U.S. patent application number 11/370879 was filed with the patent office on 2006-07-20 for methods for producing human antibodies in scid mice.
Invention is credited to Peter Brams, Marco Anthony Coccia.
Application Number | 20060161997 11/370879 |
Document ID | / |
Family ID | 22012997 |
Filed Date | 2006-07-20 |
United States Patent
Application |
20060161997 |
Kind Code |
A1 |
Coccia; Marco Anthony ; et
al. |
July 20, 2006 |
Methods for producing human antibodies in SCID mice
Abstract
An improved method for producing human antibodies in SCID mice
is provided. The improvement includes the use of dendritic cells
pulsed with antigen-antibody complexes and antigen-antibody
complexes as immunizing agents.
Inventors: |
Coccia; Marco Anthony; (San
Diego, CA) ; Brams; Peter; (San Diego, CA) |
Correspondence
Address: |
PILLSBURY WINTHROP SHAW PITTMAN, LLP
P.O. BOX 10500
MCLEAN
VA
22102
US
|
Family ID: |
22012997 |
Appl. No.: |
11/370879 |
Filed: |
March 9, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09798525 |
Feb 21, 2001 |
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11370879 |
Mar 9, 2006 |
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09149479 |
Sep 8, 1998 |
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09798525 |
Feb 21, 2001 |
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60057831 |
Sep 8, 1997 |
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Current U.S.
Class: |
800/6 ;
435/70.21 |
Current CPC
Class: |
A61K 2039/5154 20130101;
C07K 16/00 20130101; C07K 16/40 20130101; A61K 2039/5158 20130101;
A61K 39/00 20130101; C07K 16/3069 20130101; C07K 2317/21
20130101 |
Class at
Publication: |
800/006 ;
435/070.21 |
International
Class: |
C12P 21/04 20060101
C12P021/04; A01K 67/027 20060101 A01K067/027 |
Claims
1. A method for producing human antibodies in SCID mice which
comprises immunizing SCID mice with dendritic cells which have been
contacted (pulsed) in vitro with an antigen-antibody complex.
2. The method of claim 1, wherein the antigen is prostrate specific
antigen.
3. The method of claim 2, wherein the antibody is a mouse
IgG.sub.2a antibody.
4. The method of claim 1, wherein the dendritic cells comprise
autologous peripheral blood dendritic cells.
5. The method of claim 1, which includes EBV transformation during
the immunization step.
6. The method of claim 1, wherein the antibody is anti-PSA
IgG.sub.2, monoclonal antibody and the antigen is PSA.
7. The method of claim 1, wherein said SCID mice are immunized with
antigen-antibody complex prior to a immunization with dendritic
which have been contacted (pulsed) with antigen-antibody
complex.
8. The method of claim 1, wherein the second immunization is
effected about 1 to 15 days after the first.
9. The method of claim 8, wherein said second immunization is
effected about 7 days after the first immunization.
10. The method of claim 7, which further includes at least one
additional immunization ("boosting") wherein SCID mice is
administered antigen or antigen-antibody complex.
11. The method of claim 10, wherein a boosting step is effected
about a week after the first immunization.
12. The method of claim 11, wherein another boosting step is
effected about two weeks after the first immunization.
13. The method of claim 12, wherein another boosting step is
effected about three weeks after the first immunization.
Description
FIELD OF THE INVENTION
[0001] The subject invention provides a novel and reproducible
method for producing human monoclonal antibodies to desired
antigens, e.g. prostate specific antigen. These monoclonal
antibodies, because of their human origin, should be useful
therapeutic agents, e.g. for the treatment of human prostate
cancer.
BACKGROUND OF THE INVENTION
[0002] Antibodies (Ab) that recognize and adhere to proteins on the
surface of bacteria, virus or parasites help immune system cells
identify, attack and remove them from the body. Similarly,
monoclonal Ab (MoAb) that adhere to cancer cells but not to normal
cells can be an effective therapy for human cancers. Such MoAbs are
generally murine Abs genetically modified to contain human constant
regions ("humanized"). However, fully human MoAb are potentially
superior to humanized murine MoAb as therapies for human cancer
because of their absence of immunogenicity in humans. Human B cells
can be stimulated to produce Abs that recognize specific human
target proteins. However, previous methods are typically very
complex and yield inconsistent results. Therefore, there exists a
need in the art for improved methods for producing human monoclonal
antibodies.
OBJECTS OF THE INVENTION
[0003] It is an object of the invention to obviate the problems of
the prior art.
[0004] It is a specific object of the invention to provide a novel
method for producing human antibodies in SCID mice.
