Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~'~.91/17769 PCT/US91/0~406
~2~
NOVEL METHODS FOR PRODUCING ANTIGEN-SPECIFIC,
HIGH-AFFINITY HUMAN HONOCLONAL ANTIBODIES
Reference
This is a continuing application under 35 U.S.C.
120/121 of application U.S.S.N. 07/527,203 filed 22
May 1990.
Acknowledaement
This invention was made with U.S. government support
in the form of a grant from the National Institutes
of Health. The government has certain rights in this
invention.
Field of the Invention
This invention relates to the field of biotechnology,
and more specifically, is directed to a novel method
of preparing and culturing in vitro, human
lymphocytes having particular characteristic~ ~uch
1~ that antigen-specific IgG as ~ell as IgM human ' ~`
monoclonal ~ntibodies are obtainable with useful
affinities suitable for clinical application.
Backqround of the Invention
Much research ~ctivity has focused on ondeavor~ to
produce monoclonal antibodies for evident industrial
applicability in the field of medicine. Xohler ~nd
~il6tein pioneered the production of ~pecific
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2cl -2- Pcr/us9l/o34ot
mono onal antib~dies by somatic cell hybridization
techniques (~ature 356, 495 (1976)). That technique
involved fusing lymphocytes that ~ad been immunized
in vivo against a particular antigen with n fusion
S partner 6uch as myeloma cell6. The fu~ed cell~ were
then expanded in a selection medium. The surviving
hybridoma~ were nssayed for the production of the
desired monoclonal ~ntibodies and the ~elected
positives were ~ubcloned, expanded and frozen.
Initial research concentrated on in vivo i~munization
and those efforts continue. Sub~tantially les~
effort needs to ~e expended for the production of
monoclonal antibodies if a reproducible method for
vitro induction of immune responses were to become
available. Accordingly, in vitro immunizations were
pursued .
Borrebaeck and coresearchers at the University of
Lund in Sweden are believed to be the fir6t to have
reported the use of mixed lymphoeyte cultures tMLC)
by co-culturing histoxenogeneic mouse lymphocytes.
In a first paper, they cultured in ~uch a way 50 as
to prevent cell aggregation. See Borrebaeck, ~cand.
J. Immunol. 18, 9 (1983).
In a later paper, they extended their analysis of the
requirements for B cell growth and differentiation
factors derived from ~uch ~ixed lymphocyte cultures.
Again, they worked with lymphocytes obtained from
histoxenGgeneic ~ice 6pleen tis~ue. Although it i8
not clear exactly how they prepared their spleen
ti~sue, they do report removing non-adherent cells
and recovering bound cells. They report the
importance of ~upplementing their MLC lymphocytes
with derived lymphokines and these were the
preparations used in their ~tudies. They report that
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0 91/17769 P~-r/US91/03406
without the ~upport of growth factors, 6uch AS 2 ~
lymphokines, no significant antigen-specific immune
response could be recorded from the in vitro
immunization.
U.S. Patents 4661586 and 4816249 are relatively
recent additions to the art but are limited in
example to the use of immunized mice spleen cell6
which were then fused to immortal lines.
These Kohler ~nd Milstein procedures and in vitro
immunization methods used lymphocytes of rodent
origin. In human clinical applications, the use of
such non-human monoclonal antibodies is not optimal
in that rodent antibodies are foreign to the human
host, and therefore could be expected to induce host
immunity responses and subsequent reductions of
therapeutic efficacy.
Consequently, research efforts included endeavors to
produce human monoclonal antibodies. However, human
cells are difficultly cultured in vitro. A 1986
review is provided by Larrick, et al., Journal of
Bioloaical Response Modifiers 5, 379 (1986). See
also James et al., J. Immun._Methods 100, 5 (1987).
This review article concluded by speculating on
whether human monoclonal antibodies would prove to be
efficacious replacements of the rodent monoclonal
~ntibodies, depending largely on the ready
availability of ~uch human m~noclonal ~ntibodie~ in
clinically useful purified form, the problems of
maintaining a stable culture of human cell6
effectively immunized again6t a specific antigen
being the major 6tumb1ing block.
Croce, et al., Nature ~, 488 (1980) ~nd Olson and
Raplan, FNAS 77, 5429 (1980) reported on applying the
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W091/17769 ~1~X ~O il -A- PCT/US91/0340~ j
Kohler and Milstein techniques to human cell~. See
also European Patent Application Publication No.
44722 and U.S. Patent 4668629. In the latter
documents is di6closed an alternative mean~ of
producing splenocyte6 in either an in ~ivo or n
vitro immunization. However, the experimental data
provided for the preparation of spleen tissue by
Ficoll-Hypaque centrifugation, with apparent removal
of adherent cells, and the lymphocyte ~ononuclear
cell suspension product~ were fused with ~ specific
human myeloma cell line.
Similar procedures are disclosed by Larrick, et al.
in U.S. Patent 4624921. See also European Patent
Application Publication No. 157574 where Ficoll
preparation of peripheral blood lymphocytes was al~o
used and the lymphocytes were transformed with
Epstein Barr Virus (EBV), a means engaged in by many
prior researchers to produce cells that can be grown
continuously, albeit often lo6ing their ability to
secrete immunoglobulins (Ig) in a relatively short
period of time. European Patent Application
Publication No. 62409 describes a human
lymphoblastoid cell line capable of acting as a
fusion partner in the preparation of hybridomas in a
rather specific manner.
U.S. Patent 4451570 similarly di6closes use of human
cell lines for the production of human monoclonal
nntibodies but emphasizes t~e use of ~on-~dherent
lymphoid cell culture for fusion and the preparation
of the lymphocyte cells with Ficoll techniques.
Patent Application W085/02413, corresponding to
European P~tent Application Publication No. 0162918
report on the preparation of human monoclonal
antibodies specific to human Rh(D) antigen, using
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.... . i
peripheral blood lymphocytes (P8Ls) 6eparated by
Ficoll-Hypaque gradient. See al~o European Patent
Application Publication No. 174204.
European Patent Application No. 292965 report6 on the
pro~uction of a stable, continuous human cell line
that secretes less than 40 ng/ml of endogenous IgM
antibodies from a Epstein-Barr virus tr~nsformed
human lymphoblastoid B cell line fusion.
Yamaura et al, J. Immunol. Methods 84, 105 (1985)
report on primary immunization of 6pleen cells
combined with monoclonal antibody production of both
IgM and IgG. They ~tate that the EBV transformation
step is essential for the expansion of antigen
specific clones, isotope ~witching and subsequent
hybridoma production.
For related systems, attention may also be directed
to the following: Ho, et al., J. I~munol. 1~, 3831
(1985); Cavagnaro, et ~1., Biotechniques 1, 30
(1983); Strike, et al., J. Immunol. 132, 1798
(1984); Wasserman, J. Immunol. Methods 93, 275
(1986); Hoffmann, PNAS 77, 1139 (1980); and Pollock,
et al., in vitro Immunization in Hybridoma
Technology, Elsevier Science Publishers, Amsterdam,
1988, page 277. The Strike, et al paper uses an
25 immunization of an allogeneic culture of tonsillar -
lymphocytes.
Borrebaeck, et al., PNAS 85, 3995 (1988) later
reported on the preparation of human-human hybridomas -~
for the production of human monoclonal ~ntibodies
from peripheral blood lymphocytes immunized in vitro
against T cell dependent antigens. Again, their n
vitro immunizations required support by various known
growth and differentiation factors. See ~l~o the
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WO91/17769 ` -6- PCT/~'S91/0~40
related PBL papers Danielsson, et ~l., I~munology 61,
51 (1987) and Borrebaeck, J. Immuno. ~ethods l~, 157
(1989).
In contrast to the results herein, the Banchereau et
~l-, Science ~1, 70 (1991) lymphocytes die after
about lO weeks.
Finally, the coinventors of the present application
disclosed certain preliminary findings of their
research which led to the present invention. Those
preliminary findings were published in ~ meeting held
23 to 30 April 1988 and were followed by proceedings
published in early 1989 in Human Tumor Antiqens and
Specific Tumor TheraDv, Alan R. Liss, NY, NY (1989),
p. 147. The conditions described were insufficient
to produce high-affinity, nntigen-specific monoclonal
antibodies presumably because the numbers of adherent
cells, now known to be essential, were depleted.
Since publishing ~hese findings, the present
inventors expanded their research considerably and
have now produced methodology enabling the
preparation of human monoclonal antibodies that
exhibit useful affinities for specific antigens,
including human antigens, and is the subject of the
present invention. An effective number of these
monoclonal antibodie are IgG, An antibody class not
frequently obtained from primary ~in vi~o
immunizations. The present invention i6 believed to
represent a major step toward the production in
~ufficient quantities of stable efficacious human
30 monoclonal antibodies befitting their use in ~ -
clinical ~etting for the assaying or treatment (of
human beings) against antigens specific for a
particular di6ease ~tate.
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Summary of the Invention
The present invention provides in vitro cultures
comprising human lymphocytes that are obtained from
lymphoid ti~sue, having essential numbers of
autologous accessory, including adherent, cells.
The6e lymphocyte~ ~re i~munized in y~t~o against a
specific antlgen. I~ particular, the lymphocyte
preparations and culturing condition~ are ~uch that
the resultant monoclonal antibodies obtained after
immortalization include effective numbers of IgG
having useful affinities to the ~pecific antigen,
i.e., of ~t least about S x 107 liters per mole tan
expression representative of an affinity constant, K,
that is equal to l/C where C i5 concentration or
M (mole) per L (liter). The present ~ystem produces
effective numbers of such monoclonal antibodies
without requiring supplementation by various growth
or other factors that have heretofore been reported
as necessary for production of antigen-specific
antibodies. A particular embodiment of the present
invention involves coculturing allogeneic lymphocyte
cell~, that is, for example cpleen cells taken from
histoincompatible individuals.
The present invention is directed to such ly~phocyte
preparations ~nd culturi~g methods useful to produce
such monoclonal antibodies in all aspects, including
particularly, the methods of lymphocyte prepar~tion,
the resultant cultures themselves, and e~senti~l
components, ~nd the stable continuous cell line~ t~at
produce the antigen- pecific monoclonal ~ntibodles
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prepared, for example, by fusion with an immortal
fusion partner or by means of reco~binant DNA. For
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example, cell line~ can be operatively transfected
with DNA encoding the vari~ble region of the antibody
desired. It also i~ directed to the human monoclonal
antibodies produced from such ~table continuous llnes
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W091/17769 ~2~ 8- PCT/US91/0~4
as well as a process for preparing such antigen-
specific monoclonal antibodies, such as by co-
culturing allogeneic spleen lymphocytes.
Further, the present invention is directed to
~ntibodies obtained a6 de~cribed herein linked to ~
material capable of ~odulating cell growth, or with a
reporter molecule. In the former case, ~he
monoclonal ~ntibody hereof derived by immunization
with, for example, ~ tumor antigen, is useful to site
~pecifically target a tumor cell receptor for
treatment of the cell with a material capable of
killing it or arresting its growth. Similarly, where
a monoclonal antibody hereof would be linked to a
reporter molecule it could serve as a diagnostic tool
identifying presence of cells producing ~ntigen
specific to a disease state.
It has been found that the use of three specific
means of preparation of lymphocytes provide the
minimum number of accessory cells that support this
invention. One method is to discard the commonly
used method of Ficoll treatment to isolate
lymphocytes, instead using a hypotonic lysis of red
blood cells, 6uch as with ammonium chloride. The
second means is to minimize conditions that are
conducive to cell binding to exposed vessel 6urf~ces
~y minimizing room temper~ture incubations, thereby
reducing loss of adherent ~ccessory cells during
preparation. The third ~eans i6 to retain, ~nd/or
supplement with, ~mall fragments of lymphoid tissue
in the final cell Eu~pension prior to freezing.
These expedients, ~pecifically det~iled inf~a. and
their equivalents, nre the enabling process
predicates of the present invention.
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The presently described invention is further
specifically characterized by the following: l. The
process described will support a primary immunization
response by human ly~phocytes in vit~o. The novel
5 aspect is t~t the conditions which underlie the
specific immune responses support development of
antigen induced IgG- as well as IgM-secreting B
cells. 2. This process, when applied to human
lymphocytes in good condition, will 6upport
development of a human immune response to human as
well as to foreign antigens. 3. Human lymphocytes,
immunized and fused according to the protocols
described herein, are capable of producing hybrid
clones at a frequency (~ hybrids produced/million
lymphocytes fused) 5-lO-fold higher than previously
reported for in vitro immunized human lymphocytes. Of
the hybridomas produced, a minimum of about 3 to lO
percent are antigen-reactive, depending upon t~e
nature of the antigen. Of the antigen-reactive
monoclonal antibodies produced, about 20 - 90% have
been IgG.
Thus, it will be apparent that following the
teachings of the present invention, one endeavors to
use techniques that ensure the presence of high
numbers of accessory, including adherent, cells in
the human lymphocyte culturing. The end points of
buch successful techniques, or equivalents to those
~pecifically taught herein, are the production of
monoclonal antibodies being antigen 6pecific and
ex~ibiting useful affinities in the order of at least
about 5 x 107 liters per mole hnd having effective
numbers of the IgG class. These endpoint~ are
measured by means known in the art, e.g., via
competition ELISA assay.
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W091/17769 2~8~ lO- PCT/VS91/0~40
Present teachings emphasizing the es6ential presence
of large numbers of ~ccessory, including ~dherent,
cells in t~e culture ~uspension are ~otably followed
by 6pecifically including fragments of lymphoid
ti~sue. One can ~ccomplish this by u6ing techniques
which will not exclude such fragment~ and/or by
~pecifically supplementing the culture with lymphoid
tissue fragments. While not intending to be bound to
any specific theory, experimental observations with
sy~tems containing deliberate quantitie6 of lymphoid
tissue reveal consequential multiple accessory cells.
