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WO1990011779A1 - Heteroconjugates - Google Patents

Heteroconjugates Download PDF

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Publication number
WO1990011779A1
WO1990011779A1 PCT/GB1990/000476 GB9000476W WO9011779A1 WO 1990011779 A1 WO1990011779 A1 WO 1990011779A1 GB 9000476 W GB9000476 W GB 9000476W WO 9011779 A1 WO9011779 A1 WO 9011779A1
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WIPO (PCT)
Prior art keywords
antigen
antibody
tumour
patient
conjugate according
Prior art date
Application number
PCT/GB1990/000476
Other languages
French (fr)
Inventor
Peter Julius Lachmann
Original Assignee
Medical Research Council
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Filing date
Publication date
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Publication of WO1990011779A1 publication Critical patent/WO1990011779A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/44—Antibodies bound to carriers
    • 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
    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6056—Antibodies
    • 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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to novel targetting agents and to their use in the treatment of cancers. Radio- and chemotherapy are now well established as cancer treatments, as is surgery, for clearly defined tumours. However these techniques are less effective at destroying small lesions due to metastasis and, with surgery, there is always a risk of leaving behind small areas of cancerous tissue.
  • the present invention is particularly concerned with providing a means to clear up such small pockets of cancerous cells and is therefore considered mainly as an adjunct to the already established therapeutic and surgical techniques.
  • the invention aims to recruit aspects of the patient's own immune system and to target this against the tumour cells.
  • the present invention in one aspect provides a method for the treatment of the human or animal body comprising administering an effective, non-toxic amount of a targetting agent which is a conjugate of an antigen and an antibody, or fragments thereof, the antigen being selected such that the patient already has immunity to the antigen and the antibody being selected to bind specifically to the tumour cells.
  • a targetting agent which is a conjugate of an antigen and an antibody, or fragments thereof, the antigen being selected such that the patient already has immunity to the antigen and the antibody being selected to bind specifically to the tumour cells.
  • Figs, la & lb show the clone 3-PPD conjugate mediated lysis of C3 coated MC6A tumour cells in cytotoxicity assays.
  • Figs. 2a & 2b show the MM2-9B6-PPD conjugate mediated lysis of B16-F10 tumour cells in cytotoxicity assays.
  • Figs. 3a & 3b show the tumour cytostasis mediated by clone supernatant.
  • Figs. 4a & 4b show lymphokine assays.
  • the antigen may be any antigen to which the patient has previously been exposed, or any antigen which cross-reacts with lymphocytes in the patient's blood.
  • suitable antigens include those of the childhood illnesses such as measles, chickenpox or mumps and other antigens to which the population in general is likely to have induced immunity, for instance, tetanus, typhoid and tuberculosis. The latter is particularly relevant to the present invention as the vast majority of the population have been immunised using BCG vaccine against Mvcoplasma tuberculosis. the cross-reacting PPD (purified protein derivative) from M.
  • PPD purified protein derivative
  • tuberculosis may be used in the present method and is particularly preferred because of the very strong immune reaction which it elicits. Fragments of such antigens may also be used in the invention provided that they retain the epitope which will be recognised by the patient's immune system. In the case of PPD, which consists of a number of different polypeptide sub-units, any antigenic sub-unit or indeed any antigenic domain of one of the sub-units, may be used as the antigen.
  • the antibody used in the conjugate may be any antibody or fragment thereof which retains an antigen-binding site, such as the Fab' fragment, which will bind specifically to the tumour cells to be destroyed.
  • tumour specific antigens are now known and others will be discovered in the future; antibodies, whether polyclonal or monoclonal (the latter are preferred) against such antigens may be used in the present invention.
  • Certain tumours do not express tumour-specific antigens but these may be targetted using antibodies against neo-antigens in bound components of complement, such as C3 which are often found to accumulate on the surface of those tumour cells which activate the alternative complement pathway.
