FR2527928A1 - PROCESS FOR THE PREPARATION OF MONOCLONAL ANTIBODIES CONJUGATED WITH A METAL CHELATE AND SOLUTION CONTAINING THEM - Google Patents

Abstract

THE INVENTION RELATES IN PARTICULAR TO PROCESSES FOR THE PREPARATION AND PURIFICATION OF MONOCLONAL ANTIBODIES CONJUGATED WITH A METAL CHELATE, IN WHICH THE METAL CHELATE EMITS ALPHA RADIATION. CONJUGATES ARE USEFUL FOR THERAPEUTIC PURPOSES FOR THE TREATMENT OF CELL CONDITIONS AS THEY ARE PRACTICALLY FREE OF NON-CHELATED RADIOACTIVE METAL.

Description

In general, the invention relates to monoclonal antibodies

  The invention also relates to a method for the treatment of ailments.

  cells, especially cancer, which uses an anti

  monoclonal body conjugated with a radioactive metal chelate. Effective therapeutic procedures for treatment

  cell conditions such as cancer have been

  jet of intensive research Classic therapy uses surgery, radiation and chemotherapy Each of these procedures suffers from a serious disadvantage in that it is not

  very selective towards healthy cells and can-

  To be effective, these processes kill or eliminate

  significant amounts of healthy tissue In addition, the chiminthé-

  rapie adversely affects the immune system, so

  death or serious illness often comes from

  fungal, bacterial or viral fections.

  The development of monoclonal antibodies has opened up the possibility of selectively providing therapeutic agents

  or diagnostic agents to specific target cells.

  Monoclonal antibodies are structural immunoglobulins

  well-defined chemical ture An important characteristic of monoclonal antibodies is the reproducibility of the function

and the great specificity.

  For diagnostics and therapy,

  radioactive iodine attached directly to monoclonal antibodies

  Iodine-131 has had some therapeutic success with

  large tumors, but antibodies labeled with radioactive iodine

  active have been shown to be ineffective in treating foci of small tumors or metastases In addition, antibodies

  specifically fixed are relatively quickly catalyzed

  bolised by the target cell Catabolism therefore leads to the incorporation of metabolized iodine into the excretory organs, that is to say the kidney, the gallbladder and the stomach. In addition, effective tests - goose to transport toxins to tumor cells using mnoclonal antibodies

  have not D 2 rmnis to establish a satisfactory therapeutic process

s ant.

  It has been indicated in the literature that diethylene triariine pentaacetic acid (DTPA) could form stable metal complexes when attached to a protein, z rejcarek et ai, Biochem, & Biophys Communes 77, 581 (19 Y 77) 7 Visualization of target sites in vivo with

  polyclonal conjugated antibodies -with radioactive metal-

  DTPA prepared according to the method of Krejcarek has been reported by Lhaw et al, Science 29, 295 (1980) Despite the separation,

  by gel chromatography and dialysis, of free and expensive metal

  latated from polyclonal antibodies conjugated with a ché

  metallic late, gamma images included in the article mon-

  trent that a large proportion of the radioactive metal is local

read in the liver.

  The object of the invention is to provide therapeutic methods

sophisticated ticks.

  The invention also aims to provide a means ef-

  effective for the treatment of cellular ailments, useful

health of monoclonal antibodies.

  Another object of the invention is to provide a method for the selective distribution of lethal doses of radiation to affected cells, which does not or only cause very little

of destruction of healthy cells.

  Another object of the invention is to provide a means of

  selective treatment of foci of small tumors and

tastases.

  : Snfin, the invention also aims to provide a pro-

  yielded from the introduction of selectively "projected" radioactive metals

  3 In vivo development, which appreciably avoids the incorporation

  tion of radioactive metal in the healthy organs of the body.

  According to one of its aspects, the invention provides a means

  treatment of cellular ailments, including the con-

  tact of a target cell with monoclonal antibodies con-

  judged with a metal chelate, in which the radioactive metal

: 3

  active is a metal nuclide emitting an alpha particle.

  In another embodiment, the invention relates to a method comprising the introduction into a body fluid of monoclonal antibodies conjugated with a metal chelate, in which the conjugated chelate is a derivative of di-acid.

  thylene triamine penta-acetic, the conjugate being practical-

  mentally free from ions of said metal accidentally attached, and re-

  holding practically all the activity and selectivity of the antibody. Such a technique is desired both for

  diagnostic and therapeutic purposes The invention provides

  also a method for the production of a monoclonal antibody conjugated with a metal chelate, the metal ra-, di (active emitting alpha particles; method in which the antibody conjugated with a metal chelate passes through a chromatography column consisting of one or more layers chosen from ion-retardant resins,

  anion exchange resins, carbon exchange resins

  and a chelating ion exchange resin, and a

  final layer comprising a classification matrix.

  The invention uses monoclonal antibodies conjugated with a metal chelate for therapeutic techniques,

especially in vivo.

  The invention also provides monoclonal antibodies.

  conjugated with a metal chelate, which retain their ac-

  biological activity and their specificity, and which are practical-

  free of metals accidentally attached Metals

  accidentally fixed levels are not stable and cause free metal to enter the blood Metals that are released into the blood can be fixed by transferrin or other metal-binding proteins (by

  ferritin) which are present in the blood These

  fixed rates are retained in the circulatory system for long periods of time and are evacuated by the system

  reticuloendothélial (RES) Such an evacuation (clearance).

  results in a concentration of the metal in the liver and the

  rate i 1 is obvious that a long-term circulation, at the ha-

  sard, of radioactive metals in the crros or a concentration

  d (metals ra inactive in the liver and spleen are Darfaite-

  nt undesirable The implementation of the invention can avoid

  ter these s Serious problems. Nonoclonal antibodies are immunoglobulins with a well-defined chemical structure, unlike: antibodies

  polyclonal which are heterogeneous mixtures of immunoglobulins

  lines An important characteristic of monoclonal antibodies

  e nals is the reproducibility of function and specificity, and these antibodies can be, and have been developed for,

  wide variety of target antigens, including tu-

  Methods for obtaining monoclonal antibodies have been the subject of serious discussion and are well known in the art. As useful text, mention will be made of "ononnclonal

  Antibodies "P H Kennett, T J Mic Kearn and K 9 Bechtol, eds.

  lg '), see also Koprowski et al, US Patent 4,196,265.

