Clinical experience with CD64-directed immunotherapy. An overview

 The class I IgG receptor (FcγRI or CD64 receptor), which is present on key cytotoxic effector cells, has been shown to initiate the destruction of tumor cells in vitro and has been hypothesized to play a role in the destruction of antibody-coated cells such as platelets in idiopathic thrombocytopenia purpura (ITP). This overview summarizes the clinical experience with CD64-directed immunotherapy in cancer patients with the bispecific antibodies MDX-447 [humanized Fab anti-CD64 × humanized Fab anti-(epidermal growth factor receptor, EGFR)] and MDX-H210 (humanized Fab anti-DC64 × Fab anti-HER2/neu), and with the anti-CD64 monoclonal antibody (mAB) MDX-33 (H22) in the modulation of monocyte CD64 in vivo. In an ongoing phase I/II open-label trial with progressive dose escalation (1–15 mg/m²), patients with treatment refractory EGFR-positive cancers (renal cell carcinoma (RCC), head and neck, bladder, ovarian, prostate cancer and skin cancer) are treated weekly with intravenous MDX-447, with and without granulocyte-colony-stimulating factor (G-CSF). MDX-447 has been found to be immunologically active at all doses, binding to circulating monocytes and neutrophils (when given with G-CSF), causing monocytopenia and stimulating increases in circulating plasma cytokines. MDX-447 is well tolerated, the primary toxicities being fever, chills, blood pressure lability, and pain/myalgias. Of 36 patients evaluable for response, 9 have experienced stable disease of 3–6 month’s duration. The optimal dose and the maximal tolerated dose (MTD) have yet to be defined; dose escalation continues to define better the dose, toxicity, and the potential therapeutic role of this bispecific antibody. Three MDX-H210 phase II trials are currently in progress, all using the intravenous dose of 15 mg/m² given with granulocyte/macrophage (GM-CSF). These consist of one trial each in the treatment of RCC patients, patients with protrate cancer, and colorectal cancer patients, all of whom have failed standard therapy. At the time of writing, 11 patients have been treated in these phase II trials. Four patients have demonstrated antitumor effects. Patients demonstrating responses include 2 with RCC and 2 with prostate cancer. One RCC patient has had a 54% reduction in size of a hepatic metastatic lesion and the other has had a 49% decrease in the size of a lung metastasis with simultaneous clearing of other non-measurable lung lesions. Regarding the two patients with prostate cancer, one has had a 90% reduction in serum prostate-specific antigen (PSA; 118–11 ng/ml), which has persisted for several months; the other patient with prostate has had a 70% reduction of serum PSA (872 ng/ml to 208 ng/ml) within the first month of treatment. Both patients have also demonstrated symptomatic improvement. In a completed phase I and in ongoing phase I/II clinical trials, patients with treatment-refractory HER2/neu positive cancers (breast, ovarian, colorectal, prostate) have been treated with MDX-H210, which has been given alone and in conjunction with G-CSF, GM-CSF, and interferon γ (IFNγ). These trials have been open-label, progressive dose-escalation (0.35–135 mg/m²) studies in which single, and more often, multiple weekly doses have been administered. MDX-H210 has been well tolerated, with untoward effects being primarily mild-to-moderate flu-like symptoms. The MTD has not yet been defined. MDX-H210 is immunologically active, binding to circulating monocytes, causing monocytopenia, as well as stimulating increases in plasma cytokine levels. Furthermore, some patients have evidence of active antitumor immunity following treatment with MDX-210. Antitumor effects have been seen in response to MDX-H210 administration; these include 1 partial, 2 minor, and 1 mixed tumor response; 15 protocol-defined stable disease responses have occurred. In a completed phase I trial, MDX-33 was administered as a single intravenous dose to 17 normal subjects in order to assess its potential as an immunomodulator for the treatment of idiopathic thrombocytopenia purpura and other immune disorders. Doses of 1.5, 3.0, 5.0, and 7.5 mg/m² were administered. The variables evaluated in response to MDX-33 were circulating monocyte and neutrophil counts, monocyte CD64-mediated phagocytosis, monocyte CD64 modulation, MDX-33 pharmocokinetics, and various safety paramenters. MDX-33 is well tolerated at doses of 5.0 mg/m² or less, the primary toxicities being chills, low-grade fever, headache, and muscle aches. Persistent binding of MDX-33 to 80–99 % of circulating monocytes is seen for at least 6 days; down-modulation of monocyte CD64 occurs and also lasts more than 6 days. Monocyte CD64-mediated phagocytosis is significantly inhibited at all doses of MDX-33. At the 3.0 mg/m² and 5.0-mg/m² dose, phagocytosis is fully inhibited for at least 6 days, returning to baseline levels by 20 days after dosing. These results clearly demonstrate that immunomodulation of monocyte CD64 by the mAB MDX-33 can be accomplished with minimal clinical toxicity, and further indicate the potential of MDX-33 in the treatment of ITP and other auto-immune disorders. In conclusion, the results from completed and ongoing clinical trials with the CD64-directed bsAB MDX-447 and MDX-H210 demonstrate excellent tolerability in association with promising antitumor effects in tumors that have become refractory to all available therapies. Also promising are the results from the trial of the CD64-directed mAB, MDX-33, which show the ability to modulate monocyte CD64 in the clinical setting. Studies are currently being conducted to elucidate the full potential of these and other approaches using CD64-directed immunotherapy. Accepted: 14 October 1997     

