CA1232838A - Immunotoxin conjugates - Google Patents

Immunotoxin conjugates

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Publication number
CA1232838A
CA1232838A CA000456816A CA456816A CA1232838A CA 1232838 A CA1232838 A CA 1232838A CA 000456816 A CA000456816 A CA 000456816A CA 456816 A CA456816 A CA 456816A CA 1232838 A CA1232838 A CA 1232838A
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Prior art keywords
chain
toxin
conjugate
cell surface
antibody
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CA000456816A
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French (fr)
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Ellen S. Vitetta
Jonathan W. Uhr
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University of Texas System
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University of Texas System
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G18/00—Polymeric products of isocyanates or isothiocyanates
    • C08G18/06—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen
    • C08G18/28—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen characterised by the compounds used containing active hydrogen
    • C08G18/40—High-molecular-weight compounds
    • C08G18/54—Polycondensates of aldehydes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6829—Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6819—Plant toxins
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G18/00—Polymeric products of isocyanates or isothiocyanates
    • C08G18/06—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen
    • C08G18/28—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen characterised by the compounds used containing active hydrogen
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/811—Test for named disease, body condition or organ function
    • Y10S436/813—Cancer
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/819—Multifunctional antigen or antibody
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/866—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof involving immunoglobulin or antibody fragment, e.g. fab', fab, fv, fc, heavy chain or light chain

Abstract

Abstract The present invention provides a means for potentiating the cytotoxic activity of immunotoxin con-jugates. The compositions of the present invention include a selective binding agent such as an antibody coupled to a toxin B chain moiety such as ricin B chain.

Description

I

~~65~4 -1-IMPROVEMENTS IN OR RELATING TO II~.MUNOTOXIN CONJUGATES

This invention relates to im~lunoto~in coinage-gates arid their use to delete selectively a target population of cells. In particular, a toxin B chain moiety coupled to a cell surface affinity binding agent is useful in potenti2ting the cytotoxicity provided by a cell surface affinity binding agent coupled to a -toxin A chain moiety.
Ricing is one of a number of plant proteins which, in minute quantities, exhibits considerable toxicity toward eukaryotic cells. Ricing is composed of two glycoprotein chains covalently linked via a single disulfide bond. The A chain of ricing having an apparent molecular weight (Awry) of about 30,000, is responsible four the e~pressioll of toxicity, and acts enzymatic ally upon the 60S ribosomal subunit to produce irreversible abrogation of protein synthesis ~Olsnes, et at., FOBS Let 28, 48 50 ( 197G ) ] . I' gin B chain _ _ AMY 32,000) functions as a pectin with specificity for galactose and serves to bind the -toxin to the cell membrane [see, e.cJ., Banger, et at., J_ of. Chum.
254, 9'795-97'`39 ( 197C.3 ) ] .
The use of ricing or thy purified x-icin A
chain, in conjunction with antibodies, has been the subject of great interest as poten'iallv-useful reagents in tumor therapy. Antibody-ricin and antibody-A chain conjugates, an "i~munotoxins", have been used in a number of systems with varying degas of success see, e.g., Vitetta, et at., Science 219, 6~4-650 (,983);

I, X-656~ -2-Thorpe, et at., Immunol. Rev. 62, 120-158 (1982);
Seville, et at., Immunol. rev. 62, 75-91 (1982); and Jensen, et at., Immunol. Rev. 62, 185-216 (1982)].
Procedures for deleting selected populations of cells by ricing A chain-antibody conjugates are well-recognized. The antibodies of choice are those which react with antigens on tumor cells or on subsets of normal lymphocytes. By deletion of the tumor cells, one may reduce, for example, tumor burdens in viva [Krolick, et at., J. Exp. Med. 155, 1797 (1982)] and remove tumor cells from bone marrow for autologous marrow transplantation [Thorpe, et at., Nature (London) 271, 752 (1978); and Krolick, et at., Nature (London) 295, 604 (1982)].
Also, by deleting normal subsets of lymph-cites, one may be able to "up" or "down" regulate the immune response. The advantage of immuno~oxins is that they are highly specific for their target cell and that small doses can eliminate unwanted cells. Ricing A
2C chain-antibody conjugates have been used primarily to-delete normal and neoplastic B cells, both in viva and in vitro. Certain laboratories have also used coinage-gates of ricing A chain and monoclonal antibody to eliminate neoplastic cells of T cell origin and a variety of other cancerous cells.
However, ricing A chain-antibody conjugates are not active when used against certain types o tumor cells (e.g., some T cell tumors) novel, et at., Immunol. Rev. 62, 75 (1982); and Thorpe, et at., Immunol. Rev. 62, 119 ~1982)~.