These cells appear to form part of loose aggregates
of undefined material that within weeks develop into
tight forms having a well-defined border. These are
thought to be putatiYe germinal centers and are
defined as "splenoids. n These ~plenoids develop out-
pockets from the membrane, "blebs", that themselves
may migrate to form additional accessory cell
aggregates and so forth. It has further been
observed that such systems support IgG secretion for
months and depict class 6witch from IgM to
predominantly IgG within that time.
Thus, use of lymphoid fragments in the culturing
systems herein results in predominantly IgG-secreting
culture that are long-lived upwards of si~ months and
beyond.
Detailed Descri~ticn
1. particularly Preferred Embodiments
As a contemplated ~est mode di~closure, there are
described successful results producing human
monoclonal antibodies specific to the hu~an and horse
ferritin ~ntigens. The specific culturing conditions
that enable the production of the antigen-specific,
high-affinity monoclonal antibodies hereof are
similarly detailed. It will be understood, however,
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that one of ordinary skill in the art having been
provided the present disclosure will well enough know
how to employ the present invention in the
preparation of other human high-affinity ~onoclonal
antibodies having ~pecificity to other antigens.
Similarly, alternative human lymphocytes other than
splenocytes may be employed by following the
teachings of the present invention which were
initially applied with that particul~r lymphoid
tissue. Moreover, other fusion partners may be
employed by following the teaching~ hereof employing
a particular fusion immortal line.
It is also foreseeable from the teachings of the
present invention that having provided methodology
for producing antigen-6pecific monoclonal antibodies,
that means are available in the art to characterize
and sequence the variable re~ion. Armed with that
information, one can apply known recombinant DNA
techniques to clone the DNA of the variable region or
of the entire Ig molecule bearing ~uch a variant
region and introduce such DNA operatively into stable
recombinant host cells for expression and collection
of the encoded Ig molecule. Similarly, the DNA may
be amplified using, for example, polymerase chain
reaction (PCR) techniques prior to or simultaneously
with its expression in a suitable stable ~ost. All
of this i~ enabled by virtue of the present
disclosure which provides the means for generating
immunized B lymphocytes bearing high-affinity,
antigen ~pecific human immunoglobulins.
The present, ~pecific in vitro i~muniz~tion culturing
~ethod, which (as noted above) can be expanded to
equivalent means by those skilled in the ~rt
followin~ the teachings hereof, essentially provides
lymphoid tissue preparations that contain essential
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W091/17769 ~ 8 ~ O ~ ~ - l2- PCT/US91/0340
numbers of accessory, including adherent, cells.
This result in effect produces cultures that
functionally reproduce in vivo environments capable
of supporting primary immunizations and generation of
antigen-specific IgG-bearing B cells and IgG-
secreting plasma cells.
In the in vivo late primary or secondary re6ponse
~booster), antigen exposure is immediately
encountered with ~pecific antibody forming antigen-
antibod~ complexes referred to as iccosomes (immunecomplex coated body) and is trapped in the lymphoid
organs on the surface of the follicle dendritic
cells. Expansion and maturation of antigen-specific
B cells is thought to occur within so-called germinal
centers and include the phenomena of class switch
(from immunoglobulin IgM to IgG, IgA and IgE), as
well as affinity maturation (through point mutations
in tbe variable region). These two phenomena nre
considered to be the essence of effective late
primary and secondary responses.
As noted, the above-described in vivo immune re6ponse
system is believed to be functionally reproduced by
the present invention in vitro. Conditions
reproducing, at least in part, follicle and germinal
center formation are suspected to be required for the
important phenomena of class switching and ~ffinity
maturation for most effective production of higher - -
affinity, antigen-specific (preferably IgG)
antibodies. The elements thought to be required for
optimal responses in vitro are the presence of: B
cells bearing surface immunoglobin through which they
c~n recognize And bind to specific antigens, T helper
cells that stimulate tbe proliferation of B cells by
localized secretion of various factors, macrophages
tbat ingest, pr~cess and present antigens,
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~ 91/17769 2 ~ ~ 2 ~ ~ ~ pcr/us91/o34o6
appropriate lymphokines that are produced by
macrophage and T helper cells, and the all important
presence of dendritic cell~ that promote homologous
and heterologous mixed lymphocyte response (HAR)
reactions and cellular aggregation.
In the preferred embodiments of the present
invention, spleen cells are employed. These spleen
cell cultures are prepared in a mild fashion using,
for example, ~mmonium chloride lysis instead of the
common prior art use of ficolling that ~erves to
~eparate red cells from white cells. The preparation
and culturing of lymphocytes will minimize conditions
that are conducive to cell binding to vessel
surfaces. In an embodiment, mixed instead of ~ingle
spleens are employed in allogeneic cultures,
providing initially highly responsive systems. These
combined processes, that are described in respect of
the preferred embodiments hereof, provide all of the
elements thought to ~e essential for the successful
in vitro primary followed by secondary responses at
least in part representative of those that are
produced in vivo in a human organism upon
encountering antigen. As noted above, aspects of
these essential elements are provided in the
preferred embodiments by the use essentially of human
lymphocytes obtained from lymphoid tissue via
methodology that preserves essential num~ers of
autologous ~cce~sory, including dendritic and
~dherent cells.
This i5 made po~sible primarily because of specific
elements that may be employed in the method of
ly~phoid tissue, e.g. ~plenocyte, preparation:
a. gentle dissociation of lymphocytes and
accessory cells from cut pieces of lymphoid tissue by
manual manipulation, e.g., with forceps ~nd the
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WO9l/17769 ~ 8æ ~ 14- PCT/US91/~40
(rubber-tipped) ~lunt end of a plunger from an
~ppropriate ~terile pla6tic ~yringe.
b. removal of red blood cell~ by gentle
hypotonic lysis instead of by ficolling.
c. prevention of 106s of adherent cells by
minimizing exposure to flat surfaces ~t temperatures
above about 4O C.
d. retention of, and/or supplementation with,
small (less than or ~bout 1 cubic millimeter)
fragments of ~plenic connective tissue within the
final cell preparation.
Collectively, actions a-d give rise to a mixture of
healthy splenocytes and accessory cells, including
macrophages, follicular and lymphoid dendritic cells,
and fibroblasts. This combination of cells, in the
state of activity provided by their gentle
dissociation from whole spleen, more closely
reproduces the in vivo splenic environment than any
combination of lymphocyte preparation steps or
culture methods previously described.
The systems hereof manifest ~plenoids that are
thought to be in vitro equivalents to in vivo
germinal centers of the late primary or secondary
stages of immune response, and manifest the results
of such response by class switch and long-lived IgG
secreting cultures.
A major use of the described process is the
production of immortal, continuously secreting
hybridoma clones which produce human monoclonal
antibodies ~pecifically reactive with al~ost any
antigen of interest, including human antigens.
Thus, the present invention provides for the first
time an in vitro immunization culturing ~ystem that
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~'~91/17769 2 ni ~` 2 9 ;11 PCT/US91/03406
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produces relatively high numbers of high-affinity,
antigen-specific monoclonal ~ntibodies when converted
into stable continuous cell lines, that ~re useful in
a clinical setting.
S 2. De~ri~tion of Parametç~s.
The present invention i5 predicated on the finding
that the method of preparing the lymphocytes for
culture and immunization in vitro i~ es6ential. The
essential aspects employ methodology ensuring the
retention of essential numbers of accessory cell~
within the lymphocyte cultures. Such accessory cells
are necessary for reproducing at least in part the
lymphoid follicle type of environment that 6upports
efficient antigen presentation, class switching and
affinity maturation. The presence of essential
numbers of autologous accessory cells is ~anifested
by the presence of an "essential number~ of adherent
cells in the culture. It has been found from
research results that optionally the nu~bers of non-
lymphocyte (non-T, non-B) cell6, i.e., that
population of cells containing essential nccessory
cells, in the final preparation should not be less
than about 10% of the total cell number. It has also
been observed that the numbers of essential adherent
~onocyte/macrophage cells in the lymphoid tissue
chosen for culturing should not be less than about 2%
of the total cell population. The~e numbers were
arrived at by labelling monocytes and macrophages
with a specific ~onoclonal antibody, tagging with a
fluorescent lAbel and determining the percent of
labelled cells by fluorescence activated cell sorting
(FACS) analy6is.
It will be understood that culturing techniques can
be varied from those ~pecifically disclosed herein so
as to ensure the retention of effective nu2ber6 of
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W09~/17769 2(I82~ 16- PCr/us91/o34
accessory cells thought to be essential. Further, it
may be found with further re~earch not requiring
undue experimentation that t~e number~ of adherent
cells disclo6ed as essential herein may vary somewhat
outside of the minimum range given; hence, the use of
the term "about~ when defininy that limit.
It has been found via the re6earch herein that the
culturing conditions that use essential numbers of
adherent cell6 provide effective numbers of IgG class
immunoglobulins. It i~ believed, based upon the
present research, that a minimum of about lO percent
of the monoclonal ~ntibodies hereof being of the IgG
class would be an ~effective number"; a 50 to 80
percent level would be considered most effective for
identification of clinically useful monoclonal
antibodies. Again, it will be understood that
~arying the culturing conditions within the general
scope of the present invention may provide numbers of
IgG molecules that are effective but 60mewhat outside
of the range as specified. It is considered that
where those numbers are effective, there are
generated sufficient antigen-specific IgG monoclonal
antibodies ~uch that antibodies having useful
affinities and qood ~pecificity can be 6elected.
Hence, the use of the term "about" when expressing
those values.
3. Definitions and General ~rocedures
In a 6pecific process hereof, cplenocytes, obtained
by careful dissection from ~uman pleen tis~ue, are
i~munized, i.e., ~pecifically ~timulated, by
~icrogram quantities of either xenogeneic or
allogeneic antigen. The stimulated B cell~ are then
collected at a ~pecific time after exposure to
antigen and fused at high efficiency to a
heteromyeloma human fusion partner, K6H6/B5 (publicly
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UO91/17769 -17- ~ ~ 8 2 0 ~i 1 PcT/ls9l/03406
available; see also J. Immunol. Meth. 89:6l-72,
1986), to produce hybrid clones which secrete human
monoclonal antibodies ~pecifically reactive with the
immunizing antigen. Specifically immunized cultures
may also be depleted of antigen ~nd allowed to
secrete antibody which ~ay then be detected by
conventional ELISA techniques. Po~t-immunization
~ssay allows semi-guantitative evaluation of the
responses and of the classes of immunoglobulin
pxoduced under a wide variety of immunization
conditions. These results are then used to identify
optimal conditions for immunization prior to fusion
and to estimate the results of subsequent fusions.
By the term "in vitro immunization culture" is meant
a culture in which the immunization with specific
antigen is not done in man or any other animal;
instead, a culture derived from tissue of human
origin is cultured ex vivo.
By the term ~lymphoid tissue" from which the human
lymphocytes hereof are obtained for culturing is
meant any human lymphoid tissue that can be expected
to have large numbers of T-/B-cells, and accessory
cells, including spleen tissue, tonsil6 and lymph
nodes.
By the term "essential numbers" of autologous
adherent cells herein is meant numbers that prove to
produce IgG monoclonal antibodies in accord herewith
that have useful affinities ~pecifically to A given
antigen, both non-human and notably human. These
results are preserved herein by the expedi~nt of
using lymphoid tissue fragments in the culture
system. Hence, the term has a functional definition
wit~in an approximated range defined above ~nd can be
attained by specific, preferred means.
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2~ L8- PCT/US91/0~406
By the term "autologous accessory cells" herein is
meant non-T, non-B cells which support or enhance
i~munization xesponses, derived from the 6ame
individual from which the T and B lymphocytes ~re
obtained.
By the term ~specific antigen" i5 ~eant nny antigen
that i8 desired for specific-reaction with a human
monoclonal ~nti~ody hereof including various tumor
antigens ~uch ~s ferritin, carcinoembryonic antigen
(CEA), TAG 72, etc.
By reciting herein that the culturing of the present
invention does not require ~supplementation with
growth or other factors", such as ly~phokines, that
have heretofore been considered essential is meant
that the conditions described herein make it
unnecessary to ~upplement exogenously the cultures
with these materials. That is not to imply that the
invention proscribes the supplementation with such
materials, only that it is unnecessary by virtue of
the advance of the present invention.
In the term "at least about" in respect of the
affinity of the monoclonal antibodies hereof, by the
component "at least" is meant that the value
specified represents a currently believed threshold
value ~bove which the monoclonal ~nti~odie~ would
find utility in a clinical 6etting. The component
"about" in respect of thi6 term i6 intended to convey
~ome latitude in the interpretation range o~ the term
in question, given the inherent variances of
biological systems such as differences in h3st cells
employed, fusion partner6 employed, ~ntigens chosen
and in culturing conditions from those specifically
disclosed herein. It will be understood that it i6
considered within the 5kill of the art to vary the
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.
-19-2~82~
~wn 91/17769 PCT/US91/0~406
procedures and produce clinically useful human
monoclonal antibodies in ~ccord herewith that may
differ in value from that qiven for the optimum
threshold affinity provided herein.
The term ~effective number6" in re6pect of the IgG
class antibodies is defined as discu~sed above.
The term "multiple macroscopic ~ggregate forms" i6
used to define the observation that clump5 of cells
derived from lymphoid tissue, which can include B ~nd
T lymphocytes and a~cessory cells, ~egin forming
within hours of incubation of culture, ~nd ~re
visible without magnification.
"Splenoids" develop within one to two or more weeks
of culturing and have been observed where fragments
of lymphoid tissue are specifically included in the
cultures. They may be mimics of the in viVo germinal
center of late primary or secondary immune response.
By the term "allogeneic coculture" is meant a
culturing of histoincompatible tissues, e~g., spleen
or other lymphoid tissue from more than one non-
identical individual.
The reference to "IgG", etc. i~ the ~tandard
reference to immunoglobulins of one of the five ~nown
major classes of constant regions that determine the
class of the immunoglobulin molecule ~nd are referred
to as IgG, IgM, IgA, IgD and IgE.
