On the morning of the final day of the European Antibody Congress, attendees were greeted with a snow-covered Geneva (and a closed airport), but the indomitable Chairman Clive Wood (Bayer Schering Pharma) kept the conference on track with some help from fast-working IT personnel who arranged teleconferencing for a few speakers who could not travel to Geneva. The topics for the final day included the review of antibody products, antibody-drug conjugates and radio-immunoconjugates, bispecific and recombinant oligoclonal antibodies, as well as domain antibodies and new scaffolds.
Quality Review for Antibody Products
Marjorie Shapiro (US Food and Drug Administration) gave a perspective on the development of therapeutic mAbs, next generation antibodies and antibody alternatives. She discussed the time line for FDA's approval of 34 therapeutic and diagnostic mAbs and 5 Fc-fusion protein products between 1986 and 2010, with first approvals of an Fc-fusion protein (etanercept) in 1998, an antibody-drug conjugate (ADC; gemtuzumab ozogamicin) in 2000, and a human mAb derived from phage display (adalimumab) in 2002. Dr. Shapiro noted that more than half (6 of 11) of the antibody therapeutics approved during 2000–04 were treatments for cancer indications, but only two anticancer antibodies (panitumumab, ofatumumab) were among the ten mAbs approved during 2005–2010. Instead, five of these ten recent approvals were treatments for rheumatoid arthritis and other autoimmune disorders.
Dr. Shapiro highlighted the main themes of mAb development in the 21st century, which include an emphasis on Fc engineering to reduce or enhance effector function, exploration of mAb fragments and novel mAb constructs such as bispecific mAbs, antibody cocktails and antibody conjugates. A wide variety of formats are now possible, e.g., the antibody portion of conjugates may be whole IgG, Fab, single chain or a single variable domain, while the “payload” may be a drug, radioisotope, cytokine, toxin or peptide. She mentioned that, although gemtuzumab ozogamicin was approved as a drug, i.e., the new drug application (NDA) filed for the product was approved under the Federal Food, Drug and Cosmetic Act, currently the FDA is determining the regulatory pathway for ADCs on a case-by-case basis and some ADCs will likely be regulated as biologics, i.e., a biologics license application (BLA) filed would be approved under the Public Health Service Act, in the future.
The mechanisms of action (MOA) for mAbs were then discussed by Dr. Shapiro. She reviewed the main elements required to understand binding specificity: (1) map the epitope on the target antigen, (2) examine the reactivity with related antigens and include a histochemistry screen on human/animal tissues, (3) determine cellular localization of the epitope, (4) determine the accessibility of the epitope to the antibody under conditions of the intended use and (5) assess the binding affinity and kinetics, e.g., by surface plasmon resonance and calorimetry. The functionality of the Fc portion should be assessed through demonstration of binding or lack of binding to Fc receptors, complement fixation and C1q binding, and binding to FcRn. Assays such as those for antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and phagocytosis should be done to determine whether the Fc function is related to the MOA. The potential for side effects related to the Fc function, with an emphasis on the potential for cytokine-release syndrome and cell depletion, should also be assessed. In discussing MOA, Dr. Shapiro emphasized some facts recently learned, including that fucose inhibits ADCC,1,2 but differentially impacts cytotoxicity on different effector cells;3 the anti-inflammatory properties of IVIG are dependent on sialylation of N-linked Fc glycan;4 IgG2 mAbs can trigger ADCC with neutrophils and monocytes, but not NK cells, via FcγRIIA;5 IgG2 has disulfide isoforms;6–8 IgG4 engages in dynamic Fab exchange;9 and the presence of non-consensus N-linked glycosylation on CH1 and VL (although the impact is unknown).10
The choice of isotype as part of rational design was described as critical by Dr. Shapiro. IgG1 is suitable when effector function is desirable, but IgG2 and IgG4 mAbs should be considered when the objective is to block ligand/receptor interactions or pathogen/receptor interactions, and may be desirable for antibody-drug conjugates that are internalized or use direct/bystander killing as a MOA. She noted that sponsors should provide evidence that Fab arm exchange is not occurring when IgG4 isotypes are used as product candidates. She also stressed that it is important to understand potential differences in potency for charge variants that can arise during production of mAbs. A final point was that patient selection can be a critical factor in the design of clinical trials. Knowledge of biomarkers for intended mAbs and the extent to which Fcγ receptor polymorphism might predict response to immunotherapy would help with selection of patient who will likely benefit from treatment from those who might not respond.
Dr. Shapiro's final comments touched on immunogenicity and affinity. Regarding immunogenicity, she noted that in general immunogenicity assays should be in place and appropriately qualified at the time of initiation of clinical studies, but that for most mAbs it is acceptable to bank samples from early clinical studies and test them all together when an immunogenicity assay is ready. On a case-by-case basis there may be some mAbs that require immunogenicity testing at the initiation of clinical studies. She discussed the approximate ranges of immunogenicity for various types of mAbs, e.g., murine whole Ig (55–80%, with ibritumomab an exception at 3%), murine Fab and Fab2 (<1–8%), chimeric (<1–13%), humanized (<1–10%), human (<1–4% for mAbs derived from “humanized” mouse, 12% for mAbs derived from phage display administered as monotherapy). Regarding affinity, Dr. Shapiro questioned whether affinities in the picomolar to femtomolar range are really necessary for efficacy, while noting that such high affinity may be desirable for some indications, while very high affinity mAbs might be more likely to bind to normal cells and thus cause an unwanted safety signal.
Antibody-Drug Conjugates and Radio-immunoconjugates
The session on antibody-drug conjugates (ADCs) and radio-immunoconjugates began with discussion of the development of advanced antibody-based therapeutics in oncology by Robert Lutz (ImmunoGen). He noted that ADCs in development today solve some of the challenges of antibody therapy by arming antibodies with cytotoxic effector molecules, improving potency while ensuring the conjugate is inactive in the blood compartment. At least seven ADC utilizing ImmunoGen's maytansinoid technology are in clinical study, including trastuzumab emtansine (T-DM1; Genentech), lorvotuzumab mertansine and IMGN388 (ImmunoGen), SAR3419 and SAR 566658 (sanofi-aventis), BIIB015 (Biogen Idec) and BT-062 (Biotest). In addition, at least 9 other ADCs are undergoing clinical development, with two of these, brentuximab vedotin (Seattle Genetics/Takeda) and inotuzumab ozogamicin (Pfizer) in Phase 3 studies.
Dr. Lutz then discussed the four key components of an ADC program: an appropriate and disease-relevant target, the tumor-targeting antibody, the potent cancer cell killing agent, and the engineered linker. The target should be expressed selectively on tumor rather than on normal tissue and should have favorable properties, e.g., density on target cells, internalization rate. He noted targets that are also expressed on normal cells may also be considered if there are relevant toxicity models or appropriate clinical monitoring is available to evaluate potential toxic effects. Another potential issue is that the target on normal cells may act as an ‘antigen sink’ and thus limit exposure of the tumor to the ADC. The antibody itself must be non-immunogenic. Dr. Lutz noted that intrinsic activities such as ADCC are maintained in most ADC strategies, but low potency antibodies can also be appropriate for payload delivery.
With regard to the drug and linker, Dr. Lutz discussed ImmunoGen's maytansinoid agents, which inhibit tubulin polymerization and have potent anti-mitotic activity. The agents thus selectively inhibit proliferating cells. Dr. Lutz mentioned that ImmunoGen uses two classes of maytansinoids that differ in the way they are attached to the antibody, but that the company also has a new class of drug for use in ADCs. The IGNs comprise indolino-benzodiazepine dimers that alkylate and crosslink DNA. IGNs are highly potent and are not substrates for multi-drug resistance protein pumps. He noted that there is a need for variety in the types of drugs used in ADCs because of the variable sensitivity of different tumor types to the mode of action of the drugs. A variety of strategies to link drugs to the antibody carriers have been used, e.g., selective reduction of intrinsic disulfide bonds, introduction of amino acids for selective chemical modification. The linkers can be designed as cleavable and non-cleavable. ImmunoGen's approach involves modification of intrinsic lysine residues, which allows flexibility in payload optimization. The attachment sites are identifiable through peptide mapping. The modular design of drugs and linkers allows optimization of the therapeutic window for a particular antigen. Dr. Lutz noted that controlling exposure is an important feature of ADC development as the clinical toxicity profiles for maytansinoid ADCs, as well as other current generation ADCs, correlate most closely with conjugate exposures, not free toxophore exposure.
Dr. Lutz briefly reviewed the results for T-DM1 as first-line therapy in HER2-positive metastatic disease that were presented at the 35th Congress of the European Society for Medical Oncology conference in Milan, Italy in October 2010. In the trial, 137 women were randomly assigned to treatment with either trastuzumab (8 mg/kg dose, then 6 mg/kg once every three weeks) and docetaxel, or only T-DM1 (3.6 mg/kg once every three weeks until disease progression). The overall response rates were 45% in patients administered T-DM1 and 41% in those administered both trastuzumab and docetaxel after a median follow up of approximately 6 months. Clinically relevant adverse events occurred in 37% of T-DM1 treated patients compared with 75% of those administered both trastuzumab and docetaxel. The three most common adverse events (AE) were nausea, fatigue and pyrexia; incidence of grade 3 or higher AEs was 37% in the T-DM1 arm compared with 75% in the trastuzumab and docetaxel arm.
