Disabling erbB receptors with rationally designed exocyclic mimetics of antibodies: structure-function analysis

Overexpression of the HER2 receptor is observed in about 30% of breast and ovarian cancers and is often associated with an unfavorable prognosis. We have recently designed an anti-HER2 peptide (AHNP) based on the structure of the CDR-H3 loop of the anti-HER2 rhumAb 4D5 and showed that this peptide can mimic some functions of rhumAb 4D5. The peptide disabled HER2 tyrosine kinases in vitro and in vivo similar to the monoclonal antibody (Park, B.-W. et al. Nat. Biotechnol. 2000, 18, 194--198). AHNP has been shown to selectively bind to the extracellular domain of the HER2 receptor with a submicromolar affinity in Biacore assays. In the present paper, we demonstrate that in addition to being a structural and functional mimic of rhumAb 4D5, AHNP can also effectively compete with the antibody for binding to the HER2 receptor indicating a similar binding site for the peptide and the parental antibody. To further develop AHNP as an antitumor agent useful for preclinical trials and as a radiopharmaceutical to be used for tumor imaging, a number of derivatives of AHNP have been designed. Structure--function relationships have been studied using surface plasmon resonance technology. Some of the AHNP analogues have improved binding properties, solubility, and cytotoxic activity relative to AHNP. Residues in the exocyclic region of AHNP appear to be essential for high-affinity binding. Kinetic and equilibrium analysis of peptide-receptor binding for various AHNP analogues revealed a strong correlation between peptide binding characteristics and their biological activity. For AHNP analogues, dissociation rate constants have been shown to be better indicators of peptide biological activity than receptor-binding affinities. This study demonstrates a possibility of mimicking the well-documented antibody effects and its applications in tumor therapy by much smaller antibody-based cyclic peptides with potentially significant therapeutic advantages. Strategies used to improve binding properties of rationally designed AHNP analogues are discussed.     

Application and potential limitations of animal models utilized in the development of trastuzumab (Herceptin): a case study

The preclinical and clinical development of trastuzumab, a humanized monoclonal antibody directed against a juxtamembrane epitope in the HER2 receptor ectodomain, relied heavily on the use of animal models to validate HER2 as a potential MAb target. The identification of HER2 (neu) as a proto-oncogene was first established in a carcinogen-induced brain tumor in the rat. Transgenic mouse technology led to an understanding of the role of HER2 in pathogenesis of breast cancer. Transfection studies of human HER2 cDNA into murine xenograft models further explored the role HER2 plays in tumor progression and metastasis. A murine subrenal capsule fresh human tumor explant assay was utilized to test efficacy of various murine monoclonal anti-HER2 antibodies, and the data were helpful in choosing the most efficacious for subsequent human engineering for clinical use. HER2-overexpressing xenograft models in athymic mice were used to test the efficacy of anti-HER2 antibodies, develop dose-response relationships, measure drug interactions between trastuzumab and chemotherapy, and optimize dosing schedules of chemotherapeutics combined with trastuzumab. In this work, we will highlight the utility of animal models exploited in the development of trastuzumab - noting not only their contribution to drug development but also their limitations in translation of preclinical data into the clinic. It is likely that the experience we gained in the case of preclinical animal models to study in vivo effects of trastuzumab have parallels in the development of other monoclonal antibodies since overcoming the species boundaries (i.e. cross-reactivity with antigenic determinant, development of cross-species neutralizing antibodies, and cross-species interaction with activating Fc receptors on immune effector cells) are major limitations in the design and interpretation of preclinical/translational experiments designed to fulfill various regulatory requirements prior to initiation of phase I human clinical trials.     