[0005] It is an even more specific object of the invention to
provide a novel method for producing human antibodies in SCID mice
wherein the immunizing protocol includes the administration of
dendritic cells which have been pulsed in vitro with
antigen-antibody complexes and/or antigen-antibody complexes.
[0006] It is a more specific object of the invention to provide a
novel method for producing human antibodies specific to human
prostate specific antigen (PSA).
[0007] It is an even more specific object of the invention to
provide a novel method for producing human antibodies to human PSA
in SCID mice wherein the immunization protocol includes the
administration of dendritic cells which have been pulsed in vitro
with PSA-anti-PSA antibody complexes and/or PSA-anti-PSA antibody
complexes.
[0008] It is still another object of the invention to provide a
novel immunization protocol for producing human antibodies in SCID
mice that includes in vivo transformation with EBV during
immunization.
BRIEF DESCRIPTION OF THE INVENTION
[0009] As discussed in greater detail infra, by judicious
experimentation, the present inventors have developed an improved
method for producing human antibodies in SCID mice. Specifically,
it has been found that immunization of SCID mice with autologous
dendritic cells, e.g., autologous peripheral blood dendritic cells
that have been pulsed in vitro with a desired antigen, more
preferably an antigen-antibody complex, yields high antibody titers
wherein such antibodies possess the desired specificity.
[0010] Also, it has been found that immunization with
antigen-antibody complexes yields improved results, i.e., high
serum antibody titers wherein such antibodies exhibit the desired
specificity.
[0011] Still further, the present invention provides in particular
a novel immunization protocol for producing human monoclonal
antibodies to prostate specific antigen (PSA). These antibodies,
because of their specificity and human origin, should be useful for
the treatment of prostate cancer. Because of their human origin,
they should possess human antibody effector functions and should
elicit no immunogenicity.
DESCRIPTION OF THE FIGURES
[0012] FIG. 1 schematically depicts the immunization strategy of
the invention.
[0013] FIG. 2 is a flow chart summarizing the engraftment and
immunization of SCIDhu PBL mice.
[0014] FIG. 3 is a FACS analysis of peripheral blood dendritic
cells cultured in serum free media. DC were grown in triplicate
cultures, harvested on day 7, pooled and subjected to FACS analysis
as described in "Materials and Methods". The DC generated from PBMC
used to reconstitute the SCIDhu PBL mice were 65% large, MHC class
II.sup.+/CD33.sup.+/CD40.sup.+/CD1a.sup.lo/CD14.sup.- cells with
dendritic morphology. The remaining cells were mostly T cells and
some B cells. These results are similar to those obtained from
cultures generated from 8 individual PBMC donors. All donors
generated cultures that were between 50 and 75%
CD11c.sup.hi/CD32.sup.+/CD33.sup.+/CD40.sup.+/CD45RO.sup.+/-ClassII.sup.+-
/B7.1.sup.+/B7.2.sup.+DC. DC generated from different donors were
heterogeneous for CD1a, CD4, CD14, and CD64 expression (Data not
shown).
[0015] FIG. 4 is a comparison of MHC and T cell co-stimulatory
surface Ag expression by DC cultures. DC were grown in triplicate
cultures, harvested on day 7, pooled and subjected to FACS analysis
as described in "Materials and Methods". Results show MHC class II,
B7.1, B7.2 and CD40 expression was significantly enhanced on DC
pulsed with soluble PSA but not PSA-mIG.sub.2a. Similar results
were obtained with DC cultures generated from another donor and
pulsed with Tetanus toxoid.
[0016] FIG. 5 is a quantitation of human IgG in sera of SCIDhu PBL
mice. Sera was collected on days 14 and 28. Total and PSA specific
human IgG were quantitated by ELISA. Results shown are the average
of 8 mice per group. Error bars represent .+-.Std. Dev. A. Total
human IgG (mg/ml). Group H mice IgG sera concentrations ranged
between 0.56 and 2.19 mg/ml IgG by day 28. IgG sera concentrations
in groups F and G control mice ranged between 8 and 840 .mu.g/ml
IgG by day 28. B. PSA specific IgG (.mu.g/ml). PSA specific IgG was
quantitated using a mouse monoclonal IgG specific for human PSA as
a standard. Only Group H produced PSA specific IgG. C. Percent PSA
specific IgG. The relative quantity of PSA specific IgG was
calculated as follows: [PSA specific IgG]/[total
IgG].times.100.
