Anti-CD18 mAb Genentech/Roche William M Brown

953

Anti-CD18 mAb Genentech/Roche William M Brown Address Taro Pharmaceuticals USA Inc Hawthorne NY USA Email: [email protected]

Licensee Roche Holding AG

IDrugs 2000 3(8):953-960 © Current Drugs Ltd ISSN 1369-7056

Indication Hemorrhagic shock, Myocardial infarction, Reperfusion injury

Genentech and Roche are collaborating on the development of an anti-CD18 humanized monoclonal antibody (rhuMAb). Inhibition of leukocyte adhesion with antibodies to CD18 is proposed as a therapeutic intervention for indications with an inflammatory component resulting from a reperfusion injury. In June 2000, the company reported that the primary objectives of its phase II trial of the antibody were not met according to preliminary results of the 415-patient trial for the treatment of myocardial infarction [371173], although the company is currently undecided about the implications of the data, and plans to meet with investigators.

Action Immunosuppressant

In 1999, phase II studies for reperfusion injury in patients successfully treated with thrombolytics were planned; enrollment was expected to finish in the third quarter of 1999 [317276]. In February 1999, Lehman Brothers predicted the drug had a 30% probability of reaching market. Assuming a successful launch, Lehman Brothers analysts estimated peak sales would occur in 2009, with sales at that time of $100 million [319225].

Introduction Until quite recently, cell loss in myocardial infarction was thought to be caused wholly by ischemia [334206]. However, despite this general consensus, there were some observations in the literature pointing to other causes [334207,334236]. Specifically, necrosis was seen in areas of successful revascularization following cardiac bypass surgery, whereas there was no evidence of similar injury in nearby occluded vessels that did not undergo bypass [334236]. It is now recognized that in addition to the damage undoubtedly caused by ischemia, both reperfusion and the immune system's response to the tissue death caused by ischemia also contribute significantly to the total damage suffered in myocardial tissue [334160,334177,334186, 334205,334206,334245]. This damage is now referred to as reperfusion injury and is believed to be the result of an acute inflammatory process [334206,334254]. Of course, the issue of reperfusion injury has come to the fore since the advent of thrombolytics [300338,334230, 334231,334232,334233] and the recent expansion in the use of balloon angioplasty [334230,334232]. Many studies have demonstrated that circulating polymorphonuclear leukocytes (PMNs) are involved in the pathophysiology of myocardial infarction and the ensuing ischemic and reperfusion injury [334186,334205,334259]. Interactions between PMNs and endothelial cells are mediated by adhesive interactions between the PMN cell surface complex CD11/CD18 and coronary endothelial cell intracellular adhesion molecule (ICAM)-1 [334186,334206, 334259]. Once the PMNs have infiltrated the infarct tissue,

Originator Genentech Inc

Status Phase II Clinical

Synonym anti-CD18 mAb (Genentech/Roche) they release oxygen radicals, proteolytic enzymes, and arachidonic acid metabolites, including leukotriene B4 [334186]. While not all the details are known, the process involves the attraction of neutrophils to the site of inflammation by chemotactic factors, including complement C5a, interleukin (IL) 8, and tumor necrosis factor (TNF) [334206,334211]. Two endogenous factors counter reperfusion injury; adenosine and prostaglandins. Obviously, it is a major concern to reduce cardiac tissue damage in myocardial infarction and the area has received great attention, both from academics and pharmaceutical and biotechnology companies. At a broad level, two fundamental approaches are to stimulate the activity of natural factors, such as adenosine and prostaglandins, which reduce reperfusion injury, or to inhibit agents that cause or worsen reperfusion injury. No drug currently marketed specifically addresses reperfusion injury. Invading PMNs also metabolize arachidonic acid by a lipoxygenase (LO) enzyme; some of the products are potent chemotactic agents and others are coronary vasoconstrictors [334180]. Inhibitors of this LO enzyme reduce tissue injury [334180]. Adhesion molecules are believed to play a pivotal role in reperfusion injury, because without them (or with defects in them), the PMNs never accumulate at the site of insult [334153,334157,334179,334201,334209,334220,334224,334229, 334234]. As a result, monoclonal antibodies to adhesion molecules are a major focus of preclinical and clinical research and development [21028,155649,162429,248297, 334150,334151,334153, 331733,334157]. Three families of adhesion molecules are involved in adhesion of PMNs to endothelial cells: selectins (eg, Lselectin, P-selectin [284042,334193]), integrins (including CD18/CD11a,b,c), and immunoglobulin superfamily proteins (eg, ICAM-1, vascular cell adhesion molecule (VCAM)-1). Selectins are involved in the initial contact between PMNs and endothelial cells [284042,334193]. Monoclonal antibodies against P-selectin can reduce the extent of tissue injury accompanying reperfusion after a period of ischemia [334209,334210,334237,334239]. After PMNs have made contact with endothelial cells via selectins, integrins are responsible for much tighter binding [262255,334179]. Proteins of the integrin family are expressed on the surface of many cells, most of which

