Carl V. Hamby

Characterization of anti-HLA class II monoclonal antibody LGII-612.14 reacting with formalin fixed tissues.

mAb LGII-612.14 derived from a BALB/c mouse immunized with interferon-gamma (IFN-gamma) treated cultured human B lymphoid cells LG-2 has been shown with serological and immunochemical assays to recognize a monomorphic determinant expressed on the beta chain of HLA-DR, -DQ and -DP antigens. The linear nature of the determinant, which is likely to be formed by residues 19-25, is indicated by the reactivity of mAb LGII-612.14 with HLA-DR, -DQ and -DP beta chains purified by electrophoresis in presence of SDS. An unusual characteristic of mAb LGII-612.14 is its reactivity with fixed tissue sections. The intensity of staining is affected by the incubation temperature, the incubation time and the fixative used. Maximal intensity of staining of formalin fixed, paraffin embedded tissue sections required an incubation time of 16 h. The intensity of staining of paraffin embedded tissues initially fixed with Bouins solution, formalin or ethanol was similar to that of frozen tissue sections and stronger than that of tissues fixed with B5 solution. No staining was detected of paraffin embedded tissues fixed with glutaraldehyde or Zenkers solution. Comparison of the staining patterns with mAb LGII-612.14 of frozen and fixed tissue sections showed that the latter substrates provide a superior detail of tissue architecture and cellular morphology without significant loss of sensitivity. Furthermore, comparison of the characteristics of mAb LGII-612.14 with the few previously published anti-HLA class II mAb reacting with fixed tissues indicates that mAb LGII-612.14 stains formalin fixed, paraffin embedded tissues, while mAb 910D7 and TAL-1B5 stain tissues fixed with less commonly used fixatives. Furthermore, mAb LGII-612.14 is likely to yield more sensitive staining results than anti-HLA-DR, -DQ and -DP mAb KUL/05. The present results indicate that mAb LGII-612.14 represents a useful probe to apply immunohistochemical techniques to the analysis of the distribution of HLA class II antigens in fixed tissues. This will greatly facilitate the use of readily available collections of fixed tissue specimens in retrospective studies to assess the clinical significance of changes in HLA class II antigen expression which occur in various disease states.

Differential clinical significance of αvΒ3 expression in primary lesions of acral lentiginous melanoma and of other melanoma histotypes

Despite its potential clinical relevance, αvβ3 expression has been analyzed only in a limited number of melanoma lesions, mostly nodular melanoma (NM) and superficial spreading melanoma (SSM). Therefore, in the present study, we have correlated αvβ3 expression in 33 acral lentiginous melanomas (ALMs), 6 lentigo maligna melanomas, 7 mucosal melanomas, 12 NMs and 9 SSMs with their antigenic profile, with their histo‐pathological characteristics and with the clinical course of the disease. Furthermore, we have compared αvβ3 expression in ALM lesions with that in NM and SSM lesions since this information helps to clarify the relationship of the latter 2 histotypes with ALM. Such a relationship is uncertain since ALM has a clinical course similar to that of NM and SSM despite different antigenic profiles and biological characteristics. The level of αvβ3 expression in primary lesions was not correlated with that of high‐m.w. melanoma‐associated antigen and intercellular adhesion molecule‐1, with lesion thickness and with disease recurrence in ALM but was significantly correlated with these 4 parameters in the other melanoma histotypes analyzed. Therefore, αvβ3 expression appears to have a differential clinical significance in ALM and in the other histotypes of melanoma we have analyzed since it appears to play a significant role in the progression of the disease only in non‐ALM histotypes. Int. J. Cancer (Pred. Oncol.) 89:153–159, 2000.

