Recombinant anti-human HER2/neu IgG3-(GM-CSF) fusion protein retains antigen specificity and cytokine function and demonstrates antitumor activity

Anti-HER2/neu therapy of human HER2/neu-expressing malignancies such as breast cancer has shown only partial success in clinical trials. To expand the clinical potential of this approach, we have genetically engineered an anti-HER2/neu IgG3 fusion protein containing GM-CSF. Anti-HER2/neu IgG3-(GM-CSF) expressed in myeloma cells was correctly assembled and secreted. It was able to target HER2/neu-expressing cells and to support growth of a GM-CSF-dependent murine myeloid cell line, FDC-P1. The Ab fusion protein activated J774.2 macrophage cells so that they exhibit an enhanced cytotoxic activity and was comparable to the parental Ab in its ability to effect Ab-dependent cellular cytotoxicity-mediated tumor cell lysis. Pharmacokinetic studies showed that anti-HER2/neu IgG3-(GM-CSF) is stable in the blood. Interestingly, the half-life of anti-HER2/neu IgG3-(GM-CSF) depended on the injected dose with longer in vivo persistence observed at higher doses. Biodistribution studies showed that anti-HER2/neu IgG3-(GM-CSF) is mainly localized in the spleen. In addition, anti-HER2/neu IgG3-(GM-CSF) was able to target the HER2/neu-expressing murine tumor CT26-HER2/neu and enhance the immune response against the targeted Ag HER2/neu. Anti-HER2/neu IgG3-(GM-CSF) is able to enhance both Th1- and Th2-mediated immune responses and treatment with this Ab fusion protein resulted in significant retardation in the growth of s.c. CT26-HER2/neu tumors. Our results suggest that anti-HER2/neu IgG3-(GM-CSF) fusion protein is useful in the treatment of HER2/neu-expressing tumors.     

Regulation of antibody-dependent cellular cytotoxicity by IgG intrinsic and apparent affinity for target antigen

Unconjugated mAbs have emerged as useful cancer therapeutics. Ab-dependent cellular cytotoxicity (ADCC) is believed to be a major antitumor mechanism of some anticancer Abs. However, the factors that regulate the magnitude of ADCC are incompletely understood. In this study, we described the relationship between Ab affinity and ADCC. A series of human IgG1 isotype Abs was created from the anti-HER2/neu (also named c-erbB2) C6.5 single-chain Fv (scFv) and its affinity mutants. The scFv affinities range from 10(-7) to 10(-11) M, and the IgG Abs retain the affinities of the scFv from which they were derived. The apparent affinity of the Abs ranged from nearly 10(-10) M (the lowest affinity variant) to almost 10(-11) M (the other variants). The IgG molecules were tested for their ability to elicit ADCC in vitro against three tumor cell lines with differing levels of HER2/neu expression using unactivated human PBMC from healthy donors as the effector cells. The results demonstrated that both the apparent affinity and intrinsic affinity of the Abs studied regulate ADCC. High-affinity tumor Ag binding by the IgGs led to the most efficient and powerful ADCC. Tumor cells expressing high levels of HER2/neu are more susceptible to the ADCC triggered by Abs than the cells expressing lower amounts of HER2/neu. These findings justify the examination of high affinity Abs for ADCC promotion. Because high affinity may impair in vivo tumor targeting, a careful examination of Ab structure to function relationships is required to develop optimized therapeutic unconjugated Abs.     

Secreted antibody/granzyme B fusion protein stimulates selective killing of HER2-overexpressing tumor cells