954 IDrugs 2000 Vol 3 No 8

express several integrins [262255]. They appear to be the major receptors by which cells attach to extracellular matrix [262255]. Integrins are also involved in many cell-cell interactions [262255]. Because these proteins are the major receptors by which cells attach to the extracellular matrix, they are heavily involved in many physiological processes in addition to their role in reperfusion injury [262255]. All integrins are heterodimers, comprising one α and one β chain. The α chains vary from 120 to 180 kDa; many have now been characterized and sequenced [262255,334179]. The β chains are attached non-covalently to the α chains. Many β chains have also been characterized [262255,334179]. Additionally, several α and β subunits are known to be alternatively spliced [262255,334179]. Both the α and β chains are transmembrane proteins, each crossing the cell membrane a single time. Typically, the cytoplasmic carboxyl-terminal domains are short and the amino-terminal extracellular domains much larger [262255]. Cleaved extracellular domains associate to form functional α-β heterodimers; thus, the transmembrane regions and the cytoplasmic domains are not required for dimerization [262255]). Divalent cations (Mg2+ and Ca2+) are also required for the α-β subunit interaction [262255,334201]. Leukocyte-specific integrins are commonly referred to by their CD number including CD18 (β2 integrin) and CD11a,b,c (the three α subunits with which CD18 interacts) [262255,334179]. These three α subunits are distinct proteins encoded by separate genes, all of which are located in a cluster on human chromosome 16 [334201]. In the literature, CD18/CD11a is also referred to as LFA-1, CD18/CD11b as Mac-1, and CD18/CD11c as p150,95 [334205]. Two subfamilies of integrin are particularly involved in inflammatory conditions, β1 and β2. The β1 integrins are also referred to as very late antigen (VLA) molecules. These proteins are believed to play a role in chronic disorders, including rheumatoid arthritis and psoriasis, but do not appear to be involved in reperfusion injury. The β2 integrin (CD18) is involved in neutrophil extravasation (migration through tissues) and the binding of neutrophils to inflamed tissue [262255,334179]. The β2 integrin is also involved in many acute inflammatory processes, including septic shock and adult respiratory distress syndrome. As a result, β2 integrins, their ligands, and antibodies that bind to and block them have received considerable attention in recent years as potential therapeutic targets [21028,155649,162429, 248297,334150,334151,334153, 331733,334157]. The normal ligand for CD18 is the endothelial cell marker ICAM-1. As might be expected, antibodies to it cause similar effects to antibodies to CD18 [334251]. Much information on the role of CD18/CD11a,b,c comes from the study of human patients with a leukocyte adhesion deficiency [334153,334179,334201,334209,334241,334234, 334248]. A related disease has been extensively studied in Holstein cattle [334220,334224,334229]. The absence of or presence of defective β2 integrins leads to the lifethreatening leukocyte adhesion deficiency-1 (LAD1), whereas impaired glycosylation of intact β2 integrins leads

to the less severe leukocyte adhesion deficiency-2 (LAD2) [334221,334222,334223,334225,334226]. LAD1 patients suffer recurrent life-threatening infections without pus formation [334201], which are often fatal within the first two years of life [334209]. It is believed that the absence of CD18/CD11 prevents the adherence of PMNs to the endothelium and prevents the cells from accumulating at an inflammatory locus [331708,334234,334209]. With this significant theoretical background and many animal experiments using various anti-CD18 antibodies, Genentech undertook a clinical program to assess the safety and efficacy of a humanized anti-CD18 mAb in hemmorrhagic shock and myocardial infarction patients.

Synthesis and SAR Monoclonal antibodies produced by traditional means are, of course, mouse proteins and will likely be recognized as foreign when used in human therapy. Various attempts have been made to humanize the antibodies used clinically to reduce the so-called human anti-mouse antibody (HAMA) response [334150,334151,334153]. Humanization involves making a chimeric antibody containing murine variable regions and human constant regions. However, while such humanization leads to a decrease in the human response to the foreign protein, it also typically leads to a decrease in the binding affinity of the antibody [162429,334153].

Pharmacology An avenue of investigation has developed with the availability of CD18 knockout mice [334157,334210,334255]. Mice deficient in CD18 are protected in reperfused but not non-reperfused (permanently occluded) stroke [334210]. PMN accumulation was also greatly reduced [334210]. This points to the role of PMN in reperfusion injury [334210] and suggests that anti-PMN measures should be taken with thrombolytic therapy to re-establish blood flow. Similar findings were made in a myocardial ischemia and reperfusion injury. In CD18-deficient and ICAM-1-deficient mice, myocardial necrosis and PMN accumulation was greatly reduced relative to wild-type animals [334157]. Wildtype and CD18- and ICAM-1-deficient mice were subjected to 30 min of myocardial ischemia followed by 120 min of reperfusion. The size of the resulting infarct was much reduced in the CD18- and ICAM-1-lacking mice, as was PMN infiltration [334157]. Anti-CD18 antibodies effect reductions in tissue injury in a variety of animal models of ischemia and reperfusion in several tissues. In Langendorff perfused rat hearts subjected to 20 min of global ischemia followed by 45 min of reperfusion, administration of an anti-CD18 antibody significantly reduced PMN accumulation and reperfusion injury [334184]. Similarly in a rabbit ear model of ischemia and reperfusion, treatment with an anti-CD18 antibody before or after ischemia, but prior to reperfusion, brought about protection from endothelial, microvascular, and tissue damage [21028] In dogs, treatment with anti-CD18 antibodies prior to reperfusion greatly limited the damage inflicted by 90 min of circumflex coronary occlusion, followed by 48 h of reperfusion [334160]. Similar results were obtained in other studies [334177,334205].