By the term n immortal fusion cell p~rtner~ i~ ueant n
cell line con6isting of cells which reproduce
themselves indefinitely and which, upon fu~ion with n
ly~phocyte-bearing functional immunoglobulin gene,
provides a vehicle for indefinite ~nd constant
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, . ... . . , , . ~ . . ... .
wo 9l,l7769 2 ~ 0~ 20- PCr/US91/0~40 ~
secretion of immunoglobulin molecules coded by said
gene. Suoh fusion partners include myeloma and
plasmacytoma cell lines as well as heteromyelomas and
heterohybridomas.
By the term ~via recombinant means~ and Wharboring
operatively" is meant the generally known and
published ~ethodology by which suitable host cells
~re transfected with DNA, preferably within vector~
where the encoded DNA is linked to DNA element that
induce expression to form encoded polypeptide. Such
expression vectors are generally replicable ~nd may
remain as episomes or as an integral part of the host
chromosome.
See also, generally, ~anitias, et al., Molecular
Cloning: A Laboratory ~anual, Cold Spring Harbor
Laboratory, New York, 1982, and various references
cited therein, and in particular, Colowick, et al.,
Methods in EnzYmolo~v, Vol. 152, Academic Press
(lg87) .
By the term "material capable of modulating cell
growth~ is meant a material that ~atisfies two
criteria, namely, that it be specifically linkable to
the antibndy such as via a chelate or a direct
covale~t link, and that it have a cytocidal or
cytostatic effect cellularly in vivo. Examples of
such ~aterial~ would include radio~ctive 6ubstance6
~uch as yttrium~, iodine ll~ and toxins ~uch as ricin.
.
By the term ~reporter moiety" is ~eant a material
that ~atisfies two ~riteria, namely, that it be
specifically linkable to the antibody, ~uch as via a
chelate or a covalent link, and that it provide 60me
identification of its existence such as in a
bioassay, or more commonly, in a chromaphore assay.
- . , . ,~ - : " .
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.. . ... . . .. . ..
~ - . : . - , : .
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W~91/l776~ PCT/US91/03406
Examples of ~uch moieties would include a radioactive
label ~uch as indium"" or enzymes ~uch as alkaline
phosphatase.
Reporter moieties may be attached to the antlbodies
S hereof ~ccording to conventional techniques known per
6e in the art. For example, nucleophilic groups on
the antibodies 6uch as primary hmine groups ~ay react
with a fluorescent or enzymatic reporter group to
form a covalent bond, or bifunctional coupling
reagen~s ~nown per 6e in the art may be employed.
Other useful reporter moieties are biotin,
fluorophores, chemiluminescent moieties, enzymes or
colloidal compounds. Examples of fluorophore groups
are fluorescein-5-isothiocyanate, diacyl fluorescein-
5 and/or 6 carboxylic acid pentafluorophenyl ester,tetramethylrhodamine-5 (and 6) isothiocyanate, eosin-
isothiocyanate, erythrosin- 5-isothiocyanate, 4-
chloro-7-nitrobenz-2-oxa-1,3-diazole, succinimidyl
12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-
yl)aminododecanoate, 7-hydroxycoumarin-4-acetic acid,
4-acetamido-4'-isothio-cyanatostilbene-2-2'-
disulfonic acid, 9-chloroacridine, p-nitrophenyl 1-
pyrenebutyrate, 9-anthracenepropionic ~cid, or 2-
anthracenesulfonyl chloride.
,
Enzy~ic reporter moieties include ~-galactosidase,
horse radish peroxida6e, ~lkaline phosphatase,
dehydrogenases, lucifera6e and carbonic nnhydrase.
4. Fi~ure Leqends
Fiqure 1. Ferritin-reac$ive IgM nntibody ~ecretion
by in vitro primed human lymphocytes: compari60n of
6ingle (A,B) and allogeneic, mixed (A+B) cultures.
"
.
WO91/17769 PCT/US91/0~40~ !
2~ 22-
Human 6plenocytes were prepared without depletion of
adherent cells as described infra and cultured at a
density of 3 x lO(6) cells/ml in the presence of
absence of l ug/ml horse spleen ferritin. Allogeneic
cultures consi~ted of a l:l mixture of cells prepared
from ~pleens A and B.
Figure 2. Ferritin-reactive IgM antibody ~ecretion
~y in vitro primed human ly~phocytes: ~nfluence of
inactivating X-radiation on specific i~mune responses
by single and allogeneic mixed cultures.
Human splenocytes were prepared without depletion of
adherent cells as described nfra and exposed to 2000
rays of x-radiation within 1-2 hours of preparation.
Within one hour of irradiation, 6yngeneic or l:l co-
cultures of normal and/or irradiated cells wereprimed with 0 or 2 ug/ml horse spleen ferritin for 3
days. After removal of ferritin by washing, the
cells were cultured for an additional 2 days. The
supernatants were collected and analyzed by ELISA for
reacti~ity with adsorbed ferritin.
Figure 3. Ferritin-reactive IgM antibody ~ecretion
by in vitFo primed hu~an lymphocytes: time dependence
of ferritin priming and secretion of ferritin-
reactive antibody.
Human spleno~ytes were prepared without depletion of
adherent cells and cultured at 1.5 x 10(6~ cells/ml
with the concentrations of hor~e ~pleen ferritin
indicated in the ~igure (0 - l.0 ug/ml). The top row
of figures represent~ cell6 which had been primed for
l day, washed free of antigen, and cultured for an
additional l, 2, 3, or 5 day6, as indicated, to allow
antibody ~ecretion in the absence of ferritin. The
6econd, third and fourth horizontal rows of figures
. .
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,
.
~91/17769 2 ~ ~ 2 ~ PCT/~IS91/0~406
-23-
represent antigen priming for 2, 3, and 4 days,
respectively, after which antigen was removed ~nd the
cells were cultured without ferritin for an
additional 2, 3, or 5 days, as indicated. Culture
~upernatants were collected and analyzed by ELISA for
reactivity with adsorbed ferritin.
Figure 4. Ferritin-reactive IgM and IqG antibody
~ecretion by in vitro primed human lymphocytes: time
dependence of ~ecretion ~fter a 3 day immunization.
Human splenocytes were prepared without depletion of
adherent cells. Duplicate wells were cultured at 3 x
lO6 cells/ml with or without l ug/ml horse spleen
ferritin for 3 days. Ferritin was removed by washing
and the cell were cultured for an additional 13 days
without ferritin. Supernatants were collected for
ferritin-reactivity analysis by ELISA at days 5, 7,
9, ll, 13, and 16. After each collection the
cultures were refed with 1.5 ml culture medium.
Culture supernatants collected on a given day
contained primarily immunoglobulins ~ecreted between
the previous refeeding ~nd the time of supernatant
collection. Open and ~haded areas repre~ent IgM and
IgG responses, respectively.
Figure 5. Affinity/avidity analysis of ferritin-
reactive IgM antibodies secreted by in vitro primed
human lymphocytes. Human ~plenocytes were prepared
without depletion of adherent cells. l:l co-cultures
were primed with O or l ug/ml horse ~pleen ferritin
for 3 days followed by washing to remove ferritin and
2 additional days of culture in the absence of
ferritin. Ferritin reactivity of 6ecreted antibody
was analyzed by ELISA and relative affinity/avidity
was estimated. St~ndard error bars not shown are
contained within the 6ymbols.
.,- . . . , , :,
~ ' ... ~ . . '
.
~; :
WO91/17769 ~ 3~ 24- PCT~US91/0340 ~ ~
Figure 6. Ferritin-reactive IgM antibody secretion
by human lymphocytes primed in vitro in the presence
or nbsence of adherent cells: compari~on of
responses to horse and to human ferritin.
~uman splenocytes were prepared without depletion (B
~nd D) or with depletion (A and C) of adherent cell6.
1:1 co-culture~ of cell~ from 2 spleens were primed
with the indicated concentrations of hor6e ~pleen
ferritin (A ~nd B) or w~th human (C and D) ferritin
for 3 days. After removal of ferritin by washing the
cells were cultured for 2 additional days and the
supernatants were collected and analyzed by ELISA for
reactivity with adsorbed ferritin.
Figure 7. Ferritin-reactive antibody 6ecretion by n
vitro primed human lymphocytes: comparison of IgM
and IgG responses to horse and human ferritin in the
presence and absence of adherent cells. Human
6plenocytes were prepared with or without depletion
of adherent cells. 1:1 co-cultures were primed at 3
x 106 cells/ml with the indicated concentrations of
horse or human ferritin for 3 days. After removal of
ferritin, the cells were cultured for 2 days and
6upernatants were analyzed by ELISA for ferritin
reactivity.
Figure 8. Antigen-reactive IgM antibody ~ecreted by
human lymphocytes primed in vitr~ with purified
~urine monoclonal antibodies, No. 1 or No. 2.
Human 6plenocytes were prepared without depletion of
adherent cell~ and cultured ~t 3 x 10(6) cells/ml for
3 day6 with the concentration6 of purified ~urine
~onoclonal antibody indicated in the figure. After
removal of ~ntigen by washing, the cells were
cultured for an additional 2 days. The 5upernatants
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2Q~2~
W~91/17769 -25- PCT/US91/0~406
.` :`!
were analyzed for reactivity with the immunizing
antigen by ELISA. Antibodies 1 and 2 differed in
their light chain components (~1 contained a lambda
light chain and ~2 contained a kappa li~ht chain).
Figure 9. Comparison of reactivity by ELISA of ~
ferritin-reactive, specific monoclonal ~ntibody (A),
with a ferritin-reactive, non-specific monoclonal
antibody (B).
Culture supernatants were harvested from terminal
cultures of each clone grown in 24 well plates and
assayed by ELISA as described infra. Immunoglobulin
concentrations of culture supernatants were in the
range of l-lO~g/ml. Reactivities with increasing
concentrations of ferritin coated to the assay plate
(values shown along the X-axis) were compared with
reactivities to increasing concentrations of
ferritin-unrelated proteins as shown in the figure.
Figure lO. Titration of the ferritin reactivity of
two purified anti-ferritin human monoclonal
antibodies. Monoclonal antibodies were purified by
affinity chromatography on protein G ~nd ELISA
analysis of the indicated concentrations of antibody
were carried out as described infra.
Figure 11. Affinity analysis of selected ferritin-
specific IgG human monoclonal antibodies by
competition ELISA.
This ~nalysis was per~ormed as described inf ra . The
first graph represents the degree of inhibition of
~ntibody binding to the ELISA plate (Y-axi~) as A
function of the concentration of competitive ferritin
in colution (X-axis). Ao represents antibody
reactivity in the absence of soluble ~ntigen (in the
- .
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.
WO91/17769 ~ 26- PCT/US91/0340
form of absorbance at 490 nm produced by ELISA
analysis of the incubation mixture) and A represent~
reactivity of the antibody incubated in the presence
of the indicated concentrations of 601uble ~ntigen.
The ~econd graph represents a Rlotz plot ~nalysis of
the data presented in the first panel where l/v
corresponds to Ao - A/Ao and ao represents the total
concentration of free nntigen. The open circles
represent monoclonal ~ntibody 14.2.2.59, produced
from lymphocytes immunized with horse ferritin,
having a calc~lated Rd ~ 0.86 x lO~ M. The filled
~quares ~how monoclonal antibody 2101B.9, produced
from ly~phocytes immunized with human ferritin,
having a calculated Kd = 1.90 x 10~ M.
Fi~ure 12 represents a time course of cumulative Ig
secretion from a long-term splenocyte culture in a
24-well plate prepared in accord with the present
invention preserving ~ultiple numbers of accessory
cells, for example by insuring presence of lymphoid
fragments during culturing. The mature immune
responses of class ~witch (from total IgM to IgG) and
longevity are evident.
Figure 13 represent~ the antigen reactivity
(reactivity with ferritin) of the immunoglobulin (Ig)
secreted by a long-term splenocyte culture in a T25
flask, ~howing ~pecific antigen reactivity of the Ig
~ecreted. The data in Figures 12, 14 and 15
represent cumulative, total Ig secretion. The data
in Figure 13 represents only that component of the
~ecreted Ig which reacts with the immuni~ing antigen,
~erritin, and it is not cumulative. The OD values
shown represent the difference in ferritin reactivity
between primed ~nd non-primed control cultures.
These data ~how that ferritin ~timulated the
~ecretion of ferritin-re~ctive IgM at early time
- ........ : - .. . , , . - - . .....
~ . - . . . . . ~ . . ~, . . - . .. .
- ., . ~ . , , . - -
. . , . : , .- , .. . . ..... . .. .. .. . . .
... . .. .
.. -' .' . '' ~.............. :. . ': ' . " . . :, ' ~ .'- :
:
.
~-)91/17769 2 ~ 3 2 ~ ~ ~ PCT/US91t03406
points, 0-25 days, and that ferritin significantly
stimulated ferritin-reactive IgG only after the
~econd boost, at ~0-85 days. The fact that antigen-
reactive IgM secretion was 6timulated by ferritin
early in the life of the culture, whereas antigen-
reactive IgG 6ecretion was stimulated by ferritin
only ~fter boosting constitutes additional evidence
of in vitro cla~s switching.
Figure 14 represents a ~ime course of cumulative,
general IgM secretion from a long-term splenocyte
culture in a T25 flask. The influence of a priming
antigen, horse ~pleen ferritin, on the rate of
~ecretion i~ ~hown. The ferritin-primed cultures
showed an increased rate of IgM ~ecretion over the
first 0-30 days relative to the control culture.
After 30 days the rates of both cultures appeared
fairly ~imilar until shortly after the 6econd boost
when the rate of the primed/boosted culture again
appeared to increase relative to the control culture.
Shortly after 100 days, the IgM secretion rate of the
control culture showed a ~harp increase. It is
thought that this increase in the control culture
resulted from a general contamination with mold which
became evident 2t approximately 110 days and the
culture had to ~e di6carded. (The rate of 6ecretion
is calculated from the 610pe of the line, which is
defined ~s the vertical rise di~ided by the
horizontal distance spanning the rise. The steeper
the ~lope, the greater the rate.)
