JPS58201717A - Tumor immunological medicine and manufacture - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION This question relates to immunotherapeutic and chemotherapeutic products and methods of treating a host with cancer.・A, Forced flow 1a
Forced flow electrophoresis
rophore+sim) was maintained in extracorporeal perfusion,
This is a technique that electrophoretically separates a fraction rich in gamma globulin from a heparinized surface (Reference 1
~5). The numbers indicate the references listed at the end of the detailed description of the invention. The general technique is similar to that used in artificial kidneys, and more particularly to electrodialysis of blood (6). Forced-flow electrophoresis (FFE) was first initiated as a large-scale electrophoretic method for the rapid fractionation of biochemical materials to purify isoelectric protein components (
Reference 5). The mechanism principle of forced flow electricity is that at neutral pH,
All plasma proteins except gamma globulin move in an electric field. This principle is used to recycle all plasma proteins and separate 1 gamma pzolin. 9- Electrofiltration and its application to water purification (Reference 7)
, electroabsorption of Nonocutilia phage (Reference 8), electrodialysis of blood, electroosmosis, desalination of Tan, Tsuku solution (Reference 9)
) has been found to be a suitable method for studying various Ω-membrane phenomena such as FFE can be designed for on-line operation and has been used to remove immunoglobulin from dogs and prevent or delay kidney transplant rejection (10). To the inventor's knowledge, the use of this device to treat a host with cancer has not been previously done.

During the course of FFE, an effluent 7 (EIE) is obtained which is rich in gamma globulin and also contains other plasma proteins. Prior to the present invention, this effluent was discarded without further treatment.

The present invention takes advantage of the collection of this effluent by an FFE device, its subsequent processing and readministration to a host with cancer.

Protein A is produced by Staphylococcus aureus cowans 1 (ataphyloaoaus aura).
usCowanm 1), which reacts with the Fo fragment of immunoglobulins from many mammalian species to form immune complexes that fix complement and consume complement components. (References 11 to 16). Previous studies on dogs with spontaneous mammary carcinoma, which are good models for human chest pain, have shown that a continuous flow plasma cell separator (a continuous plasma cell separator) was fixed in a microporous membrane filtration system.
flou plammm calls sparato
Staphylococcus (SpA) with protein A placed online so that the plasma comes out from
), plasma was circulated (Reference 17). After circulating one plasma volume into Staphylococci containing protein Ai, tumor necrosis followed by objective tumor regression was observed. These effects were not observed when plasma was circulated in Staphylococci lacking protein A (Reference 17). The finding that abscess regression occurs in this experimental canine system was confirmed in a separate study (18).

The plasma perfusion was then purified and treated with purified protein A immobilized on a collodion-charcoal matrix.
A collodlon charcoals PAC
A similar tumor necrosis reaction was observed after perfusion with plasma in C) (ref. rs).

This mode of treatment was applied to five human patients with thymic carcinoma, and a sharp tumoricidal response was observed shortly after perfusion into the PACC, and an objective tumor regression effect was observed in four patients. (Reference 20).

Previous studies on dogs and humans with spontaneous thymic carcinoma showed that when treated with immobilized 8pA or PACCK perfused plasma (ref. 17, 1920). PACC a small volume of plasma
After K perfusion, the chest wall tumor showed morphological changes and rapid onset of pain, suggesting that the effect was achieved by factors generated in the plasma after passage of the PACC. (References 17, 19.20).

This method was technically simplified as follows. That is, the on-11n extracorporeal circulation system is removed and a small amount of autologous or allogeneic aliquots, previously collected by phlebotomy, is instead transferred to PACC off-11n.
and then directly injected into the patient (Reference 2).
0). Patients treated completely with this extracorporeal plasma perfusion showed acute pain with morphological responses and tumor regression effects similar to those seen with the intracorporeal extracorporeal perfusion system (ref.
). Additionally, tumoricidal responses in both dogs and humans were accompanied by serological and immunohistochemical changes. That is, solid-phase C1q binding and C5a levels increased, and C3 in series with 03 and IgG deposition in visceral cell membranes decreased (
Reference 17.20.21).

8pA has the ability to bind to the IgGOFa region, and in
Not only does vltr6 produce complement activation and other effects, but 1nv1ma0 produces effects that resemble the immune response produced by antigen-antibody complexes. Some of these appear to contain complement activated by the 8pA-IgG13-complex in solution.

For example, when 8 pA is administered intradermally to a normal human using a small dose of 10 μg, swelling occurs, an erythematous reaction is detected after 30 minutes, and the strongest reaction is reached after 24 to 48 hours. Similarly, when a small amount of 10 μg of apA is given intradermally to guinea pigs, a pronounced Alluras-like reaction occurs, and a 300-4001JJl TetB1m1 reaction occurs, which is strongest within 12-24 hours (Reference 22).
.

Guinea pigs given 500-1000 μg intracardially receive a fatal anaphylaxis. This can be prevented by administering antihistamines in advance. H@asko et al. (Reference 2
3) showed that guinea pigs sensitized with as little as 50 μl of 8pA in complete 70% adjuvant showed a delayed response that was maximal at 24 hours, whereas guinea pigs sensitized with 8pA as low as 50μI in complete 70% adjuvant showed a delayed response that reached a maximum at 24 hours, whereas guinea pigs sensitized with 8pA as low as 50μI in complete 70% adjuvant showed a delayed response that was maximal at 24 hours, whereas guinea pigs sensitized with 8pA as low as 50μI in complete 70% adjuvant showed a delayed response that reached a maximum at 24 hours, whereas guinea pigs sensitized with 8pA as low as 50μI in complete 70% adjuvant exhibited a delayed response that reached a maximum at 24 hours, whereas guinea pigs sensitized with 8pA as low as 50μI in complete 70% adjuvant showed a delayed response that was maximal at 24 hours; When the same amount is given, the Alluras reaction reaches its maximum in 6 hours. In contrast to the spA
When given alone, no hypersensitivity reactions were observed. Rabbits fed up to 6 da SPA by injection alone and lO
Mice given ~500 μg showed no early onset or delayed response (refs. 24,25). However, 1
00-180 Da human IgG was administered intravenously, followed 10-15 minutes later by 0.025-21.51 N9 8p.
Within 5 to 10 minutes of intradermal administration of A, a direct Alluras reaction occurs in rabbits, and preformed human IgQ and S
Hemorrhagic necrosis was observed 18 to 24 hours after intradermal injection of the precipitate with PA (Reference 24).The soluble complex of rabbit IgG and 1!1I8PA was released into the circulation of rabbits through the liver and spleen. It was promptly removed. Also, up to 49% of the label complex was removed during one pass in 1nVitro.
The overflow of physiological saline was taken up by the rabbit's liver.

Sp added to humans, guinea pigs, rabbits or pigs
A is 08PA, which is known to weaken or even significantly decrease complement activity, depending on the species, and human normal or bone f4jm! IgG (normal guinea pigs collected with IgG or Fo fragments or SpA) had a similar effect (ref. ao,
at ). 8pA and guinea pig subclass IgG1
, the complex with both IgG1 fixes complement, while I
The antigen-antibody complex ri containing gGB was not immobilized (References 30 and 31). Adding 89 μm to whole human serum resulted in a complement titer of 3 for all 9 of complements, except for C7, which was not determined.
s % (C3) decreased from 0% (C8), with optimal suppression occurring in IgG excess. Fe7 fragment B of complement fixing myeloma IgG prepared in maximally active conditions
The preformed complex with p^ reduces the overall complement titer to 6
4%, increasing individual complement titers from 4% to 99% (C
3) K decreased (Reference 14). spA also suppresses the binding between C1 and heat-aggregated IgG or IgGf myeloma protein, or has no effect on the binding between C1 and IgG (Reference 14). Decrease in complement activity is associated with 5 pk (D Ig
Depends on the amount relative to G.

As the dose of 8pA serum was increased, there was a maximum level of complement activity, which decreased at concentrations up to 89% (ref. 14).
26.2g). This effect is explained as meaning that high concentrations of 8pA suppress C1 binding. Experiments by Langon et al. revealed the effect of SPA on antibody activity. IgG and 8p formed with relatively high or low doses of SPA
The complex with A behaves like authentic 1gG. Experimental formula (
(IgG) map person] n'f: 8pA-IgG 9
M-conjugation is important in terms of hemolytic ability, as well as total complement or purified CI.
It behaves like IgM in that it interacts with Rabbit Fe is a monovalent 7-ragment BK by fluorescence quenching method and light scattering method.
It was found that IgG and SpA had two binding sites, and that the complex was formed of 2 to 4 F(l fragments) cross-linked by SpA (Reference 35). complexation with a
ttachm@nt) (Reference 1)
3).

17- Human polymorphonuclear leukocytes promptly ingest and disassemble the insoluble complex formed by human IgG and SPA (References 36 and 37). Leukocytes release histamine in the presence of SpA (Reference 38). The degree of phagocytosis depended on the amount of 8 pA and optimally on the precipitate formed. The release equivalent of myeloperoxidase was synonymous with the phagocytosis of the complex. 12
Using 3I-labeled IgG, uptake was determined by neutrophil concentration,
It was clarified that this depends on the properties of the complex, the incubation time, and serum factors (Reference 36).

The present invention provides protein A after plasma instillation into the PACC.
- Demonstrate that an IgG complex is formed. IgG or serum and protein Alt- in various molar ratios.
When the homogeneous complex was cultured and then given intravenously to a tumor-bearing host, a tumoricidal response and tumor regression were observed. This provides a new product for cancer treatment.

For many years, it has been recognized that the antigen-antibody complexes in question, whether naturally occurring or artificially produced, have a tendency to react effectively at extreme pH ranges, either acidic or alkaline. It has been found to completely decompose and break down into its corresponding constituent components. As first shown by Camp-B@11 et al., when artificially produced bovine serum albumin-antibiotic serum albumin is made acidic, it decomposes into its constituent components (
Reference 39). This concept has been used in the field of immunology for the separation of antigens and antibodies that are present in the form of immune complexes. The method of decomposing the antibody from the antigen-antibody complex by acidification, removing the antigen by ultrafiltration at low pn, and then isolating the antibody was described by Schigren (8jogr@n) et al. in a mouse tumor system. It has been described (Reference 40). More recently, cytotoxic antibodies directed against leukemia-associated antigens, with latent potential, have been found in the serum during the acute phase of acute myeloid leukemia. After filtration, significant complement-dependent cytotoxicity was detected in most of the sera tested (ref. 41).
Dors@tt) et al. (Reference 42), salted out, and then
After culturing at pH 2.8, a tumor-specific antibody was isolated from the ovarian diagnostic fluid of ovarian cancer by slowly adding sodium hydroxide to the sample for neutralization. The liberated antibodies were tested by indirect immunofluorescence and showed a substantial increase in titer. The idea of alkalinizing solutions to break down immune complexes was first described by Kabat and May (43) and has been used for this purpose for several years. . □ To the inventor's knowledge, the acidification and alkalinization of forced-flow electrophoresis effluent or tumor-bearing plasma, or its application to cancer therapy, has not been performed before, and , has not been reported.