Anti-CD18 mAb Brown 955

Anti-CD18 antibodies also significantly reduce tissue damage in animal models of hemorrhagic shock. In rabbits subjected to hemorrhagic shock (mean blood pressure of 45 torr and cardiac output decreased to 30% of normal) for 1 h followed by resuscitation, treatment with an anti-CD18 antibody greatly increased survival to 5 days and greatly reduced organ damage [331733].

one phase I, uncontrolled, dose-escalation study using ICOS Corporation's humanized anti-CD18 antibody Hu23F2G has been published [324614]. This antibody is under investigation for use in treating multiple sclerosis [324614], and was well-tolerated with few adverse effects occurring [324614].

Anti-CD18 antibodies may also have utility in treating multiple sclerosis [334242,324614]. In an animal model of the disease, anti-CD18 antibodies significantly prolonged survival and even apparently reversed the disease [334242].

Side Effects and Contraindications

Metabolism/Pharmacokinetics

Myocardial infarction and, in particular, the ensuing ischemia and reperfusion injury is, at the present, an unmet clinical need; no drug specifically addresses reperfusion injury. The involvement of PMNs in reperfusion injury is now established, and pharmaceutical and biotechnology companies are focusing attention on reducing PMN accumulation. Of particular interest are adhesion molecules that are involved in the initial contact between endothelial cells; the β2 integrin, CD18, is involved in neutrophil extravasation and binding in inflamed tissues. With this in mind Genentech has targeted CD18 for the potential treatment of reperfusion injury associated with myocardial infarction.

No pharmacokinetic or metabolism data have been formally published, although preliminary data were reported at a recent conference [363362]. The plasma half life was estimated to be 6 to 8 h, the clearance 5 to 7 ml/h/kg and the volume of distribution was 45 to 55 ml/kg.

Toxicity Although no data are available for Genentech's anti-CD18 antibody, a few adverse effects were observed with ICOS Corporation's humanized anti-CD18 antibody, Hu23F2G (rovelizumab); in the study there were two cases of urinary tract infection and two cases of gingivitis. In one subject, a transient effect on leukocyte numbers developed at the higher doses, and a worsening of MS was observed in one patient, although this was associated with infection [324614].

Clinical Development Phase I trials began in 1996 [243712], [226593], and a 415patient phase II trial for myocardial infarction began at the end of 1998 [314374,312273]. The company has stated that preliminary data from the latter did not meet its primary objectives; these will be presented during the European Society of Cardiology meeting in August 2000, in Amsterdam, The Netherlands [371173]. Although no previous clinical data from the use of Genentech's anti-CD18 antibody have yet been published,

No data are currently available.

Current Opinion

Given the large number of successful animal studies in so many different models, listed previously, and the few adverse effects occurring in phase I studies with ICOS' anti-CD18 monoclonal antibody, rovelizumab, one can only be encouraged. However, recently rovelizumab was discontinued for the treatment of stroke after failing to meet the company's criteria for success and is no longer in active development [365543]. In light of similarly disappointing results with its anti-CD18 therapy, it is currently unclear whether Genetech will pursue the development of this antibody. Nevertheless, preclinical data remain favorable and, and it is hoped that further studies will help to establish the validity of this approach in the treatment of diseases arising from PMN accumulation.

Licensing Roche Holding AG Licensed anti-CD18 Mab from Genentech [226593].

Development History DEVELOPER Genentech Inc

COUNTRY US

STATUS C2

INDICATION Myocardial infarction

DATE 10-FEB-99

REFERENCE 310464

Genentech Inc

US

C2

Hemorrhagic shock

01-AUG-98

310464

Roche Holding AG

Switzerland

C1

Hemorrhagic shock

01-AUG-96

243712

Literature classifications Key references relating to the drug are classified according to a set of standard headings to provide a quick guide to the bibliography. These headings are as follows: Biology: References which disclose aspects of the drug's pharmacology in animal models.


Biology STUDY TYPE In vitro

EFFECT STUDIED Ability to inhibit leukocyte function.

EXPERIMENTAL MODEL Homotypic aggregation of a lymphoblastoid cell line, inhibition of T-cell proliferation, and binding of human mononuclear cells to bovine aortic endothelial cells.

RESULT The anti-CD18 and some of the antiCD11 antibodies inhibited PMA-induced homotypic adherence; some anti-CD11 and anti-CD18 antibodies inhibited antigen-specific T-cell proliferation; antiCD11 antibodies had no effect, whereas anti-CD18 antibodies inhibited binding of human mononuclear cells to bovine aortic endothelial cells.

REFERENCE 334151

In vitro

PMN motility.

Movement of PMNs through three-dimensional collagen type I gels, with or without stimulation by N-formyl peptides.

In low concentration collagen gels (< 0.6 mg/ml), an anti-CD18 monoclonal antibody, R15.7, increased motility in unstimulated PMN. In higher concentration gels (> 0.6 mg/ml), the antibody had no effect.

334152

In vitro

Homotypic aggregation.

JY human lymphoblastoid cell line.

An anti-CD18 monoclonal antibody, KIM185, stimulated homotypic aggregation by a CD11a pathway and induced adherence of neutrophils to protein-coated plastic by a CD11b mechanism.