[0017] FIG. 6 is an analysis of PSA specificity of group H sera
IgG. A. Relative PSA specificity. Human IgG in group H sera binds
PSA ten times greater than the nonspecific binding generated by
group F sera with an equivalent concentration of IgG or by an
equivalent concentration of purified human IgG. B. Soluble PSA
competition ELISA. Soluble PSA inhibits the binding of group H sera
IgG in a concentration dependent manner.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The present inventors have developed a novel and
reproducible method to stimulate human B cells to make Ab that
adhere to desired antigens, e.g. prostate specific antigen (PSA), a
protein on the surface of prostate cancer cells. Using these
methods, specific human monoclonal antibodies to desired antigens
can be cloned, which have applicability in human treatments, e.g.,
the treatment of prostate cancer.
[0019] The advantages of the subject invention are significant.
[0020] In particular, these methods are advantageous for the rapid
production of fully human monoclonal antibodies for immunotherapy
of human diseases.
[0021] The major distinguishing differences of the subject
protocols compared to prior practices are the use of Ab-antigen
(Ag) complexes and autologous dendritic cells (DC) as immunizing
adjuvants.
[0022] Still another non-obvious distinguishing difference of the
subject methods in relation to previous methods is the inclusion of
intentional EBV transformation in vivo during the unique DC/Ab-Ag
complex immunization steps. Also, the present inventors have
determined optimal conditions for Ag boosting SCIDhu PBL mice
(using PSA as a model antigen), and high affinity antibodies to PSA
using two different donors.
[0023] In order to generate human anti-PSA specific IgG responses
that could be immortalized we have developed a novel SCIDhu mouse
immunization protocol (see FIG. 1). See, also, FIG. 2, which is a
flow chart summarizing the engraftment and immunization of SCIDhu
mice. Briefly, we hoped that complexing antigen to the Fc receptors
of dendritic cells (DC) would increase the immunogenicity of the
antigen. (However, given the inherent unpredictability with
monoclonal antibody manufacture, this result was not assured.)
Therefore, we pulsed dendritic cells isolated and expanded from a
particular donor with PSA complexed to a mouse anti-PSA IgG.sub.2a
monoclonal Ab (Ab-PSA complex).
[0024] On day 0, SCID's were reconstituted with 10.sup.8 female
PBL's and immunized with 25 mg Ab-PSA complex. Simultaneously,
autologous, DC cultures were initiated. The DC were pulsed with 25
mg/ml Ab-PSA complex on day 6, and injected i.p. on day 7. The mice
were boosted with 25 mg of Ab-PSA complex on days 7 and 14 and with
25 mg of soluble PSA on day 21. Mice immunized by this method
generated PSA specific IgG sera concentrations that were comparable
to those induced to Tetanus Toxoid using standard immunization
methods (TT in alum). Moreover, these results were reproduced in
two separate experiments using different PBMC donors. Therefore,
the present immunization protocol is reproducible and therefore
should be applicable to different antigens, in particular those
involved in human diseases.
[0025] As noted, the subject method uses dendritic cells which have
been pulsed in vitro with antigen or antigen-antibody complexes as
immunizing agents. Dendritic cells (DC) are professional antigen
presenting cells (APC) that initiate immune response (see 1, 2 for
review). Recently, several methods have been developed to generate
human DC from peripheral blood mononuclear cell (PBMC) derived
progenitor cells), ill vitro. These different culture methods yield
several DC subtypes with heterogeneous morphology, phenotype and
function. However, all of these DC subtypes have been shown to be
potent stimulators of naive Ag specific T cells (3-5). This is due
in large part to the fact that DC express class I and II MHC and
co-stimulatory cell surface molecules B7.1 and B7.2 (6, 7). In
addition, human DC pulsed with weakly immunogenic, tumor associated
antigens (TAA) are capable of stimulating TAA specific cytotoxic T
lymphocyte (CTL) proliferation and cytotoxicity, in vitro, thus
illustrating both their potency as APC and their potential utility
as tumor specific vaccines (8-10).
[0026] DC derived from PBMC and cultured with GM-CSF and IL-4
express both the high affinity IgG receptor Fc.gamma.RI (CD64) and
the low affinity IgG receptor Fc.tau.RII (CD32) at varying levels
(3, 11). Both CD64 and CD32 have been shown to mediate uptake of Ag
by DC (11, 12). Targeting Ag to Fc.gamma.R on human monocytes and
DC via monoclonal antibody (mAb)-Ag complexes reduces the amount of
Ag required for Ag specific T cell activation as much as 1000-fold
(13, 14).