Figure 15 repre~ent~ a ti~e course of cumul~tive,
general IgG ~ecretion from the 6ame long-term
splenocyte culture ~hown in Figure 13. The influence
of the priming antigen, horse spleen ferritin, on the
rate of IgG ~ecretion i~ ~hown. ~erritin had no
3~ ~ignificant effect on the rate of IgG 6ecretion 0-40
.. . ., - - ,, -. , . ~
3 .~
: , . . . ' ~:
w091/17769 2~138`2~ 8- PCr/US91/034
days, in contrast to the time when ferritin had the
greatest effect on general IgM secretion. At
approximately 44 days the rate of IgG secretion in
the primed culture showed a small but significant
increase rel~tive to the control culture. At 72
day~, just ~fter the second boost followed by ~ w~h,
the rate of IgG secretion in the primed culture
increased dramatically relative to the control
culture. The fact that ferritin primarily stimulated
IgM secretion at early times and primarily stimulated
IgG secretion at later times i6 evidence of in vitro
class switching.
Figure (photomicrograph) 16 shows underlying ~dherent
cells, the outgrowth of which supports develop~ent of
secondary structures. Also shown are three different
secondary structures: (l) a medium sized one on the
right having a large, dense center, (2) a medium
sized one on the upper left having a 6mall dense,
dark center, from which looser material radiates
outwardly, and (3) a ~mall one on the lower left
having a small dense, dark center and a 6mall amount
of looser material radiating outwardly.
Figure (photomicrograph) 17 shows two examples of
secondary structures: (l) on the right is ~ denser,
darker, bounded structure with light blebs appearing
on the top, right; and (2) n lefis den~e, lighter,
non-bounded ~tru~ture, apparently consisting of n
loose collection of larger, brown cell~ i~ fihown on
the left, with individual brown cells on the bottom
and to the left, either spreading out or in the
process of aggregating.
~igure (photomicrograph) 18 shows a secondary
fitructure on the right, which has not yet developed a
dense, dark center and which rests on an underlying
: ', . . , ~ :
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~91/17769 ~ ~ ~ 2 ~ '~ i` PCT/US91/03406
layer of adherent cells (not visible under the layer
of lymphocytes) and surrounded by many small, either
round or irregularly shaped cells which ~ppear to be
lymphocytes. Dense collections of these small cells
are occasionally observed surrounding or ~djacent to
the depicted secondary structure6 sugge~ting that the
secondary structure may be a site of nultiplic~tion
of these small lymphoid cells.
The variety of cells and structures shown in
Figures 16 to 18 develop with time as the cultures
mature. Outgrowth of ~dherent cells and development
of secondary structures begins at 1-2 weeks after
splenic fr~gments are seeded into flasks or plates
and continues for a minimum of several months.
Antibody secretion is not observed without outgrowth
of adherent cells and development of the type of
secondary structures shown. It is believed that
these 6econdary structures are aggregates of
different types of cells which represent or perform
functions of in vitro germinal centers.
5. Examples
The following include protocols for the preparation,
culturing, and primary immunization of human
splenocytes with protein antigens. Horse spleen
ferritin was used for development of these method6
because this protein has been well characterized,
human immune responses to hor~e ferritin c~n be
compared with human responses to human ferritin, and
human ferritin, in the onco-fetal form, has been
characterized as a tumor-associated antigen. Thus,
ferritin-reactive human ~onoclonal antibodies could
have therapeutic application. It has ~lso been found
that conditions developed for ferritin can be applied
to immunizations with other protein ~ntigens. It i~
demonstrated that lymphocytes immunized as described
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. . . ~ . . . .
. .
.
... .
.
WO9~/17769 2~ ~30- PCT/US91/0340
can be fused at high efficiency with a human/~ouse ;
heteromyeloma fusion partner to produce ~ntigen-
specific IgG as well as IgM huma~ monoclonal
antibodies.
Human splenocytes i~munized in vitro with protein
antigens were immortalized by high efficiency fu~ion
with one of the heterohybrids constructed by Carroll
et al., J. Trmuno. Methods 89, 61 (1986), K6H6/B5, to
produce ~pecific antigen-reactive human monoclonal
antibodies. Results evidenced good fusion efficiency
and growth of the hybrid progeny, level of
immunoglobulin secretion in the range of 0.5-50
ug/ml, stability of immunoglobulin secretion in
approximately 50% of the hybrids, and production of
IgG as well as IgM class antibodies.
A. Materials and Methods
Preferred ~bodiments
l. Splenocyte Preparation: All steps are performed
under sterile conditions:
a. Spleen tissue, obtained from accident victims
within hours after surgery, was provided by the
University of California San Diego (UCSD) Tissue
Ban~.
b. The tissue is cut into approximate 1 inch square
fragmentfi.
c. A single cell suspension in RPMI growth medium i6
generated by forcing 6pleen fragments through ~ 50
mesh wire screen.
d. As the cell suspension is generated, it is
collected into ~ sterile bottle on ice and then it
can be filtered through several layers of sterile
, , , . :,- : : ... .: , . . .. ... .
.; , . . ., .. - ,, .. , . :,.:, ., , ... : . . . : , , . ::, ~ , - .. :
: - ,. . .. - , .. : . , : : :: ::, . - .. : : . : . ~, ~ : :
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: ........ . . . . . .
~V~91/17769 2 ~ PCT/US91/0~406
-31-
cheesecloth into a sterile glass beaker to remove
larger tissue fragments.
e. The cell ~uspension is then transferred into 250
ml centrifuge bottles and the cells are collected by
centrifugation at lO00 rpm for lO ~in; the
~upernatant discarded and the loose pellet
resuspended with a minimal volume of RPMI and
transferred to a lO0 ml 6terile ~ottle.
f. The cells are expo~ed to hypotonic ammonium
chloride for 30-90 seconds to lyse red blood cells
(RBCs) and washed ~everal times.
g. Preferably, fragments of lymphoid tissue of
about O.l to l.0 millimeter in size are deliberately
retained in the cell suspension.
h. After the final wash, the cells are counted,
resuspended into freeze medium, aliquoted into
cryotu~es, and frozen at about 100-600 million cell~
per milliliter per vial.
2. Immunization: All steps are performed under
sterile conditions:
a. 100-300 million cells each from 2 separate spleen
preparations are thawed by gentle ~haking in a 37C
water bath; these ~re washed twice with RPMI.
b. The cells are resuspended in 5 ml of medium and
counted.
c. The cell concentration is immediately adjusted to
between 0.5-5.0 X lO(6) cellslml and the cell
~uspension is then transferred to a 24 well costar
plate at 2 ml/well.
.. . . .
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WO91/17769 -32- PCT/US91/0340
d. ~ added at the desired concentration
(O.l-lO.0 ug/ml has been found ~s ~n effective
range).
e. The composition of RPMI growth ~edium
(commercially available) included the following: 10%
fetal calf serum (FCS), 2 mM glutamine, nnd Gibco
~mino acids and pyruvate (l:lO0 dilution).
f. Exposure to nntigen is allowed to proceed for at
least 2 days.
g. Control cultures are al~o establi6hed, as
described above, but are not exposed to antigen.
3. Elisa,,~otocols: Analysis of the immunization
response:
a. After 3-5 days, the antigen is removed from the
cultures as follows: the cells from each culture ~re
resuspended by aspiration with lO ml of RPMI ~ 2S FCS
and transferred to a 15 ml centrifuge tube.
b. The cells are collected by centrifugation at
about lO00 rpm for lO min, and may be washed and the
20 cells are then resuspended by aspiration with a lO ~l `
pipet.
c. The cells are resuspended into 2 ~l of new growth ,,
~edium and returned to the original culture wells to
allow secretion of ~ntibody in the ~b~ence of
25 antigen. ~ '
d. After 2-4 days, l.5 ml of supernatant i~
collected by ~spiration with a pipet ~nd stored at ,~
4C with 0.01% azide until assayed for immunoyen-
~pecific reactivity by conventional ELISA techniques.
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W~91/17769 ~33- 2 0 ~ 2 0 ~ 1 PCT/US9l/03406
e. The level of response measured for nonimmunized
cultures (this represents background reactivity and
i~ highly dependent upon culture condition6) i6
subtracted from the response measured using immunized
cells; this difference represents n semi-guantitative
measure of the Antigen-driven reaction and may be
used as a rough predictor of the number and
predominant immunoglobulin class of the hybrid clones
obtainable from fusions of lymphocytes immunized
under similar condition~.
4. Immortalization: Production of hybrid clones
from in vitro immunized lymphocytes.
a. After a minimum of 2 days exposure to the
immunizing antigen, the cells are resuspended into 5
1~ ml of RPMI, counted, and combined with K6F6/BS
heteromyeloma cells.
b. These cells are diluted to 50 ml with RPMI
containing glutamine, pyruvate, and non-essential
amino acids as des~ribed above and centrifuged at
lO00 rpm for lO min.
c. The cells are washed l more time with the above
solution and fused and hybrids selected according to
conventional fusion protocols.
SPecific Protocol
Materials.
Hor~e spleen ferritin (F-4503), BSA (A-7906), ~ween
20 (P-1379) and potassium thiocyanate (P-3011) were
purchased from Sigma. Human ferritin, purified from
liver c~rcinoma tissue, was generou~ly provided ~y
Dr. Jerry Rlein of Johns Hopkins University,
Baltimore, MD. Murine monoclonal antibodies, were
generously provided by Dr. Malaya Bhattacharya-
. . .
WOgl/17769 ;~ 34_ PCT/US91/0~40
Chatterjee (4DC6) and Dr. Ben K. Seon (SN2) of
Rossell Park Memorial Institute, Buffalo, NY. Goat
anti-human IgM (4102), IgG (4100), IqM-HRP (2392),
and IgG-HRP (2390) were purchased from TAG0,
Burlingame, CA. Primary antibodies were either
purchased as purified monoclonal antibodies from
Coulter Immunology, Hialea, Florida, or used as
culture ~upernatants pr~duced from the cell lines CRL
8001 (anti-CD3) and CRL 8014 (anti-CD8), purchased
from the ATCC.
SplenocYte preparation.
Spleen tissue, obtained from accident victims within
hours after surgery, was provided by the UCSD Cancer
Center Tissue Bank. A single cell 6uspension was
prepared by forcing fragments through a wire screen.
The cells were collected ~y centrifugation at 1000
rpm for 10 minutes and RBCs were removed by Ammonium
chloride lysis. The remaining cells were washed,
resuspended in a freezing medium consisting of 40%
RPMI, 50% FCS, and 10~ DMS0 at a concentration of
100-300 million cells/ml, frozen in 1.5 ml aliquots,
and stored in liquid nitrogen. For establishment of
long-term cultures, it is essential that fragments of
spleen tissue be retained in the cell suspension
prior to freezing.
In vitro immunization.
All steps were performed under sterile conditions.
Frozen cells from each spleen preparation were thawed
by gentle ~haking at 37C and washed 2 times with 15
ml RPMI.
Two ml. immuniz~tion cultures were set up immediately
after thawing and washing. For mixed cultures, each
spleen contributed an equal number of cells to the
final concentrntion. The cell suspension was then
, .: . ........... . .
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W~91/17769 _35_ 2. ~ PCT/~S91/0~40fi
transferred to the inner wells of a 24 well tissue
culture dish at 2 ml/well. Ferritin or other
antigens were immediately added at O-lO ug/ml, as
indicated. At least 4 wells were set up for each
condition tested. If multiple ~ntigen concentrations
were used, one well received no antigen. This was
the non-immunized, control sample, analogous to the
pre-immune sera of i~_yivo immunizations. The
remaining 3 well~ each received a different ~mount of
antigen. If only l concentration of antigen was to
be used, 2 wells received no antigen and the
remaining 2 wells received the indicated
concentration.
Unless otherwise indicated, the cells were cultured
with antigen for 3 days. After priming, cells from
each well were gently transferred to a 15 ml
centrifuge tube, washed l time with l5 ml RPMI +
2%FCS to remove the antigen, and then returned to the
well from which they had been removed for ~n
additional 2 days, unless otherwise indicated.
Culture supernatants were collected at 5 days and the
cells either discarded or refed for ~ ~econd analysis
of anti~ody production at day 7. Standard growth
medium consisted of RPMI ~upplemented with lO % FCS,
1% non-essential amino acids, (Irvine Scientific) 2
mM glutamine, 1 mM sodium pyruvate, 15 mM HEPES, and
gentamicin.
Anti-Ferritin Assay
Anti-ferritin reactivity of antibody in the cultuse
6u?ernatants was estimated as follows. Puri~ied
ferritin was diluted to lO ug/ml in .05 M ~odiu~
carbonate buffer, pH 9.3. 0.05 ml/well were incubated
overnight at 4 degrees C. using 96 well round bottom
Immulon I plates (Dynatech). Each ~upernatant was
~ssayed in triplicate on ferritin and in duplicate on
- : . . .. .
. . .
,, ',
,
WO9l/l7769 ~ PCT/US91/0340
36-
bovine serum ~lbumin tBSA). OD-490 values observed
with BSA were considered non-specific protein
reactivity ~nd were subtr~cted from the OD-490 values
observed with ferritin. The difference in OD values
between ferritin and BSA binding is referred to as
ferritin reactivity. Difference in ferritin
reactivity between non-immunized and primed ~amples
is referred to as ferritin-induced, ferritin
reactivity. After coating overnight, the plates were
washed 3 times with phosphate buffered 6aline (PBS)
and blocked with 1% BSA in PBS. An alternative method
giving similar results involved omission of the BSA
blocking step and dilution of samples and reagents in
0.05% Tween 20/P8S. After 2 hours at 37 degrees C,
the blocking solution was removed and 0.05 ml of
supernatant were added to the appropriate wells.