155649

In vitro

Leukotriene (LT) C4 generation on PMNendothelial cell interaction.

GM-CSF-primed PMN and endothelial cells cultured from kidney glomeruli.

Both PMN-endothelial cell adhesion and transcellular LTC4 generation were inhibited by an anti-CD18 monoclonal antibody, R15.7.

130723

In vitro

Tissue damage after reperfusion.

Langendorff-perfused rat hearts.

Treatment with an anti-CD18 monoclonal antibody, R15.7, significantly reduced the PMN-induced contractile dysfunction by limiting PMN accumulation

334184

In vivo

Infarct volume.

Cerebral ischemia (middle cerebral artery occlusion) for 2 h followed by reperfusion for up to seven days.

Treatment with the anti-CD18 F(ab')2 fragment of monoclonal antibody, CL26, significantly reduced infarct volume and inhibited the increase in brain myeloperoxidase activity seen in controls.

334158

In vivo

Neurological outcome, efficacy of thrombolysis.

New Zealand white rabbit cerebral embolism stroke model.

Treatment with an anti-CD18 monoclonal antibody, R16.7, administered 5 min after embolization increased the quantity of clots required to produce neurological damage.

240901

In vivo

PMN accumulation.

Inflammatory reactions in guinea pig skin induced by injection of complement fragment, C5a desArg.

Treatment with an anti-CD18 monoclonal antibody, 6.5E, reduced by up to 89% the accumulation of eosinophils and neutrophils. However, edema was not reduced.

248297

In vivo

PMN accumulation.

Chemically-induced colitis in rats; treatment with antiCD18 antibody 2 h before or 3 days after induction.

Treatment with an anti-CD18 monoclonal antibody, ED7, reduced clinical signs of inflammatory bowel disease by reducing the infiltration of macrophages and granulocytes.

334174

In vivo

PMN accumulation.

Liver oxygen consumption in pigs with induced fecal peritonitis.

Treatment with an anti-CD18 monoclonal antibody, IB4, 0 to 15 min before induction of peritonitis did counteract the CD18-dependent leukopenia but did not affect liver oxygen consumption.

334168

In vivo

PMN accumulation.

Adjuvant arthritis in rats.

Treatment with an anti-CD18 monoclonal antibody, WT.3, reduced the severity of adjuvant arthritis even after joint inflammation had developed. PMN and T-lymphocyte migration to the joint were also inhibited.

265817


Biology (continued) STUDY TYPE In vivo

EFFECT STUDIED Allergen-induced late airway response (LAR), airway hyperresponsiveness (AHR), and cellular recruitment.

EXPERIMENTAL MODEL New Zealand white rabbits immunized with Alternaria tenuis within 24 h of birth, challenged with inhaled allergen as adult rabbits.

RESULT Treatment with an anti-CD18 monoclonal antibody, R15.7, reduced allergeninduced infiltration of eosinophils and neutrophils and abolished LAR and AHR.

REFERENCE 334162

In vivo

Basal release of nitric oxide from the left anterior descending (LAD) coronary artery.

Adult male cats; occlusion of LAD coronary artery for 90 min followed by 270min reperfusion.

Treatment with an anti-CD18 monoclonal antibody, R15.7, almost completely abolished PMN adherence to endothelial cells.

132043

In vivo

PMN accumulation.

New Zealand white rabbits; transorbital occlusion of the left middle cerebral, anterior cerebral, and internal carotid arteries for 2 h, followed by 6 h of reperfusion.

Treatment with an anti-CD18 monoclonal antibody, Hu23F2G, significantly reduced hemispheric ischemic neuronal damage and PMN accumulation.

324616

In vivo

Myocardial infarct size.

New Zealand white rabbits; coronary artery occlusion for 30 or 45 min followed by 3-h reperfusion.

Treatment with an anti-CD18 monoclonal antibody, IB4, 10 min prior to reperfusion significantly reduced infarct size following 30, but not 45 min, of ischemia.

334155

In vivo

PMN accumulation and PMN-dependent plasma leakage.

Inflammatory lesions on the shaved backs of male New Zealand white rabbits.

Pretreatment with an anti-CD18 monoclonal antibody, 60.3, abolished PMN accumulation and PMN-dependent plasma leakage.

331708

In vivo

'No-reflow' phenomenon (histological evaluation).

Adult mongrel dogs; carotid-coronary extracorporeal circulation, perfused with whole blood or leukocyte-depleted blood during 1 h of ischemia (LAD coronary artery occlusion).

With whole blood reperfusion, 27% of capillaries were occluded; with leukocyte-depleted blood, only 1% of capillaries were occluded. Leukocyte depletion also prevented edema and decreased the incidence of ventricular arrhythmias.

334177

In vivo

Leukocyte adherence.

Female New Zealand white rabbits; hemorrhagic shock for 1 h followed by resuscitation.

Treatment with an anti-CD18 monoclonal antibody, 60.3, increased survival to 5 days and organ damage was absent or greatly reduced relative to control animals.

331733

In vivo

Myocardial infarct size and left ventricular function.

Adult mongrel dogs; 90 min cirumflex coronary artery occlusion followed by 48-h reperfusion.

Treatment with an anti-CD18 monoclonal antibody, R15.7, 10 min before reperfusion significantly limited myocardial infarct size and preserved left ventricular function.

