Robert E. Jordan

Pharmacodynamic Profile of Short-term Abciximab Treatment Demonstrates Prolonged Platelet Inhibition With Gradual Recovery From GP IIb/IIIa Receptor Blockade

BACKGROUNDnThe glycoprotein (GP) IIb/IIIa receptor antagonist abciximab is approved for use in high-risk percutaneous coronary interventions. The purpose of the present study was to establish the pharmacodynamic profile and platelet-bound life span of abciximab.nnnMETHODS AND RESULTSnThe pharmacodynamics of abciximab (inhibition of ex vivo platelet aggregation and GP IIb/IIIa receptor blockade) were measured in 41 individuals who were randomized to receive a 0.25-mg/kg bolus and a 12-hour infusion of either 10 microg/min (EPIC regimen) or 0.125 microg x kg(-1) x min(-1) (EPILOG regimen) of the antiplatelet agent. At extended times, the amount and distribution of platelet-bound abciximab were monitored by flow cytometry. The EPIC and EPILOG infusion regimens exhibited equivalent blockade of both GP IIb/IIIa receptors and platelet aggregation throughout the duration of abciximab treatment. Flow cytometry revealed a single, highly fluorescent platelet population during treatment, consistent with complete saturation and homogeneous distribution of abciximab on circulating platelets. For 15 days after treatment, the fluorescence histograms remained unimodal with gradually diminishing fluorescence intensity, indicating decreasing levels of platelet-bound abciximab. At 8 and 15 days, which exceeds the normal circulating life span of platelets, median relative fluorescence intensity corresponded to 29100 (29% GP IIb/IIIa receptor blockade) and 13300 (13% GP IIb/IIIa receptor blockade) abciximab molecules bound per platelet, respectively.nnnCONCLUSIONSnThese results are consistent with continuous reequilibration of abciximab among circulating platelets and may explain the gradual recovery of platelet function and long-term prevention of ischemic complications by abciximab after coronary intervention.

Abciximab (ReoPro, Chimeric 7E3 Fab) Demonstrates Equivalent Affinity and Functional Blockade of Glycoprotein IIb/IIIa and αvβ3 Integrins

Background—Large, randomized, and blinded clinical trials (EPIC, EPILOG, and CAPTURE) have demonstrated that abciximab (ReoPro, chimeric 7E3 Fab) markedly reduces thrombotic events associated with percutaneous transluminal coronary interventions. The marked early benefits at 30 days were sustained at 6 months and 3 years. Initially developed because of its efficacy in blocking GP IIb/IIIa (αIIb/β3) receptors on platelets, abciximab also binds with equivalent affinity to αvβ3. Methods and Results—This study presents a detailed characterization of the αvβ3 interaction, including the ability of abciximab to (1) bind with comparable affinity to αvβ3 and GP IIb/IIIa, (2) inhibit αvβ3 and GP IIb/IIIa–mediated cell adhesion in vitro with IC50 values approximating binding KD values, and (3) redistribute between GP IIb/IIIa and αvβ3 integrins in vitro. Conclusions—As an antagonist of not only GP IIb/IIIa but also αvβ3, abciximab may provide additional clinical benefit in preventing αvβ3-mediated effects such as th...

Differential inhibition of platelet aggregation induced by adenosine diphosphate or a thrombin receptor-activating peptide in patients treated with bolus chimeric 7E3 Fab: Implications for inhibition of the internal pool of GPIIb/IIIa receptors