  • Antibodies against C3 or other complement components may also be used when the conjugates of the invention are to be administered after conventional monoclonal antibody treatment of cancers.
  • tumour specific antibodies results in the tumour cells becoming covered first in the anti-tumour antibodies and then in complement which binds to the anti-tumour antibodies.
  • anti-complement antibodies it is preferred that they are directed against neo-antigenic sites, i.e, sites which are only formed or exposed once complement binding has occurred, in order that binding to unbound complement is avoided.
  • the antibodies should preferably be derived from antibody-producing cells of the same species as the patient or should be modified to mask or remove any species-specific determinants other than those of the same species as the patient.
  • the antigen (or fragment thereof) and antibody (or fragment thereof) may be coupled by any conventional method for covalently binding such materials.
  • linking groups may be bound to the antigen and to the antibody and the linking groups are then coupled together.
  • one linking group is formed using the reagent SMCC (succinimidyl4-(N-maleimidomethyl)cyclohexane-
  • Coupling techniques should be selected so as to avoid impairing either the antigenicity of the antigen or the affinity of the antibody for the tumour cells; where necessary the product may be fractionated to obtain quantities of effective conjugate.
  • the present invention also provides a conjugate comprising an antigen and an antibody, or fragments thereof, covalently coupled via linking groups. Processes for coupling the antigen and antibody to form such a conjugate form a further aspect of the invention.
  • the invention further provides such conjugates for use in a therapeutic method for the treatment of the human or animal body and the use of such conjugates in the manufacture of a medicament for use in the treatment of cancer.
  • the conjugates of the invention may be administered as such but are preferably administered as pharmaceutical compositions also comprising a pharmaceutically acceptable diluent or carrier.
  • Typical diluents and carriers include water for injection and other injection media.
  • compositions may be presented in unit or multi-dosage form.
  • the compositions may be presented in ready-to-use form or as a concentrate or dry powder for reconstitution, e.g. using water for injection, prior to use.
  • the compositions will generally be sterile and pyrogen free.
  • the compositions may also comprise accessory ingredients such as antibacterial and antifungal agents, buffers, salts, agents to adjust the tonicity of the composition, anti-oxidants, wetting agents and suspending agents to improve the solubilisation or suspension of the conjugates and analgesics or anaesthetics to reduce pain at the injection site.
  • conjugates and compositions of the invention will usually be administered by injection, preferably by the intravenous route, or by infusion. Where appropriate, injection or infusion directly into a tumour or lesion is also contemplated.
  • the dosage amounts will be depend on the patient- for instance body weight, age, sex and general state of health - the size, location and nature of the tumour and the rate of clearance of the agent as well as the antigenicity of the conjugate and the level of the patient's immunity to the antigen or fragment thereof.
  • the dosage of a conjugate of PPD and an antibody would be in the range of from 10 to 50 ⁇ g PPD per injection.
  • Dosage regimes also depend upon a number of factors including those outlined above.
  • the administration may take place over the period of several hours, possibly repeated daily, or may continue uninterrupted for days.
  • the conjugates are preferably administered either daily or at longer intervals, for instance of a few days. Administration may be repeated as necessary.
  • the conjugates of the invention will bind to the tumour cells by virtue of the interaction between the antibody part of the conjugate and the corresponding tumour specific antigen or complement component. This results in the tumour cells being labelled with the antigen part of the conjugate. This is then recognised by the cellular immune system of the patient leading to the development of a local delayed-type hypersensitivity (DTH) reaction with destructive effects against tumour cells, and possibly adjacent cells, effected by T helper cells and their secreted products. The generation of cytotoxic T cells should also be enhanced as a result.
  • DTH delayed-type hypersensitivity
  • Treatment involving the use of conjugates according to the invention may therefore involve testing samples of tissue or body fluid from the patient to identify a suitable antigen against which the patient has T-cell immunity and selection of a conjugate of such an antigen.
