JPH0720884B2 - Immune response to tumors and viruses elicited by anti-idiotypic antibodies - Google Patents

Description

TECHNICAL FIELD The present invention relates to an antigen, particularly a tumor, which induces an immunological reaction against a virus. More specifically, the invention relates to the use of anti-idiotypic antibodies for the induction of such immunological reactions, as well as the antibodies and cell lines producing them.

[Background of the Invention]

Amino acid sequences in the variable regions of the heavy chain (V _H ) and the light chain (V _L ) of immunoglobulin (Ig) allow a specific antigen to interact with the antibody in the antigen-binding site (ie parotope). Form a conformation. Injecting immunoglobulin into a heterologous recipient animal produces anti-xenotype (species-specific), anti-isotype (immunoglobulin class-specific) and anti-idiotype (specific to antibody variable regions) antibodies It will be. Anti-idiotype antibodies can exist in two functional classes, one that acts on the parotope and the other that acts on the framework of the H and / or L chains. (Framework determinants). In general, Jieha, New England Journal of Medicin (N.Engl.J.Med.) 305, 25-28 (1981), Jean, Anales de Iminology (Ann.Immunol.) 125. See Volume C, pages 373-389 (1974).

[Outline of Invention]

The present invention aims to provide a method of eliciting an immunological response against a tumor, in particular a tumor such as a solid tumor or a virus such as rabies virus.

The present invention is also directed to the use of anti-idiotypic antibodies in raising the immunological response as described above.

Yet another object of this invention is a monoclonal anti-idiotypic antibody useful for inducing an immunological response against tumors, particularly solid tumors and viruses such as rabies virus, and immortal B-lymph for said antibodies. To provide a ball source.

The above and other objects of the invention are achieved by one or more embodiments described below.

As one of the embodiments, the present invention provides a method for inducing an immunological response to an antigen selected from a tumor or a virus, which comprises (a) an anti-idiotype antibody, wherein the epitope identified by the anti-idiotype antibody is An anti-tumor or anti-virus antibody parotope, which comprises (b) stimulating a patient to produce an anti- (anti-idiotype) antibody that identifies an atope on tumor cells or viral particles by administering the anti-idiotype antibody to a human. To provide a way to become.

The present invention also provides a polyclonal anti-idiotype antibody, wherein the epitope identified by the anti-idiotype antibody is a parotope of anti-tumor and anti-virus antibody which is substantially free of anti-isotype antibody. is there.

In another embodiment, the present invention provides an immortal B lymphocyte that produces an anti-idiotype antibody, wherein the epitope identified by the anti-idiotype antibody is an antitumor and antiviral antibody parotope. It is a thing.

The present invention also provides a monoclonal antibody produced by the above Immortal B lymphocytes and substantially free of other antibodies.

Suitable tumors are solid tumors such as solid digestive system tumors. Suitable viruses are influenza, herpes simplex, hepatitis, and especially rabies virus.

Administration of anti-idiotype antibodies in accordance with this invention stimulates subjects, particularly mammals, and even humans to produce anti- (anti-idiotype) antibodies that identify epitopes on tumor cells or viral particles.

[Detailed Description of the Invention]

The present invention provides a unique strategy for cancer therapy and viral immunity. Traditionally, strategies in tumor therapy have involved administering anti-tumor antibodies (ie, antibodies that identify epitopes on solid tumor cells) to patients who intend to destroy the tumor. Traditionally, antiviral therapy has meant immunization with prepared vaccines. However, Applicants have discovered that anti-idiotypic antibodies to antibodies that recognize tumor or viral antigens can elicit an immune response against the patient's own tumor or virus. Inducing this immune response is beneficial as a therapeutic and, at least in the case of viruses, as a prophylactic.

Although the applicant does not wish to be bound to any particular theory of practice, the immune response achieved by the present invention results from the interaction between the anti-idiotype antibody molecule and the patient's immune system. I think that the. The idiotype (ie, variable) region of an anti-idiotype antibody molecule contains antigenic determinants (epitopes) that are considered by the patient to be antigens. This is anti- (anti-idiotype) in patients
Induces the production of antibodies. Some of the anti- (anti-idiotype) antibody sets are directly complementary to the anti-idiotype antibody parotope. The anti-idiotypic antibody parotope reveals an "internal" image of the tumor cell or viral epitope identified by the idiotypic antibody (i.e., selectively bound), and thus the anti- (anti-idiotype) antibody. Is also believed to bind to tumor or viral antigens. In fact, the method elicits an immune response against tumors and viruses by allowing some of the antibodies produced by the patient to develop antigens (a palotype of anti-idiotype antibody) that are essentially indistinguishable from tumor or viral antigens. .

Surprisingly, the method is an effective way to suppress tumor growth or elicit an immune response against the virus. It has some advantages over many more conventional methods. First, most foreign antigens need not be administered to the patient. Second, the patient's anti-idiotypic response favors and is decisive for the desired effect. Third, the patient's own antibodies are antitumor or antiviral antibodies, thus obviating the need for repeated administration of exogenous antitumor antibodies. Other advantages will be readily appreciated by those skilled in the art.

