The present invention relates to the use of novel antibody-aminodextran
conjugates for use in inducing the activation and proliferation of leukocytes.
In particular, the invention relates to the use of an anti-CD3 monoclonal antibody
covalently coupled to an aminodextran. The covalently coupled antibody-aminodextran
conjugates have application in the analysis of immune cell functions in patients
with various medical conditions, such as AIDS and other immunodeficiencies, infectious
diseases, cancers, autoimmunity, and atopic diseases. In addition, the leukocytes
from recipients of transplanted cells and/or tissues can be functionally evaluated
utilizing this technology.
Immunosuppression, whether induced by drugs or disease, can lead
to alterations in T cell and/or accessory cell function. For example, it has been
demonstrated that AIDS patients manifest defective responses to mitogens, autoantigens,
alloantigens, and soluble antigens. This altered immunoreactivity is attributable
to defects in both responding (T) and stimulating (monocyte and dendritic) cell
populations. Decreased CD4 expression on the monocytes obtained from AIDS patients
has been demonstrated, yet no decrease in monocyte count has been observed. Immunosuppressive
drugs can also alter antigen presenting cell function. As a result of these observations,
it has been determined that a method for specifically stimulating T cells which
did not rely on the presence of monocytes or dendritic cells would be desirable.
While there are numerous methods of activating T cells, the optimal
method appears to require the multivalent interaction of antibodies, or other receptor
binding species such as lectins, with the T cell antigen receptor/CD3 complex
[hereafter TCR/CD3] on the surface of T cells. [A. Altman et al., Crit. Revs. in
Immunol. 10:347-391 (1990)]. CD3 specific monoclonal antibodies can induce highly
purified, resting T cells to proliferate, provided, however, that there is present
a mechanism for crosslinking the antibody-bound TCR/CD3 complexes. Numerous authors
have shown that the crosslinking requirement can be met by binding the antibody
to a substrate, for example, Sepharose® beads [S. Meuer et al., J. Exp. Med.
158:988-999 (1983) and D.A. Hafler et al., J. Immunol. 142:2590-2596 (1989)],
polystyrene beads [S. Panzer et al., Scand. J. Immunol. 32:359-371 (1990)], or
tissue culture dishes. International Patent Publication WO 90/04633 describes
solid-state supported monoclonal antibodies for induction of T cell activation
and the growth of T cells. Accessory cells, for example, monocytes, can also fulfill
the need for crosslinking by means of Fc receptor mediated binding of the T cell
bound anti-CD3 monoclonal antibodies to the monocyte cell surface.
The parameters used to assess cellular activation were reviewed by
A. Altman et al., Crit. Revs. in Immunol. 10: 347-391 (1990). Cell activation has
been measured by changes in nucleic acid synthesis, protein or glycoprotein synthesis,
cellular size and morphology, membrane integrity, expression of cellular constituents,
cell function, cell growth, cell differentiation and the release of cellular components.
These cellular changes have been detected by numerous different methods, many of
which are described in the patents and publications cited herein. Historically,
the diagnosis of immune deficient conditions has been done using laboratory tests
in which various stimuli are applied to T cells in order to determine if they
can be activated in vitro. Deficient T cell reactivity has been tested by stimulation
with mitogens, alloantigens and soluble antigens [R. Hong in Manual of Clinical
Immunology, 2nd Ed., N.R. Rose and H. Friedman, eds. (American Society for Microbiology
1980), Chapter 111, pages 833-849]. However, these methods are not specific for
all T cells. Mitogens activate both T and B cells. Alloantigens activate only those
selected T cells which have the appropriate receptor type. The response to soluble
antigens, for example, tetanus toxoid, can be effected by a patient's immunization
history. The anti-CD3 aminodextran conjugates of the claimed invention specifically
activate all CD3 positive cells and thus avoid these problems. The CD3 antigen
is found on virtually all mature peripheral T lymphocytes. This antigen is a component
of the T cell receptor complex and is non-covalently linked to a polymorphic,
clonotypic structure termed Ti. Antibodies to the CD3 surface structure serve as
probes for constant regions of the T cell receptor which is exclusively expressed
on immunocompetent T lymphocytes. Consequently, quantifying immunocompetent T
cells using this antibody is rapid and efficient.
In addition to the forementioned copending applications, particles
coated with dextran or dextran derivatives have been described by R.J. Mrsny et
al., Eur. J. Cell. Biol. 45:200-208 (1987) (ouabain-aminodextran-gold particles);
J.W.M. Bulte et al. Magn. Reson. Med. 25:148-157 (1992) (biotinylated dextranmagnetite
particles). The use of antibody-dextran type substances as carriers has been described
by U. Manabe et al., J. Lab Clin. Med. 104:445-454 (1984) (antibody-polyaldehyde
dextran-methotrexate); A.R. Oseroff et al., Proc. Natl. Acad. Sci. USA 83:8744-8748
(1986) (antibody-aminodextran-chlorin); and S. Rakestraw et al., Proc. Natl. Acad.
Sci. USA 87:4217-4221 (1990) (antibody-dextran hydrazide-Sn(IV) chlorin). Other
conjugated and crosslinked species have been described by S.S. Wang in "Chemistry
of Protein Conjugation and Crosslinking" (CRC Press, Boca Raton, Florida 1991)
and H. Maeda et al., Bioconjugate Chem. 3: 351-362 (1992). The standard procedure
for the introduction of amine groups into dextran has been to first cleave the
sugar rings to form polyaldehyde-dextran. The second step is to react the cleaved
rings with a diamine such as ethylenediamine or 1,3-diaminopropane to form a Schiff's
base complex. The Schiff's base is then stabilized by reduction with sodium borohydride.
The "aminodextran" compounds as described in the above cited art were ill-described,
typically lacking either elemental analyses or even average molecular weight determinations.
Furthermore, the periodate oxidation method of preparing aminodextrans as described
in these publications resulted in a low percentage of amino groups per molecule.