Dr. Lutz concluded by noting that encouraging clinical results have also been obtained with SAR3419 and lorvotuzumab mertansine. The anti-CD19 SAR3419 is in Phase 1 studies sponsored by sanofi-aventis for the treatment of relapsed/refractory CD19-expressing non-Hodgkin's lymphoma and other B-cell malignancies. The anti-CD56 lorvotuzumab mertansine (IMGN901, BB-10901) has demonstrated impressive single agent efficacy tolerability in dose-escalating trials in Merkel cell carcinoma (MCC) patients. ImmunoGen received orphan medicinal product designation for lorvotuzumab mertansine as a treatment of MCC in both the US and European Union in 2010. The ADC is also in Phase 1 studies of multiple myeloma patients and a Phase 1/2 study of patients with small cell lung cancer.
Veronique Blanc (sanofi-aventis) provided practical advice regarding the development of ADCs. She noted that the goal of ADC development is to design a therapeutic that will target very potent cytotoxins to tumor cells. However, ADCs are complex molecules, and the selection of an appropriate combination of target, antibody, drug and linker is difficult because each of these elements has its own selection criteria, but each element interacts and influences the others. Dr. Blanc mentioned that there are 17 ADCs in clinical development, but it is difficult to draw general rules or to define specific criteria for the different elements because most ADCs are in early clinical studies and they target different antigens and are composed of different drugs and linkers. As a consequence, there is too much variability to make general rules regarding what works and what does not work with the information available to date. Importantly, clinical benefit proof of concept for ADCs has been shown, e.g., results for trastuzumab emtansine and brentuximab vedotin, which are in Phase 3 studies.
The effects of the various elements of ADCs on the safety, efficacy and pharmacokinetics (PK) were then discussed by Dr. Blanc. She noted that the target influences safety in a number of ways. For example, differential expression is critical. If the antigen is expressed on normal and tumor cells at the same level, then a targeted therapeutic would have no safety margin. Dr. Blanc mentioned that skin toxicity was observed in patients treated with the anti-CD44v6 maytansinoid ADC bivatuzumab. Although the antigen was abundantly expressed in tumors, the ADC also bound antigen on skin keratinocyles, which mediated the skin toxicity. She explained that target-associated properties that affect efficacy include density and the rate of internalization and processing. Accurate quantification of the antigen density on cells that are as similar as possible to the tumors encountered in patients is critical for ADC development. Dr. Blanc noted that studies indicate a clear relationship between antigen density and the level of ADC internalized, but the rate of internalization and processing is also important. Low density antigens that internalize and process ADCs quickly may be suitable. The target can also affect PK if it is shed. High levels of shed antigen have been found to increase the rate of antibody clearance, e.g., huC242 and huC242-DM4. Dr. Blanc also emphasized that it is important to understand how the target is modulated by the ADC. She shared preclinical data that showed reductions in the expression of a target that were dependent on the dose of ADC administered, and she suggested that such information may inform decisions regarding dosing schedules in clinical studies.
Dr. Blanc then discussed three points regarding the antibody portion of ADCs. Her first point was that not all antibodies make good ADCs. Studies from Genentech have shown that ADCs comprising the same linker and drug targeting the same antigen and administered at the same dose can behave differently. These results may be due to differences in the capacity of the specific antibodies to be internalized and allow processing. Finding the right antibody is thus still an empirical process. The second point involved efforts to improve the homogeneity of ADCs, which are complex molecules with an average of 3–5 drug molecules per antibody. Dr. Blanc discussed Genentech's ThioMAbs, which have engineered cysteines that are introduced by single point mutations. Formation of a ThioMAb drug conjugate results in a homogeneous ADC with the number of drug molecules defined by the number of engineered cysteines. Such a homogenous ADC may have improved safety, although this must be proven in clinical studies. Her third point was that alternate antibody formats may provide improvement over full-size IgG-based ADC, especially with regard to distribution in tumors. A smaller antibody such as Seattle Genetic's diabody-drug conjugates may hold promise. Seattle Genetics has designed diabody-drug conjugates that have similar activity (3- to 4-fold lower) compared to IgG-based ADC in a mouse xenograft model despite the faster clearance (25- to 34-fold faster) of the diabody.
Dr. Blanc then discussed differences between cleavable and non-cleavable linkers that may affect safety and efficacy. She presented data on weight loss in mice administered an antibody conjugated to DM4 through either a cleavable or non-cleavable linker. The difference in the highest non-toxic dose was nearly 3-fold, suggesting that the non-cleavable linker could increase the safety margin for an ADC. However, she also presented data on antitumor activity that suggested the cleavable linker may be active against a broader array of tumor types compared to the non-cleavable linker. Taken together, the data for the two types of linkers suggests that a compromise between safety and activity may be required.
The impact of the cytotoxic element on the safety and efficacy of ADCs was Dr. Blanc's final topic. She discussed clinical data for ADCs that included either a DNA-binding drug or a maytansinoid. These ADCs had differences in the maximum tolerated dose of 20- to 80-fold. She noted that no conclusions can be drawn from the data on adverse events. Dr. Blanc emphasized that achieving a sufficient pharmacological index was critical to ADC development, and that ADCs that included both types of drug have had issues with toxicity or efficacy, e.g., gemtuzumab ozogamicin, AVE9633. In the case of AVE9633, there were no dose limiting toxicities at 260 mg/m2, but the ADC was not effective despite the fact that all the sites on tumor cells were saturated. Dr. Blanc reiterated the point also made by Dr. Lutz that diversity of mechanism of action is desirable because not all tumors respond to the same drug. Dr. Blanc concluded by noting that the challenge of ADC development is to find the right targets, especially regarding the safety profile and capacity to internalize the drug; select the right cytotoxic agent based on the indication; select the best antibody, likely based on an empirical approach; and find a linker that is adapted to the type of tumor and provides the desired safety profile.
Calicheamicin antibodies and beyond were discussed by Hans-Peter Gerber (Pfizer BioTherapeutics). He noted that ADCs combine the best of two worlds by utilizing a targeting system to deliver potent drugs. Clinical proof of concept for ADCs has occurred in the past few years, with impressive results reported for trastuzumab emtansine (T-DM1), brentuximab vedotin (SGN-35) and inotuzumab ozogamicin (CMC-544). The number of ADC clinical programs is expected to substantially increase over the next 3–5 years as more companies become involved in ADC development.
Pfizer's development pipeline focuses on calicheamicin as the “payload” drug. The anti-CD22 IgG4 inotuzumab ozogamicin is in Phase 2 studies as a treatment for non-Hodgkin lymphoma, and a second ADC that targets 5T4, a rapidly-internalized tumor surface antigen that is associated with poor prognosis in colorectal, ovarian and gastric cancer, is undergoing preclinical evaluation. Gemtuzumab ozogamicin (Mylotarg™) was approved by FDA in 2000 for treatment of acute myeloid leukemia, but was withdrawn in June 2010 after no improvement in clinical benefit was observed, and after a greater number of deaths occurred in the group of patients who received the mAb compared with those receiving chemotherapy alone in a confirmatory, post-approval clinical trial.
Dr. Gerber explained that calicheamicin is an extremely potent drug that is attached to an antibody through an acid-labile, hydrazone linker. After a calicheamicin-containing ADC is internalized in a targeted cell, the drug is released via the activity of a reductive group such as glutathione and causes DNA strand breaks. Calicheamicin-containing ADCs are potent at doses that are 50–100-fold lower compared to those containing tubulin-binding drugs. The extreme potency can potentially pose challenges to achieving an appropriate therapeutic index.
Safety, tolerability and preliminary efficacy data for anti-CD22 inotuzumab ozogamicin as monotherapy in a Phase 1 study and in combination with rituximab in a Phase 1/2 study were presented by Dr. Gerber. In the Phase 1 study of 50 patients, the maximum tolerated dose (MTD) was determined to be 1.8 mg/m2, which is approximately 50 µg/kg based on the mAb, administered every four weeks. The half-life was 12–30 h based on data for the first dose, and this increased with dose and cycle. Activity was observed in follicular (68.8% overall response rate, 30% complete response) and diffuse large B-cell lymphoma patients (33% ORR). Adverse events (thrombocytopenia, bone marrow suppression, liver enzyme elevation) were manageable and reversible. The Phase 1/2 study of the inotuzumab ozogamicin/rituximab combination is on-going. The study is designed with two parts: (1) dose-escalating, with 3–6 patients per cohort and (2) two-arm, expanded cohort with inotuzumab ozogamicin administered at the MTD to 30 evaluable follicular B-cell lymphoma patients (arm 1) and 30 evaluable diffuse large B-cell lymphoma patients (arm 2). Preliminary results in non-rituximab refractory patients are encouraging, with overall response rates in the 75–85% range and complete responses in the 50–60% range for both FL and DLBCL patients.
Dr. Gerber then discussed the properties of successful ADCs and the challenges associated with ADC development. He emphasized that successful ADCs show a trend toward longer half-life, higher copy number of the antigen and faster rate of internalization. In particular, the increased antigen copy number and rate of internalization are important because both affect the amount of drug entering the cell. He noted that the location of the release of the drug in the cell is important for success also. Dr. Gerber noted that challenges in ADC development include the lack of “clean” targets, i.e., those with no or limited expression on normal tissues, which ensures that the potent drugs used in ADCs are delivered selectively, target expression levels that are not predictive of pharmacology, liver metabolism and the empirical ADC lead selection process that results from a limited understanding of factors affecting pharmacology and safety. He suggested several strategies to address key ADC limitations, such as use of in vitro and in vivo biopanning and function first assays to identify tumor selective, fast internalizing ADC targets, use of site-specific conjugation technology to minimize off-target toxicity/liver metabolism of ADCs and sequential use of high throughput screening and rational drug design for payloads and linkers to help optimize the therapeutic index.