D. The use of immunoglobulin in oncology immunoglobulin single-bed drugs is based on basing (
nehrtng) and Kitasato's use of antivenoms, progress has been made to the clinical use of animal antivenoms for the prevention of disease in HiF. The use of human immunoglobulins for passive immunization began with the work of Mekhan et al., who isolated the glupurin fraction from placental extracts using ammonium sulfate (refs. 45, 46). In 1936, this fraction, which was found to be effective in preventing or mitigating measles, was called immunoglobulin (human) (47).

Major advances in this field came with the development of cold ethanol to fractionate immunoglobulins from human plasma, as well as in the treatment of immune deficiencies (ref. 47) and hepatitis (ref. 5).
0.51), with the introduction of a fraction, namely immune serum globulin, that is effective in the treatment of measles (References 48 and 49). The development of immunoglobulins in the United States consists primarily of specific immunoglobulins.

Products such as tetanus immune glusolin, anti-antibody glusolin, and rabies immune glusolin have firmly established themselves in clinical medicine. The newest of these products is hepatitis B immunoglobulin, while others, such as varicella21-, are still under investigation. Current formulations for intravenous use of gamma globulin□ include reduced, alkylated, salt fractionated, β-propiolactone-treated, and pfl-treated preparations (52-57). These formulations have shown efficacy and all exhibit a wide range of side effects. Side effects appear to be substantially reduced with formulation at pH 4, and there are also suggestions that slow infusion may avoid side effects. The ideal properties of gamma globulin are the absence of subdivision, the absence of aggregation and IgA, and the stability of the formulation. It is suitable for both intramuscular and intravenous use, and there is no risk of hepatitis.

The use of tumor-specific antibodies for specific active immunotherapy has followed the following steps. Attempts have been made to use xenogeneic serum, although this requires administration of xenogeneic serum for long periods of time with the risk of consequent allergic reactions.
In an early attempt, Llndstroom
rom) confirmed the response to administration of serum made in rabbits after immunization with bone marrow leukemia cells (Reference 58). Decal d Ruho (Daaarvalha) is
A normal antigen in tumor tissue was precipitated with an antibody against normal tissue, and hyperimmune globulin in horses against the antigen isolated from this antigen was prepared. Thirteen of the 15 leukemia patients showed a 2θ increase in immune serum globulin starting from 4 weeks.
There was a remission of Kezuki (Reference 59). Sekura (8)
were unable to find remission in five patients using heterologous immunoglobulin prepared using a slightly different method (Reference 60). Chirin/? vinegar(
Tslrimbas et al. treated five chronic lympholeukemia patients with equine antilymphocyte serum. Peripheral cell numbers fell to approximately 40% of their initial values (ref. 61). Using a globulin prepared for leukemia, Decarpalho observed objectively significant responses in patients with solid tumors. Marsh et al. administered rabbit antitumor serum to patients with solid tumors of Yuzu (Reference 62). As a result, although the antibody was selectively localized in the cadaveric sarcoma cell area, no clinical effect was observed. In recent attempts using serum prepared from myeloma-immunized chimpanzees, regression was incomplete and complicated by fatal thrombocytopenia in one patient;
Acute necrosis was observed in visceral disease in two of the three patients (63).

As with leukemia, allogeneic sera have been studied for therapeutic efficacy. Several attempts have been made to treat lymphoma and leukemia with serum containing antibodies against human lymphocytes. In a study by Laslow et al., antiphosphorus, e-cell plasma was prepared by immunizing normal subjects against normal lymphocytes (64). Then, IgG
was isolated from this plasma and administered to three patients with chronic lymphoid 9-cell leukemia. In each patient, there was a transient lymphopenia and a decrease in the size of the phalangeal nodes after the infusion. Djarasai treated four patients with acute lymphocytic leukemia using serum from patients with thalassemia, many of whom were sensitized to both granulocytes and phospho/4 cells. Peripheral leukocyte counts decreased in all 04 patients (Reference 65).

Hernoseman (H Rbsrman) used serum containing cytotoxic antibodies from pregnant women to treat seven patients with phospho-A proliferative disease (L).
ymphoprolifaratlv@dls+ord
sr@) was administered to the patient. In this patient, the number of phosphorus cells decreased to 11, and the platelet count averaged 48.8 to 2, compared to the pretreatment value.
8.5%, which lasted for one day. Two of the five patients showed shrinkage of peripheral lymphoid tissue, four showed minor side effects such as fever and chills, and two showed hyaline membrane disease (Reference 66).

(Left below) 25- Allogeneic sera in which the donor is purposely immunized against tumor cells are also being investigated. Printing Ham (B
Chaplin
) immunized normal donors with lymphocytes from patients with chronic lymphocytic leukemia. Normal subjects were reimmunized 3 years later with whole blood from a second patient with chronic myeloma leukemia. Anti-leukocyte antibodies active on donor cells were found in normal subjects. Gamma globulin was obtained from this donor, whose plasma was administered to leukemia patients whose cells had already been used for reimmunization. No clear positive effect was observed regarding the patient's illness (Reference 67). Skirkovich (5k
immunized patients with leukemia cells from other patients. Plasma was exchanged 2-3 times between donors in patient pairs 8-15 days after immunization. Six children were treated, complete in three and partial in five.
Hematological symptoms were observed. Seven of these patients received steroid and cell therapy, and one patient achieved complete clearance with immunotherapy alone (Ref. 68). Remission plasma was also given for passive therapy. Meholkovitz and colleagues collected autologous plasma and white blood cells during early remission and administered these to patients in late remission. It appears that the remission period was extended by this skillful method (Reference 69).
.

Using late remission serum from patients with Burkltta lymphoma, Ngn showed regression in patients with active disease (Reference 70).
. Serum from patients with regression of malignant melanoma,
Administration to other patients sometimes caused regression.

Researchers have attempted to develop antibody specificities that deliver cytotoxic agents directly to tumor cells in animal models. Chlorambucil
and daunomycin (Daunayain) are conjugated with antitumor antibodies (References 72, 7a, ).
, Antibody ha! "N.

It was labeled with a radioactive isotope such as (Reference 74).

They also contain toxins such as diphtheria (ref. 75).
and an enzyme with cytotoxic ability (Reference 76). A possible synergistic effect between antibodies and chemotherapy was described by Segling et al., in which Solmot's hepatoma cells, which normally resist killing by antibodies and complement, respond to treatment with chemotherapeutic agents. after,
It has been shown to cause sensitization (Reference 77). The cross-sectional considerations in the prior art regarding administering immunoguprines to tumor-bearing animals and humans are clear and the results of these experiments are combined. First, it should be noted that these treatments have not been consistently effective in treating tumors. A variety of treatments have been employed, ranging from the use of the patient's own serum to the use of serum from other patients with similar diseases, or even from different species of animals.

Recently, attempts have been made to treat specific tumors in patients using monoclonal antibodies generated in Max. As described in the above documents, various efforts have clearly not met with general success in the prior art. Contrary to the results obtained by the prior art, the tumor-associated immunoglobulin preparations of the present invention, when injected into a tumor-bearing host, whether human or animal,
- produces tumoricidal activity throughout. Furthermore, the raw material of this material poses few complications. Any immunoglobulin obtained from any tissue of the species can be used; this material can be extracted from a host, for example by plasmapheresizing the host, which has immune complexes containing the desired antibodies. Cohn of the obtained plasma
) was easily obtained by obtaining antibodies from fractionation. The technology disclosed herein allows for the preparation of a rich and generally effective anti-tumor agent material that has been prepared according to the prior art.

Here, we disclose a novel tumor immunoglobulin obtained from the serum of a tumor-bearing host and a method for producing the same.

This substance exhibits tumoricidal efficacy when administered to patients with cancer.

29- E, complement activity The human complement system consists of various classes of plasma proteins.

More than 20 of all complements participate in a regular series of reactions that result in complement activity. Accumulating evidence supports that the complement system serves as a fundamental component of normal host defense mechanisms and, as a result, complement activity is commonly associated with a variety of pathological conditions. (Reference 78
．． 79.80).

Two primary modes, called the classical pathway and the alternative pathway, activate complement.

These two pathways are generally distinguished by the process by which their initiation occurs. Regardless of the mode of complement activation, it is the conversion of central C3 complement into C3a and C3b fragments that indicates that activation has occurred.

As a result of the activity of the classical or alternative pathway, C3 compertase is formed. C3 conversion results in the formation of an enzyme complex (05 convertase), which in turn splits C5 and converts C
Generate f5a and C3b-30-7 rungments. 05m is subsequently released into solution, and the csb portion of the molecule is converted into a lytic metabolite complex (Iyt
le membrane attack complete
plays a fundamental role in initiating the formation of

03m and 05m are molecules called anaphylatoxins, which are extremely powerful biologically active substances that play an important role as mediators of acute inflammatory responses (Reference 81.8).
2). Both 05a and C5b share certain spasmogenic properties;
03m is effective in the nanomolar concentration range, and 05m is effective in the subnanomolar concentration range.
olar) is effective in the concentration range (Reference 83). Both factors have been shown to increase vascular permeability (ref. 84
．． 85), and has also been shown to cause histamine to be released from mast cells (Reference 86). 05a is a chemotactic factor, and this glycopolypeptide appears to be the main mediator of neutrophils induced by inflammation (Reference 87).

A. Several mechanisms have been proposed to be effective in limiting the biological activity of naphylatoxins.

Serum exopeptidase effectively removes the basic C-terminal arginyl residue from the anaphylatoxin molecule, leaving 0
3m and 05m make spasmogeic reactions virtually impossible during the short time they are released into the circulation (ref. 88).

Arg non@th@1ess product 05m retains the ability to promote chemotaxis.

03m is a single chain polypeptide with 77 amino acid residues and a molecular weight of approximately 9,000. Human 05m anaphylatoxin is a single chain glycopezotide of 74 amino acid residues containing a single oligosaccharide unit attached to the asA lagine residue at position 64. The binding molecular weight between the polypeptide and the oligosaccharide moiety is 11,000 (Reference 90.91).

In previous studies in humans, serum levels of 05m (and analogues) rose immediately following plasma perfusion to activated charcoal.

The appearance of 05m is associated with peripheral vasodilation and the appearance of interstitial fluid (ref. 20.21). For example, when C5m was detected, pulmonary congestion was observed (Reference 21). Furthermore, the acute inflammatory response in this patient was associated with the appearance of a small fluid-filled fluid over the tumor site in the skin containing polymorphonuclear leukocytes (20). C51 may contribute to the physiological effects seen after Zoptin perfusion, as C5 increases vascular permeability and vasodilation and is known to be chemotactic for neutrophils. , presumably facilitating the movement of circulating tumoricidal factors into and out of tumor sites.

Kass et al. have clearly shown that when pure Cl1a is injected into leukemic mice, significant regression of leukemia is observed (Reference 92). In AKR mice with spontaneous leukemia, , injected with normal serum from various species.

Leukemic cell destruction occurred with serum from a mouse strain with a full range of complement components, but not with serum from a strain of Nessidae genetically determined to be C5-deficient. Morseat, horse, and human serum also caused destruction of leukemia cells. The mechanism by which C5 destroys leukemia cells in AKR mice is devised as follows.