  The choice of a monoclonal antibody for the implementation of

  the invention depends on the end use to which the mono- antibody

  clonal conjugated with a metal chelate is intended.

  The anticor Ds are generally stored in a solution.

  aqueous solution which contains an ionic compound A solution

  physiological line, normal, is very often used and is very readily available. But other ionic solutions can also be used, such as those containing sodium or potassium phosphate, sodium carbonate and

  others, well known in the art.

  A wide variety of chelating agents or gold ligands

  ganics can be conjugated with monoclonal antibodies.

  3 e The organic ligands to be conjugated with nmono- antibodies

  clonal can be chosen from natural or synthetic amines, porphyrins, aminocarboxylic acids, iminocarboxylic acids, ethers, thiols,

  phenols, glycols and alcohols, or among the p Dlya-

  mines, polyaminocarboxylic acids, polyimino acids

  carboxylic, aminopolycarboxylic acids, acids

  iminopolycarboxylic, nitrilocarboxylic acids, a-

  dinitropolycarboxylic acid, polynitrilopolycar-

  boxylics, ethylene diamine tetraacetates, diethyl-

  lene triamine penta or tetraacetates, polyethers,

  polythiols, cryptands, polyetherphenolates, pol: -

  otherthiols, ethers of thioglvcols or alcohols, po-

  lyaminephenols, all acyclic, macrocyclic ligands,

  cyclic, nmacrobicyclic or polycyclic or other similar

  wheat that produces very stable metal chelates or cryptates Naturally, the choice of ligands depends on the metal

  to chelate and is left to the specialist.

  The ligand used in certain embodiments of the invention has an organic functional group which is not attached to a metal, capable of binding to the antibody

  monoclonal The functional groups can be chosen by-

  mi carboxylic acid groups, diazota amine groups

  wheat, succinimide esters, anhydrides, anhydrides

  mixed, benzimidates, nitrene, isothiocyanates,

  azides, sulfonamides, bromoacetamides, iodoaceous-

  tamides, carbodiimides, sulfonyl chlorides, hy-

  drazides, thioglycols or any reactive functional group

  known in the art as a conjugation or coupling agent

biomolecular range.

  The invention preferably uses a derivative of diethylene triamine pentaacetic acid (DTPA). It has been found that the ligands of DTPA firmly bind the metal ions and that

  the DTPA derivative (hereinafter called "chelate") forms an anti

  monoclonal body conjugated with a chelate, very stable, both

  from the point of view of fixing the metal chelate as

  jugat chelate-antibody These properties are very important.

  lift particularly in in vivo applications By

  example, if the chelate releases the metal ion after the intro-

  duction in the blood, c: s ion U ert to be fixed by transferrin or other, and to be distributed generally in o

  l 1 circulatory sacer of the body In addition, the ions have

  eventually launches to come together and stay in organs

  such as the liver and spleen These effects have serious consequences.

  depending on the toxicity of the fetus and its radioactivity In addition, if the chelate does not form a very stable conjugate with the antibody, there is a significant decrease in the

  quantity of metal supplied to the target site and a corresponding reduction

responsible for efficiency.

  When preparing conjunct monoclonal antibodies

  fords with a metal chelate according to the invention, it is im-

  to avoid metallic contamination from external sources Laboratory equipment should be

  plastic or glass, free of all exo-metals

  gene All stock solutions must be cleared

  metallic traces, for example by chromatography

  on a column with an appropriate resin.

  To facilitate its presentation, the invention will be

  written on the basis of DTPA chelate The preferred chelate is

  prepared from an amine salt of DTPA The ter-

  2 N amine in the broad sense and it includes primary, secondary and tertiary amines which completely eliminate the protons

  DTPA The choice of an appropriate amine is left to the specialist.

  list of technique and effectiveness of any amine (including ammonia) can be easily determined A

  23 particularly preferred anine is triethylamine.

  te at least 5 equivalents of the amine to an aqueous solution of DTPA and heating to complete the reaction The reaction

  produces a DTPA pentakis (amine) salt according to the following equation

  te in which the amine is triethylamine: (CH 3 C t) 3 N + DP 4 \ (CI 13 C Hi) 3 N 7;) TPA The snlide salt of DTPA-amine can be recovered by evaporation or linphilisate Ln from the solution for removing water and excess

of amine.

  The real chc'late is a derivative of 2 DTPA We add a group

  eg functional with DPTFA and fi: e the D Ti A to the amine groups on the monoclonal antibody, by means of this group

  functional The esters of a haloformic acid are reacted

  that with the salt of DTPA-amine to obtain the chelate used in the invention By ester of a haloformic acid is meant an ester of general formula XC (O) -OR in which X is a halogen, preferably the chlorine, and R is an appropriate functional group, advantageously containing a maximum

  of about 6 carbon atoms The choice of R and X and leaves

  specialist who must consider the stability of the chelate

  and steric hindrance when the chelate is brought to react

  gir with monoclonal antibody A preferred ester is chlo-

isobutyl roformate.

  In a sample preparation, we dissolve quantities

  approximately equimolar tees of the halo acid ester

  formic acid and DTPA-amine salt in a polar organic solvent such as dry, pure acetonitrile Avoid all

  excess haloformic acid ester so as not to block the

  chelation with the metal on the modified DTPA ligand.

  The reaction temperature is generally not critical

  and can be chosen so as to obtain a salt which is par-

  partially or practically precipitated The reaction is carried out

  preferably at a temperature low enough to pre-

  cipitate practically all of the haloamine salt,

  reaction product When the amine used is tri-

  thylamine and the ester is an ester of chloroformic acid, the temperature must be between approximately -20 C and -70 C Maintaining the temperature in this range moves the equilibrium reaction to the right, giving a high yield into a mixed carboxycarbonic anhydride of DTPA according to the following equation: XC (O) -OR + 1 (CH 3 CH 2) 3 N 75 DTPA Z =

0 O '

  Il Il l (CH 3 CH 2) 3 N 74 DTPA-C, o ,, C-O-R + P (CH 3 CH 2) 3 NHX n leading to the above reaction in the range of

  specified temperatures, a high concentration can be obtained

  vee in chelate, practically free of halnami salt: e sub-

  product For example, about 0.25 m M of the pentakis (triethylamine) salt DTPA can be dissolved in 0.5 ml of acetonitrile

  and reacted with 35 µl of isobutyl chloroformate.