Targeted cell killing is required for effective treatment of cancers. We previously described the generation of a chimeric immunocasp-3 protein and its potent selective antitumor activity (Jia, L. T., Zhang, L. H., Yu, C. J., Zhao, J., Xu, Y. M., Gui, J. H., Jin, M., Ji, Z. L., Wen, W. H., Wang, C. J., Chen, S. Y., and Yang, A. G. (2003) Cancer Res. 63, 3257-3262). Here we extend the repertoire of another chimeric pro-apoptotic protein immunoGrB, which comprises an anti-HER2 single-chain antibody, a Pseudomonas exotoxin A translocation domain and active granzyme B. Human lymphoma Jurkat cells transfected with the immunoGrB gene expression vector were able to produce and secrete the chimeric protein. The immunoGrB molecule selectively recognized and destroyed HER2-overexpressing tumor cells both in vitro and in nude mouse after intramuscular injection of the immunoGrB expression plasmid. Further in vivo study showed that intravenous administration of immunoGrB gene-modified lymphocytes led to suppression of HER2-overexpressing tumor growth and prolonged animal survival because of continuous secretion of immunoGrB molecules into blood and lymph fluid. These results demonstrate that the chimeric immunoGrB molecule, which is capable of antibody-directed targeting and granzyme B-mediated killing, has therapeutic potential against HER2 tumors, especially in cases in which caspase-dependent apoptosis is inhibited.     

Synergistic antitumor effect of interferon and anti-idiotype monoclonal antibody in murine lymphoma

Both IFN-alpha and anti-idiotype monoclonal antibody therapy have significant antitumor activity in vivo in a murine B cell lymphoma model. Combination therapy with syngeneic anti-idiotype antibody of the IgG2a or IgG2b isotype (a single i.p. injection of 100 micrograms) and recombinant human hybrid interferon-alpha A/D (10(4) to 10(6) U three times weekly for 3 wk) synergistically increased median survival time in mice challenged with a lethal dose of tumor cells compared with the sum of the median survival times of the two individual treatments. IFN-alpha has direct antiproliferative activity against 38C13 in vitro and enhances in vitro macrophage anti-idiotype antibody-specific cytolysis for IgG2a, IgG2b, and IgG1 isotypes.     

Idiotype vaccination against murine B cell lymphoma. Humoral and cellular requirements for the full expression of antitumor immunity

The roles of humoral and cellular antitumor immune responses induced by immunization with tumor-derived idiotypic IgM were studied in a syngeneic, transplantable B cell lymphoma (38C13) of C3H mice. Id vaccination with keyhole limpet hemocyanin-conjugated Id induced protection against a subsequent lethal tumor challenge. Such immunizations elicited anti-idiotypic antibodies that were cytotoxic in in vitro antibody-dependent cellular cytotoxicity assays as well as in vivo passive transfer experiments. L3T4+ T cells, which proliferated in vitro in response to the specific Id protein, were also induced. However, cells mediating direct cytotoxicity, either in vitro or in vivo, were not observed in the lymph nodes, spleens, or peritoneal cavity of immune mice or at the site of tumor regression as demonstrated by using a tumor sponge implantation model. In addition, in vitro sensitization of immune lymphocytes against 38C13 tumor cells failed to induce cytotoxicity. Immunization with lipid conjugated Id also elicited a T cell proliferative response but failed to induce anti-idiotypic antibodies and did not confer resistance to tumor growth. These results suggest that anti-idiotypic antibodies play the major role in the destruction of 38C13 tumor cells. However, in vivo depletion of L3T4+ or Lyt-2+ cells from 38C-Id-keyhole limpet hemocyanin-immunized mice resulted in diminished protection against a tumor challenge. Thus, although humoral responses appear to play the predominant part in tumor destruction, cellular responses are also required for the full expression of antitumor immunity in this system.     

Enhanced circulating half-life and antitumor activity of a site-specific pegylated interferon-alpha protein therapeutic

