Efficient and reproducible generation of tumour-infiltrating lymphocytes for renal cell carcinoma.

BACKGROUND: Tumour-infiltrating lymphocyte (TIL) therapy is showing great promise in the treatment of patients with advanced malignant melanoma. However, the translation of TIL therapy to non-melanoma tumours such as renal cell carcinoma has been less successful with a major constraint being the inability to reproducibly generate TILs from primary and metastatic tumour tissue. METHODS: Primary and metastatic renal cell carcinoma biopsies were subjected to differential tumour disaggregation methods and procedures that stimulate the specific expansion of TILs tested to determine which reliably generated TIL maintained antitumour specificity. RESULTS: Enzymatic or combined enzymatic/mechanical disaggregation resulted in equivalent numbers of TILs being liberated from renal cell carcinoma biopsies. Following mitogenic activation of the isolated TILs with anti-CD3/anti-CD28-coated paramagnetic beads, successful TIL expansion was achieved in 90% of initiated cultures. The frequency of T-cell recognition of autologous tumours was enhanced when tumours were disaggregated using the GentleMACS enzymatic/mechanical system. CONCLUSION: TILs can be consistently produced from renal cell carcinoma biopsies maintaining autologous tumour recognition after expansion in vitro. While the method of disaggregation has little impact on the success of TIL growth, methods that preserve the cell surface architecture facilitate TIL recognition of an autologous tumour, which is important in terms of characterising the functionality of the expanded TIL population.

Final results from the large sunitinib global expanded-access trial in metastatic renal cell carcinoma.

BACKGROUND: We report final results with extended follow-up from a global, expanded-access trial that pre-regulatory approval provided sunitinib to metastatic renal cell carcinoma (mRCC) patients, ineligible for registration-directed trials. METHODS: Patients ⩾18 years received oral sunitinib 50 mg per day on a 4-weeks-on-2-weeks-off schedule. Safety was assessed regularly. Tumour measurements were scheduled per local practice. RESULTS: A total of 4543 patients received sunitinib. Median treatment duration and follow-up were 7.5 and 13.6 months. Objective response rate was 16% (95% confidence interval (CI): 15-17). Median progression-free survival (PFS) and overall survival (OS) were 9.4 months (95% CI: 8.8-10.0) and 18.7 months (95% CI: 17.5-19.5). Median PFS in subgroups of interest: aged ⩾65 years (33%), 10.1 months; Eastern Cooperative Oncology Group performance status ⩾2 (14%), 3.5 months; non-clear cell histology (12%), 6.0 months; and brain metastases (7%), 5.3 months. OS was strongly associated with the International Metastatic Renal-Cell Carcinoma Database Consortium prognostic model (n=4065). The most common grade 3/4 treatment-related adverse events were thrombocytopenia (10%), fatigue (9%), and asthenia, neutropenia, and hand-foot syndrome (each 7%). CONCLUSION: Final analysis of the sunitinib expanded-access trial provided a good opportunity to evaluate the long-term side effects of a tyrosine kinase inhibitor used worldwide in mRCC. Efficacy and safety findings were consistent with previous results.

CD3ζ-based chimeric antigen receptors mediate T cell activation via cis- and trans-signalling mechanisms: implications for optimization of receptor structure for adoptive cell therapy.

Chimeric antigen receptors (CARs) can mediate redirected lysis of tumour cells in a major histocompatibility complex (MHC)-independent manner, thereby enabling autologous adoptive T cell therapy for a variety of malignant neoplasms. Currently, most CARs incorporate the T cell receptor (TCR) CD3ζ signalling chain; however, the precise mechanisms responsible for CAR-mediated T cell activation are unclear. In this study, we used a series of immunoreceptor tyrosine-based activation motif (ITAM)-mutant and transmembrane-modified receptors to demonstrate that CARs activate T cells both directly via the antigen-ligated signalling chain and indirectly via associated chains within the TCR complex. These observations allowed us to generate new receptors capable of eliciting polyfunctional responses in primary human T cells. This work increases our understanding of CAR function and identifies new avenues for the optimization of CAR-based therapeutic interventions.

Ligation of the CD2 co-stimulatory receptor enhances IL-2 production from first-generation chimeric antigen receptor T cells.