  • MAbs were used which were specific for a tumour associated antigen of CS7/BL6 melanomas, or for the human complement component C3d, which was fixed de novo to the surface of tumour cells.
  • the ability of the conjugate to induce PPD-specific T-cell activation, lymphokine secretion and tumour cell cytolysis/cytostatis in vivo was determined with a view to focusing a DTH response against selected tumour target in BCG immunized animals.
  • Effectors Synergistic non-adherant spleen cells from BCG immunized mice or a L3T4+, Lyt-, PPD reactive T-cell clone (PPD-MW1) .
  • MAb was covalently linked to PPD using the hetero- bifunctional cross linkers SPDP and SMCC.
  • Tumour cells were pretreated (45 mins, 4°C) with optimal concentrations of either MM2-9B6-PPD or Clone 3-PPD conjugates. Control cells were treated with equivalent concentrations of the MAbs or PPD alone. Tumour cells were then co-cultured with effectors for 16 hrs. at various E.T. ratios. Specific cytotoxicity was determined using a standard 51Cr release assay.
  • TNF Tumour necrosis factor alpha/beta
  • Clone PPD-MWl was activated by B16-F10 cells pretreated with MM2-9B6-PPD and TNF production determined using the TNF sensi .ti.ve cell li.ne L929. 51Cr labelled L929 cells were incubated with serially diluted control and activated clone supernatant for 16 hrs in the presence of actinomycin D. Susceptibility of the tumour targets to human recombinant TNF (rTNF) alpha was measured using a 16 hr
  • Control and MM2-9B6-PPD conjugate treated B16-F10 cells were injected SC into BCG immunized or normal C57/BL6 mice (3 x 10 5 cells/animal) . Tumours were excised on day 11 and weighed. Significance levels were determined using the Mann-Whitney U test.
  • Clone 3-PPD conjugate mediated significant levels of cytoxicity against C3 coated MC6A tumour cells using both immune spleen cells (Fig. la) and the clone (Fig. lb) at high E:T ratios. MAb or PPD alone did not increase cytotoxicity.
  • MM2-9B6-PPD conjugate failed to mediate significant levels of cytotoxicity against B16-F10 tumour cells when immune spleen cells were used as a source of effectors.
  • Fig 2a Marginal cytotoxicity above control levels was, however, evident when the PPD-reactive clone was used.
  • Tumour cells pretreated with MM2-9B6-PPD conjugate were able to stimulate clone PPD-MWl to produce significant levels of TNF alpha/beta (Fig 4a) .
  • Significant MAF activity at a supernatant titer of 1/32 was also found.
  • Susceptibility of the tumour lines of rTNF alpha correlated well with the levels of cytotoxicity observed in vitro with PPD-reactive T-cells (Figs, l & 2) .
  • MAb-PPD heteroconjugates specific for a tumour associated antigen, or a de novo fixed complement component can be used to focus PPD-specific T-cells onto tumour targets in vitro.
  • Heteroconjugate treated tumour cell targets activate PPD-specific T-cell clones, resulting in concomitant release of significant levels of TNF alpha/beta and MAF.
  • Susceptibility of the tumour targets to rTNF alpha correlated well with the levels of cytotoxicity achieved over 18 hours in vitro with PPD-specific T-cells.
  • the high levels of cytostatis achieved over 72 hours could not, however, be attributed solely to the activity of rTNF alpha and may reflect synergy between the TNF alpha/beta and MAF.
  • the activation of PPD-specific T-helper cells at sites of tumour growth in vivo may result in the recruitment of other tumourcidal effects, and ultimately induce a DTH response.
  • Heteroconjugates directed to complement components should further allow the activation of T-cells at sites of complement fixation, and this may be exploited to enhance the effectiveness of conventional MAb therapy.
  • MM2-9B6-PPD conjugate can give a significant reduction in the growth of B16-F10 cells in BCG immunised animals.