The percentage was less than 4-5 percent. Higher degrees of amine substitution
were not possible under the usual conditions of the prior art because high diamine
concentrations caused extensive aminolysis of the glucosidic linkages between
the sugar rings in dextran which resulted in very low molecular weight fragments.
As a result, the yields of polymeric aminodextran derivatives were low and decreased
drastically as higher and higher degrees of amine substitution were pursued.
An alternative method of producing amlnodextrans is by carboxymethylation
of sugar residue hydroxyl groups in chloroacetic acid, followed by carbodiimide
coupling of a diamine such as ethylenediamine. M. Brunswick et al., J. Immunol
140:3364-3372 (1988) and P.K.A. Mongini et al., J. Immunol. 148:3892-3902 (1992)
used this method to produce an aminodextran having about one amine group per sixty-seven
glucose residues (1/67). These authors then used the aminodextrans to prepare anti-Ig
conjugates for use in inducing B cell activation and proliferation.
The claimed invention teaches the use of anti-CD3 monoclonal antibodies
conjugated to aminodextrans as a method of providing for the specific stimulation
of T cells. Analysis of T cell function is critical to the diagnosis of immunodeficiency.
For example, the CD3-aminodextran conjugates described herein provide a uniquely
specific method for activating the T cells used for T cell analysis in AIDS patients.
M. Clerci et al., J. Clin. Invest. 84:1892-1899 (1988) found that an in vitro
T helper cell (TH) assay "can detect multiple stages of immune dysregulation
early in the course of HIV infection". Tetanus toxoid and alloantigens, which have
selectivity problems as stated above, were used as stimuli for T cells. S.C. Muluk
et al., Transplantation Proceedings 23:1274-1276 (1991) have shown that T cell
monitoring can be useful for determining the efficacy of immunosuppressive agents
in transplant patients.
The claimed invention teaches the use of aminodextrans, particularly
aminodextrans having a high degree or percentage of amine substitution, as a means
of crosslinking antibodies and of using the resulting antibody-aminodextran conjugate
to induce activation and proliferation of T cells. Aminodextrans have been used
in the copending applications cited herein to coat polystyrene microspheres and
magnetic and non-magnetic particles such as ferrite and metallic gold particles.
The aminodextran coated particles are then used to covalently link various monoclonal
antibodies. Both the degree of amine substitution and the degree of polymerization
of the dextran can be varied to determine the optimal form of the resulting coated
particle to which an antibody can be conjugated. Non-specific interactions between
antibody-aminodextran coated particles and cells are minimized by blocking amine
groups with excess crosslinking agent which, in turn, is also blocked.
DISCLOSURE OF THE INVENTION
The invention relates to the use of aminodextrans having 7-20% by
weight amine groups as crosslinking agents for monoclonal antibodies to produce
antibody-aminodextran conjugates that are useful in the induction and activation
of mammalian cells and especially human cells. In particular, the conjugates find
utility for inducing the activation and proliferation of human T and B cells.
A preferred embodiment of the invention is the preparation and use of anti-CD3
monoclonal antibodies conjugated to aminodextrans to induce T cell activation
and proliferation. The invention further describes the use of novel aminodextrans
having a high degree of amine substitution (greater than 10%) in the formation
of such conjugates and compares the results with conjugates formed using aminodextrans
generally known in the art which typically have about 4-5% amine substitution.
Comparative results indicate that the use of aminodextrans with high amine content
The invention also relates to a method of analyzing mammalian T cells,
particularly human cells. A sample containing or thought to contain T cells is
reacted with an aminodextran/anti-T-cell monoclonal antibody prepared as described
herein. The resulting aminodextran-antibody-cell complex, after any appropriate
incubation time, may be analyzed to evaluate T cell functions or changes in T
cell function. Tests which may be used in such analysis comprise changes in nucleic
acid (DNA or RNA) synthesis, protein or glycoprotein synthesis, cellular size
and morphology, membrane integrity, expression of cellular constituents and the
release of cellular components into the medium containing the cells undergoing
analysis. Typical diseases or disorders which are amenable to this type of analysis
are AIDS, other non-AIDS immunodeficiency diseases, infectious diseases, cancer,
autoimmunity disorders and atopic disorders. The method also may be used to test
T cells from patients who are the recipients of tissue, organ or cell transplants.
In those cases involving transplants, prior to the T cell analysis, non-T leukocyte
cells and their immature precursors may be stimulated to facilitate additional
testing related to conditions arising from the transplant. The non-T cells include
B cells, macrophages/monocytes, dendritic cells, neutrophils, eosinophils, basophils,
cytotoxic effector cells, hematopoietic stem cells and the immature precursor
cells of each of these cells. Methods stimulating such non-T cells and their precursor
cells are described in the publications cited in this application.
BRIEF DESCRIPTION OF DRAWINGS
BEST MODE FOR CARRYING OUT THE INVENTION
- FIG. 1 illustrates the distribution of cells in the forward versus side scatter
histogram of a sample containing unactivated, control T cells.
- FIG. 2 illustrates the distribution of cells in the forward versus side scatter
histogram of a sample containing T cells activated by the use of an anti-CD3/1X-Amdex
- FIG. 3 illustrates the distribution of cells in the forward versus side scatter
histogram of a sample containing T cells activated by the use of an anti-CD3/5X-Amdex
- FIG. 4 graphically compares the effect of selected anti-CD3/Amdex conjugates
on the formation of activated blast cells.
- FIG. 5 illustrates the DNA content of control cultured T cells which were not
stimulated with anti-CD3/Amdex conjugates.
- FIG. 6 illustrates the DNA content of T cells activated with anti-CD3/1X-Amdex
- FIG. 7 illustrates the DNA content of T cells activated with anti-CD3/5X-Amdex
- FIG. 8 graphically illustrates the effect of selected anti-CD3/Amdex conjugates
on the percentage of CD25, CD71, and PCNA positive cells.
