The identification and use of antibodies dominates the biologic, clinical diagnostic, and therape... more The identification and use of antibodies dominates the biologic, clinical diagnostic, and therapeutic landscapes. In particular, antibodies have become essential tools in a variety of protein analytical experiments and to study the disposition of biologic therapeutics. One emerging class of peptide biologics is known as the Elastin-like polypeptide (ELP), which are repetitive protein polymers inspired by human tropoelastin. A major limitation in the clinical translation of ELP biologics has been a lack of a monoclonal antibody (mAb) to characterize their identity during expression. To facilitate these studies, we successfully generated a new mAb that is specific towards ELPs and ELP fusion proteins. Purified antibody was evaluated in ELISA, Western Blotting, and immunofluorescence assay. The optimal anti-ELP mAb proved highly reactive and specific towards ELPs. Moreover, they were able to detect ELPs with a variety of aliphatic guest residues. ELPs phase separate in response to heating; furthermore, when incubated at great excess to ELP the anti-ELP mAb partially blocks phase separation. These findings are direct evidence that novel murine mAbs can be raised against purified ELPs. This new reagent will enable purification and experimental detection, and characterization of these biopolymers.
International journal of molecular sciences, Jan 20, 2017
T cells expressing chimeric antigen receptors (CARs) recognizing CD19 epitopes have produced rema... more T cells expressing chimeric antigen receptors (CARs) recognizing CD19 epitopes have produced remarkable anti-tumor effects in patients with B-cell malignancies. However, cancer cells lacking recognized epitopes can emerge, leading to relapse and death. Thus, CAR T cells targeting different epitopes on different antigens could improve immunotherapy. The Lym-1 antibody targets a conformational epitope of Human Leukocyte Antigen-antigen D Related (HLA-DR) on the surface of human B-cell lymphomas. Lym-1 CAR T cells were thus generated for evaluation of cytotoxic activity towards lymphoma cells in vitro and in vivo. Human T cells from healthy donors were transduced to express a Lym-1 CAR, and assessed for epitope-driven function in culture and towards Raji xenografts in NOD-scidIL2Rgammanull (NSG) mice. Lym-1 CAR T cells exhibited epitope-driven activation and lytic function against human B-cell lymphoma cell lines in culture and mediated complete regression of Raji/Luciferase-Green fluo...
The ‘pre-S’ parts of the envelope protein of hepatitis B virus (HBV) have been proposed to be inv... more The ‘pre-S’ parts of the envelope protein of hepatitis B virus (HBV) have been proposed to be involved in the infection of hepatocytes by HBV. In order to facilitate the study of these processes, we have developed an expression system to allow the production and purification of large quantities of the pre-S protein. To obtain a protein containing all of the pre-S sequence and only this sequence, mutations were introduced into the HBV(ayw) genome to create an NdeI restriction site at the initial ATG of the large surface protein gene. Also, stop codons and a BglII restriction site were introduced after the last codon of pre-S2. This fragment was then cloned into the high-expression vector pET-3A. A protein of the expected Mr was expressed at a level of up to 10% of the total soluble protein in HMS174 (DE3) cells, as judged by SDS/PAGE. A rapid purification method has been developed for this protein. The protein retains the polyalbumin-binding activity ascribed to the pre-S2 sequence, ...
Public reporting burden for this collection of information is estimated to average 1 hour per res... more Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON
LEC chemokine promotes TH1 responses and recruits immune cells to inflammatory sites. By linking ... more LEC chemokine promotes TH1 responses and recruits immune cells to inflammatory sites. By linking LEC to an antibody targeting tumor necrosis, LEC/chTNT-3 can be used for the immunotherapeutic treatment of tumors. The primary objective of this study was to determine the safety profile of LEC/chTNT-3 and toceranib phosphate (Palladia(®)) combination therapy in dogs with spontaneous malignancies. Secondary purpose was to determine objective responses to treatment. Twenty-three dogs with cancer were enrolled, covering nine different malignancies. In this dose escalation study, dogs received LEC/chTNT-3 for five days, and toceranib every 48 hours for the remainder of the study. Dogs received physical exams, chemistry panel, urinalysis, and complete blood counts on days 0, 10, 28 of the study, and every 6-8 weeks thereafter. Lethargy was noted in 13% dogs. There were no statistical differences in the prevalence of anorexia, diarrhea, thrombocytopenia, renal toxicity, or hepatic toxicity b...