After 2 hours at 37 degrees C, the plate was washed 5
times with PBS - 0.1~ Tween 20 and 0.05 ml of a
1:1000 dilution of a 1 mg/ml solution of peroxidase
conjugated goat anti-human IgM were added. This
secondary reagent was diluted into 10% FCS in PBS.
After 45 minutes at 37 degrees C, the plates were
washed 5 times with PBS-Tween and 0.150 ml/well of
0.4 mg/ml o-phenylenediamine (Sigma P-1526),
dissolved in 0.05M sodium citrate buffer, pH 5.0, ~nd
0.0175~ hydrogen peroxide was added. Color
development was terminated after 15-60 minutes by
addition of 0.025 ml of 2.S M sulfuric acid. OD-490
values were recorded using a Molecular Devices
(Mountain View, CA) V max kinetic plate reader. Human
IgG anti-ferritin reactivity was a6sayed Eimil~rly
except that the assay wells were coated with 50 ug/~l
ferritin or BSA, the protein W~5 adhered overnight at
37, and the secondary re~gent was peroxidase-
conjugated goat anti-human IgG.
Anti-murine immunoalvbulin ~ssav.
~ - . ~ ,- , . . . --. - - - - , -
- . - , . . -. . ~ , - . - ~.
. . , . , ;. ... :. .. : , .
- : ~, . ., . .. : .
.
.: ... .. . . . ......... .. ... . .. .... ..... .. . . .. ...
2~(~2~Lq 1
WO91/17769 PCT/US91/03406
_37_ ;
Polyclonal human anti-mouse immunoglobulin reactivity
was assayed by ELISA using the basic procedure
described above for detection of anti-ferritin
reactivity, except that S ug/ml murine monoclonal
immunoglobulin wa used ~ the capture antigen.
Ouantitation of human IqG and Ig~.
Human IgG ~nd IgM were quantitated by ELISA as
follows. 0.05 ml/well of l:lOO dilution~ of l Dg/ml
~olutions of goat anti-human IgM or IgG (Tago) ~ere
adsorbed at 4C overnight to 6erve a~ capture for the
human IgG or IgM. Supernatants containing unknown
quantities of immunoglobulin were serially diluted
until reactivity was undetectable. Assay protocols
were similar to those described for anti-ferritin
reactivity, except that peroxidase-conjugated
secondary reagents, goat anti-human HRP-conjugated
IgM or IgM, were used at a dilution of 1:5000 instead
of l:l000. Concentr~tions of unknown samples were
calculated from dilutions where OD values fell within
~he linear range of the standard curve, which was
defined by standard 6amples of purified polyclonal
human IgM or IgG, used in a range of 0.0l to 2.0
ug/ml.
~na~ysis of relati~e anti~en binding a~finitv/
avidity values of polyclonal antibodies produced
in vitro.
An assay utilizing disruption of antigen-antibody
binding by thiocyanate was adapted from MacDonald et
~1-, J Imm~no. Methods 1~6, l9l (1988), integrated
with the anti-ferritin ELISA described ~bove, and
used to characterize the quality of the ~n vitro
immune response. After immunization, culture
~upernatants were incubated with ferritin bound to
the assay plate ~nd the plates washed 5 times with
PBS-Tween, 0.l ml of the indicated concentrations of
WO9l/17769 2. d ~ PCT/ Us9 1/0~40
-38-
potassium thiocyanate, dissolved in PBS, were added
to each well and the plate was incubated at room
temperature for 15 minutes. The plates were washed 5
times with PBS-Tween and the anti-ferritin ~ssay
concluded as described above. Supernatants having
high binding activities were diluted prior to assay
so that ferritin binding activity would be relatively
uniform from sample to ~ample. Each supernatant was
assayed in duplicate at each thiocyanate
concentration and supernatants from two ~eparate but
identical immunizations were analyzed for each
condition tested. tKSCN]-50 values were defined as
the concentration of thiocyanate required to decrease
the amount of antibody bound to ferritin by 50%.
Lymphoc~te marker analysis.
Approximately 1 million cells were used/test. The
cells were harvested at the indicated times after the
initiation of priming, washed 1 time in growth medium
and resuspended in 1 ml cold PBS + % BSA + 0.02 M.
sodium azide (washing buffer). Lymphocytes were
incubated for 30 minutes at 4 degrees C with murine
monoclonal antibodies reactive with human heavy chain
mu, heavy chain gamma, PCA-1, B1, CD3, CD4, CD8, or
CD25. The cells were washed 3 times and resuspended
in washing ~uffer with fluorescein i~othiocyanate
(FITC) - labeled affinity isolated, human absorbed
goat F(ab')2 anti-murine IgG (TAG0, Burlingame, CA)
for 30 minutes at 4 degrees C. The cells were then
washed 4 times and resuspended in 0.4 ml PBS I 1%
para-formaldehyde. Quantit~tion of fluorescence for
all ~amples was done on the same day with An EPICS
profile flow cytometer. Dead cells were excluded by
forward and 90 degree light ~catter ~easured-.
Sensitivity and amplificAtion of the signal was set
~o that recorded labeling of the fluorescence control
~ample (cells labeled with nonspecifis mouse
- . : . .. ; - . . . , . .: : . . .
.
.: . .- :. : . . : . .: - .. .
2 ~ 8 2 ~ ~ ~
~91/17769 PCT/US91/0~406
-39-
immunoglobulin as the primary antibody) was 5%. The
data presented were calculated by subtraction of the
backqround values of cell labeled with non-specific
mouse Ig from the value~ recorded with s~mple6
reacted with cell ~urface 6pecific antibodies.
B. ~esults
Comparison of i~mune responses by sinqle
and allogeneic. ~ixed cultures.
Antigen-dependent immune re~ponses were measured by
ELISA as the difference in antigen reactivity of
polyclonal antibody in ~upernatant~ from cultures
primed with antigen and supernatants from control
cultures primed with antigen and supernatants from
control cultures not exposed to antigen. Co-
cult~ring of lymphocytes prepared from two unrelatedspleens provided one of the best methods for ~upport
and for detection of antigen-induced production of
antigen-reactive immunog~obulin. Lymphocytes from 8
~pleens were examined individually and in
combination. ~ow IgM responses were observed when
certain spleen preparations were cultured
individually, as shown in Figure 1 for spleen A and
spleen B. In contrast, ~ome spleen preparations were
unresponsive under most conditions tested; for
example, spleen D in Table I. However, when cells
from 2 spleens, respon~ive or unresponsive, were co-
cultured, a ferritin-dependent IgM response was
consistently observed (Figure 1; Table I). Non-
~pecific binding activity, estimated from cultureE
not exposed to antigen, was also increased under
nllogeneic culture condition~ (Figure 1). Howe~er,
in ~pite of increased reactivity of non-immunized
cultures, nllogeneic ~timulation in the presence of
~ntigen alway~ led to an antigen-induced signal
greater than the signal expected from the ~um of the
observed contributions of each ~pleen alone.
.. ~. ., . ., ~
' ,'.: ,': ~ ;; '"' ' . ' ; ''' , '- . , :
' ~: .~ . : : :
.
W091/177~9 ~ 8~ x~ 4~) PCT/US91/0~40,~
Calculated and observed responses ~re shown in Table
I for all combinations of spleens A, B, C, And D. In
general, a greater anti~en-induced 6ignal was
observed with two spleens than with cultures of ~ore
than two or with only one 6pleen. It ~as therefore
concluded that u~e of two ~pleen~ provided an optimal
balance between activation in~ufficient to induce
detectable 6pecific responses ~nd allogeneic
activation EO intense that low quality, non-specific
antibody responses overwhelmed antigen-driven
reactions.
Ferritin-dependent production of ferritin-
reactive antibody by irradiated sin~le a~d
allogeneic mixed spleen cultures.
Non-immunized syngeneic cultures prepared from either
~pleen l or ~pleen 2 (Figure 2) produced negligible
levels of antibody which cross-reacted with ferritin.
After priming with ferritin, induction of low levels
of ferritin-reactive antibody was observed. Co-
culture resulted in increased non-specific ~nd
~pecific reactivity. Irradiation of ~ingle and
allogeneic mixed cultures abrogated all responses.
However, irradiation of only one component of the
mixed culture prior to antigen priming resulted in no
loss of specific reactivity. In the case of
irradiation of spleen 2, 6pecific induction by
antigen was increased approximately two-fold. These
results indicated that ~uccessful ~llogeneic
~timulation required only one partner with functional
B cell~, that either spleén in an allogeneic culture
could be 6timulated by antigen to produce cpecific
antibody, and that B cells from 6pleen 2 of the
mixture ~hown in Figure 2 produce ~ore non-6pecific
antibody cros6-reactive with ferritin than B cell6
from 6pleen l.
.. . . . . . . : : . -
.: ` , ' . . ' . ' ~: ': ., .:
, . ' ' ' ~. . , ' ~' ':
WO9l/17769 41~ ~ 2 ~ ~ ~ PCT/US~1/0~406
. :
Time reouirements for antiaen primina ~nd
for detection of antiqen-induced. antiqen-
reactive an~ibody.
Co-cultures of lymphocytes from 2 spleens were
exposed to 4 concentrations of horse ferritin, from 0
to l.O ug/ml, for 1, 2, 3, or 4 days to determine the
optimal time required for initial exposure to ~ntigen
(priming). After priming, cells were cultured in
horse ferritin-free medium to determine the optimal
time required for 6ecretion of detectable ferritin-
reactive anti~ody (Figure 3). Cultures primed for 1
day required the longest antigen-free incubation, 3-5
days, to detect even low levels of secreted ferritin-
reactive antibody. Furthermore, cultures primed for
one day were not ~ensitive to the lowest
concentration of ferritin tested, O.1 ug/ml. Cells
primed for 2 days began to produce detectable
ferritin-reactive antibody after 2-3 days incubation
and were sensitive to O.l ug/ml ferritin, especially
when incubated for 5 days after priminy. Cultures
primed for 3 and 4 days produced detectable ferritin-
reactive antibody after a one day incubation in horse
ferritin-free medium.
In general, the longer the priming, the sooner
ferritin-induced, ferritin-reactive antibody became
detectable after removal of antigen. If cultures
were primed for 3-4 days ~nd allowed to 6ecrete
antibody in the ab~ence of ferritin for ~ore than 2
day~, the higher the background reactivity, even when
only two ~pleen cultures were used, prevented
di~rimination ~etween ~pecific induction by antigen
and excessi~e, non-6pecific B cell activat~on. The
most consistent ferritin-dependent ~ignals were
observed when 3 day~ were allowed for priming and 2
days ~or ~ecretion, or when 4 days were allowed for
priming and 1 day for 6ecretion. Three days of
- . .- . . , ~ .: . . :
., : ~ .
: : . ~ , , . - ,
WO9l/17769 PCT/US9t/0340 ,1
2~8~ 42- 6~
priming followed by two day6 of secretion in the
~bsence of antigen constituted the immunization and
detection conditions used for sub6equent experiments.
After priming of allogeneic culture6 for 3 days,
ferritin-reactive antibody production continued for
longer than 1 week. In the experiment shown in
Figure 5, the culture medium was changed ~nd antigen-
reactive antibody produced during each 2-3 day
interval following priming was measured. The highest
level of production occurred on days 5-7, followed by
a steady decline in specific and non-specific
reactivity. ~y days 11-13, antigen-induced respon6es
were barely detectable, and by days 13-16, no
ferritin-reactivity was observed. The ratio of
ferritin-induced to non-specific reactivity was
fairly constant, with a slight optimum in specificity
observed on days 3-5.
Antigen-induced, antigen-reactive IoG s,ecretion.
Culture supernatants were monitored for antigen-
induced IgG as well as IgM secretion. Ferritin-
induced, ferritin-reactive IgG responses were of
lower frequency than antigen-reactive IgM class
responses and their detection required a more
sensitive ELISA than used for IgM responses. Vnder
the culture conditions used, IgG responses were
observed in 10-30~ of the experiments. Thi~ low
frequency of antigen-induced IgG antibodies is
characteristic of a primary respon~e; however, other ' '
factors may be involved. When monoclonal antibodies
were produced after fusion of ~n vitro i~munized
lymphocytes, effective numbers of IgG class
monoclonals were always obtained even if a polyclonal
IgG response was not observed.
,:
2 i~ ~ 2 ~
W~91/17769 _43_ PCT/US91/0~406
IgG responses were more consistently observed with
60me spleens ~nd spleen combinations than with
other~, but cell marker analy~i6 of cells from
different spleen6 revealed no difference~ which might
S support IgG ~ecretion by one 6pleen ~nd not by
~nother. When IgG response~ were observed, ~heir
expression wa6 consi~tent within one experiment. A~
6een in Figure 4, the kinetics of appearance of
ferritin-induced, ferritin-reactive IgG ~ntibody
paralleled the expression of IgM re~ponse6 By day~
9-ll, however, non-~pecific IgG reactivity was not
observed ~nd detection of ferritin-reactive IgG wa~
completely dependent upon exposure to ferritin.
Prolongation of either IgM or IgG responses by
allogeneic cultures significantly beyond lO days wa6
neither observed nor expected, since the cellular
death rate from cytotoxic killing by allogeneic cells
increased significantly by this time.
Analysis of relative affinity/aviditv
roperties of polyclonal ferritin reactive
antibody produced under different
~onditions.
Quantitation of ease of antibody elution from antigen
bound to ELISA plates by exposure to thiocyanate has
been used to evaluate relative strength6 of binding
of different antibody preparations. Thi6 method was
applied to determine if ferritin-induced, ferritin-
reactive ~ntibody produced in vitro could be
qualitatively di~tinguished from the non-specific,
ferritin-reactive immunoglobulin detected ln non-
immunized cultures. The ease of elution of
polyclonal antibody from solid phaEe bound ferritin
was evaluated u~ing ~upernatants from ferritin-pri~ed
and from non-immunized, control cultures. As fieen in
Figure 5, nntibody from primed cultures required
higher levels of thiocyanate for elution than
2 ~ ~ 2 Q p CT / u s 91/0~40~ '
supernatants from non-immunized cultures. The molar
concentrations of thiocyanate required to elute 50%
of the ~inding activity ([XSCN]-50) produced by non-
immunized and primed cultures, respectively, were 2.0
M and 2.6 M.