Che-Leung Law (Seattle Genetics) gave an update on the preclinical and clinical development of auristatin-based ADCs. In his presentation, Dr. Law summarized clinical data to date for brentuximab vedotin (SGN-35), and discussed the preclinical studies and the ongoing Phase 1 clinical study of the anti-CD70 ADC SGN-75. As Dr. Law explained, brentuximab vedotin comprises an anti-CD30 mAb with the payload, antitubulin agent monomethyl auristatin E (MMAE), attached via a protease cleavable linker. The ADC targets CD30, which is strongly expressed on the surface of malignant cells in Hodgkin and certain T-cell lymphoma patients. After binding and internalization, the CD30-ADC complex is internalized and traffics to the lysosome where the peptide linker is proteolytically degraded to release the MMAE payload; released MMAE disrupts the cellular microtubule network, induces G2/M phase cell cycle arrest, and ultimately leads to apoptosis. Brentuximab vedotin is undergoing evaluation in a Phase 2 pivotal clinical trial in patients with relapsed or refractory Hodgkin lymphoma (NCT00848926), a Phase 3 study in patients at high risk of residual Hodgkin lymphoma following autologous stem cell transplant (NCT01100502), a Phase 2/3 study in patients with progression of Hodgkin lymphoma (NCT01196208), a Phase 2 study in patients with relapsed or refractory systemic anaplastic large cell lymphoma (ALCL; NCT00866047), a Phase 2 study in patients with CD30-positive hematologic malignancies who previously participated in an SGN-35 study (NCT00947856), as well as a number of Phase 1 studies.
Dr. Law reviewed the recently published results of a Phase 1 dose escalation study of brentuximab vedotin.11 The overall response rate was approximately 39%, and a clear trend toward dose dependency was observed. The maximum tolerated dose (MTD) was 1.8 mg/kg administered every three weeks. Of 12 patients who received the MTD, 50% had an objective response. Dr. Law also noted that encouraging results have been observed in the pivotal study in Hodgkin lymphoma and the Phase 2 study in ALCL. Patients were administered brentuximab vedotin at 1.8 mg/kg every three weeks in both studies. The overall response rate was 75%, and the median duration was more than 6 months, in the Hodgkin lymphoma study. The overall response rate was 86%, and the median duration has not yet been reached, in the ALCL study. In clinical studies to date, the ADC has generally been well-tolerated. Adverse events (AEs) in the Phase 1 study were primarily grade 1 or 2, with the most common being fatigue, pyrexia, diarrhea, nausea, neutropenia and peripheral neuropathy; the safety profile has been similar in other studies. Submission of a BLA based on results from the pivotal trial in relapsed or refractory Hodgkin lymphoma patients is planned for the first half of 2011.
Dr. Law then presented data on the expression profile of CD70 in various tissues. The antigen is restricted to activated B and T cells and mature dendritic cells and is not found on normal tissues outside the hematopoietic system. It binds CD27, a member of the TNF receptor family, to regulate lymphoid proliferation, differentiation and apoptosis. CD70 is abundantly expressed in hematopoietic malignancies such as non-Hodgkin lymphoma and multiple myeloma, but also in solid tumors, e.g., subsets of clear cell renal carcinoma, pancreatic cancer, lung cancer. Heterogeneity of expression is observed, with patient-to-patient variation. Dr. Law noted that it will therefore be important to understand the relationship between target expression and response in clinical studies, and whether there is a threshold level of expression necessary to achieve a clinical benefit.
The components and mechanism of action of Seattle Genetics' anti-CD70 SGN-75 were explained by Dr. Law. The antibody is a humanized IgG1. The drug portion is the antitubulin agent monomethyl auristatin F (MMAF) that is conjugated onto the antibody through a maleimidocaproyl (mc) linker. When the ADC is degraded in the lysosomal compartment, Cys-mc-MMAF is released and kills the cell by disrupting the microtubule network in a manner similar to MMAE. In in vitro cytotoxicity studies, SGN-75 has shown potent activity against CD70-expressing cells, e.g., renal cell carcinoma and lymphoma cell lines. The conjugate has also been tested in xenograft models of solid tumors and hematological malignancies and has demonstrated convincing antitumor activity at doses that were ≤3 mg/kg. Dr. Law noted that a Phase 1 dose-escalation (range 0.3–4.5 mg/kg) study (NCT01015911) to evaluate the safety and tolerability of SGN-75 in patients with CD70-positive relapsed or refractory non-Hodgkin lymphoma or metastatic renal cell carcinoma was initiated in November 2009. Preliminary clinical data suggests the ADC is generally well-tolerated; no MTD has yet been reached.
Erik Merten (Bayer Schering Pharma) discussed the technical, regulatory and logistical challenges to bringing radio-immunotherapy (RIT) into clinical practice. RIT combines cancer killing radiation with precise targeting capacity and enables delivery of a high dose of radiation to tumors while normal tissues receive minimal doses. Two RITs, ibritumomab tiuxetan (Zevalin) and tositumomab-I131 (Bexxar) are currently approved by FDA.
Dr. Merten explained that an RIT product must be easily applicable and have a good therapeutic window for use in clinical practice. The manufacturing process must be robust, reproducible and yield a stable product with an appropriate shelf life. In this context, he discussed the development of I-131-L19 small immunoprotein (SIP). L19 is an 80 kDa covalent dimer of a human single-chain antibody fragment with high affinity to extra domain B of fibronectin. The antigen is selectively expressed in solid tumors, lymphomas and metastases. The SIP format has a molecular mass above the renal filtration threshold and shows high total tumor uptake, rapid clearance from circulation, low bone marrow uptake and improved tolerability. Thus, SIP molecules can provide an optimized therapeutic window for RIT compared to single chain and full-size formats.
Use of an appropriate isotope and labeling method are important aspects of RIT development. Dr. Merten explained that I-131 was selected because the isotope emits high energy beta radiation, has a half-life (8.04 days) appropriate for radiotherapy and can be introduced via direct iodination. Direct iodination is a high yield reaction providing product with medium-to-high specific activity, no precursor or preliminary chemistry is needed and the reaction is selective for tyrosine residues. Disadvantages include the possibility of oxidative damage to proteins and the need for effective purification methods.
Dr. Merten discussed radiolysis as a key challenge for scale up. A protein damaged by radiolysis may have decreased or no binding affinity to its target, thereby causing a loss of efficacy and reduced therapeutic window. Radiolysis also reduces shelf life, which may result in loss of flexibility in manufacturing, shipment and clinical use. Therefore, formulation of RITs should include suitable scavenging agents such as ascorbic acid, gentisic acid, maltose, inositol, methionine or human serum albumin. These agents have high anti-oxidizing potential, good tolerability, low toxicity and are available in appropriate quality and amounts for RIT formulation.
Supply concepts for the commercial phase need to be considered early in the development of an RIT because they determine critical technical development goals. Dr. Merten thus discussed the use of a decentralized vs. central labeling approach for supply of the radiolabeled product. He noted that the decentralized approach is used for yttrium-90 labeled ibritumomab tiuxetan, i.e., the product is compounded as a radiopharmaceutical at hospitals, whereas tositumomab-I131 is centrally labeled, i.e., the radiolabeled product is produced by a contract manufacturing organization (CMO) for distribution to hospitals. He explained that each approach has advantages and disadvantages. The decentralized approach provides more flexibility in patient scheduling due to the flexibility of producing the “hot” product at the hospitals, but infrastructure and authorization to use radioactive material is needed at all sites, availability of sterile isotope is required and there is substantial effort involved in the site set up. Central labeling is less complex to set up, there is more control of the product quality and the radiolabeling, filling, and product packaging and distribution are done by a CMO. However, formulation and stability of the product are more challenging, distribution may require frozen shipments and a suitable CMO must be identified.
Dr. Merten noted that I-131-L19SIP will be commercialized using the central manufacturing concept. The first results using the process have been encouraging. Scale up has provided material of >1 Ci in >90% radiochemical yield and with a radiochemical purity of >>90%. The immunoreactivity was >90% and the radiolabeled material was stable for a minimum of five days. The goal is to develop a manufacturing process for batch sizes at activity level of 10 Ci or greater, identify appropriate packaging, and design a worldwide supply chain concept based on shipment of the “hot” I-131-L19SIP product. In conclusion, Dr. Merten stated that I-131 radiolabeled proteins are suitable to give broad access to RIT in clinical practice.
The uses of radiolabeled antibodies for tumor imaging and as anticancer agents were discussed by Jacques Barbet (University of Nantes) in a thorough and well-referenced presentation. He emphasized that RIT has evolved over the course of more than 20 years and that numerous improvements have been made, such as use of new stable chelates, humanized antibodies and pretargeting methods. Dr. Barbet noted that William H. Beierwaltes was a pioneer in the use of nuclear medicine because he was the first to administer radiolabeled (I-131) antibodies to a melanoma patient and he invented meta-iodobenzyl-guanidine (MIBG). Early work with radiolabeled antibodies emphasized imaging (immunoscintigraphy) with isotopes of iodine; development of chelating agents enabled use of other radiolabels.12 Immunoscintigraphy has largely been replaced by 18F-deoxyglucose positron emission tomography (18F-FDG PET) for detection of cancer, and the focus for use of radiolabeled antibodies has shifted to development of therapies.