In contrast, immunotoxins coupled to the whole ricing toxin are much more potent cytocidal agents.
Unfortunately, the presence of the galactose binding site of ricing s in intact ricing prevents its use in Volvo because its target cell specificity thereby is lost. Attempts to overcome the non specificity of ricing containing immunotoxins by blocking the galactose bind-in site are ongoing however their use in Volvo has not been described yet.
Others have described studies in which ricing A chain-antibody conjugates can be potentiated by the addition of free B chain to cell cultures (Seville et at., a). Researchers have postulated, therefore, that the B chain of ricing has two functions: (1) to facilitate entry of ricing into the cell by virtue of its galactose-binding properties, and (2) to allow the A chain to gain rapid access to the cytoplasm, perhaps by formation of a pore in the endocytic-vesicle rem-brine.
In a recent unpublished study, it has keen discovered that injection of mice with nontoxic ricing A
chain, followed 4-8 hours later by injection with non-toxic ricing B chain, produces ricin-induced death. This probably occurs by reformation of the intact ricing molecule in the serum or on the surface of circulating cells. It is believed, therefore, that the B chain plays an active role in potentiating the toxic activity of the ricing A chain.

12~Z1~8 In accordance with the invention, there are provided compositions and a method for potentiating the cytotoxic activity of cell surface binding agent-toxin conjugates while at the same time retaining target cell specificity. The compositions provided by the present invention include a selective binding agent coupled to a toxin B chain moiety.
The present invention, then, in one aspect, resides in a process for preparing a toxin B chain conjugate which comprises covalently coupling a cell surface affinity binding agent to a toxin B chain moiety.
Further, there is provided a composition come prosing, in combination, a first conjugate including a selective binding agent coupled to a toxin chain moiety together with a second conjugate including a cell surface binding agent coupled to a toxin A chain moiety. The selective binding agent of the first con-gigawatt can be either a cell surface binding agent or a binding agent specific for the cell surface binding agent of the second conjugate.
In one aspect of the i~Yention, there is pro-voided a conjugate which encompasses an antibody as the cell surface binding agent coupled to a ricing B chain moiety. Further, there is provided a composition come prosing a combination a first conjugate of an antibody coupled to a ricing B chain moiety together with a second conjugate of an antibody coupled to a ricing A
chain moiety.
In yet another aspect of the invention there is provided a method for eliminating target cells from a population of cells containing such target cells by contacting the population of cells with a first con-gigawatt comprising an affinity binding agent which is specific for an antigenic determinant on such target cells and which is coupled to a toxin B chain moiety ~,~

1;232~

and a second conjugate comprising a cell surface aft finite binding agent specific to a different determi-Nat on the same target cell and which is coupled to a toxin A chain moiety. The mixture of such conjugates potentates the selective cytotoxic activity provided by the conjugate comprising a cell surface affinity binding agent coupled to toxin A chain moiety alone.
In addition, there is provided a process for preparing a toxin B chain conjugate which comprises covalently coupling a toxin B chain moiety to an anti-body or cell surface binding fragment thereof.
As a preferred embodiment, this invention provides a conjugate comprising an antibody coupled to a toxin B chain moiety. Further, this invention provides a method for eliminating target cells using, in concert, a composition comprising a first conjugate containing ricing B chain moiety and a second conjugate containing ; ricing A chain moiety.
! Ricing is one of a number of toxin proteins which, in minute quantities, exhibits considerable toxicity toward cells. Ricing toxin is composed of two different glycoprotein chains covalently linked via a single disulfide bond. The A chain of ricing (AM
30,000) is responsible for the toxicity caused by irreversible abrogation of protein synthesis. Ricing B
chain (AM 32,000) functions as a pectin which binds to galactose-containing glycoproteins or glycolipids ox-posed on cell surfaces. The general structure and mode of action exhibited by ricing is present in a variety of plant toxin proteins such as auburn, modeccin, puked X-65~4 -6-mitogen factor, and viscumin, and bacterial toxin pro-loins such as cholera, E. golf. heat-labile, pertussis, tetanus, botulinum, pseudomonas, shaggily, and diphtheria toxins.
The ricing s conjugate and the ricing A conjugate used in the methods of this invention each comprise two active moieties: a cell surface or selective binding agent and a toxin A or B chain subunit covalently joined, preferably via a coupling agent. In each composition, lo one of the active moieties is represented by a molecule having binding affinity to a surface structure of a target cell or binding affinity to cell surface binding agent of the toxin A chain conjugate. Typically such molecules may be substances such as hormones, growth factors, pectins, or antibodies. The molecules of choice are antibodies or fragments thereof (in portico-far, Fob fragments) having cell surface binding affinity.
Monoclonal antibodies are preferred but not essential. Immunoglobulin fractions from serum can be used, albeit with a lesser degree of target specificity.
Since the immunoglobulin fraction of an antiserum con twins a multitude of antibodies directed to a wide range of divergent antigens, a practical usefulness of the compositions of this invention and the defined method for eliminating target cells dictates the need to isolate a desired collection of antibodies, each directed to a surface antigenic determinant present on the particular target cell.