Trastuzumab,a humanized anti-HER2 monoclonal antibody,for the treatment of breast cancer

The HER2/neu gene encodes a 185 kDa transmembrane receptor (HER2) that belongs to the epidermal growth factor receptor family and has intrinsic tyrosine kinase activity. HER2 is overexpressed in 25-30% of breast cancers and is suggested to have a direct role in the pathogenesis and clinical aggressiveness of HER2 overexpressing tumors. A murine monoclonal antibody, 4D5, directed against the extracellular domain of HER2, is a potent inhibitor of growth of human breast cancer cells overexpressing HER2 in vitro and in xenograft models. To facilitate clinical investigation, 4D5 was humanized by inserting the complementary determining regions of 4D5 into the framework of a consensus human IgG1. The resulting recombinant humanized anti-HER2 MAb, trastuzumab, was found to inhibit the growth of human cancer cells and tumor xenografts overexpressing HER2. Data from phase II trials in women with breast cancer whose tumors overexpress HER2 have shown that trastuzumab has a favorable toxicity profile, is active as a single agent and induces long-lasting objective tumor responses. In combination studies, there was no evidence that trastuzumab enhanced the toxicity of cisplatin and the pharmacokinetic parameters of trastuzumab were unaltered by coadministration of cisplatin. Furthermore, clinical response rates were higher than those reported with either agent alone in a similar patient population. Results of a multicenter, phase III clinical trial of chemotherapy (doxorubicin- or paclitaxel-based) plus trastuzumab as compared to chemotherapy alone in patients with advanced breast cancers overexpressing HER2 showed a significant enhancement in the effects of chemotherapy on time to disease progression, response rates and survival with coadministration of trastuzumab, without increases in overall severe adverse events. Myocardial dysfunction syndrome, similar to that observed with anthracyclines, was reported more commonly with chemotherapy plus trastuzumab. Positive results from clinical studies led to the approval of trastuzumab in the U.S in October 1998 for the treatment of metastatic breast cancer in patients with tumors overexpressing HER2. Since then, the MAb has also been marketed in Switzerland and Canada.     

New target antigens for cancer immunoprevention

Prevention of cancer through the activation of the immune system has been explored in recent years in preclinical systems thanks to the availability of several new transgenic mouse models that closely mimic the natural history of human tumors. The most thoroughly investigated model of cancer immunoprevention is the mammary carcinoma of HER-2/neu transgenic mouse. In this system it has clearly been shown that the activation of immune defences in healthy individuals can effectively prevent the subsequent onset of highly aggressive mammary carcinomas. A complete prevention was obtained using a combination of three signals (the so called "triplex" vaccine) that included the specific antigen (p185, the product of HER-2/neu) and nonspecific signals like allogeneic histocompatibility antigens and interleukin 12. The analysis of protective immune responses in models of cancer immunoprevention revealed some unexpected features, in particular the central role of antibodies in immunoprevention, at variance with conventional immuno-therapy which is firmly based on cytotoxic T cells. In the HER-2/neu system anti-p185 antibodies, in addition to immunological functions leading to tumor cell lysis, inhibit p185 dimerization and induce its internalization, resulting in the inhibition of mitogenic signaling. Most current tumor antigens appear to be unsuitable targets for cancer immunoprevention. An ideal antigen should have a crucial pathogenetic role in tumor growth to avoid the selection of antigen loss variants. Downregulation of major histocompatibility complex (MHC) expression during tumor progression frequently limits antigen recognition by MHC-restricted T cells. Thus an ideal antigen for cancer immunoprevention should be recognized both by T cells and by antibodies. Antibody binding to cell surface oncogenic determinants, in addition to complement- and cell-mediated tumor cell lysis, can block mitogenic signaling and induce internalization, resulting in tumor growth arrest. A search for new tumor antigens should be conducted among molecules that are directly involved in neoplastic transformation and are recognizable by the immune response also in MHC loss variants. Novel tumor antigens fulfilling both conditions will be crucial for the development of cancer immunoprevention and will provide new targets also for cancer immunotherapy.     