Contortrostatin is a unique dimeric disintegrin isolated from southern copperhead snake venom. Th... more Contortrostatin is a unique dimeric disintegrin isolated from southern copperhead snake venom. Through antagonism of integrins αIIbβ3, α5β1, αvβ3, and αvβ5, contortrostatin inhibits platelet aggregation and disrupts cancer cell adhesion and invasion. We cloned cDNA from a library made from the venom gland cells of Agkistrodon contortrix contortrix using polymerase chain reaction. We found that the contortrostatin gene is part
LEC/chTNT-3, a chemokine fusion protein generated previously in our laboratory, produces a 40-60%... more LEC/chTNT-3, a chemokine fusion protein generated previously in our laboratory, produces a 40-60% reduction in well-established solid tumors of the BALB/c mouse. In this study, CD25(+) T-cell depletion was used in combination with LEC/chTNT-3 treatment to enhance the therapeutic value of this approach. In two tumor models (Colon 26 and RENCA), this combination immunotherapy produced complete regression of established s.c. tumors after 5 consecutive days of i.v. treatment. To show that targeted LEC is critical to these results, similar combination studies were performed with chTNT-3/cytokine fusion proteins consisting of human interleukin 2, murine IFN-gamma, and murine granulocyte macrophage colony-stimulating factor using identical treatment regimens. These studies showed no significant improvement indicating that combination therapy with anti-CD25(+) antisera requires LEC localization to tumor to produce complete regression. To study the mechanism of this remarkable response, immu...
Tissue Factor (TF) is a cell membrane receptor protein that is the initiator of the extrinsic pat... more Tissue Factor (TF) is a cell membrane receptor protein that is the initiator of the extrinsic pathway of the blood coagulation cascade and normally released from damaged tissues. By substituting the attachment site with a tumor delivery agent, this potent thrombogenic protein in its truncated form (tTF) can be targeted to the tumor where it can initiate clotting, thereby occluding the tumor's blood supply and causing rapid tumor destruction. To test the therapeutic potential of this vascular targeting approach, three fusion proteins, chTNT-3/tTF, chTV-1/tTF, and RGD/tTF, which target DNA exposed in degenerative areas of tumors, fibronectin on the tumor vascular basement membrane, and alpha nu beta 3 on the luminal side of tumor vessels, respectively, were developed and tested for their antitumor effects. Antigen binding and clotting assays demonstrated that each of the fusion proteins retained their antigen binding and thrombogenic activities. In vivo studies in mice bearing est...
Clinical cancer research : an official journal of the American Association for Cancer Research, 1999
The efficacy of molecular therapies for human malignancies is limited by inadequate accumulation ... more The efficacy of molecular therapies for human malignancies is limited by inadequate accumulation within solid tumors. Our laboratory has developed a novel approach that uses monoclonal antibodies (MAbs) to direct vasoactive proteins to tumor sites to increase local vascular permeability and, in turn, improve the delivery of therapeutic reagents. Previously, we demonstrated that pretreatment with immunoconjugates containing interleukin-2 (IL-2) enhances specific tumor uptake of radiolabeled MAbs without affecting normal tissues. In the present study, we describe a fusion protein consisting of a chimeric antinuclear antibody and IL-2 (chTNT-3/IL-2) and illustrate its potential for improving the delivery of both MAbs and drugs. The ability of pretreatment with chTNT-3/IL-2 to increase specific tumor uptake of the MAb B72.3 was demonstrated in LS174T colon tumor-bearing mice. Tumor accretion of B72.3 increased nearly 3-fold, with no changes in normal tissues. Abrogation of this effect w...
Promising results from clinical trials have led to renewed interest in effector mechanisms operat... more Promising results from clinical trials have led to renewed interest in effector mechanisms operating in antibody-based therapy of leukemia and lymphoma. We tested a panel of B-cell antibodies from the Sixth Human Leukocyte Differentiation Antigen workshop for their capacity to mediate antibody-dependent cellular cytotoxicity, often considered to be one of the most potent effector mechanisms in vivo. As effector cells, mononuclear cells and polymorphonuclear (PMN) cells from healthy donors were compared with Fc gammaRI (CD64)-expressing PMN cells from patients receiving granulocyte colony-stimulating factor (G-CSF) treatment. Of the 29 IgG workshop antibodies binding most strongly to the tested malignant human B-cell lines, only 3 consistently induced target cell lysis. These three antibodies were determined to be HLA DR reactive. Experiments with a panel of HLA class II antibodies showed the involvement of individual Fc gamma receptors on effector cells to be strongly dependent on t...