~:232~

x-6s64 -7-An effective collection of such antibodies can be obtained by passing the immunoglobulin fraction over a column containing the respective antigen chemically coupled to a matrix. Antibody specific to the antigen will be retained on the column while unrelated immune-globulin passes through. The retained antibody then can be collected by elusion from the column using suit-able eluding agents, such as acidic buffers of chaos tropic agents. One should note that the isolated immunoglobulin, although directed to a single antigen, is not homogeneous. It comprises antibodies directed to a variety of antigenic determinants present on the antigen molecule. Consequently, the possibility exists for cross-reaction with other related antigens.
Therefore, use of monoclonal antibodies in preparing the compositions of this invention is highly preferred because they are directed to only one of posse-by many antigenic determinants present on an antigen.
Monoclonal antibodies are available by recognized methodology from hybridomas derived from lymphocytes present in the spleen or other organ Moreover, the use of monoclonal antibodies in the compositions used in the method of this invention carries the highly desirable feature of enhanced selectivity. Therefore, that both the ricing B conjugate and the ricing A conjugate be comprised of a monoclonal antibody is highly preferred. This ensures a high level of target cell specificity.

of The preceding paragraphs have reference to owe aspect of the present invention, the use of the same cell surface affinity binding agent for construction of both the ricing A chain conjugate and the ricing B chain conjugate. A greater degree of specificity, however, can be attained. Because a normal or tumor cell bears several different surface markers, one may be able to target the ricing A and the ricing s chains to such cells by coupling each to an antibody directed against a different determinant on the same cell. For example, in the case of a neoplastic B cell bearing both surface It (spa) and surface Ig(sIg), immunotoxins against the spa and the swig idiotype can be prepared with the ricing B chain and ricing A chain, respectively. In the case of T cell tumors, a number of monoclonal antibodies exist which are reactive with subsets of human T cells. By using selected combinations of antibodies, one may target the ricing A and ricing B chains to specific sub-sets of such culls. Preferably, one antibody (coupled to ricing A chain) would define the subset, and the second (coupled to ricing B chain) would be a more general marker common to many subsets of cells. The B chain immunotoxin, directed against the more common marker, would bind also to normal cells; however, they would not be deleted. In contrast, the A chain-immunotoxin would be focused only on the tumor cell and would be potentiated by B chain-containing immunotoxin.
Another approach contemplated by the present invention involves first directing a tumor cell reactive antibody-ricin A chain conjugate to tumor cells in viva.
The antibody preferably is univalent, e.g. Phoebe, ~'~32~