This method was also used to evaluate the binding
properties of ferritin-induced antibodies ~enerated
under different culture conditions. If prcduction of
high affinity antibodies i6 desired, evaluation of
relative quality without regard for the quality of
the response may be misleading. Analysis by
thiocyanate elution of ferritin-binding strengths of
polyclonal antibody mixtures produced under different
culture conditions revealed a significant dependence
of antibody binding strengths on immunization
conditions. Further, this dependence could not have
been predicted from the magnitude of the ferritin-
induced responses revealed by ELISA analysis above.
[KSCN]-50 values of duplicate cultures primed with or
without antigen under different conditions are shown
in Table II. Although ~ingle cultures of cells from -
a responsive spleen B, produced ferritin-induced,
ferritin-reactive IgM, [KSCN]-50 values were ~imilar
for immunized and control cultures. In contrast, co-
cultures of spleens A I B primed with ferritin in
medium containing 10% FCS produced antibody with
significa~tly higher ~KSCN]-50 values than antibody
produced by non-i~munized cultures. Differences
between immunized and control cultures reached a peak
for ~ntibody produced days 7-9, but ~ecame
insignificant with antibody produced ~fter day g.
Exposure of allogeneic cultures to antigen for 10-20
hours in the absence of serum usually increased the
level of ferritin-induced, ferritin-reactive Gntibody
measured by ELISA, ~ecause background activity of
~, , ~ ,',
...
:
W~91/17769 ~ PCT/US91/03406
non-immunized cultures was lower. However,
differences in [KSCN~-50 values between immunized and
control cultures were not as lRrge nor a6 consistent
when FCS was absent during initial priming a when
cultures were continuously supplemented with FCS
(Table II). The6e result~ illustrate the importance
of ~chieving a balance among different forms of
activation for induction of antibodies of the desired
quality as well as quantity.
Effects of non-lymphokine ~actors on
antiaen-induced ~ntiqen-reactive Iq
secretion and on total Ia secretion.
Effects of the non-lymphokine factors, muramyl
dipeptide (MDP) and pokeweed mitogen (PWM), on
ferritin-induced, ferritin-specific responses were
tested using both single and allogeneic mixed
cultures (Table III). PWM stimulated overall Ig
secretion, but did not enhance specific induction by
antigen. ~DP also stimulated Ig secretion in general
and, in the experiment shown in Table III, enhanced
the effects o~ exposure to antigen. However,
specific ~timulation by MDP was not consistent, and
was usually observed only under sub-optimal culture
conditions (data not presented). These results
further indicated that allogeneic two-spleen cultures
provided sufficient stimulation for induction of
antigen-specific responses, and that additional non-
specific mitogenic stimulation was either inhibitory
or was not usually required.
~0 Influence of adherçnt cells o~ tke i~ ro
resDonse ~c_~or6e ~er~itin.
An initial protocol for establi6hment of im~unization
cultures included overnight incubation in large
flasks to allow recovery from thawing prior to
~timulation with antigen. This step resulted in
- ~ , .. .. .
- ,
' . . . : : .
W091/17769 ~ f9~t~l PCT/US91/0~40
-46-
depletion of adherent cells. Comparison of depleted
and non-depleted cultures revealed that non-depleted
cultures responded better to hor~e ferritin than
depleted culture (Figure 6). Spleen 2 ~lone was not
significantly responsive under either condition but
~pleen l, which was unresponsive in depleted
cultures, gave a limited respon6e to low
concentrations of antigen and a large response to
high levels when adherent cell~ were not depleted.
In mixed cultures of spleens l ~nd 2, reactivity of
non-primed cells was reduced and sensitivity to low
levels of antigen was increased when adherent cells
were not depleted. These results demonstrate that
antigen may be presented more efficiently when
immunizations are carried out in the presence of
optimal numbers of adherent cells.
In vitro human i~mune responses to_human
ferritin.
Responses to human ferritin were also tested to
determine if in vitro human immune responses would be
detected to human as well as to foreign antigens.
Responses to human ferritin were not observed in
adherent cell depleted cultures under conditions
which supported consistent responses to horse
ferritin (Figures 6C, 7). However, when freshly
thawed spleen cells were not preincubated overnight,
the cells not only responded better to horse
ferritin, they also now responded to human ferritin
(Figures 6D, 7). When depleted cultures were primed
for 2 days with human ferritin and then incubated for
a ~ecretion perivd of 5 days instead of for 3 d~ys, a
small response to human ferrit$n was obser~ed. If
depleted cultures were reconstituted with adherent
cells at the time of priming, ~ response to hu~an
ferritin was then again observed after secretion for
2 days. ~uman ferritin-induced IgG responses by non-
2~2~ ~
~'~91/17769 47 PCT/US91/03406
depleted cultures were als~ observed in some
experiments (Figure 7A). Specific IgG response~ were
lower, but paralleled IgM responses.
In vitro antigen ~riminq with foreian
l~munoglobulins.
In order to a~sess the ~pplicability of in vi~o
i~munization conditions developed for ferritin to
other protein antigen6, reactivity to priming with
murine monoclonal IgG ~ntibodies was tested under
conditions which ~upported re~ponses to hor~e and tohuman ferritin. Antigen-induced, antigen-reactive
IgM antibody responses to two monoclonal prep~rations
were detected (Figure 8). In general, background
reactivity of non-immunized ~upernatants was lower
when assayed on a monoclonal murine immunoglobulin
than on ferritin. Polyclonal, Antigen-dependent IgG
reactivity was not observed with these antigens, but
fusion of lymphocytes primed with a murine monoclonal
antibody produced antigen-specific IgG ~s well as IgM
class monoclonal antibodies.
Cellular aggreqation
10-20 hour6 ~fter initiation of priming many cell
aggregates appeared, which were visible to the naked
eye from the bottom of the culture dishes. If
disaggregated by vigorous aspiration, the aggregates
began reforming within ~everal hours. Their ~ize
depended upon the presence, concentration and the
nature of the ~ntigen, on cell density, And on the
presence of allogeneic lymphocytes. ~ntigen-free
cultures formed aggregates which were barely
detectable by the naked eye. Exposure to horse
ferritin induced larger ~ggregates than human
ferritin. Less complex antigens of lower molecular
weight, such as the murine immunoglobulins, induced
aggregates barely larger than those seen with non-
.
. , . . . .: ~. .
. , . - . . : .~ . .~ : :
.
WO91/]7769 ~ j PCT/US91/0~40~ 1
~, 3 8 ~ 48~
primed cultures. Larger aggregates were also induced
in allogeneic than in single cultures. Cellular
aggregation also depended upon the presence of FCS.
Supplementation with FCS could be delayed during
priming for 15-20 hours witbout reduction of antigen-
dependent respon~es, but ~acroscopic ~ggregates did
not begin to form until several hours after addition
of FCS.
Çell marker analYsis.
Cell marker analysis indicated that human 6plenocytes
could differentiate to a limited extent when cultured
under appropriate conditions. The following cell
surface markers were ~nalyzed using ~pleens B + D at
day 0, 3, 5, 7 and lO after initiation of priming:
IgG, IgM, Bl, PCA-l, T3, T4, T8, and the IL-2
receptor (CD25). Spleens B and D did not differ
significantly except that spleen D preparations
contained 17~ fewer 6urface IgM bearing cells than
spleen B. Changes observed with allogeneic cultures
as a function of time were approximately 50%
decreases in the percentages of cells bearing 6urface
IgG, IgM, and Bl (Table IV). The percentage of T8
positive cells increased ~lightly, whereas T3
positive cells were relatively unchanged and T4
positive cells decreased slightly. Striking time-
dependent changes included a 6harp, approximately
ten-fold increase in the percent of IL-2 receptor
bearing cells beginning before day 3, ~ollowed by an
approximate two-fold increa6e in the percent of PCA-l
positive cells between day~ 5 ~nd 7, which paralleled
the decrease of Bl bearing cells. Cell surf~ce
changes were not significantly influenced by
ferritin; 6imil~r ~ut less ~triking changes were
observed with single cultures.
,
.. ; :
. ' :
~'~91/17769 ~ PCT/US91/0~406
Monoclonal antibodies produced aaainst
horse ferritin.
Allogeneic cultures of human lymphocytes were primed
wit~ hor6e spleen ferritin for l, 2, or 5 day6 and
then fused [Table I). ~wo separate but identical
fusions, 4 and 5, were performed with ~dentical 5
day-primed cultures to estimate fusion-to-fu~ion
reproducibility. An additional 6et of cell~ was
maintained in culture for one day but was not
intentionally exposed to horse ferritin tfusion l).
Fusion frequency, Ig ~ecretion, antigen-reactive
monoclonal antibody production, and antigen-specific
monoclonal antibody production were monitored for
each fusion.
Fusion frequencies were similar for cells primed with
ferritin for l through 5 days (35-50 clones/million
lymphocytes~, but non-immunized cells fused at a
lower frequency (17 clones/million lymphocytes) than
primed cells. Non-iDmunized cells appeared less
activated, forming smaller macroscopic aggregates
than primed cells. The number of cells in ferritin-
primed cultures did not ~ignificantly increase
relative to control cultures.
A large percentage of the hybridoma clones produced
in these fusions secreted immunoglobulin (20-60%).
In the 6et of fusions described in Table I, IgG-
secreting clones were 5-lO times more numerous than
IgM-~ecreting clones. The majority of the fusion~ in
our laboratory have produced approxi~ately equal
number~ of IgG and IgM secreting hybrids (see
Table II ~nd III).
In general, t~e pattern~ of induction of ferritin~
specific and ferritin-reactive monoclonal antibodies
were ~imilar (cee Figure 9 for comparison of binding
- , ~........ . . .
- : - ~ . . : ,. ~ ., .,:: .: . . , , :. . . -
.. . ...........
- . ~ . . :.
WO91/17769 2 ~ 8 ~ 1 PCT/US91/0~40
specificities of ferritin-reactive versus ferritin-
~pecific monoclonal antibodies). Exposure to
ferritin for only one day did not significantly
increase the percent of ferritin-reactive or of
ferritin-specific hybridomas compared with those
produced from the non-immunized, control cultures. A
greater number of ferritin-reactive hybrids were
produced from cells immunized for one day, but fusion
freauencies were greater. After 2 days of priming,
the percentage of ferritin-reactive clones rose from
5-6% to 16%. After 5 days, the level dropped to lO-
12%, but was still ~ignificantly higher than the
background observed with cells cultured for one day.
Cells exposed to ferritin for as long as 8 or 15 days
fused with similarly high frequencies. However, out
of 93 tested, none secreted 6table, ferritin-reactive
monoclonal antibody.
All antibody-secreting clones which initially reacted
with ferritin by ELISA were tested further for
ferritin specificity. Although many of the
monoclonal anti~odies initially identified cross-
reacted with other proteins (Figure 9), and would not
ordinarily ~e of interest, we have monitored their
incidence to determine if certain conditions or
antigens induced higher percentages of specific
antibodies than others. No 6uch culture conditions
have been identified, but ~llogeneic ~ntigens induced
a higher percentage of cpecific ~ntibodies than horse
ferritin (Table VIII).
Monoclonal antibodies ~roduced ~a~i~st
human erritin.
A 6imilar 6eries of fusions used lymphocyte6
immunized with human instead of with hor6e ferritin
tTable YI). Fusions ~-3 u~ed lymphocytes ~aintained
in culture for 2 days. Fusion 1 used lymph~cytes not
, . . . ..
, .,, ~, :
. . ' : -:
. . , . . -
. ............. . . . .
.,.
.. .- ., ~ . . . ~ '.
.: ' '~ :. ,
~91/17769 -51- PCT/US91/0~406 !-
,....,
intentionally exposed to ferritin, while fusions 2
and 3 were primed with 0.25 ug/ml ferritin. Fu~ions
2 and 3 were identical, but were maintained
separately to asse6s fusion-to-fusion variation.
Fusions 5 and 6 were performed on days 4 and 6,
respectively, ~fter initiation of pr~ming with 2.5
ug/ml human ferritin in6tead of wit~ 0.25 ug/ml.
The fusion frequency of cells primed for 2 days with
0.25 ug/ml human ferritin was higher than the fusion
frequency of control non-i~munized cells, but was not
as high as cells immunized with 0.25 ug/ml hor6e
ferritin. Cellular aggregates were al50 smaller when
cultures were primed with human than with horse
ferritin. Cultures immunized for 4 or 6 days with a
lO-fold higher level of human ferritin fused with
frequencies equivalent to cultures primed with 0.25
ug/ml horse ferritin. Since fusion frequencies of
horse ferritin primed cells were not influenced by
time of priming, the higher frequencies of fusions 4
and 5, relative to fusions 2 and 3, may have resulted
rom the higher concentration of human ferritin used,
although fusion-to-fusion variation cannot be
excluded.
In these fusi~ns, 30-45~ of the hybrids prcduced by
lymphocytes maintained in culture for 2-4 days
6erreted im~unoglobulin. Ferritin did not influence
the number of Ig secreting hybrid~ produced.
However, comparison of fusions 4 and 5 revealed th~t
cell~ primed for 6 days produced far fewer ~ecreting
clones than cells primed for 4 days (8~ ver~us 44S).
Thi~ result is sim~lar to the low productivity of
fusions of cell~ i~munized ~ith horse ferritin for 8
and l5 days. According to cell marker ~nalysi6, the
- percent of Bl+ cells decreased and the percent of
plasma (PCA-l+) cells increased two-fold between days
- ,- . --. - , . -- . ....... ... ., ~.-, . . - . . - . - .... .