  After about 45 minutes at -70 C, the solution can be concentrated

  trifuged to separate the precipitate, leaving a liquid over

  swimming containing the desired chelate at a concentration of about 0.5 M The chelate is introduced in a desirable manner

  in the chelate-antibody conjugation reaction to a

  centering at least about 0.25 M in the organic solvent.

  Such concentrations of chelate allow the use of relatively small amounts of organic solvents in the conjugation reaction mixture. Excessive amounts of organic solvent in the reaction mixture should be avoided, as the solvent can have adverse effects on

  to the biological activity and the specificity of the antibody.

  The chelate-antibody conjugation takes place by adding the

  chelate in organic solvent to saline aqueous solution

  no antibody it is important to drive the reaction of the

  Modified DTPA and the antibody to a p H at most equal to 7.2 en-

  viron The chelate-joint reaction involves the decomposition of the chelate, due to its reaction with water If the p H is too low, the chelate then undergoes a decomposition catalyzed by

  an acid and the biological activity and the specificity of the anti

  bodies are reduced The p H is advantageously between approximately 6.0 and 7.2, preferably as close as possible to 7.0 In this range, the reaction of the DTPA chelate with

  water is less detrimental to the chelate-antibody reaction.

  Although the above discussion is focused on DTPA, the technique also allows conjugation with other liganus see, for example, Proc Natl Acad.

Sci USA 73, 3803 (Nov 1976).

  To preserve the maximum biological activity of the anti

  body, avoid using strong acids or

  its strong to adjust the fold value in the preparation of any chelate-antibody The use of a strong acid or a strong base can cause localized denaturation in the solution The pi can be adjusted in the aqueous solution of the monoclonal antibody by incorporating an appropriate buffer Par

  example, Nal ICO 3 can be used at a concentration of approx.

  ron 0.1 Mr Other buffers such as MES (2- (N-morpho-

  lino) ethane sulfonic) are known in the art and can

  can also be used Choosing an appropriate tampon

is left to the specialist.

  When adding the chelate solution to the aqueous solution of the antibody, the two solutions must be at a temperature close to O OC The temperature of the solution must generally not be able to exceed approximately 4 OC for

  the course of the reaction The application of temperatures including

  its between about O and 4 O C tends to avoid decomposition

  tion of the antibody and therefore also reduces the decomposition of the chelate The duration of the reaction is not critical since the reaction can be complete and the solution can be left cold overnight The molar ratio chelate antibody can vary in

  wide limits depending on the intended use of the spouse

  gat The chelate antibody molar ratio may vary by approx.

  0.1 to 10 or more, preferably about 0.25 to 5 In many cases, the molar ratio of chelate to antibody is

between approximately 0.5 and 3.

  In general, an excess of chelate is used in the reaction, because the chelate decomposes to a certain degree

  in the aqueous solution The number of chelates fixed per molar

  antibody count is a function of the concentration of the chelate

  and the concentration of the antibody in the reaction mixture

  nel, high concentrations tending to provide more chelates per anticrps Si La qu? tity of art-ic,) ps used

  is relatively weak and if we use a relative solution-

1. ('

  n-2 nt diluted, a substantial excess of chelate may be necessary

  saire For example, a molar excess of about 6 ': 1 do ch-

  lat may be necessary to react with an antibody solution before a protein concentration of approximately 5 A 1 O ng / nl, so as to provide approximately 1.5 chelate fixed by

  antibody molecule The addition of too many mo-

  chelate molecules per antibody molecule can reduce the ac-

  biological activity and specificity of the antibody.

  When the addition of the chelate to the antibody is complete, substantial amounts of decomposed chelate can be

  present in the solution This can happen so that

  which chelate-antibody conjugate The decomposed chelate must be eliminated while maintaining the biological activity and the specificity of the antibody. Dialysis or chromatography can be used, for example, if necessary, a first dialysis against acid diluted ascorbic and an EDTA solution can be made to remove any residual iron that may be present in the chelate or protein An antibody conjugated with a purified chelate can

  2 be obtained by dialysis of the reaction mixture for 48 hours

  res counter 3 times a liter of an aqueous solution at about 4 C and a p H of about 6, containing 50 m M of citrate and m M of sodium chloride with 1 ml of Chelex 100 resin (Bio-Rad) in the dialysis tank A final dialysis in i liter of solution containing 10 m IM of MIS and 200 m M of sodium chloride A at 4 C and A p H of 6 completes the purification of the protein Variants of the dialysis processes are known and

applicable by specialists.

  The metal chelation is carried out in aqueous solution.

  se and again avoid using strong acids or strong bases Metal chelation for any chelate-antibody conjugate is performed at p H which does not significantly reduce the biological activity or the specificity of the antibody generally, the acceptable range of

  p HI is between approximately 3.2 and 9, but for certain anti

  1 l particular bodies this range may be narrower At a

  p H value less than about 3.5, accidental fixation

  such as metal ions on antibodies is substantial-

  slightly reduced for many metals A range of p H pre-

  is often between 3.2 and 3.5 approximately. But, factors particular to the solutions of the metal used,

  can allow a p H greater than 3.5 The choice of p H ap-

  property in the above range is left to the specialist.

  In the invention, an acid is advantageously used

  or a base with little chelating power, as a buffer.

  Citric acid or glycine are useful buffers.

  very buffers are, of course, known in the art.

  The invention includes a solution of antibodies conjugated with

* a chelate, adjusted to the desired p H with an acid buffer or ba-

  sic with low chelating power and without the addition of an acid

  strong or strong base To this solution we add a salt

  metallic If the metallic salt is in solution, the p H of this-

  the solution is also adjusted with a chelating buffer The p H of the metal solution can however be adjusted with

  strong acids or strong bases before addition to the so-

  lution of antibody conjugated with a chelate.

  Any acceptable metal salt can be used to prepare the monoclonal antibodies conjugated with a metal chelate. Typical salts include halides (for example chlorides), nitrates, perchlorates.

  or the like Metal salt is used at a concentration

  as high as possible The radiation exposure of individuals handling the preparation generally sets a

  lower limit of a metal equivalent per fixing site

tion of the chelate.