BCG immunotherapy for recurrent malignant melanoma

Summary A study was made of immunologic parameters obtained from patients with stage IIIB malignant melanoma who were treated with BCG. Patients with the longest disease-free interval and survival times were those who had small initial skin test reactions and developed larger reactions during the course of BCG treatment. Of these patients, those with less than five involved nodes had the longest disease-free interval and survival times. Patients who had increases in skin test reactivity generally showed these increases by the first visit after initiation of BCG therapy.     

BCG in the immunotherapy of malignant lymphomas

Summary The effectiveness of a nonspecific immunostimulation in related human or animal diseases incited us to do a study of nonspecific immunotherapy by BCG in Hodgkin's disease, and then in other malignant lymphomas. Seventy patients, each one fulfilling at least 2 criteria of poor prognosis, were initially put in complete remission by a combination of radio-chemotherapy, followed by a reinforcing chemotherapy. These patients were then randomized into two groups. The first group received no further treatment; the second received BCG in weekly cutaneous scarifications. Eight patients were excluded from the study. The rate of relapses is significantly lower in the treated group. The results are discussed. Other therapeutic studies are necessary to fix the indications and modalities of this immunotherapy.Communication to the Medical Oncology Society, Nice, December 7, 1976     

Reinitiation of DNA Synthesis and Cell Division in Senescent Human Fibroblasts by Microinjection of Anti-p53 Antibodies

In human fibroblasts, growth arrest at the end of the normal proliferative life span (induction of senescence) is dependent on the activity of the tumor suppressor protein p53. In contrast, once senescence has been established, it is generally accepted that reinitiation of DNA synthesis requires loss of multiple suppressor pathways, for example, by expression of Simian virus 40 (SV40) large T antigen, and that even this will not induce complete cell cycle traverse. Here we have used microinjection of monoclonal antibodies to the N terminus of p53, PAb1801 and DO-1, to reinvestigate the effect of blocking p53 function in senescent human fibroblasts. Unexpectedly, we found that both antibodies induce senescent cells to reenter S phase almost as efficiently as SV40, accompanied by a reversion to the “young” morphology. Furthermore, this is followed by completion of the cell division cycle, as shown by the appearance of mitoses, and by a four- to fivefold increase in cell number 9 days after injection. Immunofluorescence analysis showed that expression of the p53-inducible cyclin/kinase inhibitor p21^sdi1/WAF1 was greatly diminished by targeting p53 with either PAb1801 or DO-1 but remained high and, moreover, still p53 dependent in cells expressing SV40 T antigen. As previously observed for induction, the maintenance of fibroblast senescence therefore appears to be critically dependent on functional p53. We suggest that the previous failure to observe this by using SV40 T-antigen mutants to target p53 was most probably due to incomplete abrogation of p53 function.     

Present status of BCG immunotherapy of malignant melanoma

Summary BCG systemic adjuvant immunotherapy may be effective for improving both the recurrence and survival rates in patients with regional metastases from malignant melanoma. Clinical trials show that many of the principles derived from the study of animal tumor systems are applicable to human cancer in that immunotherapy is most effective for a small residual number of tumor cells. BCG treatment fulfills many of the ideal criteria for adjuvant treatment following surgery when disease burden is lowest. It is relatively nontoxic; it is effective for disseminated melanoma; it has systemic activity in the adjuvant treatment of subclinical metastases. However, until clinical trials are complete, BCG adjuvant therapy must be considered investigational.Supported by USPHS grants CA05252, CA12582, and NIH 0732001 CB43852.     

