CA2082041A1 - Methods for producing antigen-specific, high-affinity human monoclonal antibodies - Google Patents

Abstract

The present invention relates to advances in monoclonal antibody production, and particularly, in culturing methodology preparatory to hybridoma formation. Specifically, retention of lymphoid fragments in the culture systems results in predominantly IgG-secreting, long-lived cultures.

Description

~'~.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 . . : .. ... : ., ~ . . .

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 : : :, ~ .

~.~9l/17769 5~ ? Q ~ 1 PCT/USgl/03406 .... . 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 ~ ~ 8 '~
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 .. ...--. ,.. ., " ... ,~ ,....~ .... . .. ....
prepared, for example, by fusion with an immortal fusion partner or by means of reco~binant DNA. For _ , .. .. _, .. ... ..... .. . .
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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~ 91/17769 PCT/US91/03406 9 2~2~
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.

. . : ' ' , , ~ .:. '' :., . . : ,: . : :: -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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W~91/17769 PCT/US91/03406 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, :, :
,, .

~ 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 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 .
., . . , , ~
.,. . , -, ,- . .. ' ~- ' , ~ " ,,.' ~ ',. , 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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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 . .

', ' ' : - . ~:

, .

~91/17769 2 ~ ~ 2 ~ PCT/~IS91/0~406 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 - - . . ' . :

. . , - , :, . , ' 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 - .

- . .. ..
- . ' . .' ' : - . . ~ . ~

.

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 : ', . . , ~ :

- . . . , ~ .:

~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 .. . . .
.. . , ~ . . ; .
. . . ~ . . . .

. .
.

... .
.

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 , , , . :,- : : ... .: , . . .. ... .
.; , . . ., .. - ,, .. , . :,.:, ., , ... : . . . : , , . ::, ~ , - .. :
: - ,. . .. - , .. : . , : : :: ::, . - .. : : . : . ~, ~ : :

.. .. .. . . . . ................... .
: ........ . . . . . .

~V~91/17769 2 ~ PCT/US91/0~406 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.

.. . . .
. . ` .
.

, ' : ': ' ' ' ' 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.

- -. ~, . , ~ . , . : -.

- - : .. 1 . :

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 , .: . ........... . .
..

~.
., . . . ' . . . '.. ~

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 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 _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 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 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 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, .
.. . . , :, .............. . , ~. . ....... . ..

:
' ' ' `:'' 6~ 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 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 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 - _ ____ 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.

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~ 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 .
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.. . . , :~ ' ', '' ' ' 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.

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Claims (33)

1. An in vitro immunization culture comprising lymphoid tissue derived human lymphocytes having essential numbers of autologous accessory cells, immunized with a specific antigen under culturing conditions not requiring supplementation with growth or other factors, such that effective numbers of human monoclonal IgG antibodies having affinities with said specific antigen of at least about 5 x 107 liters/mole are derived from said lymphocytes.

2. A culture according to Claim 1 wherein said lymphoid tissue is human spleen.

3. A culture according to Claim 1 wherein said lymphocytes are human splenocytes.

4. A culture according to Claim 1 wherein said antibodies are produced from in vitro immunization of lymphocytes having multiple macroscopic aggregate forms.

5. A culture according to Claim 4 wherein said lymphocytes are splenocytes.

6. A culture according to Claim 4 that is an allogeneic coculture.

7. A culture according to Claim 4 containing multiple splenoids.

8. A stable continuous cell line that produces antigen specific human monoclonal antibodies defined according to Claim 1.

9. A stable continuous cell line according to Claim 8 wherein said lymphocytes are immortalized via fusion with immortal fusion cell partners.

10. A stable continuous cell line according to Claim 8 prepared via recombinant means and harboring operatively DNA encoding the variable region of said antibody.

11. A stable continuous cell line according to Claim 8 wherein nucleic acid encoding the variable region of said antibody is amplified and identified via application of polymerase chain reaction.

12. A hybridoma according to Claim 9 wherein said immortal fusion cell partners are selected from the group consisting of an immortalized lymphoid, myeloma and heterohybridoma cell line.

13. A hybridoma according to Claim 9 capable of producing antigen specific human monoclonal antibodies having an antigen affinity of at least about 5 x 107 liters/mole.

14. A hybridoma according to Claim 9 wherein said monoclonal antibodies are IgG antibodies.

15. A hybridoma according to Claim 9 that secretes a human monoclonal antibody to a human antigen.

16. A hybridoma according to Claim 9 that secretes a human monoclonal antibody to a non-human antigen.

17. A human monoclonal antibody when prepared from a cell line according to any one of Claims 8, 9, 10, 11, 12, 13 or 14.

18. A human monoclonal antibody according to Claim 17 specific for a human antigen.

19. A human monoclonal IgG antibody according to Claim 18.

20. A process of preparing an antigen-specific human monoclonal antibody comprising culturing is vitro lymphoid tissue derived human lymphocytes having essential numbers of autologous accessory cells, immunized with a specific antigen under conditions not requiring supplementation with growth or other factors, such that effective numbers of human monoclonal IgG antibodies having antigen affinities of at least about 5 x 107 liters/mole are derived from said lymphocytes, means for producing said human monoclonal antibodies from said lymphocytes and recovering said human monoclonal antibodies.

21. A process according to Claim 20 wherein said culturing is of an allogeneic coculture.

22. A process according to Claim 20 wherein said human lymphocytes are prepared and cultured by a) using hypotonic lysis of red blood cells, b) minimizing room temperature incubations to avoid loss of accessory cells, c) retaining small fragments of lymphoid tissue in the final cell suspension prior to freezing, and d) including or supplementing small fragments of lymphoid tissue in the cell suspension.

23. A process according to Claim 20, wherein said means comprises fusing said lymphocytes with immortal fusion cell partners.

24. A process according to Claim 23 wherein said immortal fusion cell partners are selected from the group consisting of an immortalized myeloma, plasmacytoma, heteromyeloma and heterohybridoma cell line.

25. A human antigen-specific human monoclonal antibody having an antigen affinity of at least about 5 X 107 liters/mole when prepared by the process according to Claim 22.

26. An antibody according to Claim 25 that is an IgG antibody.

27. An antibody according to Claim 25 linked with a material capable of modulating cell growth.

28. An antibody according to Claim 27 wherein said material is yttrium90.

29. An antibody according to Claim 25 linked with a reporter moiety.

30. An antibody according to Claim 29 wherein said moiety is indium111.

31. A method of treating cancer, bacterial or viral disease comprising administering to an individual suffering therefrom an antibody according to Claim 27.

32. A method of testing an individual for presence of disease comprising administering to said individual an antibody according to Claim 29.

33. A human monoclonal antibody that is an antiidiotype of an antibody according to Claim 25.