As a source of recombinant antigen, soluble constant fragment (Fc) fusion proteins have become va... more As a source of recombinant antigen, soluble constant fragment (Fc) fusion proteins have become valuable reagents for immunotherapy and laboratory investigations. Additional applications for these reagents include flow cytometry, immunohistochemistry, and in vitro activity assays. To aid investigators in the generation of these reagents, the materials and methods required for producing Fc fusion proteins are described. The investigator's protein moiety of interest is genetically linked to the N-terminus of murine Fc and subsequently expressed in large quantity using a mammalian cell expression system. The resulting Fc fusion proteins are purified on a protein A column and may be stored for at least one year at -20 degrees C. The availability of easily purified, soluble Fc fusion proteins in such quantity can facilitate research in multiple fields of medicine and biotechnology.
We used cDNA microarrays to identify differentially expressed genes in mice in response to infect... more We used cDNA microarrays to identify differentially expressed genes in mice in response to infections with influenza virus A/PR/8/34 (H1N1) and Streptococcus pneumoniae. Expression microarray analysis showed up-regulation and down-regulation of many genes involved in the defense, inflammatory response and intracellular signaling pathways including chemokine, apoptosis, MAPK, Notch, Jak-STAT, T-cell receptor and complement and coagulation cascades. We have revealed signature patterns of gene expression in mice infected with two different classes of pathogens: influenza virus A and S. pneumoniae. Quantitative real-time RT-PCR results confirmed microarray results for most of the genes tested. These studies document clear differences in gene expression profiles between mice infected with influenza virus A and S. pneumoniae. Identification of genes that are differentially expressed after respiratory infections can provide insights into the mechanisms by which the host interacts with different pathogens, useful information about stage of diseases and selection of suitable targets for early diagnosis and treatments. The advantage of this novel approach is that the detection of pathogens is based on the differences in host gene expression profiles in response to different pathogens instead of detecting pathogens directly.
JNCI Journal of the National Cancer Institute, 2003
Background: The cytokine interleukin 2 (IL-2) is involved in the activation of T cells and has be... more Background: The cytokine interleukin 2 (IL-2) is involved in the activation of T cells and has been shown to play a central role in cancer immunotherapy. The full therapeutic potential of IL-2, however, has not been realized because of its doselimiting systemic toxicity. We sought to identify a region of IL-2 that is responsible for the induction of vasopermeability (leaky tumor endothelium), a property associated with the toxicity of the molecule. Methods: Intact IL-2 or overlapping synthetic peptides of IL-2 that were chemically conjugated to tumor-targeting monoclonal antibodies (TNT-1 or Lym-1) were injected into groups of mice (n = 4) that had previously been xenotransplanted with human tumor cells (ME-180 cervical carcinoma and Raji lymphoma). Two hours later, mice received intravenous injections of radiolabeled tracer antibody, and 3 days later they were subjected to biodistribution analysis to measure the ability of each immunoconjugate to enhance tumor uptake of the tracer antibody (i.e., vasopermeability activity). The cytokine activity of the immunoconjugates was determined by assaying their ability to promote the proliferation of a mouse IL-2-dependent cell line. Results: Pretreatment of mice with an antibody/IL-2 immunoconjugate resulted in an approximately fourfold increase in radiolabeled tracer antibody uptake in the xenograft tumor as compared with uptake in mice injected with antibody alone. One synthetic fragment consisting of amino acids 22-58 contained 100% of the vasopermeability activity of IL-2 and was designated permeability-enhancing peptide (PEP). PEP had vasopermeability activity only when conjugated to a tumor-targeting antibody, had maximal activity as a dimer, and was devoid of cytokine activity. Conclusions: The identification of PEP should aid in the discovery of ways to decrease the toxicity of IL-2. Moreover, PEP is a promising candidate for the generation of agents that can enhance the delivery of antibodies and drugs to tumors. [
Despite the growing number of preclinical and clinical trials focused on immunotherapy for the tr... more Despite the growing number of preclinical and clinical trials focused on immunotherapy for the treatment of malignant gliomas, the prognosis for this disease remains grim. Although some promising advances have been made, the immune response stimulated as a result of immunotherapeutic protocols has been inefficient at complete tumor elimination, primarily due to our lack of understanding of the necessary effector functions of the immune system. We previously demonstrated that a tumor lysate vaccine/Fc-OX40L therapy is capable of inducing enhanced survival and tumor elimination in the GL261 mouse glioma model. The following experiments were performed to determine the mechanism(s) of action of this therapy that elicits a potent antitumor immune response. The evidence subsequently outlined indicates a CD8 + T cell-independent and CD4 + T cell-, NK cell-, and B cell-dependent means of prolonged survival. CD8 + T cell-independent tumor clearance is surprising considering the current focus of many cancer immunotherapy protocols. These results provide evidence for CD8 + T cell-independent means of antitumor response and should lead to additional examination of the potential manipulation of this mechanism for future treatment strategies.