and, therefore, is unable to cap and modulate. After the antibody-ricin A conjugate has been injected into a cancer-bearing patient and the excess eliminated from the recipient by excretion or degradation, a ricing B
chain-containing immunotoxin directed against the antibody of the ricing A conjugate is injected. Only those cells which had bound the first immunotoxin would focus the second immunotoxin on the first. Therefore, such cells would be selectively deleted. The second - 10 immunotoxin preferably is a diva lent anti-antibody, such as a F(ab')2-B, which would not bind to macro-phases, mainstays, or other cells bearing Fc receptors.
Furthermore, since the B chain-containing immunotoxin would be innocuous if nonspecifically bound to a cell which had not previously bound the first immunotoxin, any side effects caused by the administration of the second immunotoxin would be eliminated. In contrast, cells binding both immunotoxins would be killed.
As noted, the ricing B-containing composition of this invention and the ricing A-containing compost-lion used in the method of this invention each come proses at least two separate active moieties, one of which affords binding affinity (BY) and the other of which is a ricing subunit (US), whether ricing A (RAY) or ricing B (RUB). These are joined through a coupling reagent, the requirements of the resulting composition being (a) the presence of at least one of each class of moiety, and (b) the retention of the innate activity of at least one of each class of moiety.

I

Other toxin proteins may be similarly coupled to the binding agent component for use in accordance with the present invention. Due to the similarity in their structure and mode of action, plant or bacterial toxin proteins such as auburn, modeccin, poXeweed mitogen factor, viscumin, and cholera, E. golf. heat-labile, pertussis, tetanus, botulinum, pseudomonas, shigella and diphtheria toxins may be utilized. Fur then, it may be advantageous to couple the A chain from auburn, for example, to a cell surface binding moiety to form the first conjugate of the invention and the B chain from viscumin, for example, to a selective binding agent moiety to form the second conjugate. It may be advantageous to use a plant protein toxin such as gelonin, which consists only of an A chain, as the A chain to be coupled to the cell surface binding moiety to form the first conjugate. This first conjugate may be used then with a conjugate comprising a selective binding moiety coupled to a B chain selected from any one of the toxins ricing viscumin, modeccin or auburn.
In accordance with these limitations, the compositions of this invention and those used in the method of this invention can be dim Eric (BARS), i. e., contain one of each class of moiety; trimeLic BUYERS) or (BUYERS)], lye., contain two of one class of moiety and one of the other; tetrameric [(BYPASS), (BUYERS), or (BUYERS)]; and the like.
As noted, highly preferred compositions for use in the method of this invention are those in which the binding moiety is antibody or an antigen binding I

fragment of antibody; and preferably a monoclonal anti-body or an antigen binding fragment thereof. Typical compositions may be Ab-P~B, Ab2-RB, Ab-RB2, Ab3-RB,
2 2' A RB3, Abhor, Abhor, Ab~P~2, Brie by ERA
or Abhor.
In preparing the compositions of this invent lion, the BY and US moieties are joined via a suitable coupling reagent. A wide variety of coupling agents is reported in chose, T., and Blair, A. I., J. Tuttle. Cancer lo Inst. 61, 657-676 (1980). These authors report the use of carbodiimides as well as other bifunctional reagents, such as glutaraldehyde, ~-b~nzoquinone, deflower-m,_'dinitrodiphenylsulfone, or dim ethyl adipimidate, for coupling antibody to cytotoxic agents. Because it is highly desirable to preclude formation of homopolymers, e.g., By or Run use of a heterobifunctional reagent is preferred, ensuring formation of compositions having at least one of each class of moiety. Examples of such heterobifunctional reagents may be N-succinimidyl-3-~2-pyridyldithio)propionate (SPDP), _-maleimidobenzoyl-N-hydroxy-succinimidyl ester, bromoacetyl-_-aminobenzoyl-N-hydroxy-succinimidyl ester, or iodoacetyl N-hydroxy-succinimidyl ester.
For example, using SPDP as coupling agent, a process for preparing a composition of this invention comprises (a) separately modifying both A and US by reaction with SPDP, (b) reducing the Ab-containing pro-duct, (c) causing formation of the composition by mixing the Ab-containing and RS-contailling products, and (d) separating non-reacted monomers Joy gel filtration.
3~3 The conjugates of this invention containing ricing B, when used in concert with ricing A conjugates, have general applicability in the specific and select live killing of a cell type defined by particular anti-genie markers. By appropriate selection of the anti~genic marker the cell surface binding agent can be directed to either a set of normal cells or to a subset of neoplastic cells bearing a distinguishing determinant. As such, they are useful, for example, in the immunotherapy of cancer, for treating parasitic infections, and for treating a wide range of autoirnmune diseases. Moreover, the compositions have several in vitro applications, including, for example, elimination of leukemia cells in bone marrow prior to autologous bone marrow transplantation; elimination of T cells in bone marrow prior to allogeneic bone marrow trays-plantation; and killing of wild types for selection of mutants .
The compositions of this invention can be used in a variety of pharmaceutical formulations and can be administered by a variety of conventional routes, such as intramuscular, intravenous, subcutaneous, and intraperitoneal. As used, the term "pharmaceutically acceptable" means those agents useful in the comma-therapy of warm-blooded animals.
When administering the conjugate compositions parenterally or intraperitoneally, pharmaceutically acceptable forms for injection may include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions I