., . .. .. . :
W091/]7769 ~ 2~ 52- PCT/US91/0~40
5 and 7. These results sugge~ted that Ig-secreting
hybrids were more likely to be produced from B cells
fused before rather than after differenti~tion to
plasma cells, ~nd that the most productive fusions
resulted from cells immunized for more than one but
for no more than five days.
Monocl~nal antibodies produced ~ainst a
murine monoclonal i~E~ungalobulin.
Human in vit~o immune respon~e6 to murine monoclonal
antibodies were tested in efforts to produce human
anti-idiotypic monoclonal antibodies to murine
monoclonal antibodies. Allogeneic cultures of human
lymphocytes were primed with 2 ug/ml of a murine
monoclonal antibody for 2, 3, or 4 days (Table VII,
fusions 2, 3, 4, and 5). Cells used for fusion l
were cultured for 2 day6 but were not primed with
specific antigen. Fusions 3 and 4 were identical
except that fusion 4 cells were cultured at l.5
million cells/ml instead of at the 6tandard 3 million
cells/ml. Similar fusion frequencies were observed
with each of the immunizations. Non-primed cell~
were again less fusogenic. Of the antigen-reactive
clones identified, 63% were antigen-~pecific. 92S of
antigen-reactive IgG clones were antigen-specific,
whereas only 50% of the IgM antigen-reactive clones
were ~pecific. This result i8 in agreement with the
predicted greater ~pecificity of IgG ~ntib~dies.
Fusion ~ummarY
The re~ults of fusions of lymphocytes immunized with
either ~orse ferr~tin, human ferritin, or with ~
murine monoclonal IgG i~munoglobulin are summarized
in Table VIII. Of the hybrid5 produced from cells
immunized with either of the foreign proteins, 9-10%
were antigen-reacti~e. Of the clones produced from
cells immunized with human ferritin, only 3~ were
. . . , - :
:: ., , ~ :
. .
~ ~ ,, 2 ~
~V~ 9t/17769 53 PC~r/US91/0~406
antigen-reactive, a result which might be expected
from the lower immu~ogenicity of the human protein.
of the antigen-reactive hybrids produced from cells
primed with horse ferritin, 29% were highly ~ntigen
specific. Relative to the number of ~ntigen-reactive
hybridoma, antigen ~pecificity wa~ Duch higher for
monoclonal ~ntibodies produced from cell6 primed with
either human ferritin (65%) or with the murine IgG
~olecule (63%). The greater apparent lymphocyte
activation by horse ferritin than by human ferritin
or by the murine IgG may be related to the greater
production of cross-reactive antibodies from horse
ferritin-primed cells. Comparison of antigen-
specific IgM versus IgG production showed that the
highest ratio of IgM:IgG antibodies resulted from
immunizations with the allogeneic, human ferritin.
Immunizations with xenogeneic antigens showed less
bias towards production of IgM class antibodies.
~tability of human ~onoclonal ~ntibody
produ~tion.
Ferritin-reactive ~onoclonal antibody production wac
initially monitored with hybrids growing in 96 well
plates. When a hybrid clone was fir6t identified as
cecreting ferritin-reactive antibody, it was expanded
to a 48 and then to a 24 well plate, for a minimum
number of 3 passages. ~he presence of ferritin-
reactive monoclonal antibody was monitored at each
passage. Approximately 50% of the clones ~nitially
positive for ferritin-reactive antibody secretion
lost production within 3 passages (T~ble IX).
Hybrids used for further study, a~ well ~8 those used
to compile the data presented in T~bles V-VII, were
selected from those which were 6table beyond 3
passages. Most of these selected clones rem~ined
~5 6table for at least several months. Unstable hybrids
could therefore be eliminated early in the procedure,
.
.. . . , :, .............. . , ~. . ....... . ..
:
' ' ' `:''
WO91/1776~ aS,~ 54_ PCT/~S91/0340
with the remaining hybrids having a high probability
(>90%) of maintaining antibody production.
Quantitation_of anti-ferritin IgG
ÇCretiPn.
Antibody concentrations of supernatants from
confluent 24 well terminal cultures, 6ecreting
ferritin-reactive IgG antibody, ~ere measured by
quantitative ELISA. Levels of immunoglobulin
~ecreted under these conditions ranged from l to 50
ug/ml (Table X). The scale-up of two relatively high
affinity anti-ferritin antibodies to spinner flasks
resulted in levels of production from 0.5-2.0 ug/ml.
These hybrid cells often grew faster in spinner
culture than in flasks, but antibody production
levels were lower. Preliminary results indicate that
growth of cells in a coreactor (Synbiotics,
Incorporated, San Diego) leads to 5-lO times higher
antibody concentrations than growth in ~pinner
culture.
Characterization of ~erritin-sDecific
~onoclonal antibodies.
IgG class human monoclonal antibodies were purified
in one step by affinity chromatography on Protein G.
SDS-polyacrylamide gel electrophoresis revealed heavy
and light chain bands. Western blotting using anti-
human heavy ~nd liqht chain and ~nti-mouse heavy and
light chain reagent~ confirmed that the monoclonal
antibodies tested, products of a human ly~phocyte
fused with a hu~an X mouse heteromyeloma, were of
hu~an and not of murine origin.
Light chain analy~is of 16 IgM class anti~odies
revealed 6 with a lambda light chain, 9 with a kappa
light chain, and l with lambda + kappa reactivity.
Analysis of 95 IgG class antibodies showed 61 with a
.
.. . . .
~2~
~W;~9l/17769 PCT/US9l/0~406
-55-
lambda light chain, 31 with a kappa light chain, and
3 with lambda + kappa light chains. Since hybrids
had not been subcloned at this stage, some of the
cultures were not mon~clonal.
Affinity ~easurements ~y competition ELISA, using two
cubcloned, purified highly epecific anti-ferritin IgG
~ntibo~_es revealed dissociation constants in the
ranqe of 1-2 X lO(-8) M (Figure 10). Competition
assay6 utilized horse ferritin, the monoclonal
antibody 14-2-2-59 was raised against horse ferritin
and anti~ody 21-lB-9 was made against human ferritin.
Of the IgM supernatants tested for ferritin
specificity, 13-5-3-18 was the most specific and most
highly reactive. This monoclonal antibody was tested
for reactivity with ferritin-containing tissues by
immunohistological analysis. Figure 11 shows
reactivity 13-5-3-18 with human liver tissue in
comparison with reactivity of a preparation of
polyclonal human IgM antibodies used as a negative
control. A second c~ntrol, co~sisting of a human IgM
monoclonal antibody produced by the ~ame procedure as
13-5-3-18 but produced against a di~ferent antigen,
was also negative.
C. ~iscussion
The present work focuses on conditions that will
~upp~rt primary immunization reactions in vit~o. The
results were achieved without Ts cell depletion or
adjustment of T:B cell ratios, and suggest that an
initial level of 30-40% Ts cells in the presence of
15-25~ Th cells, 40-60~ B cells (Table IV), ~nd ~ore
than ~inimal levels of adherent cellz w~ll support
antigen priming when ly~phokines are gener~ted by
allogeneic st;imulation. These cell number~ result in
a ~:T cell ratio of æpproximately 1:1.
Supplementation with adherent cells or with factors
: , . .: ~ . ' ' . -,: , . '
: , -. - . . , : :. .
~, lr 8 2 1~ 56- PCT/US91/0340 ~
secreted by ~dherent cells, such as IL-l, has been
another variable requirement. The pre~ent protocols
using spleen or tonsil preparation~ did not
specifically require supplementation with either
S adherent cells or ~onocyte-~ecreted factors. The
present finding that immunizations with ferritin,
especially with human ferritin, were better when
adherent cells were retained, indicate that these
cells and/or their products are helpful (hor6e
ferritin) or are an absolute requirement (human
ferritin) for efficient priming with T-cell
dependent, protein antigens.
A minimal level of lymphokine stimulation is required
for ~uccessful in vitro priming and it is likely that
the appropriate ~timulation can be nchieved for most
tissue preparations in a variety of ways. The method
of generation of lymphokines may not be nearly as
important as the achievement of optimal level~ at the
appropriate times. For spleen tissue prepared
according to the present protocol, ~yngeneic culture
without supplementation was suboptimal for certain
spleens and not at all effective for others.
However, allogeneic culture of two ~pleens appeared
sufficient for any combination of two spleens tested,
while culture of more than two appeared to lead to
excessive non-specific ~timulation, similar to
effects observed when PWM was included in the
immunization cultures.
Another potentially critical factor in the ~upport of
antigen priming by lymphokines is the timing of
exposure of the cell~ to individual f~ctors.
According to cell marker studies (Table IV), many
cells differentiate in culture as the response
proceeds. The response to ~ lymphokine by a cell in
one state may differ significantly from its response
' '.
.
wn 9l/17769 2 ~ (~ 2 0 '1~ PCT/US91/Ot406
-57-
during or after differentiation. In the present
experiments, addition of exogenously generated
lymphokines in the form of a supernatant (MICS) or
endogenous generation by non-specific ~itogenic
stimulation of T cells were less effective than
~llogeneic co-culturing. These results ~uggest that
the levels and kinetics of lymphokine exposure
generated by allogeneic 6timulation during priming
may most closely mimic exposure during ~n ~ivo
immunization responses.
Post-immunization culture supernatants by ELISA
analysis revealed low, but detectable IgG responses
in 10-30% of our experiments when cells were primed
with ferritin, but in none of the experiments when
cells were primed with murine immunoglobulin
proteins. However, fusion of lymphocytes immunized
with either antigen led to the production of antigen-
specific IgG as well as ~g~ monoclonal antibodies.
The highest numbers of antigen-reactive IgG
monoclonal antibodies were derived from fusions of
lymphocytes immunized with horse ferritin, for which
polyclonal antigen-induced IgG responses were most
easily detected prior to fusion. The early
appearance of the antigen-driven IgG response in
experiments where observed, was surpri~ing.
Polyclonal IgM antibody produced ~fter antigen
priming under ~ppropriate conditions (Table II) bound
6ignificantly better to ferritin than polyclonal
antibody produced from non-immunized culture6. Less
complex antigens than ferritin ~howed lower levels of
non-specific binding. Results of the thiocyanate
~naly~is, however, did indicate that ferritin-induced
maturation of the anti-ferritin i~mune response
occurred in cultures primed under appropriate
conditions. The maturation of the IgM response
.: . . , : : - : .............................. : ,.
, :. .. ..
WOgltl776g 2 ~ ~ 2 ~ 58- PCT/US91/0~40
observed was probably primarily due to specific ~'
binding of ferritin to naive B cells, activation, and
their subsequent maturation to ~ntibody-secreting
plasma cells and possibly also to memory cells.
Production of antigen-specific IgG monoclonal
antibodies from fusion of in vitro immunized
lymphocytes ~uggests that antigen-driven cl~ss ~witch
~nd affinity maturation might occur in vitro, if the
IgG ~ecreting hybrids were not generated from cross-
reactive hybridoma clones.
The present results demonstrate that in v~oactivated human lymphocytes can be efficiently fused
with a mouse:human heteromyeloma to produce a high
percentage of IgG and IgM secreting heterohybridoma
cell lines.
The present invention describes speci~ically the
fusion of the K6H6/B5 cell line, developed by Carroll
et al. J. I~muno, Methods 89, 61 (1986), to human
splenocytes cultured in vitro for 1-6 days. The
average fusion frequency was 35 and the range for 15
separate fusions was 17-50 hybrid containing
wells/million lymphocytes. In vitro culture of
lymphocytes, especially if combined with allogeneic
stimulation, leads to a highly fusogenic state of
activation. The larger cellular aggregates and the
higher fusion freguencies observed for immunized
relative to the control cultures (Tables V-VII)
suggested that fusion efficiency could be inf luenced
by the degree or by the nature of lymphocyte
activation.
The level of Ig production was fairly consistent for
each hybrid but the range for 24 hybrids v~ried by
50-fold. Production by some of these cell lines
compared well with other human hybrids and with
- :- . . .................. ~.' . - .
. .
2 ~ 2 ~
~'~91/17769 PCT/US91/03406
l -59-
murine monoclonal ~ntibody production in culture. Ig
production was unstable in ~pproximately 50% of the
hybrids produced, but the lnrge number6 obtained
permitted early elimination of un~table hyhrids,
leaving a large 6election for evalu~tion of secretion
levels, ~peciflcity and affinity. Prel~minary
karyotypic analysis with G-banding of the ~6H6/B5
fusion partner revealed an average chro~osome number
of 9l (range - 77 to 97), surpri~ingly few
identifiabie mouse or human chromosome~, and many
~tructures which appeared to be chimeric rouse:human
chromosomes. Fusion of thi6 geneti~ background with
human lymphocytes ~ay allow better retention of human
chromosomes than fusion with B cells of murine
lineage, but the initial instability of secretion in
50~ of the hybrids was understandable in view of the
karyotypic complexity and abnormality of the K6H6/B5
partner.
The results also revealed that many antigen-specific
IgG ~ecretinq hybrid6 could be produced from fusions
of in vitro immunized lymphocytes. The IgG-secreting
clones observed in these experiments may have
developed from in vit~o primary responses, 6ince
activated lymphocytes can give ri6e to IgM-secreting
hybrids or can undergo class 6witch to become IgG-
secreting plasma cells as part of the primary
response. Fu6ion of either type of cell at an as yet
undetermined point or points during their development
could, therefore, produce either IgM or IgG-secreting
hybrid cell6. Pos~ibly, the allogeneic Etimulation
induced by co-culture of 6plenocyte~ from different
individual~ provided more ~upport for class ~witching
than ha~ previou~ly been observed in vit~o.
An alternative explanation for the recovery of
antigen-~pecific IgG class antibodies from the
.. . . . , , . .. . ~ .
- .. . ,: . . . . . ... . .
.
. :: : . . .. . : .
- . . ,. .. , . ~.