  The duration of the reaction is not critical unless the p H is close to the extreme limits of p H acceptable for the antibody At these limits or near these limits

  p H, reaction times should generally be less than

  laughing at 1 hour and preferably about 30 minutes or less 3 N fact, from the point of view of saving time, time

  reaction within these limits are desired We also prefer

  ment conduct chelation in the presence of a reducing agent soluble in water, incapable of being chelated, biologically harmless, such as ascorbic acid to avoid chelation

  tion of any trace of iron The reaction is normally completed

  by adding enough trisodium citrate so that the p H of the solution increases to a point at which the metal conjugate is no longer labile. It has been determined that most of the DTPA complexes are particularly stable at a p H of approximately 6 Other weak bases or acids, when the reaction has a p H greater than 6, can be used as long as they do not harm the antibodies. Their choice is

left to the specialist.

  The reaction solution generally requires a purification

  tion before its use in vivo, and may also require purification before being used in vitro Any non-fixed metal and any metal accidentally fixed must be

  eliminated The discussion here concerns metal ions ac-

  cidentially fixed But, part of the metal can be retained

  naked unsafe by chelates and act the same

  way that metal ions accidentally attached.

  When using a radioactive metal that has a short

  half-life period, it is particularly important that the o-

  purification process is as quick as possible, The

  vention includes a relatively rapid purification by chro-

  matography and this characteristic of the invention is applied

  cable to chelate-antibody conjugates in general By using one or more ion-exchange, retarding or chelating resins together with a classification matrix (for example a gel), the monoclonal antibodies conjugated with a metal chelate according to the invention can be quickly and

suitably purified.

  Different ion exchange resins can be used alone, or any combination of an ion retarding resin, a cation exchange resin, an anion exchange resin or a chelating ion exchange resin Le

  choice of one or more suitable resins, their degree of reti-

  culation of their chemical form and their granular size

  l-metric is left to the specialist.

  The cation exchange resins used in the invention

  vention are often strongly acidic polystyrene gel resins (for example Dowex 50 WX) or other strongly acidic resins, but which are not a polystyrene, such as

  than 7 eocarb 215 (Permutit Co) Like other acid resins

  suitable dity, mention will be made of polystyrene gel resins

  weakly acidic, macropolystyrene polystyrene gel resins

  rous, or carboxy-cation exchange resins

  macroréticuliques lique The anion exchange resins can

  may be highly bastated polystyrene gel resins

  sics (eg Dowvex 1 X 8) or other less basic resins

  such as resins of the pyridinium polymer type

  or of the phenolic polyamine type The chelating resins can

  can be Chelex 100 or any resin of the copolymer type

  styrene-divinyl benzene containing iminodiacetate ions ap-

  bet (eg Dowvex A-1) Used delay resins

  are resins containing paired anion and cation exchange sites (eg Pio-Iad AG 11-A 8) These resins

  They are normally obtained by polymerizing acrylic acid.

  that inside a strongly basic resin such as

  resin containing quaternary ammonium groups in a re-

  bucket of styrene-divinyl benzene copolymers The discussion above

  above concerns only representative examples of each resin; many other resins are also known in the

  technique A list of resins marketed with a brief

  ve description of their properties and applications is contained,

  among others, in "Bio-Rad Laboratories 1982", Price List H. The choice and combination of resins depends on the problems

  separation papers encountered and fate left to the specialist

  cialiste A useful reference is J Khym, "Analytical Ion-Ex-

  change Procedures in Chemistry and: iology "(1974).

  The classification matrices are also well known in the art. They include pnlyacrylanides,

  agarnses, polvsaccharoses or the like A matrix of classes-

  What is particularly useful is a polysaccharide gel (for example Sephadex G-5 n gel) As an example of

  polvacrylamide, mention will be made of Series 31 o-Gel P (in-2 'ad Labs).

  The classification matrix ch-ix depends on the orotein to

  purify and is left to the specialist.

  In the implementation of the invention, the different

  resins can be deposited as layers in a co-

  lonne and the solution to be purified can be loaded from top to

  bottom in the column or bottom up in the column An ali-

  top-down mentation is a pre-

  13 when radioactive compounds are used, because the

  Gravity also requires little or no additional equipment or instruments The choice of bed heights,

  debits and the like is easy to do by a specialist.

  It seems that very charged metals are fixed ac-

  cidentially, sometimes, by the antibody on the ionic sites

  along the surface of the protein In other cases, accelerally attached metals appear to be included with the folds of the antibody protein These metals may

  be released in solution but can also be reabsorbed

  bés starting from the solution c ians a process of type in equilibrium

  bre Retardant or ion exchange resins used

  sées in the purification according to the invention are used to shift the balance and allow the metal to be separated

  of the antibody For example, when the antibody goes through

  -3 to a resin delaying the ions, the passage of the metal ions-

  liques in the solution is slowed down, but that of the protein is not slowed down The very charged metal ions (+ 3 or more), accidentally fixed, are then released in the solution to restore the balance When these ions are released in the solution, they are in turn delayed by the resin for

  cause a continuous release of metal ion by the anti

body.

  A rate of ion exchange resin can be used.

  lower than that of the ion-delaying resin to firmly fix the separated, highly charged ions, and continue the separation process When the resin depletes the solution of

  protein in free ions, very charged, the balance is again

  calf restored between free and accidental metal ions-

  However, during this process, metal ions

  spikes inside the chelate are retained with the antibody.

  However, it has been determined that the simple use of an ion retardant resin or an ion exchange resin is not satisfactory for providing effective removal.

  of virtually all of the attached metal ions accidentally

  To make sure to complete the purification, we use a classification matrix. The antibody solution which enters the matrix is already partially used up.

  free, highly charged metal ions In the matrix of -class-

  Another depletion occurs When the solution passes through the matrix, the antibodies are not delayed, while the ions are delayed The resulting solution at the exit of the classification matrix is practically free of metals accidentally fixed These metals fixed so loose can

  be reduced to less than about 6% o of the total metal content

  tal of the conjugate, so that at least about 94% of the metal entering the conjugate is stably fixed by the chelate Desirably, at least about 97% o of the total metals are fixed by the chelate It is possible to get rates of metals in which 98% or more of the metal is

  fixed by the chelate Dialysis can be used to

  finish the fixed metal content stably.