334160

In vivo

Myocardial infarct size.

Adult mongrel dogs; open chest, coronary artery occlusion for 90 min, followed by 3.5-h reperfusion.

Treatment with an anti-CD18 monoclonal antibody, R15.7, 10 min before reperfusion significantly limited myocardial infarct size. Two other antiCD18 antibodies were much less effective.

334205

In vivo

Leukocyte adherence following reperfusion.

New Zealand white rabbit ears, temporary ischemia and reperfusion.

Treatment with an anti-CD18 monoclonal antibody, 60.3, before or after ischemia, but before reperfusion led to a significant degree of protection from endothelial, microvascular and tissue injury.

21028

In vivo

Neurological deficits, infarct volume and hemorrhage.

Male Wistar rats, subjected to middle cerebral artery occlusion; the anti-CD18 antibody was used as an 'adjuvant' to recombinant tPA treatment 2 and 4 h after occlusion.

Treatment with the antibody alone did not reduce infarct size or neurological deficits, compared with the control group. The combination of recombinant tPA and the anti-CD18 antibody may lengthen the window for successful thrombolytic treatment.

324612


Bibliography

243712 Genentech Inc ANNUAL REPORT 1996

•

248297 Role of CD18 in the accumulation of eosinophils and neutrophils and local oedema formation in inflammatory reactions in guinea-pig skin. Teixeira MM, Reynia S, Robinson M, Shock A, Williams TJ, Williams FM, Rossi AG, Hellewell PG BR J PHARMACOL 1994 111 3 811-818

of special interest

21028 Inhibition of leukocyte adherence by anti-CD18 monoclonal antibody attenuates reperfusion injury in the rabbit ear. Vedder NB, Winn RK, Rice CL, Chi EY, Arfors K-E, Harlan JM PROC NATL ACAD SCI USA 1990 87 7 2643-2646 21033 Cytokine-induced respiratory burst of human neutrophils: Dependence on extracellular matrix proteins and CD11/CD18 integrins. Nathan C, Srimal S, Farber C, Sanchez E, Kabbash L, Asch A, Gailit J, Wright SD J CELL BIOL 1989 109 1341-1349 • Cytokine-induced respiratory burst of human neutrophils shown to be dependent on extracellular matrix proteins and CD11/CD18 integrins. 130723 Adhesion promotes transcellular leukotriene biosynthesis during neutrophil-glomerular endothelial cell interactions: inhibition by antibodies against CD18 and Lselection. Brady HR, Serhan CN BIOCHEM BIOPHYS RES COMMUN 1992 186 1307-1314 132043 Diminished basal nitric oxide release after myocardial ischemia and reperfusion promotes neutrophil adherence to coronary endothelium. Ma XL, Weyrich AS, Lefer DJ, Lefer AM CIRC RES 1993 72 403-412 155649 KIM185, a monoclonal antibody to CD18 which induces a change in the conformation of CD-18 and promotes both LFA1- and CR3-dependent adhesion. Andrew D, Shock A, Ball E, Ortlepp S, Bell J, Robinson M EUR J IMMUNOL 1993 23 9 22172222 156316 Genentech Inc ANNUAL REPORT 1992 160589 Biosynthesis and function of membrane bound and secreted forms of recombinant CD11b/CD18 (Mac-1). Berman PW, Nakamura GR, Riddle L, Chiu H, Fisher K, Champe M, Gray AM, Ward P, Fong S J CELL BIOCHEM 1993 52 2 183-195 162429 X-ray structures of fragments from binding and nonbinding versions of a humanized anti-CD18 antibody: structural indications of the key role of VH residues 59 to 65. Eigenbrot C, Gonzalaz T, Mayeda J, Carter P, Werther W, Hotaling T, Fox J, Kessler J PROTEIN STRUCT FUNCT GENET 1994 18 1 49-62 162430 sICAM-1 enhances cytokine production stimulated by alloantigen. McCabe SM, Riddle L, Nakamura GR, Prashad H, Mehta A, Berman PW, Jardieu P CELL IMMUNOL 1993 150 2 364375 162431 Ling Zhi-8: a novel T cell mitogen induces cytokine production and upregulation of ICAM-1 expression. HaakFrendscho M, Kino K, Sone T, Jardieu P CELL IMMUNOL 1993 150 1 101-113 169689 Genentech Inc ANNUAL REPORT 1993 171991 Robust growth continues at Genentech. SCRIP 1995 1995 13 176953 Genentech Inc ANNUAL REPORT 1994 226593 Genetech's partners bolster product pipeline. Van Brunt J BIOWORLD FINANCIAL WATCH 1996 4 44 1-2 & 10 240901 Monoclonal antibodies preventing leukocyte activation reduce experimental neurologic injury and enhance efficacy of thrombolytic therapy. Bowes MP, Rothlein R, Fagan SC, Zivin JA NEUROLOGY 1995 45 4 815-819