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Abstract

An antigen-antibody conjugate for treatment of a tumour in a patient wherein the antigen is an antigen against which the patient has immunity and the antibody is an antibody capable of specifically binding the cells of the tumour.

Description

HETEROCON UGATES The present invention relates to novel targetting agents and to their use in the treatment of cancers. Radio- and chemotherapy are now well established as cancer treatments, as is surgery, for clearly defined tumours. However these techniques are less effective at destroying small lesions due to metastasis and, with surgery, there is always a risk of leaving behind small areas of cancerous tissue. The present invention is particularly concerned with providing a means to clear up such small pockets of cancerous cells and is therefore considered mainly as an adjunct to the already established therapeutic and surgical techniques. In particular, the invention aims to recruit aspects of the patient's own immune system and to target this against the tumour cells. Accordingly the present invention, in one aspect provides a method for the treatment of the human or animal body comprising administering an effective, non-toxic amount of a targetting agent which is a conjugate of an antigen and an antibody, or fragments thereof, the antigen being selected such that the patient already has immunity to the antigen and the antibody being selected to bind specifically to the tumour cells.
Figs, la & lb show the clone 3-PPD conjugate mediated lysis of C3 coated MC6A tumour cells in cytotoxicity assays. Figs. 2a & 2b show the MM2-9B6-PPD conjugate mediated lysis of B16-F10 tumour cells in cytotoxicity assays.
Figs. 3a & 3b show the tumour cytostasis mediated by clone supernatant. Figs. 4a & 4b show lymphokine assays.
The antigen may be any antigen to which the patient has previously been exposed, or any antigen which cross-reacts with lymphocytes in the patient's blood. Examples of suitable antigens include those of the childhood illnesses such as measles, chickenpox or mumps and other antigens to which the population in general is likely to have induced immunity, for instance, tetanus, typhoid and tuberculosis. The latter is particularly relevant to the present invention as the vast majority of the population have been immunised using BCG vaccine against Mvcoplasma tuberculosis. the cross-reacting PPD (purified protein derivative) from M. tuberculosis may be used in the present method and is particularly preferred because of the very strong immune reaction which it elicits. Fragments of such antigens may also be used in the invention provided that they retain the epitope which will be recognised by the patient's immune system. In the case of PPD, which consists of a number of different polypeptide sub-units, any antigenic sub-unit or indeed any antigenic domain of one of the sub-units, may be used as the antigen. The antibody used in the conjugate may be any antibody or fragment thereof which retains an antigen-binding site, such as the Fab' fragment, which will bind specifically to the tumour cells to be destroyed. Many tumour specific antigens are now known and others will be discovered in the future; antibodies, whether polyclonal or monoclonal (the latter are preferred) against such antigens may be used in the present invention. Certain tumours do not express tumour-specific antigens but these may be targetted using antibodies against neo-antigens in bound components of complement, such as C3 which are often found to accumulate on the surface of those tumour cells which activate the alternative complement pathway. Antibodies against C3 or other complement components may also be used when the conjugates of the invention are to be administered after conventional monoclonal antibody treatment of cancers.
(Such treatment, using tumour specific antibodies, results in the tumour cells becoming covered first in the anti-tumour antibodies and then in complement which binds to the anti-tumour antibodies) . When anti-complement antibodies are used it is preferred that they are directed against neo-antigenic sites, i.e, sites which are only formed or exposed once complement binding has occurred, in order that binding to unbound complement is avoided.
In addition to being specific for the tumour cells to be destroyed, the antibodies should preferably be derived from antibody-producing cells of the same species as the patient or should be modified to mask or remove any species-specific determinants other than those of the same species as the patient.
