AU7835294A - Peripheralization of hematopoietic stem cells - Google Patents

Description

PERIPHERALIZATION OF HEMATOPOIETIC STEM CELLS

BACKGROUND OF THE INVENTION

Field Of The Invention

The invention relates to the inhibition of interactions between reticulocytes and activated endothelial cells. More particularly, the invention relates to treating human diseases associated with abnormal reticulocyte-endothelium interactions, by inhibiting these

interactions.

Summary Of The Related Art Sickle cell anemia is a disease that is associated with various debilitating complications, including hemolytic anemia, increased infection, ischemic organ damage and episodes of severe pain. Pauling et al. (Science 10:543 (1949)) and Ingram (Nature 180:326 (1957)) teach that this disease and its complications are manifestations of homozygosity for a single amino acid substitution of valine for glutamic acid in the B6 position of the globin chain. Various researchers (including Hahn and Gillespie, Arch. Intern. Med. 39:233 (1927);

Harris et al., Arch. Intern. Med. 97:145 (1956); Charache and Conley, Blood 42:25 (1964); and Ham and Castle, Trans. Assoc. Amer. Phys. 55:127 (1940)) have postulated that the primary pathologic event may be formation of mechanically rigid "sickled" cells with deoxygenation. The sickled cells, which cannot normally traverse the microvasculature, occlude small and large blood vessels, causing ischemic organ damage and episodes of pain.

However, others (including Hofrichter et al., Proc. Natl. Acad. Sci. USA 71 :4864 (1974); Eaton et al, Blood 47:621 (1976); Eaton and Hofrichter, Blood 70: 1245 (1987); Mozzarelli et al., Science 237:500 (1987); and Ferrone, Ann. Ny Acad. Sci. 565:63 (1989)) have noted that capillary obstruction is an uncommon occurrence because hemoglobin S polymerization does not occur immediately after deoxygenation, and most erythrocytes traverse the capillary bed before sickling occurs. The fraction of erythrocytes that sickle in the microvasculature and cause obstruction may be determined by other factors that increase capillary transit time or decrease the delay in polymerization.

Hebbel et al. ( N. Engl. J. Med. 302:992 (1980)), Kaul et al. (Proc. Natl. Acad. Sci. USA 86:3356 (1989)), and Fabry et al. (Blood 72:1602 (1992)) teach that one factor that potentially increases transit time may be abnormal adherence of erythrocytes from patients with sickle cell anemia (SSRBC) to vascular endothelial cells, and that the less dense cells, primarily reticulocytes, are the subpopulation of SSRBC most adherent to rat post-capillary venule endothelium ex vivo. Wick et al. (J. Clin. Invest. 80:95 (1987)) extends this finding to in vitro adherence to human venous endothelium, and suggests that factors in sickle plasma, including high molecular weight multimers of von Willebrand factor and thrombospondin, mediate binding of SSRBC to endothelial cells cultured from large and small vessel, utilizing GPIb and integrin receptors. However, Brittain et al., J. Lab. Clin. Med. (in press) teaches that adherence of SSRBC to cultured microvascular endothelium is not promoted by high molecular weight von Willebrand factor, thus suggesting phenotypic variation in endothelial receptor-ligand interactions. Francis and Johnson, Blood 77:1405 (1991); and Hebbel, Blood 77:214 (1991) point out that despite considerable study, the complex underlying mechanisms for the interaction of SSRBC with vascular endothelial cells are only partially defined. There is therefore, a need for greater understanding of the specific interactions that are involved in increasing transit time and initiating vascular occlusive complications. Patel and Lodish, J. Cell. Biol. 105:3105 (1987) and 102:449 (1986); Tsai et al., Blood 69:1587 (1987); Papayannopolou and Brice, Blood 7:1686 (1992); and Seligman, Progress in Hematology 13:131 (1983) together teach that various integrins, including LFA-1, VLA-4 and VLA-5, as well as receptors for transferrin and fibronectin, are present on erythrocyte precursors, but that their expression is usually lost from erythrocytes prior to entry into the peripheral circulation. Elices et al., Cell 60:577 (1990); and Pulido et al., J. Biol. Chem. 266:10241 (1991) suggest

that the VLA-4 receptor may mediate binding to the inducible endothelial cell adhesion

molecule VCAM-1 on cytokine-activated endothelial cells in vitro. Unfortunately, the particular integrin involved in SSRBC binding, if any, is not elucidated by these previous reports. In two recent abstracts, Blood 80: Supplement 1, Abstract 36 (1992); and Clin. Res. 4J.:262A (1993), the present inventor and co-workers implicate VCAM-1 and CD36 in

binding of a subpopulation of SSRBC to endothelial cells stimulated with TNF-α. Tracy and Cerami, Proc. Soc. Exp. Biol. Med. 200:233 (1992) teaches that acute illnesses are often accompanied by elevated circulating cytokine levels, resulting in endothelial cell activation and VCAM-1 induction. There is, therefore, a need to develop means for inhibiting the interactions between SSRBC and activated endothelial cells that result in transit time increase and vascular occlusive conditions.

BRIEF SUMMARY OF THE INVENTION

The invention demonstrates the mechanism by which a subpopulation of SSRBC bind to activated endothelial cells, thereby leading to increase in transit time and resulting in vascular occlusion in sickle cell disease. The invention further provides a method for inhibiting such binding, thus preventing such vascular occlusion and its resulting complications. This method comprises the step of administering a blocking agent of VLA-4 antigens on the surface of reticulocytes that express VLA-4, including sickle reticulocytes. Various agents can be used to mediate such blocking, including anti-VLA-4 or anti-VCAM-1 antibodies which may optionally be single chain, humanized or chimeric, Fab, Fab', F(ab')₂ or F(v) fragments thereof, heavy or light chain monomers or dimers thereof, or intermixtures of the same, soluble fibronectin, or soluble VCAM-1, bifunctional VCAM-1/Ig fusion proteins or VCAM-1 peptides.

It is an object of the invention to provide a method for blocking the interactions between VLA-4 expressing reticulocytes and activated endothelial cells as an experimental model for understanding the mechanism of vascular occlusion and its resulting complications in sickle cell anemia and other diseases. It is a further object of the invention to provide a method of treating the complications of sickle cell disease by preventing the vascular occlusion mediated by interactions between sickle reticulocytes and activated endothelial cells in patients suffering from sickle cell disease. The invention satisfies these objects by providing a method for blocking the binding interactions between VLA-4 expressing reticulocytes, including sickle reticulocytes and activated endothelial cells by administering a blocking agent of VLA-4 antigen on the surface of reticulocytes. The invention clearly demonstrates, for the first time, that administration of such blocking agents is sufficient to prevent binding between sickle reticulocytes and activated endothelial cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of assays for Bi integrin expression by cells in venous blood, as described in Example 1.