X-65~4 -13-or dispersions. The carrier can be a solvent or disk pursing medium containing, for example, water, ethanol, polyol for example glycerol, propylene glycol, or fig-rid polyethylene glycol) suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. van-out antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like may also be used. In many cases, including isotonic agents, for example, sugars, sodium chloride, and the like will be desirable. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum moo-Stewart and gelatin.
Sterile injectable solutions can be prepared by associating the conjugate composition refined earlier in the required amount of the appropriate solvent with any other ingredient as desired.
If desired, for more effective distribution, the compositions can be incorporated into slow release systems such as polymer matrices, liposomes, and micro spheres. Moreover, the compositions can be administered either alone or as a mixture of a plurality of active ingredients.
Doses of the compositions are administered Jo the recipient for a period during which a therapeutic response is desired. The weight of the recipient and mode of administration will determine the size of the dose necessary to induce the desired response.

~Z3~3~

Especially advantageous is to formulate the conjugate compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form refers to a physically discrete unit suited as unitary dosages for the subject to be treated. Each unit contains a predetermined quantity of the compost-lion calculated to produce the desired therapeutic effect in association with the pharmaceutically-acceptable carrier. The specific unit dosage form is dependent upon (a) the unique characteristics of the particular composition and by the particular Thorpe-tic effect to be achieved.
The following non-limiting examples are pro-voided to further illustrate the invention.
I. PREPARATION OF IMMUNOTOXINS

A. RICING A AND B CHAIN
The A and B chain subunits of ricing were pun-chased from Coma Corporation, San Francisco, California Prior to use, the A and B chains were dialyzed extent lively at 4~C against phosphate buffered saline (PBS), pi 7.2. The recovery of the A and B chains were 50%
and 80%, respectively.
B. ANTIBODY
The selective and cell surface binding agent of this embodiment is affinity purified rabbit anti-human immunoglobulin (Rug) made according to protocol described in the literature tree, e q. Myriad, et at., Blood, 42, 327 (1983); Vitetta, et at., Science, 219, 644 ~19~3~).

. .

`" ~L2~2~
~-6564 -15-C. CONJUGATION
10 I of 60 my dithiothreitol (DOT) in PBS was added to each my of the dialyzed A or B chain. The mixtures were incubated at 25C for 60 minutes and the reduced chains were separated from the DOT by gel lit-traction at 25C on a Sephadex G-25 *column (18 x 1.5 cm) in PBS, put 7.2. Antibodies were coupled as described in Vitetta, et at., Immunol. Rev. 62:159-183 (1982), and Carlsson, et at., Become. J., 173, 723 (1978), In particular, the antibody in PBS is treated with SPDP, N-succinimidyl-3-(2-pyridyl)dithio)propionate at a PI of ~uccinimydyl-3-(2-pyridyl)dithio)propionate at a pi of about 7.0 to 7.5 at a temperature of about 20C to about 25C. The antibody derivative then is treated with dithiothreitol in buffer solution. The thiolated antibody may be purified by gel filtration, if desired.
The activated coupled antibodies were mixed with the freshly reduced A or B chains at an antibody chain or antibody chain molar ratio of 5:1. The mixture was incubated for 15 minute at 4C with gentle shaking and then dialyzed overnight against ABS at 4C. The immunotoxins were concentrated to 1 mg/ml by pervapora-lion, dialyzed for 2-16 hours at 4C against PBS, and centrifuged to remove insoluble material. The swooper-lion of the immunotoxin from the majority of free Chain, B chains and antibody was performed by gel lit-traction at 25C on a Sephacryl S-200*c*olumn equal-brazed with PBS, pi 7.2. Material with an apparent molecular weight of greater than 200,000 was pooled.
Reduced and alkylated bovine alpha globulin, fraction * Trade Mark for an ion exchange resin formed of highly cross linked dextran containing functional ionic groups attached to the glucose units of the polysaccharide chains by ether linkages.
** Trade Mark .