Target-independent Immune-cell Activation by Aggregates of T Cell-redirecting Bispecific Antibodies

T cell-redirecting bispecific antibodies (bsAbs) have been under development as a new class of biotherapeutics for cancer immunotherapy. T cell-redirecting bsAbs simultaneously bind tumor-associated antigens on tumor cells and CD3 on T cells, resulting in T cell-mediated cytotoxicity against tumor cells. In this study, we prepared a tandem scFv-typed bsAb targeting HER2 and CD3 (HER2-CD3), and evaluated the impact of aggregation of HER2-CD3 on the in vitro immunotoxicity. A cell-based assay using CD3-expressing reporter cells revealed that the aggregates of HER2-CD3 directly activated CD3-expressing immune cells in the absence of target antigen (HER2)-expressing cells. Comparison of the aggregates generated under various stress conditions indicated the possibility that insoluble protein particles, which were detected by qLD analysis and contained non-denatured functional domains, contributed to the activation of CD3-expressing immune cells. In addition, HER2-CD3 aggregates stimulated hPBMCs and strongly induced the secretion of inflammatory cytokines and chemokines. The cytokine/chemokine-release profiles suggested that the aggregates could induce inflammatory responses not only by CD3-mediated T cell activation but also by other immune cell activations. These results indicated the potential risk of aggregation of T cell-redirecting bsAbs, which could induce unwanted immune cell activation and inflammation and thereby immune-mediated adverse reactions.     

Differential tumor cell targeting of anti-HER2 (Herceptin) and anti-CD20 (Mabthera) coupled nanoparticles

Two types of antibody-labeled nanoparticles (mAb-NPs) were prepared with the aim to achieve specific tumor targeting. Anti-HER2 and anti-CD20 monoclonal antibodies (mAb) were used as model ligands. Small poly(dl-lactic acid) nanoparticles (PLA NPs) with a mean size of about 170 nm were prepared by the salting out method. Thereafter, the coating of PLA NPs with mAbs was performed in two steps. First, thiol groups (-SH) were introduced on the surface of PLA-NPs by a two-step carbodiimide reaction. The number of -SH groups on the surface of NPs increased from 150 to 400 mmol-SH/mol PLA when cystamine concentrations of 25-1518 mol cystamine/mol PLA were used during the thiolation reaction. In the second step, covalent coupling of antibodies to thiolated NPs (NPs-SH) was obtained via a bifunctional cross-linker, m-maleimidobenzoyl-N-hydroxy-sulfosuccinimide ester (sulfo-MBS). For both mAbs anti-HER2 and anti-CD20, respectively, the number of -SH functions on the NPs had no influence on the amount of mAb coupled to the NPs. Approximately, 295 anti-HER2 and 557 anti-CD20 molecules, respectively, were covalently coupled per nanoparticle. The NPs size after the coupling reactions was about 250 nm. The specific interaction between tumor cells and mAb-NPs was determined by confocal microscopy using two cell lines: SKOV-3 human ovarian cancer cells (overexpressing HER2) and Daudi lymphoma cells (overexpressing CD20). The results showed the selective targeting of mAb-NPs to tumor cells overexpressing the specific antigen. While anti-CD20 labeled NPs (anti-CD20 NPs) bound to and remained at the cellular surface, anti-HER2 labeled NPs (anti-HER2 NPs) were efficiently internalized. The mAb-NPs represent a promising approach to improve the efficacy of NPs in active targeting for cancer therapy while the choice of the antibody-target system defines the fate of the mAb-NPs after their binding to the cells.     

Disitamab vedotin: a novel antibody-drug conjugates for cancer therapy

Human epidermal growth factor receptor 2 (HER2) regulates cell mitosis, proliferation, and apoptosis. Trastuzumab is a HER2-targeted monoclonal antibody (mAB), which can prolong the overall survival rate of patients with HER2 overexpression in later periods of gastric cancer and breast cancer. Although anti-HER2 monoclonal antibody has a curative effect, adjuvant chemotherapy is still necessary to upgrade the curative effect maximumly. Antibody-drug conjugate (ADC) is a kind of therapeutic drug that contains antigen-specific antibody and cytotoxic payload, which can improve the survival time of tumor patients. To date, there are several HER2-ADC products on the market, for which two anti-HER2 ADC (trastuzumab emtansine and trastuzumab deruxtecan) have been authorized by the FDA for distinct types of HER2-positive carcinoma in the breast. Disitamab vedotin (RC48) is a newly developed ADC drug targeting HER2 that is comprised of hertuzumab coupling monomethyl auristatin E (MMAE) via a cleavable linker. This paper aims to offer a general insight and summary of the mechanism of action and the currently completed and ongoing clinical studies of RC-48 in HER-2 positive solid tumors.     