Recombinant interferon alpha-2 (IFN-alpha2) has proven useful for treating a variety of human cancers and viral diseases. IFN-alpha2 has a short circulating half-life in vivo, which necessitates daily or thrice weekly administration to patients. It is possible to extend the circulating half-life of IFN-alpha2 by random modification of lysine residues in the protein with polyethylene glycol (PEG); however, such preparations have heterogeneous structures and low specific activities, and may not provide optimal therapeutic benefits to patients. A long-acting, site-specific, monoPEGylated IFN-alpha2 protein has now been created by targeted attachment of a 20 kDa or a 40 kDa maleimide-PEG to a cysteine analogue of IFN-alpha2, M111C. In vitro bioactivities of the purified 20 kDa and 40 kDa PEG-M111C proteins were within 2- to 3-fold of those of wild type IFN-alpha2 and 7- to 10-fold better than that of a 40 kDa PEG IFN-alpha2 protein created using nontargeted, amine-PEGylation methodology. The 20 kDa and 40 kDa PEG-M111C proteins demonstrated 26- to 38-fold longer half-lives, respectively, than IFN-alpha2 following subcutaneous administration to rats. The 20 kDa PEG M111C protein inhibited growth of human NIH:OVCAR-3 cells transplanted into nude mice by >90%, as measured by tumor size, tumor weight, and number of animals with detectable tumors at necropsy, and was significantly more effective than a comparable dose of IFN-alpha2. These data extend our previous findings that bioactivity of IFN-alpha2 can be largely preserved by targeted attachment of PEG moieties to nonessential sites in the protein and provide evidence that site-specific PEGylated IFN-alpha2 proteins possess enhanced tumoricidal properties in vivo.     

A murine B cell lymphoma expressing human HER2/neu undergoes spontaneous tumor regression and elicits antitumor immunity

 In the present study we describe a novel murine tumor model in which the highly malignant murine B cell lymphoma 38C13 has been transduced with the cDNA encoding human tumor-associated antigen HER2/neu. This new cell line (38C13-HER2/neu) showed stable surface expression but not secretion of human HER2/neu. It also maintained expression of the idiotype (Id) of the surface immunoglobulin of 38C13, which serves as another tumor-associated antigen. Surprisingly, spontaneous tumor regression was observed following s.c. but not i.v. injection of 38C13-HER2/neu cells in immunocompetent syngeneic mice. Regression was more frequently observed with larger tumor cell challenges and was mediated through immunological mechanisms because it was not observed in syngeneic immunodeficient mice. Mice that showed complete tumor regression were immune to challenge with the parental cell line 38C13 and V1, a variant of 38C13 that does not express the Id. Immunity could be transferred with sera, suggesting that an antibody response mediated rejection and immunity. Continuously growing s.c. tumors as well as metastatic tumors obtained after the i.v. injection of 38C13-HER2/neu maintained expression of human HER2/neu, which can serve as a target for active immunotherapy. As spontaneous tumor regression has not been observed in other human murine models expressing human HER2/neu, our results illustrate the enormous differences that can exist among different murine tumors expressing the same antigen. The present model provides a useful tool for the study of the mechanisms of protective immunity to B cell lymphoma and for the evaluation of different therapeutic approaches based on the stimulation or suppression of the immune response. Received: 2 August 2000 / Accepted: 20 September 2000     

In vivo properties of three human HER2/neu-expressing murine cell lines in immunocompetent mice.

BACKGROUND AND PURPOSE: Expression of the HER2/neu proto-oncogene, a receptor-like transmembrane protein expressed at low levels on some normal cells, is markedly increased in a subset of human breast, colon, lung, and ovarian cancers. A humanized HER2/neu antibody has been tested as a therapeutic agent in several clinical trials, with promising results. We have developed a family of anti-HER2/neu fusion proteins. To evaluate the immunologic efficacy of these proteins, it is critical that tumors expressing the target antigen can grow in immunologically intact mice. METHOD: To produce murine tumors expressing human HER2/neu on the surface, CT26, MC38, and EL4 murine cell lines were transduced by use of a retroviral construct containing the cDNA encoding the human HER2/neu gene. RESULTS: Histologic features and kinetics of tumor growth in subcutaneous space of the human HER2/neu-expressing cells were similar to those of the respective parental cell lines. Intravenous inoculation with these cells induced disseminated malignant disease. Flow cytometric and immmunohistochemical analyses of freshly isolated tumors revealed in vivo expression of human HER2/neu. Secretion of antigen was not detected by use of an ELISA. CONCLUSION: Although an antibody response against the human HER2/neu antigen was observed, this response does not affect the growth rate of the HER2/neu-expressing cells. These murine models may be useful tools for evaluation of anti-cancer therapeutic approaches that target human HER2/neu.     

