JPH0820545A - Antibody conjugate having improved property - Google Patents

Abstract

PURPOSE: To obtain an antibody conjugate formed by the covalent bonding of a carrier protein with a monoclonal antibody, and useful for a human imunopotentiation therapy. CONSTITUTION: This antibody conjugate is obtained by the covalent bonding of one antibody such as a monoclonal antibody, preferably an anti-idiotype antibody, e.g. an antibody against antigens of a virus, a fungus, a parasite, a bacterium or against a human antigen with a carrier protein derived from a bacterial toxin, e.g. CRM197, a tetanus toxoid, etc., by a coupling method. For example, the bonding with the antibody is performed by a suitable activation of the carrier protein, e.g. by reacting a hetero bifunctional cross-linking agent having an end of N-hydroxysuccinimide(NHS), and then reacting the activated carrier protein with the antibody. The conjugate is soluble and non- aggregating, and has improved properties for clinical uses.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a soluble conjugate of a monoclonal antibody and a carrier protein.
ate). The invention further relates to methods for producing such antibody-carrier protein conjugates, pharmaceutical preparations comprising the conjugates and methods for inducing an antibody response in a mammal.

[0002]

Monoclonal anti-idiotype antibodies are antibodies that react with antibody variable region epitopes (idiotopes. Therefore, anti-idiotype antibodies are often referred to as Ab2 (antibody 2), while A2
The immunizing antibody used to produce Ab1 is Ab1
(Antibody 1).

Antibodies (Ab1) have many idiotopes, some of which bind their antigen binding sites (paratopes) and others of which the variable region domain framework.
k) Since it is a determinant, the complementary antibody response thereto is functional and structurally heterogeneous and can be divided into three major categories (HCJ Ertl and CABon
a, Vaccine 6, 80-84 (1988)). One of these categories is referred to as Ab2, which recognizes a paratope-binding idiotope as Ab2β. Antibodies in this category are the internal image of the antigen for Ab1 and are therefore commonly referred to as essential image-type (anti-idiotype) antibodies. "Normal", a criterion that distinguishes essential image-type anti-idiotypic antibodies from non-essential image-type anti-idiotype antibodies, is immunochemical criteria, such as Ab2 vs. Ab1 by antigen. Inhibition of binding, as well as functional criteria, such as eliciting an antigen-specific immune response in mammals that have not previously encountered the antigen (HCJHtrl and CABone, ibid.). On the basis of its ability to resemble an antigen, essential image antibodies are capable of eliciting antigen-specific antibodies in a variety of systems, and also anti-anti-idiotypic antibodies or Ab3.
(Antibody 3). Systems in which an Ab3 response can be generated are, for example, human and animal test systems, such as mice and goats.

Anti-idiotype-based methods include the use of idiotypes for passive immunotherapy of T-cell leukemia or B-cell lymphoma (S. Meller, H. Keller, D. Anderson in Recombinant DNA Vaccines: Lashonare and Strategy, RE Isacoson (editor), Marcel Decker, New York, 1992, 345-
346) and, as vaccines for inducing immunity against, for example, viral parasitic or bacterial pathogens, as cancer immunotherapeutic agents, as therapeutic agents in immunomodulatory disorders and agonist / andagonist in antigen-antibody binding studies The use of anti-idiotypic monoclonals as is (eg, JR Schleiber, Curren
t Drugs Ltd., 1641-1648 (1993); ECJ Ertl and CA Bona, ibid.). For example, an intrinsic image anti-idiotype antibody may be used as a surrogate antibody and thus has great promise for octyne injection against an antigen, and is appropriately reactive with that antigen. Antibodies are present, but the antigens are poorly defined or cannot be purified. Furthermore, the use of essential image antibodies may be appropriate in cases where the antigen itself is incapable of inducing a suitable immune response, for example certain carbohydrate antigens which do not induce a memory response. Intrinsic image antibodies may also be useful in breaking tolerance to "self" antigens, and thus are considered a suitable immunological method for the treatment of tumors, such as the treatment of melanoma.

The strong immunogenicity of such antibody vaccines is a condition, which would require formulation with respect to carrier proteins in order to elicit a T-cell dependent (memory) response, for example. In accordance with the state of the art today, aggregated and / or poorly water-soluble or water-insoluble conjugates of carrier proteins and antibodies, such as keyhole limpet hemocyanin (KLH, molecular weight> 1,000,000 Da), are applied to produce anti-idio Type (Ab
2) or an anti-anti-idiotype (Ab3) immune response is obtained (eg, M. Kusama et al., J. Immunology 14).
3, 3844 to 3852 (1989); VK Li et al., Biochim.Biophys.A
cta 865, 127-139 (1986); Mitterman et al. Proc.
Natl. Acad. Sci. USA 89, 466-470 (1992); McNamara et al., Science 226, 1325-1326)). Random conjugation, including protein aggregation, is typically achieved by homobifunctional cross-linking agents such as glutaraldehyde or disuccinimidyl suberate.

[0006]

However, aggregated and / or insoluble antibody-protein conjugates for clinical use suffer from drawbacks such as preparations that are fairly heterogeneous and considerably difficult to standardize. Have. Therefore,
There is a need for antibody-based preparations with improved properties, such as more uniform preparations that facilitate standardization of commercial products.

[0007]

It is an object of the present invention to provide this need by providing antibody-carrier protein conjugates that are soluble and non-aggregated and have improved properties for clinical use. Is achieved. Surprisingly, the following was found: that such soluble, non-aggregating conjugates have strong antigenicity, which antigenicity is specific for the original antigen upon in vivo administration. Suitable anti-idiotype (Ab
2) or an anti-idiotype (Ab3) antibody response.

With respect to the in vivo testing of the soluble conjugates of the present invention, the specific anti-idiotope humoral immune response is at least comparable to the commonly used aggregate-evoked response. Preferably, the immunogenicity of the soluble conjugates of the invention comprising a unique antibody, e.g., an anti-idiotype antibody, is determined to be poorly soluble or insoluble (in a water-based solution) comprising the same specific antibody. so)
It is superior to the immunogenicity of aggregates. This excellent immunogenicity results in higher antigen-specific antibody (Ab2 or Ab3) titers.

