WO1992004912A1

WO1992004912A1 - Composition for inducing granulocyte production or b cell production in peripheral blood

Info

Publication number: WO1992004912A1
Application number: PCT/US1991/006850
Authority: WO
Inventors: Joseph Carlino; Naina Singh; Larry Ellingsworth
Original assignee: Celtrix Laboratories, Inc.
Priority date: 1990-09-21
Filing date: 1991-09-20
Publication date: 1992-04-02
Also published as: EP0502171A4; EP0502171A1; JPH05502244A; AU645619B2; AU8637991A; CA2067498A1; CA2067498C; JP2625033B2

Abstract

CIFs (TGF-βs) are used to induce B cell production in peripheral blood or to induce granulopoiesis.

Description

COMPOSITION FOR INDUCING GRANULOCYTE

PRODUCTION OR B CELL PRODUCTION IN PERIPHERAL BLOOD

Description Technical Field

This invention relates to using polypeptides called cartilage-inducing factors (CIFs) and functionally related polypeptides as factors for inducing B cell proliferation in peripheral blood or granulocyte

proliferation.

Background

Commonly owned copending U.S. patent 4,806,523 describes two bovine bone-derived CIFs, designated CIF-A and CIF-B. Both have molecular weights of approximately

26,000 daltons by SDS-PAGE and are dimers. They each exhibit in vitro chondrogenic activity by themselves, as measured by cartilage specific proteoglycan (PG)

production in an agarose gel culture model using fetal rat mesenchymal cells. Neither, however, is

chondrogenically active in vivo by itself. Amino acid sequencing of the CIF-A showed that it has a partial (30 amino acids) N-terminal sequence identical to that reported for a human placenta-derived polypeptide called beta-type transforming growth factor (TGF-β). The partial N-terminal sequence of CIF-B is different from that of TGF-β. Both CIFs exhibit activity in the TGF-β assay (ability to induce anchorage-independent growth of normal rat kidney cell colonies in soft agar). TGF-β derived from bovine kidney, human

placenta, and human platelets is described in International Patent Application PCT/US83/01460, published 29 March 1984 under no. WO84/01106, and EPA 84450016.5, published 19 December 1984 under no. 0128849. These applications present data showing that such TGF-β (1) promotes cell proliferation in the above mentioned soft agar culture assay and (2) promotes cell proliferation and protein deposition in a rat soft tissue wound healing model. The applications characterize the TGF-βs as being dimers having a molecular weight of approximately 26,000 daltons by SDS-PAGE. CIF-A is now referred to as TGF- β1, and CIF-B is referred to as TGF-β2. Disclosure of the Invention

The present invention is based on the finding that CIFs (or TGF-βs) induce the production of B cells in peripheral blood or induce the production of

granulocytes. For convenience, the term CIF is used in this section and the claims as a generic term to

encompass CIF-A, CIF-B, the TGF-βs and functional

equivalents thereof.

Accordingly, the invention is the use of a CIF for the manufacture of a medicament for inducing

production of B cells in peripheral blood or for inducing production of granulocytes.

Brief Description of the Drawings

Figure l is the amino acid sequence of platelet-derived human TGF-β monomer.

Figure 2 is a graph of the optical densities (absorbances) (280 nm) of the gel filtration fractions of the example 1 ( C) . Figure 3 is a graph of the optical densities (280 nm) of eluate fractions from the preparative ion exchange chromatography of the example 1 (

D) .

Figure 4 graphically depicts the increase in peripheral white blood cells following in vivo

administration of TGF-β1, as reported in the example.

Figures 5A and 5B graphically depict the increase in peripheral B cells following in vivo

administration of TGF-β1, as reported in the example.