(a) C5 provides the missing complement link, allowing cytotoxic antibodies produced by the host to cause cell lysis and can have a direct effect on leukemia patients.

(b) C5 reacts with antigen-antibody complexes in the blood or kidney at sites other than the surface of leukemic cells, and is itself a lymphokine (l) that mediates destruction of leukemic cells.
ymphoklH@s).

(a) The antileukemic effect of CB is not related to pre-existing immune responses suggesting other mechanisms of killing of leukemic cells.

Construction The present invention provides tumor-associated immunoguprines that are effective on their own or in combination with other modalities (therapies) for the treatment of cancer.

One aspect of the present invention is to collect immunoglobulins containing 1 (a) 6m rings in the serum of the subject to be treated or from other animals (including humans) bearing tumors of the same type. , separated from circulating soluble tumor antigens with which it is complexed, and (b) collects this free immunoglobulin to produce a substance that exhibits enhanced killing activity when injected into a cancer-bearing host. It is processed as follows.

This separation step can be performed using any separation technique that can sufficiently separate circulating immune complexes into the desired immunoglobulin and soluble tumor antigens without denaturing the desired immunoglobulin. Substantial pH variations, large changes in ionic strength (as addition of salt), heating, and any number of other conventional separation methods may be used.

One preferred method for accomplishing this dissociation is to treat the resulting immune complexes under dissociative conditions with an acid or base for a time sufficient to effectively dissociate the complexes.

As is known in the art, a pH of about 3.2 to 1.5 is generally sufficient for this dissociation. The time period that is expected to effectively carry out this dissociation can be as long as about 2 minutes to 8 hours, and may be longer if necessary. The inventors have found that treating with acid to lower the pu to about 2.6 for about 5 minutes, followed by rapid neutralization to about pH 7.4, is effective in producing the preferred material. When using other dissociation methods, it is necessary to sequentially treat the above substances in order to induce the preferable 1) aggregation of immunoglupulins; however, when using acid treatment, aggregation occurs during the treatment. occur virtually simultaneously.

In other embodiments of the invention, various conjugates of immunoglupulins and proteinaceans are disclosed.

These complexes may be complexes of proteins and immune glupulins from either tumor-bearing hosts or normal humans or animals.

These complexes are most easily produced by mixing protein with serum collected from normal or tumor-bearing human or animal hosts; It can also be prepared by passing serum through a column in which staphylococcus has been attached to a solid phase. Furthermore, these complexes can also be obtained by combining the protein with a pure IgG preparation obtained from the serum of a tumor-bearing host or a normal body. This purified immunoglupulin preparation can be obtained, for example, by Cohn fractionation, electrophoresis, or by methods further disclosed herein or by other methods known to those skilled in the art. • These soltin-immunoglobulin complexes can be prepared in a wide range of effective ratios of soltin to immunoglobulin if the conjugate is effective in treating tumors. For example, these ranges may be 1:000 to 1:2 protein A/globulin. A ratio of about 11,100 has been found to be particularly effective. These complexes can be conveniently obtained by precipitation, as is well known in the art.

Useful precipitation methods are those using materials well known in the art. Of course, the immunoglobulin may also be a single antibody raised in a mouse or other animal to the same tumor that the host had, or to a homologous tumor in another tissue of the same species of animal. .

Another aspect of the invention is an article of manufacture useful for treating a host with cancer.

Product 1: A tumor-associated immunoglobulin preparation with tumor-enhancing activity and derived from plasma from normal or tumor-bearing hosts, which preparation contains predominantly IgG and IgM and has a titer of tumor-associated antibodies. is at least HI from the host
twice the titer of E, and the ciq binding is in the precipitated 7-lactone above 78 in the silosaccharide density gradient analysis. The term tumor-associated refers to immunoglobulins that react with tumor-associated or congener antigens and are also present in other normal body tissues. It is now recognized that tumor-associated immunoglobulins are useful in detecting or treating tumors, even if they are not completely specific to tumors.

This tumor-associated immunoglobulin preparation is prepared using immunoglobulin-enriched effluent (EIE) obtained from forced-flow electrophoresis of host plasma, which is sufficient for separation as described in detail elsewhere in this invention. This can be conveniently obtained by subjecting EIE to a pH that is substantially increased or decreased compared to normal plasma pi for a period of time. The material prepared in this way has a higher concentration of IgG*I compared to untreated EIE.
gM and immune complexes, but increased titers of tumor-associated antibodies and C in the heavy sediment fraction.
1q binding increases.

Product 2: A tumor-associated antibody globulin preparation with tumor-enhancing activity and derived from plasma from normal or tumor-bearing hosts, which contains IgG and small amounts of IgAt- and substantially frees IgM and complement. the titer of tumor-associated antibodies was at least 1.5 times that of the cone gamma globulin fraction and increased C1q-bound IgG in the fraction that precipitated above 7S in sucrose density gradient analysis.
'ji and exhibits very little anti-complement activity. When administered to a host bearing a tumor, particularly a tumor histologically similar to the host tumor from which the antibody was derived, the tumor produces a tumoricidal response.

As used herein, tumoricidal response means that the substance in question promotes or assists in killing tumor cells. One way to obtain product 2 is to obtain gamma globulin 7 lactis by cone@7 lactylation or other modified methods. Once this material is obtained, it is then processed by separation methods such as acidification followed by prompt neutralization as described above. Compared to untreated gamma globulin cone fraction,
The tumor-associated antibody globulin preparations described herein include Ig
It reduces the levels of G and IgM, increases C1q binding in the heavy sediment fraction, increases the level of tumor-associated antibodies, and reduces anti-complement activity.

Product 4 is a protein A-IgG&il agent with the following properties.

(a) Protein A and IgG H region (heavy fR) and L chain (light region) by polyacrylamide gel electrophoresis
Mainly includes.

...) with increased C1q binding, identified in the 78 and above sedimentation fractions on the sucrose density gradient.

(a) Dissociates into lower molecular weight C1q binding fragments under acidic conditions.

(d) Precipitation with 5% polyethylene glycol.

(,) Shows anti-complement activity.

(f) Fa dependence 9 pa 2 d@p@nd@nt lymphoeyt@rovet
t@formatton).

(x) Causes neutrophils to release mini-p-peroxidase, cathepsin, and peroxide anions.

(h) Causing agglutination of dog red blood cells.

0) Non-specific complement (non-sp*cifia co
mplement) causes a shortage.

(j) Protein A by a complement-independent mechanism
- Occurs in normal and tumor-bearing serum after perfusion through a cordion-charcoal column and is absent in serum during processing.

When serum containing specific antibodies is perfused with protein A, cordion, and activated charcoal, protein A-I in the serum after perfusion
The gGl1 fusion maintains the above properties (1) to (j) and also acquires the following remarkable properties.

These formulations were found to have tumor-associated antibody activity in the PEG-precipitated 7-lactone. When protein A-IgG preparations were administered to tumor-bearing hosts at tumoricidal concentrations, tumoricidal responses and tumor regression were observed. Specific antibodies in the PEG-precipitated 7-lactone were found in the serum of dogs immunized with human chest pain (Mcp-7) 1 and also immunized with human red blood cells. The latter experiment has been used as a model system to study antibody responses, even though the antigen used was not a tumor antigen.

Product 4: A tumor-associated immunoglobulin preparation having tumoricidal activity and derived from the plasma of a tumor-bearing host, which primarily contains I
gG and IgM, the titer of tumor-associated antibodies is at least twice the titer of serum from the host, and C1q binding is increased in Fracciales above 78 in sucrose density gradient analysis; Compared to the serum, the amount of zeidase increases and the concentration of fraxinases containing Sephadex G-200 increases. This can be achieved by using the serum of a tumor-bearing host and subjecting the serum to a substantial increase or decrease in pm relative to normal plasma for a period of time effective for dissociation.

Compared to unprocessed serum, tumor-immune plasma has increased titers of tumor-associated antibodies, increased C1q binding in heavy precipitated hooksions, and decreased levels in the containing fractions, as well as in zeidase volumes. The C-2001 protein levels increased.

Product 5: Thymosan-activated plasma (a) does not contain thymosan and produces peripheral vasodilatory effects in vlvo and spasmogienic (ipasmog@nle) effects consistent with complement activation by the product, and also Objects and embodiments reside in methods of producing and isolating these novel products useful in the treatment of the cancers described above. These include, for product 1, collecting the product from the enriched immunogloupin fraction from forced flow electrophoresis of a tumor-bearing host and then acidifying or alkalinizing it. Regarding product 2, the immunobrepurin formulation involves the acidification of corn fluxilonated gamma globulin obtained from a tumor-bearing host.

or by precipitating them with polyethylene glycol or ammonium sulfate, or by precipitating them with polyethylene glycol or ammonium sulfate, or by precipitating them with ethylene glycol precipitation and other known precipitants. It can also be obtained by law.

In addition to the above objects and features, the present invention also includes the combination of unique steps that exhibit tumoricidal effects, such as the following.

Administration 1 Product 1 is administered with protein A, followed by L-asparaginase and/or a cytotoxic drug such as cyclophosphamide or adriamycin.

Regimen 2 Complement activated serum (Product 6) and acidified plasma (Product 4)
) was injected for two consecutive days, and on the fourth day KL-asparaginase or cycle phosphamide was injected.

3. Inject product 2 and complement activation serum (product 5) for 2 days,
On day 4, L-asparaginase and cyclophosphamide were injected.

Regimen 4 Products 3 and 4 were infused for two days, followed by cycle phosphamide and/or adriamycin on the fourth day.

5 L-asparaginase and cyclophosphamide'f
, on day 1, followed by infusion of product 5 and autologous acidified plasma (product 4) on days 2 and 3.

Regimen 6: Inject product 5 and xenogeneic antitumor antiserum into Putin AK.

Sesame raw 7 Inject product 3 and xenogeneic antitumor antiserum.

Sesho 8 Inject protein A alone.

Additionally, Products 1, 2, 3, 4 and 5 and Protein A, alone or in combination, eg, as described above, are useful in treating hosts with cancer.

Other objects, configurations, functions, and effects of the present invention will become apparent from the description of the specification, including the claims.

PREFERRED EMBODIMENTS The present invention provides novel products of manufacture, their preparation and
Includes methods of treating cancers such as solid tumors, which result in significant tumor reduction.

Product 1 was subjected to forced flow electrophoresis, followed by acidification,
Produced by alkalization or other dissociation methods.

As shown in FIG. 1, the forced flow electrophoresis cell 8 includes a plastic end plate 10 supporting together a series of spacers 12 (see FIG. 2) and means for blood and buffer circulation. and DC power supply (not shown) connector 1
It has 4. Parallel membranes 16 suitably spaced by spacers 12 form a series of narrow passages across which an electric field is formed.

Four compartments are recognized. That is, the two recesses 1B in the end plate 10 accommodate electrodes (not shown) and serve for external buffer circulation. The added passageway aids in internal buffer circulation. The added passageway contains flowing blood, plasma or serum, separated from the glupulin outlet by a membrane or filter 16. The membrane 16 used is cell p-
Vlsklng, which has regenerated membranes, is used in cuprophan, which is used in artificial kidneys.
n), but mechanically more resistant.