Crosslinked antibodies show enhanced ability to induce resting T
cells in the G0/G1 phase to progress into the readily observable
S,G2/M phases of the cell cycle, including DNA synthesis. Soluble polymers
such as aminodextran, which have been used to coat a variety of colloidal particles,
should be suitable crosslinking agents for antibodies used to initiate and sustain
T cell proliferation. While aminodextrans have been used in conjugates keyed to
the stimulation of B cells, they have not been used with T cells. We describe herein
improved methods for preparing aminodextrans, particularly those with greater
than 10% amine substitution, and their use in the formation of antibody-aminodextran
conjugates which are subsequently used for stimulating the activation and proliferation
of mammalian cells and especially for stimulating T cell activation and proliferation.
Both the degree of amine substitution and the degree of dextran polymerization
can be varied to find the optimal form of the aminodextran to be used as an antibody
carrier and crosslinking agent.
The examples provided herein are for illustrating the invention and
are not to be construed as limiting the invention. For example, while the examples
herein describe the induction of human T cell activation and proliferation, the
proper choice of antibodies may extend the utility to other cells, such as a B
cell, and other mammalian species, such as a cat, dog or horse.
Two forms of aminodextran were used to form the conjugates described
herein. The first, which is described below as 1X-aminodextran (abbreviated 1X-Amdex),
degree of substitution equal to about 1/32 (two 1,3-diamino-propane groups per
sugar residue) and an average molecular weight of about 1,000,000 daltons. The
second, described below as 5X-aminodextran (abbreviated 5X-Amdex), has a degree
of substitution equal to about 1/7 and an average molecular weight of about 350,000
The anti-CD3 monoclonal antibody used herein was obtained from coulter
Corporation, Miami, Florida and was activated for conjugation to the aminodextran
by iminothiolane using standard procedures. The source of the anti-CD3 monoclonal
antibody or other T cell activating monoclonal antibodies is not critical to the
invention and other sources of such antibody may be used in place of that described
herein. The aminodextrans were activated with the heterobifunctional reagent sulfo-SMCC
[sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate] prior to conjugation
with the activated antibody. The invention is not limited to the use of iminothiolane
and sulfo-SMCC as activating agents. Those skilled in the art will recognize that
other reagents, such as those described in U.S. Patent No. 5,169,754 and the other
related applications, may be used in place of the iminothiolane and sulfo-SMCC
The Methods A-C described herein and in US-A-5 248 772 and US-A-5
466 609 may be used to prepare aminodextrans having an amine content in the range
of greater-than-zero to about twenty percent. Method C is preferred for preparing
aminodextrans having more than 5% amine groups; particularly, for aminodextrans
having more than 10% amine groups. The use of the hollow fiber cartridge described
herein places a lower molecular weight limit of 5,000 daltons on the aminodextrans
prepared using such cartridge. This lower limit may be raised or lowered by changing
the choice of cartridge used in the process. The maximum molecular weight of the
aminodextran products is limited to that of the starting dextran materials. Aminodextrans
prepared by oxidative cleavage methods known in the art have a maximum of 4-5%
amine group. The methods described herein allow for the preparation of aminodextrans
having 300% to 400% more amine groups than the aminodextrans described in the
Antibodies other than the Coulter T3 antibody described herein have
also been used according to the invention. These include an anti-CD2 antibody (Coulter
T11, IgG1), and an antibody against an 85,000 dalton species expressed on the
gamma chain of the T cell receptor (Coulter TiGamma, IgG1) among others. An anti-CD3
monoclonal antibody such as T3 is preferred.
When the invention is used in conjunction with methods of stimulating
non-T cells and their precursor cells the methods used are those found in the technical
literature and known to those skilled in the art. For example, if one wishes to
analyze and/or evaluate B cells as well as T cells, one may use anti-IgD and anti-IgM
monoclonal antibodies as described by M. Brunswick et al. in J. Immunology 140:
3364-3372 (1988). In such a case, the dextran or Ficoll used by Brunswick et al.
may be replaced by the aminodextrans disclosed herein. If one wished to stimulate
and activate macrophages, one may use a poly(styrene-co-maleic n-butyl ester)-conjugated
neocarzinostatin as taught by H. Maeda et al. in Bioconjugate Chemistry 3: 351-362
I. Preparation of Aminodextrans.
Method A. Small scale preparation of aminodextran.
Aminodextran was prepared by partial cleavage and oxidation of the
glucopyranose rings in dextran to give aldehyde functional groups, coupling of
the aldehyde groups with 1,3- diaminopropane to form Schiff base linkages and
reduction of the Schiff's base linkages to form stable carbon-nitrogen bonds. In
a typical procedure, 20 g of dextran were dissolved in 150 ml of 50 mM potassium
acetate buffer, pH 6.5. A solution of 2.14 g of sodium periodate in 25 ml of distilled
water was added dropwise to the dextran over about 10 minutes using vigorous magnetic
mixing. The resulting solution was stirred at room temperature, 15-27°C, for about
1.5 hours and then dialyzed against distilled water. 20 ml of 1,3-diaminopropane
were mixed with 20 ml of distilled water, cooled in an ice bath, vigorously stirred
and pH adjusted from about 11.5 to about 8.7 over about 15 minutes by the addition
of glacial acetic acid. Typically, 15-20 ml of glacial acetic acid were used. The
dialyzed dextran solution was added dropwise over about 15-20 minutes to the chilled
diamine solution. After the addition was completed, the resulting solution was
stirred at room temperature for about 2.25 hours. A reducing solution of 0.8 g
sodium borohydride in 10 ml of 0.1 mM sodium hydroxide was added to the dextran
reaction mixture at room temperature over about 15 minutes. The reaction mixture
was stirred during the borohydride addition to expel most of the effervescence.
The crude aminodextran solution was exhaustively dialyzed against distilled water
until the conductivity of the effluent was 3-4 µmho/cm. The dialyzed solution was
then filtered through a 0.2 µm filter and freeze-dried over 24 hours in a model
TDS-00030-A, Dura-Dry® microprocessor controlled freeze-dryer (FTS Systems,
Inc.) to produce 4.25g of flaky, pale yellow crystals in 21% yield.