The multiple modes of action of RIT were then discussed by Dr. Barbet. The effects of RIT result from both radiobiological (e.g., direct radiation of bound cell, cross-fire irradiation) and immunological (e.g., ADCC, CDC, apoptosis) mechanisms.13 Two radiolabeled antibodies are currently approved for treatment of cancer: ibritumomab tiuxetan (Zevalin®), which is an anti-CD20 murine IgG1 mAb conjugated to the yttrium-90 or indium-111 chelator tiuxetan, and tositumomab (Bexxar®), which is an anti-CD20 murine IgG2a mAb linked to iodine-131. Differences in clinical outcomes have been demonstrated in comparisons of (90)Y ibritumomab tiuxetan and rituximab, which also targets CD20 on B cells, with statistically and clinically significant higher overall response rate and complete responses observed in non-Hodgkin lymphoma patients treated with (90)Y ibritumomab tiuxetan compared with those who were administered rituximab.14,15
Dr. Barbet noted that better efficacy has been shown when RIT is given for minimal disease14,16–18 and that RIT consolidation therapy after initial tumor reduction may maximize treatment efficacy and significantly improve progression-free survival. In consolidation therapy, RIT is given to treat minimal residual disease that exists after initial treatment of patients with immuno-chemotherapy that is intended to reduce tumor burden. In a Phase 3 study of patients with advanced-stage follicular lymphoma, consolidation of first remission with (90)Y-ibritumomab tiuxetan was shown to prolong progression-free survival by two years and result in high rates for conversion of partial responses to complete responses compared to patients who received no treatment other than the first-line therapy.19
Dr. Barbet then discussed improvements in radio-immunotherapy based on use of better radionuclides and methods such as pretargeting.20 In addition to the well-studied beta/gamma emitting iodine and yttrium isotopes, researchers are also investigating alternate isotopes such as the alpha-emitters actinium-225, bismuth-213 or astatine-211. Dr. Barbet explained that α particle radiation has advantages because of its limited range and high linear energy transfer, which may make α-emitters better suited for the treatment of residual disease in cancer patients.21
As discussed by Dr. Barbet, pretargeting methods involve administration of an unlabeled, bifunctional (anti-tumor and anti-chelate) antibody or other cell-targeting agent followed by administration of a radionuclide construct that binds to the pre-localized antibody. This approach was pioneered by David A. Goodwin and colleagues.22–25 An example of a pair of agents used in the method include a bispecific antibody that localizes to tumor cells and then binds a radiolabeled hapten-peptide that is subsequently administered.26–28 Streptavidin-antibody constructs paired with biotin-radionuclide conjugates have also been used in pretargeting approaches to the treatment of cancer. Clinical studies of pretargeting radio-immunotherapy have produced encouraging results.29–31
In conclusion, Dr. Barbet remarked that targeted radionuclide therapy is a workable therapeutic option against disseminated tumors, and specified that radio-immunotherapy is efficient in lymphoma. In contrast, nuclear medicine therapy is proving its efficacy against small tumor masses in solid tumors. He noted that the therapy has been tested mostly as a single therapeutic agent injected only once. As a consequence, repeated injections and combined therapy should be more thoroughly investigated. Dr. Barbet also reiterated the point that better radionuclides must be tested, including the alpha particle emitters that are ideally suited to treat diseases where tumor cells are isolated. Because development and advancement of the field requires highly multidisciplinary approaches, Dr. Barbet called for greater cooperation between academia and industry.
Bispecific and Recombinant Oligoclonal Antibodies
The afternoon session was opened by Scott Glaser (Biogen Idec) who discussed advances in the preclinical development of IgG-like bispecific antibodies. He noted that next generation antibodies are designed to improve clinical outcomes, e.g., reduce immunogenicity, provide improved binding/access to target, increase potency by more effectively engaging immune effector functions, target two or more biological pathways or engage multiple cell types. Bispecific antibodies (BsAb) are not a new format, but manufacturability and protein stability posed substantial hurdles that took several decades to overcome. A number of second generation designs are using protein engineering strategies to address manufacturing issues. Dr. Glaser explained that Biogen Idec has been developing tetravalent IgG-like BsAbs32–35 that bind one antigen via Fab and a second antigen via single chain variable fragments (scFv) attached to the Fc while retaining key antibody features such as IgG-like PK, an option for immune effector function, adaptability to current mAb manufacturing processes, and acceptable product quality.
Dr. Glaser noted that scFvs, which are key building blocks of IgG-like bispecific antibodies, often do not have sufficient intrinsic stability to permit scale-up production of molecules with acceptable biopharmaceutical properties. CHO cells produce heterogeneous mixtures of products, with monomer present as approximately 60% and soluble aggregates as 40% of the product. The monomer fraction is also unstable and will eventually degrade and aggregate during storage. Biogen Idec has applied a number of methods for designing stabilized scFvs, including statistical analyses, structure-guided design and knowledge-based design, to yield information regarding which scFv positions to target for mutagenesis. They synthesized mutagenic oligonucleotides, constructed focused plasmid libraries, transformed E. coli and then screened the libraries for mutants that demonstrated elevated thermal stability in a thermal challenge screen. An iterative stability screening process yielded stability engineered scFvs that met their goals (Tm of at least 65° C) for production of stable BsAbs.
Based on their experience with stabilization of scFvs, the wild-type variable light (VL) domain has typically shown greater stability than variable heavy (VH) domain and first focusing on stabilizing VH has been productive. However, Dr. Glaser described a BsAb designed to target two soluble ligands, designated as M1 and M2, in which the scFv VL hits destabilized VH to some extent. They resolved the problem by determining the Tm values of individual VL designs by differential scanning calorimetry (DSC), finding hits and combining those with the best VH combinations to yield a stable scFv. This led them to a new library work flow, which included DSC on wild-type scFv while assuming VH is the least stable domain, screening of the top 10–20 VH libraries in a thermal challenge assay, iterative combination of VH mutations, construction of VL libraries in the context of most stable VH and finally iterative combination of VL mutations. He noted that splitting the iterations into two steps increases speed due to the factorial nature of possible combinations. To date, seven scFvs have been successfully stabilized using their design tools, which have proven robust enough to manually make high-scoring mutants to rescue misbehaving scFvs.
Dr. Glaser noted that Biogen Idec's BsAb can be produced in CHO cell lines that provide titers in the 2–4 g/L range and that production can be scaled to a 1,000 L bioreactor. Platform purification that provides an acceptable product quality can be performed, e.g., main peak 97.9% by size exclusion chromatography (SEC) analysis. The purified BsAb is stable for a minimum of 5 months at a concentration of 10 mg/mL in phosphate buffered saline. In some examples, the bispecific antibody protein has been concentrated to over 100 mg/mL in research formulations without inducing aggregates or protein loss. Stability studies performed with 10, 50 and 100 mg/mL solutions indicate that BsAb are amenable to standard antibody formulation.
In concluding his presentation, Dr. Glaser discussed characteristics of the M1 × M2 BsAb. Biacore analysis indicated that the BsAb binds both M1 (target of scFv) and M2 (target of Fab) simultaneously, but the magnitude of the M1 binding signal suggested a different stoichiometry of binding to the BsAb compared to an anti-M1 mAb. Specifically, the stoichiometry of M1 binding to anti-M1 mAb decreases as the density of mAb increased, but the bispecific antibody was relatively insensitive to this effect. The length of the linker between the scFv and the Fc was observed to have an effect on BsAb activity, e.g., 1:1 binding of a M1 or M2 trimer to a mAb required a long, flexible linker. The behavior of the reverse BsAb (C-terminal anti-M2 scFv) suggested that this effect is not unique to the original BsAb. These results imply that valency, mobility and distance between binding sites are important considerations for optimizing BsAb activity. In experiments in mice, the M1 × M2 BsAb was found to simultaneously inhibit early and late phase activation of signaling pathway by M1 and M2 mediators. The M1 × M2 BsAb is thus functional in vivo and capable of neutralizing the two mediators.
Tariq Ghayur (Abbott Laboratories) discussed Abbott's dual variable domain (DVD)-Ig™ technology platform. The DVD-Ig™ format combines the antigen binding domains of two mAbs into a single entity by adding a binding domain to each Fab arm.36 It preserves the dual functional specificities of the parental mAbs, but behaves like conventional mAbs in many aspects. DVD-Ig™ molecules can be made to a variety of target pairs (e.g., two soluble ligands, soluble/cell surface antigens, two cell surface antigens) and using different variable domains (e.g., two human, human/humanized, human/mouse). Dr. Ghayur described several examples of DVD-Ig™ molecules that have been tested in preclinical animal models, including a DVD-Ig™ construct targeting IL-12/IL-18 that inhibited Staphylococcus aureus Cowan I (SAC)-induced IFNγ in a HuPBMC-SCID mouse model and a DVD-Ig™ construct targeting IL-1α/IL-1β that inhibited disease progression in a collagen-induced rheumatoid arthritis model.37 Dr. Ghayur also showed in vitro re-directed toxicity data for several CD3+X DVD-Ig™ constructs (IC50 in pM), and mentioned that two of these constructs (CD3/CD20 and CD3/EGFR) tested in vivo showed tumor growth inhibition activity in xenograft animal models.
The features to consider in making a DVD-Ig™ drug were then discussed in detail by Dr. Ghayur. Specifically, four key questions were addressed: (1) can the two variable domains (VD) retain function, e.g., affinity, potency, specificity? (2) can DVD-Ig™ constructs with desired physicochemical and drug-like properties be identified early in discovery? (3) do DVD-Ig™ possess desired PK properties? and (4) can DVD-Ig™ be expressed and manufactured in CHO cells? Dr. Ghayur explained that, in general, protein expression correlates with protein stability and physicochemical properties, and expression profiles have been used as a tool to identify stable VH/VL combinations for mAbs. In a similar fashion, expression profiles may allow selection of stable variable domain combinations/orientations in DVD-Ig™ constructs. He emphasized use of an ‘anchor’ concept, i.e., keeping one domain constant while altering domain combinations and orientations and the length of the heavy chain and light chain linkers.