3.0 to 0.45 for Fraxi Nishitan (separation)
A Millipore filter with a pore size of μ is used. Electrophoretically, all negatively charged blood components are transported in a direction opposite to the direction of fluid flow. It is the noselance of these two vexiles that determines the quality of the fractionation. The purity of the removed fraction depends primarily on the filtration rate of the globulin and the applied voltage. It is usually preferable to remove gamma globulin as quickly as possible, resulting in contamination with other plasma proteins. If no voltage is applied, filtration of plasma proteins will not occur. In all experiments, a cell member with four blood spacers was used;
The flows were parallel. Each spacer has an effective area of 500d. The internal buffer spacer is injection molded with a protruding rib structure and has an average plasma membrane thickness of approximately 0.6 mm.
Compress the plasma inlet spacer to 0.30 inches;
This limits the total plasma volume of the device to 200-215 (ltJ). The V xar sieve prevents membrane breakdown in the meppurin exit compartment. The voltage gradient of 6-8 v/cn1 is 0.02 It produces a current density of ~0.4 h/cr/I. Plasma flow is kept at 15-20111j/spacer, while internal blood flow is about 5 times as large. The gamma globulin withdrawal rate is 1. It ranges from 5 to 2.5 ml/spacer.

FIG. 1 is a 70-diagram of a forced flow electrophoresis cell illustrating the operating sounds of the device. A plasma parator 20 of the conventional or preferred type is coupled to the cell 8 by line 22 through an air trap filter 24 . The external buffer is
A pump 26 pumps it from an external buffer container 28 into the cell 8 and back again to the binding line as shown by the arrow. Internal buffer is pumped by pump 30 from internal buffer reservoir 32 into cell 8 and back onto the binding line as indicated by the arrow. The external buffer autoflow line 34 serves to return the overflow a=l internal circulating fluid to the internal circulating fluid container 32.

Enriched EIE is collected in line 36 from cell 8 and pumped to container 38 by pump 40.

Plasma is returned from cell 8 in line 42 by pump 44 and preheated by plasma preheater 46 .

The use of this device in dogs with spontaneous values is as follows. That is, arteries and veins are cannulated with catheters or needles, and whole blood is pumped into a continuous flow plasma-cell separator. Here, whole blood is plasma-
The cell separator 20 separates the morphological elements and plasma.

The plasma is then pumped into the FFE cell 8 at a rate of 5 to 15 g/m. In FFE cell 8, a current is applied to the plasma as described above and EIE is collected in container 384C. The plasma exiting the FFE cell 8 is then recombined with morphological elements (not shown) and returned to the host. In a typical process, approximately 1 caloulated blood cap was passed through this unit at a flow rate of 10-15 me/vm, and approximately 40 ηlb of canine EIE was collected.

The EIE collected from FFE was then subjected to the following biochemical procedures. That is, the pH of EIE is 10N-1 (
Titrated to 3.0 with CI, held for 5 minutes, then 1
ON7NaOH was added dropwise to quickly neutralize to 7.4.

The precipitate that appeared was removed by centrifugation at 1 fSOOxg for 3 minutes at 4C. The EIE was then passed through a 0.45-5μ Breat membrane (pl@at@d m@mbrane) filter and then returned to the host intravenously at a flow rate of 2 ml/v*.

After treatment with acid and neutralization, there was a significant increase in tumor-associated antibodies as measured by indirect immunofluorescence using the dog's own tumor as a substrate.

The results of this treatment are shown in Table 1A. The biochemical properties after acidification were shown as a decrease in the levels of IgGSIgM and immune complexes measured by laser turbidimetry (ref. 93) compared to samples without acidification (Table 1B). . Furthermore, when untreated and acidified samples were subjected to sucrose density gradient fractionation and various fractions were analyzed for C1q, it was found that the precipitated fractions exceeding 7S contained untreated EI.
%i increased C1q levels in acidified samples compared to E.

(Left below) Table 1A 1 Lymphoma 20,0.00
10.0002 Lymphoma 10,00
0 13,0003 Lymphoma
20,000 12,000*) Numbers indicate the end point dilution that gave positive fluorescence against the dog's own tumor matrix. The response of untreated and treated serum to canine vascular endothelial layer Zaku p-÷ as a substrate is 1:10 to 1:5.
It was in the range of 0. The above reaction was performed using unconjugated goat anti-dog IgG before addition of fluorescently labeled goat anti-dog IgG.
It decreased to 1:50 by keeping warm.

(Left below) 53- Table 1B 1 64.0 30.2 73.6 71.9 10.
8 1.8 (*) Immunoreactants were measured by laser turbidimetry.

In all dogs tested, the forced flow electrophoresis device 1
Within 4 hours after passing a single plasma volume through and collecting approximately 400 ml of effluent, the tumor became warm and edematous. After 12 hours, acidification E I E (t
o μg/kIP) and PuE17inA (8 μg/lb
) was injected. Within 4 hours, the lymph nodes became even more hyperhyperemic and edematous after 0.48 hours when the animal was given L.
-asnoglaginase and cyclotetraphos-7amide were given.

Tumor regression seen over subsequent days decreased from preperfusion levels to levels of 66-95%, with an average of 45%.
It lasted for five months starting from the 15th. 20 dogs were treated with FFE and acidified EIE plus plutin A.
It showed ~35% regression and lasted an average of 6-8 days. Animals that received only L-asparaginase and cycle phosphamide showed 18-25% degeneration, which lasted only 8-10 days. Dogs receiving Zoltin A alone showed no significant regression.

Therefore, the tumoricidal response was substantially greater in animals receiving the full regimen than with the immunotherapy program or chemotherapy program alone.

The results are shown in Table 2.

103-1 immediately after electrophoresis treatment in all dogs.
An increase in temperature of 0.7C was observed, which is usually 6.0C after perfusion.
relieved within hours. Cultures of blood taken while the four dogs had fevers showed no bacterial growth.

At various times after treatment after FFE, EIE (acidification) and chemotherapy, tumor 9-isocy is obtained from physiological activity. When the lymph nodes were edematous after treatment with FFE, nuclear aggregation,
Granulation of the cytoplasm and widening of intercellular spaces were observed.

At this time, inflammatory cells were not significant in this sample. Samples obtained after administration of acidified EIFi and plutin A are exemplified by spheroidization of the cytoplasm, hyperbrightness of the cytoplasm, and aggregation of chromatin and nuclei with blurring of intercellular spaces. Significant tumor changes were observed.

Some findings indicated lethal or sublethal changes in cells. There was minimal associated inflammatory cell infiltration. Samples obtained approximately 7 days after initiation of treatment showed necrosis extending to the malignant lymph nodes with the appearance of tumor cells and infiltration of the lesions by neutrophils.

Tumor-Associated Antibodies C'3 and Immune Glusolin Levels After Treatment Before treatment, tumor-associated antibodies are undetectable or only rarely detected in the serum of hosts with cancer.

Within 12 hours after treatment by forced flow electrophoresis, there was an increase in tumor-associated antibodies as measured by immunofluorescence. This was not further evaluated as the animals were being fed ExEt-olfactory containing tumor-associated antibodies. In all dogs tested, serum C3 and IgG levels decreased 12 hours after forced-flow electrophoresis treatment, but after 4 hours.
In 8 hours, the level rose to above the level before treatment. Immunoglobulin M levels did not change significantly.

No significant changes were observed in white blood cell and platelet counts or hematocrit before and after the 4-day regimen.

Furthermore, no significant changes in sodium, potassium, chloride, calcium, phosphorus, 80PT, 8GOT, BUN or creanitine were observed in the serum after treatment.

Preferred Embodiments: Product 2 Tumor Immunoglobulin Preparation Product 2 (Tumor Immunoglobulin Preparation) Suitable for intravenous use, using serum from a tumor-bearing host, described by Cohn et al. (94), Kigtler.

It is prepared by applying a modification of the method already disclosed by Nitsehmann (95).

An outline of the method is shown diagrammatically in Table 3. The plasma fractionation method is performed as follows.

Table 3 Buffer (A) pH 14.0 Buffer r 0.05 M-NalH
PO4, 1 volume: 0.05M-acetic acid; 6 volumes (B) pH 14,8 buffer: 0.05M-Nal
HPO, 1 volume: 0.05M-acetic acid, 1.615 volumes (C), pfl6.2 buffer: O,O! ! M-Nal
HPO,,1 capacity? 0.05 M-acetic acid, 0.833 volumes Q)) 9115.1 buffer: 0.05 M-Nal
HPO4j 1 volume: o, o5M-acetic acid 1.2 volumes Plasma source plasma was derived from peripheral whole blood of human patients.

Approximately 150 collected in acid-citric acid-dextrose
0 mJo plasma was induced and 2600 g, 20 min, OC
was purified by centrifugation.

Preparation of Precipitate #1 Determine the pH of the purified plasma, add O,SM-sodium acetate pH 4.0 buffer if necessary, and adjust to pH 7.2.
Adjusted to. Meanwhile, the temperature was maintained at OC and 0.95% ethanol was slowly added to the 461+1jt-purified plasma to give a final ethanol concentration of 8%. During this operation, the suspension was cooled to oc. With main binding
2600 ml of visible precipitate consisting of physolinogen
g, centrifuged for 20 min at θ~4c. The removed paste was called Precipitate 1.

Preparation of Precipitate 2 Titrate 2 ml of the supernatant of Precipitate #1 with 0.8 M sodium acetate pH 4.0 to pH 4.0 and add enough sodium acetate to bring the supernatant suspension to pi Is, 8. The amount of buffer was determined.

Approximately 72 d of cold 95% ethanol was added to the pfl 5.8 suspension to give a final concentration of 19 arc. During this addition the suspension was cooled to -5C. After addition of ethanol 3
The suspension was gently stirred for 0 min to maintain the solution pit at 5.8. The suspension was then centrifuged at 6000xg for 25 minutes at OC. The meat was removed from the centrifuge bowl and weighed. This Pace F was named Precipitate 2.

Preparation of Supernatant 3 The precipitate-paste was resuspended in 10 d of cold water per gram of precipitate. Approximately 28% of the water was in the form of fine ice 1. This precipitate became quite homogeneous. l capacity of 0.06M
The pn of the suspension was adjusted to 4.8 by adding a pH 4.0 buffer (buffer A) consisting of -NalHPO4 and 6 volumes of 0,OIsM-acetic acid. The ionic strength was then increased by adding 1 volume of 0.05 M acetic acid at pH 4, $ buffer (Buffer B). The required amount of this buffer is 4.85 mA per gram of precipitate2! Kara, front K
pHIN l! +7) pH 4,0 added at 7e
minus the volume of buffer A.

Separate ivy, pH 6 consisting of 1 volume of 0.05 M-NalHPO4 and 0.833 volume of 60.05 M-acetic acid.
, 3 buffer solution (buffer C) was added to adjust the pH of the suspension to 5.
Raised it to 1. Then, 1 container of 0.05 M −N *
Add pHIs, 1 buffer (Buffer D) consisting of @HP 04 and 1.25 volumes of O,OIs M-acetic acid;
The ionic strength was readjusted. The amount of buffer solution required is 1g.
% per precipitate 2 minus the volume of buffer C used for pH adjustment. Finally, the suspension was diluted with cold water to a total volume of 19,5111 per duram of sediment #2. Then, 95% cold ethanol was added to the am solution to make the ethanol concentration 12%. The suspension was kept in the OC for 30 min and 14,00 (lcg, 25
Centrifuge for 1 min at oc. After removing the precipitate, the supernatant was obtained and named supernatant 3.