[0027] Although many recent studies have analyzed human T cell
activation by DC, what is not clear is whether in vitro generated
DC are capable of stimulating a primary humoral immune response. DC
isolated from mouse spleen and pulsed with myoglobin were capable
of stimulating a primary humoral immune response in syngeneic mice,
but mouse splenic DC may have different immunostimulatory effector
functions than DC derived from human peripheral blood (15).
Follicular DC (FDC)-lymphocyte clusters isolated from human tonsil
enhanced growth and Ig production by CD40 activated human B cells,
in vitro (16). However, tonsillar FDC are phenotypically and
morphologically distinct from peripheral blood derived DC and,
therefore, are likely to have different effector functions as well
(17).
[0028] SCID mice are deficient in mature lymphocytes, Ig production
and lymphocyte mediated immune responses due to defective Ig and T
cell receptor gene rearrangement (18). SCID mice reconstituted with
human peripheral blood lymphocytes (SCIDhu PBL mice) can be
effective models of recall antigen directed Ig production by human
B cells (19, 20). However, it is very difficult to stimulate
neo-Ag, self-Ag or TAA specific primary immune response and IgG
production in SCIDhu PBL mice (21).
[0029] In this study we characterized the phenotype of DC generated
from PBMC in low protein, serum free media. We then assessed the
ability of serum free cultured DC to stimulate a prostate specific
antigen (PSA) specific, primary humoral immune response by SCIDhu
PBL mice. We showed that DC pulsed with PSA complexed to a mouse
IgG.sub.2a specific for human PSA (PSA-mIgG.sub.2a) can induce PSA
specific human IgG production in SCIDhu PBL mice. SCIDhu PBL mice
immunized with soluble PSA pulsed DC did not produce PSA specific
IgG. These results suggest that the mechanism by which DC acquired
Ag altered DC expression and immunostimulating effector functions.
Different Ag acquisition mechanisms yield different co-stimulating
molecule surface expression and subsequent immunostimulatory
effector functions by DC.
EXAMPLE
[0030] The following materials and methods were used.
DC Generation in Serum Free Cultures
[0031] PBMC were obtained from healthy donors by leukophoresis or
by venapuncture into heparinized tubes. RBC were removed from
residual PBMC by hypotonic lysis in Gey's lysis buffer prior to
freezing in 50% human serum, 40% Iscoves complete media (Iscove's
modified Delbucco's media (Irvine Scientific, Santa Ana, Calif.)
plus sodium pyruvate, minimal essential amino acids, L-glutamine
(Sigma, St. Louis, Mo.) and gentamicin (Gibco BRL, Grand Island,
N.Y.)) and 10% DMSO (Sigma). Frozen PBMC were stored in LN.sub.2.
DC were grown essentially as described by Romani et al, except that
Iscove's complete was supplemented with 2% Nutridoma.RTM. HU
(Boehringer Mannheim Corporation, Indianapolis, Ind.) instead of
10% fetal bovine serum. Freshly isolated and thawed PBMC were
purified by Histopaquel (Sigma) gradient separation, washed and
plated at 5.times.10.sup.6 cells/ml in IN2 at 37.degree. C. for 2
hrs. Non-adherent cells were gently removed with the media,
additional 37.degree. C. IN2 was added and the cells were incubated
at 37.degree. C. for 5 additional minutes. Non-adherent cells were
again gently removed and the residual cells were cultured in IN2
supplemented with 500 U/ml IL-4 and 800 U/ml GM-CSF (Genzyme, Inc.,
Cambridge, Mass.). Cultures were fed with additional cytokines on
day 3. Human PSA specific mouse monoclonal IgG.sub.2a (Clone
10-P20; Fitzgerald Industries International Inc., Concord, Mass.)
was complexed with >99% pure PSA (Fitzgerald Industries
International) at equimolar ratios at 4.degree. C. overnight
(PSA-mIgG.sub.2a). The DC enriched cultures were pulsed with 25
.mu.g/ml (final concentration) PSA, PSA-mIgG.sub.2a or an
equivalent volume of IN2 on day 6 and non-adherent cells were
harvested on day 7.