The antigen (or fragment thereof) and antibody (or fragment thereof) may be coupled by any conventional method for covalently binding such materials. For instance, linking groups may be bound to the antigen and to the antibody and the linking groups are then coupled together. Typically one linking group is formed using the reagent SMCC (succinimidyl4-(N-maleimidomethyl)cyclohexane-
1-carboxylate described in Yoshitake, S. et al (1979) Eur. J. Biochem, 101, 395-399 and Mahan, D.G. et al (1987) Anal. Biochem, 162, 163-170) and the other linking group is formed using SPDP (N-succinimidyl-3-(2-pyridylditho) propionate described in Walden, P. et al (1986) J. Mol. Cell. Immunol. 2, 191-197 and Gordon, R.D. et al (1987) Proc. Natl. Acad. Sci. 84, 308-312), both available commercially from Pierce U.K. Ltd, and the two linking groups are then coupled, or conjugated, by the formation of thiomaleimide bonds.
Coupling techniques should be selected so as to avoid impairing either the antigenicity of the antigen or the affinity of the antibody for the tumour cells; where necessary the product may be fractionated to obtain quantities of effective conjugate. The present invention also provides a conjugate comprising an antigen and an antibody, or fragments thereof, covalently coupled via linking groups. Processes for coupling the antigen and antibody to form such a conjugate form a further aspect of the invention.
The invention further provides such conjugates for use in a therapeutic method for the treatment of the human or animal body and the use of such conjugates in the manufacture of a medicament for use in the treatment of cancer.
The conjugates of the invention may be administered as such but are preferably administered as pharmaceutical compositions also comprising a pharmaceutically acceptable diluent or carrier. Typical diluents and carriers include water for injection and other injection media.
Compositions may be presented in unit or multi-dosage form. For administration by injection the compositions may be presented in ready-to-use form or as a concentrate or dry powder for reconstitution, e.g. using water for injection, prior to use. The compositions will generally be sterile and pyrogen free. The compositions may also comprise accessory ingredients such as antibacterial and antifungal agents, buffers, salts, agents to adjust the tonicity of the composition, anti-oxidants, wetting agents and suspending agents to improve the solubilisation or suspension of the conjugates and analgesics or anaesthetics to reduce pain at the injection site. These compositions form a further aspect of the invention.
The conjugates and compositions of the invention will usually be administered by injection, preferably by the intravenous route, or by infusion. Where appropriate, injection or infusion directly into a tumour or lesion is also contemplated.
The dosage amounts will be depend on the patient- for instance body weight, age, sex and general state of health - the size, location and nature of the tumour and the rate of clearance of the agent as well as the antigenicity of the conjugate and the level of the patient's immunity to the antigen or fragment thereof. As a guide the dosage of a conjugate of PPD and an antibody would be in the range of from 10 to 50μg PPD per injection.
Dosage regimes also depend upon a number of factors including those outlined above. For infusion, the administration may take place over the period of several hours, possibly repeated daily, or may continue uninterrupted for days. When given by injection, the conjugates are preferably administered either daily or at longer intervals, for instance of a few days. Administration may be repeated as necessary.
Without wishing to be bound by theory, it is believed that the conjugates of the invention will bind to the tumour cells by virtue of the interaction between the antibody part of the conjugate and the corresponding tumour specific antigen or complement component. This results in the tumour cells being labelled with the antigen part of the conjugate. This is then recognised by the cellular immune system of the patient leading to the development of a local delayed-type hypersensitivity (DTH) reaction with destructive effects against tumour cells, and possibly adjacent cells, effected by T helper cells and their secreted products. The generation of cytotoxic T cells should also be enhanced as a result.
It is therefore important that the patient has T-cell immunity against the antigen to be used in the conjugate. Treatment involving the use of conjugates according to the invention may therefore involve testing samples of tissue or body fluid from the patient to identify a suitable antigen against which the patient has T-cell immunity and selection of a conjugate of such an antigen.
The invention will now be illustrated by the following Example:
EXAMPLE 1
Preparation of PPD-monoclonal antibody (MAb) heterocon ucrates.