Method B. Large scale preparation of aminodextran.
The procedure of Method A was modified for the large scale preparation
of aminodextran and for increasing the number of amine groups introduced into dextran.
Hollow fiber membrane filtration replaces dialysis and a smaller diamine-periodate
molar ratio was used to avoid further cleavage of the sugar polymer into lower
molecular weight fragments. These changes also serve to shorten the contact time
between excess diamine and substituted dextran during the removal of excess low
molecular weight reagents. Without these changes, the aminolysis of the glucosidic
linkages in high molecular weight dextran (e.g. dextran T-2M) was very extensive
and drastically reduced the yield of aminodextran above a cut-off molecular weight
of 5,000 daltons. A hollow fiber cartridge (polysulfone, 3 ft2 membrane
surface area, 1 mm diameter fibers and 5,000 MW cut-off model UFP-5-E-6, A/G Technology
Corp.) was mounted vertically with an input power pump (two pump heads, maximum
flow rate of about 4.56 liters/minute with No. 18 Norprene® food grade tubing)
delivering 15-20 psi which corresponds to 5-10 psi in the retentate line. The filtrate
was collected at 50-100 ml/min. Washing was done using 20-30 liters of distilled
water over about 6-8 hours. The specific conductance was reduced to about 3-4 µmho-cm-1
and the pH was 6.0-6.5. The feed volume was maintained at 2 liters during desalting
and then concentrated to 800 ml in the first washing of oxidized dextran and to
400 ml in the second washing of aminodextran.
In a standard scaled-up preparation, 80 g of dextran were transferred
to 1 quart [liter] glass blender bowl containing 600 ml distilled water. The solid
was blended for about 2-5 minutes at medium speed to dissolve all the dextran.
8.56 g of sodium periodate were dissolved in 100 ml of distilled water and the
resulting solution was added dropwise to the dextran solution over about 10 minutes
using vigorous magnetic stirring. After the addition was completed, the resulting
mixture was stirred at room temperature for an additional 3 hours. The resulting
viscous reaction mixture was then diluted to 2 liters with distilled water and
desalted using a hollow fiber cartridge. The initial specific conductance was
1.5 mmho-cm-1 or higher and the initial pH was 4.0. About 18-22 liters
of distilled water were used to obtain a solution having a final pH of 6.0-6.5.
The final volume of washed, oxidized dextran solution was 800 ml.
To the washed, oxidized dextran solution, 80 ml of colorless, liquid
1,3-diaminopropane were slowly added over about 10 minutes at room temperature.
The resulting mixture was then stirred at room temperature for an additional 3
hours. After the stirring was finished, 3.2 g of sodium borohydride dissolved in
40 ml of 1 mM aqueous sodium hydroxide were added to the room temperature aminodextran
reaction mixture over about 5 minutes with magnetic stirring. After the completion
of the sodium borohydride addition, the resulting mixture was stirred for an additional
1 hour and then desalted using a hollow fiber cartridge. The initial specific
conductance was 5.0 mmho-cm-1 or higher and the initial pH was about
12.0. About 20-25 liters of distilled water were needed to reduce the specific
conductance to about 3-4 µmho-cm-1 and the pH to 6.0-6.5. The final
volume of aminodextran solution was 400 ml. This solution was passed through a
0.2 µm sterile cellulose acetate filter unit and then freeze-dried over 48 hours
to obtain 48 grams of flaky, pale yellow crystals, a 52% yield.
Elemental analyses (C,H,N) were obtained for two samples of aminodextran
prepared from dextran T-2M by the methods described above. The analyses are:
Sample 1. 20 g dextran scale, Method A (desalting by dialysis).
- C, 43.04; H, 6.60, N, 1.09;
O (by difference), 49.27.
- Calculated for C46H79NO37&peseta;3H2O:
- C, 42.76; H, 6.63; N, 1.08; O, 49.53.
Sample 2. 80 g dextran scale, Method B (desalting by membrane filtration).
- C, 42.53; H, 6.52 N, 1.01;
O (by difference), 49.94
- Calculated for C49H84NO40&peseta;3H2O:
- C, 42.61; H, 6.57; N, 1.01; O, 49.81
The analyses for aminodextran in the two preparations were very similar,
thus indicating that the same product was obtained whether desalting was done by
dialysis or by membrane filtration and whether acetate buffer was used or not
used. The yield of aminodextran, however, was raised by 31% in Method B over that
in Method A. The empirical formula obtained for Sample 1, C46H84NO40,
is very similar to the formula C46H79NO37&peseta;3H2O
based on 29 units of glucose (C6H10O5), 1 unit
of fully diamine-substituted sugar ring (C12H28N4O3
two mol diamine per mol sugar unit,) and twelve units of water. Therefore, the
degree of diamine substitution of sugar residues in dextran was 1/30 in Sample
1 in contrast to a theoretical value of 1/12 based on 100% periodate cleavage and
diamine substitution according to the balanced oxidation-reduction equation. The
empirical formula obtained for Sample 2, C49H90NO43,
is very similar to the formula C49H84NO40&peseta;3H2O
based on 31 units of glucose, 1 unit of fully diamine substituted sugar ring and
twelve units of water. The degree of substitution in dextran by diamine was 1/32
for Sample 2.
Similar results were obtained using aminodextrans having average
molecular weights of 10,000, 40,000 and 2,000,000 daltons (T-10, T-40 and T-2M)
with 1X (1X= 3.3% substitution of sugar residues), 2X (6.6%) 3X (9.9%) and 5X
(16.5%) molar amounts of amino groups. All the aminodextrans were initially prepared
according to methods A and B using 2 and 3 times the amount of sodium periodate
used in the 1X oxidation of dextran. The amount of 1,3-diaminopropane used for
Schiff base formation was kept constant.