Figures 6A and 6B graphically depict the increase in the percentage of both small and large B cells following in vivo administration of TGF-β1, as reported in the example. Modes for Carrying Out the Invention

The term "functional equivalent" as used to describe a polypeptide is intended to mean polypeptides, whether native or synthetic and regardless of species or derivation, that have the same amino acid sequence as the referenced polypeptide, and polypeptides of substantially homologous (i.e., at least 90% identity in amino acid sequence) but different amino acid sequence, which difference (s) does not affect biological activity (i.e., the ability to reduce B cell and granulocyte proliferation) adversely.

CIF-A, CIF-B and TGF-βs exhibit activity in the TGF-β assay described in Methods for Preparation of

Media, Supplements, and Substrate for Serum-Free Animal Cell Culture (1984) pp 181-194, Alan R. Liss, Inc. That assay determines ability to induce anchorage-independent growth in non-neoplastic normal rat kidney (NRK)

fibroblasts by measuring the formation of cell colonies in soft agar. Procedures for obtaining TGF-βs from platelets, placenta and kidney tissues are described in International patent publication WO84/01106 and EPA 128,849. Briefly, they involve extracting the source material with acid-ethanol, sizing the extract by gel filtration, and isolating the TGF-β from the filtrate by high performance liquid chromatography (HPLC).

A procedure for isolating CIFs from bovine bone is described in commonly owned, U.S. patent 4,806,523.

It involves extracting demineralized bone (DMB) with an extractant (e.g., ≥4M guanidine hydrochloride, 8M urea) that solubilizes nonfibrous proteins, gel filtering the extract to obtain a < 30 kDa fraction, chromatographing the fraction on carboxymethyl cellulose (CMC) at pH

4.5-5.5, preferably 4.8, eluting the CMC-adsorbed

fraction with an NaCl gradient, and purifying the

proteins from the portion eluting at about 150-250 mM NaCl by RP-HPLC or gel electrophoresis.

CIF-A, CIF-B, and the TGF-βs isolated to date from natural sources are polypeptide dimers of

approximately 25 to 26 kDa molecular weight as determined by SDS-PAGE. Nature (1985) 316:701-705 reported a cDNA nucleotide sequence and deduced amino acid sequence for platelet-derived human TGF-β. Mature

platelet-derived human TGF-β is characterized as a homodimer of a 112 amino acid-long monomer.

Platelet/placenta/kidney-derived TGF-β and CIF-A and CIF-B are non-species specific as regards TGF-β activity. It is believed, therefore, that these polypeptides have been highly conserved among animal species (i.e., a given polypeptide from different mammalian species has an amino acid sequence that varies, if at all, in one or more amino acid residue additives,

deletions, or substitutions that do not affect the non-species specific activity of the molecule adversely) and have cross-species functionality. Accordingly, CIF-A, CIF-B, and the TGF-βs may be derived from cells or tissue of diverse animal origin or may be obtained by recombinant DNA technology. Correlatively, CIF (TGF-β) from one vertebrate species may be used to treat another vertebrate species. The most common usage of CIF (TGF-β) as an anti-inflammatory agent will be in the treatment of humans, domestic animals such as cattle, sheep, and pigs, and sports or pet animals such as dogs, cats, and horses. CIF-A and CIF-B are preferred for use in the invention method. Examples

The following examples are intended to illustrate specific embodiments of the invention. They are not intended to limit the invention in any manner. Example 1

(Preparation of CIFs from Bone) A. Preparation of Demineralized Bone

Bovine metatarsal bone was obtained fresh from a slaughterhouse and transported on dry ice. The bones were cleaned of marrow and non-bone tissues, broken in fragments smaller than 1 cm diameter, and pulverized in a mill at 4°C. The pulverized bone was washed twice with 9.4 liters of double distilled water per Kg of bone for about 15 min each, and then washed overnight in 0.01 N HCl at 4°C. Washed bone was defatted using 3 X3 volumes ethanol, followed by 3 X 3 volumes diethylether, each washed for 20 min and all at room temperature. The resulting defatted bone powder was then demineralized in 0.5 N HCl (25 L/Kg defatted bone) at 4°C. The acid was decanted and the resulting DMB washed until the wash pH was greater than 4, and the DMB dried on a suction filter. B. Extraction of Noncollagenous Proteins