The present invention further provides methods for producing such conjugates, pharmaceutical formulations comprising such conjugates and therapeutic methods comprising administering such conjugates to mammals. . More specifically, the invention relates to defined, soluble conjugates, wherein a monoclonal antibody molecule, preferably an anti-idiotype antibody molecule, is covalently attached to at least one carrier protein molecule. The conjugate of the present invention can elicit an antibody response (Ab2 or Ab3 response) in mammals. Conjugation of components is targeted in that the coupling of the carrier protein is restricted to the constant region part of the antibody. To this end, the coupling method is chosen such that it contains only the appropriate groups in multiple constant regions, which are available or accessible in the variable region. For example, such site-selective conjugation to an antibody is reacted with a heterobifunctional crosslinker, such as such a crosslinker comprising an N-hydroxysuccinimide (NHS) endo, by appropriate activation of a carrier protein. And then subsequently reacting the activated carrier protein with the antibody. The conjugation of the antibody to the carrier protein should not affect the conformation and / or accessibility of the variable region of the antibody.

Further, the conjugate of the invention is defined as follows: it comprises only one antibody molecule to which a limited number of protein molecules are covalently attached; The gate itself is non-aggregated. As used herein, "soluble" means that the conjugates of the invention are soluble in water, or are water-based solutions such as suitable buffers, such as conjugate components individually. Soluble means soluble in a buffer.

In the conjugates of the invention, the antibody component can be derived from the antibody, and the antibody in its cell-bound form is a unique tumor-specific antigen such as B.
-Serves as an antibody that binds to cell lymphoma. Preferably, the antibody component is an anti-idiotype antibody, particularly a monoclonal essential image-type antibody (Ab2β), ie, the antibody component is an antigen-binding structure on an immune antibody (Ab1) which is complementary to the antigen. It is reactive. Such antibodies represent a mirror image of the antibody's conformation and can be used as antigen surrogates. Thus, a conjugate of the invention comprising an antibody of the essential image anti-idiotype will inhibit the binding of immune antibodies to target cells in vivo, and anti-anti-in vivo.
An idiotype antibody (Ab3) can be elicited, which antibody (Ab3) is specific for the antigen and has the same reactivity pattern as Ab1. The anti-idiotype may be from a mammalian, eg, murine, origin.

Anti-idiotypes suitable for the purposes of the invention are, for example, antibodies which resemble a viral, fungal, parasitic, bacterial or mammalian, especially human, antigen. The advantages of monoclonal anti-idiotypes as viral vaccines include the escape of potentially pathogenic virus-derived antigens, as well as the escape of genomic material from attenuated viruses that can revert to wild type or transform host cells. Is included. Suitable viral pathogens include, for example, Sendai virus, herpes virus, such as cytomegalovirus (CMV), human immunodeficiency virus (HI).
V), polio, influenza A, SV40, retroviruses, and virus-derived antigens such as hepatitis B.
Surface antigen (HBsAg) is included. It has been shown that: Anti-idiotypic antibodies are able to mimic essential bacterial or parasitic antigens and are associated with serious or lethal infections such as Streptococcus pneumoniae, E. coli, Pseudomonas aeruginosa, Infection with Neisseria meningitidis, Trypanosoma or Schistosoma mansoni can be induced.

The conjugates according to the invention are preferred as vaccines, eg the conjugates are human lymphocyte antigen (HLA; eg European patent application EP-A-049.
8767) comprising an anti-idiotypic component which mimics an epitope with respect to 8767), preferably as a tumor vaccine, eg the conjugate is a cancer embryo antigen (CE).
A) or a tumor-binding antigen (TAA), in particular comprising an anti-idiotypic component which mimics an epitope with respect to melanoma-binding antigen (note, eg S. Muller, H. Keller, D. Anderson, ibid., P343-351). Become. Melanoma-binding antigens are, for example, p97 (eg M. Kahn et al., Cancer
Res. 49, 3157-3162 (1982) and high molecular weight-melanoma binding antigen (HMW-MAA) (eg European patent application EP-A-0428485 and international application WO89 /
11296). In a particularly preferred embodiment of the invention, the antibody component of the conjugate is eg MAb76.
3.74 (P. Gearcominie, J. Immucol. 135, 696
(1985)), the HMA
-Essential image of the determinants recognized by the monoclonal antibody on MAA. An example of such a particularly preferred conjugate is the mouse anti-idiotype monoclonal antibody MK2-23 (European patent application EP-A-0428485, which is incorporated herein by reference in its number) as the antibody component. Or an antibody derivable therefrom, eg an isotype switch variant thereof,
Or a chimeric antibody such as MK2-CHγ1 (International Patent Application WO 93/19180, which is incorporated herein by reference to that number). The antibody MK2-CHγ1 is
Light chain human constant region kappa (κ), heavy chain human constant region γ and mouse anti-idiotype monoclonal antibody MK
It comprises a variable part derived from -23. Antibody MK2-
The amino acid sequences of the variable domains of the CHγ1 heavy and light chains are set forth in SEQ ID NO: 1 and SEQ ID NO: 3, respectively. MK2-CHγ1 is a hybridoma cell line MK
-CHγ1-6, which was deposited with the European Collection of Animal Cell Culture (ECACC) (Patron Gunung, Salisbury, Witz SP40JG, UK) on March 6, 1992 under deposit number 92030462. It was

The antibody component of the preferred conjugates of the present invention comprises a heavy chain variable domain comprising a polypeptide of the amino acid sequence set forth in SEQ ID NO: 1, or a polypeptide of the amino acid set forth in SEQ ID NO: 2. Become. From the understanding of the disclosure it will be clear to the person skilled in the art that: instead of a mammal, for example a mouse,
Monoclonal antibodies, or chimeric monoclonal antibodies, wherein the variable and constant regions are from different species, fragments of such antibodies, eg F
The (ab ') ₂ fragment, or, if appropriate, the reshaped (humanized) antibody can be used to form the conjugate of the invention.