Dr. Ghayur discussed the stepwise approach to building DVD-Ig™ generate hundreds of DVD-Ig™ molecules in a short time period; establishment of processes to fully characterize multiple DVD-Ig™ in parallel; and features that determine the desired drug-like properties (DLP) and PK for a DVD-Ig™ molecule. Initially, over a thousand DVD-Ig™ constructs were made, expressed, purified and preliminary functional characterization of both VDs by ELISA, Biacore, FACS or bioassays was performed. The contribution of VD combination, VD orientation and linker lengths to mammalian cell (HEK293) expression was evaluated and described. Dr. Ghayur then described detailed biophysical/chemical and DLP analysis, PK in rats (half-life typically in the 3–15 day range), and CHO cell expression and MTX amplification (100 nM) for 20–26 DVD-Ig™ molecules. Selected DVD-Ig™ were then additionally amplified; yields of ∼1 g/L or more have been achieved. Dr. Ghayur provided the identity of some of the assays used for biophysical/biochemical evaluation of DVD-Ig™ constructs, e.g., intact and reduced LC-MS analysis; peptide mapping by LC-MS, SDS-PAGE under reducing and non-reducing conditions, Western blot analysis, DSC, universal buffer platform assays at pH 4, 6 and 8; assessment of freeze/thaw stability, sedimentation assay by SV-AUC; FTIR and near UV spectroscopy; capillary zone electrophoresis, analytical HIC chromatography and size exclusion chromatography—multi-angle laser light scattering (SEC MALLS).
Dr. Ghayur then described how their high throughput processes can be applied to a program. As an example, he showed data from a factorial study involving 96 possible DVD-Ig™ composed of combinations of three anti-VEGF VDs paired with four anti-Delta-like 4 (DLL4) VDs in two orientations (inside and outside positions) via four linkers (long and short linkers each for VH and VL). Using the anchor concept, the anti-DLL4 VD or anti-VEGF VD was held constant in the inside position, while the linkers and various sequences for the anti-VEGF or anti-DLL4 domain were varied. Of the 96 constructs, 24 constructs with various VD combinations, VD orientations and linker lengths were selected and tested in vitro for functional activity. From this analysis, three DVD-Ig™ with different features were selected for further characterization.
The final topic of Dr. Ghayur's presentation was the external (independent) validation of the DVD-Ig™ format. In this study, a DVD-Ig™ derived from anti-VEGF-A bevacizumab and anti-OPN hu1A12 effectively bound and inhibited both targets.38 Biacore analysis indicated that the Kds were 9.43 and 1.64 nM for OPN and VEGF, respectively, which were similar to the KDs of parental mAbs. In addition, VEGF/OPN DVD-Ig™ effectively suppressed HCCLM3 tumor growth and spontaneous lung metastasis in nude mice when dosed sc at 20 mg/kg, and was more effective than either bevacizumab or hu1A12 alone in the two animal models studied.
HESylation® of new scaffolds to improve drug characteristics was discussed by Frank Feller (Fresenius Kabi). The process involves coupling hydroxyethyl starch (HES) derivatives to drug substances in order to modify drug properties such as half-life. HES is a poly-disperse amylopectin polymer that is isolated from corn (waxy maize); HES products are marketed as plasma expanders and have a long history of safe use (e.g., intravenous doses up to 210 g per day can be administered to adults without side effects). Allergic reactions are known to occur in only a very small percentage (0.058%) of patients, and immunogenicity was demonstrated to be very low.39
The HES polymer is modified by acid hydrolysis to achieve the desired molecular weight (MW) and glucose residues are hydroxyethylated (molar substitution, MS) to control the rate of degradation caused by alpha-amylase. The MW and the MS can be tailored to achieve the required pharmacokinetic properties and these parameters also affect the degradation of a HESylated® therapeutic agent. Clearance is decreased with increasing MW of the HES derivative, while the degree of molar substitution directly interferes with degradation caused by alpha-amylase.
Mr. Feller explained that HESylation® enables prolongation of the circulation half-life of therapeutic agents by increasing the stability of the molecules, as well as by reducing renal clearance, resulting in increased biological activity. HESylation® also reduces immunogenicity or allergenicity and reduces viscosity (up to 4-fold compared to PEG). He concluded by describing two examples of drugs that demonstrated improved pharmacodynamics or PK after HESylation®. In mice, HESylated® erythropoietin was shown to substantially increase hematocrit compared with the same dose of unHESylated® product and the hyperglycosylated erythropoietin darbepoetin alfa (Aranesp®; Amgen). The percent change of hematocrit was shown to increase with increasing MW of three HESylated® erythropoietins (HES 30, 60, 100 kDa). In dogs, the HES100-erythropoietin demonstrated a thrice amplified half-life compared to darbepoetin alfa. In rabbits, HESylated® interferon alpha showed higher in vitro activity, longer half-life and earlier onset of antiviral effect compared to peginterferon alfa-2a (Pegasys®; Genentech).
Beyond Antibodies: Domain Antibodies and New Scaffolds
The final session of the meeting began with a presentation from Ruud de Wildt (Domantis), who provided an update on domain antibodies (dAbs) that comprise the pipeline and technology of Domantis. GlaxoSmithKline (GSK) acquired Domantis in 2007, then formed the biopharm units in 2008 and the biopharm discovery units in 2009. The units are responsible for defined areas of research and they function on a three year funding model based on specific business plans. Non-vaccine biopharmaceuticals currently comprise approximately 20% of GSK's overall clinical pipeline.
Dr. de Wildt described the properties of domain antibodies (dAbs) and highlighted the advantages of dAbs compared to full-size mAbs. DAbs are composed of human VH or VL and thus are the smallest functional binding units of antibodies. The molecules are derived from phage display libraries and bind to either protein A or protein L for detection, immobilization and purification. DAbs are soluble, stable and heat resistant, and they can be made to target a wide variety of antigens, including cell surface receptors. A comparison of dAbs with conventional human VH-VL structure has shown that the frameworks and complementarity-determining regions (CDR) have fully human conformations. At 11–13 kDa, dAbs are approximately one-tenth the size of IgG1. They can be designed to be either single or dual specific, as well as either mono- or bi-valent; they can also be derivatized, e.g., by conjugation to toxins or drugs. The half-life can be adjusted as desired and effector functions can be added.
Dr. de Wildt explained that dAbs can be delivered via a number of routes, e.g., intravenous, subcutaneous, inhaled, ocular, dermal, and he then discussed the pulmonary route in detail. The lungs are well-suited for local delivery because of their large surface area (>100 m2), highly permeable membrane and lack of mucociliary clearance. The small size of dAbs allows substantially higher doses to be delivered locally (approximately 12 times the equivalent mAb dose), efficient tissue penetration, and relatively slow clearance from the lung (half-life approx 4 h in mice). The molecules are also resistant to the proteases that exist in diseased lung, and can withstand the shear and thermal stress of nebulization. Lead dAb candidates have been developed that are thermodynamically stable (Tm >65° C), maintain activity after 14 days at 50° C, resist degradation by proteases such as trypsin, elastase and leucozyme, and remain stable after nebulization at 20 mg/mL. There are currently three human dAb-based molecules in clinical studies (anti-inflammatory dAb, dAb targeted multicomponent vaccine, anti-TNF dAb-Fc fusion ART621).
The validation of TNFR1, also known as the p55 receptor, as a target in pulmonary disease was then discussed by Dr. de Wildt. Studies of TNF receptor pathways have identified divergent functions of TNF and suggested that selective inhibition of TNFR1 signaling may be more beneficial than total TNF blockade. For example, TNFR1 knock-out mice were protected from ventilator-induced lung injury (VILI) whereas TNFR2 knock-out mice died more quickly than wild-type mice.40 In a murine model of tobacco smoke-induced lung inflammation, daily intranasal administration 1 mg/kg of an anti-TNFR1 dAb demonstrated a greater percent inhibition of response to smoke compared with a 10 mg/kg dose of PEGylated anti-TNFR1 dAb intraperitoneally (ip) administered once every two days and twice daily 10 mg/kg doses of PDE4 inhibitor given orally. The PEGylated and native dAbs had half maximal inhibitory concentrations (IC50) of approximately 1 nM. Dr. de Wildt explained that the duration of action of the dAb targeting TNFR1 is greater than 6 h, as determined in a mouse study of the dAb delivered intranasally at 0.3 mg/kg at various times prior to intranasal TNF challenge. This suggests that the dAb may be suitable as a therapeutic with daily dosing. He additionally described experiments that suggested that selective blockade of TNFR1 with the dAb attenuates VILI, whereas total blockade of TNF with an anti-TNF mAb does not attenuate VILI.
The in vitro/in vivo profile of an anti-human TNFR1 lead dAb was the final topic presented by Dr. de Wildt. The ability of dAbs to stimulate IL8 release from human lung fibroblast cells was examined, and no agonist activity was observed using monomeric dAbs tested at concentrations up to 16 µM, whereas the conventional anti-TNFR1 control mAb 225 showed agonist activity. The effect of the dAb on neutrophil cell influx in a cynomolgus lipopolysaccharide (LPS) challenge model was assessed. The nebulized dAb was administered 1 h prior to LPS challenge. Medium and high doses of dAb significantly inhibited LPS-induced bronchoalveolar lavage (BAL) neutrophil influx at 6 and 24 h (p < 0.005). The inhaled dAb also inhibited inflammatory mediators such as IL6, MIP1b and IL8 when tested in the cynomolgus LPS challenge model. In a PK study of inhaled vs. intravenous administration of similar doses of dAb in cynomolgus monkeys, the lung/plasma ratios indicated lung retention and low systemic bioavailability (<1%). The observed PK profile may support once or twice daily dosing in man.