The DMB as prepared in

A was extracted with 3.3 L of 4 M guanidine-HCl, 10 mM ethylenediamine- tetraacetic acid (EDTA), pH 6.8, 1 mM PMSF, 10 mM NEM per Kg for 16 hr, the suspension suction filtered and the non-soluble material extracted again for 4 hr. The soluble fractions were combined and concentrated at least 5-fold by ultrafiltration using an Amicon ultrafiltration (10K) unit, and the concentrate dialyzed against 6 changes of 35 volumes cold deionized water over a period of 4 days, and then lyophilized. All of the procedures of this paragraph were conducted at 4°C except the lyophilization which was conducted under standard

lyophilization conditions.

C. Gel Filtration

The extract from

B, redissolved in 4 M guanidine-HCl, was fractionated on a Sephacryl^® S-200 column equilibrated in 4 M guanidine-HCl, 0.02% sodium azide, 10 mM EDTA, pH 6.8. Fractions were assayed by their absorbance at 280 nm and the fractions were combined as shown in Figure 1. Fraction F2 of Figure 1, constituting a low molecular weight (LMW, 10,000-30,000 daltons) protein fraction was dialyzed against 6 changes of 180 volumes of deionized water and lyophilized. All operations except lyophilization and dialysis (4°C) were conducted at room temperature.

D. Ion Exchange Chromatography

Fraction F2 from

∬C was dissolved in 6 M urea,

10 mM NaCl, 1 mM NEM, 50 mM sodium acetate, pH 4.8 and centrifuged at 10,000 rpm for 5 min. The supernatant was fractionated on a CM52 (a commercially available CMC) column equilibrated in the same buffer. Bound proteins were eluted from the column using a 10 mM to 400 mM NaCl gradient in the same buffer, and a total volume of 350 mL at a flow rate of 27 mL/hr. Three major fractions, designated CM-1, CM-2 and CM-3, were collected as shown in Figure 2. CM-2 and CM-3 eluted at approximately

150-250 mM NaCl. Each fraction was dialyzed against 6 changes of 110 volumes of deionized water for 4 days and lyophilized. All of the foregoing operations were conducted at room temperature except dialysis (4°C). E. RP-HPLC

The combined lyophilized fractions CM-2 and CM-3 from

∬D were each dissolved in 0.1% trifluoroacetic acid (TFA) and aliquots of the solutions loaded onto a Vydac^® C18 RP-HPLC columns (4.6 mm ID X 25 cm) and washed with 0.1% TFA for 5 min at 1 mL/min. The eluting solvent was a 0%-60% acetonitrile gradient in 0.1% TFA at a rate of 2%/min.

Two peaks were obtained from the RP-HPLC of combined CM-2 and CM-3: peak A at about 29.5 min and peak B at about 31.2 min. The proteins of these peaks are the subject of said U.S. patent application serial no. 630,938, and are designated CIF-A and CIF-B,

respectively.

The proteins were stored in 0.1% TFA/acetonitrile eluting solution at -20°C until used.

F. Characterization of CIF-A and CIF-B

Table 1 below gives the partial amino acid compositions of CIF-A and CIF-B.

SDS-PAGE analysis of CIF-A and CIF-B indicate that both have a molecular weight of approximately 26,000 daltons. Both proteins exhibited activity in the TGF-β assay referred to above comparable to that reported for TGF-βs derived from human platelets, human placenta, or bovine kidney.