Flow Cytometric Analysis
[0032] The following FITC and PE labeled monoclonal antibodies
(mAb) were used: anti-HLA DR, DP, DQ, anti-CD 1a, anti-CD3,
anti-CD11c, anti-CD 16, anti-CD32w (Fc.gamma.RII), anti-CD33,
anti-CD40, anti-CD45RO, anti-CD64 (Fc.gamma.RI), anti-CD86 (B7.2),
(Pharmingen, San Diego, Calif.), anti-CD4, anti-CD 14, anti-CD80
(B7.1), PE-labeled isotype controls (Becton and Dickinson, San
Jose, Calif.), anti-ABC, and FITC labeled isotype controls (Harlan
Bioproducts for Science, Inc., Indianapolis, Ind.). Day 7 DC
enriched cultures and single cell isolates from SCIDhu PBL mouse
tissues were washed and resuspended in 4.degree. C. FACS buffer (1%
BSA, 1.times.PBS, 0.1% Na Azide and 40 .mu.g/ml human IgG) at
1.times.10.sup.6 cells/ml. The cells were then aliquoted and
stained for 45 minutes with mAb diluted to the manufacturers'
recommended concentration. The cells were washed twice in FACS
buffer and data was acquired on a FACScan.RTM. (Becton Dickinson).
Data was analyzed using Lysis 1.RTM. ((Becton Dickinson) or F cap
List.RTM. (Soft FlowHungary, Inc., Pecs, Hungary) software.
Specific reactivity data shown as .DELTA.MFI is calculated as
follows: MFI of FITC or PE labeled specific mAb--MFI of isotype and
fluorochrome matched mAb control. These results are contained in
FIGS. 3 and 4.
SCID Mouse Engraftment and Immunization
[0033] PBMC were obtained from healthy female donors by
leukophoresis. RBC were removed by hypotonic lysis in Gey's lysis
solution. Residual PBMC were frozen and stored as described above.
Four to six week old male Fox Chase ICR SCID.TM. mice (Taconic,
Germantown, N.Y.) were housed, fed and handled according to
established protocols for immunodeficient strains. Mice were
engrafted with 10.sup.8 PBMC, i.p., on day 0. Autologous DC
cultures were initiated on day 0 as described above. Group F mice
were immunized with 25 .mu.g of PSA-mIgG.sub.2a complex weekly,
7.times.10.sup.6 thawed autologous PBMC on day 7 and then boosted
with 25 .mu.g PSA on day 21. Group G Mice were immunized with 25
.mu.g of soluble PSA weekly and 7.times.10.sup.6 PSA pulsed DC
enriched cells on day 7. Group H mice were immunized with 25 .mu.g
of PSA-IgG.sub.2a complex weekly, 7.5.times.10.sup.6
PSA-mIgG.sub.2a pulsed DC enriched cells on day 7 and then boosted
with 25 .mu.g PSA on day 21. Sera was collected on days 14 and 28.
Mice were sacrificed and spleens and lymph nodes were collected on
day 28. Some spleens were laterally bisected and single cells
isolated from one half were analyzed by flow cytometry as described
above. The remaining spleens and LN were embedded in OCT compound
(Sukura Finetek, Inc., Torrance, Calif.) and then simultaneously
frozen and fixed in LN.sub.2 chilled 2-methylbutane (Sigma) for
immunohistochemical staining.
ELISAs
[0034] Human Ig sera concentrations were assayed by quantitative
ELISAs. ELISAs were performed in 96 well Immulon 2' "U" ELISA
plates (Dynatech Laboratories, Inc., Chantilly, Va.). Human IgG and
IgM ELISA plates were coated with 2 .mu.g/ml polyclonal goat
anti-human IgG or goat anti-human IgM (Southern Biotechnology
Associates, Inc., Birmingham, Ala.) in bicarbonate buffer (pH 9.3)
overnight. PSA specific IgG plates were coated with 99% pure PSA at
4 .mu.g/ml in bicarbonate buffer. PSA specific IgG was quantitated
using a mouse monoclonal IgG.sub.1 specific for PSA (clone ERPR8,
ICN, Costa Mesa, Calif.) as a standard. Incubations were done at RT
in serially diluted duplicate wells. Binding of Ig was detected by
horseradish peroxidase (HRP) conjugated polyclonal goat anti-human
IgM-HRP, polyclonal goat anti-human IgG-HRP or polyclonal goat
anti-mouse IgG-HRP secondary antibody (Southern Biotechnology
Associates) incubation and subsequent enzymatic development of
o-phenylenediamine dihydrochloride (Sigma) substrate. Reactions
were quenched with 4N H.sub.2SO.sub.4 and the plates were read on a
ELISA plate reader at OD.sub.490. The concentration of human Ig in
SCIDhu PBL sera was quantitated by comparison of SCIDhu PBL serum
OD.sub.490 values with serially diluted standard curves. These
results are contained in FIG. 5.