~Z3Z~
4 (Sigma), was added to a final concentration of 1 mg/ml to the pooled samples. The samples were stored 1~-20 hours at 4C prior to affinity purification.
The Rug chain (Rug) and Rugby chain (Rugby) immunotoxins were purified by affinity chromatography on Sepharose~Ig. For purification of the A chain-containing immunotoxins, the columns were equilibrated and washed in PBS, pi 7.2 and for the puff-ligation of the B chain-containing immunotoxins, the columns were equilibrated and washed in PBS, pi 7.6 con-twining elm galactose. Samples were applied to the column and the fall-through was discarded. The columns were washed extensively in PBS (or PBS-O.lM gal) lot-lowed by 0.85% Nail. Immunotoxins were eluded batch-wise at 37C with 2-3 column volumes of 3.5M McCoy.
The McCoy was removed by dialysis and the samples were concentrated by pervaporation to approximately 200-300 gel Reduced and alkylated bovine alpha globulin was added to a final concentration of 1 mg/ml. Samples were sterilized by filtration Jon a filter preheated with PBS
2% fetal calf serum (FCS)], and then allocated into sterile vials and stored at -20C. Samples stored in - this manner were stable for up to 4-6 months.
The recovery of the immunoto~in following affinity purification was 26-45% and each immunotoxin contained one or two A (or B) chain subunits per mole-cute of antibody. When immunotoxins were analyzed on SDS-PAGE slab gels using a sensitive silver staining technique, no free A or B chain in any of the immune-toxins was detected.
* Trade Mark for Agrees ~2~2~

II. USE OF RICING B CHAIN CONTAINING CONJUGATES
AND RICING A CHAIN CONJUGATES It THE KILL-IN OF NEOPLASTIC HUMAN B CELLS _ A. NEOPLASTIC CELL CULTURE
The human Berkowitz lymphoma cell line, Saudi, as described in Houston, LO Become. Boyce. Rest Commune 92:319-326 (1980), was maintained in suspension -culture in RPMI-1640 supplemented with 10% (v/v) fetal calf serum (FCS), 20 my fresh glut amine, and antibiotics.
Cultures were maintained at 37C in a humid incubator with a 95% 2/5% C2 atmosphere. All cultures were used two days following subculture.

B. TREATMENT OF SAUDI CELLS
It days after subculture, Saudi cells were harvested and washed in buffered saline solution (BUS).
105 cells in BUS were distributed into micro titer wells.
Dilutions of each of the two immunotoxins, Rug and Rugby, or combination of both, were added to triple-gate wells for 15 minutes at 4C. Cells were centric fused and washed three times in BUS. 200 I of RPMI
lacking Lawson and containing 10% FCS were added to each well and the cells were resuspended by gentle ago-station. Plates were cultured for 22 hours at 37 in a% C2 incubator. Cells were then pulsed for 6 hours at 37 in 5% COY with 5 Sue per well of Lawson (New England Nuclear). Cells were harvested onto glass fiber discs and counted in a liquid scintillation spectrometer.
Cells were treated with each of the immunotoxins India ~3Z~

visually as well as the following combinations of immunotoxins:
1. A nontoxic amount of Rug (0.3 gig plus different amounts of Rugby, or S 2. A nontoxic amount of Rugby (0.5 gel followed by different amolmts of Rug.
The results are tabulated in TABLE 1.

Table 1 - % of Cells Remaining After Treatment vs.
Control Conjugate Used Concentration gel 0.03 0.05 0.3 0.5 1.3 2.6 IT* 83 90 100 100 90 82 IT** 100 90 82 - 22 18 IT -I IT (0.5 gel 75 40 12 - 6 5 IT + IT (0.3 gel 95 64 50 - 20 10 *IT = immunotoxin B conjugate (Rugby) **IT = immunotoxin A conjugate (Rug) As indicated in the Table, wren Saudi cells were treated with 0.3 go of Rug chain/105 cells, little toxicity was observed. No concentration of Rugby was toxic. However, when 0.3 go of the Rug was mixed with various combinations of Rugby, there was significant cytoxicity. It should be noted that this treatment of the Saudi cells with the mixture of immunotoxins was performed in BUS lacking galactose.
As shown in TABLE 1, when Saudi cells were treated with 0.5 go of Rugby, no toxicity was observed. In con-tryst, the Rug willed the Saudi cells in a dose-related manner. However, treatment of cells with 0.5 go ~3;~33~