  A preferred purification process uses a resin

  retarding ions (Bio-Rad AG 11-18) on an exchange resin

  cation (Bio-Rad AG 50 MX 8) and a gel (Pharmacia Sephadey G-50).

  In the purification of monoclonal antibodies conjugated with a technicium chlate for the invention, the preferred column contains an ion-retardant resin (Bio-Rad AG 11-18) on

  a catinns exchange resin (Bio-Rad 501 v X 8), on a re-

  anion exchange sine (Bio-Rad AG IX 53) and a classification matrix in the form of a gel (Pharmacia Sephadex G-50). In the process of the invention, the antibody is retained in a non-aggregated form The aggregation of the antibodies, by agglutination or crosslinking, results in a loss of

  specificity of the antibody which, naturally, is undesirable

  ble Aggregation may be due to excessive concentrations

  antibodies in a vehicle, or in contact with pro-

  chemical products which entered a crosslinking of the protein,

  such as for example carbodiimides Sediment tests

  Conventional tation, with classification or other matrices, can be used to determine if the antibodies are

  aggregated In fact, the antibody specificity tests dis-

  cut here reflect aggregation as a loss of specifi-

  cited. The activity and specificity of the conjugated antibodies of the invention are maintained at a rate of at least about 80%

  and preferably at least 90% of the activity and the specific

  specificity of the antibody used to produce the conjugate Particularly preferred solutions are characterized by an activity and an antibody specificity of at least about

  95%, and in fact we got products that hold the ac-

  tivity and specificity of the original antibody virtually

  unchanged The activity and specificity of the antibodies are

  usually safe in the art by fixing anti

  body, in vitro, on an epitope The degree of activity and

  3 o specificity of the final antibody produced can be easily determined

  finished by simply repeating the initial test with the product

final conjugate.

  The invention relates to an in vivo therapeutic process in which monoclonal antibodies conjugated with a radioactive metal chelate are introduced into the body and caused to

  focus in the target region There are a variety of

  radioactive metal sotopes that form complexes of

  DTPA stable and emit cytotoxic alpha particles.

  The therapeutic effect occurs when the conjugates are close to, or in contact with the target cells and become

  xent on them It is believed that cell death is a re-

  direct or indirect result of radiation due to radioactive metal

  active located near the cell.

  The advantages of this aspect of the invention are manifold.

  1 C First of all, the great specificity of the conjugated monoclonal antibody minimizes the total dose of radiation We only use

  enough radiation for the target cells.

  In addition, radioactive metal chelates are generally quickly evacuated from the body in the event that the conjugated antibody is ruptured. The isotope may have a short period of

  life and the affinity constant by which the isotope is re-

  held in the DTPA chelate is very high, hence a metal fixed stably Finally, since the amount of radioactive metal used is minimized, the risk of irradiation

  personnel preparing and administering the antibody conjugate a-

  with a metal chelate, is reduced significantly.

  Due to the properties of the monoclonal antibody con-

  judged with a radioactive metal DTPA chelate, according to the

  vention, tissue breakdown or total body dose

  the during therapy is considerably reduced compared to

  port to that resulting from the therapeutic processes by ray-

  currently applied, such as isotope implbnts

  pes, external radiation therapy and immunoradio-

  therapy using polyclonal or autologous antibodies

  additionally to iodine-131 In addition, the biological and physical half-periods of the radiobiological products supplied to the targets can now be verified, minimizing the effects of radiation on the whole body Since the radiation specifically targets cell types (nar example of neoplastic cells), a therapeutic dose is provided specifically to malignant cells, either localized or dispersed in the form of metastases The ability of an antibody

  mon-clonal designed with a radioactive metal chelate

  nir spicifiq ', ernent an effective dose of therapeutic radiation

  tick to scattered cells in the form of metastases is

  also remarkable and particularly useful in tea-

cures cancer.

  The invention uses the monoclonal antibody conjugated with a metal chelate containing a radioactive metal emitting

  alpha particles, to treat ailments in cells.

  In most applications, it is desirable that the radioactive metal has a half-period of less than about 4 days and rapidly disintegrates into a stable isotope as soon as the alpha particles have been emitted. The preferred isotopes used in the invention are bismuth -211, bismuth-212,

  bismuth-213 and bismuth-214 bismuth-212, with a de-

  mid-period of 60.6 minutes is particularly preferred.

  The monoclonal antibody used is specific for the

  sick cell that needs to be destroyed The destruction of a cell

  It is caused by the decay of the radioactive metal and can occur in two different ways. If the alpha particle is emitted towards the diseased cell, a single blow in the cell nucleus can be cytotoxic. The isotope that results from the disintegration radioactive metal after the emission of the alpha particle, is ejected from the chelate on a trajectory opposite to that of the alpha particle The fixed cell can therefore still be reached even when the particle

  alpha is emitted on a path far from the cell.

  A simple blow in the cell membrane by the disinfecting isotope

  tégré can cause irreparable damage to the cell,

  leading to cell death Relatively high efficiency

  Vee of the alpha particle implies the use of less radioactive metal The selectivity of the antibody

  monoclonal and short amplitude (from a few diameters of this

  lules) alpha particles minimize tissue destruction

healthy on a cellular level.

  Bismuth-212 decays in two different ways

  tes About 64% of bismuth-212 disintegrates with an emis-

  sion of beta particle into polonium-212 which has a half-period of 0.3 microseconds Polonium-212 decays into stable lead- 203, after having emitted an alpha particle of the order of 90 microns The other 36 o of bismuth-212 disintegrate into thallium-208 by emitting an alpha particle of the order of at 50 microns Thallium-208, with a half-period of 3 minutes, then disintegrates by emission of beta particle

lead-208.