T cells bearing chimeric antigen receptors (CARs) are broadly categorised into first- and second-generation receptors. Second-generation CARs contain a co-stimulatory signalling molecule and have been shown to secrete IL-2, undergo greater proliferation and to have enhanced persistence in vivo. However, we have shown that T cells bearing a first-generation CAR containing a CD19-targeting scFv (single-chain variable fragment) and the CD3ζ-signalling domain are able to produce IL-2 upon co-culture with CD19(+) B-cell lymphomas independent of CD28 activity. Here, we report that signalling through endogenous CD2 following ligation with its ligands, CD48 in mouse and CD58 in humans, drives IL-2 production by first-generation CD19-specific CAR. Moreover, the high levels of IL-2 produced by human T cells engrafted with a second-generation CD28-containing CAR during target-cell recognition are dependent to a degree upon CD2 receptor activity. These observations highlight the fact that the functional activity induced by T-cell-expressed CARs is dependent upon endogenous 'natural' receptor interactions. A deeper understanding of the role of these activities will serve to further refine the design of future CARs to either exploit or avoid these interactions.

Clinical evidence of efficient tumor targeting based on single-chain Fv antibody selected from a combinatorial library.

We present a system for cancer targeting based on single-chain Fv (scFv) antibodies selected from combinatorial libraries, produced in bacteria and purified by using an engineered tag. Combinatorial libraries of scFv genes contain great diversity, and scFv antibodies with characteristics optimized for a particular task can be selected from them using filamentous bacteriophage. We illustrate the benefits of this system by imaging patients with carcinoembryonic antigen (CEA)-producing cancers using an iodine-123 labeled scFv anti-CEA selected for high affinity. All known tumor deposits were located, and advantages over current imaging technology are illustrated. ScFvs are produced in a cloned form and can be readily engineered to have localizing and therapeutic functions that will be applicable in cancer and other diseases.

Impact of laparotomy and liver resection on the peritoneal concentrations of fibroblast growth factor 2, vascular endothelial growth factor and hepatocyte growth factor.

PURPOSE: Some data have suggested that major surgery is associated with the post-operative growth of residual tumour masses but the mechanism of this is unknown. This study was designed to determine the relationship between intraperitoneal (IP) cytokine levels, and laparotomy in benign and malignant settings. METHODS: Intraperitoneal fluid specimens were obtained at the start and at the end of laparotomy in patients with benign conditions (n=10) and in others undergoing resection of hepatic metastases from colorectal cancer (n=10). Using ELISA the concentration of the angiogenic cytokines, HGF, VEGF-A, VEGF-C, VEGF-D and FGF-2 was determined. RESULTS: The data show that in 16 of 20 patients there was a significant increase (P=0.006) in the IP concentration of hepatocyte growth factor (HGF) but not in the other growth factors by the end of the operation. The mean increase in HGF concentration was 821.5 pg/ml (95% CI: 11.0-6,426.0). Neither the groups (malignant and non-malignant) nor the length of operation correlated with greater or lesser increases in HGF. CONCLUSION: The observation that the increase in HGF occurred in both the cancer and non-cancer groups suggests that it is the surgery rather than the disease that is associated with the increased cytokine concentration. As HGF is a potent endothelial, epithelial and mesenchymal mitogen the data highlight HGF as a potential target for anti-cancer treatments in the peri-operative period. However, investigators should closely monitor wound healing as this may be compromised by this new class of drugs.

High-dose interleukin2 - a 10-year single-site experience in the treatment of metastatic renal cell carcinoma: careful selection of patients gives an excellent outcome.