Purpose Tumor necrosis treatment (TNT) uses degenerating tumor cells and necrotic regions of tumo... more Purpose Tumor necrosis treatment (TNT) uses degenerating tumor cells and necrotic regions of tumors as targets for radioimmunotherapy. Previous studies in animal tumor models and clinical trials have demonstrated that when linked to the therapeutic radionuclide iodine-131, recombinant chimeric TNT antibody (131I-chTNT) can deliver therapeutic doses to tumors regardless of the location or type of malignancy. Therapeutic efficacy and toxicity of 131I-chTNT in advanced lung cancer patients were studied in this pivotal registration trial. Patients and Methods Patients with advanced lung cancer were treated with systemic or intratumoral injection of 131I-chTNT in eight oncology centers in China. The objective response rate (ORR) was assessed as the primary end point. Results All 107 patients who were entered onto the study and completed therapy had experienced treatment failure after prior radiotherapy or chemotherapy a mean of three times. The results showed an ORR of 34.6% (complete re...
Purpose: We tested the combination of a tumor lysate vaccine with a panel of costimulatory molecu... more Purpose: We tested the combination of a tumor lysate vaccine with a panel of costimulatory molecules to identify an immunotherapeutic approach capable of curing established murine gliomas. Experimental Design: Glioma-bearing mice were primed with a tumor lysate vaccine, followed by systemic administration of the following costimulatory ligands: OX40L, CD80, 4-1BBL, and GITRL, which were fused to the Fc portion of human immunoglobulin. Lymphocytes and mRNA were purified from the brain tumor site for immune monitoring studies. Numerous variations of the vaccine and Fc-OX40L regimen were tested alone or in combination with temozolomide. Results: Lysate vaccinations combined with Fc-OX40L led to the best overall survival, yielding cure rates of 50% to 100% depending on the timing, regimen, and combination with temozolomide. Cured mice that were rechallenged with glioma cells rejected the challenge, showing immunologic memory. Lymphocytes isolated from the draining lymph nodes of vaccine...
Purpose: Previously, we have shown that the attachment of interleukin 2 (IL-2) to a tumor-targeti... more Purpose: Previously, we have shown that the attachment of interleukin 2 (IL-2) to a tumor-targeting antibody can produce a 4-fold enhancement in the uptake of antibodies and drugs in tumors. More recently, we discovered that a 37-amino-acid linear sequence of IL-2 designated vasopermeability-enhancing peptide (PEP), contained the vasopermeability activity of IL-2, and could be used after linkage to tumor-targeting antibodies to produce the same enhancement of drugs and antibodies in tumors. We now describe the generation of a fully human antibody fusion protein, designated NHS76/PEP2, which can be used in patients to enhance the therapeutic potential of chemotherapy. Methods: NHS76/PEP2 was expressed in NS0 cells using the glutamine synthetase gene amplification system. To show its clinical potential as a pretreatment to chemotherapy, NHS76/PEP2 was given i.v. 2 hours before the injection of suboptimal doses of etoposide, doxorubicin, Taxol, Taxotere, 5-fluorouracil, or vinblastine ...