The idiotype of an antibody is defined by the individual overt antigenic determinants in the variable or idiotype regions of the antibody molecule. Some of these idiotypic determinants are located on or near the antibody parotope, others in the variable region framework. Each antibody has its own idiotype, but in the following, specific antibodies are indicated by the following terms. “Idiotype antibody” or “Id Ab” is an anti-tumor or anti-virus antibody (ie, the epitope identified by the idiotype antibody is present on tumor cells or viral particles)
Means "Anti-idiotype antibody" or "anti-Id A
"b" means an antibody that identifies an epitope on the variable region of an idiotype antibody. Some of these antibodies are
An epitope that is a parotope of the idiotype antibody was identified, and thus represents an "internal" image of the epitope on the tumor cells identified by the idiotype antibody.
"Anti (anti-idiotype) antibody" or "anti-anti-Id) Ab
Is a resistance that identifies an epitope on the variable region of an anti-idiotype antibody. Some of the anti- (anti-idiotype) antibodies identify (i) an anti-idiotype antibody parotope and (ii) an epitope corresponding to an epitope on tumor cells.

As described below, the methods of this invention contemplate administering anti-idiotypic antibodies to a host organism. The anti-idiotype antibody is administered to the host in any physiologically appropriate carrier (eg, sterile pyrogen-free saline), the formulation of which is known in the art. The choice of carrier is not limited, and the antibody can be administered by any method that introduces it into the circulatory system (eg, intravenous, intramuscular, or subcutaneous injection).

The host organism can be any animal, but most commonly is a human and, in the case of viruses, also joins cats or dogs. The amount of most antibodies administered to a host will vary widely depending, for example, on the particular antibody used and the patient being vaccinated. It is only necessary to administer an anti-idiotype antibody in an amount sufficient to stimulate the production of anti- (anti-idiotype) antibodies by the patient's immune system. However, the total amount of antibodies used need not be very high, as only small amounts of antibody are required to elicit an immune response. In many cases, antibody doses will be from a few micrograms to a few milligrams (eg, about 50-200 μg to about 1).
A range of -5 mg) is sufficient. Determination of the proper dosage can be readily accomplished by one of ordinary skill in the art.

In one embodiment, the invention provides that a human is administered an anti-idiotypic antibody-containing preparation to treat a tumor, eg, a solid tumor (ie, a cancer, a sarcoma, a melanoma, etc. It is intended to elicit an immune response against a solid mass of malignant cells produced by In a preferred embodiment, the tumor is a gastrointestinal tumor.

As described above, the subclass of anti-idiotype antibody selectively binds (ie, identifies) to the anti-tumor antibody parotope (idiotype antibody). Formulations containing anti-idiotype antibodies can be administered to hosts where the anti-idiotype antibody parotope is the internal image of the viral antigen. Such antibodies recognize an epitope that is a parotope of the corresponding idiotype antibody. Anti-idiotypic antibodies that display an internal image of a tumor or viral antibody can be distinguished from anti-idiotypic antibodies that recognize framework determinants in the variable regions of the idiotypic antibody by any of several methods.
One method of identifying a desired anti-idiotype antibody is a competitive binding assay between a tumor or viral antigen (or hapten, if available), an idiotype antibody, and an anti-idiotype antibody. If the antigen interferes with the binding of the anti-idiotype antibody to the idiotype antibody, the epitope identified by the anti-idiotype antibody is closely related to the parotope of the idiotype antibody. Another test method is to determine whether antisera to anti-idiotype antibodies are also anti-tumor or viral antibodies. The above and other methods of identifying suitable anti-idiotypic antibodies are within the range known to those of skill in the art. Formulations for administration to a host can include anti-idiotypic antibodies to framework determinants, as well as subclasses of parotopes of idiotypic antibodies. It is only necessary that the formulation include a subclass for the parotope of the idiotype antibody.

The anti-idiotype antibody used may be a homologous or heterologous antibody against the host. However, the preferred antibody for humans is a human antibody with minimal immune response to the C region of the antibody molecule. However, since a relatively small amount of anti-idiotype antibody is required in the present invention, a heterologous antibody (eg mouse, rat, goat, rabbit etc.) may be used. Further, when there is no significant reaction to the heterologous anti-idiotype antibody, such antibody may be preferable in terms of easiness of production and cost. Further, a polyclonal antibody idiotype antibody can be used as well as a monoclonal anti-idiotype antibody.

Polyclonal antibodies Idiotypic antibodies can be prepared by conventional methods known in the art. For example, polyclonal anti-Id Ab can be produced by immunizing an animal with a monoclonal anti-tumor or viral antibody (eg, Id Ab). Immunized animals produce anti-Id Ab. This subclass of anti-idiotypic antibodies in this antiserum identifies epitopes that are parotopes of antitumor or viral antibodies. For example, antisera collected from animals are
(I) an immobilized antibody of the same isotype but different idiotype as monoclonal Id Ab for removing anti-isotype antibody from antiserum, and (i
i) Purified by serial absorption with an immobilized monoclonal Id Ab, whose subclass removes the anti-Id Ab that represents the internal image of tumor and viral antigens. The anti-Id Ab is then eluted from the bound monoclonal anti-tumor and viral antibody to obtain a solution substantially free of anti-isotype antibody. This solution can be analyzed for the presence of anti-Id Ab, which identifies the Id Ab parotope.