Adjuvant BCG immunotherapy for malignant melanoma.

A total of 199 patients with stage I malignant melanoma at Clark''s level 3 to 5 of invasion were entered into a prospectively controlled randomized clinical trial that attempted to assess the value of local and systemic immunotherapy with BCG (bacille Calmette-Guérin) after surgery. The patients were randomly assigned, with stratification by Clark''s level, to receive either routine follow-up or immunotherapy with BCG, administered intradermally with a Heaf gun around the site of wide excision and then given orally for 2 years. Intradermal administration of BCG was repeated after 1 year''s oral therapy with BCG. Of the 99 patients in the treatment group 66 had Clark''s level 3, 28 had level 4, and 5 had level 5 invasion. Of the 100 patients in the control group, 61 had level 3, 36 had level 4, and 3 had level 5 invasion. Other prognostic factors, such as sex, depth of invasion, histologic features, site of disease and type of surgery, were evenly distributed. There were 57 recurrences of the melanoma, 24 in the treatment group and 33 in the control group. However, this trend was not statistically significant (p = 0.194). The suggestion that BCG may reduce the likelihood of local/regional recurrence has not been confirmed with longer follow-up. There were 13 such recurrences in the BCG group, compared with 21 in the control group; the proportions of patients in each group who had such a recurrence were not significantly different. Of the 199 patients 41 died, 24 in the control group and 17 in the treatment group; again, this difference was not significant. While there may be minor activity in selected patients, there appeared to be no benefit from this form of adjuvant BCG therapy in patients with malignant melanoma.     

The immunotherapy of Alzheimer's disease

Only a small percentage of patients with Alzheimer's disease benefit from current drug therapy and for only a relatively short time. This is not surprising as the goal of these drugs is to enhance existing cerebral function in Alzheimer patients and not to block the progression of cognitive decline. In contrast, immunotherapy is directed at clearing the neurotoxic amyloid beta peptide from the brain that directly or indirectly leads to cognitive decline in patients with Alzheimer's disease. The single trial of active immunization with the amyloid beta peptide provided suggestive evidence of a reduction in cerebral amyloid plaques and of stabilization in cognitive function of half the patients who developed good antibody responses to the amyloid beta peptide. However, 6% of actively immunized Alzheimer patients developed sterile meningoencephalitis that forced the cessation of the clinical trial. Passive immunotherapy in animal models of Alzheimer's disease has provided similar benefits comparable to those seen with active immunotherapy and has the potential of being effective in the half of Alzheimer's disease patients who do not make a significant anti-amyloid beta peptide antibody response and without inducing T-cell-mediated encephalitis. Published studies of 5 patients with sporadic Alzheimer disease treated with intravenous immunoglobulin containing anti-amyloid beta peptide antibodies showed that amyloid beta peptide was mobilized from the brain and cognitive decline was interrupted. Further studies of passive immunotherapy are urgently required to confirm these observations.     

Pathogenicity of Autoantibodies in Anti-p200 Pemphigoid

Recently, the C-terminus of laminin γ1 has been identified as target antigen in anti-p200 pemphigoid and the disease was renamed as anti-laminin γ1 pemphigoid. However, the pathogenic relevance of these autoantibodies has not yet been demonstrated. Therefore, we employed an ex vivo model of autoantibody-mediated leukocyte-dependent neutrophil activation and dermal-epidermal separation (DES) using cryosections of human skin. We showed that anti-p200 pemphigoid sera (n = 7) induced DES in a time-dependent manner, in contrast to sera from healthy controls. Furthermore, laminin γ1-specific IgG and serum depleted from anti-laminin γ1 reactivity were generated using the recombinant C-terminus of laminin γ1 (LAMC1-term; amino acids 1364 to 1609). Interestingly, both fractions labeled the dermal-epidermal-junction (DEJ) by indirect immunofluorescence microscopy on human foreskin and recognized a 200 kDa protein by immunoblotting with dermal extract. Human and rabbit IgG against LAMC1-cterm failed to attract neutrophils at the DEJ and to induce DES. In contrast, patient serum depleted from LAMC1-cterm reactivity led to the same extent of DES as non-depleted IgG. Repeated injection of rabbit anti-murine LAMC1-cterm IgG into both neonatal and adult C57BL/6mice as well as repetitive immunization of various mouse strains with murine LAMC1-cterm failed to induce macro- and microscopic lesions. In all mice, circulating anti-LAMC1-cterm antibodies were present, but only in some mice, IgG deposits were seen at the DEJ. We conclude that autoantibodies in anti-p200 pemphigoid sera are pathogenic while pathogenicity is not mediated by autoantibodies against laminin γ1. Further studies are needed to identify the pathogenically relevant autoantigen in anti-p200 pemphigoid.     