The identification and use of antibodies dominates the biologic, clinical diagnostic, and therape... more The identification and use of antibodies dominates the biologic, clinical diagnostic, and therapeutic landscapes. In particular, antibodies have become essential tools in a variety of protein analytical experiments and to study the disposition of biologic therapeutics. One emerging class of peptide biologics is known as the Elastin-like polypeptide (ELP), which are repetitive protein polymers inspired by human tropoelastin. A major limitation in the clinical translation of ELP biologics has been a lack of a monoclonal antibody (mAb) to characterize their identity during expression. To facilitate these studies, we successfully generated a new mAb that is specific towards ELPs and ELP fusion proteins. Purified antibody was evaluated in ELISA, Western Blotting, and immunofluorescence assay. The optimal anti-ELP mAb proved highly reactive and specific towards ELPs. Moreover, they were able to detect ELPs with a variety of aliphatic guest residues. ELPs phase separate in response to heating; furthermore, when incubated at great excess to ELP the anti-ELP mAb partially blocks phase separation. These findings are direct evidence that novel murine mAbs can be raised against purified ELPs. This new reagent will enable purification and experimental detection, and characterization of these biopolymers.
International journal of molecular sciences, Jan 20, 2017
T cells expressing chimeric antigen receptors (CARs) recognizing CD19 epitopes have produced rema... more T cells expressing chimeric antigen receptors (CARs) recognizing CD19 epitopes have produced remarkable anti-tumor effects in patients with B-cell malignancies. However, cancer cells lacking recognized epitopes can emerge, leading to relapse and death. Thus, CAR T cells targeting different epitopes on different antigens could improve immunotherapy. The Lym-1 antibody targets a conformational epitope of Human Leukocyte Antigen-antigen D Related (HLA-DR) on the surface of human B-cell lymphomas. Lym-1 CAR T cells were thus generated for evaluation of cytotoxic activity towards lymphoma cells in vitro and in vivo. Human T cells from healthy donors were transduced to express a Lym-1 CAR, and assessed for epitope-driven function in culture and towards Raji xenografts in NOD-scidIL2Rgammanull (NSG) mice. Lym-1 CAR T cells exhibited epitope-driven activation and lytic function against human B-cell lymphoma cell lines in culture and mediated complete regression of Raji/Luciferase-Green fluo...
The ‘pre-S’ parts of the envelope protein of hepatitis B virus (HBV) have been proposed to be inv... more The ‘pre-S’ parts of the envelope protein of hepatitis B virus (HBV) have been proposed to be involved in the infection of hepatocytes by HBV. In order to facilitate the study of these processes, we have developed an expression system to allow the production and purification of large quantities of the pre-S protein. To obtain a protein containing all of the pre-S sequence and only this sequence, mutations were introduced into the HBV(ayw) genome to create an NdeI restriction site at the initial ATG of the large surface protein gene. Also, stop codons and a BglII restriction site were introduced after the last codon of pre-S2. This fragment was then cloned into the high-expression vector pET-3A. A protein of the expected Mr was expressed at a level of up to 10% of the total soluble protein in HMS174 (DE3) cells, as judged by SDS/PAGE. A rapid purification method has been developed for this protein. The protein retains the polyalbumin-binding activity ascribed to the pre-S2 sequence, ...
Public reporting burden for this collection of information is estimated to average 1 hour per res... more Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON
LEC chemokine promotes TH1 responses and recruits immune cells to inflammatory sites. By linking ... more LEC chemokine promotes TH1 responses and recruits immune cells to inflammatory sites. By linking LEC to an antibody targeting tumor necrosis, LEC/chTNT-3 can be used for the immunotherapeutic treatment of tumors. The primary objective of this study was to determine the safety profile of LEC/chTNT-3 and toceranib phosphate (Palladia(®)) combination therapy in dogs with spontaneous malignancies. Secondary purpose was to determine objective responses to treatment. Twenty-three dogs with cancer were enrolled, covering nine different malignancies. In this dose escalation study, dogs received LEC/chTNT-3 for five days, and toceranib every 48 hours for the remainder of the study. Dogs received physical exams, chemistry panel, urinalysis, and complete blood counts on days 0, 10, 28 of the study, and every 6-8 weeks thereafter. Lethargy was noted in 13% dogs. There were no statistical differences in the prevalence of anorexia, diarrhea, thrombocytopenia, renal toxicity, or hepatic toxicity b...