The antibody may be of any immunoglobulin class / subclass, preferably class IgG. As a carrier protein component, the conjugates of the invention may be any protein useful in humans, such as a non-toxic, non-pyrogenic, water-soluble, pharmaceutically acceptable carrier protein, polypeptide or oligopeptide, preferably exposed. Such a protein molecule having a selected amino group,
For example, it may include a mutant diphtheria toxin lacking a toxin molecule (Uchida et al., J. Biol. Chem. 218, 3838-3844 (1973). Polymorphic HLA class I and classes.
Any protein molecule containing a hyperimmune promiscuous T-cell epitope that will bind extensively to the II gene product is suitable, eg a microbial, especially bacterial protein, polypeptide or oligopeptide.

Bacterial toxins such as tetanus elements (eg B. Bi
zzini in Bacterial Vaccines, Academic, 198
4: Tetanus, pp. 38-68), a carrier protein component which is particularly preferred, which protein component lacks toxin activity, but in particular retains antigenicity against the toxin, eg strong immunogenicity. Such non-toxic carrier protein components can be obtained, for example, by detoxification of the toxin in the case of tetanus toxin, or by mutation in the case of diphtheria toxin.

Detoxification can be performed chemically according to methods known to those skilled in the art, for example by treating crude or purified toxin under formaldehyde (formalin) under conditions known to produce toxoids. . For example, the toxin can be toxoidized by adding formalin to the toxin solution and then incubating at about 35 ° to about 37 ° C for about 1 month. Toxoid is
It should meet all the requirements stated by the World Health Organization for toxoids for human use. The preferred carrier protein for forming the conjugates of the invention is tetanus toxoid.

The diphtheria toxin is RK Homes, Infect.I.
It can be obtained from the culture supernatant of Corynebacterium diphtheriae according to the method disclosed by mmun. 12, 1392 (1975). Preferred carrier proteins for forming the conjugates of the invention are non-toxic mutant diphtheria toxins, especially mutant diphtheria toxin CRM1.
97. CRM197 is a non-toxic protein,
This protein immunologically cross-reacts with diphtheria toxin and C.I. It can be obtained from the culture supernatant of diphtheria C7 (RK Homes, supra; US Pat. No. 4,92).
5,792, which are incorporated herein by reference). The CRM197 protein is composed of fragment B having the same molecular weight as the diphtheria toxin and identical to those of the toxin in terms of their function and structure and fragment A which is non-toxic and differs by one amino acid from the original fragment. To be done.

In the conjugates of the present invention, at least 1, preferably about 1 to about 5, ie about 1,
About 2, about 3, about 4 or about 5 carrier protein molecules,
Covalently bound to one antibody molecule. Carrier-antibody conjugates, where the molar ratio is about 3: 1 are preferred. Said ratio of carrier protein to antibody of about 1 to 5 carrier protein molecules per carrier should be understood as an average ratio.

The average number of carrier protein molecules bound to one antibody molecule can be determined by methods known to those skilled in the art, for example by measuring the molecular weight of the conjugate. From the molecular weight of the conjugate, the average number of carrier proteins can be calculated as follows: the molecular weight measured for the conjugate and the molecular weight of the antibody component (eg about 150 for IgG by gel filtration or free flowing fractionation). The difference from kilodaltons (kDa) is divided by the molecular weight of the carrier protein (eg about 50 kDa for CMR197) to give the average number of carrier protein molecules.

For the conjugate comprising IgG and CRM197, the preferred molecular weight is about 200 kDa.
It is about 400 kDa, especially about 300 kDa. The carrier protein is covalently attached to the antibody via a bond containing selected functional groups of the carrier. Typically, conjugations involving antibody amino groups will not be considered suitable for the purposes of the invention. Conveniently, the carrier protein molecule is linked, for example, via a link containing the rare carbohydrate structure of the antibody, a link containing the functional group of the C-terminal amino acid, especially a carboxy group, a disulfide link, or most preferably a thioether link. It may be covalently attached to the antibody molecule.

Preferentially, the antibody reacts in pure form. Purification of antibodies is accomplished by methods known to those skilled in the art, eg by one or more chromatographic steps, eg affinity chromatography, in particular chromatography by protein A or protein G affinity medium, ion exchange chromatography, hydrophobic interactions. This can be accomplished by chromatography or gel filtration, or a procedure involving a precipitation step, such as ammonium sulfate precipitation.

If appropriate, the particular antibody may be subject to isotype switching, eg to facilitate purification. Such isotype switch variants of a particular antibody can be produced by conventional methods; eg, Igs having structurally identical variable regions but different heavy chain constant regions.
Matched sets of hybridoma Ig heavy chain class switch variants (which often occur spontaneously at frequencies of about 10 ⁻⁴ to about 10 ^{−10 in} growth medium).
Can be obtained by isolation of For example, mouse antibody M
Instead of using K2-23, which is a G1 type immunoglobulin, it may be advantageous to use a G2a or G2b type switch variant; because
Contrary to the IgG1 antibody, this variant can be affinity purified with protein A or protein G affinity media. To isolate such IgG1-IG2a or IG1-IgG2b heavy chain isotype switch variants of hybridoma MK-23, continuous sublining is used in combination with IgG2a- or IgG2b-specific ELISA.

In one aspect, anti-idiotypic antibodies, such as mutated non-toxic diphtheria toxin,
For example, CRM197, or HMW conjugated to tetanus toxin
-A mouse IgG2b anti-idiotype antibody having an internal image of the epitope of MAA.
In a preferred embodiment of the invention, the conjugate is CMR197 or tetanus toxin as a carrier protein component and an antibody or MK2-23 designated MK2-CHγ1.
The antibody component comprises the variable region of the L chain and / or the H chain of the antibody.

IgG-2-, which comprises CRM197 or tetanus toxin and a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 2 and a light chain variable domain having the amino acid sequence set forth in SEQ ID NO: 3 as protein components. More preferred is a conjugate comprising a type of antibody component. Anti-idiotype anti-HMW-MAA mouse I characterized by an H chain variable domain having the amino acid sequence set forth in SEQ ID NO: 2 and an L chain variable domain having the amino acid sequence set forth in SEQ ID NO: 3
Particularly preferred is a conjugate comprising the g2b: kappa monoclonal antibody, as well as the mutated diphtheria toxin CMR197.