BACKGROUND: VEGF-targeted therapy has become the mainstay of treatment for majority of mRCC patients. For most patients, benefit is short-lived and therefore treatment remains palliative in intent. HD IL2 is an effective immunotherapy treatment capable of durable remission in some patients but its unselected use has been difficult due to its modest response rate and considerable adverse effects. Using set pathology criteria as a selection tool in clinical practice, we have been able to show improved outcomes in our previous report. Here, we present an updated and extended report of this treatment and seek to explore any pathological, clinical and treatment variables likely to predict better outcomes. METHODS: This is an extension of a previously reported clinical audit, which includes mRCC cases treated with HD IL2 between 2003 and 2013. Since 2006, tumour specimens of potential candidates were routinely reviewed prospectively and stratified into Favourable or Other categories based on constitution of histological growth pattern, namely alveolar or solid versus papillary and/or sarcomatoid architecture; clear cell versus granular cell cytoplasmic morphology. HD IL2 was preferentially offered to patients with Favourable pathology. Outcome evaluation includes response rates, survival, and treatment tolerance. Multivariate analysis was performed to explore potential prognostic and predictive factors. RESULTS: Among prospectively selected patients with Favourable pathology (n = 106), overall response rate was 48.1 % (51/106) with CR rate of 21.6 % (23/106). Median OS was 58.1 months. Factors associated with significantly better response and/or survival includes favourable pathology pattern, higher cycle 1 tolerance and lower number of metastatic organ sites (<3). CAIX (Carbonic anhydrase 9) has prognostic value but is not predictive of response. Toxicities were those expected of IL2 but were manageable on general medical wards, with no treatment-related death. Importantly most complete responses were durable with 76 % (23/30) cases remained relapse-free (median 39 months follow up) and 2 of the seven who relapsed had had long-term disease free survival after resection of oligometastatic relapse. CONCLUSIONS: Our experience shows that HD IL2 remains an effective and safe treatment in well-selected cases of mRCC. The result in this single-institution patient series confirms similar outcomes to our previously reported retrospective series. Given the prospect of long-term remission, fit patients with Favourable histology and low disease burden should be considered for HD IL2 in an experienced centre. Better understanding has been gained from this in-depth analysis especially the examination of possible response predictors and strategies that can improve treatment outcome.

Selective targeting of gene transfer to vascular endothelial cells by use of peptides isolated by phage display.

BACKGROUND: Gene transfer to vascular cells is a highly inefficient and nonselective process, defined by the lack of specific cell-surface receptors for both nonviral and viral gene delivery vectors. METHODS AND RESULTS: We used filamentous phage display to isolate a panel of peptides that have the ability to bind selectively and efficiently to quiescent human umbilical vein endothelial cells (HUVECs) with reduced or negligible binding to nonendothelial cells, including vascular smooth muscle cells and hepatocytes. By direct biopanning on HUVECs and a second approach involving preclearing steps before panning on HUVECs, we isolated and sequenced 140 individual phages and identified 59 peptides. We selected 7 candidates for further investigation by secondary screening of homogeneous phages on a panel of cell types. Using adenovirus-mediated gene transfer as a model gene delivery system, we cloned the peptide SIGYPLP and the positive control peptide KKKKKKK upstream of the S11e single-chain Fv ("adenobody") directed against the knob domain of the adenovirus to create fusion proteins. Adenovirus-mediated gene transfer via fiber-dependent infection was blocked with S11e, whereas inclusion of the KKKKKKK peptide retargeted gene transfer. The peptide SIGYPLP, however, retargeted gene delivery specifically to endothelial cells with a significantly enhanced efficiency over nontargeted adenovirus and without transduction of nontarget cells. CONCLUSIONS: Our study demonstrates the feasibility of using small, novel peptides isolated via phage display to target gene delivery specifically and efficiently to HUVECs and highlights their use for retargeting both viral and nonviral gene transfer to vascular endothelial cells for future clinical applications.

Selection of phage antibodies by binding affinity. Mimicking affinity maturation.