WO91/17769 , PCT/US91/0340
2 ~ r 1 - 60- ~ l
fusions involves the possibility that ~uch hybrids
arose from ~econdary ~timulation of memory cell~. Ig
receptors on certain memory cell~ in the splenocyte
preparations may recognize determinant6 on the
priming antigens ~imil~r enough to previously
encountered determinants to lead to ~ctivation.
The nature of the antigen used for priming influenced
the percent of antigen-reacti~e hybrid6 derived/total
hybrids produced. The foreign proteins, horse
ferritin and mouse IgG, induced 9-lOS antigen-
reactive clones/total hybrids, while the all~geneic
human ferritin induced only 3-4S ferritin-reactive
clones/total hybrids (Table VIII). The response to
human ferri~in, ~lthough lower than to foreign
proteins, indicates that monoclonal antibodies can be
produced to highly conserved and even to self-
proteins by in vitro immunization. The similarity of
the affinity of 21-lB-9, produced against human
ferritin, to the affinity of one of the best
antibodies produced against horse ferritin, 14-2-2-59
(Figure ll), indicated that in vitro antibodies
produced against self could be similar guality to
antibodies produced ~gainst foreign proteins.
The IgG antibodies selected were affinity purified on
Protein G. Immunoblot analysis on ferritin ~nd on
extracts of ferritin-containing tissue showed
reactivity patterns 5imilar to those observed with a
high affinity murine anti-ferritin monoclonal
antibody, produced by Hybritech, Inc., reactivity
which also requires binding affinitie~ of
lO(7)-lO(8)/mol. Direct analy5is of the strength of
antigen ~inding of two of the purified Anti-ferritin
IgG nntibodies by competition E~ISA (Figure ll) also
indicated that affinities wherein the range of
lO(7)-lO(8)/mol. The induction of IgG monoclonal
;
- . . . - ,
: .
.
: . . .
U'~-~9l/17769 -61~ 2 ~ PCT/US91/03406
antibodies having ~pparent affinitie~ of thi6
strength is compatible with ~ specific primary
response in combination with class switching, but
probably not associated with extensive somatic
5 mutation. Alternatively, the antigen-reactive, IgG-
secreting hybrid~ could be products of cross-reactive
memory re~pon6es, where affinitie6 to primary
antigen~ are high, but affinities to cross~reactive,
inducing ~ntige~s are usually, but not always, lower.
Tablo I. Analysis of ferritin-reactive IgM antibody
secretion in vitro primed 6yngeneic and allogeneic
lymphocyte cultures: comparison of predicted with
observed responses.
~ ,,
Ferritin-dependent Response
. (~ OD4~ )
Spleen Observed Calculated
A ,15 __
D ND _~
A+B 34 10
A+C 33 25
A+D 13 08
B+C 2B 10
B+D 05 03
C~D .18 .07
A+B+C 21 12
B+C+D .16 07
A+8+D 15 10
A+8+C+D 19 D9
Numan 6plenocytes were prepared without depletion of
adherent cells and cultured in the presence or
absence of 1 ug/ml horse ~pleen ferritin as described
.. : ,- ,: , . - - ~ ,
, . : , - . . - .: : ~, ,' ' ' ......... , ' ', ' ' ......... . , . .:
.
W091/17769 ~$'~ 62- PCT/US91/0~40 ~
above. Observed values repre~ent the difference in
OD~ value~ between control ~nd ferritin-primed
cultures. Calculated values for mixed cultures were
derived from the ~ummation addition of the
appropriate fractions of the respon6es of ~yngeneic
cultures (i.e., calculated value for
A+B~C -- 3 ( .15) + 3 (.06) + 3.14 = .12.
ND = not detected
Table Il. Use of affinity/avidity estimations to
evaluate different culture conditions for in vitro
antigen priming.
Serum Secretion tKSCN]~
Spleen time Control
_
(%) (day5) exp 1 exp 2 exp 1 exp 2
..__
B 10% 3-5 2.0 2.1 1.8 2.3
A+B 10% 3-5 2.0 2.0 2.6 2.6
5-7 2.6 2.6 2.9 2.8
, ._ . __
7-9 2.2 1.7 2.9 2 9
9-ll 1.5 1.2 2.2 1.4
A+B 0% 3-5 2.1 2.2 2.3 2.3
5-7 1.8 2.4 2.9 2.5
. _
7-9 2.1 2.4 2.7 2.4
9~ 1.5 1.8 1.5 2.0__
Human plenocyte~ were prepared without depletion of
adherent cells ~s described ~bove. Eitber syn~eneic
or l:1 co-culture~ of 2 ~pleens were pri~ed with 0 or
l ugtml horse spleen ferritin for 3 days. The
. . . ........... ......... . . . , .- . .- .
: . . . .
.. . . . ~, . -
. . .- . . . ..
2~2~
~'~91/27769 -63- PCT/US9l/0~406
ferritin was removed by washing and supernatants were
collected at the indicated times. The ferritin
reactivity of the secreted immunoglobulin was
analyzed by ELISA and relative affinity/avidity
estimations were made as described ~bove. The
numbers shown represent the Molar concentrations of
RSCN required to decrease the amount of antibody
bound to ferritin on the assay plates by 50
([KSCNJ-50).
Table III. Non-specific mitogen effects on (A)
6ecretion of total Ig and (B) on ferritin induction
of ferritin-reactive antibody.
, . . , ~
I Spleen 1 I Spleen 2 ! Spleen 1 & 2
A. ~g/ml total im~unoglobulin
No additions 2.4_0.4 0.6+0.1 12.3+1.5
+ PWM 30.8+3.7 2.6+l.0 20.1l0.8
+ MDP 5.8+1.4 4.0+1.0 21.5+1 8
B. ferritin-induced, ferritin-reactive OD
___ - ~49D
No additi~ns 0 0 0.28+0 14
.
+ PWM O O 0.13+0.08
+ MDP O.29+0.8 o,04+0.02 O.46+0.09
~_ . . . .... _
, . ,
wo ~ 7769 ~ ~ 8 2 ~ 64- PCT/US9~/034 ~
Table IV. Cell marker analysis of human ~plenocytes
as a function of time in culture.
.. ~..... ~_ _ _ . , ................. _ _
MarkerDay o 1 2 1 2 1 2 1 2
IgG =49 17 16 15 14 _ 12 11 8
IgM 30 22 20 17 15 810 7 10
_ _
Bl 51 44 41 37 35 1715 11 12
PCA-l 14 14 13 17 15 3124 33 23
T3 37 40 42 45 42 463S 46 40
T4 20 17 18 17 16 129 20 16
T8 34 38 40 47 46 4344 49 46
- _ ____
IL-2 3 17 15 32 3035 35 24 22
receptor
__ _ _ _ , ,. _ _
Human splenocytes were prepared without depletion of
adherent cells and cultured as discussed above.
Parallel experiments were performed for each surface
marker on days 3, 5, 7, and 10 and depicted on the
table as 1 or 2. Cultures wer~ harvested analyzed as
described above.
- ' :
, : . : , ,
.. . . .
.
:- . .:: :
.
~ 91/1~769 ~ ~ ~ 2 ~ PCT~uS9l~03406
~able V. Hybridoma clone production from human
lymphocytes immunized m_vitro with horse 6pleen
ferritinO
. . .. . _ .. _ __ ~ _
¦Fusion 1 2 3 4 5 -
5 Antigen _ + + + +
(.25 ug/ml)
Culture Time 1 1 2 5 5
~ cells 5 5 5 5 5
10 fused (x 10~ ._
~ hybrid- 86 249 230 177 199
wells .
Fusion 17 50 46 35 40
15 Frequency
# Secretors
_ _
IgM 1/59 5/173 14/137 7/88 6/90
.(~%) (3%) (10%) (8%) ~7%)
IgG 12/5949/173 80/137 46/88 48/90
(20%)~28%) (58%) (52%) (53%)
Reconfirmed ferritin-reactivl
IgM ¦ 1 ¦ 0 4 ¦ 6 ¦ 5
IgG ¦ 4 10 33 16 14
~ __ . ,.
IgM ¦ 0 ¦ 0
IgG ¦ 2 ¦ 4 ¦ 11 i _5 ~_3._
Immunizations, fusions, and hybridoma screening were
performed as described above. Lymphocytes were
primed with either 0 or 0.25 ug/ml hor6e spleen
ferritin for 1 (fusion~ 1 ~nd 2), 2 (fusion 3), or 5
(fusions 4 and 5) days. the number of IgM or IgG
~ecreting hybrid6 i6 6hown ~s the number of ~ecretors
per the number of clones tested for IgM or IgG
~ecretion.
~'", ' ~' ''' ':
''
~, , ".: ~, : ''
WO 91/17769 ~ 3 2 l~ 66-- PCI/US91/034~,
Table VI. Hybridoma clone production from human
lymphocytes immunized in vitro with human ferritin
... .,~ , , ... , __ ..... ._~ ~_ _=
Fusion 1 2 3 4 5
._ .
Ferritin 0 .25 .25 2.5 2.5
(ug/ml)
Culture Time 2 2 2 4 6
(days) .
~ lympho- 3.8 4 4 4 4
cytes fu~ed
(x 106)
~ hybrid- 31 49 64 266 268
containing
wells .
Fusion 8 12 16 32 39
Frequency
(clones/l06
lymphocytes) . .
Ig 6ecreting
_. .. . _
IgM 3 4 12 62 6
(10%) ~8%) (9%) (23%) (2%)
.... _ .
IgG 11 12 19 55 15
(35i) (24S) (3~i) (2li) (6i)
Ferritin-reactive
IgM 0 2 4 8 l
IgG 1 1 1 3 2
Ferritin-specific
(v~. ~-galactosidase And BSA)
. _
IgM _ 1 1 8 l
IgG 1 1_~ 0 2 0
Immunizations, fusion6 and hybridoma ~creening were
performed ~s described aboYe. Lymphocytes were
cultured at 3 x 106 cells/ml for 2 (fusions 1-3), 4
(fusion 4), or 6 days (fusion 5) in the presence of
O.25 or 2.5 ug/ml human ferritin a~ indicated.
. .' . : ' :
: , . ... . .
9l/17769 -~7- ~s~l~2 ~ pCT/US91/0~406 `
,
T~bl- ~II. Hybridoma clone production from human
lymphocytes immunized in vitro ~ith murine monoclonal
IgG
.. .... .. .. .. - ~ . v __ . .. _. .. ., . __ - _
¦Fusion 1 2 3 4 5
5 Culture Time 2 2 3 3 4
Antigen 0 2 2 2 2
(ug/ml)
Cell density 3 3 3 1.5 3
(x 106'~ .
~ hybrid- 27 107 116 98 1.26
containing .
Fusion 7 27 29 25 32
Frequency
Antigen-reactive
IgM 1 10 12 5 2 : :
IgG 0 4 8 1 0
Antigen-specific
IgM I 1 S 1 4 4 1
. I I
IgG ¦ 0 4 ¦ 7 1 . _
Immunizations, fusions, and hybridoma screening were
performed as described above.
,
: . ; , .
.
W O 91/17769 2 0 ~ 2 D ~ 6~- PC-r/US91/034
~able VIII. Compari~on of hybrid production from
lymphocytes immunized with horse ferritin, human
ferritin, or with murine monoclonal IgG.
..____ , ,..~ _ " __.,_,_;""",~_ _~ _ .._ _S~e
~ Clones Antigen-
Antigen reactive specific Antigen- IgM IgG
Horse 941 93 27 2 25
ferritin
.
Human 678 23 15 11 4
ferritin
Murine 474 43 27 15 12
10 monoclonal __ __ ~ _ _ _
Immunizations, fusions, and hybridoma ~creening were
performed as described above. Lymphocyte6 were
cultur~d and immunized as described in Tables Y-VII.
~able I~. Stability of immunoglobulin production by
human hybridomas.
-- . .. _ .. .. _ . -- .AI __ . __._ _ _~A~
Immunogen ~ initial ~ reconfirmed
posit_ve clones (passaged 3 x~
horse ferritin 192 92 (48%)
human ferritin 40 24 (60%)
Immunizations, fusions, and hybridoma screening were
performed as described above.
-
...
- . .
.. .. . .
~ 91/17769 2 ~ ~ 2 ~ PCT/us91/o34o6
Table ~. Quantitation of immunoglobulin production
by IgG ~ecreting ~uman hybridoma clone~.
. . . _ ._ _ . .. . __.. _,.. ~
Clone Ig Ferritin-reactivity
(ug/ml) (O.D.)
1 50 + 11 1.15
2 42 + 9 1.65
3 38 ~ 6 .2
.
4 31 + 13 .2
28 + 5 .8
6 27 + 6 .6
7 23 + 6 7
8 20 + 5 6
9 19 + 4 1.5
. 10 17 + 7 2
11 14 + 4 l
12 12 + 3 .9
,
: 13 12 + 3 .2
. :
_ 14 10 + 2 .1
9 + 1 .
16 7 + 2 .2
17 6 + 1 2
: 18 6 + 1
19 5 + 1 1.3
.
: 20 5 + 1 .1
. . _
21 3 + 1 2
22 2 + 1 1
2 1.4+0.3 1
24 1.3+0.3 .4
.,
~ ybridoma 6creening and quantitation a6say~ were
performed as described above.
,.
Concludinq Remarks
.
.
.. . . , :~ ' ', '' ' '
WO91/17769 ~ PCT/US91/0~40
The foregoing description detail~ ~pecific ~ethods
that can be employed to practice the present
invention. Having detailed such specific methods
initially used to prepare, isolate, characteriz~ ~nd
use the antigen 6pecific high affinity monoclonal
antibodies hereof, and a further di6clo6ure as to
~pecific model procedure6 and entities, the art
skilled will well enough know how to devise
alternative reliable methods for arriving ~t the same
information and for extending this information to
other related preparation~ of ~uch monoclonal
antibodies. Thus, however detailed the foregoing may
appear in text, it should not be construed as
limiting the overall scope hereof; rather, the ~mbit
of the present invention is to be governed only by
the lawful construction of the appended claims.
- .. ~ . . ... : :