Monoclonal anti-idiotype antibodies that are substantially free of other antibodies are isolated from the supernatant of substantially pure immortal B lymphocyte cultures. "Immortal B lymphocytes"
Includes hybridomas (somatic cells hybridized with normal and malignant lymphocytes), and normal lymphocytes transformed with a virus (eg, Epstein-Barr virus) or oncogenic DNA, It includes relatively stable continuous antibody-producing cells that can be maintained in culture for several months (preferably indefinitely). Techniques for producing immortal B lymphocytes from normal B lymphocytes that produce anti-idiotype antibodies are known in the art. "Monoclonal Anti-Bodies" (edited by H. H. Kennett, T. J. McLean and K. Beetle, 1980); M. Sirayayer et al., "Hybridoma Technic" (Cold Spring Harbor Ladatory). , 1980); "Monoclonal Anti-Body's and Tassel Hybridomas," GJ Harmaring, You Harmaring and JF Keeney, 1981); Kosber et al., Proceeding of National Academy of Sciences (USA, 1982) Volume 79 6651
-6655; Jyona et al., "Hybridoma" (1983)
Volume 2, p. 124; "Monoclonal Antibodies and Funeral Cell Lines" (R.
Etch Kennett, KB Beetle and Tay
See Jay Matsukin, 1983); Kosber et al., "Immunology Today" (1983), Vol. 4, pp. 72-79.

Normal B lymphocytes that produce anti-Id Ab and are suitable for the production of Immortal B lymphocytes are provided by modifications of methods known in the art. For example, an animal such as a rat or mouse can be immunized with a monoclonal anti-tumor or anti-viral antibody and anti-Id Ab producing B lymphocytes can be harvested from the spleen of the animal. Human B lymphocytes producing anti-Id Ab are obtained by immunizing patients with monoclonal anti-tumor or viral antibodies. Peripheral blood lymphocytes are harvested from patients and the cultures are stimulated with monoclonal anti-tumor or viral antibodies to induce in vitro growth of anti-Id Ab producing B lymphocytes. Defreytes et al., Proceeding of National Academy of Science (USA, 1982), 79 volumes,
See pages 6646-6650. Thus, animal or human B lymphocytes producing anti-Id Ab can be harvested and fixed by methods known in the art. Of course, those lymphocytes that produce anti-Id Abs that reveal an internal image of tumor cells or viral antigens need to be distinguished from B lymphocytes that produce anti-Id Abs for framework determinants on the idiotype region. Is.

The method of the invention for application to tumors is also described in US Pat.
It can be practiced with other methods of eliciting an immune response against a tumor, such as the viral tumor cell debris vaccine described in 4108983. With respect to viruses, in the case of viruses, if desired, the immune response generated in the host mammal will be exacerbated by immunization with known viral vaccines in addition to administration of anti-Id Ab as described above. After the productivity of anti- (anti-Id Ab) is induced in the host mammal by administering anti-Id Ab (eg, about 2 weeks post-administration), the host is infected with the virus using conventional techniques known in the art. Give a single dose of vaccine (eg, an inactive viral vaccine such as the HDC rabies vaccine). Protkin et al., American Journal of Epidemiology (1976), 103, 75-80; Victor et al., Rabbie's Waxin Huo Human Youth, No. 40.
See page 9 (1978). Vaccine types for administration and experimental methods are known in the art. The following examples are illustrative of the invention and do not limit the scope of the invention.

In Example 1 tumor antibodies (monoclonal idiotype antibody) following experiment, mouse monoclonal antibody 17-1A that binds human digestive system cancer cells, was used C ₄ 2032 and C ₄ 1472. These are described in Harlin et al., "Producing of National Academy of Sciences (USA, 1979), Vol. 76, pp. 1438-1442, and in Koprovsky, Monoclonal Antibodies in Canon. Monoclonal antibodies in the body "; in the Proceedings of the Forth Armand Harmer Canceler Symposium (B-Boss Earl Langman, I. Troublitz and Dalbetzko, 1983). Monoclonal antibody (MAb), C ₄ 2032, has specificity for Mr 180, 160, 50 and 40K colorectal carcinoma (CRC) binding antibodies. _{MAb C 4 1742 (IgG 2 a} ) has specificity for CRC associated antigens Mr50K. MAb A ₅ C ₃ against hepatitis virus
This is also used, but this is described by Vans et al., Proceeding of National Academy of the
Science (USA, 1981), 78, 1214-1218. Mab17-1A (IgG ₂ A, XL chain) and C ₄ 20
32 (IgG ₂ a, xL strand), Ai et al., Immunochemistry Chemistry (1978) Vol. 15, Protein A · Sepharose column as described in pp. 429-436 (Pharmacia, Pisukatoauei, Niyujiyaji) Africa in It is purified from the ascites obtained from the hybridoma-bearing mouse by subjecting it to initial chromatography.

Patients All patients had metastatic and recurrent colorectal adenocarcinoma and were treated ascites in Balb / C mice using the method described by Sears et al., Lansett (1982) 762-765. A purified, pyrogen-free, sterile product of MAb 17-1A concentrated from was injected systemically in a single dose. Mab17-1A 192m
Seven out of nine patients injected with g or less developed anti-myoglobulin antibodies. Monoclonal antibody 200-1000mg
Of the 20 patients who received the treatment, 3 developed anti-mouse globulin antibody. To isolate anti-idiotypic antibodies from the sera of the first group of 3 patients (patient nos. 07, 08 and 09) and the second group of 2 patients (nos. 14 and 23) that developed anti-myoglobulin molecules. To choose or processed. The sera used to isolate anti-idiotypic antibodies from patient numbers 07, 08 and 23 were obtained when all three patients showed high levels of anti-myoglobulin antibodies. Patient No. 08 received a second injection of 130 mg of the monoclonal antibody 20 months after the first injection, and the serum obtained after the second injection was used for a screening test for the presence or absence of anti-idiotype antibody.