  Generators for Bi-212 have been described in the literature by Gleu et al, Z Anorg Allg Chem 290, 270 (1957) and by Zucchini et al, Int J Nucl Med & Biol (June 1982) (the abstract of the manuscript has been distributed at the ACS Congress

  August 1981 in New York) A useful generator consists of Th-

  228 in the tetravalent state absorbed on a 3 x 5 mm bed

  sodium tanate contained in a quartz column above

  of a roughly sintered glass disc sealed in the colon-

  ne The titanate hermetically retains Th-228 and its daughter Ra-224 When water passes through the titanate, the Rn-220 daughter of the isotope Ra224 dissolves in water, crosses the sintered disc and is collected in a cm glass tank filled with water The aqueous solution of Rn-220 flows from the

  tank in a 10 mm diameter column containing in-

  about 1 ml of a strongly acidic ion exchange resin such as

  the Bio-Rad AG-50 WX 8 Le cation exchange resin

  Rn-220 decays practically in 5 minutes in the reservoir

  see, in Pb-212 which is absorbed when passing over the resin.

  3 N At a flow rate of approximately 1.5 ml / min through the resin, approximately% of the Pb212 produced is collected in the column where it

disintegrates into his daughter the Bi-212.

  When the desired amount of Bi-212 has been formed on the

  resin, it can be eluted by an acid according to operating modes.

  roofs well known to specialists A useful 2nd elution process for Pb-212 and Bi-212 consists in passing 5 ml ae IC 1 2 N through the resin Or else, if only the

  Bi-212, 1.5 ml of 0.5 ml HC 1 is passed through the resin.

  Although metals which emit beta particles or Auger electrons can be used for therapy, radioactive metals emitting alpha particles are preferred for several reasons.

  alpha nucleotide typically has a short am-

  tissue fullness and linear energy transfer

  very high compared to beta radiation or Auger La ra-

  alpha diation can kill a cell with only one hit on the nucleus and kill practically any cell with 10 hits or less In addition, the decay also emits an isotope (e.g. TI-208 or Pb-208 in the case of Bi-212) which can also cause cell destruction The amplitude of alpha particles is normally less than about 150 microns in tissue On the contrary, beta or Auger particles require hundreds of hits in the nucleus before causing the death of a cell and have amplitudes in them

  fabrics on the order of tenths of a millimeter to centimeters.

  When using beta particles, a larger dose

  you need it, and the disintegration of substantial antibodies

  so more radioactively labeled is necessary to ob-

  hold the death of a cell This is how antibodies fi-

  xed in a specific way are catabolized, releasing

  radioactive metals emitting beta particles into the blood.

  Radioactive metals emitting alpha particles kill re-

  laterally quickly, so that fewer antibodies are

  tabolized. The antibodies conjugated with a metal chelate according to the invention can be administered in vivo in any pharmaceutically acceptable vehicle. As indicated above, a normal physiological saline solution can be used in an appropriate manner. Often, the vehicle contains a minor amount of a protein. carrier such as serum albumin

  human to stabilize the antibody The concentration of anti

  bodies conjugated with a metal chelate in the solution is a matter of choice Rates of 0.5 mg / ml can be readily obtained, but the concentrations can vary considerably depending on the specificity of any device.

  determined application Appropriate concentrations of material

  biologically active ingredients in a vehicle are determined

  routinely in the art.

  The effective dose of radiation or the metal content to be used for any application also depends on the

  particularities of the application For example in the

  Ment of tumors, the dose depends, among other things, on the size, accessibility of the tumor, and others In a similar way, the use of antibodies conjugated with a metal chelate for diagnostic purposes depends , among others, of

  the sensitive device used, from the location of the site to be examined

  ner et al. In case the patient has a circulating antigen

  In addition to those located on the site, the antigens in

  circulation can be eliminated before treatment Such-

  elimination of antigens can be done, for example, using

  reading unlabeled antibodies or by plasmaphoresis in

  which the patient's serum is processed to separate the anti

  Genoa. The following nonlimiting examples are given as

of illustration of the invention.

Example 1

  We weigh 100 mg of DTPA in a flask and add 1 ml

  of water This solution is reacted with 0.125 g of triethyl-

  redistilled amine The reaction solution is heated to

  undermine the reaction and, by lyophilization, a pro-

solid.

  The lyophilized product is dissolved in 0.5 ml of acetonitrile.

  pure, dry trile and 35 "l of isobu- chloroformate are added

  tyle at a temperature of around -20 ° C, then cooled to

  about -70 C After about 45 minutes, the so-

  lution in a vial of Eppendorf (n collects the liquid on-

  swimming which contains the desired mixed DTPA carbocxycarbonic anhydride, at a concentration of about 0.5 M. The monnclonal antibody used is deionized by 103 A 5 and obtained by fusing mouse myelone P 3 cells. 3 Ag 3 with spleen cells isolated from C 56 B 1/6 mice innunized with purified retrovirus glycoprotein, O; O daltons (gp 70) obtained according to the method described by M Strand and J T August, J Biol Chem 251, 559 (1976) Cn performs the fusion as described by M Strand, Proc.

Natl Acad Sci USA 77, 3234 (1980).

  114 μl of a solution containing 2 mg of anti-

  monoclonal body 103 A 5 in Na HCO 3 0.1, 1 to a p H it is approximately 7.2 and 150 m M of sodium chloride and using a pipette is

  transfer to a Nunc flask Then to the flask, we add

  te 33 Pl of a 0.1 M Na HCO 3 solution has a p H of 7.0 Finally, after having cooled the chelate and antibody solutions to O C, 26.4 pl of the carboxycarbonic anhydride are added

  of mixed DTPA (0.5 M in acetonitrile) The reaction is left to

* tion continue overnight.

  The product is first dialyzed at 4 ° C., against a liter containing 30 m M of ascorbic acid, 5 m M of EDTA, 200 m M of Na Cl and 20 ml of sodium citrate (p H of 7, O) We dialyze

  resulting solution at 4 C against three times a liter contained

  with 50 me citrate, 200 m M sodium chloride at p H

  ce 6.0 and 1 ml of Chelex 100 resin (Bio-Rad) for 48 hours

  res Finally, the resulting solution is dialyzed for S hours against one liter of a solution having a concentration

  of 10 m 1 of MES and 200 m M of sodium chloride at a p H of 6.0.

  About 1.7 mg of monoclonal antibody conjugated to

  chelate Similar trials using carp-labeled DTPA

  bone-14, analyzed by scintillation counting, show a

  content of approximately 1.5 chelates per antibody molecule.