Preparation of gamma globulin precipitate Using a salt conductivity meter, the ionic strength of supernatant 3 was increased with concentrated NaCl.

Titrate 2 m of supernatant 3) with lN-NaOH to 7.2 to adjust the pn of the entire mfm solution.
-The amount of NaOH was determined. NaOH was then added to the suspension in an appropriate form such that the pH was 7.2.

Then, 95% ethanol was added to make the ethanol concentration 25%. During this addition the suspension was cooled to -70. Gently stir the gM solution at 7c for 30 minutes, then
Centrifuge at -7G for 30 min at 15,000xg.

Then, the suspension was kept warm at 27C for 5 minutes, and then 0. l
Neutralized to pH 7.a with N-NaOH. Preparation is next 2
Pass through a micro-filter, 1. ■, administered at 5ω per minute.

Physical and chemical properties of tumor immunoglobulin Tumor immunoglobulin (acidified Cohn 7 lactation) analyzed by laser nephelometer (ref. 93) contained IgG and a small amount of IgA, but no IgM e C3. Compared to the non-acidified corn fraction, IgG and I
There was a decrease in gA (Table 4).

Table 4 Before After Before After Before After Before After - Me■----■1~-111----■-1-Me one loaf---1-1+----- - Breast cancer chest pain 032
960 25.1 19 0 0 0 0 Normal
5904 3192 41.0 32.8 0 0 0
0*) Examined using a laser nephelometer as described previously (Reference 96).

Tumor-associated antibodies were analyzed on each of two doses of tumor immunoglobulin obtained from two humans with thymic carcinoma. Indirect immunofluorescence was used on the chest pain matrix, with end point fluorescence titers of 1 nea 0,000e for both Sansols tested.
Beyond. Non-acidified samples of the same patch were 1:I50,
showed an end point titer of less than 0.000.

Tumor immunoglobulins were tested for tumor-specific binding of MCF-7 thymic carcinoma by radioimmunoassay in fractions obtained on a silosaccharide density gradient compared to non-acidified Cohn fractions. Tumor immunoglobulin showed increased tumor-specific binding activity in the fraction above the 7S marker. Similarly, C1q binding increased in fractions greater than 78 compared to non-acidified fractions. (
Figure 3). Therefore, both tumor-associated binding and C1q binding are heavy (>7
8) Move to the sedimentation fraction. Rome (Rom@
The anti-complement activity of tumor immunoglobulin tested by the method of K. R. et al. (96) was in the commercial range and lower in the non-acidified fraction compared to other intravenous gamma globulin preparations (Table 5). ).

Table 5 10 3 75 2 100.5 1
0 81$ 4 350.25 12
The study was carried out by the 14 538 0-mer method (ref. 96).

A positive control without serum produced 100 hours of hemolysis. Kao IgG produced anti-complement activity of less than 1.

The functional capacity of tumor immunoglobulins prepared from autologous tumor-bearing serum was demonstrated by perfusing tumor-bearing hosts at 400-5 o Omf with thymosan activated with normal dog serum (regimen 3. Table 9). 2
Immediately after injection in two dogs (one had spontaneous thymic flI
Jwomochi, one has melanoma 8), tumor! 68 - There was acute necrosis of the ulcer, characterized by visible necrosis and hyperemia. Microscopically, the tumor site showed widespread necrosis of the tumor, with neutrophil infiltration in the focal area.

Protein A-IgG complex (Product 3) was prepared by preparing protein A-IgG complexes from Staphylococcus aureus cowans l by immobilizing 5 to 50 plasma from normal or cancer-bearing hosts on collodion charcoal. (containing immobilized protein A%LOtfif). Alternatively, it can be isolated from the effluent by addition of 5% polyethylene glycol or other high molecular weight tan microprecipitation methods. The resulting precipitate is resuspended in normal saline, dialyzed against phosphate buffered saline, and then injected into tumor-bearing hosts 1. V, was administered.

This method is shown in Table 6.

Table 6 For isolating protein A-IgG complexes? Lietelene glycol precipitation method (1) 20% P of 311/ml of serum or plasma flowing out of the protein group/collodion/charcoal column
EG (PEG6000.Fisher Scientific Can/E Knee (Fisher 8cie)
ntificCo, ), Fairtavn,
NJ] and kept at 25°C for 1 hour.

Qli 8000Xf, centrifuge at 4℃ and PEG
A precipitate was obtained, washed twice with 5-PEG, dissolved in PE8,
Dialyzed against PE8 at 4°C.

Protein A-IgG complex can be produced without using protein groups, collodion, or activated carbon columns.
10-1000 f) and normal serum or plasma (1-1000 f) and
3 resistance] or IgG (1 to 10 inches) at 27°C for 30 minutes.
It can be prepared by keeping warm at
01) Add normal saline solution and then add this mixture to 1.
V.

injected into a tumor-bearing host. Table 7 below shows other methods of preparing protein A-IgG complexes.

Table 7 (1) Protein A (10-1000/7f) is incubated with 1-10 my IgG at pH 8 for 30 minutes.

(2) Add PBG to a concentration of 5% at 27°C, and centrifuge the mixture at 5000xf for 210 minutes at 4°C.

(3) Resuspend the precipitate in 20-saline solution and dialyze against PE8 for 2 hours to remove PBG.

(4) Inject the solution intravenously to a volume of 5-10114.

Protein-collodion-charcoal (P-OC) column or immobilized Staphylococcus aureus cowans I
(5pA) The serum from a dog with spontaneous thymic carcinoma flowing out from the column (8AO) was filtered through a microporous membrane filter.
(12 micron) immobilized 8 pA or protein A-collodion-activated charcoal. The following was found.

71- Results 01q in serum after 8 AO or PAOO perfusion. Plasma from 5 normal dogs and 6 dogs with thymic carcinoma was SA
Pre- and post-perfusion samples were assayed for levels of O1q polymerization by radioimmunoassay as described. There was a consistent increase in OIQ binding relative to pre-treatment values (Table 8). The initial decrease in O1q binding is
Dilution effect and/or protein A-collodion-
This appears to demonstrate removal of IgG-bound immune complexes by activated charcoal (PACO). Generally, O1q binding levels in pre-perfused tumor-bearing serum are higher than in pre-perfused normal serum. And the net increase in tumor-bearing serum was (mean increase: 882±3788.D, nt A(3
G na ii / ILl), normal serum (average enhancement 1 de 1
14±1868. D, ntAOGi amount). An increase in O1q polymerization oiliness was observed, whereas SAC
In perfused serum, canine IgM levels, such as post-perfusion IgM levels.
Gfi degree decreased uniformly below the preperfusion level (Table 8) Table 8 1 40 8 482 2 16 4 22 3 16 4 62 4 19 4 -5 5 14 4 6 Average 114 ± 186 2 87 6 978 3 37 8 4 4 41 4 182 5 48 4 103 6 70 4 248 Mean 382±378 *) Perfusion value was multiplied by the number of fractions collected in the effluent and subtracted from the cumulative AOG weight of these fractions. 100-8A of normal or tumor-bearing serum
GtC perfusion, collect fraction IIIj, 01q
tested for activity.

To confirm that protein A on the SAC is important for the O1q polymerization increase to occur, serum from normal dogs and dogs with thymic carcinoma was deficient in 8% AO or Staphylococcus aureus Strainwood 4. 6 (8taphytococcui
Aureus 8traInWood
46) by the method described above, and the effluent was irrigated with O1q.
The polymerization tube was tested. The sample effluent from the aAO column showed an increase over pre-perfusion O1q binding levels, while the effluent sample from the SAW column showed minimal change from pre-treatment levels.

Regarding the above studies, the initial decrease in 014 reflects dilution effects and removal of immune complexes by 8AO or 8AW.

Sera from normal dogs and dogs with thymic carcinoma were perfused into the PACO as described above.

An increase in O1q polymerization was observed in the effluent serum of 7 of the 7 tumor-bearing sera and 5 of the 7 normal sera (Table 9). In contrast to the perfusion to the SAC, no significant changes were observed in the IgG and IgA levels in the serum perfused to the PkCC (Table 9). was passed through PkC (3) and solid phase otq binding was analyzed before and after treatment. Olq binding was expressed as nanograms of anti-dog IgG binding. Numbers represent average volumes of 2x or 3x samples. Figures in parentheses The numbers indicate the range of values from samples multiplied by 2 or 3.

Characteristics of the complex formed in the serum after perfusion to PAO (!) Since protein A is known to bind to canine IgM and IgA, these components in the complex after perfusion, as well as IgG, 03 For this purpose, serum 101q was kept warm in a tube covered with blood before and after perfusion.
The complex was combined. As a control, serum perfused with canine alzomin instead of protein immobilized on collodion-activated charcoal was similarly incubated in an Olq@'-covered tube. Wash the tubes for canine IgG, IgM, IgA, C! +1
A specific 1111 F(ab')* fragment was added and the bound radioactivity was measured. The post-perfusion binding levels of anti-1gG and anti-IgM increased by approximately 20- compared to pre-perfusion values.

No significant changes were observed in anti-IgA and anti-C3 binding76
- It happened. The results were similar to tumor-bearing and normal sera and also to the increase in anti-IgG binding detected in post-perfusion plasma from tumor-bearing and normal dogs. Conversely, IgG binding activity in serum perfused with canine aldimine was reduced by 15 degrees, while binding of other components remained virtually unchanged.

Normal or tumor-bearing serum was similarly passed through the PAOO, incubated in otq-coated tubes, and incubated with human IgG, I
gA, the binding of heavy “IP(ah’)H specific to CI was tested. As a result, anti-IgG, IgM and C3
showed an increase in IgA binding, but no increase in IgA binding.

PA (O1q-binding IgG generated in serum after 30 perfusions)
To examine the sieving properties of the sieves, pre- and post-perfusion fluids from normal or tumor-bearing serum were ultracentrifuged in a sucrose density gradient and the fractions were evaluated for O1q-bound IgG.

Both plasmas showed increased levels of O1q-bound IgG in fractions >78 compared to the corresponding fractions from pre-perfusion serum. 4 in the gamma chain of dog IgG
A similar increase in 01q binding in these gradient fractions was seen when bound IgG was detected using IA-like F (ab')1 fragments.

High molecular weight IgG complexes in dog serum after perfusion were
It was isolated by fractionation on 00 column chromatography and passage of the void volume peak fraction through an immunoadsorbent containing a gamma chain specific for rabbit anti-dog IgG. The retained protein was eluted, neutralized, concentrated, and returned before analysis by PAGE.

The Coomassie blue stained PAGE profile shows prominent io, ooo and 4 lipeptides of molecular weight of 2400 G that co-migrate with the gamma and light chains of dog IgG. A lightly stained 74ooo molecular weight polypeptide was also observed that migrated with the mu immunoglobulin chain of canine IgM, indicating the possible presence of canine IgM in the complex isolate.