(Production of Polyclonal Anti-Idiotype Antibody) Purified MAb 17-1A (300 μg) emulsified with Freund's admixture was subcutaneously injected into multiple sites of New Zealand white rabbits, and 30 days later, 100 μg of monoclonal antibody was reinforced by intramuscular injection. Serum was collected 10 days after the second reaction.

Antisera were absorbed with immobilized MAbs C ₄ 2032 and MAbs 17-1A. Purified monoclonal antibodies (30 mg each)
Gel 10 [Bio-Rad Laboratories, Richmon
d, CA)] 2 ml. Then, the antiserum was the immunosorbent MAb C ₁ 2032 and MAb17-1A is continuously absorbed to remove anti-isotype and anti-idiotype antibody, respectively. Absorbed antibody in 0.1 M glycine buffer (pH
Elute with 2.8), immediately neutralize with phosphate buffer and dialyz with phosphate buffered saline to remove protein. It was quantified by the absorbance of.

The antiserum obtained from the patient after a single injection of MAb17-1A was also purified as described above. MAb17 respectively
-1A 750 mg patient number 23, and MAb 17-1A
Serum from Patient Nos. 08 and No. 07 administered 133 and 125 mg was collected at various times after the first injection of antibody.

Samples that were found to contain anti-murin IgG antibodies by the radioimmunoassay method were treated with immunoadsorbents MAb C ₄ 2032 and MAb 17 to remove anti-isotype and anti-idiotype antibodies, respectively, as described above. Absorbed well at -1A. The anti-idiotype antibody isolated from serum was shown to be an immunoglobulin by binding to ¹²⁵ I-labeled anti-human F (ab ') ₂ fragment. The yields of anti-idiotype proteins obtained from serum samples were different, 13 μg / ml from No. 08 and 8.9 from No. 07.
μG / ml, and 43 μg / ml from No. 23. Serum No.23 showing the highest level of anti-mouse immunoglobulin antibody
The highest amount was obtained.

(Serum screening method for determining the presence / absence of anti-idiotype antibody) In order to examine the presence / absence of anti-idiotype antibody in a serum sample, a competitive analysis was carried out by using ¹²⁵ I-labeled MAb 17-1A and four preincubated human sera and house Rabbit anti-idiotype antibody was used.

Quarter-inch (6.35 mm) particle size polystyrene beads [Precision Plastics Corporation, Chicago, Illinois (Precision Plastic Ball C
, Chicago, IL)] was washed 3 times with 95% ethanol.
Air-dry the beads with 0.02 M sodium tetraborate (pH
Incubated with rabbit or human anti-idiotype antibody diluted in 8.2). After overnight culture at 4 ° C. with gentle shaking, the beads were washed 3 times with phosphate buffered saline, 2% bovine serum albumin and 0.04% NaN ₃
Incubated for at least 3 hours at room temperature with phosphate buffered saline containing The beads were then exposed to a source of human anti-idiotype antibody and MAb 17-1A labeled with ¹²⁵ I as a reference for pre-incubated idiotype. That is, human serum was diluted with Ca ²⁺ and Mg ²⁺ free phosphate buffered saline to a concentration of 25% and supplemented with 2% bovine serum albumin and 0.04% NaN ₃ . After further culturing overnight, the beads were washed and the bound radioactivity was measured with a gamma ray measuring instrument.

Three of the sera obtained after single injection of monoclonal antibody (No. 23,09 or 14) showed higher binding inhibition to MAb 17-1A than before injection of monoclonal antibody. Patient number 1
The sera obtained after injection of the monoclonal antibody of 4 had lower binding inhibition values than those of the other two sera, but were higher than those of the same patients before administration of the monoclonal antibody. Patient number 7 days after the second injection of monoclonal antibody
The inhibition value of the serum obtained from 08 was already high.

(Competitive analysis of anti-idiotype detection) As described above for measuring the binding of the separated anti-idiotype antibody to MAb 17-1A as well as to monoclonal antibodies C ₄ 2032, C ₄ 1472 and A ₅ C ₃ . Competitive analysis was performed by various methods.

The results showed that the binding of anti-idiotype antibodies from three sera (Nos. 08, 07 and 23) to MAb17-1A was significantly higher than that of the other three monoclonal antibodies, and that of the other two Monoclonal antibody (C ₄ 2032 and
C ₄ 1472) also detected antigenic sites on colorectal carcinoma cells. However, these sites became NAb17-1A recognition sites. Immunoglobulins isolated from sera of all three patients prior to exposure to MAb17-1A were concentrated to approximately 2.5 μg / ml and bound to polystyrene beads. However,
The product did not bind any of the monoclonal antibodies tested, indicating the absence of anti-idiotypic antibodies in the serum prior to treatment.