MAbs were used which were specific for a tumour associated antigen of CS7/BL6 melanomas, or for the human complement component C3d, which was fixed de novo to the surface of tumour cells. The ability of the conjugate to induce PPD-specific T-cell activation, lymphokine secretion and tumour cell cytolysis/cytostatis in vivo was determined with a view to focusing a DTH response against selected tumour target in BCG immunized animals.
Materials used
Effectors: Synergistic non-adherant spleen cells from BCG immunized mice or a L3T4+, Lyt-, PPD reactive T-cell clone (PPD-MW1) .
Monoclonal antibodies and tumour lines: IqG MAb Specificity Tumour Target
Clone 3 Human C3d Fibrosarcoma MC6A (MC6A cells coated with C3 via the classical pathway)
MM2-9B6 C57/BL6 B16-F10 melanomas
Methods used
MAb-PPD Conjugation
MAb was covalently linked to PPD using the hetero- bifunctional cross linkers SPDP and SMCC.
Cvtotoxicity Assay
Tumour cells were pretreated (45 mins, 4°C) with optimal concentrations of either MM2-9B6-PPD or Clone 3-PPD conjugates. Control cells were treated with equivalent concentrations of the MAbs or PPD alone. Tumour cells were then co-cultured with effectors for 16 hrs. at various E.T. ratios. Specific cytotoxicity was determined using a standard 51Cr release assay.
Cvtostasis Assay
Serially diluted supernatant from PPD activated clone PPD-MWl was added to both tumour cell lines and cytostasis measured by the inhibition of 3H TdR incorporation on day 3.
Lvmphokine Assays
Tumour necrosis factor (TNF) alpha/beta
Clone PPD-MWl was activated by B16-F10 cells pretreated with MM2-9B6-PPD and TNF production determined using the TNF sensi .ti.ve cell li.ne L929. 51Cr labelled L929 cells were incubated with serially diluted control and activated clone supernatant for 16 hrs in the presence of actinomycin D. Susceptibility of the tumour targets to human recombinant TNF (rTNF) alpha was measured using a 16 hr
15 Cr release assay, and by inhibition of J-*H TdR incorporation on day 3. Macrophage activation Factor (MAF)
Serially diluted supernatant from PPD activated clone PPD-MWl was added to purified peritoneal macrophages in the presence of LPS for 18 hrs. Macrophage activation was determined by lysis of 51Cr labelled P815 cells following an 18 hr co-culture.
Tumour Growth In vivo
Control and MM2-9B6-PPD conjugate treated B16-F10 cells were injected SC into BCG immunized or normal C57/BL6 mice (3 x 105 cells/animal) . Tumours were excised on day 11 and weighed. Significance levels were determined using the Mann-Whitney U test.
Cytoxicity assays
Clone 3-PPD conjugate mediated significant levels of cytoxicity against C3 coated MC6A tumour cells using both immune spleen cells (Fig. la) and the clone (Fig. lb) at high E:T ratios. MAb or PPD alone did not increase cytotoxicity.
MM2-9B6-PPD conjugate failed to mediate significant levels of cytotoxicity against B16-F10 tumour cells when immune spleen cells were used as a source of effectors. (Fig 2a) . Marginal cytotoxicity above control levels was, however, evident when the PPD-reactive clone was used.
Cytostasis assay
The supernatant from PPD activated clone PPD-MWl was able to induce high levels of cytostasis in both tumour cell lines MC6A and B16-F10 (Figs 3a & 3b) . This cytostasis could not be attributed soley to the action of rTNF alpha.