Modifications have been made to the Methods A and B of preparing
aminodextrans which were originally disclosed in US-A-5248772. These modifications,
disclosed in US-A-5466609 involve the oxidation and cleavage of the dextran glucose
rings with periodate anion, diamine addition and sodium borohydride reduction
of the Schiff's base. The modifications have resulted in increased yield of the
aminodextrans, particularly the 5X-aminodextran which was produced in less than
5% yield by the old procedures. Generally, the first modification was to use only
a ten percent (10%) excess of diamine over the stoichiometric 2:1 diamine:periodate
molar ratio previously disclosed. Second, the diamine addition reaction was conducted
at a temperature in the range of about 5-10°C. Third, the diamine addition reaction
was spectroscopically monitored in the near ultraviolet (UV) region for Schiff
base formation. Schiff's base formation was deemed completed when successive spectral
analyses indicated a plateau was reached. The reaction was then quenched by sodium
borohydride addition which reduces the Schiff's base linkages to carbon-nitrogen
single bonds and reduces any unreacted aldehyde groups to alcohol groups. These
modifications reduced aminolysis of the polymeric sugar groups into lower weight
fragments and thus gave higher yield of product after purification and concentration
by hollow fiber membrane filtration. The hollow fiber filtration was done using
polysulfone cartridge of 3 ft.2 membrane surface area, 1 mm diameter
fibers having a 5,000 molecular weight cut off. The cartridge was mounted vertically
in an input power pump having two pump heads delivering 15-20 psi with a maximum
flow rate of 4.56 liter/minute when using No. 18 Norprene® food grade tubing.
With this configuration, the pressure in the retenate line was about 5-10 psi.
The filtrate was collected at 50-100 ml/min. Washing was done using 20-30 liters
of distilled water over about 6-8 hours. The following method for preparing 5X-aminodextran
is given to illustrate the modified procedure which is applicable to the preparation
of all aminodextrans.
Method C. Preparation of 5X-Aminodextran.
T-2M dextran (50g, 0.308 mol, obtained from Sigma, St. Louis, Missouri,
or Pharmacia, Piscatawny, New Jersey) was added to a 1-quart or 1-liter glass blender
bowl containing 300 ml of distilled water. The mixture was blended at maximum
speed until all the dextran dissolved, typically about 3-5 minutes. A solution
of 26.75g (0.125 mol) of NaIO4 in 300 ml distilled water was added
to the dextran solution over about a 10 minute period using vigorous magnetic stirring.
After the periodate addition was completed, the reaction mixture was stirred at
room temperature for about an additional three hours. After the three hours, the
600 ml reaction volume had an initial specific conductivity of 9.7 mmho-cm
-1 and an initial pH of 2.5. The reaction mixture was diluted to two
liters with distilled water and desalted using the hollow fiber cartridge. Washing
was done using 15-18 liter of distilled water to obtain 600 ml of washed, oxidized
dextran solution having a specific conductance of 10 µmho-cm-1 and pH
The solution of oxidized dextran was cooled to about 8°C using an
ice bath and 23.2 ml (0.275 mol) of 1,3-diaminopropane was added over about 10
minutes to the oxidized dextran solution. The resulting reaction mixture was stirred
and maintained at the ice bath temperature. The formation of the yellow Schiff's
base was monitored ever 10-15 minutes by measuring the 335 nm near-UV absorbance
of an extracted sample. In a typical experiment, the measurements at 335 nm using
a 1 mm path length cell were:
After the absorbance had reached a plateau, 19.3 g (0.500 mol) of
sodium borohydride in 19.3 ml of 1 mM aqueous potassium hydroxide were added to
the reaction mixture over about 10 minutes at ambient room temperature using magnetic
stirring. After the sodium borohydride addition was completed, the reaction mixture
was stirred at ambient room temperature for about an additional two hours. After
the stirring was completed, spectroscopic measurement at 335 nm using a 1 cm path
length cell gave an absorbance value of 0.067 units which indicates that the Schiff's
base compound had essentially disappeared. The reaction mixture, about 1000 ml
volume, was then desalted using the hollow fiber cartridge. The initial specific
conductance was 43 mmho-cm-1 and the initial pH was 11.0. About 18-20
liters of distilled water were used as wash liquid to produce about 300 ml of 5X-aminodextran
solution having a specific conductance of about 4-6 µmho-cm-1 and a
pH of 6.5-7.0. The 5X-aminodextran solution was filtered through a 0.2 µm cellulose
nitrate filter and freeze-dried over 48 hours in a model TDS-00030-A, Dura-Dry®
microprocessor-controlled freeze-dryer (FTS Systems, Inc.) to produce 24g (48%
yield) of flaky, pale yellow crystals.
Elemental analysis: C= 45.83%, H= 7.00%, N=4.49%, 0 (by difference) = 42.68%. Calculated
analysis for C12H22O8.25N: C=46.15%, H= 7.10%,
N= 4.48%, 0=42.26%.
The empirical formula based on actual analysis is C12H22O8.3N,
which is very similar to the formula C12H22O8.25N
based on 6 units of glucose per one unit of fully diamine-substituted sugar ring
(C12H28N4O3). Therefore, the degree
of diamine substitution in dextran was 1/7 in contrast to a theoretical value of
1/2.5 based on 100% periodate cleavage and diamine substitution.
Repeat experiments using an initial charge of 100g and 300g dextran
produced a 5X-Amdex product having a similar degree of substitution. The preparation
of 1X-Amdex was also scaled up to the 100g and 300g levels using Method C. At
the 300g scale the hollow fiber cartridge was changed to a polysulfone membrane
of 8.5 ft.3 surface area, 1 mm diameter fibers having a 5,000 nominal
molecular weight cut-off (model UFP-5-E-35, A/G Technology Corp.). The cartridge
was used for ultrafiltration of both the oxidized dextran and the final aminodextran
product to remove salts and low molecular weight reagents or by-products. At the
300g scale, the yields of the 5X-Amdex and 1X-Amdex products were 135g (45%) and
162.1g (57%), respectively. The elemental analysis of the products were:
- 1X-Amdex, 300g dextran scale.