Figure 2 shows the results of assays for reticulocyte counts and β, integrin expression

by cells in venous blood, as described in Example 2.

Figure 3 shows the results of assays for alpha and beta integrin chain by SSRBC, as described in Example 3.

Figure 4 shows the results of assays for βj or α₄ integrin and glycophorin A expression by venous blood cells, as described in Example 4.

Figure 5 shows the results of assays for binding of erythrocytes to TNF-α-stimulated endothelial cells, as described in Example 5. Figure 6 shows anti-VLA-4 and anti-VCAM-1 antibody inhibition of binding of

SSRBC to activated endothelial cells, as described in Example 6.

Figure 7 shows the nucleotide sequences encoding the variable regions of the heavy and light chains of anti-VLA-4 murine monoclonal antibody HP 1/2. Panel A is the nucleotide sequence encoding the variable heavy region, with the first nucleotide representing the beginning of the first codon. Panel B is the nucleotide sequence encoding the variable light region, with the first nucleotide representing the beginning of the first codon. Figure 8 shows the nucleotide sequences of V_H and V_κ -encoding regions having CDR-encoding sequences from murine HP 1/2 transplanted therein. Panel A shows the transplanted V_H sequence. Panel B shows the transplanted V_κ sequence.

Figure 9 shows the nucleotide sequences encoding the variable regions of the heavy and light chains of the humanized anti-VLA-4 antibody hHPl/2. Panel A is the nucleotide sequence encoding the V_H region. Panel B is the nucleotide sequence encoding the V_κ region.

Figure 10 shows the expression of adhesion proteins on erythrocytes from an infant with necrotizing enterocolitis. Figure 11 shows the expression of adhesion proteins on erythrocytes from an infant with no known risk of necrotizing enterocolitis.

DETAILED DESCRIPTION OF THE INVENTION The invention relates to inhibition of binding interactions between VLA-4 expressing reticulocytes and activated endothelial cells. More particularly, the invention relates to the inhibition of _.such binding interactions that are involved in the complications associated with human disease, especially sickle cell disease. The invention provides an experimental model for studying the role of increased capillary transit time of sickle reticulocytes in common complications of sickle cell disease, such as vascular occlusion, hemolytic anemia, increased infection, ischemic organ damage and episodes of severe pain. The invention further provides a method for preventing such complications in patients suffering from sickle cell disease.

In a first aspect, the invention provides an experimental model for studying the role of increased capillary transit time of sickle reticulocytes in the complications associated with sickle cell disease. Previously, the mechanism of such increased transit time has been poorly understood. Moreover, unclear or conflicting reports on the molecules involved in the binding interactions leading to increased transit time have not facilitated the discovery of means for blocking such binding. For the first time, the invention provides the necessary information for producing an experimental model for studying the involvement of increased capillary transit time in complications arising from sickle cell disease.

In this experimental model, increased capillary transit time is mediated by binding of sickle reticulocytes to activated endothelial cells via a VLA-4/VCAM-1 interaction.

Supporting this model, it is demonstrated herein that a subpopulation of SSRBC expresses the

β] integrin chain, but do not express either the β₂ or β₃ integrin chain. The expression of the β, integrin chain is found almost exclusively on reticulocytes and the population of reticulocytes demonstrating the greatest expression of β, integrin stain brightest with thiazole orange, suggesting that only the youngest reticulocytes express β, integrin. No detectable

erythrocyte integrin expression is found in normal controls with reticulocyte counts less than 5%, although a small population of βj expressing reticulocytes is detected in blood from non- sickle patients with reticulocyte counts greater than 5%, particularly in neonates with elevated reticulocyte counts. Reticulocyte counts in 25 samples obtained from 22 sickle cell disease patients range from 4% to 45% with a mean and standard deviation of 18±11%. Since β, expression is limited to the reticulocyte fraction, the total SSRBC expressing β, ranges from 0.3% to 11.2%. There is a clear correlation (r² = 0.62) between the per cent reticulocytosis and the per cent of cells expression β[ integrin. When analyzed for cell surface α chains normally associated with β,, erythrocytes from patients with sickle cell anemia clearly express the α₄ integrin chain, and this expressio

is limited to the reticulocyte fraction. The per centage of α₄ expressing erythrocytes is

essentially identical to the per centage of β_! expressing cells. Expression of α,, a^, α₃, α₅, α₆ or α-, integrin chains is not detected on SSRBC by flow cytometry. Double staining with bot anti-α₄ and anti-β, shows that these integrins are co-expressed by the same cells.

Samples double stained to examine co-expression of the erythrocyte membrane protein glycophorin A and the β_j or α₄ integrin chain demonstrate that the integrin expressing cells also clearly express glycophorin A. The percentage of βj and α₄ expressing cells is similar to that found in the reticulocyte population.

These data demonstrate that α₄ and β, expressing cells are erythrocytes, rather than leukocytes or platelets, and that reticulocytes in the peripheral circulation may express the α₄β, integrin complex commonly known as VLA-4.

Tumor necrosis factor (TNF)α stimulation of endothelial cell monolayers, which is associated with VCAM-1 induction, results in markedly increased binding of SSRBC to the monolayers, relative to unstimulated endothelial cell monolayer controls, with only a small number of SSRBC or normal RBC binding to unstimulated human umbilical vein endothelial cells. The increase in SSRBC binding parallels VCAM-1 induction. Under identical conditions, stimulation of the endothelial cells by TNFα causes only a small increase in binding of RBC from normal individuals.

The role of VLA-4 expressed by sickle reticulocytes in the binding of these cells to activated endothelium is supported by monoclonal antibody (mAb) blocking studies. Preincubation of TNFα-stimulated endothelial cells with anti-VCAM-1 mAb, or of SSRBC ■ with anti-α₄ integrin mAb causes up to 75% inhibition of binding of SSRBC to endothelial cells. In contrast, a control mAb that recognizes intercellular adhesion molecule 1 (ICAM-1) does not inhibit SSRBC binding to activated endothelial cells. Taken together, these data strongly support a model for capillary transit time for sickle reticulocytes in which the transit time is increased by binding of the reticulocytes to activated endothelial cells via a VLA-4/VCAM-1 interaction. This model should provide insights into the pathophysiology of vaso-occlusive complications in sickle cell anemia, by allowing in

vitro and in vivo testing of the effect of administering one or more blocking agents of VLA-4

antigen present on the surface of sickle reticulocytes, then analyzing the effect on endothelial cell binding, the resultant capillary transit time and vascular occlusion, as well as their effect upon the various complications associated with sickle cell anemia.