Lymphokine assays
Tumour cells pretreated with MM2-9B6-PPD conjugate were able to stimulate clone PPD-MWl to produce significant levels of TNF alpha/beta (Fig 4a) . Significant MAF activity at a supernatant titer of 1/32 was also found. Susceptibility of the tumour lines of rTNF alpha (Fig. 4b) correlated well with the levels of cytotoxicity observed in vitro with PPD-reactive T-cells (Figs, l & 2) . Tumour growth inhibition in vivo
Pretreatment of B16-F10 tumour cells with MM2-9B6-PPD conjugate, but not PPD or the MAb alone, resulted in significant tumour growth inhibition in BCG immunised animals. Treatment Non-Immune (g) BCG Immune (g)
PPD 2.8 + 0.7* 2.2 + 0.6
MAb 2.7 + 0.8 1.7 + 0.9
MAb-PPD 1.5 + 1.0 0.9 + 0.8**
* Mean wet weight + 1SD (n=10)
** Significantly different from control groups (p<0.05).
Experimental Conclusion
MAb-PPD heteroconjugates specific for a tumour associated antigen, or a de novo fixed complement component, can be used to focus PPD-specific T-cells onto tumour targets in vitro.
Heteroconjugate treated tumour cell targets activate PPD-specific T-cell clones, resulting in concomitant release of significant levels of TNF alpha/beta and MAF. Susceptibility of the tumour targets to rTNF alpha correlated well with the levels of cytotoxicity achieved over 18 hours in vitro with PPD-specific T-cells. The high levels of cytostatis achieved over 72 hours could not, however, be attributed solely to the activity of rTNF alpha and may reflect synergy between the TNF alpha/beta and MAF. The activation of PPD-specific T-helper cells at sites of tumour growth in vivo may result in the recruitment of other tumourcidal effects, and ultimately induce a DTH response. Heteroconjugates directed to complement components should further allow the activation of T-cells at sites of complement fixation, and this may be exploited to enhance the effectiveness of conventional MAb therapy. MM2-9B6-PPD conjugate can give a significant reduction in the growth of B16-F10 cells in BCG immunised animals.

Claims

1. An antigen-antibody conjugate for treatment of a tumour in a patient wherein the antigen is an antigen against which the patent has immunity and the antibody is an antibody capable of specifically binding the cells of the tumour.
2. A conjugate according to claim 1 wherein the antigen is an antigen to which the patient has previously been exposed, an antigen which is capable of cross-reacting with lymphocytes in the patient's blood or a fragment of such an antigen containing an epitope which will be recognised by the patient's immune system.
3. A conjugate according to claim 2 wherein the antigen is a measles, chickenpox, mumps, tetanus, typhoid or tuberculosis antigen.
4. A conjugate according to claim 3 wherein the antigen is the cross-reacting purified protein derivative (PPD) of Mycoplasma tuberculosis or an antigenic sub-unit or antigenic domain of a sub-unit of PPD.
5. A conjugate according to any one of claims 1 to 4 wherein the antibody is an antibody which binds specifically to a tumour specific antigen or an antibody which binds specifically to a neo-antigenic site of a bound complement component, or a fragment of such an antibody containing an antigen-binding site.
6. A conjugate according to claim 5 wherein the antibody is an antibody against a tumour specific antigen or a neo-antigenic site of bound C3 or a Fab* fragment of such an antibody.
7. A conjugate according to any one of claims 1 to 6 wherein the antibody is a polyclonal antibody.
8. A conjugate according to any one of claims 1 to 6 wherein the antibody is a monoclonal antibody produced by a cell derived from an antibody-secreting cell of the same species as the patient or which has been modified to mask or remove species-specific determinants of species other than those of the same species as the patient.
9. A conjugate according to any one of claims 1 to 8 wherein the antibody and antigen are coupled by the linked residues of a linking group on the antibody and a different linking group on the antigen.
10. A conjugated according to claim 9 wherein the antibody and antigen are coupled by the thiomaleimide- linked residues of succinimidyl 4-(N-maleimidomethyl)cyclo- hexane-1-carboxylate and N-succinimidyl-3-(2- pyridylthio) ropionate.