The coupling reaction is carried out according to methods known to those skilled in the art in such a way that aggregation is avoided, the antibody is not denatured and the non-covalent bonds within the antibody are essentially retained. The chemistry should be chosen to minimize the formation of undesired side reaction products (eg homoconjugates) while maximizing the yield of the functionally active conjugate. Moreover, the conjugate should be stable and well defined with respect to the actual binding function. The method using a heterobifunctional coupling agent is preferred. Heterobifunctional crosslinkers have two selective reactive groups that can be used to attach proteins in a stepwise and specific manner, so that undesired side reactions occur, for example homoprotein (homopr).
traits) are preferably avoided. The number of carrier protein molecules bound to each antibody molecule can be controlled by changing the molar introduction ratio of the reaction in the opposite direction. The binding of the carrier protein will be across the heavy and light chain constant regions of the antibody.

Links containing the carbohydrate structure of an antibody can be prepared by methods known to those skilled in the art (Note, eg DJ Ochansee, RH Cures, J. Immunological Methods 99, 153-161 (198).
According to 7)), for example mild oxidations and groups of carbohydrates can be formed by subsequent modification. For example, such conjugates of the invention can be formed with a coupling agent having a hydrazide moiety.

Coupling involving the C-terminal carboxy group of an antibody can be accomplished by conventional methods, eg US Pat. No. 5,234,820.
The method disclosed in No. Coupling of a carrier protein to an antibody is accomplished by reacting the carrier protein with a suitable heterobifunctional coupling agent, such as tetanus toxoid or CR.
It may include activation of M197. Suitable heterobifunctional coupling agents have two different reactive groups, one of which is capable of reacting with the functional groups of the carrier protein, especially the amino groups of the carrier protein. Preferably, the reactive group in the coupling agent is the N-hydroxysuccinimide ester moiety.

Carrier protein molecules to antibody molecules via disulfide or thioether bonds require at least one accessible thiol (sulfhydryl) group in the carrier. Directional site-selective conjugation to an antibody can be performed at the hinge of the antibody and through the reaction of an interchain cysteine with an activated carrier, and the thiol group released by reduction of the interchain disulfide bond is removed. To use.

Such reduction is carried out under mild conditions, for example, using a suitable reducing agent, and under conditions without significant denaturation of the protein. A suitable reducing agent is, for example, if used in sufficient molar excess (above the antibody):
Agents that help achieve disulfide exchange, such as dithiols, such as dithiothreitol (DTT).
Depending on their sensitivity to disulfide exchange, disulfide bonds within antibodies can be divided into three categories: shielded bonds (usually intrachain), assisted bonds and unassisted bonds. (Both are normal chains)
(GT Stephenson et al. In Protein Engineering of
Antibody Molecules for Prophylactic and Therapeuti
c Applications in Man, Editor M. Clark, Academic Title, Bram Court Nottingham, UK, 1993, 127-141). The latter type is most preferred for the preparation of the conjugates of the invention. Preparation of the conjugates of the invention containing one or more disulfide or thioether bonds requires that at least one disulfide bond within the antibody be reduced.

For example, a heterobifunctional lysine / cysteine crosslinker, such as sulfosuccinimidyl-4- (p-maleimidphenyl) butyrate (sulfo-SMPB), is used to attach the carrier protein to the antibody. The carrier protein can react with such cross-linking agents via accessible amino acid residues having a reactive primary amine, such as a surface exposed lysine residue, thus enabling activation of a reactive thiol. Forming a carrier protein, this activated carrier protein having a surface-exposed cysteine-reactive moiety is then exposed to an antibody partially reduced, for example with dithiothreitol. Exposure to some (but not all) of the disulfide bonds present in the. The conjugate is purified and analyzed according to methods known to those of skill in the art.

Suitable heterobifunctional coupling agents are commercially available or can be prepared according to methods known to those skilled in the art, and include, for example, amine reactive groups such as N-hydroxysuccinimide esters, and sulfhydryl reactive groups, Crosslinking agents containing N- (3- [2'-pyridyldithio] propionoxy) -succinimide (SPDP) for coupling via, for example, a haloacetyl group, a maleimide group or a reactive disulfide such as a disulfide bond are included. Suitable agents for the alkylation of antibody thiol groups resulting in the formation of thioether bonds include N-succinimidyl bromoacetate or maleimido-N-hydroxysuccinimide ester type crosslinkers. Maleimide-N-hydroxy-succinimide ester type crosslinkers are aromatic,
For example, m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfosuccinimidyl-4- (p-maleimidophenyl) butyrate (S-
SMPB or sulfo-SMPB) and m-maleimidobenzoyl sulfosuccinimide ester (S-MBS),
Or an aliphatic compound such as N-γ-maleimidobutyryloxysuccinimide ester (GMBS) (DE Meller et al., J. Im.
munological Methods 121, 129-141 (1989)). Reaction of such a coupling agent with a carrier protein produces an activated carrier protein, which is capable of reacting with an antibody molecule due to the introduction of reactive groups that are essentially non-reactive with the carrier protein. Prior to reacting the antibody with the activated carrier protein, excess coupling agent should be removed, for example by gel filtration or dialysis.

Most preferred is the attachment of the conjugate component via a thioether bond. Preferred conjugates of the invention are essential image-type anti-idiotype monoclonal antibodies, particularly anti-idiotype antibodies, which are the essential image of the determinants recognized by the monoclonal antibody on HMW-MAA and CRM197. A soluble, non-aggregated conjugate with a protein or tetanus toxin, wherein the conjugate is characterized in that one antibody molecule has an average of 1 to 5, preferably an average of 3 of said carrier protein. The protein is site-selective and is covalently attached to the constant region of the antibody via a thioether bond. Particularly preferred is such a conjugate obtained by use of sulfo-SMPB as a heterobifunctional coupling agent.