Immunology and immunotherapy of Alzheimer's disease

Although Alzheimer's disease is considered to be a degenerative brain disease, it is clear that the immune system has an important role in the disease process. As discussed in this Review, immune-based therapies that are designed to remove amyloid-beta peptide from the brain have produced positive results in animal models of the disease and are being tested in humans with Alzheimer's disease. Although immunotherapy holds great promise for the treatment of Alzheimer's disease, clinical trials of active amyloid-beta vaccination of patients with Alzheimer's disease were discontinued after some patients developed meningoencephalitis. New immunotherapies using humoral and cell-based approaches are currently being investigated for the treatment and prevention of Alzheimer's disease.     

Adoptive immunotherapy of malignant diseases with IL-2-activated lymphocytes

Lymphokine activated killer cells (LAK cells) or interleukin 2 (IL-2)-activated killer cells were induced by recombinant IL-2 (TGP-3) for clinical adoptive immunotherapy of malignant diseases. After incubation of peripheral blood lymphocytes (PBL) with IL-2 and normal human plasma for 1-2 weeks LAK cells were obtained that showed a maximum cytotoxicity against target cells, and did not need a toxic dose of IL-2 to enhance or maintain their cytotoxicity. Both autologous and allogeneic LAK cells were used in five clinical cases without any immune side effects, and were effective in three cases.     

Recurrent malignant melanoma: effect of adjuvant immunotherapy on survival.

Twenty-nine patients referred consecutively to a cancer clinic because of recurrent metastatic malignant melanoma were given 5 mg of Connaught Laboratories bacillus Calmette-Guérin (BCG) by multiple cutaneous puncture at weekly and later at monthly intervals. Eight were also treated with autologous tumour vaccine and three with intralesional BCG. This group was compared with a retrospective control group of 54 patients treated with surgery and radiotherapy alone after recurrence. Prognostic features such as site of primary and of first metastasis, disease-free interval, age and sex were similar in the two groups. However, the median survival from the time of first recurrence was 12 months in the control group but 21 months in the BCG-treated group. The major improvement was in patients with disease limited to the regional lymph nodes: the median survival was 16 months in the control group but over 32 months in the BCG-treated group. Autologous tumour vaccine appeared to have no effect on survival. Serial testing of immunocompetence did not offer any prognostic advantage, although the results of some tests correleated well with extent of disease.     

Phage-selected primate antibodies fused to superantigens for immunotherapy of malignant melanoma

 The high-molecular-weight melanoma-associated antigen, HMW-MAA, has been demonstrated to be of potential interest for diagnosis and treatment of malignant melanoma. Murine monoclonal antibodies (mAb) generated in response to different epitopes of this cell-surface molecule efficiently localise to metastatic lesions in patients with disseminated disease. In this work, phage-display-driven selection for melanoma-reactive antibodies generated HMW-MAA specificities capable of targeting bacterial superantigens (SAg) and cytotoxic T cells to melanoma cells. Cynomolgus monkeys were immunised with a crude suspension of metastatic melanoma. A strong serological response towards HMW-MAA demonstrated its role as an immunodominant molecule in the primate. Several clones producing monoclonal scFv antibody fragments that react with HMW-MAA were identified using melanoma cells and tissue sections for phage selection of a recombinant antibody phage library generated from lymph node mRNA. One of these scFv fragments, K305, was transferred and expressed as a Fab-SAg fusion protein and evaluated as the tumour-targeting moiety for superantigen-based immunotherapy. It binds with high affinity to a unique human-specific epitope on the HMW-MAA, and demonstrates more restricted crossreactivity with normal smooth-muscle cells than previously described murine mAb. The K305 Fab was fused to the superantigen staphylococcal enterotoxin A (D227A) [SEA(D227A)], which had been mutated to reduce its intrinsic MHC class II binding affinity, and the fusion protein was used to demonstrate redirection of T cell cytotoxicity to melanoma cells in vitro. In mice with severe combined immunodeficiency, carrying human melanoma tumours, engraftment of human lymphoid cells followed by treatment with the K305Fab-SEA(D227A) fusion protein, induced HMW-MAA-specific tumour growth reduction. The phage-selected K305 antibody demonstrated high-affinity binding and selectivity, supporting its use for tumour therapy in conjunction with T-cell-activating superantigens. Received: 9 September 1999 / Accepted: 21 October 1999     