- C, 43.58; H, 6.50; N, 0.82;
O (by difference), 49.10
Calculated for C62H105NO50 &peseta;2H2O:
C, 43.79; H, 6.46; N, 0.82; O, 48.92
- 5X-Amdex, 300g dextran scale.
- C, 45.67; H, 6.90; N, 4.04
O (by difference), 43.39
Calculated for C13 H24 O9 N:
C, 46.15; H, 7.15; N, 4.14; O, 42.56
The empirical formula obtained for 5X-Amdex, C13H24NO9,
is very similar to the formula C13.2H24NO9.5 based
on 6,8 units of glucose and one unit of fully diamine-substituted sugar ring.
The degree of substitution of sugar residues in dextran was, therefore, about 1/7.
The empirical formula C62H110NO52 obtained for
the 1X-Amdex is similar to the formula C62H105NO50&peseta;2H2O
based on 39.3 units of glucose, one unit of fully diamine-substituted sugar ring
and two units of water. The degree of substitution of sugar residues in the dextran
was, therefore, about 1/40.
II. Preparation of Anti-CD3 Antibody-Aminodextran Conjugate
1. Activation of aminodextran with sulfo-SHCC.
25 mg of 1X-Amdex and 5X-Amdex were dissolved in 6.667 ml portions
of 1X PBS in separate. sealable 15 ml tubes to give solutions having a concentration
of 3.75 mg/ml. 1X PBS is made by dilution of a 20X PBS solution comprising 53.8g
K2HPO4, 12.8g KH2PO4 and 340g NaCl in
2 L distilled water as described in US-A-5369620. The 1X-Amdex was activated by
the addition of 13.5 µL of 10 mg/ml sulfo-SMCC solution per milliliter 1X-Amdex
solution (a total of 0.090 ml sulfo-SMCC solution). The 5X-Amdex was activated
using five times the amount of sulfo-SMCC solution (0.450 ml). The sulfo-SMCC
was pipetted into the respective tubes, vortexed to mix well and then roller mixed
for about two hours. After the mixing was completed, each reaction mixture was
chromatographed on a separate 100 ml G-50 Sephadex ® column (2.5 cm x 20 cm)
equilibrated with 1X PBS. The samples were eluted using 1X PBS and collected in
about 4 ml fractions. Fractions of the first band absorbing at 280 nm contain the
high molecular weight activated aminodextran as was verified by Tyndall scatter
with a focused light beam. These fractions were pooled to give about 10-11 ml
total sulfo-SMCC activated aminodextran in each case. A second, larger band eluted
from the column gave no Tyndall scatter. This second band was determined to contain
excess low molecular weight sulfo-SMCC reagent.
2. Activation of Antibody.
T3 monoclonal antibody (an anti-CD3 antibody sold by Coulter Corporation)
was activated by the addition of 1.423 ml of T3 concentrate (35.14 mg/ml) to a
solution comprising 0.323 ml of 2 mg/ml iminothiolane in 1X PBS and 1.587 ml 1X
PBS. The resulting solution which had an antibody concentration of 15 mg/ml and
an iminothiolane molar concentration fifteen-fold larger was mixed at ambient
temperature for about one hour. The entire reaction mixture was then applied to
a 100ml G-50 Sephadex® column equilibrated with 1X PBS and the sample was
eluted using 1X PBS. First band peak fractions of about 5 ml volume were combined
to give about 10.2 ml of 4.887 mg/ml antibody solution which contains a total of
49.847 mg iminothiolane-derivatized T3 antibody (abbreviated as IT-T3).
3. Conjugation of sulfo-SMCC-aminodextran and iminothiolane-T3 antibody.
5 ml of 4.887 mg/ml IT-T3 solution (about 24.435 ml antibody) were
mixed with 10.4 ml of sulfo-SMCC-lX-Amdex (about 25 mg 1X-Amdex) solution for about
two hours. In a like manner, 5 ml of 4.887 mg/ml IT-T3 solution were mixed with
11.2 ml of sulfo-SMCC-5X-Amdex (about 25mg 5X-Amdex) solution. After the mixing
was ended, the total volume of each mixture was determined and 0.120 times this
volume of 5 mg/ml L-cysteine in 1X PBS was added to each conjugation mixture. The
L-cysteine containing mixtures were then mixed for an additional 15 minutes to
effect blocking of any unreacted sulfo-SMCC moieties. Lastly, 20 mg/ml iodoacetamide
in 1X PBS solution in the amount of 0.120 times the total mixture volume and 1M
borate buffer solution, pH 9.8, in the amount of 0.020 times the total mixture
volume were added to each mixture. The resulting mixtures were mixed for about
30 minutes to block any unreacted sulfhydryl groups.
4. Purification of anti-CD3-aminodextran conjugates.
The total volume of each conjugation mixture was reduced to about
7.5 ml by centrifuging Amicon Centripep-30 tubes containing the samples for about
20 minutes at about 2500 rpm using a refrigerated Beckman J-6B centrifuge. After
centrifuging, the reduced volume mixtures were placed on a Bio-Gel® A-5m agarose
column (2.5 cm x 48 cm) equilibrated with 1X PBS and chromatographed using 1X
PBS as eluent. Eluent fractions of about 4 ml volume were collected using a Pharmacia
LKB Frac-100 collector operating in the drop collection mode. The fractions were
monitored using a LKB 2138 Uvicord S monitor operating at 280 nm. The first broad
band eluted from the column contained the T3 antibody-aminodextran conjugate.
A well-separated weaker band of less than half the intensity of the first band
was attributed to excess IT-T3 since it showed no Tyndall effect and it had the
same retention time on the column as the γ-globulin (Bio-Rad®) standard
for gel filtration columns. A strong, well separated third band was assigned to
low molecular weight excess blocking reagents. Comparing conjugates made with
1X-Amdex and 5X-Amdex, the T3 antibody-5X-Amdex conjugate had a narrower first
band which showed a longer retention time and a very weak second band.