OBJECTIVESnThis study sought to describe in detail the pharmacokinetics and pharmacodynamics of chimeric monoclonal 7E3 Fab (c7E3 Fab) and to compare platelet responses to adenosine diphosphate (ADP) and the 11-amino acid thrombin receptor-activating peptide (TRAP [SFLLRNPNDKY-NH2]) in patients undergoing elective coronary angioplasty.nnnBACKGROUNDnInhibition of platelet aggregation with monoclonal antibody c7E3 Fab directed against glycoprotein (GP) IIb/IIIa has been shown to reduce ischemic complications after angioplasty and is being considered for treatment of other acute ischemic syndromes.nnnMETHODSnPatients undergoing elective coronary angioplasty received aspirin (325 mg orally), heparin (12,000 U intravenously) and a bolus of c7E3 Fab (0.25 mg/kg body weight). Surface GPIIb/IIIa receptor blockade and aggregation in response to 20 mumol/liter ADP, 5 micrograms/ml collagen and 7.5 and 15 mumol/liter TRAP were assessed.nnnRESULTSnSurface GPIIb/IIIa receptor blockade by c7E3 Fab was 80% 2 h after injection and decreased to 50% at 24 h. Platelet aggregation in response to 20 mumol/liter ADP was inhibited by 73% at 2 h, and this inhibition decreased to 27% at 24 h. Platelet aggregation in response to 7.5 mumol/liter TRAP was inhibited by 53% at 2 h and 30% at 24 h. In contrast, aggregation in response to 15 mumol/liter TRAP was inhibited only 37% at 2 h and 10% at 24 h (p < 0.001 and p = 0.006, respectively vs. 20 mumol/liter ADP). Addition of exogenous c7E3 Fab to platelet-rich plasma led to more complete inhibition of 7.5 mumol/liter TRAP-induced aggregation.nnnCONCLUSIONSnAfter c7E3 Fab treatment, inhibition of platelet aggregation depends on the agonist and can be overcome by increased thrombin activity but is restored if additional c7E3 Fab is added to block additional GPIIb/IIIa receptors. This phenomenon may be related to an internal pool of GPIIb/IIIa receptors joining the surface membrane and has implications concerning the duration of therapy with c7E3 Fab for patients with unstable angina or acute myocardial infarction.

Rapid assessment of platelet function with a modified whole-blood aggregometer in percutaneous transluminal coronary angioplasty patients receiving anti-GP IIb/IIIa therapy.

BACKGROUNDnThe glycoprotein (GP) IIb/IIIa receptor antagonist abciximab (c7E3 Fab, ReoPro) is approved for use in high-risk percutaneous transluminal coronary angioplasty (PTCA). At present, no point of care exists for measuring pharmacological GP IIb/IIIa blockade. To address this need, the Chrono-log Whole Blood Aggregometer, which measures platelet aggregation by electrical impedance, was adapted to test platelet function at the bedside.nnnMETHODS AND RESULTSnGP IIb/IIIa receptor blockade, impedance (5 microg/mL collagen), and turbidimetric aggregation (5 and 20 micromol/L ADP) measurements were obtained on 14 PTCA patients who received the standard bolus plus a 12-hour infusion of abciximab. During abciximab administration, mean GP IIb/IIIa receptor blockade was > 91%, and both impedance and turbidimetric aggregation were inhibited by > or = 90%. At 12 hours after abciximab treatment, the mean inhibition of turbidimetric platelet aggregation to 5 and 20 micromol/L ADP was 65+/-20% and 49+/-14%, respectively, and inhibition of impedance aggregation was 69+/-12%. GP IIb/IIIa receptor blockade was 67+/-8%. At 36 hours after abciximab treatment (n=8), the mean inhibition of turbidimetric platelet aggregation to 5 and 20 micromol/L ADP was 44+/-21% and 30+/-14%, respectively, whereas impedance aggregation was inhibited by 60+/-14%. GP IIb/IIIa receptor blockade was 57+/-7%.nnnCONCLUSIONSnDuring and at 12 hours after abciximab therapy, impedance and turbidimetric platelet aggregation to 5 micromol/L ADP were comparable and closely correlated with GP IIb/IIIa receptor blockade. However, at 36 hours after abciximab treatment, impedance platelet aggregation more closely paralleled GP IIb/IIIa receptor blockade and indicated a slower recovery of platelet function than turbidimetric aggregometry.

The immunogenicity of the 7E3 murine monoclonal Fab antibody fragment variable region is dramatically reduced in humans by substitution of human for murine constant regions

A murine monoclonal antibody (7E3) directed against the platelet glycoprotein IIb/IIIa was engineered to reduce immunogenicity by substituting human for murine constant regions. The chimeric antibody is functionally identical to the murine antibody in vitro. Results from clinical trials with 7E3 Fab antibody fragments, however, show that the 7E3 variable region, which elicits the vast majority of the immune response to murine 7E3 Fab, is rendered dramatically less immunogenic (incidence reduced from 17% to 1%) when the identical variable region is linked to human rather than murine constant regions. Neither murine nor human constant regions were highly immunogenic themselves. We conclude that the constant regions of the Fab fragments are critical in modulating the immune response elicited by the linked 7E3 variable region. Because naturally occurring anti-human Fab fragment antibodies are prevalent both in the normal human population and in the patient population studied here, murine 7E3 Fab and chimeric 7E3 Fab may be fundamentally different in their interactions with the human immune system. This difference may be related to the dramatic difference in immunogenicity observed between murine 7E3 Fab and chimeric 7E3 Fab.