262255 Integrins: versatility, modulation and signaling in cell adhesion. Hynes RO CELL 1992 69 11-25 265817 Treatment of established adjuvant arthritis in rats with monoclonal antibody to CD18 and very late antigen-4 integrins suppresses neutrophil and T-lymphocyte migration to the joints and improves clinical disease. Issekutz AC, Ayer L, Miyasaka M, Issekutz TB IMMUNOLOGY 1996 88 569-576 275395 Genentech's year-end results show growth plan on track: Earnings increase nine percent on revenues exceeding $1 billion. Genentech Inc PRESS RELEASE 1998 January 22 284042 Increased soluble form of P-selectin in patients with unstable angina. Ikeda H, Takajo Y, Ichiki K, Ueno T, Maki S, Noda T, Sugi K, Imaizumi T CIRCULATION 1995 92 1693-1696 284047 Roche Holding AG ANNUAL REPORT 1997 December 31 299645 Protein Design Labs and Genentech announce agreement for antibody patents. Protein Design Labs Inc PRESS RELEASE 1998 September 28 300961 Cell adhesion molecules and extracellular matrix proteins: Potential therapeutic applications. Mousa SA EXP OPIN INVEST DRUGS 1998 7 7 1159-1171 301144 Genentech Inc ANNUAL REPORT 1997 December 31 310464 Genentech Inc, corporate backgrounder. Genentech Inc COMPANY WORLD WIDE WEB SITE 1999 January 04 • Overview of Genentech product and R&D pipeline. 312273 Genentech reports 41% increase in year-end net income. Genentech Inc PRESS RELEASE 1999 January 21 314374 Drug development pipeline: Genentech anti-CD18 antibody. Genentech Inc COMPANY COMMUNICATION 1999 February 10 • Lise Dumont, Genentech Corporate Communications, confirmed that the company's anti-CD18 antibody was in phase II trials for myocardial infarction. 317276 Genentech VEGF research to add third arm: opthalmic IND planned. FDC REPORTS PINK SHEET 1999 61 7 29 319225 Healthcare Global: PharmaPipelines - Volume 1 of 2. Lehman Brothers ANALYST REPORT 1999 February 324612 Increased therapeutic efficacy with rt-PA and anti-CD18 antibody treatment of stroke in the rat. Zhang RL, Zhang ZG, Chopp M NEUROLOGY 1999 52 2 273-279 324614 Phase I study of a humanized anti-CD11/CD18 monoclonal antibody in multiple sclerosis. Bowen JD, Petersdorf SH, Richards TL, Maravilla KR, Dale DC, Price TH, St John TP, Yu AS CLIN PHARMACOL THER 1998 64 3 339-346 324616 Hu23F2G, an antibody recognizing the leukocyte CD11/CD18 integrin, reduces injury in a rabbit model of transient focal cerebral ischemia. Yenari MA, Kunis D, Sun GH, Onley D, Watson L, Turner S, Whitaker S, Steinberg GK EXP NEUROL 1998 153 2 223-233 • Hu23F2G bound to leukocyte CD11/CD18 integrin and reduced injury in a rabbit model of transient focal cerebral ischemia.


324618 Inhibition of CD18 or CD11b attenuates acute lung injury after acid instillation in rabbits. Folkesson HG, Matthay MA J APPL PHYSIOL 1997 82 6 1743-1750 • Results from a phase I study of humanized anti-CD11/CD18 antibody in patients with multiple sclerosis. 331609 Genetech Inc product pipeline. Genentech Inc COMPANY WORLD WIDE WEB SITE 1999 July 12 • Website provides details on stage of development of 14 of Genetech's compounds in clinical trials and late-stage development. 331708 A monoclonal antibody to the membrane glycoprotein complex CD18 inhibits polymorphonuclear leukocyte accumulation and plasma leaskage in vivo. Arfors KE, Lundberg C, Lindbom M, Lundberg K, Beatty PG, Harlan JM BLOOD 1987 69 338 331733 A monoclonal antibody to the adherence-promoting leukocyte glycoprotein, CD18, reduces organ injury and improves survival from hemorrhagic shock and resuscitation in rabbits. Vedder NB, Winn RK, Rice CL, Chi EY, Arfors KE, Harlan JM J CLIN INVEST 1988 81 939-944 334150 Optimal humanization of IB4, an anti-CD18 murine monoclonal antibody, is achieved by correct choice of human V-region framework sequences Singer II, Kawka DW, DeMartino JA, Daugherty BL, Elliston KO, Alves K, Bush BL, Cameron PM, Cuca GC et al J IMMUNOL 1993 150 2844-2857 334151 A humanized CD18 antibody can block function without cell destruction. Sims MJ, Hassal DG, Brett S, Rowan W, Lockyer MJ, Angel A, Lewis AP, Hale G, Waldmann H, Crowe JS J IMMUNOL 1993 151 2296-2308 334152 Antibodies of CD18 influence neutrophil migration through extracellular matrix. Saltzman WM, Livingston TL, Parkhurst MR J LEUKOCYTE BIOL 1999 65 356-363 334153 Humanization of 60.3, an anti-CD18 antibody: importance of the L2 loop. Hsiao K, Bajorath J, Harris LJ PROTEIN ENG 1994 7 815-822 334155 Effect of duration of ischaemia on reduction of myocardial infarct size in inhibition of neutrophil accumulation using an anti-CD18 monoclonal antibody. Williams FM, Kus M, Tanda K, Williams TJ BR J PHARMACOL 1994 111 1123-1128