Contortrostatin is a unique dimeric disintegrin isolated from southern copperhead snake venom. Th... more Contortrostatin is a unique dimeric disintegrin isolated from southern copperhead snake venom. Through antagonism of integrins αIIbβ3, α5β1, αvβ3, and αvβ5, contortrostatin inhibits platelet aggregation and disrupts cancer cell adhesion and invasion. We cloned cDNA from a library made from the venom gland cells of Agkistrodon contortrix contortrix using polymerase chain reaction. We found that the contortrostatin gene is part
LEC/chTNT-3, a chemokine fusion protein generated previously in our laboratory, produces a 40-60%... more LEC/chTNT-3, a chemokine fusion protein generated previously in our laboratory, produces a 40-60% reduction in well-established solid tumors of the BALB/c mouse. In this study, CD25(+) T-cell depletion was used in combination with LEC/chTNT-3 treatment to enhance the therapeutic value of this approach. In two tumor models (Colon 26 and RENCA), this combination immunotherapy produced complete regression of established s.c. tumors after 5 consecutive days of i.v. treatment. To show that targeted LEC is critical to these results, similar combination studies were performed with chTNT-3/cytokine fusion proteins consisting of human interleukin 2, murine IFN-gamma, and murine granulocyte macrophage colony-stimulating factor using identical treatment regimens. These studies showed no significant improvement indicating that combination therapy with anti-CD25(+) antisera requires LEC localization to tumor to produce complete regression. To study the mechanism of this remarkable response, immu...
Tissue Factor (TF) is a cell membrane receptor protein that is the initiator of the extrinsic pat... more Tissue Factor (TF) is a cell membrane receptor protein that is the initiator of the extrinsic pathway of the blood coagulation cascade and normally released from damaged tissues. By substituting the attachment site with a tumor delivery agent, this potent thrombogenic protein in its truncated form (tTF) can be targeted to the tumor where it can initiate clotting, thereby occluding the tumor's blood supply and causing rapid tumor destruction. To test the therapeutic potential of this vascular targeting approach, three fusion proteins, chTNT-3/tTF, chTV-1/tTF, and RGD/tTF, which target DNA exposed in degenerative areas of tumors, fibronectin on the tumor vascular basement membrane, and alpha nu beta 3 on the luminal side of tumor vessels, respectively, were developed and tested for their antitumor effects. Antigen binding and clotting assays demonstrated that each of the fusion proteins retained their antigen binding and thrombogenic activities. In vivo studies in mice bearing est...
Clinical cancer research : an official journal of the American Association for Cancer Research, 1999
The efficacy of molecular therapies for human malignancies is limited by inadequate accumulation ... more The efficacy of molecular therapies for human malignancies is limited by inadequate accumulation within solid tumors. Our laboratory has developed a novel approach that uses monoclonal antibodies (MAbs) to direct vasoactive proteins to tumor sites to increase local vascular permeability and, in turn, improve the delivery of therapeutic reagents. Previously, we demonstrated that pretreatment with immunoconjugates containing interleukin-2 (IL-2) enhances specific tumor uptake of radiolabeled MAbs without affecting normal tissues. In the present study, we describe a fusion protein consisting of a chimeric antinuclear antibody and IL-2 (chTNT-3/IL-2) and illustrate its potential for improving the delivery of both MAbs and drugs. The ability of pretreatment with chTNT-3/IL-2 to increase specific tumor uptake of the MAb B72.3 was demonstrated in LS174T colon tumor-bearing mice. Tumor accretion of B72.3 increased nearly 3-fold, with no changes in normal tissues. Abrogation of this effect w...
Promising results from clinical trials have led to renewed interest in effector mechanisms operat... more Promising results from clinical trials have led to renewed interest in effector mechanisms operating in antibody-based therapy of leukemia and lymphoma. We tested a panel of B-cell antibodies from the Sixth Human Leukocyte Differentiation Antigen workshop for their capacity to mediate antibody-dependent cellular cytotoxicity, often considered to be one of the most potent effector mechanisms in vivo. As effector cells, mononuclear cells and polymorphonuclear (PMN) cells from healthy donors were compared with Fc gammaRI (CD64)-expressing PMN cells from patients receiving granulocyte colony-stimulating factor (G-CSF) treatment. Of the 29 IgG workshop antibodies binding most strongly to the tested malignant human B-cell lines, only 3 consistently induced target cell lysis. These three antibodies were determined to be HLA DR reactive. Experiments with a panel of HLA class II antibodies showed the involvement of individual Fc gamma receptors on effector cells to be strongly dependent on t...