In a second aspect, the invention provides a method for preventing increased capillary transit time and the resultant vascular occlusion in patients suffering from sickle cell anemia. Although the irreversibly sickled cell was initially believed to play a central role in the development of vascular complications, more recent clinical studies suggest that the frequency and severity of vascular complications is inversely related to the per centage of these cells and instead is closely correlated with the per centage of young deformable cells (see e.g.. Ballas et al., Blood 72:1216 (1988)). Furthermore, ex vivo studies utilizing SSRBC infused into rat mesocecum demonstrate binding of the less dense reticulocyte fraction to post- capillary venules (see Hebbel et al.; Kaul et al.; and Fabry et al., citations provided in the Background of the Invention). Finally, similar in_. vitro studies utilizing SSRBC incubated with high molecular weight multimers of vWF also show preferential binding of the sickle reticulocytes fraction to endothelial cells (see Wick et al., citation provide in the Background of the Invention). Thus, the present demonstration that VLA-4/VCAM-1 interactions are responsible for binding of sickle reticulocytes to activated endothelial cells, and that administration of blocking agents of VLA-4 antigen on the surface of sickle reticulocytes can prevent this binding, indicates that the method according to this aspect of the invention can prevent vascular occlusion in patients suffering from sickle cell anemia.

The method according to this aspect of the invention comprises the step of administering to a patient suffering form sickle cell anemia a blocking agent of VLA-4 on the surface of sickle reticulocytes. For purposes of the invention, the term "blocking agent of VLA-4 antigens" is intended to mean an agent that is capable of interfering with interactions between VLA-4 antigens and ligands on endothelial cells or in the extracellular matrix, including but not limited to either VCAM-1 or fibronectin on the surface of activated endothelial cells. As demonstrated herein, such blocking of VLA-4 antigens inhibits binding of sickle reticulocytes to endothelial cells. This demonstration utilized a monoclonal antibody against VLA-4 as a blocking agent. Those skilled in the art will recognize that, given this demonstration, any agent that can block VLA-4 antigens can be successfully used in the method of the invention. Thus, for purposes of the invention, any agent capable of blocking VLA-4 antigens on the surface of sickle reticulocytes is considered to be an equivalent of the monoclonal antibody used in the examples herein. For example, the invention contemplates as equivalents at least peptides, peptide mimetics, carbohydrates and small molecules capable of blocking VLA-4 antigens on the surface of sickle reticulocytes. In a preferred embodiment, the blocking agent that is used in the method of the invention to block VLA-4 antigens on the surface of sickle reticulocytes is a monoclonal antibody or antibody derivative. Preferred antibody derivatives include humanized antibodies, chimeric antibodies, single chain antibodies, Fab, Fab', F(ab')₂ and F(v) antibody fragments, and monomers or dimers of antibody heavy or light chains or intermixtures thereof. The successful use of monoclonal antibody OKT3 to control allograft rejection indicates that, although humanized antibodies are preferable, murine monoclonal antibodies can be effective in therapeutic

applications. Monoclonal antibodies against VLA-4 are a preferred blocking agent in the

method according to the invention. Human monoclonal antibodies against VLA-4 are another preferred blocking agent in the method according to the invention. These can be prepared using in vitro-primed human splenocytes, as described by Boerner et al., J. Immunol. 147:86- 95 (1991). Alternatively, they can be prepared by repertoire cloning as described by Persson

et al., Proc. Natl. Acad. Sci. USA 882432-2436 (1991) or by Huang and Stollar, J. of Immunol. Methods 141:227-236 (1991). Another preferred blocking agent in the method of the invention is a chimeric antibody having anti-VLA-4 specificity and a human antibody constant region. These preferred blocking agents can be prepared according to art-recognized techniques, as exemplified in U.S. Patent No. 4,816,397 and in Morrison et al., Proc. Natl. Acad. Sci. USA £1:6851-6855 (1984). Yet another preferred blocking agent in the method of the invention is a humanized antibody having anti-VLA-4 specificity. Humanized antibodies can be prepared according to art-recognized techniques, as exemplified in Jones et al., Nature 321:522 (1986); Riechmann, Nature 332:323 (1988); Queen et al., Proc. Natl. Acad. Sci. USA 86:10029 (1989); and Orlandi et al., Proc. Natl. Acad. Sci. USA 86:3833 (1989). Those skilled in the art will be able to produce all of these preferred blocking agents, based upon the nucleotide sequence encoding the heavy and light chain variable regions of HP1/2 [SEQ.

ID. NOS. 1 and 2], as shown in Figure 7, using only well known methods of cloning, mutagenesis and expression (for expression of antibodies, see e.g.. Boss et al., U.S. Patent No.

4,923,805). Two other preferred blocking agents are single chain antibodies, which can be prepared as described in U.S. Patent No. 4,946,778, the teachings of which are hereby incorporated by reference; and biosynthetic antibody binding sites, which can be prepared as described in U.S. Patent No. 5,091,513, the teachings of which are hereby incorporated by reference. Those skilled in the art will recognize that any of the above-identified antibody or antibody derivative blocking agents can also act in the method of the invention by binding the receptor for VLA-4, thus acting as agents for blocking the VLA-4 antigen on the surface of sickle reticulocytes, within the meaning of this term for purposes of the invention. Thus, antibody and antibody derivative blocking agents according to the invention, as described above, include embodiments having binding specificity for VCAM-1 or fibronectin, since these molecules are likely to be important in the adhesion between sickle reticulocytes and activated endothelial cells.

In another preferred embodiment, the blocking agents used in the method according to the invention are not antibodies or antibody derivatives, but rather are soluble forms of the natural binding proteins for VLA-4. These blocking agents include soluble VCAM-1, bifunctional VCAM-1/Ig fusion proteins, or VCAM-1 peptides as well as fibronectin, fibronectin having an alternatively spliced non-type DI connecting segment. These blocking agents will act by competing with the activated endothelial cell-bound binding protein for VLA-4 on the surface of sickle reticulocytes.

In this method according to the first aspect of the invention, blocking agents are preferably administered parenterally. The blocking agents are preferably administered as a sterile pharmaceutical composition containing a pharmaceutically acceptable carrier, which may be any of the numerous well known carriers, such as water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, or combinations thereof. Preferably, the blocking agent, if an antibody or antibody derivative, will be administered at a dose between

about 0.1 mg/kg body weight/day and about 10 mg/kg body weight/day. If non-antibody or

antibody analogs are used, administration should be at a molar equivalency to that of the antibody binding sites at the above-described concentrations.