Immune response (Ab3 response), eg anti-HM
Induction of a W-MAA immune response, followed by vaccination with an anti-idiotype conjugate of the invention is described, for example, in E.
It can be analyzed according to methods known to those skilled in the art using LISA, especially "live-cell" ELISA procedures, eg by measuring anti-melanoma cell antibody titers. HMW-
The MAA positive human melanoma cell line, eg A375met, and the HMW-MAA negative melanoma cell line, eg S-7, can be selected as cell substrates. Test cells with test Cera (Ab3), or positive and negative (non-HMW
-MAA-reactive) exposure to a dilution of control monoclonal antibody. Cell-bound Ab3 antibody can be measured by a suitable enzyme-bound antibody conjugate, eg, anti-IgG peroxidase conjugate.

The present invention further provides a method for producing the conjugates of the present invention, such that an average of 1 to 5 carrier protein molecules are site-selectively covalently attached to one antibody molecule. Reacting the antibody with a carrier protein under different conditions and then isolating the conjugate of the invention. The product obtained can be purified to give conjugates with different proportions of antibody and carrier protein. If desired, the conjugates of the invention are subjected to further analysis, eg for the purpose of “quality control”.
Such an analysis is eg according to methods generally known to those skilled in the art, eg immunoassays, eg assays including immunocytochemistry or flow cytometry, methods aimed at measuring binding kinetics, eg measuring association or dissociation rates. Analysis, for example, biospecific interaction analysis (U. Yonson et al., Bio Techniques 11, 620-627 (1991)) and the like, and comparison with non-conjugated antibody. Preferred are methods of producing the preferred conjugates of the invention.

For the preparation of the conjugates of the invention in which a carrier protein, eg CRM197, is linked to the antibody via a thioether bond, the reaction is carried out in a two-step procedure. Carrier protein containing no free cysteine,
First, it is reacted with a maleinimide-N-hydroxysuccinimide ester type crosslinking agent. This will result in the addition of cysteine-reactive maleimide groups to the surface of the carrier protein and will not result in cross-linking of the carrier protein molecule. After removal of excess cross-linking agent, eg by gel filtration or dialysis, the activated carrier protein together with the antibody, eg the antibody MK-CHγ1 or MK2-
Incubation with an antibody containing the Ig variable region of 23 (which was previously partially reduced, eg with dithiothreitol), forms the free reactive thiol without disrupting the entire protein structure. The reduced form of the antibody is separated from the unreacted reducing agent. The coupling reaction forms a covalent bond between the activated carrier protein and the reduced carrier,
It results in the formation of antibody conjugates. In the coupling reaction, the molar excess of carrier protein is chosen such that the desired number of carrier proteins will bind to one antibody molecule. For example, to generate a conjugate in which about 3 CRM197 molecules are bound to one mouse IgG molecule, eg, MK2-23, 5 activated CRM197 molecules are used.
The reaction is carried out in a 2-fold molar excess. Preferably, the conjugate according to the invention is purified by chromatographic methods and optionally sterilized, for example by filtration under sterile conditions. Conjugates of the invention may be used in vitro (absent) or live for antibodies directed against a particular antigen, eg, for diagnostic purposes, eg to which the anti-idiotypic antibodies of the conjugates of the invention are very similar. For quantitative and qualitative measurements in the body, as agonists / andagonists in the study of antigen / antibody binding, or for therapeutic purposes,
It can be used, for example, as a vaccine to induce immunity to viral parasites or bacterial pathogens, and for the control, prevention, (adjuvant) treatment and monitoring of tumors such as melanoma.

The present invention also relates to the use of a conjugate of the invention as a vaccine and a method of vaccination of a mammal, in particular a human, which comprises administering a conjugate of the invention. Such a method also relates to a method of eliciting an anti-tumor response in a mammal comprising administering a conjugate of the invention comprising a suitable antibody.

The conjugates of the present invention are useful for many therapeutic and diagnostic purposes. For example, instead of HMW-MAA itself as the antibody component, HMW-M
The advantages of using such a conjugate comprising an anti-idiotypic monoclonal antibody that is an essential image of a determinant recognized by a monoclonal antibody on AA are, for example: High immunogenicity; -Stimulation of β-cell clones that are normally unresponsive to HMW-MAA by conjugation; -Potential for development of anti-HMW-MAA T-cell responses; -Relevant to application of HMW-MAA Elimination of possible side effects; Easy production of large amounts of conjugate.

Due to their immunomodulatory function within the idiotype-anti-idiotype reaction network, the conjugates according to the invention are eg tumor-binding antigens such as HM.
It can be used to modulate the immune response to W-MAA. Modulation is specific and can be governed by the choice of monoclonal antibody used to produce anti-idiotype antibodies, ie, the specificity of immunizing MAbs (Abl) and the isotype of anti-idiotype MAbs; Conjugates of anti-idiotype antibodies would then have different immunomodulatory effects. This is because different isotypes of anti-idiotype antibodies have different immunomodulatory effects. The conjugates of the invention have immunomodulatory functions, such as the stimulation and immunity of Homoln.

The conjugates according to the invention are therefore agents which are useful, for example, for the control, treatment or adjuvant treatment of tumors, for example melanoma, ie the conjugates of the invention lead for example to tumor regression and / or Alternatively, it can be used successfully to prevent tumor recurrence. The anti-idiotypic monoclonal antibodies that make up the conjugates of the invention can be "tailor-made" to the specific determinants of the TAA structure by appropriate selection of antibody immunization. Such anti-idiotypic antibody-conjugates of the invention are also useful in preventing tumors by eliciting immunity to the TAA determinants recognized by the immunizing monoclonal antibody.

The anti-idiotype antibody-based conjugates of the present invention are useful as vaccines for concomitant treatment of cancer. For example, Ab3 that reacts with HMW-MAA
Conjugates of the invention that induce antibodies, for example, after surgery for the primary lesion, leave the risk to those with metastatic disease,
It is useful as a vaccine for concomitant treatment of malignant melanoma in patients with early stage melanoma.