We describe a process, based on display of antibodies on the surface of filamentous bacteriophage, for selecting antibodies either by their affinity for antigen or by their kinetics of dissociation (off-rate) from antigen. For affinity selection, phage are mixed with small amounts of soluble biotinylated antigen (less than 1 microgram) such that the antigen is in excess over phage but with the concentration of antigen lower than the dissociation constant (Kd) of the antibody. Those phage bound to antigen are then selected using streptavidin-coated paramagnetic beads. The process can distinguish between antibodies with closely related affinities. For off-rate selection, antibodies are preloaded with biotinylated antigen and diluted into excess unlabelled antigen for variable times prior to capture on streptavidin-coated paramagnetic beads. To mimic the affinity maturation process of the immune system, we introduced random mutations into the antibody genes in vitro using an error-prone polymerase, and used affinity selection to isolate mutants with improved affinity. Starting with a small library (40,000 clones) of mutants (average 1.7 base changes per VH gene) of the mouse antibody B1.8, and using several rounds of affinity selection, we isolated a mutant with a fourfold improved affinity to the hapten 4-hydroxy-5-iodo-3-nitrophenacetyl-(NIP)-caproic acid (mutant Kd = 9.4(+/- 0.3) nM compared with B1.8 Kd = 41.9(+/- 1.6) nm). The relative increase in affinity of the mutant is comparable to the increase seen in the anti-4-hydroxy-3-nitrophenylacetyl/NIP-caproic acid murine secondary immune response.

The contribution of contact and non-contact residues of antibody in the affinity of binding to antigen. The interaction of mutant D1.3 antibodies with lysozyme.

We have analysed the contribution of residues of the D1.3 Fv fragment to binding of hen egg lysozyme. We altered residues at the contact interface by site-directed mutagenesis, and determined the affinity of the mutant Fv fragments for lysozyme by fluorescence quench titration. We found that a band of residues at the centre of the contact interface were much more important for binding affinity than those at the periphery. We also subjected the seFv fragment to random mutagenesis to simulate somatic mutation and affinity maturation. By display of the mutants on the surface of filamentous phages, and selection of the phages with biotinylated lysozyme, we were able to select mutants with modest improvements in binding affinity to lysozyme. By combining the mutations we obtained a scFv fragment with a fivefold improved affinity (Kd approximately 0.6 nM compared to wild-type Kd = 3.3 nM). However, none of the altered residues leading to improved affinity was located in the contact interface. This indicates that the interactions of a few residues at the centre of the contact interface are responsible for the binding affinity to antigen, but that these interactions can be modulated by alterations of residues outside the binding site. This may represent a typical mechanism for the affinity maturation of antibodies.

Clinical issues in antibody design.

Antibody genes can now be cloned and expressed in various ways to give new versions of antibodies that possess reduced immunogenicity, improved affinity, altered size, increased avidity and novel effector functions. The task for any clinical application is, first, to define a relevant target, and then to design the optimal antibody-based therapeutic molecule to react with that target. This article reviews these improved antibody-based molecules and examines their role in cancer therapy.

Purification of bacterially expressed single chain Fv antibodies for clinical applications using metal chelate chromatography.

A new procedure is described for the purification of an anti-carcinoembryonic antigen (CEA) single chain Fv (scFv), referred to as MFE-23, from bacterial supernatant. A simple insertion of a hexa-histidine tail fused at the C-terminus (MFE-23 His) provides an affinity tag which selectively binds to transition metal ions immobilised on an iminodiacetic acid (IDA) derivitised solid phase matrix. This method proved to be superior to standard CEA antigen affinity chromatography in the following ways. (1) A higher yield was produced (10 mg/l as opposed to 2.2 mg/l of bacterial supernatant). The latter figure was largely affected by the limited availability (size of the column) of immobilised CEA antigen. (2) Scale-up was relatively simple and less costly. (3) The risk of tumour derived antigen leaching from the column is eliminated. Results showed that immobilised Cu2+ ions were more effective than Ni2+ and Zn2+ ions in retaining the His tagged product giving a 90% pure product on elution. Clinical grade material was generated using size exclusion chromatography to remove aggregated material, and Detoxi gel to remove bacterial endotoxins. Validation assays to measure DNA, copper and endotoxins were performed to assess the levels of contaminants. MFE-23 His retained 84% antigen binding after 6 months storage at 4 degrees C and > 75% after radiolabelling. Further experiments confirmed that the His tail did not affect biodistribution and tumour localisation in nude mice bearing human colorectal tumour xenografts.

The in-training examination in internal medicine: resident perceptions and lack of correlation between resident scores and faculty predictions of resident performance.