In a third aspect, the invention provides an experimental model for studying other diseases involving increased reticulocyte counts and/or vaso-occlusion. One example of such a condition is severe hereditary spherocytosis, which results in a poorly understood occurrence of leg ulceration and dermatitis. These complications have previously been attributed to increased blood viscosity and reduced tissue oxygen delivery caused by rigid microspherocytes. However, these attributions are called into question by the observation that the leg ulcers improve or disappear after splenectomy. Splenectomy predictably results in increased erythrocyte counts and circulating microspherocytes, which should aggravate leg ulcers if increased blood viscosity alone is responsible for ulceration and dermatitis. A plausible alternative explanation is that interaction between VLA-4 expressing reticulocytes and vascular endothelium plays a role in the pathophysiology of skin complications in sever . hereditary spherocytosis, since splenectomy invariably decreases reticulocyte counts. The

invention thus provides a method for studying the potential role of VLA-4 expression by reticulocytes in the complications by administering to the cells blocking agents of VLA-4 antigen on the surface of reticulocytes in in vitro and/or in vivo studies.

Similarly, erythrocytes parasitized by Plasmodium falciparum bind to endothelial cells, and vascular occlusive complications characteristic of malaria are thought to be mediated by this binding (see MacPherson et al., Am. J. Pathol. 119:385 (1985); Oo et al., J. Neuropathol. Exp. Neurol. 46:223 (1987); Pongponrantn et al., Am. J. Trop. Med. Hyg. 44:168 (1991)). The factors that predispose certain individuals to the most catastrophic complication of cerebral malaria are not well understood. Recent studies of patients infected with falciparum malaria report that individuals with cerebral malaria have significantly increased reticulocyte counts, as well as increased levels of TNFα. These observations, in light of the present invention, suggest that VLA-4 expressing reticulocytes may play a role in this complication _.by binding to TNFα-activated cerebral endothelium via VLA-4/VCAM-1 interactions. The invention provides an experimental model for studying this potential role by administering blocking agents of VLA-4 antigen on the surface of reticulocytes, in in vitro and or in vivo studies.

Necrotizing enterocolitis in infants is another pathologic condition in which abnormal interactions between endothelial cells and erythrocytes have been pathophysiologically implicated. Abnormal binding of red cells to bowel endothelial cells results in vascular compromise, occlusion, and a clinical syndrome marked by colitis and bowel infarction. The following examples are intended to further illustrate certain aspects of preferred . embodiments of the invention, and are not limiting in nature.

Example 1 : Expression of β, Integrin by a Subpopulation of Reticulocytes

Venous blood samples anticoagulated with EDTA were collected from patients with sickle cell anemia during routine follow up visits to the Grady Memorial Hospital Sickle Cell

Center or from normal controls after approval of and according to the guidelines of the Emory University, Georgia Institute of Technology, and Grady Memorial Hospital

Institutional Review Boards. A sample of blood with an elevated reticulocyte count was identified and collected at the Grady Memorial Hospital hematology laboratories. The identity and medical history of this patient was not available, although this sample was identified as newborn blood. 10 μl of whole blood was washed in PBS with 5% fetal calf serum and 0.1 % azide (FACS buffer), pelleted and stained with 20 μl of mAb OKT-5 (anti- CD8, ascites 1 :100 in FACS buffer, ATCC, Rockville, MD), mAb 4B4 (anti-β_j integrin, 100 μg/ml/ Coutler, Hialeah, FL), or mAb AP3 (anti-β3 integrin, ascites 1:100 in FACS buffer, Dr. Peter Neuman, Milwaukee Blood Institute, Milwaukee, WI). Cells were washed, incubated with phycoerythrin conjugated goat and mouse IgG (1:40 in FACS buffer, Tago, Burlingame, CA) and washed again. In order to identify the reticulocyte containing fraction, they were then resuspended in FACS buffer containing 100 ng/ml of thiazole orange (Molecular Probes, Eugene, OR) and allowed to incubate at room temperature protected from light for 30 minutes. Erythrocytes were then examined on a FAC scan flow cytometer

(Becton Dickinson) using standard procedures (see e.g.. Lee et al., Cytometry 7:508 (1986)). Erythrocytes were identified by forward and side scatter and 50,000 events were collected per tube. Reticulocytes were identified by green fluorescence (FL1) and cell surface expression was detected by yellow fluorescence (FL2). The results are shown in Figure 1. Erythrocytes from the normal control (A) (reticulocyte count 1.6%:%β, expressing cells 0.2%) did not express β,. In contrast, erythrocytes from a newborn (B) (reticulocyte count 10%, %β, expressing cells 1.5%) and from a patient with sickle cell anemia (C) (reticulocyte count 16.5%, %β-, expressing cells 4%) both expressed the β, integrin chain.

Example 2 : Correlation of β, Integrin Chain Expression with Reticulocyte Counts in Sickle Cell Patients

Venous blood samples anticoagulated with EDTA were collected from patients with sickle cell anemia (25 samples from 22 patients) or from normal controls (n=4). Samples of blood with reticulocyte counts greater than 5% were identified and collected at the Grady Memorial Hospital hematology laboratories (n+10). The identities and medical histories of these patients, were not available, although four samples were identified as newborn blood. Samples were stained as described in Example 1. The percent reticulocyte count was determined by thiazole orange staining and flow cytometric analysis. The results are shown in Figure 2. (A) The mean reticulocyte counts of the sickle cell group, control group, and high reticulocyte non-sickle group were 18.9%, 2.3%, and 12%, respectively. (B) The mean percentage of β_j expressing erythrocytes of these same groups were 4.3%, 0.2%, and 1.6%, respectively. (C) Linear regression analysis (Sigmaplot, Jandel Scientific, Corte Madera, CA) clearly demonstrates a correlation between the expression of the β, integrin chain on erythrocytes and the reticulocyte count (1^=0.62).

Example 3 : Expression of VLA-4 by SSRBC

Washed erythrocytes were incubated within antibody recognizing CD8 or mAb recognizing the integrin chains β„ α_! (mAb TS 2/7 Dr. Martin Hemler, Dana Farber Cancer

Institute, Boston, MA), o^ (mAb P1E6, Telios Pharmaceuticals, San Diego, CA), o^ (mAb

P1B5, Telios), α₄ (mAb, HP2/1, AMAC, Larrabee, ME), α₅ (mAb, P1D6, Telios), or α₆ (mAb

GoH3, AMAC). They were then washed and incubated with PE conjugated goat anti-mouse IgG (Tago) followed by thiazole orange and evaluated by flow cytometric analysis. The results are shown in Figure 3. A subpopulation of reticulocytes expressed both the βj and α, integrins in equivalent amounts.