Solamargine enhances HER2 expression and increases the susceptibility of human lung cancer H661 and H69 cells to trastuzumab and epirubicin

We have previously demonstrated that solamargine (SM), the major steroidal glycoalkaloid extracted from Chinese herb Solanum plants, reveals down-regulation of HER2 and up-regulation of Fas and tumor necrosis factor receptor (TNFR) expressions, triggers the mitochondria-mediated cell apoptosis pathway, and sensitizes human nonsmall cell lung cancer (NSCLC) H441 and A549 adenocarcinoma cells to chemotherapy. The present study shows that SM enhances HER2 expression in NSCLC large cell carcinoma H661 and small cell lung cancer (SCLC) H69 cells and may increase the susceptibility of the cells to trastuzumab, the humanized anti-HER2 antibody. The combinational treatment of SM and trastuzumab synergistically augments and inhibits H661 and H69 cell proliferation. After treatment with SM, coexpression of HER2 and topoisomerase IIalpha (TOP2A) H661 and H69 cells is more sensitive to the TOP2 inhibitor, epirubicin. The combinatory use of low concentrations of SM with the low-toxic epirubicin accelerated greater apoptotic cell death than each drug did alone in H661 and H69 cells. Relevant studies have shown that HER2 overexpressing cancer cells are more resistant than HER2 low-expressing cells to the chemotherapeutic agent and tumor necrosis factor-induced apoptosis. These investigations have indicated that HER2 overexpression does not suffice to induce intrinsic and pleomorphic drug resistance. The data presented herein suggest that the expression of HER2 did not influence the SM-induced apoptosis of different types of lung cancer cells and that the SM up-regulation of HER2 and TOP2A expressions simultaneously augmented trastuzumab and epirubicin-induced deaths of lung cancer H661 and H69 cells.     

Growth differentiation factor 15 (GDF15)-mediated HER2 phosphorylation reduces trastuzumab sensitivity of HER2-overexpressing breast cancer cells

Resistance to the anti-HER2 monoclonal antibody trastuzumab is a major problem in the treatment of HER2-overexpressing metastatic breast cancer. Growth differentiation factor 15 (GDF15), which is structurally similar to TGF beta, has been reported to stimulate phosphorylation of HER2. We tested the hypothesis that GDF15-mediated phosphorylation of HER2 reduces the sensitivity of HER2-overexpressing breast cancer cell lines to trastuzumab. Gene microarray analysis, real-time PCR, and ELISA were used to assess GDF15 expression. Growth inhibition and proliferation assays in response to pharmacologic inhibitors of HER2, TGF beta receptor, or Src were performed on cells stimulated with recombinant human GDF15 or stable GDF15 transfectants. Western blotting was performed to determine effects of GDF15 on HER2 signaling. Cells were infected with lentiviral GDF15 shRNA plasmid to determine effects of GDF15 knockdown on cell survival in response to trastuzumab. Cells with acquired or primary trastuzumab resistance showed increased GDF15 expression. Exposure of trastuzumab-sensitive cells to recombinant human GDF15 or stable transfection of a GDF15 expression plasmid inhibited trastuzumab-mediated growth inhibition. HER2 tyrosine kinase inhibition abrogated GDF15-mediated Akt and Erk1/2 phosphorylation and blocked GDF15-mediated trastuzumab resistance. Pharmacologic inhibition of TGF beta receptor blocked GDF15-mediated phosphorylation of Src. Further, TGF beta receptor inhibition or Src inhibition blocked GDF15-mediated trastuzumab resistance. Finally, lentiviral GDF15 shRNA increased trastuzumab sensitivity in cells with acquired or primary trastuzumab resistance. These results support GDF15-mediated activation of TGF beta receptor-Src-HER2 signaling crosstalk as a novel mechanism of trastuzumab resistance.     