In vivo targeting of HER2-positive tumor using 2-helix affibody molecules

Molecular imaging of human epidermal growth factor receptor type 2 (HER2) expression has drawn significant attention because of the unique role of the HER2 gene in diagnosis, therapy and prognosis of human breast cancer. In our previous research, a novel cyclic 2-helix small protein, MUT-DS, was discovered as an anti-HER2 Affibody analog with high affinity through rational protein design and engineering. MUT-DS was then evaluated for positron emission tomography (PET) of HER2-positive tumor by labeling with two radionuclides, 68Ga and 18F, with relatively short half-life (t1/2<2 h). In order to fully study the in vivo behavior of 2-helix small protein and demonstrate that it could be a robust platform for labeling with a variety of radionuclides for different applications, in this study, MUT-DS was further radiolabeled with 64Cu or 111In and evaluated for in vivo targeting of HER2-positive tumor in mice. Design 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated MUT-DS (DOTA-MUT-DS) was chemically synthesized using solid phase peptide synthesizer and I2 oxidation. DOTA-MUT-DS was then radiolabeled with 64Cu or 111In to prepare the HER2 imaging probe (64Cu/111In-DOTA-MUT-DS). Both biodistribution and microPET imaging of the probe were evaluated in nude mice bearing subcutaneous HER2-positive SKOV3 tumors. DOTA-MUT-DS could be successfully synthesized and radiolabeled with 64Cu or 111In. Biodistribution study showed that tumor uptake value of 64Cu or 111In-labeled DOTA-MUT-DS was 4.66±0.38 or 2.17±0.15%ID/g, respectively, in nude mice bearing SKOV3 xenografts (n=3) at 1 h post-injection (p.i.). Tumor-to-blood and tumor-to-muscle ratios for 64Cu-DOTA-MUT-DS were attained to be 3.05 and 3.48 at 1 h p.i., respectively, while for 111In-DOTA-MUT-DS, they were 2.04 and 3.19, respectively. Co-injection of the cold Affibody molecule ZHER2:342 with 64Cu-DOTA-MUT-DS specifically reduced the SKOV3 tumor uptake of the probe by 48%. 111In-DOTA-MUT-DS displayed lower liver uptake at all the time points investigated and higher tumor to blood ratios at 4 and 20 h p.i., when compared with 64Cu-DOTA-MUT-DS. This study demonstrates that the 2-helix protein based probes, 64Cu/111In DOTA-MUT-DS, are promising molecular probes for imaging HER2-positive tumor. Two-helix small protein scaffold holds great promise as a novel and robust platform for imaging and therapy applications.     

A new vector for controllable expression of an anti-HER2/neu mini-antibody-barnase fusion protein in HEK 293T cells

Tumor-targeted vectors with controllable expression of therapeutic genes and specific antitumor antibodies are promising tools for the reduction of malignant tumors. Here we describe a new plasmid for the eukaryotic expression of an anti-HER2/neu mini-antibody-barnase fusion protein (4D5 scFv-barnase-His(5)) with an NH(2)-terminal leader peptide. The 4D5 scFv-barnase-His(5) gene was placed downstream of the tetracycline responsive-element minimal promoter in the vector using the Tet-Off gene-expression system. The Bacillus amyloliquefaciens ribonuclease barnase is toxic for the host cells. To overcome this problem, barstar gene under its own minimal cytomegalovirus promoter was used in designed vector. Barstar inhibits the background level of barnase in the cells in the presence of tetracycline in culture medium. The HEK 293T cells were transfected with the designed vector, and the 4D5 scFv-barnase-His(5) fusion protein was identified by anti-barnase antibodies in cell culture medium and after purification from cell lysates using metal-affinity chromatography. The overexpression of the anti-HER2/neu mini-antibody-barnase fusion protein decreased the intensity of fluorescence of HEK 293T cells co-transfected with the generated plasmid and a plasmid containing the gene of enhanced green fluorescent protein (pEGFP-N1), in comparison with the intensity of fluorescence of HEK 293T cells transfected with pEGFP-N1, in the absence of tetracycline in the medium. The effect of the 4D5 scFv-barnase-His(5) on EGFP fluorescence indicates that the introduced barnase functions as a ribonuclease inside the cells. The anti-HER2/neu mini-antibody could be used to deliver barnase to HER2/neu-positive cells and provide its penetration into the target cells, as HER2/neu is a ligand-internalizing receptor. This expression vector has potential applications to both gene and antibody therapies of cancer.     