  40 51 of a solution of indium chloride 111 (New England Nuclear Corp) is adjusted to a p H of 3.0 by addition of 11.4 1 l of citric acid 0.4 M z a p H of 5 .0 A separate solution is prepared containing 250 μg of antibody conjugated to the chelate in a total volume of 21.6 1 l The solution has a concentration of 200 m M of sodium chloride and 10 mil of MES at a p H of 6 , 0 We adjust the solution a p H of 4.6 by ad-

  edition of 6 μl of 0.25 M citric acid at a p H of 3.0.

  The monoclonal antibody conjugated with a

  metallic late by combining the chloride solutions of

  dium and antibody conjugated to the chelate and allowed to react

  for about 30 minutes at room temperature On ter-

  mine the reaction by adding 25 µl of a saturated solution of

  sodium citrate to adjust the p H has a value of about 6.

  The antibody conjugated with the chelate is purified by chro-

  matography on a 9 cm column containing 1.0 ml of a re-

  sine delaying the ions (AG 11-A 8 marketed by Bio-Rad)

  on 1.0 ml of a cation exchange resin (AG-5 C O VWXS, form-

  + me H, from 0.037 to 0.074 mm, marketed by Bio-2 ad), on

  7 ml Sephadex G-50 gel (Pharmacia) As eluent we use

  reads a solution with concentrations of 200 m M of chlorine

  sodium rure and 10 m M of MES at a p H of 6.0, this solution

  also used to pre-balance the column.

  The eluate is collected in 0.5 ml fractions. The two fractions with the largest part of the protein are found to be

  contain 150 µg of monoclonal antibody labeled with 157.1 micro-

  curies of Indium-11 l A dialysis at 4 C against a liter of

  aqueous solution of 20 m M of MES and 200 m M of sodium chloride

  dium at a p H of 6.0 reveals less than 6% loss of indium II

  appears that the antibody retains almost 100% of its ac-

  biological activity and its specificity in in vitro tests.

  An in vivo visualization in leukemic mice is

  leaving the tumor site in the spleen When administered to normal mice, there is no fixation by the spleen.

Example I It

  A hybridoma is prepared by fusing mye 1 ome cells from mouse P 3 653 with spleen cells isolated from C 56 81/6 mice, immunized with ferritin associated with a purified tumor, isolated from human spleen. a hybridoma which produces an antiferritin antibody called 263 D 5 The antibody is specific for human ferritin and

  does not react with ferritin from other mammalian species.

  The procedure of Example I is repeated to provide a monoclonal antibody conjugated with DTPA containing indium-11 l. Physiological saline solution is injected

  normal containing monoclonal antibody conjugated-with ché

  metallic late to normal mice and leukemia mice

  ques In normal mice and leukemia mice, a

  X-ray visualization shows that there is no concen-

  radioactive metal tration These tests demonstrate that the

  late is stable in vivo with regard to the chelate-anti conjugation

  body and retention of radioactive metal Neither the spleen nor

  the liver is only highlighted in the pictures.

Example III

  The following example uses an isotope emitting parti-

  gamma cells to demonstrate the selective localization of radioactive metals (including isotopes emitting particles

alpha) obtained by the invention.

  Monoclonal antibodies conjugated with an indium chelate 11 l are prepared from an antibody specific for human breast tumor. The hybridoma which produces the antibody is prepared by fusing a mouse myeloma with

  mouse spleen cells The hybridoma and antibody are de-

  written in Proc Natl Acad Sci 78, 3199 (1981).

  The procedure used is practically the same as the procedures of Examples I and II, with the exception of the following. First, the dialysis operation of the monoclonal antibody conjugated with the chelate is suppressed. ascorbate-EDTA Then, 10 μl of 0.1 M ascorbate is added to a 4 × 4 to the indium-III solution, before its addition to the aqueous saline solution of the conjugated monoclonal antibody

with the chelate.

  The labeling efficiency is threefold compared to the

  ceded from examples I and 1 l- The final product is marked with

about 2.1 microcuries per 9.

  10 Fg of the monoclonal antibody conjugated with the indium-III chelate, collected at the outlet of the purification column, are diluted to 100 μl with a phosphate buffered aqueous saline solution. The conjugated antibody is injected.

  with diluted indium-III, in the tail vein of a sou-

  stripped naked, athymic in which we developed a tu-

  human breast dying human breast tumor cells

  maine express an antigen for the antibody 72 hours after the injection, visualization with a good definition gamma camera demonstrates a strong localization of indium-lll

  in tumor tissue No similar localization is observed

  indium-lll in the liver or spleen.

Example IV

  The following tables demonstrate that a DTPA- chelate

  radioactive metal, unlike a free radioactive metal,

  does not localize in the liver or the spleen, and is rapid

  dementally excreted by the kidneys and stomach The fixation read m 6-

  radioactive tal in the organs of normal and leukemia mice

  miques is determined by the following method A normal mice of 6 weeks and mice of the same age made leukemic 8 days before by the injection of the leukemic virus

  of Rauscher, we inject intraperitoneally 5 ig (5 mi-

  crocurie / pg) of free Bi-207, of Bi-207 chelated with DPTA and of Sc-46 chelated 6 with DTPA 18 at 24 hours later, the mice are sacrificed, their organs are weighed and the quantity of radioactivity associated with organs To ensure that the differences in injections, body weights and times of excision of organs are normalized, the amount of radioactivity per gram of tissue is divided by

  the amount of radioactivity per gram of blood, and we

  me the results by this report The results are reported 2 to

Cdzzns legs Tables 1, 2 and 3.