αvβ3 expression on blood vessels and melanoma cells in primary lesions; differential association with tumor progression and clinical prognosis

Abstract The αvβ3 integrin has emerged as a key mediator in angiogenesis. Its role in tumor-induced angiogenesis is supported by its up-regulation in vivo in the vasculature of a number of different types of carcinoma. The potential clinical significance of αvβ3 expression on blood vessels in carcinomas is suggested by its association with tumor progression. Currently no information is available about the clinical significance of αvβ3 expression on the vasculature of lesions of melanocytic origin. Since we have previously found that αvβ3 expression on melanoma cells in primary lesions is associated with a poor prognosis, in the present study we have compared αvβ3 expression on blood vessels and on cells of melanocytic origin in nevi and in malignant melanoma lesions. In addition we have examined the lesions for expression of the αv subunit to gain information on the regulation of αvβ3 expression on endothelial cells and on cells of the melanocyte lineage. αvβ3 expression on endothelial cells and on melanocytic cells was a relatively sensitive and specific marker for malignant lesions. However, αvβ3 expression on endothelial cells in primary melanoma lesions was not associated with the prognosis of the disease. The αv subunit and the αvβ3 complex were differentially expressed on endothelial cells and on melanocytic cells, implying that different regulatory pathways control their expression. This finding may account for the differential clinical significance of αvβ3 expression on tumor vasculature and on melanoma cells we observed in our patient cohort. Lastly, αvβ3 may be a useful target for immunotherapeutic approaches in melanoma because of its high expression on the vasculature of all metastatic lesions tested and its restricted distribution in normal tissues.

Expression of a catalytically inactive H118Y mutant of nm23-H2 suppresses the metastatic potential of line IV Cl 1 human melanoma cells.

Nm23‐H1 and nm23‐H2 are putative metastasis suppressor genes that encode nucleoside diphosphate kinase (NDPK) A and B. NDPKs form oligomers distributed between soluble and particulate fractions of cells and therefore may exert their effects as either soluble or bound proteins. To determine whether metastasis‐related functions of NDPKs are mediated by their catalytic activity in membrane bound or soluble complexes, we have stably transfected highly metastatic human melanoma Line IV Cl 1 cells with wild‐type and catalytically inactive (H118Y) nm23‐H1 and nm23‐H2 genes and assayed their metastatic potential in nude mice. Transfection with wild‐type nm23‐H1 and nm23‐H2 genes and catalytically inactive nm23‐H1 did not significantly (all p > 0.10) alter the metastatic potential of Line IV Cl 1 cells while transfection with catalytically inactive nm23‐H2 significantly (p < 0.01) reduced their metastatic potential. The lack of effect of transfection with wild‐type and catalytically inactive nm23‐H1 suggests that neither soluble nor membrane bound NDPK A affect the metastatic potential of Line IV Cl 1 cells. The metastasis suppressive effect of catalytically inactive NDPK B overexpression suggests that competition with bound complexes containing catalytically active NDPK B inhibits metastasis of Line IV Cl 1 cells. These results imply that bound NDPK B promotes metastasis and suggest that inhibition of its function or of its binding to critical sites may be a useful approach to limit the development of metastases in human melanoma. Int. J. Cancer 88:547–553, 2000.

Molecular Imaging of Changes in the Prevalence of Vascular Endothelial Growth Factor Receptor in Sunitinib-Treated Murine Mammary Tumors

Several drugs targeting vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) are approved for cancer treatment. However, these drugs induce relatively modest and frequently unpredictable tumor responses. In this work, we explored whether noninvasive imaging of VEGFR, a direct target of antiangiogenic drugs, can provide real-time information on responses to the treatment with sunitinib, a small-molecule VEGFR inhibitor approved by the Food and Drug Administration. Methods: We imaged VEGFR in an orthotopic mammary tumor model during the course of treatment with sunitinib using a recently developed SPECT tracer, a 99mTc-labeled single-chain VEGF (scVEGF), that binds to and is internalized by VEGFR. Tumors from imaged mice were harvested and cryosectioned, and alternating sections were analyzed by autoradiography and immunohistochemistry to determine the expression of endothelial cell markers VEGFR-2 and CD31. Results: In vitro assays with endothelial cells overexpressing VEGFR-2 established that sunitinib does not inhibit VEGFR-2–mediated uptake of scVEGF-based tracers. SPECT and autoradiography with 99mTc-scVEGF of tumor cryosections revealed a 2.2- to 2.6-fold decrease in tracer uptake after 4 daily doses of sunitinib. However, once treatment was discontinued, tracer uptake rapidly (3 d) increased, particularly at the tumor edges. Immunohistochemical analysis of VEGFR-2 and CD31 supported SPECT and autoradiographic imaging findings, revealing the corresponding depletion of VEGFR-2– and CD31-positive endothelial cells from tumor vasculature during therapy and the rapid reemergence of VEGFR-2– and CD31-positive vasculature at the tumor edges after discontinuation of treatment. Conclusion: Our findings suggest that imaging with 99mTc-scVEGF might be useful for monitoring VEGFR responses to antiangiogenic treatment regimens.