N-terminal amino acid sequencing of the first 30 amino acids of CIF-A and CIF-B was carried out and found to be as follows: CIF-A

1 5 10

Ala-Leu-Asp-Thr-Asn-Tyr-Cys-Phe-Ser-Ser-Thr-Glu-Lys-

15 20 25

Asn-Cys-Cys-Val-Arg-Gln-Leu-Tyr-Ile-Asp-Phe-Arg-Lys-

30

Asp-Leu-Gly-Trp- . CIF-B

1 5 10

Ala-Leu-Asp-Ala-Ala-Tyr-Cys-Phe-Arg-Asn-Val-Gln-Asp-

15 20 25

Asn- ( Cys-Cys ) -Leu-Arg-Pro-Leu-Tyr-Ile-Asp-Phe-Lys-Arg-

30

Asp-Leu-Gly-Trp- .

The N-terminal amino acid sequence of CIF-A is identical to that reported for platelet-derived human TGF-β (see

Nature, supra ) .

Example 2

(Increase in Peripheral Blood B cells) This experiment demonstrates induction of B- cells in the peripheral blood using CIF-A.

TGF-β1 (CIF-A) was purified from bovine bone and concentrated in 45% EtOH, 11 mM HCl to a

concentration of about 5000 μg/mL, and stored at -20°C until required. The TGF-β1 was then diluted in PBS (0.1%

MSA) to a concentration of 125 μg/mL, 1% EtOH, neutral pH.

Male C57B1/6 mice were treated daily for 14 days with TGF-β1 (25 μg, 0.2 mL, subcutaneous). Control mice were given vehicle containing no TGF-β1. The mice were evaluated 24 hours following the last treatment for histologic assessment of spleen and bone marrow,

hematologic evaluation, body weight, and phenotypic assessment of white cells from blood, spleen, and bone marrow. White cells were typed using monoclonal

antibodies and fluorescence-activated cell sorting

(FACS). B-cells were typed using MAb B220 (ATCC No. TIB 164). Granulocytes were typed using MAb 8C5, supplied by Dr. R.L. Coffman (DNAX Research Institute, Palo Alto, CA). T_H cells were typed using anti-CD4 MAbs, and T_C/S cells using anti-CD8, both obtained from Becton- Dickinson. Propidium iodide stain was used to determine the percentage of non-viable cells.

Hematologic evaluations showed that treatment with TGF-β1 significantly elevated peripheral blood white cell counts (Figure 4), and depressed red blood cell counts. Analysis by FACS determined that this increase in white blood cells was due at least in part to an increase in B-cells (B220⁺ cell percentage doubled).

Figure 5A depicts the FACS analysis of the control, while Figure 5B depicts the FACS analysis of the TGF-β1 treated sample. There was no change in the peripheral blood for either T-cell marker. The percentage of granulocytes (8C5⁺) decreased, but the decrease in proportion was largely due to the increase in B-cells rather than a decrease in the absolute number of granulocytes. Size analysis (forward light scatter) of B220⁺ indicated that there was an increase in the percentage of both small and large B-cells (i.e., B220⁺). Figure 6A depicts size analysis of the control, while Figure 6B depicts size analysis of the TGF-β1 treated sample.

An increase in granulocytes was noted in the spleen and bone marrow by 8C5 staining. No differences were seen between control and treated mice in the percentage of B and T-cells in the spleen and bone marrow. In addition, TGF-β1 treatments did not affect propidium iodide staining, indicating that administration of TGF-β1 did not affect white blood cell viability.

Thus, TGF-β1 increased the number of peripheral blood B-cells. Accordingly, formulations of TGF-β are useful for treating indications associated with depressed B-cell counts or humoral immunity, such as irradiated bone marrow recipients, patients receiving chemotherapy, and congenital disorders of B-cell growth or function.

Modifications of the modes for carrying out the invention that are obvious to those of ordinary skill in the fields of protein chemistry, immunology, pharmaceutical formulation, and related fields are intended to be within the scope of the following claims.

Claims

1. Use of a CIF (TGF-β) for the manufacture of a medicament for inducing the production of B cells in peripheral blood or for inducing the production of granulocytes.