11. A conjugate according to any one of claims 1 to 10 for use in a method of treatment of the human or animal body.
12. A conjugate according to any one of claims 1 to 10 for use in a method for the therapeutic treatment of a patient having a tumour.
13. Use of a conjugate according to any one of claims l to 10 in the manufacture of a medicament for use in the treatment of cancer.
14. A method for the treatment of the human or animal body comprising administering an effective, non-toxic amount of a targeting agent which is a conjugate of an antigen and an antibody, or fragments thereof, the antigen being selected such that the patient already has immunity to the antigen and the antibody being selected to bind specifically to the tumour cells.
15. A method according to claim 14 comprising the use of a conjugate according to any one of claims 1 to 12 or a medicament produced according to claim 13.
16. A process for producing a conjugate comprising coupling an antigen against which a patient has immunity and an antibody capable of specifically binding to cells of a tumour.
17. A process according to claim 16 for producing a conjugate according to any one of claims 1 to 12.
PCT/GB1990/000476 1989-03-31 1990-03-30 Heteroconjugates WO1990011779A1 (en)

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LT3909B (en) 1990-07-20 1996-04-25 Kabi Pharmacia Ab New antibody-superantigen conjugates, process for preparing thereof, process for target cell lysis and use of conjugates in pharmaceutical compositions
US5858363A (en) * 1990-07-20 1999-01-12 Pharmacia & Upjohn Ab Target specific antibody-superantigen conjugates and their preparation
WO2001054731A3 (en) * 2000-01-28 2002-12-12 Univ Singapore Ligand conjugates and methods for preparing same
EP0510949B2 (en) † 1991-04-23 2003-04-02 Sangstat Medical Corporation Cytomodulating conjugates of members of specific binding pairs
US7153977B2 (en) 2000-01-28 2006-12-26 National University Of Singapore Ligands and methods for preparing same
US8105608B2 (en) 2000-03-31 2012-01-31 Purdue Research Foundation Method of treatment using ligand-immunogen conjugates
WO2015105973A1 (en) * 2014-01-08 2015-07-16 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services ANTIBODY TARGETING CELL SURFACE DEPOSITED COMPLEMENT PROTEIN C3d AND USE THEREOF

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EP0324625A1 (en) * 1988-01-12 1989-07-19 Bunge (Australia) Proprietary Limited Antigen antibody conjugate
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
LT3909B (en) 1990-07-20 1996-04-25 Kabi Pharmacia Ab New antibody-superantigen conjugates, process for preparing thereof, process for target cell lysis and use of conjugates in pharmaceutical compositions
US5858363A (en) * 1990-07-20 1999-01-12 Pharmacia & Upjohn Ab Target specific antibody-superantigen conjugates and their preparation
EP0510949B2 (en) † 1991-04-23 2003-04-02 Sangstat Medical Corporation Cytomodulating conjugates of members of specific binding pairs
WO2001054731A3 (en) * 2000-01-28 2002-12-12 Univ Singapore Ligand conjugates and methods for preparing same
US7153977B2 (en) 2000-01-28 2006-12-26 National University Of Singapore Ligands and methods for preparing same
SG148022A1 (en) * 2000-01-28 2008-12-31 Univ Singapore Novel ligands and methods for preparing same
US8105608B2 (en) 2000-03-31 2012-01-31 Purdue Research Foundation Method of treatment using ligand-immunogen conjugates
WO2015105973A1 (en) * 2014-01-08 2015-07-16 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services ANTIBODY TARGETING CELL SURFACE DEPOSITED COMPLEMENT PROTEIN C3d AND USE THEREOF
US10035848B2 (en) 2014-01-08 2018-07-31 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Antibody targeting cell surface deposited complement protein C3d and use thereof
US11384139B2 (en) 2014-01-08 2022-07-12 The United States of Americans represented by the Secretary, Department of Health and Human Services Antibody targeting cell surface deposited complement protein C3d and use thereof

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