Mitchell E Reff

Elimination of Fc receptor-dependent effector functions of a modified IgG4 monoclonal antibody to human CD4.

Several CD4 mAbs have entered the clinic for the treatment of autoimmune diseases or transplant rejection. Most of these mAbs caused CD4 cell depletion, and some were murine mAbs which were further hampered by human anti-mouse Ab responses. To obviate these concerns, a primatized CD4 mAb, clenoliximab, was generated by fusing the V domains of a cynomolgus macaque mAb to human constant regions. The heavy chain constant region is a modified IgG4 containing two single residue substitutions designed to ablate residual Fc receptor binding activity and to stabilize heavy chain dimer formation. This study compares and contrasts the in vitro properties of clenoliximab with its matched IgG1 derivative, keliximab, which shares the same variable regions. Both mAbs show potent inhibition of in vitro T cell responses, lack of binding to complement component C1q, and inability to mediate complement-dependent cytotoxicity. However, clenoliximab shows markedly reduced binding to Fc receptors and therefore does not mediate Ab-dependent cell-mediated cytotoxicity or modulation/loss of CD4 from the surface of T cells, except in the presence of rheumatoid factor or activated monocytes. Thus, clenoliximab retains the key immunomodulatory attributes of keliximab without the liability of strong Fcγ receptor binding. In initial clinical trials, these properties have translated to a reduced incidence of CD4+ T cell depletion.

A review of modifications to recombinant antibodies: attempt to increase efficacy in oncology applications

Although monoclonal antibodies have high specificity, their usefulness in the clinic, especially against solid tumors, has been limited. This arises in part from the inability of antibody molecules to penetrate into the tumor and kill the tumor cells. In addition, natural cytotoxic effects of antibodies, mediated through complement or Fc receptors, may not be sufficient to kill malignant cells. This review will present some of the antibody modifications used to increase efficacy. Modified recombinant antibodies have been designed to be more cytotoxic (immunotoxins), to increase natural effector functions (bivalent antibodies, antibody-fusion molecules, multimeric antibodies, directed mutations in Fc region), or to pretarget cells for concentration of cytotoxic drugs. This review will also focus on engineering of smaller versions of antibodies that retain specificity (single chain Fvs, Fabs, Fab(2)s, minibodies, domain deleted antibodies) and have increased penetrability of solid tumors. Many of these antibody modifications may result in antigenic compounds which can limit repeat administration. Clinical experiences will be highlighted if information is available.

Future of monoclonal antibodies in the treatment of hematologic malignancies

BACKGROUND The approval of monoclonal antibodies (MAbs) as antibody-targeted therapy in the management of patients with hematologic malignancies has led to new treatment options for this group of patients. The ability to target antibodies to novel functional receptors can increase their therapeutic efficacy. METHODS The authors reviewed improvements in MAb design to enhance their effectiveness over the existing therapeutic MAb currently approved for treating hematologic malignancies. RESULTS Three classes of therapeutic MAbs showing promise in human clinical trials for treatment of hematologic malignancies include unconjugated MAb, drug conjugates in which the antibody preferentially delivers a potent cytotoxic drug to the tumor, and radioactive immunotherapy in which the antibody delivers a sterilizing dose of radiation to the tumor. CONCLUSIONS A better appreciation of how MAbs are metabolized in the body and localized to tumors is resulting in the development of new antibody constructs with improved biodistribution profiles.

A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity

The epidermal growth factor receptor (EGFR) and the type I insulin-like growth factor receptor (IGF-1R) are two cell surface receptor tyrosine kinases known to cooperate to promote tumor progression and drug resistance. Combined blockade of EGFR and IGF-1R has shown improved anti-tumor activity in preclinical models. Here, we report the characterization of a stable IgG-like bispecific antibody (BsAb) dual-targeting EGFR and IGF-1R that was developed for cancer therapy. The BsAb molecule (EI-04), constructed with a stability-engineered single chain variable fragment (scFv) against IGF-1R attached to the carboxyl-terminus of an IgG against EGFR, displays favorable biophysical properties for biopharmaceutical development. Biochemically, EI-04 bound to human EGFR and IGF-1R with sub nanomolar affinity, co-engaged the two receptors simultaneously, and blocked the binding of their respective ligands with similar potency compared to the parental monoclonal antibodies (mAbs). In tumor cells, EI-04 effectively inhibited EGFR and IGF-1R phosphorylation, and concurrently blocked downstream AKT and ERK activation, resulting in greater inhibition of tumor cell growth and cell cycle progression than the single mAbs. EI-04, likely due to its tetravalent bispecific format, exhibited high avidity binding to BxPC3 tumor cells co-expressing EGFR and IGF-1R, and consequently improved potency at inhibiting IGF-driven cell growth over the mAb combination. Importantly, EI-04 demonstrated enhanced in vivo anti-tumor efficacy over the parental mAbs in two xenograft models, and even over the mAb combination in the BxPC3 model. Our data support the clinical investigation of EI-04 as a superior cancer therapeutic in treating EGFR and IGF-1R pathway responsive tumors.