Resistance of lymphocytic choriomeningitis virus to alpha/beta interferon and to gamma interferon.

The susceptibility to alpha/beta interferon (IFN-alpha/beta) or to gamma interferon (IFN-gamma) of various lymphocytic choriomeningitis virus (LCMV) strains was evaluated in C57BL/6 mice and in various cell lines. Anti-IFN-gamma treatment in vivo revealed that the LCMV strains Armstrong, Aggressive, and WE were most susceptible to IFN-gamma whereas Traub, Cl 13-Armstrong, and Docile were resistant. The same pattern of susceptibility to recombinant IFN-gamma was observed in vitro. In vivo treatment with anti-IFN-alpha/beta showed a sizeable increase in replication of Aggressive, Armstrong, and WE; effects were less pronounced for Docile, Cl 13-Armstrong, or Traub. Correspondingly, WE, Armstrong, and Aggressive were all relatively sensitive to purified IFN-alpha/beta in vitro, and Cl 13-Armstrong, Docile, and Traub were more resistant. Overall, there was a good correlation between the capacity of LCMV strains to establish a persistent infection in adult immunocompetent mice and their relative resistance to IFN-gamma and IFN-alpha/beta.     

Increasing the affinity for tumor antigen enhances bispecific antibody cytotoxicity

We tested the hypothesis that bispecific Abs (Bsab) with increased binding affinity for tumor Ags augment retargeted antitumor cytotoxicity. We report that an increase in the affinity of Bsab for the HER2/neu Ag correlates with an increase in the ability of the Bsab to promote retargeted cytotoxicity against HER2/neu-positive cell lines. A series of anti-HER2/neu extracellular domain-directed single-chain Fv fragments (scFv), ranging in affinity for HER2/neu from 10(-7) to 10(-11) M, were fused to the phage display-derived NM3E2 human scFV: NM3E2 associates with the extracellular domain of human FcgammaRIII (CD16). The resulting series of Bsab promoted cytotoxicity of SKOV3 human ovarian carcinoma cells overexpressing HER2/neu by human PBMC preparations containing CD16-positive NK cells. The affinity for HER2/neu clearly influenced the ability of the Bsab to promote cytotoxicity of (51)Cr-labeled SKOV3 cells. Lysis was 6.5% with an anti-HER2/neu K(D) = 1.7 x 10(-7) M, 14.5% with K(D) = 5.7 x 10(-9) M, and 21.3% with K(D) = 1.7 x 10(-10) M at 50:1 E:T ratios. These scFv-based Bsab did not cross-link receptors and induce leukocyte calcium mobilization in the absence of tumor cell engagement. Thus, these novel Bsab structures should not induce the dose-limiting cytokine release syndromes that have been observed in clinical trials with intact IgG BSAB: Additional manipulations in Bsab structure that improve selective tumor retention or facilitate the ability of Bsab to selectively cross-link tumor and effector cells at tumor sites should further improve the utility of this therapeutic strategy.     

Listeriolysin O is an improved protein carrier for lymphoma immunoglobulin idiotype and provides systemic protection against 38C13 lymphoma