IGF1R- and ROR1-Specific CAR T Cells as a Potential Therapy for High Risk Sarcomas

Patients with metastatic or recurrent and refractory sarcomas have a dismal prognosis. Therefore, new targeted therapies are urgently needed. This study was designed to evaluate chimeric antigen receptor (CAR) T cells targeting the type I insulin-like growth factor receptor (IGF1R) or tyrosine kinase-like orphan receptor 1 (ROR1) molecules for their therapeutic potential against sarcomas. Here, we report that IGF1R (15/15) and ROR1 (11/15) were highly expressed in sarcoma cell lines including Ewing sarcoma, osteosarcoma, alveolar or embryonal rhabdomyosarcoma, and fibrosarcoma. IGF1R and ROR1 CAR T cells derived from eight healthy donors using the Sleeping Beauty (SB) transposon system were cytotoxic against sarcoma cells and produced high levels of IFN-γ, TNF-α and IL-13 in an antigen-specific manner. IGF1R and ROR1 CAR T cells generated from three sarcoma patients released significant amounts of IFN-γ in response to sarcoma stimulation. The adoptive transfer of IGF1R and ROR1 CAR T cells derived from a sarcoma patient significantly reduced tumor growth in pre-established, systemically disseminated and localized osteosarcoma xenograft models in NSG mice. Infusion of IGF1R and ROR1 CAR T cells also prolonged animal survival in a localized sarcoma model using NOD/scid mice. Our data indicate that both IGF1R and ROR1 can be effectively targeted by SB modified CAR T cells and that such CAR T cells may be useful in the treatment of high risk sarcoma patients.

Enhanced fluorescence diffuse optical tomography with indocyanine green-encapsulating liposomes targeted to receptors for vascular endothelial growth factor in tumor vasculature

Abstract. To develop an indocyanine green (ICG) tracer with slower clearance kinetics, we explored ICG-encapsulating liposomes (Lip) in three different formulations: untargeted (Lip/ICG), targeted to vascular endothelial growth factor (VEGF) receptors (scVEGF-Lip/ICG) by the receptor-binding moiety single-chain VEGF (scVEGF), or decorated with inactivated scVEGF (inactive-Lip/ICG) that does not bind to VEGF receptors. Experiments were conducted with tumor-bearing mice that were placed in a scattering medium with tumors located at imaging depths of either 1.5 or 2.0 cm. Near-infrared fluorescence diffuse optical tomography that provides depth-resolved spatial distributions of fluorescence in tumor was used for the detection of postinjection fluorescent signals. All liposome-based tracers, as well as free ICG, were injected intravenously into mice in the amounts corresponding to 5 nmol of ICG/mouse, and the kinetics of increase and decrease of fluorescent signals in tumors were monitored. A signal from free ICG reached maximum at 15-min postinjection and then rapidly declined with t1/2 of ∼20  min. The signals from untargeted Lip/ICG and inactive-Lip/ICG also reached maximum at 15-min postinjection, however, declined somewhat slower than free ICG with t1/2 of ∼30  min. By contrast, a signal from targeted scVEGF-Lip/ICG grew slower than that of all other tracers, reaching maximum at 30-min postinjection and declined much slower than that of other tracers with t1/2 of ∼90  min, providing a more extended observation window. Higher scVEGF-Lip/ICG tumor accumulation was further confirmed by the analysis of fluorescence on cryosections of tumors that were harvested from animals at 400 min after injection with different tracers.

