identical sibling, followed by DLI of 1 × 107 and 5 × 107 CD3+ cells/kg on d +45 and +100 respectively. Twenty‐four patients were designated as standard risk and twenty‐seven patients with more advanced leukaemia were designated as high risk. Median recipient age was 38 years (range 10–56). Median (range) of CD34+ and CD3+ cell transplant doses were 4·6 (2·3–10·9) × 106/kg and 0·83 (0·38–2) × 105/kg respectively. The cumulative probability of acute GVHD was 39%. No patient died from GVHD or its consequences. The probability of developing chronic GVHD was 54% (18% extensive). The probability of relapse was 12% for the standard‐risk patients and 66% for high‐risk patients. In multivariate analysis, the risk factors for lower disease‐free survival and overall survival were high‐risk disease, CD34+ dose < 4·6 × 106/kg and CD3+ dose < 0·83 × 105/kg. Predictive factors for chronic GVHD were a T‐cell dose at transplant > 0·83 × 105 CD3+ cells/kg. These results further define the impact of CD34 and CD3 cell dose on transplant outcome and show that careful dosing of stem cells and lymphocytes may permit the control and optimization of transplant outcome.

Optimized clinical-scale culture conditions for ex vivo selective depletion of host-reactive donor lymphocytes: A strategy for GvHD prophylaxis in allogeneic PBSC transplantation

BACKGROUND Ex vivo selective depletion (SD) is a strategy to prevent GvHD, in which host-reactive donor lymphocytes are selectively eliminated from a PBSC allograft while conserving useful donor immune function. Prior to testing this strategy in patients, our goal was to develop a clinical-scale SD process, which involves co-culture of donor lymphocytes and irradiated recipient cells, followed by the addition of an immunotoxin (IT) directed against the alpha-chain of the IL-2 receptor (CD25), expressed on activated donor T cells. METHODS Stimulator cells were generated from immunomagnetically selected and expanded recipient T lymphocytes. Donor PBMCs from G-CSF-mobilized peripheral blood were co-cultured for 72 h with irradiated stimulator cells. Alloreactive T cells were targeted for elimination by the addition of the anti-CD25 IT, RFT5-SMPT-dgA, and the IT enhancer, NH(4)Cl. RESULTS Stimulator-cell selection/expansion yielded > 2 x 10(10) highly enriched CD3(+) cells (98.9 +/- 2.2%). After SD, cell recovery was 68.5 +/- 23.3% and viability was 84.6 +/- 6.4%. This permitted a potential T-cell dose >/= 1 x 10(8) CD3(+) cells kg(-1) to transplant recipients. Although SD donor lymphocytes retained little proliferative capacity against the original stimulator cells (2.6 +/- 0.6%), responses were conserved against third party cells (107.6 +/- 18.6%), the bacterial superantigen staphylococcus enterotoxin B (108.2 +/- 4.2%), and CMV Ag (72.1 +/- 3.8%). DISCUSSION We have demonstrated that ex vivo SD is feasible in clinical-scale culture conditions. The ability of this strategy to prevent GvHD is the subject of an ongoing clinical trial, in which the SD lymphocyte product is transplanted in conjunction with a T cell-depleted PBSC allograft.