Follicular lymphoma (FL) is a disease that responds to current treatment regimens; however, patients in general relapse with increasingly refractory disease. Idiotype-based vaccines are currently under trial for the treatment of FL. These vaccines comprise the patient’s BCR idiotype (Id) as the tumor antigen conjugated to the protein carrier Keyhole Limpet Hemocyanin (KLH); however, other protein carriers may enhance the immune response to the lymphoma Id. In this study we investigated whether an alternate carrier, Listeriolysin O (LLO), would amplify the immune response to Id protein and provide better protection against challenge by 38C13 murine lymphoma. The Id-LLO vaccine compared favorably against Id-KLH in tumor-protection studies and both vaccines provided systemic immunity against 38C13 lymphoma. However, the immune response to the two conjugates was different in that Id-LLO induced a more powerful Th1 response characterized by high titer IgG2a anti-Id antibodies after one immunization and the presence of CD4 cells secreting IFN-γ. In vivo studies demonstrated that immune serum contributed to the anti-lymphoma efficacy seen following Id-LLO immunization. Interestingly, Id-LLO immunized mice, when challenged twice with 38C13 lymphoma provided better protection against challenge by the BCR loss variant 38C13-V2, suggesting that Id-LLO immunized mice have more potential to develop epitope spreading than Id-KLH. In conclusion, Id-LLO compared favorably against Id-KLH in its anti-lymphoma efficacy. Furthermore, Id-LLO induced a more potent humoral and cell-mediated immune response and promoted epitope spreading after lymphoma challenge. Thus, anti-Id vaccines incorporating LLO may be a better therapeutic option for treatment of B-cell lymphoma. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Targeting of Drugs to Solid Tumors Using Anti-Her2 Immunoliposomes

Abstract

Cancer therapy would clearly benefit from a carrier system capable of intracellular delivery of systemically administered drugs to cancer cells in solid tumors. Sterically stabilized immunoliposomes specific to the cells expressing HER2 protooncogene (anti-HER2 SIL), were designed by conjugating Fab’ fragments of a recombinant humanized anti-HER2 MAb to the distal termini of poly(ethylene glycol) chains on the surface of unilamellar liposomes (size 90–100 nm) of phosphatidylcholine, cholesterol, and poly (ethylene glycol)—derivatized phosphatidylethanolamine. Anti-HER2 SIL avidly and specifically bound to cultured HER2-overexpressing cancer cells (8,000–23,000 vesicles per cell) and became endocytosed (k_e = 0.022–0.033 min.^?1) via the coated pit pathway. Anti-HER2 SIL showed prolonged circulation lifetime in rats (blood MRT approx. 24 hours) and significantly increased antitumor activity of encapsulated doxorubicin against HER2-overexpressing human breast cancer xenografts in nude mice. Although the accumulation of anti-HER2 SIL in HER2-overexpressing tumor xenografts was not increased over that of non-targeted sterically stabilized liposomes (SL), microscopic examination revealed abundance of anti-HER2 SIL in the interstitial spaces, as well as within the cytoplasm of cancer cells, while identical liposomes lacking anti-HER2 Fab’ were located predominantly within tumor-resident macrophages. Anti-HER2 SIL, a targeted vehicle capable of in vivo intracellular delivery of substances to HER2-overexpressing solid cancers, enhances the potential for tumor targeting and opens new avenues for better treatment of cancer.     

89Zr-mAb3481 PET for HER3 tumor status assessment during lapatinib treatment

Treatment of human epidermal growth factor receptor 2 (HER2)-driven breast cancer with tyrosine kinase inhibitor lapatinib can induce a compensatory HER3 increase, which may attenuate antitumor efficacy. Therefore, we explored in vivo HER3 tumor status assessment after lapatinib treatment with zirconium-89 (⁸⁹Zr)-labeled anti-HER3 antibody mAb3481 positron emission tomography (PET). Lapatinib effects on HER3 cell surface expression and mAb3481 internalization were evaluated in human breast (BT474, SKBR3) and gastric (N87) cancer cell lines using flow cytometry. Next, in vivo effects of daily lapatinib treatment on⁸⁹Zr-mAb3481 BT474 and N87 xenograft tumor uptake were studied. PET-scans (BT474 only) were made after daily lapatinib treatment for 9 days, starting 3 days prior to ⁸⁹Zr-mAb3481 administration. Subsequently, ex vivo ⁸⁹Zr-mAb3481 organ distribution analysis was performed and HER3 tumor levels were measured with Western blot and immunohistochemistry. In vitro, lapatinib increased membranous HER3 in BT474, SKBR3 and N87 cells, and consequently mAb3481 internalization 1.7-fold (BT474), 1.4-fold (SKBR3) and 1.4-fold (N87). ⁸⁹Zr-mAb3481 BT474 tumor uptake was remarkably high at SUV_mean 5.6±0.6 (51.8±7.7%ID/g) using a 10 μg ⁸⁹Zr-mAb3481 protein dose in vehicle-treated mice. However, compared to vehicle, lapatinib did not affect ⁸⁹Zr-mAb3481 ex vivo uptake in BT474 and N87 tumors, while HER3 tumor expression remained unchanged. In conclusion, lapatinib increased in vitro HER3 tumor cell expression, but not when these cells were xenografted. ⁸⁹Zr-mAb3481 PET accurately reflected HER3 tumor status. ⁸⁹Zr-mAb3481 PET showed high, HER3-specific tumor uptake, and such an approach might sensitively assess HER3 tumor heterogeneity and treatment response in patients.     