Inhibition of Vascular Endothelial Growth Factor Receptor Signaling in Angiogenic Tumor Vasculature

Neovascularization takes place in a large number of pathologies, including cancer. Significant effort has been invested in the development of agents that can inhibit this process, and an increasing number of such agents, known as antiangiogenic drugs, are entering clinical trials or being approved for clinical use. The key players involved in the development and maintenance of tumor neovasculature are vascular endothelial growth factor (VEGF) and its receptors (VEGFRs), and therefore VEGF/VEGFR signaling pathways have been a focus of anticancer therapies for several decades. This review focuses on two main approaches designed to selectively target VEGFRs, inhibiting VEGFR with small molecule inhibitors of receptor tyrosine kinase activity and inhibiting the binding of VEGF to VEGFRs with specific antibodies or soluble decoy VEGF receptors. The major problem with these strategies is that they appeared to be effective only in relatively small and unpredictable subsets of patients. An alternative approach would be to subvert VEGFR for intracellular delivery of cytotoxic molecules. We describe here one such molecule, SLT-VEGF, a fusion protein containing VEGF121 and the highly cytotoxic catalytic subunit of Shiga-like toxin.

Coronary artery stenosis in rats affects β-adrenergic receptor signaling in myocytes

OBJECTIVE The purpose of this study was to determine whether the early chronic ischemic cardiomyopathy produced by non-occlusive coronary artery constriction was characterized by alterations in the regulation of beta-adrenoreceptor (beta-AR) signaling. METHODS Coronary artery narrowing was surgically induced in rats and the animals sacrificed at 7 and 14 days. The changes in the biochemical properties of the multiple components of the beta-AR pathway were examined in enzymatically dissociated myocytes. RESULTS Coronary stenosis, involving an average 55% reduction in luminal diameter, was associated with left ventricular failure and right ventricular dysfunction at both time intervals. A decrease in the quantity of beta-AR was detected at 7 days and preceded the loss of high-affinity binding sites. This regulatory modification was characterized by a reduction in beta 1 and beta 2 receptors and a shift in the isoproterenol dose response curve indicating a functional correlation between the decrease in beta-AR and attenuated inotropic support of the myocardium. The percentage of beta-AR binding agonist with high affinity decreased significantly at 14 days along with a further reduction in the density of beta 1 and beta 2 receptors. Reconstitution studies with cyc S49 lymphoma cells did not detect an impairment of Gs alpha functional activity, but the quantity of Gi alpha was increased at both intervals. Finally, activation of the catalytic unit of adenylyl cyclase by forskolin and GTP was not altered by coronary stenosis, however, basal cyclic AMP in myocytes was depressed at 14 days. CONCLUSIONS Coronary stenosis induces distinct and progressive modifications in the beta-AR signaling cascade which may contribute to the impaired ventricular performance in this model of myocardial ischemia.

Improved tumor targeting of rhenium-186-labeled anti-human high-M.W. melanoma-associated antigen monoclonal antibody 763.74 following purification with anti-idiotypic monoclonal antibody MK2-23

Because of its high‐energy beta emissions and imageable gamma emissions, 186Re represents an attractive isotope to radiolabel monoclonal antibodies (MAbs) recognizing human tumor‐associated antigens (TAAs) for radioimmunoscintigraphy (RIS) and radioimmunotherapy (RIT) of patients with malignant diseases. Application of186Re, however, is hindered by the frequent denaturation of MAbs following exposure to the strong reducing conditions employed in the labeling procedures. To overcome this problem, we have utilized a direct labeling procedure and combined it with affinity chromatography over columns of immobilized anti‐idiotypic (anti‐id) MAbs which recognize idiotopes in the antigen‐combining site of the radiolabeled MAb. The validity of the procedure was demonstrated with the anti‐high‐m.w. melanoma‐associated antigen (HMW‐MAA) MAb 763.74 and its corresponding anti‐id MAb MK2–23. Utilizing this approach, MAb 763.74 was labeled to specific activities of 2.8 ± 0.6 mCi/mg with 186Re. Furthermore, its immunoreactivity, which was reduced to about 30% following labeling with 186Re, was improved to about 50% by affinity chromatography over columns of anti‐id MAb MK2–23. The improvement in immunoreactivity of 186Re MAb 763.74 resulted in a significant (p < 0.05) increase in targeting to human melanoma tumors grown in nude mice and an increase in sensitivity of RIS. Our results suggest that direct labeling of anti‐TAA MAb with 186Re coupled with purification over affinity columns of anti‐id MAb may facilitate the application of 186Re to RIS and RIT. Int. J. Cancer 78:486–490, 1998.