(Cross-reactivity of human anti-idiotype antibody comrades) A competition analysis was performed to determine whether there was a cross-reactivity of human anti-idiotype antibody comrades.

The results of the competition analysis show that patient number 07 and
There was a marked cross-reactivity between the 23 anti-idiotype antisera, with a slightly weaker (but still important) cross-reactivity between the anti-idiotype antibodies of patient numbers 08 and 23. Similar cross-reactivity was observed between pre-monoclonal antibody treated serum from patient # 08 and patient # 07 anti-idiotype serum (results not shown). The above results show that anti-idiotypic antibodies produced by other patients are directed to the same antigenic site.

(Epitope detected by anti-idiotype antibody) Papten inhibition of human anti-idiotype antibody binding to MAb 17-1A was performed to show that the anti-idiotype antibody is directed against the pariotope of the idiotype antibody.

A 3M KCl extract of SW1222, a cell line obtained from colorectal cancer, was prepared by Harlin et al. [Journal of Clinical
Immunology (J. Clin. Immunol., Vol. 2, pp. 135-140]. This formulation is MAb 17
It bound to -1A, indicating that this material contained soluble antigen. MAb17-1A labeled with ¹²⁵ I was incubated with CRC cell extract and melanoma 3M KCl extract that did not bind MAb17-1A. Then the antibody-antigen mixture,
In addition to beads coated with anti-idiotype antibody from patient no. 23, the binding was compared to the binding of only radioisotope labeled monoclonal antibody. These experiments were performed with unsaturated amounts of iodide monoclonals to detect changes in binding due to small amounts of competing haptens. As a control, iodide MAb 17-1A was mixed with an extract derived from melanoma, which is known not to bind MAb 17-1A. CRC cell extracts at concentrations of 0.1 or 0.5 mg / ml were found to inhibit binding of anti-idiotypic antibody from patient number 23 to iodide MAb 17-1A at 39% and 68%, respectively. Extract from melanoma is 0.5 mg / ml
The following concentrations have no significant effect on monoclonal antibody binding.

Hapten inhibition of this binding reaction reveals an "internal image" of the CRC epitope on the anti-idiotype antibody.
This was also confirmed by the fact that CRC cell extracts do not bind anti-idiotypic antibodies, but, as expected, bind MAb 17-1A.

(Production of anti-idiotype and anti- (anti-idiotype) antibody by human B lymphocytes) Patient number 0 at 12 and 6 months after injection of MAb17-1A, respectively
Buffy coat cells were obtained from 8 and 23. Cells at 10 ng / ml F (ab ') ₂ fragment of 17-1A as described by Defreitas et al., Proceeding of National Academy of Sciences (USA, 1982) 79: 6646-6650. Stimulated. For the following 7 days, T and B were treated by rosetting aliquots of cells with sheep erythrocytes treated with 2-aminoethylisouronium bromide.
The cells were separated into populations. [Perorino et al.,
See Clinical Immunology and Immunopathology (1975) Vol. 3, pp. 324-333. Both cell populations were stained with the F-1 (ab ') ₂ fragment of 17-1A or anti-influenza monoclonal antibody and goat anti-mouse Ig-FITC. The cell population was then analyzed by cytofluorography. In addition, peripheral blood mononuclear cells derived from the same patient were modified with 17-1A F (ab ') ₂ fragment or anti-influenza monoclonal antibody to produce specific human Ig as described in Decratus et al. (Supra). Stimulation was carried out for 9 days in Mitsui Lutton medium. Supernatants from these cultures were analyzed by solid phase enzyme-linked immunosorbent assay for specific human IgG. [KPL Laboratories, Gaithersburg, M
D)] The proportion of lymphocytes that initially bind to 17-1A F (ab ') ₂ in patient number 08 is 1.2% and that in patient number 23 is 0.2%.
It was. 17 days after incubating with MAb17-1A
The proportion of lymphocytes of patient # 23 that specifically bind to F (ab ') _{2 of} -1A increased from 0.2% to 13%. All cells that bind 17-1A were present in the B cell population. After a further 9 days, human anti-MAb17-1A IgG was detected. Incubating lymphocytes from the same patient with anti-influenza monoclonal antibody under the same conditions did not produce any detectable human Ig against MAb 17-1A or anti-influenza monoclonal antibody.

In another experiment, human B lymphocytes were stimulated to produce anti- (anti-idiotype) antibodies. B lymphocytes were harvested and stimulated in vitro using the method described above except that they were stimulated with autologous anti-idiotype antibody rather than idiotype antibody. Stimulated B lymphocytes produce anti- (anti-idiotype) antibodies, and these anti- (anti-Id) Abs produce CRC.
It has been shown to identify epitopes present on cell extracts and whole cells. Thus, the human immune system produces anti-tumor antibodies in response to stimulation with anti-idiotype antibodies.

Immortal B Lymphocytes Producing Anti-Idiotype Antibodies Many methods for producing immortal B lymphocytes secreting monoclonal antibodies are known in the art. By Kosber et al.,
[Immunology Today Volume 4 72-
Page 79]. Therefore, human B lymphocytes secreting anti- (anti-idiotype) antibodies obtained from peripheral blood lymphocytes as described above can be immortalized by one of the known techniques.

One easily available method is to immortalize with the EB virus (EBV). According to this method, the above-mentioned normal lymphocytes are infected with EBV in vitro, and an immortal cell line is established, for example, by limiting dilution on the nutrient layer. See Kozibar et al., [Cited above (1983)], and the references cited therein, pages 51-60.