Insights into HER2 signaling from step-by-step optimization of anti-HER2 antibodies

HER2, a ligand-free tyrosine kinase receptor of the HER family, is frequently overexpressed in breast cancer. The anti-HER2 antibody trastuzumab has shown significant clinical benefits in metastatic breast cancer; however, resistance to trastuzumab is common. The development of monoclonal antibodies that have complementary mechanisms of action results in a more comprehensive blockade of ErbB2 signaling, especially HER2/HER3 signaling. Use of such antibodies may have clinical benefits if these antibodies can become widely accepted. Here, we describe a novel anti-HER2 antibody, hHERmAb-F0178C1, which was isolated from a screen of a phage display library. A step-by-step optimization method was employed to maximize the inhibitory effect of this anti-HER2 antibody. Crystallographic analysis was used to determine the three-dimensional structure to 3.5 Å resolution, confirming that the epitope of this antibody is in domain III of HER2. Moreover, this novel anti-HER2 antibody exhibits superior efficacy in blocking HER2/HER3 heterodimerization and signaling, and its use in combination with pertuzumab has a synergistic effect. Characterization of this antibody revealed the important role of a ligand binding site within domain III of HER2. The results of this study clearly indicate the unique potential of hHERmAb-F0178C1, and its complementary inhibition effect on HER2/HER3 signaling warrants its consideration as a promising clinical treatment.     

Insights into HER2 signaling from step-by-step optimization of anti-HER2 antibodies

HER2, a ligand-free tyrosine kinase receptor of the HER family, is frequently overexpressed in breast cancer. The anti-HER2 antibody trastuzumab has shown significant clinical benefits in metastatic breast cancer; however, resistance to trastuzumab is common. The development of monoclonal antibodies that have complementary mechanisms of action results in a more comprehensive blockade of ErbB2 signaling, especially HER2/HER3 signaling. Use of such antibodies may have clinical benefits if these antibodies can become widely accepted. Here, we describe a novel anti-HER2 antibody, hHERmAb-F0178C1, which was isolated from a screen of a phage display library. A step-by-step optimization method was employed to maximize the inhibitory effect of this anti-HER2 antibody. Crystallographic analysis was used to determine the three-dimensional structure to 3.5 Å resolution, confirming that the epitope of this antibody is in domain III of HER2. Moreover, this novel anti-HER2 antibody exhibits superior efficacy in blocking HER2/HER3 heterodimerization and signaling, and its use in combination with pertuzumab has a synergistic effect. Characterization of this antibody revealed the important role of a ligand binding site within domain III of HER2. The results of this study clearly indicate the unique potential of hHERmAb-F0178C1, and its complementary inhibition effect on HER2/HER3 signaling warrants its consideration as a promising clinical treatment.     