of Rugby mixed with R~Hlg-A was toxic to the cells, even at those concentrations at which Rug itself was not toxic.
Although the conjugate compositions and methods have been described in terms of preferred embodiment, those skilled in the art will recognize that various changes may be made without departing from the intended : scope of the invention.

Claims (18)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
1. A process for preparing a toxin B chain conjugate which comprises covalently coupling a cell surface affinity binding agent to a toxin B chain moiety.
2. A process according to Claim 1 for preparing a toxin B chain conjugate which comprises covalently coupling an antibody or a cell surface binding fragment thereof to a toxin B chain moiety.
3. A process for preparing a conjugate as claimed in Claim 2 in which the antibody or cell surface binding fragment thereof is specific for a cell surface antigen.
4. A process for preparing a conjugate as claimed in Claim 2 in which the antibody or cell surface binding fragment thereof is directed to a cell surface antigen of a tumor cell.
5. A process for preparing a conjugate as claimed in Claim 2 in which the antibody or cell surface binding fragment thereof is directed against a second antibody.
6. A process for preparing a conjugate as claimed in Claim 1 in which the toxin B chain is selected from ricin B chain, modeccin B chain, abrin B chain, pokeweed mitogen factor B chain, viscumin B chain, cholera toxin B chain, E.coli heat-labile toxin B chain, pertussis toxin B chain, botulinum toxin B chain, Pseudomonas toxin B chain, shigella toxin B chain or diphtheria toxin B chain.
7. A process for preparing a conjugate as claimed in Claim 5 in which the toxin B chain is ricin B chain.
8. A toxin B chain conjugate whenever prepared by a process according to any one of Claims 1 to 3, or by an obvious chemical equivalent thereof.
9. A toxin B chain conjugate whenever prepared by a process according to any one of Claims 4, 5 or 6, or by an obvious chemical equivalent thereof.
10. A toxin B chain conjugate whenever prepared by a process according to Claim 7 or by an obvious chemical equivalent thereof.
11. A process for preparing a cytotoxic composition which comprises associating a first conjugate prepared in accordance with the process of Claim 1, together with a second conjugate comprising an antibody or cell surface binding fragment thereof covalently coupled to a toxin A chain moiety.
12. A process for preparing a cytotoxic composition as claimed in Claim 11 in which the second conjugate antibody or cell surface binding fragment thereof is directed against a cell surface antigenic determinant.
13. A process for preparing a cytotoxic composition as claimed in Claim 11 or 12 in which each of the first and second conjugates comprises an antibody or cell surface binding fragment thereof, having identical specificity to a cell surface antigenic determinant.
14. A process for preparing a cytotoxic composition as claimed in Claim 11 or 12 in which the first conjugate antibody is directed against a cell surface antigenic determinant different from the cell surface antigenic determinant to which the second conjugate antibody or cell surface binding fragment thereof is directed.
15. A process for preparing a cytotoxic composition as claimed in Claim 11 in which each antibody or cell surface binding fragment thereof is specific for a tumor cell antigenic determinant.
16. A process for preparing a cytotoxic composition as claimed in Claim 11 in which the second conjugate comprises an antibody or cell surface binding fragment thereof which is specific for a cell surface antigenic determinant and the first conjugate comprises an antibody or cell surface binding fragment which is specific for the antibody of the second conjugate.
17. A process for preparing a cytotoxic composition as claimed in Claim 11 in which the toxin A chain is selected from the A chain moiety of ricin, abrin, modeccin, gelonin, pokeweed mitogen factor, viscumin, cholera toxin, E. coli heat-labile toxin, pertussis toxin, botulinum toxin, Pseudomonas toxin, shigella toxin and diphtheria toxin.
18. A process for preparing a cycotoxin composition as claimed in Claim 17 in which the toxin A
chain moiety is ricin A chain and the toxin B chain moiety is ricin B chain.
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