An overview of modified single domain shark antibodies as therapeutic interventions in disease was presented by Michael Foley (AdAlta). He discussed the current trend toward development of next generation antibodies and engineered scaffolds that may have advantages as therapeutics compared to conventional full size IgG molecules. AdAlta is developing two of these types of products: Ig new antigen receptors (IgNARs),41 which are antibodies found in cartilaginous fish such as sharks, rays, skates and ratfish, and i-bodies, which are single domain human proteins that mimic IgNARs. AdAlta's IgNARS have been shown to be thermally stable, as well as stable to digestive enzymes and urea, and no immunogenicity has yet been detected in rabbits and mice.
Shark antibodies have no CDR2 and a long CDR3, which can affect the fine specificities of the antibodies. The average CDR3 loop length in sharks is 16,42 whereas the loop length averaging 12–13 in humans, 9–10 in mouse,43 and 14 in camel. AdAlta's library construction starts with blood taken from a wobbegong shark. The lymphocyte fraction is isolated and an mRNA preparation is made from the lymphocytes. cDNA corresponding to mRNA from antibody-producing cells are then used to produce libraries that contain 6 × 109 antibodies that have random combinations of amino acid sequences. The sequence of the naïve shark DNA can be modified in the CDR1 and CDR3 regions, and the loop length of the CDR3 can altered to be in the range of 10–20 amino acids. Targets may have a wide diversity and include antibodies, receptors, mitochondrial receptors, microbial transmembrane proteins, bacterial surface proteins, viral coat proteins and parasite proteins.
Dr. Foley then discussed specific binders isolated from an IgNAR library, including IgNARs targeting the Plasmodium falciparum apical membrane antigen 1 (AMA1). This antigen is involved in the invasion of red blood cells by the parasite, and has a hydrophobic trough that may serve as a binding surface for the elongated CDR3 of IgNARs. The 12Y-2 IgNAR variable domain44,45 was selected from a library of individual variable domains. The clone bound AMA1 with a Kd of 2.41 × 10−7 M and was affinity-matured by error-prone PCR, resulting in several mutants with enhanced affinity over 12Y-2 that were also shown to be potent inhibitors of invasion of Plasmodium falciparum strain 3D7. Comparison of the epitope of IgNARs targeting AMA1 indicates that it overlaps with the epitope of the anti-AMA1 mAb 1F9 and that the long CDR3 loop penetrates the trough in AMA1.
In a similar fashion, IgNARs targeting insulin-like growth factor-1 receptor (IGF-1R) have been identified and characterized. The molecules can be purified from E. coli periplasm in yields up to 2–3 mg/L of purified protein. Dr. Foley explained that the anti-IGF-R1 IgNAR AD0027 has been shown to have antagonist activity and to inhibit MCF-7 cancer cell growth with an IC50 of 10.4 µM. Affinity maturation of AD0027 resulted in identification of a mutant with improved binding (Kd = 42 nM compared with Kd = 75 nM for AD0027).
Dr. Foley mentioned that IgNARs have humanization potential because their structure is very similar to human adhesion molecules such as the neural cell adhesion molecule (NCAM), which are part of the immunoglobulin domain structural (I-SET) family. AdAlta's i-body libraries are based on a human protein scaffold that has structural homology of >95% with the IgNARs. i-bodies are humanized single domains with both CDR1 and CDR3, with the CDR3 loop ranging from 10–20 amino acids in length. AdAlta has screened an i-body library on 5G8, a monoclonal antibody to malaria, and the malaria protein AMA1. The expression yields were 1.4 mg/L and 0.4 mg/L for the 5G8 and AMA1 i-bodies, respectively. The appropriate specificity of each was verified via ELISA. In concluding, Dr. Foley noted that AdAlta's internal pipeline is currently at the discovery phase, with optimization of lead candidates in three disease areas (cancer, asthma/inflammation, HIV infection) expected in 2011.
Novel insights into IgNAR V-domain structure and function were provided by Brian Fennell (Pfizer). He explained that full-size IgNARs are homodimers of two polypeptide chains each comprising a variable (VNAR) and five constant domains (CNARs). The VNARs, which are the focus of development efforts, contain a short CDR1 loop, HV2, which is an area of natural amino acid diversity that replaces the classical CDR2 loop, and a long CDR3 loop. IgNARs have Type I and II subtypes based on the presence or absence of non-canonical cysteine residues in the VNAR Fw and CDR regions, and share structural features with some vertebrate Ig domains, including TCR Vα chain and IgG Vκ chain. Interest in developing VNARs stems from the size of the paratope, which has only 2–3 loops yet has an antigen-binding interface that is similar in size (in angstroms) compared with classical dual-domain antibodies, the ability to target clefts and advantageous physicochemical properties, e.g., tolerance to high levels of salt and urea, thermostability.
Dr. Fennell discussed Pfizer's efforts to better understand the structural features that define the qualities of the VNAR domain through a structure/function study of a Type I VNAR. The study involved identification of areas of the molecule that can tolerate mutation(s), i.e., VNAR hotspots; investigations into the importance of the cysteine residues to VNAR structure/function; affinity maturation of the anti-hen egg-white lysozyme (HEL) shark VNAR 5A746 using ribosome display technology; and determination of VNAR-specific and VNAR/Ig VL/TCR Vα overlapping hallmark residues, i.e., residues crucial to the structure/functioning of the protein. Their ‘molecular scanning’ approach included generation of a ribosome-displayed 5A7 mutant library with mutations across the entire VNAR domain, and selection of a library on the HEL antigen over three rounds with iterative mutagenesis between rounds. The library inputs and outputs were sequenced at the DNA level and they screened for HEL binding using a number of functional assays, e.g., ELISA, Biacore. A heat map/scanning map of 5A7 was then constructed based on results of sequencing and functional assays.
They identified 14 hotspots, defined as an area of the NAR protein that tolerated amino acid substitutions greater than five times, that were located mostly in the solvent exposed parts of the protein; a high level of mutational plasticity was observed across the VNAR domain. However, the 5A7 paratope was found to be non-plastic, with only conserved substitutions tolerated, e.g., Y29H, A88G, A95G, which suggested that it was unlikely that 5A7 affinity-matured variants would come from further maturation of CDR1/3. Dr. Fennel presented data for 5A7 variants that indicated that mutation in the Fw1 region appeared to result in an improved off-rate and mutation in the HV4 region appeared to result in an improved on-rate. They then explored combinations of amino acid mutations and identified one triple mutant (A1D, S61R, G62R) that showed a 20-fold gain in affinity compared with 5A7 (Kd of 0.46 nM vs. 9.33 nM). Dr. Fennel noted that a structural model of 5A7 containing beneficial mutations indicated that Asp1 formed a salt bridge with R112 and K116 of HEL, which resulted in an improved off-rate, Arg61 formed a weak ionic interaction with D101 of HEL, which resulted in an improved on-rate and Arg 62 may also contact D101 of HEL or increase VNAR solubility, thereby leading to increased stability.
The group also investigated the importance of cysteines to VNAR structure/function. Four of six variants in which wild-type cysteines were mutated and nine clones in which native amino acids were replaced with cysteines lost HEL-binding. HEL binding was retained with the variants G15C (Fw1 region), G42C (Fw2 region) and S61C (HV4 region), suggesting that these might be novel sites for covalent labeling of VNAR domains. Dr. Fennel concluded by describing the set of experiments designed to identify hallmark residues, i.e., residues crucial to the structure/functioning of the protein that may have to be retained during humanization. The group constructed a 5A7 heat map, identified framework residues that did not tolerate amino acid mutation, and conducted bioinformatic analysis of suspected VNAR hallmark residues that included a comparison with unique spiny/spotted shark clones, nurse shark clones, and Ig VL and TCR Vα, the latter of which defined residues that overlap between v-domain classes. A total of 21 residues located predominantly to the core of the NAR were classified as overlapping with VNAR/IgVL/TCR hallmarks, and three residues (L18, T34 and E57) were identified as VNAR specific hallmarks. The study findings should aid future VNAR engineering and optimization studies towards development of the proteins as viable therapeutics.
Laurent Audoly (Pieris) closed the meeting with a discussion of the proof of concept (POC) trials for new scaffolds that have VEGF and c-Met as targets. He first noted that, in general, there is a need for targeted molecules with improved pharmacological and biophysical properties compared to those of antibodies, including access to greater target space and efficacy, more flexible formatting (e.g., inhaled), and reduced cost of goods. Anticalins®, which are engineered lipocalins, are in development by Pieris as targeted therapeutics. The 12 known human lipocalins endogenously bind, store and transport a broad spectrum of molecules; they are monomeric and very stable proteins that are approximately 18–20 kDa. The natural binding pocket of wild type can be redesigned to generate highly specific hapten and peptide binding Anticalin® molecules. Lipocalins also have CDR-like loops that can be modified to generate highly potent protein binding Anticalin® molecules. Combinatorial libraries up to 1 × 1011 are produced by diversification at key amino acid positions. A platformed phage display based-screening process results in the identification of candidates with pM affinity that can be produced in E. coli and modified as desired, e.g., PEGylation or fusion to protein, to extend the half-life in plasma.
Dr. Audoly explained that the screening for Anticalins® is done with the same host and in the same final format from discovery to identification of drug candidate to manufacturing. The cycle time from discovery to in vivo POC is approximately 18 months, with the overall time from discovery to IND filing of three years. Pieris has one candidate in Phase 1 clinical studies (anti-VEGF PRS-050), two candidates at the in vivo POC stage (anti-cMET PRS-110, PRS-080 targeting an undisclosed antigen) and two projects at the anticalin selection stage (inhaled anti-IL4Ra PRS-060, and a bispecific “Duocalin” PRS-190).