Targeting activity of a TCR/IL-2 fusion protein against established tumors

We have previously reported that a single-chain T cell receptor/IL-2 fusion protein (scTCR-IL2) exhibits potent targeted antitumor activity in nude mice bearing human tumor xenografts that display cognate peptide/HLA complexes. In this study, we further explore the mechanism of action of this molecule. We compared the biological activities of c264scTCR-IL2, a scTCR-IL2 protein recognizing the aa264–272 peptide of human p53, with that of MART-1scTCR-IL2, which recognizes the MART-1 melanoma antigen (aa27–35). In vitro studies showed that c264scTCR-IL2 and MART-1scTCR-IL2 were equivalent in their ability to bind cell-surface IL-2 receptors and stimulate NK cell responses. In mice, MART-1scTCR-IL2 was found to have a twofold longer serum half-life than c264scTCR-IL2. However, despite its shorter serum half-life, c264scTCR-IL2 showed significantly better antitumor activity than MART-1scTCR-IL2 against p53⁺/HLA-A2⁺ tumor xenografts. The more potent antitumor activity of c264scTCR-IL2 correlated with an enhanced capacity to promote NK cell infiltration into tumors. Similar differences in antigen-dependent tumor infiltration were observed with activated splenocytes pre-treated in vitro with c264scTCR-IL2 or MART-1scTCR-IL2 and then transferred into p53⁺/HLA-A2⁺ tumor bearing recipients. The data support a model where c264scTCR-IL2 activates immune cells to express IL-2 receptors. Following stable interactions with cell-surface IL-2 receptors, c264scTCR-IL2 fusion molecule enhances the trafficking of immune cells to tumors displaying target peptide/HLA complexes where the immune cells mediate antitumor effects. Thus, this type of fusion molecule could be used directly as a targeted immunotherapeutic or in adoptive cell transfer approaches to activate and improve the anti-cancer activities of immune cells by providing them with pre-selected antigen recognition capability.     

Tumor cell lysate-pulsed dendritic cells are more effective than TCR Id protein vaccines for active immunotherapy of T cell lymphoma

TCR Id protein conjugated to keyhole limpet hemocyanin (KLH) (TCR Id:KLH) and injected with a chemical adjuvant (QS-21) induces a protective, Id-specific immune response against the murine T cell lymphoma, C6VL. However, Id-based immunotherapy of C6VL has not demonstrated therapeutic efficacy in tumor-bearing mice. We report here that C6VL lysate-pulsed dendritic cells (C6VL-DC) vaccines display enhanced efficacy in both the prevention and the therapy of T cell lymphoma compared with TCR Id:KLH with QS-21 vaccines. C6VL-DC vaccines stimulated potent tumor-specific immunity that protected mice against lethal challenge with C6VL and significantly enhanced the survival of tumor-bearing mice. Tumor-specific proliferation and secretion of IFN-gamma indicative of a Th1-type immune response were observed upon ex vivo stimulation of vaccine-primed lymph node cells. Adoptive transfer of immune T cell-enriched lymphocytes was sufficient to protect naive recipients from lethal tumor challenge. Furthermore, CD8(+) T cells were absolutely required for tumor protection. Although C6VL-DC and control vaccines stimulated low levels of tumor-specific Ab production in mice, Ab levels did not correlate with the protective ability of the vaccine. Thus, tumor cell lysate-pulsed DC vaccines appear to be an effective approach to generate potent T cell-mediated immune responses against T cell malignancies without requiring identification of tumor-specific Ags or patient-specific Id protein expression.     

A caspase-6 and anti-human epidermal growth factor receptor-2 (HER2) antibody chimeric molecule suppresses the growth of HER2-overexpressing tumors

Clinical studies have suggested that human epidermal growth factor receptor-2 (HER2) provide a useful target for antitumor therapy. We previously described the generation of a chimeric HER2-targeted immunocasp-3 protein. In this study, we extend the repertoire of chimeric proapoptotic proteins with immunocasp-6, a construct that comprises a HER2-specific single-chain Ab, a single-chain Pseudomonas exotoxin A, and an active caspase-6, which can directly cleave lamin A leading to nucleus damage and inducing programmed cell death. We demonstrate that the secreted immunocasp-6 molecule selectively recognizes and induces apoptosis in HER2-overexpressing tumor cells in vitro, but not in cells with undetectable HER2. The immunocasp-6 gene was next transferred into BALB/c athymic mice bearing human breast SK-BR-3 tumors by i.m. injection of liposome-encapsulated vectors, by intratumor injection of adenoviral vectors, or by i.v. injection of PBMC modified by retroviral infection. Regardless of the method used, expression of immunocasp-6 suppressed tumor growth and prolonged animal survival significantly. Our data show that the chimeric immunocasp-6 molecule can recognize HER2-positive tumor cells, promptly attack their nucleus, and induce their apoptotic death, suggesting the potential of this strategy for the treatment of human cancers that overexpress HER2.