As a source of recombinant antigen, soluble constant fragment (Fc) fusion proteins have become va... more As a source of recombinant antigen, soluble constant fragment (Fc) fusion proteins have become valuable reagents for immunotherapy and laboratory investigations. Additional applications for these reagents include flow cytometry, immunohistochemistry, and in vitro activity assays. To aid investigators in the generation of these reagents, the materials and methods required for producing Fc fusion proteins are described. The investigator's protein moiety of interest is genetically linked to the N-terminus of murine Fc and subsequently expressed in large quantity using a mammalian cell expression system. The resulting Fc fusion proteins are purified on a protein A column and may be stored for at least one year at -20 degrees C. The availability of easily purified, soluble Fc fusion proteins in such quantity can facilitate research in multiple fields of medicine and biotechnology.
We used cDNA microarrays to identify differentially expressed genes in mice in response to infect... more We used cDNA microarrays to identify differentially expressed genes in mice in response to infections with influenza virus A/PR/8/34 (H1N1) and Streptococcus pneumoniae. Expression microarray analysis showed up-regulation and down-regulation of many genes involved in the defense, inflammatory response and intracellular signaling pathways including chemokine, apoptosis, MAPK, Notch, Jak-STAT, T-cell receptor and complement and coagulation cascades. We have revealed signature patterns of gene expression in mice infected with two different classes of pathogens: influenza virus A and S. pneumoniae. Quantitative real-time RT-PCR results confirmed microarray results for most of the genes tested. These studies document clear differences in gene expression profiles between mice infected with influenza virus A and S. pneumoniae. Identification of genes that are differentially expressed after respiratory infections can provide insights into the mechanisms by which the host interacts with different pathogens, useful information about stage of diseases and selection of suitable targets for early diagnosis and treatments. The advantage of this novel approach is that the detection of pathogens is based on the differences in host gene expression profiles in response to different pathogens instead of detecting pathogens directly.
JNCI Journal of the National Cancer Institute, 2003
Background: The cytokine interleukin 2 (IL-2) is involved in the activation of T cells and has be... more Background: The cytokine interleukin 2 (IL-2) is involved in the activation of T cells and has been shown to play a central role in cancer immunotherapy. The full therapeutic potential of IL-2, however, has not been realized because of its doselimiting systemic toxicity. We sought to identify a region of IL-2 that is responsible for the induction of vasopermeability (leaky tumor endothelium), a property associated with the toxicity of the molecule. Methods: Intact IL-2 or overlapping synthetic peptides of IL-2 that were chemically conjugated to tumor-targeting monoclonal antibodies (TNT-1 or Lym-1) were injected into groups of mice (n = 4) that had previously been xenotransplanted with human tumor cells (ME-180 cervical carcinoma and Raji lymphoma). Two hours later, mice received intravenous injections of radiolabeled tracer antibody, and 3 days later they were subjected to biodistribution analysis to measure the ability of each immunoconjugate to enhance tumor uptake of the tracer antibody (i.e., vasopermeability activity). The cytokine activity of the immunoconjugates was determined by assaying their ability to promote the proliferation of a mouse IL-2-dependent cell line. Results: Pretreatment of mice with an antibody/IL-2 immunoconjugate resulted in an approximately fourfold increase in radiolabeled tracer antibody uptake in the xenograft tumor as compared with uptake in mice injected with antibody alone. One synthetic fragment consisting of amino acids 22-58 contained 100% of the vasopermeability activity of IL-2 and was designated permeability-enhancing peptide (PEP). PEP had vasopermeability activity only when conjugated to a tumor-targeting antibody, had maximal activity as a dimer, and was devoid of cytokine activity. Conclusions: The identification of PEP should aid in the discovery of ways to decrease the toxicity of IL-2. Moreover, PEP is a promising candidate for the generation of agents that can enhance the delivery of antibodies and drugs to tumors. [
Despite the growing number of preclinical and clinical trials focused on immunotherapy for the tr... more Despite the growing number of preclinical and clinical trials focused on immunotherapy for the treatment of malignant gliomas, the prognosis for this disease remains grim. Although some promising advances have been made, the immune response stimulated as a result of immunotherapeutic protocols has been inefficient at complete tumor elimination, primarily due to our lack of understanding of the necessary effector functions of the immune system. We previously demonstrated that a tumor lysate vaccine/Fc-OX40L therapy is capable of inducing enhanced survival and tumor elimination in the GL261 mouse glioma model. The following experiments were performed to determine the mechanism(s) of action of this therapy that elicits a potent antitumor immune response. The evidence subsequently outlined indicates a CD8 + T cell-independent and CD4 + T cell-, NK cell-, and B cell-dependent means of prolonged survival. CD8 + T cell-independent tumor clearance is surprising considering the current focus of many cancer immunotherapy protocols. These results provide evidence for CD8 + T cell-independent means of antitumor response and should lead to additional examination of the potential manipulation of this mechanism for future treatment strategies.