Example 4 : VLA-4 Expression by Glycophorin A Positive Cells

A) Erythrocytes incubated with mAb antibody recognizing β_j (4B4) or α₄ (mAb HP2/1), washed and then incubated with PE-conjugated goat anti-mouse IgG (Tago), then incubated with thiazole orange as previously described and evaluated by flow cytometric analysis. Similar percentages of erythrocytes express both β_! and α₄ (3.99% and 3.5% respectively) and expression is restricted to the reticulocyte population. B) Erythrocytes from the same specimen similarly incubated with mAb recognizing β] or α₄ as described above followed by FTTC goat anti-mouse IgG (Tago), then incubated with normal mouse ascites (1 :100 in FACS buffer, Accurate Scientific, Westbury, NY), washed, and then incubated with phycoerythrin conjugated anti-glycophorin A (mAb D2.10, AMAC). After washing, cells were resuspended in FACS buffer and examined by flow cytometry. This study demonstrates that the β, and α₄ expressing populations also express glycophorin A, confirming these integrin chains are expressed on erythrocytes.

Example 5 : Binding of SSRBC. but not Normal Erythrocytes to TNF-α Stimulated Endothelial Cells

Human umbilical vein endothelial cells (HUVEC) were isolated by standard procedures (see e.g.. Jaffe et al., J. Clin. Invest. 52:2745 (1973)). HUVEC were plated on to gelatin coated LabTek chambers, grown to confluence, and stimulated with tumor necrosis factor α (500 U/ml x 6 hours, Genentech, Inc., San Francisco, CA). The binding of normal of SSRBC were examined utilizing a parallel plate flow chamber apparatus at a shear stress of 1 dyne/cm² by standard procedures (see e.g.. Wick et al., J. Clin. Invest. 80:95 (1987)). The results are shown in Figure 5. Results represent the mean number of adherent erythrocytes/mm²± S.D. The expression of cell surface VCAM-1 on HUVEC stimulated in parallel was determined in parallel by ELISA as in Example 1. The results are expressed as

O.D. 450 nm and represent the mean of four data points ± S.D.

Example 6 : VLA-4 and VCAM-1 Dependence of Binding of SSRBC to Activated Endothelial Cells

The binding of SSRBC was examined under flow conditions as described above to unstimulated HUVEC or monolayers stimulated with TNFα (500 U/ml x 6 hours). TNFα stimulated endothelial cells were left untreated or preincubated with mAb recognizing VCAM-1 (mAb BBA 6, 10 μg/ml, R&D Systems, Minneapolis, MN) or ICAM-1 (mAb 84H10, tissue culture supernatants. Sickle RBC were untreated or preincubated with anti-α₄ (50 μg/ml, mAb HP2/1. The results are shown in Figure 6.

Example 7 : Preparation Of A Humanized Anti-VLA-4 Antibody

The complementarity determining regions (CDRs) of the light and heavy chains of the

anti-VLA-4 monoclonal antibody HP 1/2 were determined according to the sequence alignment

approach of Kabat et al., 1991, 5th Ed., 4 vol., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, NIH, USA. The CDRs of murine HP1/2 V_H correspond to the residues identified in the humanized V_H sequences disclosed herein as amino acids 31-35 (CDRl), 50-66 (CDR2) and 99-110 (CDR3), which respectively correspond to amino acids 31-35, 50-65 and 95-102 in the Kabat alignment. The CDRs of murine HP1/2 V_κ correspond to the residues identified in the humanized V_κ sequences disclosed herein as amino acids 24-34 (CDRl), 50-56 (CDR2) and 89-97 (CDR3), and to the same residues in the Kabat alignment. The Kabat NEWM framework was chosen to accept the heavy chain CDRs and the Kabat REI framework was chosen to accept the kappa chain CDRs. Transplantation of the CDRs into the human frameworks was achieved by using M13 mutagenesis vectors and synthetic oligonucleotides containing the HP 1/2 CDR-encoding sequences flanked by short sequences derived from the frameworks. The V_H mutagenesis vector, M13VHPCR1 contains the NEWM framework and has been described by Orlandi et al., Proc. Natl. Acad. Sci USA 86:3833-3837 (1989). The V_κ mutagenesis vector, M13VKPCR2 contains essentially the REI framework and is identical to the M13VKPCR1 , vector described by Orlandi et al., except that there is a single amino acid change from Val to

Glu in framework 4. Transplanted product was recovered by PCR and cloned into M13mpl9 for sequencing. The transplanted V_H sequence [SEQ. ID NO: 3] is shown in Figure 9, panel A. In addition to the CDR grafting, this product encodes the murine amino acids at positions

27-30 and an Arg to Asp change at position 94. The transplanted V_κ sequence [SEQ. ID NO: 4] is shown in Figure 9, panel B.

Additional modifications were introduced via the two step PCR-directed mutagenesis method of Ho et al., Gene 77:51-59 (1989). For the V_H sequence, position 24 (Kabat numbering) was changed from Vak to Ala and position 75 (Kabat numbering) was changed from Lys to Ser, then amino acid positions 27-30 and 94 were mutated back to the NEWM sequences. The final humanized V_H sequence [SEQ. ID NO: 5] is shown in Figure 8, panel A. For the V_κ sequence, the same two step PCR-directed mutagenesis approach was used to introduce additional modifications. The final humanized V_κ sequence [SEQ. ID NO: 6] is shown in Figure 8, panel B.

The entire V_H and V_κ regions of humanized HP1/2 were cloned into appropriate expression vectors. The appropriate human IgGl, IgG4 or kappa constant region was then added to the vector in appropriate reading frame with respect to the murine variable regions. The vectors were cotransfected into YB2/0 ray myeloma cells (available from ATCC), which were then selected for the presence of both vectors. ELISA analysis of cell supernatants demonstrated that the humanized antibody produced by these cells was at least equipotent with murine HP1/2. The cell line expressing this humanized antibody was deposited with the ATCC on November 3, 1992 and given accession number CRL 11 175.

Example 8: Ervthrocvte adhesive proteins in the pathogenesis of necrotizing enterocolitis in premature infants Necrotizing enterocolitis (NEC) is the most common serious acquired gastrointestinal tract disorder in the neonatal intensive care unit, responsible for an increasing number of infectious disease associated deaths in low birth weight babies. It has been estimated that

NEC affects at least 25,000 newborn children in the United States annually and carries an

overall mortality as high as 50%. The continued improvement in the treatment of hyaline membrane disease of the lung is likely to result in an increasing population of infants at risk for developing NEC. NEC is a disease of unknown origin. Despite extensive study, the initiating events and the pathogenesis of NEC have not been established. The initial clinical manifestations of necrotizing enterocolitis may be indistinguishable from those of neonatal sepsis and shock, with pneumatosis intestinalis and intrahepatic venous gas the radiographic hallmarks required to confirm the diagnosis. Epidemiological observations emphasize the potential roles of infection, feeding, and local vascular compromise of the GI tract in the pathogenesis of NEC, but it is likely that NEC represents a final common pathway of response of the immature intestine to injury which may be initiated by multiple factors.