[0035] To confirm the PSA binding specificity of group H sera IgG
pooled sera from four group H mice was diluted 1:15 (50 .mu.g/ml
total IgG final concentration) and 1:20 (50 .mu.g/ml total IgG
final concentration) into triplicate wells containing serially
diluted concentrations of soluble PSA. Soluble PSA induced
inhibition of PSA specific binding by group H sera and by an
equivalent concentration of control human IgG (Zymed, Inc.) was
assayed using polyclonal goat anti-human IgG-HRP, as described
above. These ELISA results are contained in FIG. 6.
Immunohistochemistry Analysis
[0036] Histologic and human lymphocyte specific antibody (CD3 and
CD 19) staining of frozen and fixed SCIDhu PBL mouse tissues was
contracted to BioPharMaceutical Support Services (Pharmingen).
Immunoblot Analysis
[0037] These experiments are ongoing.
Example 1
Human IgG Production in DC/Ab-PSA Complex Immunized
SCIDhu PBL Mice Mouse Monoclonal IgG.
[0038] Antibody (Cat. No. 10-P2O; Fitzgerald Industries Inc.) was
complexed with PSA at equimolar ratios at 4.degree. C. over night
(Ab-PSA complex) and then dialyzed to remove azide. Autologous
peripheral blood dendritic cells (pDC) were grown in serum free
media and pulsed with either 25 .mu.g/ml soluble PSA or Ab-PSA
complex. All mice received 10.sup.8 PBMC i.p. on day 0. Each group
consisted of 8 mice. Group F mice were immunized with 25 g of
Ab-PSA complex weekly and with 25 .mu.g PSA on Day 21. Group G mice
were immunized with 25 .mu.g of soluble PSA weekly and
7.times.10.sup.6 soluble PSA pulsed pDC on day 7. Group H mice were
immunized with 25 .mu.g of Ab-PSA complex weekly,
7.5.times.10.sup.6 Ab-PSA pulsed pDC on day 7 and then 25 .mu.g PSA
on Day 21. These results are summarized in FIG. 6. The Day 14
results, (A) Graphs, from left to right are as follows: Average PSA
specific IgG; PSA specific IgG for individual mice; Average total
human IgG; Average of percent specific IgG (specific IgG/total
IgG.times.100); percent specific IgG for individual mice. B. Day 28
results. Graphs, from left to right are as in A. All sera IgG
concentrations are expressed as mg/ml. Error bars represent
.+-.Std. Dev.
[0039] The immunization method described in FIG. 6 enhanced human
lymphocyte engraftment in SCIDhu PBL mice. On the average, six
times more human T cells were detected per spleen and more enlarged
lymph nodes (LN) were isolated from group H mice than from either
control group (see Table, below). Importantly, the enhanced
engraftment and Ig production was not induced at the expense of
enhanced xenogeneic graft versus host disease (XGVHD), as has been
reported in other "enhanced" SCIDhu systems. TABLE-US-00001 TABLE
Summary of Engraftment Total Spleen % hCD3+ % hCD19+ #Mice w. Group
Cells Number* Cells{circumflex over ( )} Cells{circumflex over ( )}
LN/group F 1.69 .+-. 0.26 .times. 10.sup.8 2.30 .+-. 0.67 <2%
2/8 G 1.58 .+-. 0.32 .times. 10.sup.8 2.93 .+-. 0.34 <2% 4/8 H
3.04 .+-. 0.60 .times. 10.sup.8 8.93 .+-. 4.32 <2% 7/8
[0040] Mice immunized, as described in FIG. 6, were sacrificed on
day 28. Spleens and LN were collected from all mice. Spleens were
divided in half. One half was used to determine cell numbers and to
do FACS analysis (3 mice each group). The other half of the spleens
and all the isolated LN were fixed and frozen for histologic
analysis. Spleen cell number and percent human T cells are shown
.+-.Std Dev. (p>0.5). *: average of 8 Mice. : average of 3
mice.
[0041] Histologic antibody staining data (frozen/fixed slides were
stained with a-hCD3 and a-hCD19) showed that Group H mice had many
more human T and B cells in enlarged peripheral LN compared to
control mice. Also, spleens from group H mice had more localized B
cell engraftment than control mice (data not shown).
[0042] The specificity of the PSA IgG response by group H mice was
confirmed I by comparison with non-responding Group F sera, control
human IgG and by competition of sera binding by soluble PSA.
Example 2
Specificity of Antibody Responses Obtained in Ab-PSA/pDC Immunized
SCIDhu PBC Mice.
[0043] In this Example, the relative PSA specificity of pooled sera
from group H mice, control hIgG and group F serum is measured and
was shown in FIG. 5.