Combination of Two Insulin-Like Growth Factor-I Receptor Inhibitory Antibodies Targeting Distinct Epitopes Leads to an Enhanced Antitumor Response

The insulin-like growth factor-I receptor (IGF-IR) is a cell surface receptor tyrosine kinase that mediates cell survival signaling and supports tumor progression in multiple tumor types. We identified a spectrum of inhibitory IGF-IR antibodies with diverse binding epitopes and ligand-blocking properties. By binding distinct inhibitory epitopes, two of these antibodies, BIIB4 and BIIB5, block both IGF-I and IGF-II binding to IGF-IR using competitive and allosteric mechanisms, respectively. Here, we explored the inhibitory effects of combining BIIB4 and BIIB5. In biochemical assays, the combination of BIIB4 and BIIB5 improved both the potency and extent of IGF-I and IGF-II blockade compared with either antibody alone. In tumor cells, the combination of BIIB4 and BIIB5 accelerated IGF-IR downregulation and more efficiently inhibited IGF-IR activation as well as downstream signaling, particularly AKT phosphorylation. In several carcinoma cell lines, the antibody combination more effectively inhibited ligand-driven cell growth than either BIIB4 or BIIB5 alone. Notably, the enhanced tumor growth–inhibitory activity of the BIIB4 and BIIB5 combination was much more pronounced at high ligand concentrations, where the individual antibodies exhibited substantially reduced activity. Compared with single antibodies, the BIIB4 and BIIB5 combination also significantly further enhanced the antitumor activity of the epidermal growth factor receptor inhibitor erlotinib and the mTOR inhibitor rapamycin. Moreover, in osteosarcoma and hepatocellular carcinoma xenograft models, the BIIB4 and BIIB5 combination significantly reduced tumor growth to a greater degree than each single antibody. Taken together, our results suggest that targeting multiple distinct inhibitory epitopes on IGF-IR may be a more effective strategy of affecting the IGF-IR pathway in cancer. Mol Cancer Ther; 9(9); 2593–604. ©2010 AACR.

Characterization of inhibitory anti-insulin-like growth factor receptor antibodies with different epitope specificity and ligand-blocking properties: implications for mechanism of action in vivo.

Therapeutic antibodies directed against the type 1 insulin-like growth factor receptor (IGF-1R) have recently gained significant momentum in the clinic because of preliminary data generated in human patients with cancer. These antibodies inhibit ligand-mediated activation of IGF-1R and the resulting down-stream signaling cascade. Here we generated a panel of antibodies against IGF-1R and screened them for their ability to block the binding of both IGF-1 and IGF-2 at escalating ligand concentrations (>1 μm) to investigate allosteric versus competitive blocking mechanisms. Four distinct inhibitory classes were found as follows: 1) allosteric IGF-1 blockers, 2) allosteric IGF-2 blockers, 3) allosteric IGF-1 and IGF-2 blockers, and 4) competitive IGF-1 and IGF-2 blockers. The epitopes of representative antibodies from each of these classes were mapped using a purified IGF-1R library containing 64 mutations. Most of these antibodies bound overlapping surfaces on the cysteine-rich repeat and L2 domains. One class of allosteric IGF-1 and IGF-2 blocker was identified that bound a separate epitope on the outer surface of the FnIII-1 domain. Using various biophysical techniques, we show that the dual IGF blockers inhibit ligand binding using a spectrum of mechanisms ranging from highly allosteric to purely competitive. Binding of IGF-1 or the inhibitory antibodies was associated with conformational changes in IGF-1R, linked to the ordering of dynamic or unstructured regions of the receptor. These results suggest IGF-1R uses disorder/order within its polypeptide sequence to regulate its activity. Interestingly, the activity of representative allosteric and competitive inhibitors on H322M tumor cell growth in vitro was reflective of their individual ligand-blocking properties. Many of the antibodies in the clinic likely adopt one of the inhibitory mechanisms described here, and the outcome of future clinical studies may reveal whether a particular inhibitory mechanism leads to optimal clinical efficacy.