New perspectives on anti-HER2/neu therapeutics

HER2/neu and the epidermal growth factor receptor (EGFR) are significantly overexpressed in several cancer cells. Overexpression of these two receptors accounts for progression of many types of cancer: breast, ovarian, skin, pancreas and brain. In recent years, several approaches to disable the receptor complexes have shown promise. Antibody-based therapy, kinase inhibitors and other inhibitors of signaling molecules are the major approaches. Our group developed the concept that an anti-p185HER2/neu monoclonal antibody might represent a therapeutic for cancer and this has culminated in a clinically useful therapeutic, the humanized monoclonal antibody Herceptin (trastuzumab). We have now developed a small-molecule form of an anti-HER2/neu peptidomimetic (AHNP) that exhibit functions comparable to those of the monoclonal antibody Herceptin. This approach may be considered a new paradigm in receptor-specific tumor therapeutics. A brief review of our approach in developing receptor-specific therapeutic agents for HER2/neu-related cancer is presented.     

HER2在膀胱肿瘤中的表达及其单抗诱导膀胱肿瘤细胞凋亡

目的为探讨膀胱肿瘤的HER2表达阳性率及其与肿瘤分级的关系,以及抗HER2单克隆抗体对HER2表达阳性细胞株的增殖影响。方法用免疫组织化学法检测43例膀胱肿瘤标本和肿瘤细胞株的HER2表达量,用不同浓度的抗HER2单抗和阿霉素作用于HER2表达阳性和阴性的细胞株,MTT法检测细胞抑制率,流式细胞仪检测抗HER2单抗作用后肿瘤细胞的凋亡率。结果43例膀胱移行细胞癌标本中,I级12例,HER2高表达者0例;II级14例,HER2高表达者2例（14％）;III级17例,HER2高表达者13例（76％）。T24细胞株HER2高表达,BIU87阴性。抗HER2单抗对HER2表达阳性的细胞株增殖抑制呈剂量和时间依赖性,并能诱导HER2表达阳性的肿瘤细胞凋亡,但对HER2表达阴性的细胞株无明显作用,P〈0．05／P〈0．01。阿霉素对四株细胞均有细胞毒作用,P〉0．05。结论分化较差的移行细胞癌HER2阳性率高,抗HER2单抗能够选择性诱导HER2高表达的膀胱肿瘤细胞株凋亡。     

Complement in vitro inhibition by a low-sulfate chondroitin sulfate (matrix).

In this study, the ability of low-sulfate chondroitin sulfate (Matrix), a glycosaminoglycan that does not exhibit anticoagulant action, to inhibit the activation of complement has been evaluated. Two different in vitro tests were employed: lysis of mouse splenocytes sensitized with monoclonal antibodies and lysis of red blood cells sensitized using a rabbit antiserum towards those cells. In both tests, lyophilized rabbit complement was used as complement source. The low-sulfate chondroitin sulfate Matrix has been compared with a hypersulfated one (Arteparon). Both compounds share anticomplementary activity in vitro although potency appears to be related to the sulfate content of the chondroitin.     

Development and Evaluation of Tri-Functional Immunoliposomes for the Treatment of HER2 Positive Breast Cancer

Purpose

Trastuzumab combined with Doxorubicin (DOX) demonstrates significant clinical activity in human epidermal growth factor receptor-2 (HER2)-positive breast cancer (BC). However, emergence of treatment resistance and trastuzumab associated cardiotoxicity remain clinical challenges. In an effort to improve patient outcome, we have developed and evaluated novel tri-functional immunoliposomes (TFIL) that target HER2-receptors on BC cells and CD3-receptors on T-lymphocytes, and deliver DOX.