[0044] FIG. 5 shows relative PSA specificity of pooled sera from
group H mice, control hihg and group F serum. In this Figure, open
circles represent pooled group H serum, and closed circles purified
hIgG. The open triangles represent mouse F1.1 serum. The data shows
that group H serum binds PSA 10 times greater than equal
concentrations of either control hIgG. Panel B shows the inhibition
of group H sera specific binding by soluble PSA. The open circles
represent pooled group H serum diluted 1:15 (120 .mu.g/ml). The
closed circles represent pooled group H serum diluted 1;20 (90
.mu.g/ml). The open tri-angles represent purified hIgG control, (90
.mu.g/ml). The data shows that pooled group H sera binding can be
inhibited by soluble PSA to OD.sub.490 values obtained by an equal
concentration of control hIgG in et dose dependent manner.
CONCLUSIONS
[0045] Therefore, these results demonstrate that DC pulsed with
antigen-antibody complexes induced PSA specific Ab responses in
SCIDhu PBL mice better than previous immunization protocols.
Moreover, the results demonstrate that when such pulsed DC are
administered in combination with Ab-antigen complexes, that
significant enhancement of total specific (>35 fold) and
relative specific (>10-fold) PSA IgG responses in SCIDhu PBL
mice is obtained (compared to either immunization strategy
performed separately). Also, this novel immunization strategy
enhanced human lymphocyte engraftment without enhancing XGVHD as in
other "enhanced" SCIDhu systems. Moreover, this method was
reproducible in three separate experiments using different PBMC
donors.
[0046] This approach and the technology developed around it is
significant as it enables rapid, reproducible production of
clinically superior products (human monoclonal antibodies) compared
to antibodies based on rodent antibodies). These human monoclonal
antibodies are useful for immunotherapy or immunoprophylaxsis,
e.g., treatment or prevention of of cancer and viral
infections.
[0047] The described methodology should be useful for generating
human MoAb specific to any relevant target antigen (e.g.,
Macrophage Inhibitory Factor, E7 antigen, CEA, HIV, etc.). However,
being a biological system, it is impossible to predict with
absolute certainty the extent of variation of the conditions or
parameters that will provide optimal results for different
antigens, e.g., the exact number of cells or the exact quantity of
Ab-PSA complex that results in optimal antibody production or
specificity. However, this can be determined by one of ordinary
skill using routine optimization.
[0048] The preferred antigens will comprise those expressed by
human diseases treatable by monoclonal antibodies (wherein
treatment includes therapeutic and prophylactic therapy), e.g.,
cancers, parasitic infections and viral infections. Examples of
diseases treatable by human monoclonal antibodies include, by way
of example, cancers such as breast, brain, cervical, ovarian,
prostate, bladder, pancreatic, myeloma, kidney, colorectal,
nasoparingeal, endometrial, lung, liver, leukemia, lymphoma, colon,
stomach, skin, among others, viral diseases, including those caused
by HIV, hepatitis, papillomavirus, respiratory syncytial virus,
herpes, etc., and parasitic diseases, e.g., malaria.
[0049] In the preferred embodiments, the antigen will be selected
from melanocytic differentiation antigens, e.g., gp100 (Kawakami et
al, J. Immunol., 154:3961-3968 (1995); Cox et al, Science,
264:716-719 (1994)), MART-1/Melan A (Kawakami et al, J. Exp. Med.,
180:347-352 (1994); Castelli et al, J. Exp. Med., 181:363-368
(1995)), gp75 (TRP-1) (Wang et al, J. Exp. Med., 186:1131-1140
(1996)), and Tyrosinase (Wolfel et al, Eur. J. Immunol., 24:759-764
(1994); Topalian et al, J. Exp. Med., 183:1965-1971 (1996));
melanoma proteoglycan (Hellstrom et al, J. Immunol., 130:1467-1472
(1983); Ross et al, Arch. Biochem Biophys., 225:370-383 (1983));
tumor-specific, widely shared antigens, e.g., antigens of MAGE
family, for example, MAGE-1, 2, 3, 4, 6 and 12 (Van der Bruggen et
al, Science, 254:1643-1647 (1991); Rogner et al, Genomics,
29:729-731 (1995)), antigens of BAGE family (Boel et al, Immunity,
2:167-175 (1995)), antigens of GAGE family, for example, GAGE-1, 2
(Van den Eynde et al, J. Exp. Med., 182:689-698 (1995)), antigens
of RAGE family, for example, RAGE-1 (Gaugler et al, Immunogenetics,
44:323-330 (1996)), N-acetylglucosaminyltransferase-V (Guilloux et
al, J. Exp. Med., 183:1173-1183 (1996)), and p15 (Robbins et al, J.