Another strategy is to fuse either a human plasmacytoma or a lymphoblastoid fusion partner with either the anti-Id Ab secreting lymphocytes or EBV transformed lymphocytes described above. For example, EBV- that secretes anti-Id Ab
Transformed B lymphocytes can be fused with human lymphoblastoid cell line KR-4 and the like. The hybridomas of interest are then selected in ouabain-containing hypoxanthine-aminopterin-thymidine medium, which excludes parental cells. The hybridomas are tested for specific antibody production. The positive hybridoma is then cloned, recloned and propagated in the peritoneal cavity or in large amounts of medium of an immunosuppressed mammal (eg, nude mouse). By Kotsuba and others,
See Proceedings of National Academy of Sciences (1982) 79: 6651-6655.

Example 2 Viral antibody (Production of anti-idiotype antibody) An anti-idiotype antibody (anti-Id Ab produced in response to various monoclonal antibodies (mAb) to reconstitute an epitope of rabies virus glycoprotein (G). )
Studied the ability of. This G is a virus neutralizing antibody (VN
Respond to important biological properties of many rabies viruses, including inducing A). It is not currently known exactly which part of G responds to the above functions. Rahun et al., Journal of General Birology (1983) 64: 843-851, suggests that there is at least three major antigenic sites for type-specific VNA, the rabies virus G challenge range virus standard. A functional epitope mat against the (CVS) strain is described.

Five anti-G mA's from a large panel of hybridomas based on the binding site and isotype on rabies virus defined by functional epitope mats (purifiable by Protein A Sepharose chromatography)
I picked out b. Anti-rabies virus G mAb 509-6,101-
1,507-1 and 719-3 have already been described. Rahun et al. [References] and Victor et al. [Journal of Experimental Medicin (19
80) 152: 99-112. These mAbs that strongly neutralize rabies virus infection have the following properties: : 509-6 (epitope Matsupu site I; IgG ₂ a), 1
01-1 (epitope Matsupu site IIb; IgG ₂ a). 507-1
(Epitope map site IIIb; IgG ₁ ) and 719-3 (epitope map site IIc; IgG ₂ a) anti-GmAb 1104-2 (Ig
G ₁ ) was obtained by additionally fusing the mutant strain 653 of P3x63Ag8 mouse myeloma cells and spleen cells derived from BALB / c mice immunized with rabies virus. Wicker et al .; [Proceeding of National Academy of Science (USA, 1978) Vol. 75, pages 3938-3942, by Karney et al .; [Journal of Immunology (19
(1979) 123: 1548-1550]. Anti-G secreting hybridoma cells are selected and cloned in limiting dilution and ascites fluid prepared as previously described by Victor et al .; (supra) (1978). 1104-2 mAb was chosen because it shows very good binding to ERA virus but poor neutralization. Anti-rabies virus nucleocapsid mAb 515-3
(IgG ₂ a) and 389-1 the (IgG _1), was isolated from BALB / c mice immunized with cherub of virus similar techniques.

The stocks of ERA or CVS strain of rabies virus used were grown in BHK-21 by standard methods. Victor et al., Laboratory Technology in Labies 101
See page 123 (M Kyaplan & H. Koproski (Edited 3rd edition, 1973). Rabies antigen mutants ERA RV194-2 and RV509-6 are anti-
It has been reported as representing a virus that is resistant to neutralization by G mAbs 194-2 and 509-6. Volume 80, Proceeding of National Academy of Science (USA, 1983)
See pages 70-74. Rabies Glycoproteins (Gs) have been reported by Dates Skold et al.
Purified from virus particle depleted medium using immunosorbent chromatography as described on page 337.

All anti-GmAbs were purified from ascites. Antibody isotypes IgG ₂ a is Britton - were diluted to 1 in approximately 30 minutes Robinson buffer (pH.8.0). Gerhard et al., Monoclonal Antibodies, pages 317-333, (R. Kennett.
Matsukeen and Kay. See Betchitor (1980). The mAb in BRB is passed through Naeildine 0.45μ paper and a Protein A Sepharose 4B adsorption column [Falmatia
Piscataway, New Jersey (Pharmacia, Pisc
ataway, NJ). The column was washed with 30 ml of BRB (pH 8.0) before eluting the antibody to BRB at pH 3.0. First, anti-GmAb of isotype IgG ₁ was precipitated with sulfuric acid to a final concentration of 18% (weight ratio). Dissolve Ig fraction, BRB (pH8.0)
It was dialyzed against and passed through a Protein A Sepharose column as before. Ig eluted from the column was used as the antigen for ERA
It was detected by radioimmunoassay (RIA) using a virus. Dates Cold et al. [Journal of
Birology (1982) Vol. 44, p. 595-602]. The antibody was concentrated by vacuum dialysis against phosphate buffered saline (PBS), (pH 7.4). The protein concentrate was quantified using bovine serum albumin as a standard. See Bramhall et al., Analytical Biochemistry (1969), Vol. 31, pp. 146-148.