TAIIMSAL:

  : *: o 1 standard errors and errors from the report / san'j ci series of leukemic mice for 14 days and normal, 13 and 42 hours after 20 18 IISSU hours l 1-eucemiguas Norriales Cr-cur 2.35 f 0, 65 4, + 0, 301 Liver 31.3 + 7.50 '29, o 33 t \ Iate 7> 18 + 20 03 d 1 + Oà Kidney 69.4 + 43.7 612, 6 + 4718, Stomach 1335 + 0.35 6.95 + r, 65 a = dose injected 7 x 1 <) 6 cpn per mouse b = approximately 0.13 of the total cpm in the heart c = about 40 of the total cpm in the liver about = 12% o of the total cpm in the spleen

143 + 0,)

-33 2, -

  s i 70 + C '6 o c 9.3 + 9-, 18, n 1 + C' 43

TABLE 2

  Average standard errors of the average ratio / blood of 5 organs of leukemic and normal mice 18 hours, after injection with a chelate Bi-207-DTPA Bi -207 -DT P Aa Leukemic tissue 'Normal Heart 2, 60 0, 33 b 2.45 N 2 Liver 19.12 + 6.32 c 10.93 + 1.24 Spleen 13.1 J 2.6 124 + 2 e, Kidney 94 + 370 281.0 + -26.0 l Stomach 7 , 52 + 2.44 7.20 + 2, u-0 a dose injected 3.8 X 106 cpmn by scruris b = about 0.003 of the total cpm in the heart 'c = about 0.21 of the total cbr in the liver d = about 0.06 cpn t-tal in the spleen

TABLE 3

  Averages and Standard Errors of the Average of 5 Organs of Normal and Leukemic Mice

  after injection with an Sc-46-DTPA chelate.

  Sc-46-DTP Aa Leukemic Tissue Heart 3.66 O 021 b Liver 9.96 0.57 c Spleen 3.17 + 0.39 d Kidney 42.1 + 0, Stomach 12.45 i 5.43 a = b = c = d = report / blood 18 hours Normal

4.49 +

7.23 +

, 69 +

29.5 +

7.76 +

(0.84

  1, O 4 0.28, 5 2.06 injected dose = 5.4 x 10 cpm per mouse approximately 0.006% of total cpm in the heart approximately 0.14% of total cpm in the liver approximately 0.08% of cpm total in the spleen

  The data in the tables above demonstrate that the bis-

  muth and scandium chelated with DTPA do not concentrate

  in the liver or in the spleen of mice unlike bis-

  muth libre High concentrations of metal chelated in the kidneys show that it is evacuated by the urine The variation of the concentrations in the kidney between leukemic mice and normal mice is attributable to a frequent evacuation

  by leukemia mice due to stress.

Example V

  The following tests are intended to determine the effect

  alpha radiation from bismuth on mammalian cells.

  F-46 leukemia cells are proliferated in a modified Dulbacco Eagle medium containing 10% fetal serum

  heat-inactivated calf to provide a cell population

  1 x 10 cells in each well

  cell cultures with bismuth-212 by adding diluents

  tions in series (as shown in Table 4) in the mid-

  place of development The cells are cultured for 96 hours and the number of state cells is determined. They are reported in Table 4.

survivors The results

below.

TABLE 4

  Dose a (rads) on 0.2 0.4 0.8 1.5 3.1 6.2 12.3 24.6

49.2

  98.4 Average number of surviving cells

(10>)

  6.9 s, r '6.1 6.7 6.0 6.0, 0 2.9 2.2 1.1 0.6 Standard deviation 1.4 0.5 1, 7 1.4 2.3 0.6 0.6 0.4 0.4 0.4 0.1 From the data in Table 4 above, using

  standardized calculation methods, D (37% survival) is eva-

  read at 38.5 rads This shows that bismuth-212 emits radia-

  very ionizing, highly cytotoxic By comparison, it takes 900 rads of weakly ionizing radiation from a cobalt-60 source to obtain the same results For a discussion of radiation doses: see the -Nm / mird null brochures (revised)

and 10.

t survival

Claims (10)

R E V E N D I C A T I O N S   1 Process for the preparation of monoclonal antibodies   judged with a metal chelate, characterized in that: a) monoclonal antibodies conjugated with S are supplied with a chelate; b) adding a metal salt to an aqueous solution   monoclonal antibodies conjugated to a chelate ain-   if collected, and a buffer to form antibodies conjugated with a metal chelate, the   metal being a radioactive metal emitting particles alpha cules;   c) passing the aqueous solution containing the anti   monoclonal bodies conjugated with the metallic chelate   that through a chromatographic column, the co-   lonne comprising one or more selected layers   among an ion retardant resin, an exchanging resin   anion geuse, a cation exchange resin and   a chelating ion exchange resin, and a cou-   final che including a classification matrix; and   d) recovering from the column a solution of an-   monoclonal tibodies conjugated with a metal chelate-   lique of which at least 80 5 of the metal contained in the so-   lution are complexed by the chelate conjugated with the monoclonal antibodies 2 Method according to claim 1, characterized in   that the monoclonal antibody conjugated with a chelate is pre-   adorned by putting a carboxycarbonic anhydride of di-   thylene triamine penta-acetic acid in contact with a solution aqueous monoclonal antibody.   3 Method according to claim 2, characterized in that   that carboxycarbonic anhydride of diethylene acid tria-   pentaacetic mine is the reaction product of an amine salt of diethylene triamine penta-acetic acid with an ester   of a haloformic acid in a crganic sclvrnt at a time   p 2 rature below about -20 C.   4 Method according to claim 3, characterized in that the ester of a haloformic acid is isobutvle chloroformate. D 5 Process according to claim 1, characterized in that the radioactive metal emitting alpha particles is   chosen from Bi-211, BI-212 and Bi-213.   6 Method according to claim 5, characterized in that the radioactive metal emitting alpha particles is 3 i-211.   7 Aqueous solution usable especially for the treatment   tment of cell disorders, characterized in that it contains monoclonal antibodies conjugated with a metal chelate, specific for a target cell, the metal   radioactive emitting alpha particles.   8 Aqueous solution of conjugated monoclonal antibodies   with a metal chelate according to claim 7, charac-   terized in that at least about 94% of the metal is complete   xed by the chelate part of the conjugate and the metal is a   radioactive tal emitting alpha particles.   9 -Solution according to claim 8, characterized in that at least about 97% of the metal is complexed by the chelate part of the conjugate.   Solution according to claim 8, characterized in that the conjugated antibodies retain at least approximately   % of their activity and specificity.   1 i Solution according to claim 8, characterized in that the antibodies retain at least approximately 95% 3 of their activity and specificity 12 Solution according to claim 9, characterized in that the antibodies retain at least approximately 97 5 of their activity and specificity.   13 Solution according to claim 9, characterized in that the radioactive metal emitting alpha particles   is chosen from Bi-211, Bi-212 and Bi-213.   14 Solution according to claim 13, characterized 6 e in that the radioactive metal emitting alpha particles is Bi-212.