Immunol., 154:5944-5950 (1995)); tumor specific mutated antigens;
mutated .beta.-catenin (Robbins et al, J. Exp. Med., 183:1185-1192
(1996)), mutated MUM-1 (Coulie et al, Proc. Natl. Acad. Sci. USA,
92:7976-7980 (1995)), and mutated cyclin dependent kinases-4 (CDK4)
(Wolfel et al, Science, 269:1281-1284 (1995)); mutated oncogene
products: p21 ras (Fossum et al, Int. J. Cancer, 56:40-45 (1994)),
BCR-abl (Bocchia et al, Blood, 85:2680-2684 (1995)), p53 (Theobald
et al, Proc. Natl. Acad. Sci. USA, 92:11993-11997 (1995)), and p185
(HER2/neu (Fisk et al, J. Exp. Med., 181:2109-2117 (1995)); Peoples
et al, Proc. Natl. Acad. Sci. USA, 92:432-436 (1995)); mutated
epidermal growth factor receptor (EGFR) (Fugimoto et al, Eur. J.
Gynecol. Oncol., 16:40-47 (19965)); Harris et al, Breast Cancer
Res. Treat, 29:1-2 (1994)); carcinoembryonic antigens (CEA) (Kwong
et al, J. Natl. Cancer Inst., 85:982-990 (1995)); carcinoma
associated mutated mucins, for example, MUC-1 gene products (Jerome
et al, J. Immunol., 151:1654-1662 (1993), Ioannides et al, J.
Immunol., 151:3693-3703 (1993), Takahashi et al, J. Immunol.,
153:2102-2109 (1994)); EBNA gene products of EBV, for example,
EBNA-1 gene product (Rickinson et al, Cancer Surveys, 13:53-80
(1992)); E7, E6 proteins of human papillomavirus (Ressing et al, J.
Immunol., 154:5934-5943 (1995)); prostate specific antigens (PSA)
(Xue et al, The Prostate, 30:70-78 (1997)); prostate specific
membrane antigen (PSMA) (Israeli et al, Cancer Res., 54:1807-1811
(1994)); PCTA-1 (Sue et al, Proc. Natl. Acad. Sci. USA,
93:7252-7257 (1996)); idiotypic epitopes or antigens, for example,
immunoglobulin idiotypes or T cell receptor idiotypes (Chen et al,
J. Immunol., 153:4775-4787 (1994); Syrengelas et al, Nat. Med.,
2:1038-1040 (1996)).
[0050] The antigen will preferably be administered to a SCID mouse
in the form of an antigen-antibody complex as described supra.
Also, as described supra, the antigen or more preferably
antigen-antibody complex will be used for in vitro priming of
autologous dendritic cells, e.g., autologous peripheral blood
dendritic cells. The amount and duration of such in vitro priming
will be that which results in an enhancement of human antibody
production, when the resultant primed dendritic cells are used as
immunizing agents in SCID mice. As disclosed, preferably SCID mice
will be immunized with autologous dendritic cells which have been
pulsed in vitro with an antigen-antibody complex and further
immunized with such antigen-antibody complex as this has been shown
to confer synergistic benefits (enhance total antisera-specific
antibody response and relative specific IgG antibody response).
[0051] Also, it is desirable that EBV transformation be effected
during immunization. After immunization, human antibody secreting
cells will be isolated from such SCID mice and used to clone human
monoclonal antibodies. This may be effected by known methods.
[0052] Monoclonal antibodies possessing desirable properties
(minimum antigen binding affinity and avidity) obtained by such
methods are useful as human therapeutics and prophylactics. These
human monoclonal antibodies will be administered by known methods,
e.g., systemically or parenterally, e.g., orally, subcutaneously,
intravenously, intramusculatory, topically, by infusion, to
patients in need of such treatment.
[0053] The administered dosage will be a dosage that results in
therapeutic or prophylactic benefits. Generally, such dosage will
range from about 0.001 to 100 mg/kg, more preferably 0.01 to 50
mg/kg, still more preferably 0.1 to 5 mg/kg body weight. Moreover,
such dosage will vary dependent upon the condition of the patient,
the disease condition, whether other therapies are also being
effected, among other factors.
[0054] Typically, the antibody will be administered in combination
with a pharmaceutically acceptable carrier or excipient, e.g.,
phosphate buffered saline, optionally in combination with adjuvants
that enhance humoral or CTL immunity.
[0055] In the case of prostate specific antigen specific
antibodies, these antibodies will be used for the treatment or
prevention of prostate cancer as this is a known antigen expressed
during prostate cancer.
* * * * *