p53 and the malignant progression of Barrett's esophagus

Barrett's esophagus (BE) is a metaplastic disorder in which specialized columnar epithelium replaces healthy squamous epithelium (intestinal metaplasia). Even though its pathophysiology and the steps of its neoplastic progression are not completely understood, BE can be considered as a complication of gastroesophageal reflux disease (GERD). Given that esophageal adenocarcinoma, which is continually increasing in the Western world, still has a poor prognosis and suffers from late diagnosis, and because BE is a precancerous lesion, there is a strong need for good molecular markers of malignant progression in Barrett's metaplasia (BM). The aim of this review is to examine the published data regarding the role that assessment of p53 may play in the management of BE, trying to understand if it may be a useful marker to early diagnose BE malignant transformation.     

Engineered antibody approaches for Alzheimer's disease immunotherapy

The accumulation of amyloid-β-peptide (Aβ or A-beta) in the brain is considered to be a key event in the pathogenesis of Alzheimer's disease (AD). Over the last decade, antibody strategies aimed at reducing high levels of Aβ in the brain and or neutralizing its toxic effects have emerged as one of the most promising treatments for AD. Early approaches using conventional antibody formats demonstrated the potential of immunotherapy, but also caused a range of undesirable side effects such meningoencephalitis, vasogenic edema or cerebral microhemorrhages in both murine and humans. This prompted the exploration of alternative approaches using engineered antibodies to avoid adverse immunological responses and provide a safer and more effective therapy. Encouraging results have been obtained using a range of recombinant antibody formats including, single chain antibodies, antibody domains, intrabodies, bispecific antibodies as well as Fc-engineered antibodies in transgenic AD mouse and primate models. This review will address recent progress using these recombinant antibodies against Aβ, highlighting their advantages over conventional monoclonal antibodies and delivery methods.     

Adjuvant BCG immunotherapy for stage I and II malignant melanoma.

Initial adjuvant immunotherapy trials have demonstrated a greater disease-free interval in patients treated with bacille Calmette-Guérin (BCG) compared with historical controls. In this study 149 patients at high risk of recurrence after surgical treatment of local or regional malignant melanoma were given BCG for 2 years and were followed up for a median of 28 months from the start of immunotherapy. The 36 patients in the comparison group had a higher rate of recurrence than the patients treated with BCG, and the rate in the treatment group was close to that reported from a similar study at the University of California at Los Angeles. The relatively long disease-free interval for the high-risk comparison patients in this study suggests that the control groups at other centres may have included patients with unrecognized additional risk. The rates of survival in the Canadian treatment group were also comparable to those reported by other centres. However, reports of a favourable BCG-mediated pattern of recurrence could not be confirmed. Therefore, the routine use of adjuvant BCG immunotherapy is not recommended.     

p53--prognostic factor of malignant transformation of Barrett's esophagus

The most significant precancerosis in the esophageal cancer is Barrett's esophagus. The risk of malignant transformation is determined primarily in accordance with the degree of dysplastic alterations of the mucosa. Indication of "preventive" extirpation of the esophagus should be supported by other factors, for example by detection of p53 mutation or expression. The study reports on the evaluation of a group of 20 patients with Barrett's esophagus treated at the 1st Department of Surgery, the p53 level and its correlation with histological findings evaluated in these patients. A good correlation was found between the grade of Barrett's esophagus dysplasia and high p53 positivity. This correlation was also confirmed by detection of early carcinoma in patients with "preventive" extirpation of the esophagus due to a high-grade dysplasia. Preliminary results show that examination of p53 level in specimens taken from the esophageal mucosa may be helpful for the estimation of malignant potential of the dysplastic mucosa.