Methods

Trastuzumab (anti-HER2) and OKT-3 (anti-CD3) antibodies were conjugated to liposomes using a micelle-transfer method. Cytotoxicity of targeted immunoliposomes loaded with DOX was examined in vitro on HER2-positive BC cells (BT474), with peripheral blood monocytic cells (PBMC) as immune effector cells.

Results

TFIL demonstrated high antibody-liposome conjugation ratios (100–130 μg protein/μmol phospholipid) and cargo capacity (0.21 mol:mol drug:lipid), highly efficient DOX loading (>90%), a particle size favorable for extended circulation (~150 nm), and good stability (up to 3 months at 4°C). In the presence of PBMCs, TFIL showed complete killing of BT474 cells, and were superior to mono-targeted trastuzumab-bearing liposomes, non-targeted liposomes, and free Trastuzumab and DOX.

Conclusions

Novel anti-HER2xCD3?+?DOX TFIL show promise as a means to both engage immune cells against HER2 positive breast cancer cells and deliver chemotherapy, and have the potential to improve clinical outcomes.

     

Overcoming treatment resistance in HER2-positive breast cancer: potential strategies

Human epidermal growth factor receptor (HER)-2 overexpression or amplification occurs in about 20% of all breast cancers and results in a worse prognosis. Nevertheless, anti-HER2 treatments have recently been developed, resulting in dramatic improvements in the clinical outcome of patients with HER2-positive breast cancer. Trastuzumab has shown efficacy in early and advanced breast cancer treatment and lapatinib is currently approved for the treatment of advanced disease. Other anti-HER2 agents are being investigated. Mechanisms of resistance to trastuzumab treatment include crosstalk with heterologous receptors and amplification of HER2 signalling; amplification of the phosphoinositide 3-kinase (PI3K)/AKT pathway; alteration in binding of trastuzumab to HER2; and loss of HER2 expression. Proposed mechanisms of resistance to lapatinib involve derepression and/or activation of compensatory survival pathways through increased PI3K/AKT or estrogen receptor (ER) signalling. Several strategies to overcome resistance to anti-HER2 treatment are in different phases of development and include treatment with pertuzumab, T-DM1 and mammalian target of rapamycin (mTOR) inhibitors.     

Imaging of HER-2 overexpression in tumors for guiding therapy

Human epidermal growth factor receptor type 2 (HER2) is a transmembrane tyrosine kinase receptor, which is overexpressed in a large fraction of breast, ovarian, urinary bladder and a number of other carcinomas. Overexpression of HER2 is associated with poor prognosis. Treatment of patients with HER2-expressing breast cancer with a humanized anti-HER2 monoclonal antibody trastuzumab has resulted in improved survival. Several kinds of other anti-HER2 therapies are under development. Radionuclide molecular imaging of HER2 expression may influence patient management by selecting patients, who would benefit form anti-HER2 therapy. Other applications, such as therapy response monitoring and follow-up are also possible. In this case, the use of radionuclide imaging may overcome problems associated with biopsies, including sampling errors and discordance of expression between primary tumors and metastases. Important preconditions for development of a successful tracer for radionuclide imaging are high affinity of a targeting agent and suitable chemistry of labeling. The paper reviews information concerning major classes of HER2-targeting agents, including full-length monoclonal antibodies, their enzymatically produced fragments, engineered immunoglobulin based tracers, and alternative high affinity binders. Available information suggests that Affibody molecules or other small non-immunoglobulin based tracers have the best potential for development of high-contrast imaging agents for visualization of HER2 in vivo.     