The invention also relates to a pharmaceutical composition comprising a conjugate according to the invention. The pharmaceutical composition comprises a therapeutically effective amount of the conjugate, for example with or in admixture with a pharmaceutically acceptable inorganic or organic, solid or liquid. Further preferred is a pharmaceutical composition comprising an adjuvant, ie a drug which further increases the immune response. Possible adjuvants are Freud's complete adjuvant (emulsion of mineral oil, water and microbial extract), incomplete Freud's adjuvant (emulsion of water and oil only),
Mineral gels such as aluminum hydroxide gel, surface active substances such as lysolentin, polyanions, peptides and BCG (Bacillus Calmette-Guerin). MF5 as a conjugate and adjuvant of the invention
9 (International Patent Application WO 90/14837) and optionally N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2- [1,2-dipalmitoyl-sn-glycero-3- (hydroxyphospho). Riloxy] ethylamide (MTP-PE: International Patent Application WO9
0/14837) is particularly preferred.

Pharmaceutical compositions for parenteral administration are preferred. Compositions for intramuscular, subcutaneous or intravenous administration are, for example, isotonic aqueous solutions or suspensions, optionally prepared just prior to use, from lyophilized or concentrated preparations. The pharmaceutical composition may be sterilized and may contain an adjuvant, eg for storage, stabilization or solubilization of the components, salts, buffers, and / or viscosity adjusting compounds for osmolality, such as sodium carboxycellulose, dextran, polyvinyl. It may contain pyrrolidone or gelatin. They can be prepared by conventional mixing, dissolving or lyophilizing by methods known to those skilled in the art and contain from about 0.01% to about 50% active ingredient. Injectable compositions are processed, filled into ampoules or vials and aseptically sealed according to known methods. For example, because of its solubility in aqueous solutions,
The conjugates of the invention are formulated as a "two vial system" with the adjuvants described above, eg, MF59-0.

Depo with an adjuvant
Pharmaceutical compositions comprising a conjugate of the invention suitable under intramuscular administration under formulation t) are preferred. Pharmaceutical compositions comprising the conjugates of the invention suitable for mucosal application are also preferred (HF Starts et al., Current Opinion in I
mmunology 6, 572-583 (1994)), or swallowed stabilized pharmaceutical compositions for oral immunization are preferred.

The specific form and dose of administration will be selected by a physician considering the individual circumstances of the patient, the condition and the disease to be treated. The therapeutic dose for mammals is 1 body weight depending on the condition of the patient and the mode of administration.
About 0.1 μg to 100 μg per kg, preferably about 0.1 μg.
1 μg to about 10 μg. Most preferably, the dose to mammals is from about 0.1 μg / kg to about 1 μg / kg of body weight.
g.

The conjugates of the invention may also include pathogens such as those mentioned above, or TAAs such as HMA-MAA.
It can be used for the qualitative and quantitative measurement of antibodies specific for. This is particularly useful for monitoring the success of processing with the conjugates of the invention. For example, the conjugates of the invention can be used in assays based on binding interactions, especially between the idiotope of Ab1 or Ab3 and the "corresponding" anti-idiotype monoclonal antibody (Ab2) of the conjugate of the invention. Examples of such analyzes include radioprecipitation, immunoprecipitation, latex agglutination,
And hemagglutination immunoassay.

The present invention is also directed to specific antigens that are coagulated by the conjugates of the present invention, and optionally other polyclonal or monoclonal antibodies, and / or antibody components of the conjugates of the present invention comprising adjuvants. And a kit for qualitative and quantitative measurement of specific antibody (Ab1 or Ab3). The invention particularly relates to embodiments, such as conjugates, processes for their preparation and formulations comprising such conjugates as described in the examples.

The present invention is illustrated by the following examples, but the present invention is not limited thereto.

[0049]

【Example】

Example 1: Conjugation of CRM197 to antibody MK2-CHγ1 1.1 CRM1 with sulfosuccinimidyl 4- (p-maleimidophenyl) butyrate (S-SMPB)
Activation of 97 PBS (25 mM sodium phosphate buffer pH 8.
245 μl of CR dissolved in 150 mM NaCl)
M197 (52 mg / ml) was added to 450 μl of PBS / ED.
TA (25 mM sodium phosphate buffer pH 8.
2, 150 mM NaCl, 1 mM EDTA).
A fresh solution of S-SMPB is made by adding 9 mg S-SMPB to 1 ml DMSO (dimethyl sulfoxide). Then 200 μl of this solution was added to CMR1.
Drop 97. The reaction mixture is then incubated on ice for 2 hours.

1.2 Unreacted S-SMPB to Activated C
Separation of MR197 Equipped with HR10 / 10 high speed desalting column (Pharmacia) and in PBS / EDTA (pH 7.2) at room temperature for 3
Operate at ml / min. The column is then filled with activated CRM197 solution. Protein elution is monitored by UV absorption at 280 nm. Activated CMR19 in equilibration buffer
Collect the first appearing peak containing 7 (about 2.2.
ml). Transfer this solution to ice.

1.3 Reduction of disulfide in antibody MK2-CHγ1 to generate reactive thiols 1 ml of MK2-CHγ1 (12 mg / m 2) dissolved in PBS
l; International application WO89 / 11296) using 2M Trizma Base (Sigma) 20 μl and pH
Adjust to 8.0. Add 50 μl of fresh 1M aqueous DDT (dithiothreitol) (final concentration of 50 mM).
The mixture is then incubated in a water bath at 37 ° C for 30 minutes.

1.4 Separation of reduced MK2-CHγ1 from unreacted DTT Equilibrate HR10 / 10 fast desalting column and then PBS
/ Operate at room temperature at 3 ml / min in EDTA (pH 7.2).
The column is filled with the reduced antibody solution. Protein elution is monitored by UV absorption at 280 nm. Collect the first appearing peak containing the reduced antibody in equilibration buffer (about 2.2 ml). Transfer this solution to ice.

1.5 Activated C for reduced antibody
Coupling of MR197 The reduced antibody solution is continuously stirred at room temperature during which activated CRM197 is added dropwise. The reaction mixture is then returned to ice and allowed to react overnight. The final solution contains a small amount of precipitate, which is removed by centrifugation and filtration through a 0.22 μm membrane filter.

Example 2: Analysis of the final conjugate of CMR197 and antibody 2.1 Gel filtration Analysis of the conjugate by Superrose 6 (Pharmacia) gel filtration chromatography showed an average molecular weight of 3
00 kDa peak (peak I) followed by unreacted CRM19
7 is shown. A molecular weight of 300 kDa corresponds to three CRM197 (Mr (molecular weight)) 47 kDa bound to each antibody.