Data on PRS-050, a VEGF targeting Anticalin® for oncology was then presented by Dr. Audoly. The molecule is specific for all major human VEGF-A splice forms (Kd = 1 nM) and has comparable affinity and efficacy to competitors in preclinical models. The binding mode is antagonistic, with an IC50 of 12 nM in competition ELISA; the molecule blocks VEGF interaction with VEGF-R1 and -R2. Half-life extension was achieved via site-directed mono-PEGylation with branched 40 k-PEG. The molecule shows cross-reactivity towards murine and cynomolgus monkey VEGF with similar potency. PK in mice, rat and cynomolgus monkeys suggested that the molecule would have a five to six day half-life in humans. PRS-050 inhibited the extravasation of dye in an assay of VEGF-induced enhanced vascular permeability in guinea pigs and also inhibited angiogenesis when dosed at 9.3 mg/kg daily ip in a Matrigel plug angiogenesis assay. In an A673 rhabdomysarcoma tumor xenograft model, PRS-050 inhibited tumor growth in a dose (2.6, 7.7 or 15.4 mg/kg dosed ip daily) and time (7.7 mg/kg dosed ip daily or every other day) dependent manner. Dr. Audoly also noted that published reports have described an unexpected increase in thromboembolic events in clinical trials of anti-VEGF bevacizumab47 and that a possible cause is platelet activation induced by bevacizumab-VEGF-heparin immune complexes. As of today, it is also important to note that this increase in thromboembolic events is controversial as this has not been consistently measured. Nonetheless, studies in FcγRIIa transgenic mice have shown that PRS-050 does not form immune complexes and does not cause significant thrombocytopenia in a preclinical model suggesting a possible path forward for differentiation. From a manufacturing “line of sight”, a robust GMP process has been developed. PRS-050 is produced in E. coli, undergoes two-step purification via chromatography, which is followed by PEGylation, another chromatography step and then sterile filtration and fill/finish. The first administration to humans with solid tumors occurred in June 2010 and preliminary human single dose PK data suggest the half-life is approximately six days.
The final topic discussed by Dr. Audoly was the development of PRS-110, a monovalent c-Met antagonist. cMet is implicated in tumor cell proliferation, metastasis and angiogenesis, and is the only receptor identified to date for hepatic growth factor/scatter factor (HGF). Dysregulation of c-MET or HGF expression has been correlated with a variety of human malignancies. At least four therapeutic proteins targeting either c-MET or HGF are in clinical studies: anti-c-MET one-armed antibody-like molecule ortuzumab (Genentech), anti-HGF rilotumumab (Amgen), anti-HGF TAK701 (Takeda) and anti-HGF AV299 (AVEO). Dr. Audoly presented data for PRS-110 indicating that the molecule is highly specific with low nM affinity against c-Met and displays in vivo efficacy. Anti-tumor activity was observed with PRS-110 dosed at 7.5 mg/kg as a single agent, and enhanced activity was observed when the agent was combined with temozolomide. PRS-110 can be produced with a fermentation yield of 8.9 g/L, and a 36% yield after 2-step purification. The PEG conversion is 88% and final purity is 95% by SDS-PAGE and 96% by SEC-HPLC.
References
-
1.Shinkawa T, Nakamura K, Yamane N, Shoji-Hosaka E, Kanda Y, Sakurada M, et al. The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J Biol Chem. 2003;278:3466–3473. doi: 10.1074/jbc.M210665200. [DOI] [PubMed] [Google Scholar]
-
2.Okazaki A, Shoji-Hosaka E, Nakamura K, Wakitani M, Uchida K, Kakita S, et al. Fucose depletion from human IgG1 oligosaccharide enhances binding enthalpy and association rate between IgG1 and FcgammaRIIIa. J Mol Biol. 2004;336:1239–1249. doi: 10.1016/j.jmb.2004.01.007. [DOI] [PubMed] [Google Scholar]
-
3.Peipp M, Lammerts van Bueren JJ, Schneider-Merck T, Bleeker WW, Dechant M, Beyer T, et al. Antibody fucosylation differentially impacts cytotoxicity mediated by NK and PMN effector cells. Blood. 2008;112:2390–2399. doi: 10.1182/blood-2008-03-144600. [DOI] [PubMed] [Google Scholar]
-
4.Anthony RM, Nimmerjahn F, Ashline DJ, Reinhold VN, Paulson JC, Ravetch JV. Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc. Science. 2008;320:373–376. doi: 10.1126/science.1154315. [DOI] [PMC free article] [PubMed] [Google Scholar]
-
5.Schneider-Merck T, Lammerts van Bueren JJ, Berger S, Rossen K, van Berkel PH, Derer S, et al. Human IgG2 antibodies against epidermal growth factor receptor effectively trigger antibody-dependent cellular cytotoxicity but, in contrast to IgG1, only by cells of myeloid lineage. J Immunol. 2010;184:512–520. doi: 10.4049/jimmunol.0900847. [DOI] [PubMed] [Google Scholar]
-
6.Wypych J, Li M, Guo A, Zhang Z, Martinez T, Allen M, et al. Human IgG2 antibodies display disulfide mediated structural isoforms. J Biol Chem. 2008;283:16194–16205. doi: 10.1074/jbc.M709987200. [DOI] [PMC free article] [PubMed] [Google Scholar]
-
7.Martinez T, Guo A, Allen MJ, Han M, Pace D, Jones J, et al. Disulfide connectivity of human immunoglobulin G2 structural isoforms. Biochemistry. 2008;47:7496–7508. doi: 10.1021/bi800576c. [DOI] [PubMed] [Google Scholar]
-
8.Dillon TM, Ricci MS, Vezina C, Flynn G, Liu YD, Rehder DS, et al. Structural and functional characterization of disulfide isoforms of the human IgG2 subclass. J Biol Chem. 2008;283:16206–16215. doi: 10.1074/jbc.M709988200. [DOI] [PMC free article] [PubMed] [Google Scholar]
-
9.van der Neut Kolfschoten M, Schuurman J, Losen M, Bleeker WK, Martínez-Martínez P, Vermeulen E, et al. Anti-inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange. Science. 2007;317:1554–1557. doi: 10.1126/science.1144603. [DOI] [PubMed] [Google Scholar]
-
10.Valliere-Douglass JF, Eakin CM, Wallace A, Ketchem RR, Wang W, Treuheit MJ, et al. Glutamine-linked and non-consensus asparagine-linked oligosaccharides present in human recombinant antibodies define novel protein glycosylation motifs. J Biol Chem. 2010;285:16012–16022. doi: 10.1074/jbc.M109.096412. [DOI] [PMC free article] [PubMed] [Google Scholar]
-
11.Younes A, Bartlett NL, Leonard JP, Kennedy DA, Lynch CM, Sievers EL, et al. Brentuximab vedotin (SGN-35) for relapsed CD30-positive lymphomas. N Engl J Med. 2010;363:1812–1821. doi: 10.1056/NEJMoa1002965. [DOI] [PubMed] [Google Scholar]
-
12.Subramanian R, Meares CF. Bifunctional chelating agents for radiometal-labeled monoclonal antibodies. Cancer Treat Res. 1990;51:183–199. doi: 10.1007/978-1-4613-1497-4_9. [DOI] [PubMed] [Google Scholar]
-
13.Sharkey RM, Brenner A, Burton J, Hajjar G, Toder SP, Alavi A, et al. Radioimmunotherapy of non-Hodgkin's lymphoma with 90Y-DOTA humanized anti-CD22 IgG (90Y-Epratuzumab): do tumor targeting and dosimetry predict therapeutic response? J Nucl Med. 2003;44:2000–2018. [PubMed] [Google Scholar]
-
14.Witzig TE, Gordon LI, Cabanillas F, Czuczman MS, Emmanouilides C, Joyce R, et al. Randomized controlled trial of yttrium-90-labeled ibritumomab tiuxetan radioimmunotherapy versus rituximab immunotherapy for patients with relapsed or refractory low-grade, follicular or transformed B-cell non-Hodgkin's lymphoma. J Clin Oncol. 2002;20:2453–2463. doi: 10.1200/JCO.2002.11.076. [DOI] [PubMed] [Google Scholar]
-
15.Gordon LI, Witzig T, Molina A, Czuczman M, Emmanouilides C, Joyce R, et al. Yttrium 90-labeled ibritumomab tiuxetan radioimmunotherapy produces high response rates and durable remissions in patients with previously treated B-cell lymphoma. Clin Lymphoma. 2004;5:98–101. doi: 10.3816/clm.2004.n.015. [DOI] [PubMed] [Google Scholar]
-
16.Wiseman GA, Gordon LI, Multani PS, Witzig TE, Spies S, Bartlett NL, et al. Ibritumomab tiuxetan radioimmunotherapy for patients with relapsed or refractory non-Hodgkin lymphoma and mild thrombocytopenia: a phase II multicenter trial. Blood. 2002;99:4336–4342. doi: 10.1182/blood.v99.12.4336. [DOI] [PubMed] [Google Scholar]
-