HER2蛋白在乳腺癌靶向治疗中的作用及意义

近年来,分子生物学在医学领域得到了充分的发展,分子靶向治疗成为治疗肿瘤的一个新方向。酪氨酸激酶受体家族调控着细胞增殖、分化以及凋亡,与肿瘤的发生与发展密切相关,是一个较为理想的特异性靶点。约有30%的乳腺癌患者出现了人表皮生长因子受体2（HER2）过表达,而HER2受体的活化直接导致了其下游的PI3K/AKT和丝裂原活化蛋白激酶（MAPK）通路被激活,而通过靶向HER2过表达的细胞对肿瘤进行控制成为了一种新的乳腺癌的治疗手段。     

Recent advances in novel targeted therapies for HER2-positive breast cancer

The monoclonal antibody trastuzumab has improved the outcomes of patients with breast cancer that overexpresses the human epidermal growth factor receptor 2 (HER2). However, despite this advancement, many tumors develop resistance and novel approaches are needed. Recently, a greater understanding of cellular biology has translated into the development of novel anti-HER2 agents with varying mechanisms of action. The small molecule tyrosine kinase inhibitor lapatinib has demonstrated activity in HER2-positive metastatic breast cancer (MBC) and in the preoperative setting. Pertuzumab is a monoclonal antibody with a distinct binding site from trastuzumab, which inhibits receptor dimerization. In recent studies, the addition of pertuzumab to combination therapy has led to improvements in progression-free survival in patients with HER2-positive MBC and higher response rates in the preoperative setting. An alternative approach is the use of novel antibody-drug conjugates such as trastuzumab-emtansine, which recently demonstrated activity in MBC. Neratinib, a pan-HER tyrosine kinase inhibitor, which irreversibly inhibits HER1 and HER2, also has proven activity in MBC. A range of compounds is being developed to attempt to overcome trastuzumab resistance by targeting heat shock protein 90, a molecular chaperone required for the stabilization of cellular proteins. Furthermore, agents are being developed to inhibit the mammalian target of rapamycin, a downstream component of the PTEN/PI3K pathway, which has been implicated in trastuzumab resistance. Finally, there are emerging data indicating that combinations of anti-HER2 agents may circumvent resistance mechanisms and improve patient outcomes. In this review, recent data on these emerging agents and novel combinations for HER2-positive breast cancer are discussed.     

Formation of multimeric antibodies for self-delivery of active monomers

Proteins and peptides have been used as drugs for almost a century. Technological advances in the past 30 years have enabled the production of pure, stable proteins in vast amounts. In contrast, administration of proteins based on their native active conformation (and thus necessitating the use of subcutaneous injections) has remained solely unchanged. The therapeutic anti-HER2 humanized monoclonal immunoglobulin (IgG) Trastuzumab (Herceptin) is a first line of the treatment for breast cancer. Chicken IgY is a commercially important polyclonal antibody (Ab). These Abs were examined for their ability to self-assemble and form ordered aggregates, by several biophysical methods. Atomic force microscopy analyses revealed the formation of multimeric nanostructures. The biological activity of multimeric IgG or IgY particles was retained and restored, in a dilution/time-dependent manner. IgG activity was confirmed by a binding assay using HER2?+?human breast cancer cell line, SKBR3, while IgY activity was confirmed by ELISA assay using the VP2 antigen. Competition assay with native Herceptin antibodies demonstrated that the binding availability of the multimer formulation remained unaffected. Under long incubation periods, IgG multimers retained five times more activity than native IgG. In conclusion, the multimeric antibody formulations can serve as a storage depositories and sustained-release particles. These two important characteristics make this formulation promising for future novel administration protocols and altogether bring to light a different conceptual approach for the future use of therapeutic proteins as self-delivery entities rather than conjugated/encapsulated to other bio-compounds.     

Complement: an inflammatory pathway fulfilling multiple roles at the interface of innate immunity and development

Complement has been long perceived as an innate immune system that plays a pivotal role in the maintenance of host defense against infectious agents and the propagation of pro-inflammatory responses in the context of human disease. Complement activation has been associated with the onset of acute inflammatory reactions leading to complications such as acute graft rejection, local tissue injury and multi-organ failure. However, recent studies have indicated that various complement activation products may exert a beneficial effect by contributing to critical developmental and regenerative processes. Appreciating this extraordinary 'versatility' of complement proteins provides a framework for revisiting the design of effective complement therapeutics. A balanced strategy will have to consider limiting the detrimental proinflammatory effects of complement while preserving those activities that promote tissue repair and regeneration, cell survival and early development.