PURPOSE: We sought to survey residents' perceptions regarding the In-Training Examination in Internal Medicine and to assess the ability of faculty members to evaluate the knowledge base of internal medicine residents. SUBJECTS AND METHODS: Residents were asked about the perceived utility of the In-Training Examination and related self-directed educational activities. Residents predicted their own performance on the examination (into upper, middle, or lower tertile). Faculty predicted housestaffs scores, and residents predicted the scores of interns. RESULTS: Most residents (35/36; 97%) believed that the examination was useful, and 91% modified their study habits or clinical rotation schedule based on its results. Approximately half of the residents accurately predicted into which tertile they would score. Faculty predictions of resident performance on the examination were accurate 49% of the time, and resident predictions of intern scores were accurate 38% of the time. The sensitivity ofa lower-tertile prediction by faculty was 34%, with a specificity of 90%. The sensitivity of a resident prediction of a lower-tertile intern score was 15%, with a specificity of 98%. Both faculty and residents were more likely to overestimate than underestimate examination scores. CONCLUSION: Residents believe that the In-Training Examination is useful and frequently initiate educational interventions based on results. Faculty and residents lack the ability to evaluate accurately the knowledge of trainees that they supervise. In particular, both groups may be unable to identify trainees who are deficient in this element of clinical competence.

Syphilis and neurosyphilis in a human immunodeficiency virus type-1 seropositive population: evidence for frequent serologic relapse after therapy.

OBJECTIVE: To describe clinical and treatment aspects of syphilis infection among patients seropositive for the human immunodeficiency virus (HIV). PATIENTS AND METHODS: Results of serologic tests for syphilis, CD4+ T-lymphocyte counts, and clinical response to therapy were retrospectively monitored in 100 HIV-infected adults with syphilis from a tertiary-care military HIV program. RESULTS: Of the 1,206 HIV-infected patients, 100 (8.3%) in the cohort had syphilis; 61 patients were treated for active syphilis. Serologic or clinical relapse eventually occurred in 10 of the 56 treated patients (17.9%) with follow-up available; 7 of the 10 who relapsed had previously received high-dose intravenous or procaine penicillin therapy. Relapse occurred more than 12 months after initial therapy in 6 of 10 patients (60%) who experienced relapse; 5 patients experienced multiple relapses. The mean CD4+ T-lymphocyte count was not predictive of relapse. Patients with reactive cerebrospinal fluid (CSF) Venereal Disease Research Laboratory (VDRL) test titers (4 of 7 patients [57%]) or the rash of secondary syphilis (4 of 14 patients [29%]) were at highest risk of subsequent relapse or treatment failure when monitored for an average of 2 years. CONCLUSION: Standard penicillin regimens, including high-dose intravenous penicillin, transiently lowered serum VDRL titers in nearly all cases, but were sometimes inadequate in preventing serologic and clinical relapse in patients infected with HIV type-1, especially among those with secondary syphilis and reactive CSF VDRL titers. Careful long-term follow-up is essential, and repeated courses of therapy may be needed for patients infected with HIV type-1 who have syphilis.

Technetium-99m radiolabeling using a phage-derived single-chain Fv with a C-terminal cysteine.

UNLABELLED: Single-chain Fv (scFv) antibody fragments have potential for clinical imaging because of their rapid tumor penetration and high tumor-to-tissue ratios at early time points. ScFvs clear rapidly from the circulation so radiolabels such as 99mTc which have short half-lives are desirable, but the free thiol groups necessary for labeling with 99mTc are not normally found on these molecules. METHODS: We constructed a vector which enabled a free cysteine to be linked to the C-terminus of scFvs. MFE-23, a scFv directed against carcinoembryonic antigen (CEA), was cloned into this vector and cys-tagged MFE-23 was labeled with 99mTc using a D-glucarate transfer method. RESULTS: The radiolabeled product was stable in vivo and in vitro and showed favorable tumor-to-blood ratios in vivo at early time points (4:1 at 24 hr and 8:1 at 48 hr), although high kidney levels were also detected. CONCLUSION: Our study demonstrates an effective method to enable scFvs radiolabeling with 99mTc and also shows the potential of using a 99mTc-labeled scFv for clinical imaging studies.

A genetic approach to idiotypic vaccination for B cell lymphoma.