10 ulP of isolated polymeric IgG was tested by double diffusion method and showed a single precipitate/1nd with goat anti-total dog serum, with a line identified as gamma chain specific for rabbit anti-dog IgG. Formed. These studies indicate that high molecular weight species, including canine IgG and IgQ, are formed during PAOO perfusion of serum.

PAOO high molecular weight O1 due to plasma and plasma components
Although q-bound IgG was formed during perfusion of serum into PAOO, PAGlif analysis of the eluate failed to reveal a band clearly characterized as protein A. □This is because proteinans migrate to locations that almost coincide with canine gamma globulins. Therefore, for protein release from serum during perfusion, 111
The study was conducted by using one protein human. Perfusion of IgG, albumin solution, and serum from three different species resulted in the release of protein A from PA (30). Furthermore, the elution was not protein specific. If so, the same solution liberated tmsl inuarzomine immobilized on collodion activated carbon in a similar manner as proteinan. Based on the amount of radioactivity liberated from PAOO, it was estimated that 0.0025-0.00 smf (0.2! S ~0
．． After perfusion with 101117 serum, protein A of 5 s) decreased to 1? It was estimated that it was desorbed from the immobilized protein A of FIF.

Protein A was further confirmed in serum perfused into PACC as follows. i.e. PAOO or 8AO
After perfusion of serum, the PEG precipitate was adjusted to pH & 0. G-
It was separated by 100. Fraction that talked about money (100,0
00M, W, ) was neutralized, concentrated, and analyzed by PAGE. Coomassie blue staining profiles of these samples revealed 2-4 prominent polypeptides/mantles,
One of these migrates with the pure proteinans.

In further studies, dog serum enriched with dog IgG was combined with PA containing protein A labeled with 011 as a label.
perfused into OO. After perfusion, the serum was treated with 80-5% PBG and 1111 protein A-IgG.
The complex was precipitated and analyzed by PAGB. Protein Ai
A radioactivity peak corresponding to @Makoto was observed.

Low molecular weight radioactive protein A'! ti is seen, which seems to indicate a fragment of 101 protein A.

In a further study, normal dog serum %PI perfused into PAOO! Protein A-IgG processed with IQ
The complex was precipitated. The solubilized precipitate was fractionated on Sephadex G-100 under dissociation conditions, and the containing fraction was precipitated with (NH4)t80a. The precipitate was solubilized and treated with IgG-conjugated Sephadex 4B.
passed through. Bounds were eluted and concentrated. The presence of protein molecules in the eluent was suggested by double diffusion analysis. This method showed a precipitin concentration of 9 for normal human serum, but not for normal chicken serum, which does not bind protein A.

The following study was performed to identify protein proteins in the high molecular weight sucrose density fraction of post-perfusion serum.

Normal dog serum was passed through PAOO, and the post-perfusion plasma containing eluted uaI protein A was fractionated using a sucrose density gradient.

1ffi proteins were found in a wide range of fractions from 78 to 198. In contrast, free 011 protein showed a peak below the 7S marker. This suggests that protein A eluted from PAOO in the form of a high molecular weight is different from free protein A.

Car after perfusion containing 1111 protein A%? lf was ultracentrifuged and its sedimentation properties were compared with purified free protein. Radioactively labeled protein A in the post-perfusion samples was distributed in the high molecular weight fraction of the gradient compared to free protein. The parallel sucrose density gradient fraction of the post-perfusion serum was 1, compared to the treated pongee serum.
O1q-bound IgG was increased. This suggests that protein A eluted from PAOO into post-perfusion serum was in a complex with immunoglobulins, contributing to the increase in Cjl binding observed in the post-perfusion sucrose density gradient fraction. It is.

(Left below) 83- To determine whether 1111 Protein A eluted from PAOO after plasma perfusion was able to bind to Olq coated on the tube, the following experiment was performed.

In other words, the plasma surrounding the 10 m long normal tumor is
Passed through PAOO containing 18II protein A.

The effluent containing eluted Protein A, 181 I Protein A, was used as a control. 11111 in the PAOO effluent compared to free protein A binding.
The results showed that protein A binding increased approximately 7-fold. Thus, protein A eluted from PAOO after plasma perfusion acquired O1q binding properties suggesting that it was released in bound form to immunoglobulins.

In addition, normal or tumor-bearing dog and human plasma were perfused with PAOOK. jcffl with 01q coated tubes with untreated and effluent samples (0,5)
It was lagooned. The tubes were washed and affinity purified 84-1211 chicken anti-protein A was added as described.

The result was ra”I 2 out of 3 of the normal dog post-processing samples.
(b) Significant increase in anti-Protein A binding in 4 of 4 post-perfusion samples from dogs with h[iM tumors and 4 of 6 from the same humans. It was seen.

In addition, 501Rt normal dog serum was added to 1! with unlabeled protein immobilized on collodion-charcoal.
Protein A or 1115: [perfused through a column containing canine albumin]. Effluent fractions with peak radioactivity were pooled. lO− of these peaks
in an aliquot of 50 NH4 1804
．． 5% PEG or 10% trichloroacetic acid was added. As a result, only 5 to 10 free protein A
%, whereas 70-80% is 1! Machi Protein A
It was shown that (NH4)*804 can be precipitated with PEG. On the other hand, less than 10% of released aldimine could be precipitated with (N)(, ), 804 or PEG. These discoveries were made with aldimine,
It has been suggested that protein A appears in the post-perfusion effluent in association with immunoglobulins.

Canine IgG from dog serum is present in serum after perfusion.
The following experiment was performed to determine whether the 1q binding complex was bound.

11111 Normal dog 10- containing dog IgG
! PAOO containing II proteins were perfused. The effluent was concentrated and kept warm in an Olq coated tube. Based on the specific activities of labeled protein A and IgG, it was calculated that the empirical formula of the Olq binding complex is close to that of IgG and PA. The approximate molecular weight of these complexes was determined to be 680.000 by Sephalo-Zero B Chromadog 2 Fee. Based on these values and the empirical formula, the molecular formula is [(IgG), FAI.

corresponds to

In the case of human serum, in addition to the high molecular weight peak corresponding to the complex seen in dog serum, a second peak appeared that was close to the molecular weight of IgG. This one, like the polymer fraction, exhibited O1q polymerization oiliness above the control level. The molecular formula of this small complex has not been determined because it is difficult to determine the molecular weight of complexes in this range using this method.

At various molar ratios of protein A and IgG, protein A
-IgG complexes were prepared, incubated at 27°C for 60 minutes, and then analyzed by nephelometric analysis. Maximum light scattering was observed at a protein A-IgG molar ratio of 1:2. Lower values were present at molar ratios of 1:10 and higher. The complement binding properties of protein A-IgG complexes prepared at various molar ratios were studied by anti-complement assay (96).
Maximum anti-complement 87 activity was observed between :100 and 1:1000. Protein A made with various molar ratios
-The IgG complex was tested for its ability to cause release of meagle clonigusase, myeloperoxidase, and acid protease from dog neutrophils. The optimal protein A-IgG ratio to release these enzymes is 1:
100, reducing the level of catezocin generation below a ratio of 1:10. .

Dog antisera specific for human red blood cells and MOF-7 human thymic carcinoma were prepared. Each of these sera and normal dog serum was treated with 51 PEG to remove immunoglobulin aggregates and endogenous immune complexes.
The PAOO was then perfused. After perfusion, add 5 pieces of serum to PFiG.
The precipitated proteins were extracted from MOF-7 cell murine 7 cells.
Antibody activity against Agrozachroma (L cells) and canine mammary adenocarcinoma cells was evaluated. Tables 10 and 11 □ show that after P A 00 perfusion, specific antibodies were detected in PE of immune serum.
The G-precipitating fraction can be recovered at 88- and thus exceeds the level of PEG-precipitating perfusion of normal serum.

Table 10 P B 8 ” 3.58±0.530 2.
51±0.206 1.9 companies 0.133 normal dog serum
20.58±3.363 23.66±2.754
9.40±0.21 dog anti-MOF-736,86
±0.066- 27.115±0.713 9.5
4±1.157*) Phosphate buffered saline solution**) Significantly higher than normal dog serum (P) 0.05
) (Margin below) Table 11 P B 8 0.84±0.037 0.063±
0.066 1.05±0181 Normal dog serum 7
．． 31±0.159 19.64±2.108 7.
Post-perfusion plasma was subjected to sucrose density gradient ultracentrifugation to investigate the sedimentation properties of antibodies containing 80±0526 aggregates, and antibody activity was evaluated by radioimmunoassay. Post-perfusion antiserum is 78
72 fractions of ˜198 showed specific binding and outperformed the binding of normal serum in these fractions. ) Post-current dog antiserum specific for human red blood cells was added to 54 Pl.
i! The specific agglutination reaction of human serum was tested and compared with the PEG precipitate from normal dog serum after perfusion. Table 12 shows that the dog anti-human red blood cell serum showed significant agglutination, in contrast to the agglutination of the same amount of precipitate from non-normal dog serum.

Table 12 Normal PE0 0 0 0 0 Precipitate immunity PE1G +++ 10 10 + 10 +
0 precipitate*) The agglutination reaction was graded from O to ++1+.

Antibody-dependent cell cytotoxicity of PEG precipitates after perfusion (An
tibody Dependent Ce1l
ular 0ytotoxicity: Post-perfusion PEG precipitates from dog anti-human RBO and normal dog serum were used to coat human erythrocyte target cells and assayed for ADOOK using canine erythrocytes as effector cells. The PBG precipitate of immune serum empty showed an effector-to-target ratio of 121 and more than 300% greater cytotoxicity than normal serum.

The role of complement in the production of O1q-binding IgG during PAOO perfusion.
To determine whether canine complement is essential for the production of G. 56
Normal dog serum heated for 60 minutes at 0C or unheated was treated with 5'I PEG to remove immunoglobulin aggregates and then perfused into the PAOO. Heated or unheated plasma had a relatively increased O1q binding in the hemorrhagic lobe after perfusion. Additionally, normal or congenital KO3-deficient dog serum was similarly treated with K PEG and perfused into PAOO, producing the same amount of (Hq binding). By mechanism, PAO
Occurs in serum after O perfusion.

92- High molecular weight PEG normal or tumor-bearing serum generated during PAOO perfusion is treated with 5 doses of PEG to remove immunoglobulin aggregates and endogenous immune complexes, then P
The effluent after 6 perfusions with AOOKm was subjected to a second PEG treatment to precipitate the high molecular weight IgG produced. Then,
For this purpose, various physiological and functional characteristics were evaluated.

To determine whether the resulting protein A-IgG-containing IgG oligomers and post-perfusion serum inhibited Fc-dependent lymphocyte rosette formation, PBG in the post-perfusion serum was analyzed.
The precipitated IgG oligomers were incubated together with dog leukocytes, and then the rosette-forming ability of sensitized human red blood cells was evaluated (Reference 99). PEG-precipitated IgG oligomers in post-perfusion serum were 47
Compared to the inhibition of steamed rice cakes, the formation of rosettes of 28 to 31 inches was inhibited.