Anti-Id Ab was prepared as described by Stout et al., Journal of Experimental Medicins (1983) 157: 687-704. Briefly, female New Zealand White Rabbit emulsified with Freund's Complete Adjuvant (FCA) Protein A Sepharose purified mAb
300 μg was injected subcutaneously at multiple sites along the breast chain. 100 μg of antibody in PBS was added to booster antigen 7 and 30 twice.
The drug was administered intramuscularly for 10 days, and serum was collected 10 days later. To remove anti-isotype Ab mAb515-3 (IgG ₂ a) or 3
Sepharose 4B bound to any of 89-1 (IgG ₁ )
Each anti-idiotype antiserum was loaded onto the column to make it specific for the idiotype region. The eluate obtained from the above column contained an antibody reactive with the idiotypic determinant. Antibodies against a certain region, 0.1M
MAbs 515-3 and 389-1 with diethylamine (pH 11.5)
It was eluted from the liquid-free absorption column. IgG derived from each anti-Id Ab
Were separated by Protein A Sepharose chromatography as described above. The reactivity of the column effluent with non-idiotypic determinants was negligible.

(Confirmation of anti-idiotype antibody) The specificity of the anti-Id Ab prepared above and the presence of the idiotype cross-reaction among various anti-GmAbs were confirmed by RIA.
The product was measured.

Solid phase RIA was used to measure the binding of anti-Id Ab to immobilized MAbs. Individual mAbs were diluted according to ascites concentration in carbonate-bicarbonate buffer (pH 8.9) as shown below. 509-6 (1: 3000), 101-1 (1: 6000), 719-3
(1: 6000), 507-1 (1: 6000), 1104-2 (1: 16000).
Next, 25 μl of each was added to the wells of a polyvinyl microtiter plate (manufactured by Dynatech Laboratories). These antibodies were incubated overnight at 37 ° C and dried in wells.
Free binding sites on the wells were blocked with 10% gamma-free horse serum (Gibco Laboratories) in phosphate buffered saline (PBSN) supplemented with 0.08% sodium azide for at least 1 hour. Add 25 μl of anti-Id Ab dilution,
After 1 hour at room temperature, the plate was thoroughly washed. Combined anti-Id
Ab is 25 μg of ¹²⁵ I goat anti-rabbit IgG (manufactured by Kuppel Laboratories) labeled by the iodination method (30000 counts / count).
Min) and then incubated for another hour at room temperature. See Markwell et al., Biochemistry (1978) 17: 4800-4817. Anti-I
d Ab diluted solution or radiolabeled probe
Made with 10% gamma-free horse serum inside. The plate was washed until there was no unbound probe, and the radioactivity bound to each well was measured with a gamma ray counter.

Titration of each anti-Id Ab product with homologous and xenogeneic mAbs revealed that each anti-Id Ab was specific for the homologous Id mAb.

Quantification of anti-idiotype antibody to pariotope of idiotype antibody A competitive RIA method was devised to test the ability of rabies virus G to inhibit the interaction between Id Ab and anti-Id Ab. Chaflin et al., Journal of Immunology (J.Immumol., 1974), 112: 1747-1756], and Sher et al., Journal of Immunology (J. Im.
mumol., 1972) 109: 176-178.

Rabies Gs was used as a competing antigen. Prior to the addition of the standard anti-Id Ab diluent, a pre-titration level of mAb was incubated with a 2-fold serial dilution of Gs for 1 hour. The amount of bound antiserum was quantified by binding a goat anti-rabbit antibody labeled with ¹²⁵ I. Corresponding anti-GmAb (509-6, 507-1,
And Gs inhibited the binding of 3 out of 5 anti-Id Abs to 1104-2). Maximum inhibition is 20-5 at 6 μg / ml Gs
Although it changed to 0%, a decrease of at least 15% was observed even at only 0.75 μg / ml Gs. The inability to totally block anti-Id Ab binding indicates that both framework and parotope site specificities are present in the three polyclonal anti-Id sera. This means that mAbs and anti-Id Abs separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a reduced or non-reduced state.
The reactivity of Id Ab to reduced mAb was specific for the framework determinants, as the antigen binding site was confirmed by reduction of mAb, which was confirmed by Western blot assay to measure the reactivity of E. coli. It will be. The absence of significant binding inhibition of anti-Id101-1Ab or anti-Id719-3Ab by Gs suggests that the parotope-specific population in the serum is weak or absent.

(Production of anti- (anti-idiotype) antibody) In the following example, anti-Id Ab having reactivity with the antigen-binding site of anti-G mAB is a sub-antibody that mimics the above-mentioned anti-G mAb-recognized viral epitope. Contains bobureucation,
It is shown that an immune response against G can be induced.

ICR mice divided into 4 groups were subcutaneously injected with FCA emulsified protein A sepharose-purified anti-Id IgG, 40 per mouse.
μg of each was inoculated. Sub-challenge subcutaneous inoculation is 7th and 32nd day
40μ in Freund's complete adjuvant
g anti-Id IgG was included. Blood was collected from the retroorbital plexus for 5 days following the final booster inoculation, and the collected serum was examined for rabies virus neutralizing antibody (VNA). As a control, another group of mice was immunized with normal rabbit IgG purified with protein A-sepharose.