2.2 SDS-PAGE In the presence or absence of a reducing agent (DTT), gel electrophoresis shows large complexes with a molecular weight (Mr) above 150 kDa. No evidence of unreacted antibody, but small amount of CRM19
7 is observed.

2.3 Free Flow Fracti
onation) This method separates molecules based on their diffusion coefficient and is an ideal complement to gel filtration because it can confirm the molecular weight (Mr) and detect large aggregates. For the Peak I eluate obtained in 2.1, this method shows the absence of large aggregates and an average molecular weight (Mr) of 3
This shows 00 kDa.

2.4 ELISA analysis ELISA for testing the binding capacity of the conjugate
Analysis shows that the complex can bind to an antibody that is specifically recognized by the unconjugated antibody MK2-CHγ1. Taken together, the conjugation reaction is found to have an average of 3 CRM197 bound to each MK2-CHγ1 molecule. Adhesions must be present across the H and L chains of the antibody; because the complex is reduced SDS-PAG
This is because it cannot be divided by E.

Example 3: CMR1 against antibody MK2-23
97 conjugation Mouse antibody MK2-23 (EP-A-042848)
The joining of CMR197 to 5) is carried out analogously to the procedure described in Example 1. In the obtained conjugate,
Three CRM197 molecules are bound to one antibody.

Example 4 Experimental Vaccination of Mice with the Conjugates of Examples 1 and 3 Conjugates of 600 μg with pyrogen-free saline.
Dilute to a concentration of antibody / ml. Equal amount of conjugate
Freund's complete supplement (FCA), Freund's incomplete supplement (IFA), or 100 μg of MTP-PE (MF
-100) or does not contain (MF
59-0) Mix with any of the MF59 supplements (Biocin). Balb / c mice are injected intramuscularly on days 0, 14 and 28 with conjugates corresponding to 15 μg of antibody. The titer to high molecular weight-melanoma binding antigen is measured by HLISA system, which is adherent cells A375 cells expressing HMW-MAA as an antigen (American type collection A
TCC CRL 1619) is used. The antibody titer is
It is obtained by reference to the standard curve of the original antibody used to obtain the Ab1 (MAb 763.74) mouse anti-idiotype, and subsequently the chimeric antibody.

Administration of a specific conjugate of chimeric antibody: CRM197 (1: 3, Example 1) gave satisfactory HW
Results in M-MAA specific antibody titers. Mouse antibody M
The conjugate obtained by reaction of K2-23 with CMR197 (Example 3) also showed strong anti-activity in mice.
Generate an HMW-MAA antibody response. To truly substantiate the surprising finding that the excellent immunogenicity of the conjugate resides within the soluble molecule, contrary to a small amount of one antigenically potent aggregate, the chimeric antibody: CMR197 (1: 3) Further purification by Superose 6 chromatography removes high molecular weight (> 6 million) components and enriches for an asserted 300 kDa molecular weight protein. Immunogenicity was found to reside in the non-aggregated 300 kDa fraction, representing a 1: 3 antibody: CRM197 conjugate.

Example 5: Pharmaceutical preparation for parenteral administration Chimeric anti-idiotype monoclonal antibody MK-
CHγ1 (Example 1) or mouse monoclonal antibody MK
10 mg conjugate comprising 2-23 (Example 2) is dissolved in 50 ml PBS. The solution is passed through a bactericidal filter, the filtrate is divided into 10 equal parts and the ampoules are filled under aseptic conditions. The ampoule is preferably kept in the cold, for example at 4 ° C. These pharmaceutical formulations are suitable for injectable use and as such or MF59 (Biocin) and optionally Muramyl peptide MTP-PE
It is applied in combination with a pharmaceutical preparation containing

Example 6: Conjugation of tetanus toxoid (II) to IgG2b-antibody The antibody referred to as IG2b antibody was prepared by the method of antibody 2-by the sequential sublining method.
Antibody IgG2b, which is an IgG switch variant obtained from a 23-producing hybridoma, contains an H chain variable domain having the amino acid sequence set forth in SEQ ID NO: 3 and an L chain variable domain having the amino acid sequence set forth in SEQ ID NO: 3. It consists of 6.1 Activity of TT with Sulfosuccinimidyl 4- (p-maleimidophenyl) butyrate (S-SMPM) A fresh solution of S-SMPM was added to 1 ml DMSO (dimethyl sulfoxide) to 11.3 mg S. -Prepared by adding SMPB. Then 38 μl of this solution was added to TBS (25 mM sodium phosphate buffer, pH
8.2, 150 mM NaCl) to TT (5.5 mg / ml)
Is dripped. The reaction mixture was incubated on ice for 20 minutes.

6.2 Unreacted S-SMPB to active TT
Equilibration of a HR10 / 10 high speed desalting column (Pharmacia) followed by PBS / EDTA (25 mM sodium phosphate buffer, 150 mM NaCl, 1 mM EDT)
A) Operate at room temperature. The column is then filled with an aliquot of active TT solution. 280 nm for protein elution
Monitor by UV absorption. Collect the appearing peaks containing active TT in equilibration buffer (about 2.2 ml) and transfer this solution to ice.

6.3 Antibody 0.72 ml of IgG2b (6.6 ml / l) dissolved in PBS which reduces disulfide in IgG2b to produce reactive thiols is treated with 50 mM E.
React with 15 μl of fresh 0.5 M aqueous DDT (dithiothreitol) containing DTA. The mixture is then incubated in a water bath at 37 ° C for 15 minutes.

6.4 Ig reduced from unreacted DTT
Separation of G2b Equilibrate HR10 / 10 fast desalting column and PBS
/ Operate at room temperature at 2 ml / min in EDTA pH 7.2. Then the column is filled with the reduced antibody solution. Protein elution is monitored by UV absorption at 280 nm. The first appearing peak containing the reduced antibody in equilibration buffer is collected (about 2.2 ml). Transfer the solution to ice.