To determine whether IgG oligomers containing protein A-IgG complexes and post-overflow serum activate and consume complement, PEG-precipitated IgG oligomers in post-perfusion serum were incubated as a source of guinea pig complement. The ability to mediate lysis was then evaluated using a complement-dependent hemolysis assay (Reference C96). P in post-perfusion plasma from normal or tumor-bearing dogs.
EG-precipitated IgG oligomers were comparable in their ability to activate and consume complement.

To determine whether protein A-IgG complexes from post-perfusion serum promote the release of peroxide anions by human polymorphonuclear cells (PMNI
After the culture medium was incubated, the production of peroxide was evaluated by chemiluminescence (References 100 and 101). in post-perfusion serum from normal or tumor-bearing dogs.
PIG precipitated IgG oligomers were comparable in their ability to promote peroxide anion production by canine PMN.

The cytotoxic function of the PEG precipitate from post-vortex serum was evaluated. PEG-precipitated oligomers containing protein A-IgG complexes from tumor-bearing or positive serum caused nonspecific cytotoxicity. Even when using target cells such as mammary adenocarcinoma, rat fL cells, and human red blood cells,
Similar degrees of dissolution were observed. However, 03-deficient serum produced significantly lower cytotoxicity than the corresponding normal serum when tested against mammary adenocarcinoma, murine L cells, and human red blood cells, indicating that the cytotoxicity is complement-dependent. There was clearly one I/C.

Progress has been made in chemiluminescence research using synthetic protein A-IgG complexes. Synthetic conjugates were initially prepared by adding protein to pure dog or human IgG or serum to generate chemorenaissance activity. Serum or IgG
The supernatant from the insoluble complex prepared from is virtually free of activity. Pure Protein Man.

95- IgG, a serum control, shows no chemoluminescence activity alone; addition of a fresh source of complement to complexes made from serum or fresh IgG does not significantly alter the chemiluminescence properties. It seemed like it couldn't be improved. In contrast, complexes made from serum in the presence of EIDTA, like complexes made from serum in the absence of FIDTA, are less susceptible to aggregometry.
) was not active. The complex supernatant obtained from serum and IgG was inactive. An unfractionated mixture in which the complex was added to the supernatant, and a mixture reconstituted from the isolated precipitate and the supernatant had 9 activity compared to only the precipitate. This indicates that there are factors in the supernatant that are not themselves active but can enhance the activity of the insoluble complex. With BDTA, significant granulocyte aggregation occurred. However, the conjugates obtained in the absence of EDTA were much more effective than those obtained in the presence of EDTA. The supernatants from these complexes were inactive, but when reconstituted into the precipitate, their activity was significantly enhanced to be equivalent to the unfractionated mixture. This finding suggests that the supernatant enhances the activity of the insoluble complex. In this respect, 03a(
High levels of :05a were detected in the supernatant from the complex by radioimmunoassay. The important points that distinguish the activities are as follows. That is, a soluble complex obtained by adding a relatively large amount of protein A to serum is aggregomethoso (ag
gregometry), with 03g and 05a levels comparable to supernatants from insoluble precipitates.

Protein A-I made from serum or pure IgG
Synthetic complexes containing gG activate total complement activity in dog or human serum. Complement deficiency occurs in dogs with a prevalence of 40 to 70 cases. Insoluble complexes prepared from human or dog serum, 03-deficient dog serum, or human or dog IgG have a total complement activity of 90% as shown in Table 817.
It consumes the people in charge and above. Insoluble or soluble complexes made by adding high levels of protein A to serum are
- yields 20-30 uy of 03a. This is approximately equivalent to 401% of the total O3 in the serum being converted to Oaa. Complexes washed and added to fresh serum will continue to generate 03a and 05a activity. Once these conjugates are prepared, they can not only initially yield 03a from the serum from which they are prepared, but also yield 03a and 05a as shown in Table 18. For example, a precipitate from pure IgG serum added to fresh serum was generated at 2500 ny 05a/d. In an experimental study.

05a is known to cause hypotension, vasodilation and high capillary permeability. A similar response was reported in vlvo under acute clinical conditions after a perfusion procedure. In a preliminary study, increased levels of 05a were observed in patient serum samples obtained when blood pressure decreased after perfusion. This finding suggests that anaphylatoxins generated from PA-IgG complexes are, in part, responsible for the observed cardiovascular effects.

99- In vivo effect of protein A-IgG complex Protein A IgG prepared by incubating 1■ protein human with 31 Ll of autologous serum at 27°C for 30 minutes
The complex was given to three dogs with spontaneous tumors (
Sesho 9. Table 18). Normal saline was then added to bring the final volume of the mixture to 51 nt, which was reduced to 11 nt.
1 at a rate of 1 tl/'till. Injected with I and V. Infusions were performed 3-5 times per week for 7-15 treatments. Immediately after injection, the visible tumor became hyperemic and edematous. As the injection was repeated, remission was observed as shown in Table 8.

Table 8 Two-dimensional products (cr/l) Boodle 11 Mammary gland a 17. ? 10
．． 0 44℃gxtoy 12 Mammary gland value 16.
2 12.7 22 Teriya Beagle 7 Rinno Kiyomiya 89.3 6
3.7 28100ug of protein A in 2011
Ll autologous 100- Protein A-IgG with addition of dog serum? JiT&
The mice were fed on two consecutive days with rabbit anti-tumor antiserum (20 m/) and tisosan-activated normal dog serum (regimen 4. Table 14). On day 3 animals were allowed to rest and on day 4 they were given cyclophosphamide and 7 year olds adriamycin. This resulted in an acute tumoricidal effect in two dogs with mammary carcinoma. With repeated dosing, two dogs showed tumor reduction of 504 or more (Table 14)
.

Table 14 Effect of protein A-IgG complexes prepared from normal dog serum on In v 1 Ma0 From physical and chemical studies on PACC effluent in which protein A-IgG complexes appear, we can confidently confirm that various The efficacy of the conjugate in dogs with spontaneous canine tumors was tested. Protein A-IgG complex was given to five dogs bearing various spontaneous tumors. PA-I used
The dose of gG was determined from studies of the amount of PA-IgG that produced macroscopic morphological changes in the tumor. Regarding the ratio of PA and IgG,
It was revealed from in vitro research that P
A: IgG ratio of 1:100 showed optimal release results for myeloperoxidase and cathepsin from dog neutrophils. These are protein A (5ug/l
b) was obtained by incubating at 37°C for 60 minutes with normal dog serum C0,1m171b). Then, normal physiological saline solution was added to bring the volume of the mixture to 107151, and 1. Injected with VL.

Infusions were given 2-4 times per week for 7-16 treatments.

Shortly after injection, the tumor became hyperemic and edematous. As the injections were repeated, regression of the tumor was observed as shown in Table 15. For example, after PA-IgG injection showed inflammatory changes in the tumor, chemotherapy was administered as a secondary treatment to the injection procedure. Even under these conditions, tumor regression was observed as shown in Table 16.

Thus, one aspect of the invention is a method of treating a host with cancer by administering a formulation of Tumoricidal I. This formulation binds to Fc receptors on leukocytes containing F6, thereby causing the release of at least one cytotoxic substance from the leukocytes.

Table 15 Two-dimensional product (crl) Inuya Tumor Number of treatments Before After Change coefficient 1 Angiosarcoma 13 96 46 51.
02 Breast cancer 7 16.2 11.8 57.23
Lymphoma 10 16G, 0 60.0 62.0
4 Breast cancer 15 45.3 30.3 33.15
Lymphoma 11 93.6 63.7 31.
9゜-103- Table 16 Protein A-IgG: Low dose of chemotherapy administered to 111 combination Tumor Number of treatments Before and after
Change 俤1 Intravascular @” 12 86.f+
4.0 96.02 Scale cell carcinoma”
15 60.0 10.0 86.0*)
After 12 treatments, 257 Q/i of adriamycin was given per treatment.

Umbrella*) After 5 treatments, 1.7 rv per treatment
/i of BaNu was given.

A dog model was developed to study the toxic effects of conjugates of free protein A incubated with dog plasma and to correlate this finding with the previously described human studies. In this study, we characterized the pathophysics of this response, investigated serological mediators, and investigated pharmacological interference (blo
ckade) effects, investigated ways to weaken the response, and finally attempted to compare it with the human response.

Four groups were studied. Group 1 received protein A-IgG complex for 3 minutes. Group 2 received protein A-IgG complex for 30 minutes. Group 3 was a dog with reduced granulocytes and was on the same regimen as Group 2. group 4
were treated with histamine blockade before receiving the same regimen as group 2.

The following parameters were considered.

(a) Left ventricular DP/DT (b) Cardiac segment length contraction
length - contractility
) (a) Mean aortic pressure (d) Peak left ventricular systolic pressure (e) Circumferential coronary artery flow (f) Renal artery flow (g) Femoral artery flow (h) Pulmonary artery flow As a result, rapid release of protein A-IgG complexes With drifting, all of the above parameters drop rapidly,
It was found that it slowly returned to baseline. Upon injection of protein A-IgG complex, all parameters decrease with a peak at 15 minutes, maintained for 45-60 minutes, and then return to baseline. The first major signs are left ventricular DP/DT and nodal length, with mean aortic blood pressure as a secondary effect. Same treatment as group 2 (
As a result, decreased cardiovascular effects were observed in dogs receiving the regimen.

The use of histamine blockade had little effect on attenuating cardiovascular responses.

After the injection, pathological examination of various tissues revealed that the most notable changes were alveolar floating shields in the lungs and cell outflow into the alveoli (c@11uralextravasa).
tion 1nto the alv*1or 5pa
ce).

In these studies, dogs showed a different pattern of cardiovascular toxicity than that seen in humans. The reduction in blood flow in the main ducts appears to be due to a reduction in cardiac nodes, whereas in acute conditions studies have shown that as a result of hypotension, cardiac blood pumping increases with a decrease in system vascular resistance. showed an increase in amount. Changes in vascular permeability were observed in both cases.

Below is the margin 107- → w&kai-109- 1 to 1 tatami 4141 tatami bone to J ” ~ ・ 7 → to △ Ichino N2 nozo V N 0 Saki et al. Is to @ screw Q Mi 岨 6kuchi 鴴
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ba * 4 [140K report 4n i −
Table 18 shows data on the effect of protein A on homologous total complement activity, and Table 19 shows data on 03m and 05m resulting from synthetic nuclear union.

Preferred embodiment: Product 4 Autologous serum or plasma from a tumor-bearing host;
4ml/1bK10N-HC1kC3b, pH2,
5 and kept at 27°C for 5 minutes. Then l0N
- Immediately neutralize the serum to pH 7.4 by adding NaOH, then 1500Xg.

Centrifuged for 30 minutes at 4°C. The supernatant serum was then taken and given I, V, at a rate of 1 ttd/m.

After acidification and prompt neutralization according to the above procedure,
The changes in the main constituents of human plasma are as follows: 18th AI%. It can be seen that total protein, glonolin fraction, and fibrinogen are reduced, while other parameters remain virtually unchanged.