Idiotypic immunoglobulin expressed by a B cell tumor presents a clear tumor antigen which could be attacked by vaccination of the host. Vaccination with idiotypic protein has been shown to induce protective immunity against lymphoma, but application to patients is limited by the requirement of "personal" vaccines for each patient. A genetic approach enables V-region sequences encoding idiotypic antigen to be rescued from tumor biopsies, and to be assembled as scFv fragments. These can be expressed in bacteria to produce recombinant protein, or used directly as naked DNA vaccines. Intramuscular injection of idiotypic DNA from a mouse B cell lymphoma induces low levels of syngeneic anti-idiotypic antibody in serum. Response can be stimulated by co-injection of DNA plasmids encoding either IL-2 or GM-CSF, and T cells which proliferate in response to idiotypic IgM are generated. However, protection against tumor appears to be blocked by continuing secretion of idiotypic antigen from the persisting vaccine vector, which forms immune complexes with serum antibody. Methods for regulating the level of scFv to engage the immune system, but not to block the effector arm are being investigated. Similar control will be applicable to the cytokine vectors, which can deliver encoded cytokines designed to activate immune pathways for tumor destruction. Experience gained in lymphoma may be extended to other tumors with defined tumor antigens.

Orogenital contact: a cause of chorioamnionitis?

BACKGROUND: Fusobacterium nucleatum and Capnocytophaga species are common oral pathogens and infrequent causes of systemic infection in patients with compromised immunity or disrupted mucosal integrity. The isolation of both organisms from a clinical specimen suggests an oral source of infection. CASE: A 23-year-old black woman was admitted at 24 weeks' gestation in preterm labor. She subsequently developed signs of clinical chorioamnionitis, including fever, fetal tachycardia, and uterine tenderness. Bacteriologic studies of the amniotic fluid and subchorionic placental cultures yielded F nucleatum and Capnocytophaga species. On review of the patient's history, a temporal relation was noted between orogenital contact and the onset of clinical infection. Thorough evaluation of the patient, including dental examination, did not reveal an obvious source of infection. However, significant periodontal disease was identified in her partner. CONCLUSION: The concomitant finding of these two organisms in the patient's amniotic fluid and a history of periodontal disease in her partner suggests that chorioamnionitis may have been due to an ascending infection after orogenital contact.

Ifosfamide with and without adriamycin in advanced uterine leiomyosarcoma.

Uterine leiomyosarcomas are rare tumours and the results of treatment of advanced disease are poor. Ifosfamide and Adriamycin are both known to be active drugs in soft-tissue sarcomas. We present our experience using ifosfamide alone and in combination with Adriamycin in advanced or recurrent uterine leiomyosarcomas. Ifosfamide alone was given as a 24-h infusion at doses ranging from 5 to 7.5 g/m2, with mesna rescue. In the combination regimen, ifosfamide was given at a dose of 5 g/m2 and Adriamycin, at a dose of 40 or 60 mg/m2. Ten patients were treated with ifosfamide alone, with only one partial response lasting 6 months. In all, 11 patients were treated with ifosfamide and Adriamycin, which resulted in 1 complete response that lasted 11 months. Although many of the patients had extensive disease and some had undergone prior treatment with other chemotherapy, ifosfamide would appear to have only modest activity at the dose and schedule used.

Simulations in the United States Medical Licensing Examination (USMLE).

Over the last several years there has been much attention focused on the detection and remediation of problems that pose potential threats to patient safety and that interfere with the provision of effective care. It has been noted that changes in medical education and assessment are integral to eventual improvement in this area. Within the assessment system used to licence physicians in the United States, there has been an evolution of assessment formats intended to improve the measurement of knowledge and skills, including the recent development of computer based patient simulations and clinical skills assessments. A number of new testing formats intended to further enhance assessment of critical knowledge and skills will be available in the near future.

Abdominal vascular catastrophes.

Abdominal vascular catastrophes are due to rupture of arterial aneurysms or arterial occlusion leading to end organ ischemia. In this article, we will discuss rupture of abdominal aortic aneurysms and visceral artery aneurysms. Arterial occlusion secondary to embolism, thrombosis and dissection will be presented. Mesenteric ischemia secondary to low flow states, venous thrombosis and other conditions will also be covered. These uncommon, but frequently fatal pathological processes, are of interest to both general and vascular surgeons.