17.Witzig TE, White CA, Wiseman GA, Gordon LI, Emmanouilides C, Raubitschek A, et al. Phase I/II trial of IDEC-Y2B8 radioimmunotherapy for treatment of relapsed or refractory CD20(+) B-cell non-Hodgkin's lymphoma. J Clin Oncol. 1999;17:3793–3803. doi: 10.1200/JCO.1999.17.12.3793. [DOI] [PubMed] [Google Scholar]
-
18.Witzig TE, Flinn IW, Gordon LI, Emmanouilides C, Czuczman MS, Saleh MN, et al. Treatment with ibritumomab tiuxetan radioimmunotherapy in patients with rituximab-refractory follicular non-Hodgkin's lymphoma. J Clin Oncol. 2002;20:3262–3269. doi: 10.1200/JCO.2002.11.017. [DOI] [PubMed] [Google Scholar]
-
19.Morschhauser F, Radford J, Van Hoof A, Vitolo U, Soubeyran P, Tilly H, et al. Phase III trial of consolidation therapy with yttrium-90-ibritumomab tiuxetan compared with no additional therapy after first remission in advanced follicular lymphoma. J Clin Oncol. 2008;26:5156–5164. doi: 10.1200/JCO.2008.17.2015. [DOI] [PubMed] [Google Scholar]
-
20.Chatal JF, Davodeau F, Cherel M, Barbet J. Different ways to improve the clinical effectiveness of radioimmunotherapy in solid tumors. J Cancer Res Ther. 2009;5:36–40. doi: 10.4103/0973-1482.55139. [DOI] [PubMed] [Google Scholar]
-
21.Miederer M, Scheinberg DA, McDevitt MR. Realizing the potential of the Actinium-225 radionuclide generator in targeted alpha particle therapy applications. Adv Drug Deliv Rev. 2008;60:1371–1382. doi: 10.1016/j.addr.2008.04.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
-
22.Reardan DT, Meares CF, Goodwin DA, McTigue M, David GS, Stone MR, et al. Antibodies against metal chelates. Nature. 1985;316:265–268. doi: 10.1038/316265a0. [DOI] [PubMed] [Google Scholar]
-
23.Goodwin DA, Meares CF, David GF, McTigue M, McCall MJ, Frincke JM, et al. Monoclonal antibodies as reversible equilibrium carriers of radiopharmaceuticals. Int J Rad Appl Instrum B. 1986;13:383–391. doi: 10.1016/0883-2897(86)90015-2. [DOI] [PubMed] [Google Scholar]
-
24.Goodwin DA, Mears CF, McTigue M, David GS. Monoclonal antibody hapten radiopharmaceutical delivery. Nucl Med Commun. 1986;7:569–580. doi: 10.1097/00006231-198608000-00002. [DOI] [PubMed] [Google Scholar]
-
25.Goodwin DA, Meares CF, McCall MJ, McTigue M, Chaovapong W. Pre-targeted immunoscintigraphy of murine tumors with indium-111-labeled bifunctional haptens. J Nucl Med. 1988;29:226–234. [PubMed] [Google Scholar]
-
26.Sharkey RM, Goldenberg DM. Use of antibodies and immunoconjugates for the therapy of more accessible cancers. Adv Drug Deliv Rev. 2008;60:1407–1420. doi: 10.1016/j.addr.2008.04.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
-
27.Goodwin DA, Meares CF. Advances in pretargeting biotechnology. Biotechnol Adv. 2001;19:435–450. doi: 10.1016/s0734-9750(01)00065-9. [DOI] [PubMed] [Google Scholar]
-
28.Goldenberg DM, Sharkey RM, Paganelli G, Barbet J, Chatal JF. Antibody pretargeting advances cancer radioimmunodetection and radioimmunotherapy. J Clin Oncol. 2006;24:823–834. doi: 10.1200/JCO.2005.03.8471. [DOI] [PubMed] [Google Scholar]
-
29.Kraeber-Bodéré F, Rousseau C, Bodet-Milin C, Ferrer L, Faivre-Chauvet A, Campion L, et al. Targeting, toxicity and efficacy of 2-step, pretargeted radioimmunotherapy using a chimeric bispecific antibody and 131I-labeled bivalent hapten in a phase I optimization clinical trial. J Nucl Med. 2006;47:247–255. [PubMed] [Google Scholar]
-
30.Kraeber-Bodéré F, Bardet S, Hoefnagel CA, Vieira MR, Vuillez JP, Murat A, et al. Radioimmunotherapy in medullary thyroid cancer using bispecific antibody and iodine 131-labeled bivalent hapten: preliminary results of a phase I/II clinical trial. Clin Cancer Res. 1999;5:3190–3198. [PubMed] [Google Scholar]
-
31.Chatal JF, Campion L, Kraeber-Bodéré F, Bardet S, Vuillez JP, Charbonnel B, et al. Survival improvement in patients with medullary thyroid carcinoma who undergo pretargeted anti-carcinoembryonic-antigen radioimmunotherapy: a collaborative study with the French Endocrine Tumor Group. J Clin Oncol. 2006;24:1705–1711. doi: 10.1200/JCO.2005.04.4917. [DOI] [PubMed] [Google Scholar]
-
32.Coloma MJ, Morrison SL. Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol. 1997;15:159–163. doi: 10.1038/nbt0297-159. [DOI] [PubMed] [Google Scholar]
-
33.Miller BR, Demarest SJ, Lugovskoy A, Huang F, Wu X, Snyder WB, et al. Stability engineering of scFvs for the development of bispecific and multivalent antibodies. Protein Eng Des Sel. 2010;23:549–557. doi: 10.1093/protein/gzq028. [DOI] [PubMed] [Google Scholar]
-
34.Dong J, Sereno A, Snyder WB, Miller BR, Tamraz S, Doern A, et al. Stable IgG-like bispecific antibodies directed towards the type I insulin-like growth factor receptor demonstrate enhanced ligand blockade and anti-tumor activity. J Biol Chem. 2011;286:4703–4717. doi: 10.1074/jbc.M110.184317. [DOI] [PMC free article] [PubMed] [Google Scholar]
-
35.Michaelson JS, Demarest SJ, Miller B, Amatucci A, Snyder WB, Wu X, et al. Anti-tumor activity of stability-engineered IgG-like bispecific antibodies targeting TRAIL-R2 and LTbetaR. mAbs. 2009;1:128–141. doi: 10.4161/mabs.1.2.7631. [DOI] [PMC free article] [PubMed] [Google Scholar]
-
36.Wu C, Ying H, Grinnell C, Bryant S, Miller R, Clabbers A, et al. Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin. Nat Biotechnol. 2007;25:1290–1297. doi: 10.1038/nbt1345. [DOI] [PubMed] [Google Scholar]
-
37.Wu C, Ying H, Bose S, Miller R, Medina L, Santora L, Ghayur T. Molecular construction and optimization of anti-human IL-1alpha/beta dual variable domain immunoglobulin (DVD-Ig) molecules. mAbs. 2009;1:339–347. doi: 10.4161/mabs.1.4.8755. [DOI] [PMC free article] [PubMed] [Google Scholar]
-
38.Kou G, Shi J, Chen L, Zhang D, Hou S, Zhao L, et al. A bispecific antibody effectively inhibits tumor growth and metastasis by simultaneous blocking vascular endothelial growth factor A and osteopontin. Cancer Lett. 2010;299:130–136. doi: 10.1016/j.canlet.2010.08.011. [DOI] [PubMed] [Google Scholar]
-
39.Dieterich HJ, Kraft D, Sirtl C, Laubenthal H, Schimetta W, Pölz W, et al. Hydroxyethyl starch antibodies in humans: incidence and clinical relevance. Anesth Analg. 1998;86:1123–1126. doi: 10.1097/00000539-199805000-00041. [DOI] [PubMed] [Google Scholar]
-
40.Wilson MR, Goddard ME, O'Dea KP, Choudhury S, Takata M. Differential roles of p55 and p75 tumor necrosis factor receptors on stretch-induced pulmonary edema in mice. Am J Physiol Lung Cell Mol Physiol. 2007;293:60–68. doi: 10.1152/ajplung.00284.2006. [DOI] [PubMed] [Google Scholar]
-
41.Greenberg AS, Avila D, Hughes M, Hughes A, McKinney EC, Flajnik MF. A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks. Nature. 1995;374:168–173. doi: 10.1038/374168a0. [DOI] [PubMed] [Google Scholar]
-
42.Nuttall SD, Krishnan UV, Doughty L, Pearson K, Ryan MT, Hoogenraad NJ, et al. Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70. Eur J Biochem. 2003;270:3543–3554. doi: 10.1046/j.1432-1033.2003.03737.x. [DOI] [PubMed] [Google Scholar]
-
43.Johnson G, Wu TT. Preferred CDRH3 lengths for antibodies with defined specificities. Int Immunol. 1998;10:1801–1805. doi: 10.1093/intimm/10.12.1801. [DOI] [PubMed] [Google Scholar]
-
44.Nuttall SD, Humberstone KS, Krishnan UV, Carmichael JA, Doughty L, Hattarki M, et al. Selection and affinity maturation of IgNAR variable domains targeting Plasmodium falciparum AMA1. Proteins. 2004;55:187–197. doi: 10.1002/prot.20005. [DOI] [PubMed] [Google Scholar]
-
45.Streltsov VA, Varghese JN, Carmichael JA, Irving RA, Hudson PJ, Nuttall SD. Structural evidence for evolution of shark Ig new antigen receptor variable domain antibodies from a cell-surface receptor. Proc Natl Acad Sci USA. 2004;101:12444–12449. doi: 10.1073/pnas.0403509101. [DOI] [PMC free article] [PubMed] [Google Scholar]
-
46.Dooley H, Flajnik MF, Porter AJ. Selection and characterization of naturally occurring single-domain (IgNAR) antibody fragments from immunized sharks by phage display. Mol Immunol. 2003;40:25–33. doi: 10.1016/s0161-5890(03)00084-1. [DOI] [PubMed] [Google Scholar]
-
47.Ferroni P, Formica V, Roselli M, Guadagni F. Thromboembolic events in patients treated with anti-angiogneic drugs. Curr Vasc Pharmacol. 2010;8:102–113. doi: 10.2174/157016110790226660. [DOI] [PubMed] [Google Scholar]