Cleavage of IgGs by proteases associated with invasive diseases

The effective functioning of immunoglobulins and IgG mAbs in removing pathological cells requires that the antigen binding regions and the Fc (effector) domain act in concert. The hinge region that connects these domains itself presents motifs that engage Fc receptors on immune effector cells to achieve cell lysis. In addition, sequences in the lower hinge/CH2 and further down the CH2 region are involved in C1q binding and complement-mediated cell killing. Proteolytic enzymes of little relevance to human physiology were successfully used for decades to generate fragments of IgGs for reagent and therapeutic use. It was subsequently noted that tumor-related and microbial proteases also cleaved human IgG specifically in the hinge region. We have shown previously that the “nick” of just one of the lower hinge heavy chains of IgG unexpectedly prevented many effector functions without impacting antigen binding. Of interest, related single-cleaved IgG breakdown products were detected in breast carcinoma extracts. This suggested a pathway by which tumors might avoid host immune surveillance under a cloak of proteolytically-generated, dysfunctional antibodies that block competent IgG binding. The host immune system cannot be blind to this pathway since there exists a widespread, low-titer incidence of anti-hinge (cleavage-site) antibodies in the healthy population. The prevalence of anti-hinge reactivity may reflect an ongoing immune recognition of normal IgG catabolism. Tumor growth and bacterial infections potentially generate hostile proteolytic environments that may pose harsh challenges to host immunity. Recent findings involving physiologically-relevant proteases suggest that the potential loss of key effector functions of host IgGs may result from subtle and limited proteolytic cleavage of IgGs, and that such events may facilitate the incursion of invasive cells in local proteolytic settings.

Galactosylation variations in marketed therapeutic antibodies

There are currently ~25 recombinant full-length IgGs (rIgGs) in the market that have been approved by regulatory agencies as biotherapeutics to treat various human diseases. Most of these are based on IgG1k framework and are either chimeric, humanized or human antibodies manufactured using either Chinese hamster ovary (CHO) cells or mouse myeloma cells as the expression system. Because CHO and mouse myeloma cells are mammalian cells, rIgGs produced in these cell lines are typically N-glycosylated at the conserved asparagine (Asn) residues in the CH2 domain of the Fc, which is also the case with serum IgGs. The Fc glycans present in these rIgGs are for the most part complex biantennary oligosaccharides with heterogeneity associated with the presence or the absence of several different terminal sugars. The major Fc glycans of rIgGs contain 0 or 1 or 2 (G0, G1 and G2, respectively) terminal galactose residues as non-reducing termini and their relative proportions may vary depending on the cell culture conditions in which they were expressed. Since glycosylation is strongly associated with antibody effector functions and terminal galactosylation may affect some of those functions, a panel of commercially available therapeutic rIgGs expressed in CHO cells and mouse myeloma cells were examined for their galactosylation patterns. The results suggest that the rIgGs expressed in CHO cells are generally less galactosylated compared to the rIgGs expressed in mouse myeloma cells. Accordingly, rIgGs produced in CHO cells tend to contain higher levels of G0 glycans compared with rIgGs produced in mouse myeloma cell lines. Despite the apparent wide variability in galactose content, adverse events or safety issues have not been associated with specific galactosylation patterns of therapeutic antibodies. Nevertheless, galactosylation may have an effect on the mechanisms of action of some therapeutic antibodies (e.g., effector pathways) and hence further studies to assess effects on product efficacy may be warranted for such antibodies. For antibodies that do not require effector functions for biological activity, however, setting a narrow specification range for galactose content may be unnecessary.

Tumor-Associated Macrophages Promote Invasion while Retaining Fc-Dependent Anti-Tumor Function