334174 Anti-CD11b/CD18 antibodies reduce inflammation in acute colitis in rats. Palmen MJHJ, Dijkstra CD, Van der Ende MB, Pena AS, Van Rees EP CLIN EXP IMMUNOL 1995 101 351-356 334177 Role of leukocytes in response to acute myocardial ischemia and reflow in dogs. Engler RL, Dahlgren MD, Morris DD, Peterson MA, Schmid-Schonbein GW AM J PHYSIOL 1986 251 H314-H322 334179 Structure and function of leukocyte integrins. Larson RS, Springer TA IMMUNOL REV 1990 114 181-217 334180 Role of leukocytes in acute myocardial infarction in anesthetized dogs: relationship to myocardial salvage by antiinflammatory drugs. Mullane KM, Read N, Salmon JA, Moncada S J PHARMACOL EXP THER 1984 228 510-522 334184 Cardioprotective actions of a monoclonal antibody against CD-18 in myocardial ischemia-reperfusion injury. Lefer DJ, Shandelya ML, Serrano CV, Becker LC, Kuppusamy P, Zweier JL CIRCULATION 1993 88 1779-1787 334186 Neutrophil function in ischemic heart disease. Mehta J, Dinerman J, Mehta P, Saldeen TGP, Lawson D, Donnelly WH, Wallin R CIRCULATION 1989 79 549-556 334189 Inflammation in acute coronary syndromes. Entman ML, Ballantyne CM CIRCULATION 1993 88 800-803 334193 Soluble P-selectin is released into the coronary circulation after coronary spasm. Kaikita K, Ogawa H, Yasue H, Sakamoto T, Suefuji H, Sumida H, Okumura K CIRCULATION 1995 92 1726-1730 334201 Structure and function of the leukocyte adhesion molecules CD11/CD18. Arnaout MA BLOOD 1990 75 1037-1050 334205 Factors modifying protective effect of anti-CD18 antibodies on myocardial reperfusion injury in dogs. Perez RG, Arai M, Richardson C, DiPaula A, Siu C, Matsumoto N, Hildreth JEK, Mariscalco MM, Smith CW, Becker LC AM J PHYSIOL 1996 270 H53-H64 334206 Reperfusion and reperfusion injury. Weisfeldt ML CLIN RES 1987 35 13-20 334207 Cerebral ischemia. II. The no-reflow phenomenon. Ames A, Wright RL, Kowada M, Thurston JM, Majno G AM J PATHOL 1968 52 437-453

334157 Myocardial ischemia-reperfusion injury in CD18-and ICAM-1-deficient mice. Palazzo AJ, Jones SP, Girod WG, Anderson DC, Granger DN, Lefer DJ AM J PHYSIOL 1998 275 6 pt 2 H2300-H2307

334209 Adhesion molecules and myelomonocytic cellendothelial interactions. Rosen H, Gordon S BR J EXP PATHOL 1989 70 385-394

334158 Postischemic treatment (2-4h) with anti-CD11b and antiCD18 monoclonal antibodies are neuroprotective after transient (2h) focal cerebral ischemia in the rat. Zhang ZG, Chopp M, Tang WX, Jang N, Zhang RL BRAIN RES 1995 698 79-85

334210 CD-18 mediated neutrophil recruitment contributes to the pathogenesis of reperfused but not nonreperfused stroke. Prestigiacomo CJ, Kim SC, Connolly ES Jr, Liao H, Yan SF, Pinsky DJ STROKE 1999 30 1110-1117

334160 An anti-CD18 antibody limits infact size and preserves left ventricular function in dogs with ischemia and 48-hour reperfusion. Arai M, Lefer DJ, So T, DiPaula A, Aversano T, Becker L J AM COLL CARDIOL 1996 27 1278-1285 334162 The effect of R 15.7/HO, an anti-CD18 antibody, on the late airway response and airway hyperresponsiveness in an allergic rabbit model. El-Hashim AZ, Jacques CA, Herd CM, Lee TH, Page CP BR J PHARMACOL 1997 121 671-678 334168 Inhibition of CD18-dependent adherence of polymorphonuclear leukocytes does not affect liver oxygen consumption in fecal peritonitis in pigs. Wolbert S, Rasmussen I, Lundberg C, Gerdin B, Arvidsson D, Haglund U CIRC SHOCK 1993 41 230-238

334211 Leukocyte adhesion - a fundamental process in leukocyte physiology. Gahmberg CG, Valmu L, Tian L, Kotovuori P, Fagerholm S, Kotovuori A, Kantorand C, Hilden T BRAZ J MED BIOL RES 1999 32 511-517 334221 Characterization of two new CD18 alleles causing severe leukocyte adhesion deficiency. Lopez Rodriguez C, Nueda A, Grospierre B, Sanchez-Madrid F, Fisher A, Springer TA, Corbi AL EUR J IMMUNOL 1993 23 2792-2798 334222 Adhesion molecule deficiencies and their clinical significance. Etzioni A CELL ADHESION COMM 1994 2 257-260 334223 Leukocyte adhesion deficiency (LAD) II. Etzioni A, Phillips LM, Paulson JC, Harlan JM CIBA FOUND SYMP 1995 189 51-62 77-78