Purpose Tumor necrosis treatment (TNT) uses degenerating tumor cells and necrotic regions of tumo... more Purpose Tumor necrosis treatment (TNT) uses degenerating tumor cells and necrotic regions of tumors as targets for radioimmunotherapy. Previous studies in animal tumor models and clinical trials have demonstrated that when linked to the therapeutic radionuclide iodine-131, recombinant chimeric TNT antibody (131I-chTNT) can deliver therapeutic doses to tumors regardless of the location or type of malignancy. Therapeutic efficacy and toxicity of 131I-chTNT in advanced lung cancer patients were studied in this pivotal registration trial. Patients and Methods Patients with advanced lung cancer were treated with systemic or intratumoral injection of 131I-chTNT in eight oncology centers in China. The objective response rate (ORR) was assessed as the primary end point. Results All 107 patients who were entered onto the study and completed therapy had experienced treatment failure after prior radiotherapy or chemotherapy a mean of three times. The results showed an ORR of 34.6% (complete re...
Purpose: We tested the combination of a tumor lysate vaccine with a panel of costimulatory molecu... more Purpose: We tested the combination of a tumor lysate vaccine with a panel of costimulatory molecules to identify an immunotherapeutic approach capable of curing established murine gliomas. Experimental Design: Glioma-bearing mice were primed with a tumor lysate vaccine, followed by systemic administration of the following costimulatory ligands: OX40L, CD80, 4-1BBL, and GITRL, which were fused to the Fc portion of human immunoglobulin. Lymphocytes and mRNA were purified from the brain tumor site for immune monitoring studies. Numerous variations of the vaccine and Fc-OX40L regimen were tested alone or in combination with temozolomide. Results: Lysate vaccinations combined with Fc-OX40L led to the best overall survival, yielding cure rates of 50% to 100% depending on the timing, regimen, and combination with temozolomide. Cured mice that were rechallenged with glioma cells rejected the challenge, showing immunologic memory. Lymphocytes isolated from the draining lymph nodes of vaccine...
Purpose: Previously, we have shown that the attachment of interleukin 2 (IL-2) to a tumor-targeti... more Purpose: Previously, we have shown that the attachment of interleukin 2 (IL-2) to a tumor-targeting antibody can produce a 4-fold enhancement in the uptake of antibodies and drugs in tumors. More recently, we discovered that a 37-amino-acid linear sequence of IL-2 designated vasopermeability-enhancing peptide (PEP), contained the vasopermeability activity of IL-2, and could be used after linkage to tumor-targeting antibodies to produce the same enhancement of drugs and antibodies in tumors. We now describe the generation of a fully human antibody fusion protein, designated NHS76/PEP2, which can be used in patients to enhance the therapeutic potential of chemotherapy. Methods: NHS76/PEP2 was expressed in NS0 cells using the glutamine synthetase gene amplification system. To show its clinical potential as a pretreatment to chemotherapy, NHS76/PEP2 was given i.v. 2 hours before the injection of suboptimal doses of etoposide, doxorubicin, Taxol, Taxotere, 5-fluorouracil, or vinblastine ...
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Papers by Peisheng Hu