The fractions collected for each conjugate were analyzed at 280 nm
using a 1 cm path length cell. Those fractions with an absorbance greater than
0.160 were pooled. The yields were 55 ml of T3-1X-Amdex conjugate having a T3
antibody concentration of 0.301 mg/ml and 54 ml of T3-5X-Amdex conjugate having
a T3 antibody concentration 0.317 mg/ml. The results indicate that 66% and 68%,
respectively, of the starting antibody was incorporated into the conjugate products.
ELISA solid phase assays for T3 in the conjugates gave a T3 antibody concentration
of 0.045 mg/ml in T3-1X-Amdex and 0.112 mg/ml in T3-5X-Amdex. The lower ELISA T3
values relative to the A280 values might indicate that either that there
is some blocking of active T3 antibody sites by conjugation to aminodextran or
that there is some interference with the A280 reading by an extra absorbance
contribution arising from the sulfo-SMCC reagent.
The T3-1X-Amdex and T3-5X-Amdex conjugates were also analyzed by
light scatter measurements (90°) using photon correlation spectroscopy. The samples
were analyzed using a Coulter® N4MD sub-micron particle analyzer operating
in the molecular weight and size distribution processor (SDP) weight analysis modes.
Samples of dextran T-2M, 1X-Amdex and 5X-Amdex at a concentration of 10 mg/ml
were analyzed. The average molecular weights were 3.1 x 106 daltons
(92%, dextran T-2M), 1.0 x 106
daltons (100%, 1X-Amdex) and 3.5 x 105
daltons (100% 5X-Amdex). The T3 antibody-aminodextrans prepared above were concentrated
to 10 ml total volume before light scatter measurements were made. The average
molecular weights were 3.6 x 106 daltons for T3-1X-Amdex and 1.4 x
106 for T3-5X-Amdex. Using these results, the T3 antibody:1X-Amdex molar
ratio was estimated as (3,600,000-1,000,000) 160,000 = 16:1 and the T3 antibody:5X-Amdex
molar ratio was estimated as (1,400,000-350,000) 160,000 = 6.6:1.
5. Saturation T3 Antibody Conjugation to Aminodextrans.
The chromatograms of the initial preparation of T3-1X-Amdex and T3-5X-Amdex
conjugates showed little, if any, excess T3 antibody when conjugations were done
using about 1:1 antibody:aminodextran weight ratios. Consequently,
III. Activation of Peripheral Blood T cells With Anti-CD3-Aminodextran
A. Isolation of Peripheral Blood Mononuclear Cells (PBMC).
additional experiments were performed using 3:1 T3 antibody: aminodextran weight
ratios and the same amount of each aminodextran. The activation, conjugation,
blocking and chromatography procedures were the same as those described above.
However, during chromatography, a narrow, intense band assigned to excess free
IT-T3 trailed the broader T3 antibody-aminodextran band in each case. The IT-T3
band was sufficiently close to the T3-aminodextran bands so that only about half
of the first band could be separated in each case. The T3-1X-Amdex preparation
thus yielded 75 ml of 0.576 mg/ml T3 in the T3-1X-Amdex solution and 40 ml of 0.466
mg/ml T3 in the T3-5X-Amdex solution. ELISA solid phase assays gave 0.392 and
0.301 mg/ml T3 antibody, respectively. Light scatter results indicate average molecular
weights of 6.9 x 106 daltons and 3.6 x 106 daltons, respectively.
The T3 antibody:aminodextran molar ratio in the conjugates were estimated as (6,900,000-1,000,000)
160,000 = 37:1 for T3-1X-Amdex and (3,600,000-350,000) 160,000 = 20:1 for T3-5X-Amdex.
The procedure used herein was performed at room temperature. Normal
whole blood was collected into tubes containing EDTA (ethylenediaminetetraacetic
acid). The tubes were centrifuged at 500g for 10 minutes. Using sterile techniques,
the buffy coat was collected, diluted 1:2 (cell:solvent) with 1X PBS and layered
over Ficoll-Hypaque®. The resulting sample was then centrifuged at 400g for
30 minutes, the PBMC interface was collected and diluted with 1X PBS. The diluted
sample was centrifuged at 300g for 10 minutes and then washed twice with 1X PBS
by resuspending the pellet, diluting with 1X PBS and centrifuging at 300g for
10 minutes. After a final resuspension in 1X PBS, the cells were counted and cell
viability was ascertained. The cells were divided into two parts, centrifuged
as above and resuspended in the appropriate media for the following steps.
B. Culture and Activation of T Cells with Anti-CD3-Aminodextran.
Non-activated cells for use as the control were cultured in Nutricyte®
medium containing 10% (v/v) CPSR-2 serum replacement (obtained from Sigma Chemical).
The initial cell concentration was 2.5 x 106 cells/ml. Cultures destined
for T cell activation were established in the same medium supplemented with 2-4
ng/ml phorbol 12-myristate 13-acetate (PMA) and 0.125-0.5 µg/ml of anti-CD3-aminodextran.
Activated cultures were established using both T3-1X-Amdex and T3-5X-Amdex. The
cell suspensions were incubated in T-150 flasks at 37°C, 5% CO2 for
C. Harvesting of Control and Activated Cells.
The control cells and the T3-1X-Amdex and T3-5X-Amdex activated cells
were harvested by scraping the bottom of the tissue culture flasks with a disposable
scraper, collecting the cells with a pipette and placing them in a centrifuge
tube. The cells were counted, centrifuged at 300g for 10 minutes and washed twice
with 1X PBS as has been described.
D. Immunofluorescent Staining and Flow Cytometric Analyses of T Cell Activation.
The degree of T cell activation was determined by immunofluorescent
staining of the cells, followed by flow cytometric analysis of the stained cells
for the expression of both activation-associated antigens (CD71, transferrin receptor
and CD25, IL2 receptor) and proliferation-associated events (DNA and proliferating
cell nuclear antigen, PCNA). Aliquots containing 1 x 106
cells of each
culture were placed in six labelled sample tubes (3 sets of six tubes). One tube
in each culture set was stained with the following reagents which are sold by
For each set of cells (control, T3-1X-Amdex activated and T3-5X-Amdex activated),
tubes 1-4 were processed for cell surface staining by incubating the cells with
the appropriate FITC conjugated monoclonal antibody for 15 minutes at room temperature.