Prematurity is recognized as the primary risk factor which predisposes infants to NEC. Disturbed hemodynamics associated with hyperviscosity has been implicated as a primary factor in the development of intestinal injury. However, explanations relying on hyperviscosity often suggest that erythrocytes are merely passive participants in vaso- occlusive disease.

Adherence of erythrocytes may initiate microvascular complications in diseases such as sickle cell anemia and diabetes. The primary event may be the binding of adhesion protein expressing reticulocytes to vascular endothelial cells. Vessels partially occluded by bound reticulocytes may be completely occluded by trapping of dense, sickled cells. Reticulocytes may bind to endothelial cells after opsonization with thrombospondin (TSP-1) or multimers of von Willebrand factor. Additionally, reticulocytes are capable of binding directly to endothelial cell surface ligands. Flow cytometric analysis of sickle erythrocytes demonstrated that a percentage of reticulocytes in peripheral circulation of individuals with sickle cell anemia abnormally express specific adhesion receptors, including CD36 and the a4bl integrin complex. The functional importance of these adhesion proteins in erythrocyte-endothelial cell interactions in sickle cell disease and malaria has been extensively examined utilizing a flow adhesion model. Reticulocyte expression of CD36 is important in TSP-1 mediated binding of erythrocytes to endothelial cells and erythrocyte express of the a4bl integrin complex can mediate binding to the inducible endothelial cell adhesion molecule VCAM-1 on cytokine activated endothelial cells. Thus the expression of adhesive proteins on population of erythrocytes may result in red cell binding to endothelium and vascular occlusion

The expression of CD36 and the a4bl integrin complex is not limited to reticulocytes of patients with sickle cell anemia. In particular, neonates with elevated reticulocyte counts also demonstrate similar, if not higher percentages of CD36 and a4bl expressing red cells. It is interesting to speculate that premature infants, particularly babies with high reticulocyte counts, would be at risk for vaso-occlusive complications. NEC may be a clinical manifestation of this predisposition. The mucosal ischemia proposed as an initiating event in NEC may not be due to an abrupt cessation of mesenteric blood flow, but rather due to vascular compromise induced by the binding of "sticky" erythrocytes to activated endothelium. Adherent erythrocytes may serve as a nidus for platelet activation of the clotting cascade with resultant complete occlusion and bowel necrosis.

The localization of pathology to bowel is not adequately understood. It is curious to note that NEC often presents after initial feeding. It has been postulated that the osmotic load

of feeding may result in depletion of intravascular fluid, hyperviscosity, and decreased blood

flow. An alternative hypothesis is that feeding results in increased bacterial carriage with concomitant increases in bacterial lipopolysaccharide (LPS) production. LPS is a potent inducer of VCAM-1 on endothelial cells. It is likely that feeding of premature infants may initially lead to expression of the adhesive ligands on vascular endothelial cells in the bowel, thus providing the ligand pair for the a4bl integrin complex on reticulocytes. To examine affected and unaffected bowel from patients with NEC for the expression of adhesion proteins which serve as ligands for adhesive erythrocytes, frozen sections of bowel were examined for the expression of vascular cell adhesion molecule -1 (VCAM-1) and CD36. These studies showed that appropriate ligands for red cell adhesion are present on blood vessels in the bowel of patients with NEC. The endothelium of the bowel expresses a variety of proteins which may serve as ligands for cell attachment. Intercellular adhesion molecule -1 (ICAM-1) is constitutively expressed protein on virtually all endothelial cells which serves as a ligand for the leukocyte adhesion proteins LFA-1 and Mac-1. The expression of ICAM-1 can be increased by a variety of cytokines inclusing tumor necrosis factor (TNF), interleukin 1 (IL-1), and interferon g (IFN- g). Additionally, two additionally adhesion molecules, E-selectin and vascular cell adhesion

molecule 1 (VCAM-1), can be induced de novo on endothelial cells. These may serve as ligands for adhesion receptors on the surface of a variety of cells. In fact, all three adhesion receptors have been shown to play a role in red cell binding to endothelial cells under certain clinical circumstances.

We have examined bowel obtained from 3 infants with NEC and 3 infants with non- infarctive bowel disease necessitating limited normal bowel resection. Normal bowel demonstrated baseline expression of ICAM-1, similar to what has been observed previously in other microvascular beds. However, normal bowel expressed essentially no expression of adhesive proteins E-selectin or VCAM-1. In all cases of NEC, bowel specimens demonstrated marked inflammatory cell infiltrates and mucosal damage. In addition, blood vessels in the submucosa and muscularis displayed striking expression of both E-selectin and VCAM-1. Also, expression of ICAM-1 was increased in the bowel from patients with NEC.

These preliminary data suggest that the expression of relevant bowel endothelial adhesion proteins is increased in NEC. In particular, the expression of VCAM-1, which is an adhesive ligand for a4bl expressing erythrocytes, is induced on blood vessels in the bowel of children affected with NEC. Red cells from infants at risk for NEC and from normal term infants were examined by flow cytometry for the expression of adhesion receptors. Erythrocytes from six neonates with congenital heart disease were examined for expression of adhesive ligands. Two of these specimens were obtained from infants with NEC. Blood specimens from four infants felt to be clinically at risk for NEC demonstrated reticulocyte counts ranging from 1-4.2% while the two infants with clinical manifestations of NEC expressed reticulocyte counts of 5.5 and 9% respectively (Table 1). Additionally, approximately half of the reticulocytes from infants with NEC expressed high levels of the adhesive proteins CD36 and the a4bl integrin complex

(Table 1 and Figure 10) and unlike expression seen in patients with sickle cell disease, adhesive proteins were also seen on non-reticulocytes as well (Table 1, Figures 10 and 11.)

However, red cells from the four at risk infants as well as infants hospitalized for other

reasons and felt to be at low risk displayed much lower adhesion molecule expression. (Table 1). Thus, within the clinical context of NEC, we have preliminary data that suggests the infants affected with NEC express adhesive ligands on both their erythrocytes and appropriate counter receptors on intestinal endothelial cells.

To examine whether erythrocytes from infants with or at known risk for NEC are more adhesive to endothelial cells under flow conditions, we measured the binding of red blood cells to microvascular endothelial cells utilizing a model which can measure binding at a shear stress resembling those found in post-capillary venules. The binding of these red cells can be blocked by antibodies or peptides which interact with the appropriate adhesion receptors. These studies demonstrate that reticulocyte adhesion was mediated by the adhesive receptors CD36 and a4bl on the red cell, and VCAM-1 on endothelial cells.