6.5 Active TT against reduced antibody
The solution of active TT (9 ml) is continuously stirred at room temperature, during which the reduced antibody (25 ml) is added dropwise. The reaction mixture is then put back on ice and reacted overnight. P for the final solution
Dialysed for 72 hours against BS, pH 7.2 and 0.22μ
Filter through an m-membrane filter.

6.6 SDS-PAGE In the presence of a reducing agent (β-mercaptoethanol), denaturing gel electrophoresis is a large complex having a molecular weight (Mr) of 150 kDa or more.

6.7 Deposit Data Hybridoma cell line producing the antibody MK2-CHγ1 was cloned into a European European Collection of Animal Cell Culture (ECACC) (Porton Down, Salisbury Wilts SP40JG, UK).
On March 6, 1992 under deposit number 92030642.

[0069]

[Sequence list]

(1) General information: (i) Applicant: (A) Name: Ciba-Geigy AG (B) City: Kleibach Street 141 (C) City: Basel (E) Country: Switzerland (F) Postal Code (ZIP) : 4002 (G) Telephone: +41 61 69 11 11 (H) Telefax: +41 61 696 797
6 (I) Telex: 962 991 (ii) Title of the invention: Antibody conjugate with improved properties (iii) Number of sequences: 3 (iv) Computer reading method: (A) Media type: Floppy disk (B) ) Computer: IBM PC compatible (C) Operating system: PC-DOS / MS-DOS (D) Software: Patent-in-release # 1.0,
Version # 1.30 (EPO)

(2) Information for SEQ ID NO: 1: (i) Sequence characteristics: (A) Length: 117 amino acids (B) Type: amino acids (C) Number of chains: 1 chain (D) Topology : Unknown (ii) Sequence type: Protein (xi) Sequence: SEQ ID NO: 1 Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Arg 1 5 10 15 Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly 20 25 30 Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Asp Ser Ser Asn Ile Tyr Tyr Ala Asp Thr 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr 65 70 75 Leu Phe Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met 80 85 90 Tyr Tyr Cys Ala Arg Ser Asn Tyr Val Gly Tyr His Val Arg Trp 95 100 105 Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr 110 115

(2) Information for SEQ ID NO: 2: (i) Sequence characteristics: (A) Length: 120 amino acids (B) Type: Amino acid (C) Number of chains: Single chain (D) Topology : Unknown (ii) Sequence type: Protein (xi) Sequence: SEQ ID NO: 2 Asp Val Val Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Phe Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu 35 40 45 Glu Trp Val Ala Tyr Ile Ser Ser Asp Ser Ser Asn Ile Tyr Tyr 50 55 60 Ala Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro 65 70 75 Lys Asn Thr Leu Phe Leu Gln Met Thr Ser Leu Arg Ser Glu Asp 80 85 90 Thr Ala Met Tyr Tyr Cys Ala Arg Ser Asn Tyr Val Gly Tyr His 95 100 105 Val Arg Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr 110 115 120

(2) Information for SEQ ID NO: 3: (i) Sequence features: (A) Length: 108 amino acids (B) Type: Amino acid (C) Number of chains: Single chain (D) Topology : Unknown (ii) Sequence type: Protein (xi) Sequence: SEQ ID NO: 3 Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr 20 25 30 Gly Ser Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro 35 40 45 Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Glu Ser Gly Val 50 55 60 Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu 65 70 75 Asn Ile His Pro Val Glu Glu Asp Asp Ile Ala Met Tyr Phe Cys 80 85 90 Gln Gln Ser Arg Lys Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys 95 100 105 Leu Glu Ile

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Terence O'Reilly Switzerland, 4057, Akerstraße, Basel 45 (72) Inventor Gerd Pruschke, Federal Republic of Germany, 79249 Merzhausen, Am Schumberg 6

Claims (16)

[Claims] 1. A soluble, non-aggregating carrier protein-antibody conjugate which elicits an immune response in vivo, wherein one or more carrier proteins are site-specifically and constant to the constant region of one antibody molecule. The soluble, non-aggregated carrier protein-antibody conjugate, which is conjugated and bound. 2. The conjugate according to claim 1, wherein the carrier protein can be derived from a bacterial toxin. 3. The conjugate according to claim 2, wherein the carrier protein is selected from the group consisting of CRM197 or tetanus toxoid. 4. The conjugate according to any one of claims 1 to 3, wherein the bond between the carrier protein and the antibody comprises a thioether bond. 5. The antibody is an anti-idiotype antibody, particularly high molecular weight melanoma binding antigen (HMW-MA).
A) an anti-idiotypic carrier which is the essential image of the determinant recognized by the above monoclonal antibody,
The conjugate according to any one of claims 1 to 4. 6. An antibody comprising an L chain variable domain having the amino acid sequence set forth in SEQ ID NO: 3, or a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 or a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2. The conjugate according to claim 5, which comprises an H chain variable domain selected from the group consisting of: 7. A method according to claims 1 to 6 for use in a diagnostic or therapeutic method carried out on the human or animal body.
The conjugate according to any one of 1. 8. A conjugate according to claim 7 for use as a vaccine in active immunotherapy. 9. A conjugate according to any one of claims 1 to 6 for use in the manufacture of a pharmaceutical composition. 10. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 6, a pharmaceutically acceptable carrier and optionally an adjuvant. 11. A method for producing the conjugate according to any one of claims 1 to 6, under the condition that one or more carrier protein molecules are covalently bound to one antibody. Reacting the antibody with a carrier protein and then isolating the desired conjugate.
The method. 12. A method of eliciting an immune response in a mammal, particularly a human, which comprises administering a soluble, non-aggregating carrier protein-antibody conjugate.
Said method, wherein one or more carrier protein molecules are site-specifically and conjugated to the constant region of one antibody molecule. 13. A method for active immunotherapy of mammals, especially humans, comprising administering a conjugate according to any one of claims 1-6. 14. A method of eliciting an anti-tumor response in a mammal, especially a human in need thereof, comprising administering a conjugate according to any one of claims 1-6. 15. Antitumor response is tumor associated antigen (TAA)
15. The method of claim 14, which is specific for. 16. The method of claim 15, wherein the antitumor response is specific for high molecular weight-melanoma binding antigen.