Tumor-associated macrophages (TAMs) have been shown to promote tumor progression, and increased TAM infiltration often correlates with poor prognosis. However, questions remain regarding the phenotype of macrophages within the tumor and their role in mAb-dependent cytotoxicity. This study demonstrates that whereas TAMs have protumor properties, they maintain Fc-dependent anti-tumor function. CD11b+CD14+ TAMs isolated from primary human breast tumors expressed activating FcγRs. To model breast cancer TAMs in vitro, conditioned medium from breast cancer cells was used to drive human peripheral monocyte differentiation into macrophages. Tumor-conditioned macrophages were compared with in vitro derived M1 and M2a macrophages and were found to promote tumor cell invasion and express M2a markers, confirming their protumor potential. However, unlike M2a macrophages, tumor-conditioned macrophages expressed FcγRs and phagocytosed tumor cells in the presence of a tumor Ag-targeting mAb, unmasking an underappreciated tumoricidal capacity of TAMs. In vivo macrophage depletion reduced the efficacy of anti-CD142 against MDA-MB-231 xenograft growth and metastasis in SCID/beige mice, implicating a critical role for macrophages in Fc-dependent cell killing. M-CSF was identified in tumor-conditioned media and shown to be capable of differentiating macrophages with both pro- and anti-tumor properties. These results highlight the plasticity of TAMs, which are capable of promoting tumor progression and invasion while still retaining tumoricidal function in the presence of tumor-targeting mAbs.

Tumor-associated and microbial proteases compromise host IgG effector functions by a single cleavage proximal to the hinge.

The successful elimination of pathogenic cells and microorganisms by the humoral immune system relies on effective interactions between host immunoglobulins and Fcγ receptors on effector cells, in addition to the complement system. Essential Ig motifs that direct those interactions reside within the conserved IgG lower hinge/CH2 interface. We noted that a group of tumor-related and microbial proteases cleaved human IgG1s in that region, and the “nick” of just one of the heavy chains profoundly inhibited IgG1 effector functions. We focused on IgG1 monoclonal antibodies (mAbs) since IgG1 is the most abundant human subclass and demonstrates robust Fc-mediated effector functions. The loss of Fc-mediated cell killing activities was correlated with diminished binding to the Fcγ family of receptors, but a similar decrease in affinity was not observed toward the FcRn receptor that maintains IgG in circulation. Endogenous human IgG cleavage products of comparable size to mAbs with the single cleavage were detected by Western blot analysis in synovial fluid from patients with rheumatoid arthritis and in breast carcinoma extracts. Their detection is problematic under physiological conditions, since there is no loss of structure, and antigen-binding capability is unaffected. These findings suggest that within the hostile proteolytic microenvironments associated with many diseases, key effector functions of host IgGs, or therapeutic Abs, may be compromised.

A single proteolytic cleavage within the lower hinge of trastuzumab reduces immune effector function and in vivo efficacy

IntroductionRecent studies reported that human IgG antibodies are susceptible to specific proteolytic cleavage in their lower hinge region, and the hinge cleavage results in a loss of Fc-mediated effector functions. Trastuzumab is a humanized IgG1 therapeutic monoclonal antibody for the treatment of HER2-overexpressing breast cancers, and its mechanisms of action consist of inhibition of HER2 signaling and Fc-mediated antibody-dependent cellular cytotoxicity (ADCC). The objective of this study is to investigate the potential effect of proteinase hinge cleavage on the efficacy of trastuzumab using both a breast cancer cell culture method and an in vivo mouse xenograft tumor model.MethodsTrastuzumab antibody was incubated with a panel of human matrix metalloproteinases, and proteolytic cleavage in the lower hinge region was detected using both western blotting and mass spectrometry. Single hinge cleaved trastuzumab (scIgG-T) was purified and evaluated for its ability to mediate ADCC and inhibition of breast cancer cell proliferation in vitro as well as anti-tumor efficacy in the mouse xenograft tumor model. Infiltrated immune cells were detected in tumor tissues by immunohistochemistry.ResultsscIgG-T retains HER2 antigen binding activity and inhibits HER2-mediated downstream signaling and cell proliferation in vitro when compared with the intact trastuzumab. However, scIgG-T lost Fc-mediated ADCC activity in vitro, and had significantly reduced anti-tumor efficacy in a mouse xenograft tumor model. Immunohistochemistry showed reduced immune cell infiltration in tumor tissues treated with scIgG-T when compared with those treated with the intact trastuzumab, which is consistent with the decreased ADCC mediated by scIgG-T in vitro.ConclusionTrastuzumab can be cleaved by matrix metalloproteinases within the lower hinge. scIgG-T exhibited a significantly reduced anti-tumor efficacy in vivo due to the weakened immune effector function such as ADCC. The results suggest that the lower hinge cleavage of trastuzumab can occur in the tumor microenvironment where matrix metalloproteinases often have high levels of expression and scIgG-T might compromise its anti-tumor efficacy in the clinic. However, further studies are needed to validate these hypotheses in the clinical setting.