TABLE 1 : Expression of adhesion proteins on erythrocytes from infants at high or low risk or affected with NEC.

Reticulocyte CD36 expression α4βl expression Clinical diagnosis

INFANT count (%) (5)

1 5.5 5.75 5.75 congenial heart disease with NEC

2 0.86 0.6 0.4 congenial heart diseasef

3 3.6 0.72 1.4 congenial heart diseasef

4 4.2 0.2 0.4 congenial heart diseasef

5 9.0 6.9 6.8 congenial heart

M σ* disease with NEC

6 1.4 2.0 4.1 congenial heart diseasef

7 4.1 0.75 .46 hyper- bilirubinemia*

8 2.9 0.16 .15 hyper- bilirubinemia*

9 3.5 0.15 .25 normal newborn

10 5.2 0.7 1.1 hyper- bilirubinemia*

f Known risk for development of NEC * No known risk for development of NEC

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: EMORY UNIVERSITY

GEORGIA TECH RESEARCH CORPORATION SWERLICK, Robert A. ECKMAN, James R. WICK, Timothy M.

(ii) TITLE OF INVENTION: Method of Inhibiting Binding of

Reticulocytes to Endothelium by Interfering with VLA-4/VCAM-1 Interactions

(iii) NUMBER OF SEQUENCES: 6

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Leslie M. Levine

(B) STREET: 14 Cambridge Center

(C) CITY: Cambridge

(D) STATE: Massachusetts

(E) COUNTRY: USA

(F) ZIP: 02142

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/122,228

(B) FILING DATE: 15-SEP-1993

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Levine, Leslie M.

(B) REGISTRATION NUMBER: 35,245

(C) REFERENCE/DOCKET NUMBER: D017CIP

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (617) 252-9810

(B) TELEFAX: (617) 252-9617

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 360 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GTCAAACTGC AGCAGTCTGG GGCAGAGCTT GTGAAGCCAG GGGCCTCAGT CAAGTTGTCC 60

TGCACAGCTT CTGGCTTCAA CATTAAAGAC ACCTATATGC ACTGGGTGAA GCAGAGGCCT 120

GAACAGGGCC TGGAGTGGAT TGGAAGGATT GATCCTGCGA GTGGCGATAC TAAATATGAC 180

CCGAAGTTCC AGGTCAAGGC CACTATTACA GCGGACACGT CCTCCAACAC AGCCTGGCTG 240

CAGCTCAGCA GCCTGACATC TGAGGACACT GCCGTCTACT ACTGTGCAGA CGGAATGTGG 300

GTATCAACGG GATATGCTCT GGACTTCTGG GGCCAAGGGA CCACGGTCAC CGTCTCCTCA 360

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 318 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

AGTATTGTGA TGACCCAGAC TCCCAAATTC CTGCTTGTTT CAGCAGGAGA CAGGGTTACC 60

ATAACCTGCA AGGCCAGTCA GAGTGTGACT AATGATGTAG CTTGGTACCA ACAGAAGCCA 120

GGGCAGTCTC CTAAACTGCT GATATATTAT GCATCCAATC GCTACACTGG AGTCCCTGAT 180

CGCTTCACTG GCAGTGGATA TGGGACGGAT TTCACTTTCA CCATCAGCAC TGTGCAGGCT 240

GAAGACCTGG CAGTTTATTT CTGTCAGCAG GATTATAGCT CTCCGTACAC GTTCGGAGGG 300

GGGACCAAGC TGGAGATC 318

(2) INFORMATION FOR SEQ ID NO:3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 429 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: sig_peptide

(B) LOCATION: 1..57

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 58..429

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..429

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "pMDR1019 insert: Stage 1 heavy chain variable region "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

ATG GAC TGG ACC TGG AGG GTC TTC TGC TTG CTG GCT GTA GCA CCA GGT 48 Met Asp Trp Thr Trp Arg Val Phe Cys Leu Leu Ala Val Ala Pro Gly -19 -15 -10 -5

GCC CAC TCC CAG GTC CAA CTG CAG GAG AGC GGT CCA GGT CTT GTG AGA 96 Ala His Ser Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg 1 5 10

CCT AGC CAG ACC CTG AGC CTG ACC TGC ACC GTG TCT GGC TTC AAC ATT 144 Pro Ser Gin Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Asn lie 15 20 25

AAA GAC ACC TAT ATG CAC TGG GTG AGA CAG CCA CCT GGA CGA GGT CTT 192 Lys Asp Thr Tyr Met His Trp Val Arg Gin Pro Pro Gly Arg Gly Leu 30 35 40 45

GAG TGG ATT GGA AGG ATT GAT CCT GCG AGT GGC GAT ACT AAA TAT GAC 240 Glu Trp lie Gly Arg lie Asp Pro Ala Ser Gly Asp Thr Lys Tyr Asp 50 55 60

CCG AAG TTC CAG GTC AGA GTG ACA ATG CTG GTA GAC ACC AGC AAG AAC 288 Pro Lys Phe Gin Val Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn 65 70 75

CAG TTC AGC CTG AGA CTC AGC AGC GTG ACA GCC GCC GAC ACC GCG GTC 336 Gin Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val 80 85 90

TAT TAT TGT GCA GAC GGA ATG TGG GTA TCA ACG GGA TAT GCT CTG GAC 384 Tyr Tyr Cys Ala Asp Gly Met Trp Val Ser Thr Gly Tyr Ala Leu Asp 95 100 105

TTC TGG GGC CAA GGG ACC ACG GTC ACC GTC TCC TCA GGT GAG TCC 429

Phe Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser Gly Glu Ser 110 115 120 (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 386 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: sig_peptide

(B) LOCATION: 1..57

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 58..386

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..386

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "pBAG190 insert: VK1

(DQL) light chain variable region"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

ATG GGT TGG TCC TGC ATC ATC CTG TTC CTG GTT GCT ACC GCT ACC GGT 48 Met Gly Trp Ser Cys lie lie Leu Phe Leu Val Ala Thr Ala Thr Gly -19 -15 -10 -5

GTT CAC TCC GAC ATC CAG CTG ACC CAG AGC CCA AGC AGC CTG AGC GCC 96 Val His Ser Asp lie Gin Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala 1 5 10

AGC GTG GGT GAC AGA GTG ACC ATC ACC TGT AAG GCC AGT CAG AGT GTG 144 Ser Val Gly Asp Arg Val Thr lie Thr Cys Lys Ala Ser Gin Ser Val 15 20 25