The incubated cells were then diluted with 1X PBS, centrifuged at 300g for 10 minutes,
washed with 1X PBS by suspension and centrifugation and finally resuspended in
1 ml of 1X PBS.
- Tube 1:
- IgG2a-FITC isotype control
- Tube 2:
- IL-2R1-FITC (IgG2a, CD25)
- Tube 3:
- IgM-FITC isotype control
- Tube 4:
- T9-FITC (IgM, CD71)
- Tube 5:
- IgG1, isotype control and propidium iodide (to label DNA)
- Tube 6:
- PCNA (IgG1) and propidium iodide.
The cells in tubes 5 and 6 were processed for the staining of intracellular
antigens by suspending the control cell or activated cell pellets in 1 ml of a
solution containing 20 µg/ml lysophosphatidyl choline in 1% paraformaldehyde and
incubating the resulting suspensions for 2 minutes at room temperature. After
incubation, the cells were resuspended in 1 ml of about -10°C absolute methanol,
incubated on ice for about 10 minutes and centrifuged. The resulting pellet was
then incubated in 1 ml of 0.1% NP-40 (available from Sigma Chemical) for about
5 minutes at about 0°C, centrifuged and then incubated again for about 15 minutes
at room temperature with IgG1, (tube 5) or PCNA (tube 6) monoclonal antibody.
The cells were washed in 1X PBS and incubated for an additional 15 minutes at room
temperature with goat anti-mouse immunoglobulin conjugated to FITC. After washing
in 1X PBS, 1 ml of propidium iodide was added to each tube.
Samples were analyzed on an EPICS® Profile I flow cytometer equipped
with a 488 nm Ar+ ion laser line and a power pack upgrade. Light scattering
and fluorescent signals were collected. FL1 represents the FITC fluorescent emission
and FL3 represents the fluorescent emission due to propidium iodide. Forward light
scatter (FS) was representative of cell size and 90° light scatter (LSS= log side
scatter) gives an indication of cell complexity or granularity. For single parameter
analysis (CD25 and CD71), a linear cursor was placed on the isotype control histogram
such that 2% of the cells were included in the cursor and the majority of cells
were excluded (negative control). As a result, CD25 and CD71 positive cells were
defined as those cells which emit sufficient fluorescent light to fall within the
area defined by the cursor. In the dual parameter analysis for PCNA and DNA, a
rectangular analysis region was drawn on the isotype control histogram such that
2% of the cells were included within the rectangle and the majority of cells were
excluded (negative control). PCNA positive cells were, therefore, defined as those
cells which emitted sufficient fluorescent light to fall within the defined region.
As the amount of DNA increases, the amount of propidium iodide taken up by a cell
also increases. The percentage of cells in the Go/G1, S,
and G2/M phases of the cell cycle were determined using the Multicycle®
DNA analysis program.
The analytical results indicate that stimulation with T3-5X-Amdex
(saturated) conjugate results in optimal T cell activation as determined by blast
cell formation, expression of cell surface activation markers (CD25 and CD71),
DNA synthesis and the expression of proliferation-associated antigen (PCNA). As
can be determined by a review of FIGS. 1-4, and Table 2, the percentage of activated
blast cells, as determined by increased forward and 90° light scatter, was maximal
after stimulation with the 5X-Amdex conjugate. The results of Multicycle®
analysis of the DNA data also revealed that stimulation with the T3-5X-Amdex conjugate
resulted in the greatest percentage of cycling cells (FIGS. 5-7 and Table 2).
Similarly, the percentage of CD25, CD71 and PCNA positive cells was optimal after
activation with T3-5X-Amdex us shown in FIG. 8 and Table 2.
Anti-CD3 induced activation and proliferation of T cells requires
crosslinking of CD3 molecules on the T cell surface. This requirement can be met
via Fc mediated
binding of anti-CD3 on the monocyte cell surface. The data shown in Table 2 and
Figures 1-8 was obtained from monocyte containing cultures. T cells were purified
and cultured with T3 monoclonal antibody alone or with the T3 antibody conjugated
to 5X aminodextran in order to verify that the enhanced T cell activation and proliferation
observed was due to the use of the 5X-Amdex conjugate. Peripheral blood mononuclear
cells (PBMC) were obtained via density gradient centrifugation over Ficoll-Hypaque®
and the majority of monocytes were removed via adherence to plastic over a period
of 90 minutes. B cells and residual monocytes were depleted with B4 and Mo2 conjugated
magnetic beads, and the essentially pure T cells (>95% as determined by immunofluorescence
analysis) were cultured as previously described. The results, given in Table 3,
reveal that while monocyte containing cultures are activated in the presence of
T3 alone, purified T cell preparations are not. For both cell preparations, similar
activation was obtained with the addition of T3-5X-Amdex.
Further experiments with purified T cells verified previous findings
as to the superiority of the 5X versus the 1X conjugate. The results of such an
experiment are given in Table 4.
The data in the following tables correspond to the indicated figures.
FIG. 4: Cell Cycle Data
Mean G1= 45.7
Mean G2= 88.1
% S = 1.7
CV G1 = 2.1
CV G2 = 2.1
G2/G1 = 1.928
% G1 = 94.7
% G2 = 3.6
Chi Sq. = 1.7
FIG. 5: Cell Cycle Data
Mean G1= 45.2
Mean G2= 95.5
% S = 11.1
CV G1 = 10.0
CV G2 = 5.7
G2/G1 = 2.115
% G1 = 86.4
% G2 = 2.5
Chi Sq. = 1.6
FIG. 6: Cell Cycle Data
Mean G1= 46.2
Mean G2= 96.8
% S = 17.0
CV G1 = 11.6
CV G2 = 6.2
G2/G1 = 2.070
% G1 = 79.4
% G2 = 3.5
Chi Sq. = 1.3