The concentration of VNA in immunized mice was measured by a modified version of the rapid fluorescence focus inhibition assay. Smith et al. Laboratory Techn Labs
iques in RabIes M. Kaplan and H. Koprowski, et al., ed., 3rd edition, 1973): 354-357. Make a 2-fold serial dilution of mouse serum on a Microtiter II plate (Falcon Plastics) (50 μl / well) and use 10 ⁴ PFU.
1 at 37 ° C with an equal volume of virus containing / 50 μl
Incubated for an hour. BHK-21 cells (2 × 10 ⁶ cells / μl) newly treated with trypsin after incubating
50 μl was added to each well and mixed. Then, 10 μl of each of the serum / virus / cell mixture was transferred (in two times) to the well of a Terasaki plate (Falcon Plastics). After incubating for 20 hours, the plate was first washed with PBS, then with 80% (volume ratio) acetone in distilled water, and fixed in 80% acetone at room temperature for 30 minutes. The plate was dried, and the cells were stained with 5 μl / well of a fluorescent conjugate rabbit-derived anti-rabies nucleocapsid antibody at 37 ° C. for 30 minutes. Victor Symposium on Series Immunobiological Standard (Symp.Serie
Immunobol. Standard) 21: 102-118. Control wells (containing virus but no antibody) showed approximately 40% of cells containing rabies virus-specific content.
The endpoint of virus neutralization was defined as the maximum serum dilution reciprocal that could reduce the number of cells infected with rabies virus by 50%.

The results of the rapid fluorescence focus inhibition test are shown in the table below.
Mice immunized with anti-Id509-6 Ab and anti-Id1104-2 Ab
The ERA strain produced a significant VNA titer against rabies virus.
The other three anti- (anti-Id) Abs did not neutralize ERA virus. Control experiments showed that mouse anti- (normal rabbit IgG) Abs as well as the anti-Id Ab used to immunize do not have rabies virus neutralizing activity. In addition, anti-Id509-6Ab
When anti- (anti-Id509-6) serum was pre-incubated with it, the neutralizing activity was lost. However, the activity was not lost even when pre-incubated with normal rabbit serum.

Other rabies viruses were used in neutralization assays to test the specificity of the resulting VNA. Rabies CVS strain is mAb 509-6
And 1104-2 both bound (data not shown here), and the table shows that CVS is neutralized by both anti- (anti-Id509-6) and anti- (anti-Id1104-2) sera. It is shown that. On the other hand, R, which is a mutant strain of ERA virus
V509-6 is the result of mutating the 509-6 epitope mAb509
Although it loses the binding and neutralizing activity by -6 (J. Ge.
n.Virol.1983) 64: 843-851), the virus was not neutralized by anti- (anti-Id509-6) serum.
Another neutralizing resistance mutant RV-194-2 is anti- (anti-Id509-6)
Neutralized by serum. The previous results show that anti-GmAb509-6
And that the antigenic sites recognized by 194-2 are completely independent. See Raton et al., Supra. This data is
Anti- (anti-Id509-6) sera reacted only with the epitope recognized by anti-GmAb509-6, thus the above epitope
It shows that it was imitated by Id509-6Ab.

The above experiments are presented for the purpose of explanation only and are not intended to limit the invention, which is defined only by the claims.

Front Page Continuation (72) Inventor Koprofsky, Hillary 19096 Pennsylvania, USA, Winwood, Fairhill Hill Road 334 (72) Inventor Regan, Calvin United States Delauea 19809, Weicliff, South Cliff Drive 21 (72) Inventor Victor, Tadeouts, USA 19096 Pennsylvania, Winwood, Downs Circle No. 9 (56) References The Journal of Immunity, vol. 121, no. 6 (1978), PP. 2358-2362 Proc. Natl. Acad. Sc i. USA, vol. 79 (1982), PP. 6651-6655 J. Exp, Med, vol. 157 (1983), PP. 687-704 Clinical Immunology and Immunopathology, vol. 21 (1981), PP. 397-406

Claims (13)

[Claims] 1. An anti-idiotype antibody which identifies an epitope that is a parotope of an anti-tumor antibody capable of inducing an immune response against a tumor in vivo. 2. The anti-idiotype antibody according to claim 1, which is a monoclonal. 3. The anti-idiotype antibody according to claim 2, which is substantially free of other antibodies. 4. The anti-idiotype antibody according to claim 1, which is polyclonal. 5. The anti-idiotype antibody according to claim 4, which is substantially free of anti-isotype antibody. 6. The anti-idiotype antibody according to any one of claims 1 to 5, which is produced by immortal B lymphocytes. 7. The anti-idiotype antibody according to claim 6, wherein the immortal B lymphocyte is a hybridoma. 8. The anti-idiotype antibody according to claim 1, wherein the tumor is a solid tumor. 9. The anti-idiotype antibody according to claim 1, wherein the antitumor antibody is a monoclonal antibody. 10. An anti-idiotype antibody for identifying an epitope that is a parotope of an anti-tumor antibody, which induces an immune response against a tumor in vivo.
A composition for inducing an immune response against a tumor. 11. An anti-idiotype antibody, which comprises administering an anti-tumor antibody to a heterologous host (excluding human) and isolating an anti-idiotype antibody that induces an immune response against a tumor in vivo from the serum of the host. A method for producing an anti-idiotype antibody for identifying an epitope that is a parotope of a tumor antibody. 12. The method according to claim 11, wherein the antitumor antibody is a monoclonal antibody. 13. The method according to claim 11, wherein the epitope is on a solid tumor.