ACT AAT GAT GTA GCT TGG TAC CAG CAG AAG CCA GGT AAG GCT CCA AAG 192 Thr Asn Asp Val Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys 30 35 40 45

CTG CTG ATC TAC TAT GCA TCC AAT CGC TAC ACT GGT GTG CCA AGC AGA 240 Leu Leu lie Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg 50 55 60

TTC AGC GGT AGC GGT AGC GGT ACC GAC TTC ACC TTC ACC ATC AGC AGC 288 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser 65 70 75

CTC CAG CCA GAG GAC ATC GCC ACC TAC TAC TGC CAG CAG GAT TAT AGC 336 Leu Gin Pro Glu Asp lie Ala Thr Tyr Tyr Cys Gin Gin Asp Tyr Ser 80 85 90

TCT CCG TAC ACG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA CGT AAG TG 386 Ser Pro Tyr Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys Arg Lys 95 100 105 (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 429 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: sig_peptide

(B) LOCATION: 1..57

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 58..429

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..429

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "pBAG195 insert: AS heavy chain variable region"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 :

ATG GAC TGG ACC TGG AGG GTC TTC TGC TTG CTG GCT GTA GCA CCA GGT 48

Met Asp Trp Thr Trp Arg Val Phe Cys Leu Leu Ala Val Ala Pro Gly

-19 -15 -10 -5

GCC CAC TCC CAG GTC CAA CTG CAG GAG AGC GGT CCA GGT CTT GTG AGA 96 Ala His Ser Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg 1 5 10

CCT AGC CAG ACC CTG AGC CTG ACC TGC ACC GCG TCT GGC TTC AAC ATT 144 Pro Ser Gin Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Asn lie 15 20 25

AAA GAC ACC TAT ATG CAC TGG GTG AGA CAG CCA CCT GGA CGA GGT CTT 192

Lys Asp Thr Tyr Met His Trp Val Arg Gin Pro Pro Gly Arg Gly Leu

30 35 40 45

GAG TGG ATT GGA AGG ATT GAT CCT GCG AGT GGC GAT ACT AAA TAT GAC 240 Glu Trp lie Gly Arg lie Asp Pro Ala Ser Gly Asp Thr Lys Tyr Asp 50 55 60

CCG AAG TTC CAG GTC AGA GTG ACA ATG CTG GTA GAC ACC AGC AGC AAC 288 Pro Lys Phe Gin Val Arg Val Thr Met Leu Val Asp Thr Ser Ser Asn 65 70 75

CAG TTC AGC CTG AGA CTC AGC AGC GTG ACA GCC GCC GAC ACC GCG GTC 336 Gin Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val 80 85 90 TAT TAT TGT GCA GAC GGA ATG TGG GTA TCA ACG GGA TAT GCT CTG GAC 384 Tyr Tyr Cys Ala Asp Gly Met Trp Val Ser Thr Gly Tyr Ala Leu Asp 95 100 105

TTC TGG GGC CAA GGG ACC ACG GTC ACC GTC TCC TCA GGT GAG TCC 429

Phe Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser Gly Glu Ser 110 115 120

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 386 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: sig_peptide

(B) LOCATION: 1..57

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 58..386

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..386

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "pBAG198 insert: VK2 (SVMDY) light chain variable region"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

ATG GGT TGG TCC TGC ATC ATC CTG TTC CTG GTT GCT ACC GCT ACC GGT 48 Met Gly Trp Ser Cys lie lie Leu Phe Leu Val Ala Thr Ala Thr Gly -19 -15 -10 -5

GTC CAC TCC AGC ATC GTG ATG ACC CAG AGC CCA AGC AGC CTG AGC GCC 96 Val His Ser Ser lie Val Met Thr Gin Ser Pro Ser Ser Leu Ser Ala 1 5 10

AGC GTG GGT GAC AGA GTG ACC ATC ACC TGT AAG GCC AGT CAG AGT GTG 144 Ser Val Gly Asp Arg Val Thr lie Thr Cys Lys Ala Ser Gin Ser Val 15 20 25

ACT AAT GAT GTA GCT TGG TAC CAG CAG AAG CCA GGT AAG GCT CCA AAG 192 Thr Asn Asp Val Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys 30 35 40 45 CTG CTG ATC TAC TAT GCA TCC AAT CGC TAC ACT GGT GTG CCA GAT AGA 240

Leu Leu lie Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg 50 55 60

TTC AGC GGT AGC GGT TAT GGT ACC GAC TTC ACC TTC ACC ATC AGC AGC 288

Phe Ser Gly Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr lie Ser Ser 65 70 75

CTC CAG CCA GAG GAC ATC GCC ACC TAC TAC TGC CAG CAG GAT TAT AGC 336

Leu Gin Pro Glu Asp lie Ala Thr Tyr Tyr Cys Gin Gin Asp Tyr Ser 80 85 90 .

TCT CCG TAC ACG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA CGT AAG TG 386

Ser Pro Tyr Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys Arg Lys

95 100 105

Claims (9)

What is claimed is:

1. A method of inhibiting binding between reticulocytes that express VLA-4 and endothelial cells that express VCAM-1, the method comprising the step of administering a blocking agent of VLA-4 antigen on the surface of reticulocytes.

2. The method according to claim 1, wherein the blocking agent is selected from the group consisting of anti-VLA-4 or anti-VCAM-1 antibody which may optionally be human, chimeric, single chain, or humanized, or Fab, Fab', F(ab')₂ or F(v) fragments thereof, fibronectin, fibronectin having an alternatively spliced non-type m connecting segment, fibronectin peptides containing the amino acid sequence EILDV or a similar conservatively substituted amino acid sequence that blocks VLA-4-mediated adhesion, soluble VCAM-1, bifunctional VCAM-1/Ig fusion proteins and VCAM-1 peptides.

3. The method according to claim 2, wherein the blocking agent is a humanized anti-VLA-4 or anti-VCAM-1 antibody.

4. The method of claim 1 , wherein the reticulocytes that express VLA-4 are sickle reticulocytes of a patient suffering from sickle cell anemia.

5. The method of claim 2, wherein the reticulocytes that express VLA-4 are sickle reticulocytes of a patient suffering from sickle cell anemia.

6. The method of claim 3, wherein the reticulocytes that express VLA-4 are sickle reticulocytes of a patient suffering from sickle cell anemia.

7. The method of claim 1, wherein the reticulocytes that express VLA-4 are sickle reticulocytes of a patient suffering from necrotizing enterocolitis.

8. The method of claim 2, wherein the reticulocytes that express VLA-4 are sickle reticulocytes of a patient suffering from necrotizing enterocolitis.

9. The method of claim 3, wherein the reticulocytes that express VLA-4 are sickle

reticulocytes of a patient suffering from necrotizing enterocolitis.