334225 Molecular characterization of leukocyte adhesion deficiency in six patients. Wright AH, Douglass WA, Taylor GM, Lau YL, Higgins D, Davies KA, Law SK EUR J IMMUNOL 1995 25 717-722 334226 Leukocyte adhesion deficiency: report of a case and review of the literature. Lipnick RN, Illiopoulos A, Salata K, Hershey J, Melnick D, Tsokos GC CLIN EXP RHEUMATOL 1996 14 95-98 334229 Bovine leukocyte adhesion deficiency: a brief overview of a modern disease and its implications. Gerardi AS ACTA VET HUNG 1996 44 1-8 334230 Primary angioplasty compared with thrombolysis: new issues in the era of glycoprotein IIb/IIIa inhibition and intracoronary stenting. Gibson CM ANN INTERN MED 1999 130 841-847 334231 Time to treatment of acute myocardial infarction revisited. Cannon CP CURR OPIN CARDIOL 1998 13 254-266 334232 Thrombolytic therapy versus primary angioplasty in the treatment of acute myocardial infarction. McErlean ES J CARDIOVASC NURS 1999 13 46-59 334233 Reteplase: a new thrombolytic for the treatment of acute myocardial infarction. Wooster MB, Luzier AB ANN PHARMACOTHER 1999 33 318-324 334234 Distinct mutations in two patients with leukocyte adhesion deficiency (LAD) and their functional correlates. Wardlaw AJ, Hibbs ML, Stacker SA, Springer TA J EXP MED 1990 172 335-345 334236 Myocardial consequences of coronary artery bypass graft surgery: the paradox of necrosis in areas of revascularization. Bulkley BH, Hutchins GM CIRCULATION 1977 56 906-913 334237 In vivo neutralization of P-selectin protects feline heart and endothelium in myocardial ischemia and reperfusion injury. Weyrich AS, Ma XL, Lefer DJ, Albertine KH, Lefer AM J CLIN INVEST 1993 91 2620-2629 334239 Monoclonal antibodies to P-selectin are effective in preventing injury to rabbit ears. Winn RK, Vedder NB, Paulson JC, Harlan JM CIRCULATION 1992 86 Suppl 1 I-80 334241 Leukocyte adhesion deficiency type 1 (LAD-1) variant. A novel immunodeficiency syndrome characterized by dysfunctional β2 integrins. Kuijpers TW, Van Lier RA, Hamann D, de Boer M, Thung LY, Weening RS, Verhoeven AJ, Ross D J CLIN INVEST 1997 100 1725-1733 334242 Resolution of CNS lesions following treatment of experimental allergic encephalomyelitis in macaques with monoclonal antibody to the CD18 leukocyte integrin. Rose LM, Richards TL, Peterson J, Petersen R, Alvord Jr EC MULTIPLE SCLEROSIS 1997 2 259-266

334245 Endothelial and leukocyte adhesion molecules in inflammation and disease. Winn R, Vedder N, Ramamoorthy C, Sharar S, Harlan J BLOOD COAGUL FIBRINOLYSIS 1998 9 Suppl 2 S17-S23 334248 A novel leukocyte adhesion deficiency caused by expressed but nonfunctional β2 integrins Mac-1 and LFA-1. Hogg N, Stewart MP, Scarth SL, Newton R, Shaw JM, Law SK, Klein N J CLIN INVEST 1999 103 97-106 334251 Monoclonal antibody to ICAM-1 preserves postischemic blood flow and reduces infarct size after ischemia-reperfusion in rabbit. Zhao ZQ, Lefer DJ, Sato H, Hart KK, Jefforda PR, VintenJohansen J J LEUKOCYTE BIOL 1997 62 292-300 334254 Cytokines and the microcirculation in ischemia and reperfusion. Frangogiannis NG, Youker KA, Rossen RD, Gwechenberger M, Lindsey MH, Mendoza LH, Michael LH, Ballantyne CM, Smith CW, Entman ML J MOL CELL CARDIOL 1998 30 25672576 334255 Mice deficient in Mac-1 (CD11b/CD18) are less susceptible to cerebral ischemia/reperfusion injury. Soriano SG, Coxon A, Wang YF, Frosch MP, Lipton SA, Hickey PR, Mayadas TN STROKE 1999 30 134-139 334259 Leukocyte-endothelial cell interaction in ischemiareperfusion injury. Winn RK, Ramamoorthy C, Vedder NB, Sharar SR, Harlan JM ANN NY ACAD SCI 1997 832 311-321 352821 Genentech Inc. HAMBRECHT & QUIST 2000 January 10-13 132 355591 Product pipeline. Genentech Inc COMPANY WORLD WIDE WEB SITE 2000 February 14 • Website detailing selected compounds in clinical development as of February 2000. 363362 Clinical PK/PD of a humanized antibody fragment to leukocyte CD18 receptors. Allison DE, Bussiere JL, Greene WL, Miller RM, Hill HM, Gourlay SG CLIN PHARMACOL THER 2000 67 2 PI-69 365543 Drug development pipeline: rovelizumab. ICOS Corp COMPANY COMMUNICATION 2000 May 09 • Lacy Fitzpatrick (ICOS Corp) stated that following interim efficacy analysis this year, ICOS had elected not to complete phase III trials and no longer had an active R&D program with rovelizumab. 371173 Genentech announces phase II trial of experimental anti-CD18 antibody did not meet its primary objectives. Genentech Inc PRESS RELEASE 2000 June 16 374809 Genentech reports 15 percent increase in product sales for second quarter - earnings driven by sales of Rituxan and Herceptin. Genentech Inc PRESS RELEASE 2000 July 17