Chromosome localization and gene-copy-number quantification of three random integrations in Chinese-hamster ovary cells and their amplified cell lines using fluorescence in situ hybridization

We have used fluorescence in situ hybridization (FISH) to localize three random Chinese‐hamster ovary (CHO) cell chromosomal integration sites and determine gene copy number in their amplified cell lines. Metaphase FISH showed all three to have integrated into different chromosome positions on different chromosomes. All three Geneticin parent cell lines were found to have single integration sites by Southern‐blot analysis, and these data were confirmed using both metaphase and interphase FISH. Following amplification, metaphase FISH showed that amplification in two of the cell lines occurred by duplication in the chromosome arm where the integration occurred. However, for one cell line, amplification resulted in the translocation of amplified genes from one marker chromosome to another. Interphase FISH showed an expected increase in gene copy number upon amplification with methotrexate.

Measurement of protein interaction bioenergetics: Application to structural variants of anti-sCD4 antibody

This chapter has described a bioenergetic analysis of the interaction of sCD4 with an IgG1 and two IgG4 derivatives of an anti-sCD4 MAb. The MAbs have identical VH and VL domains but differ markedly in their CH and CL domains, raising the question of whether their antigen-binding chemistries are altered. We find the sCD4-binding kinetics and thermodynamics of the MAbs are indistinguishable, which indicates rigorously that the molecular details of the binding interactions are the same. We also showed the importance of using multiple biophysical methods to define the binding model before the bioenergetics can be appropriately interpreted. Analysis of the binding thermodynamics and kinetics suggests conformational changes that might be coupled to sCD4 binding by these MAbs are small or absent.

Expression and characterisation of finger protease (FP); a mutant tissue-type plasminogen activator (t-PA) with improved pharmacokinetics

Summary We have constructed, expressed and purified a novel thrombolytic, finger protease (FP), composed of only the finger and serine protease domains of t-PA. This molecule, when compared to t-PA, has improved pharmacokinetics (slower plasma clearance and a longer half-life) in both the rat and rabbit. It is half as potent as t-PA in a rabbit model of peripheral arterial thrombosis. At efficacious doses, FP produced larger changes in haemostatic parameters than those produced by t-PA. In vitro studies of the purified protein showed weak fibrin binding, and low specific activity of FP in both the S-2251 and human clot lysis assays. The behavior of this molecule suggests that novel fibrinolytics with improved pharmacokinetics and equivalent in vitro activity to t-PA will be more potent in vivo.

Inhibiting Apoptosis in Cell Culture Using Multiple Inhibitors

Mammalian cell culture is widely used for the production of therapeutic proteins and for the generation of cells for cell and gene therapies. During cell culture, these cells can be exposed to a number of external and internal insults including nutrient depletion, toxin accumulation, viral infections, and external shear stress among others. These events can trigger the biochemical cascade called apoptosis in which the cells actively participate in their own demise. This program cell death (PCD) cascade decreases the viable cells in a bioreactor and lowers productivity since valuable bioreactor resources are needed to replace dead or dying cells rather than being applied to generate target proteins and cells. As a result, methodologies that limit the apoptosis pathway are desirable and represent a major application of cell engineering for mammalian culture. A number of natural anti-apoptosis genes have been identified in both eucaryotes and viruses that inhibit apoptosis at both upstream and downstream points in the apoptosis cascade. Anti-apoptosis proteins may be used to prevent the loss of mitochondrial membrane integrity or block cellular caspases from degrading the cell. Apoptosis pathways include feedback and feedforward loops that lead to amplification of the apoptotic response. Therefore, strategies that block cell death at multiple points along the cascade may limit the amplification of these apoptosis signals. As a result, combinatorial strategies that include anti-apoptosis proteins blocking apoptosis at different steps in the cascade are being implemented. In particular, the inhibitors of mitochondrial activity are being combined with proteins that block caspase activation. In this way, it may be possible to extend mammalian cell lifetimes and function for multiple biotechnology and bioengineering applications.