Table 18B shows that human immunoglobulins G, M, and A decrease after treatment of plasma with acid. Changes in human immunoglobulin samples after surface acidification IgG (
mg/di) IgM (mg/di) IgA (mg/di)
di) Untreated, 1200 100 110 Acidified 860 100 90 Autologous acidified serum from tumor-bearing hosts was free of tumor-associated antibodies as measured by indirect immunofluorescence using each dog's own tumor as a substrate. showed a significant increase in (
Table 19) Table 19 Lymphoma 10,000 20,
0002 Lymphoma 10,000
30,0003 Lymphoma 10,000
40,0004 Lymphoma 10,
000' 20.0005 Angiosarcoma
100 50.000*) The numbers indicate the end point dilution giving a positive fluorescent serum to the dog's own substrate.

Dogs with mammary adenocarcinoma were tested for changes in tumor-related antibodies in serum after acidification using a radioimmunoassay. Here, a canine mammary carcinoma tissue culture system was used as a target, and I protein A was used as an indicator for detecting canine IgGK-bound cells. 10 serum tests)
After acidification to pH 2.15, the solution was immediately neutralized. 120±63%, exceeding the value of untreated serum
An increase in tumor-associated binding levels of (S,D,) occurred.

The results of Sephadex G-200 chromatography analysis of whole serum from a human with thymic cancer before and after acidification are as follows. (a) When the serum was acidified, zyde serum brotiin increased and the proteil content in the containing fraction decreased relatively compared to the untreated level. (b) Compared to untreated serum, lung cancer-related (affinity) binding, as measured by radioimmunoassay, increased by 948 in the containing fraction and by 134% in the I-id volume fraction.

Results of analysis of acidified whole serum after sucrose density gradient ultracentrifugation:
There was an increase of up to 400% in fractions greater than 7s compared to untreated serum. Therefore, it appears that acidifying tumor-bearing serum results in increased production of tumor-associated antibodies and high molecular weight proteins.

Autologous acidified serum from a tumor-bearing dog was given with thymosan activated in normal dog serum 24 hours later (
It was given again on the second day). Animals were allowed to rest for 3 days and given cyclophosphamide I, V on the 4th day (regimen 2.
Table 18).

The results of this regimen for five dogs are shown in Figure 20.

Margin below - 115 - Table 20 Response to treatment □ Two-dimensional M relic 1 Buri Angiosarcoma 140 70 502
Labrador intervascular @ 58 32
373 Telya Myeloma50. 30 40
4 Sugerin Case)::-D) Lp Lymphoma 140 50
T.

5 Boodle Breast Cancer 250 125
50 Various effective aspects that produce effects worth considering are as follows. L-asparaginase and cyclophosphamide were given to the host on day 1, and when maximal regression of the tumor was obtained, normal dog serum (100 rnl) activated with thymosan and before acidification or alkalinization was administered. Processed autologous blood g (4rnl/lb)
The following day, L-asparaginase and cyclophosphamide were administered (Regulation 5. Table 12). Results 4 are shown in Table 21. , Table 21, 1 Subhaniel Li, Lymphoma 140 35
752 Beagle lymphoma 120 30
753 Boodle Lymphoma 70 3
5 50 thymosan is 1. After activating the complement sequence, the following results were obtained with body shaving and lnvlvo.

(a) Vasodilation, decrease in blood pressure, frequent heartbeats.

(b) 1. Smooth muscle bathmogenic effect.

These effects are due to complement accessory obstacles, namely anaphylatoxin 03a, 0.5m, in 1 s.

To activate complement in total normal dog serum, thymosan (
5g) was incubated with serum (IQOm/) at 27°C for 10 minutes. Thymosan was then removed by centrifugation at 15'00 xg for 30 minutes.

Then, the complement-activating serum was administered to the tumor-bearing host.
It was injected with I and V at a speed of 10 m/min.

In various treatment programs used to treat spontaneous canine tumors, thymosan-active normal canine plasma was used as an additional therapy. Timosare-activated dog plasma, (・) tumor immunoglobin preparation, (b) protein A-IgG
(c) When administered together with acidified or alkaline plasma, tumor killing or tumor regression effects were observed. When L-asparaginase, cyclophosphamide or adriamycin was given immediately after administration of these products, the vaginalicidal effect or regression was significant.

Preferred Embodiments Nibrotiin A 5 Protein A is a component of the cell wall of Staphylococcus aureus-cowans I and has the ability to react with Fe regions or immunoglobulins in many mammals. Antitumor activity was observed when administered to dogs with tumors. Protein A is 10-80 u
Injected at a dose of g/KH (regimen 8. Table 12)
.

As a result, 4 animals showed a slight temporary tumor reduction in 5 to 20 animals, and the other 1 showed a tumoricidal response. In addition, no effect was observed in the other two animals.

Substantial tumoricidal responses were seen using the above products alone, in combination with each other, and with a wide range of chemotherapeutic agents. The treatment method used to achieve this effect and the use of the products therein are shown in Figure 22.

The above results obtained were also described in a clever manner.

1) ΦAlthough all the examples here relate to spontaneous tumors in dogs, we have compared these tumors to the known and excellent human counterparts. Recognized as a model. A protein-collodion-charcoal system is used. Treatment in a dog model □
Clinical success (refs. 17, 19) has recently been transferred to humans, and tumor regression was obtained in 48 out of 5 patients treated (ref. 20). The data that has been thoroughly established regarding dogs with tumors may also be effective when the methods or compositions are applied to humans with spontaneous cancers. It is considered that it can be paid.

Although the present specification specifically describes the case where acid treatment is used as the dissociation technique, it is of course possible to use other dissociation techniques.

Furthermore, the disclosure herein also includes the following treatment methods.

(1) A thymosan-activated plasma preparation obtained by incubating serum from a normal or tumor-bearing host with thymosan, which does not contain thymosan and has no effect on the activity of complement byproducts. Therefore, INVID'
Treatment for a host with cancer, characterized by administering to the host a preparation that produces peripheral vasodilation and spasmodianeirectin ('Ipa+1m0g・nia') effect (in vivo). Method.゛(2. Claims 1, 2, 3, 4 or GSM) is characterized in that the preparation is administered to the host in an amount effective for treating cancer. Method of treatment.

(3) Particular requirements - The preparation described in paragraph 1 is administered to a host in a cancer therapeutically effective amount, and within 21 hours after the administration of asparaginase and asparaginase in a cancer therapeutically effective amount. A method for treating a host with cancer, which comprises administering a cytotoxic agent.
・゛(4)゛The preparation according to claim 5 and Timosashi active plasma in a cancer therapeutically effective amount! a cancer therapeutically effective amount plus L-aspar-122-laginase and a cytotoxic agent, wherein the cancer therapeutically effective amount plus L-aspar-122-laginase and a cytotoxic agent are administered within 2'4 hours after said administration. How to treat hosts that have the disease.

(5) Administering the formulations according to claims 2 and 5 to a host in a cancer therapeutically effective amount for two consecutive days, and then
A method for treating a host with cancer, comprising administering L-asparaginase and a cytotoxic agent in a cancer therapeutically effective amount within 24 hours after said administration.

(6) administering L-asparaginase and a cytotoxic agent to a host at a cancer therapeutically effective amount;
administering the formulation of claim 5 and the thymosan-activated plasma to a host, within 24 hours after said administration;
1. A method for treating a host with cancer, comprising administering L-asparaginase and a cytotoxic agent in a cancer-therapeutically effective amount.

(7) Administering the preparation according to claim 3 and thymosan-activated plasma to a host in a cancer therapeutically effective amount for two consecutive days, and then administering the same to a host within 24 hours after this administration. A method of treating a host with cancer, comprising administering therapeutically effective amounts of cyclophosphamide and adriamycin.

(8) Thymosan-activated plasma and others +/= at an effective amount for cancer treatment
Administering the derived anti-tumor IgG and protein A complex to the host for two consecutive days, and then administering L-asparaginase, cyclophosphamide or adriamycin with cancer therapeutic efficacy 1 within 24 hours after this administration. A method for treating a host having cancer, characterized by:

(9) The preparation according to claim 3 and xenogeneic antitumor IgG are administered to a host in an amount effective for cancer treatment for two consecutive days, and then, within 24 hours after this administration, cancer treatment is performed. 1. A method for treating a host suffering from cancer, comprising administering an effective amount of asparaginase and at least one cytotoxic agent.

(10) A method for treating a host with cancer, which comprises administering to the host an effective amount of Staphylococcus aurecus cowansue protein A.

(11) host (administering at least one of the formulations set forth in claims 1, 2, 3, 4, and 5 or thymosan-activated plasma in a cancer therapeutically effective amount; . A method for treating a host with cancer, comprising administering at least one chemotherapeutic agent in a cancer-therapeutically effective amount.

(12) Claims 1 and 2, whistle 3,
A method for treating a host suffering from cancer, comprising administering to the host at least two doses of the formulation described in paragraphs 4 and 5 or thymosan-activated plasma in an amount effective for cancer treatment.

(13) % Scope of Claims 1, 2, 3, and 4
At least 211I of the preparations described in Items 1 and 5 above, the active plasma of Taketimobun is administered to a host in an amount effective in treating cancer, and then at least one chemotherapeutic agent Ω is administered in an amount effective in treating cancer. -125- Fast method for treating hosts with cancer.

(14) A method for treating a host with cancer, which comprises administering a cancer-therapeutically effective amount of thymosan-activated thymosan at a time effective for activating the host's complement system.

(15) Treatment of a host with cancer, which comprises administering to the host a preparation that binds to the Fc receptor of Fc-bearing leukocytes and releasing at least one cytotoxic substance from the leukocytes. Method.

(16) The formulations described in claims 1, 2, 3, 4, and 5, as well as Staphylococcus aurecus protein A, in a cancer therapeutically effective amount to a host. 1. A method for treating a host suffering from cancer, which comprises administering at least one of the following:

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[Brief explanation of drawings]

FIG. 1 is a flow diagram of forced flow electrophoresis useful for preparing immunoglobulin-enriched effluent (EIE). FIG. 2 is an exploded partial view of a forced flow electrophoresis cell. FIG. 3 is a graph showing C1q binding of sucrose density gradient fractions after acid treatment of tumor-bearing serum. FIG. 4 shows a Coomassie blue-stained PAGE profile of high molecular weight canine IgG K isolated from serum after PACC perfusion. FIG. 5 shows the Coomassie blue-stained PAGFi profile of protein AK isolated from serum after PACC perfusion. 8... Cell 10... End plate 12... Spacer 12'... Internal buffer spacer 12'... Dialysate spacer lf'... Plasma spacer 14... Connector 16... Membrane 18... Concavity:: I11''"l':;5yly4" Pa. 28... External buffer container 32... Internal buffer container 34... External buffer overflow line 40... Globulin pump 4 ・...Plasma circulation, pump 46...Plasma preheater 137- Engraving of drawings (no change in content) Procedural amendment (method) June 1b, 1981 Kazuo Wakasugi, Commissioner of the Patent Office 1, of the case Indication 1982 Patent Application No. 61510 2 Title of Invention Tumor Immune Preparation and Process for Producing the Same 3 Relationship to the Amended Person's Case Address of Patent Applicant 77030 Ceris, Heathston, United States of America 1200 Morsand Avenue Texas Name of Medical Center Baylor Calendar of Medicine Representative Nee Oh Johnson 4 Agent 5 Subject of amendment (1) “Patent applicant” column of application (2) Drawings (3) Power of attorney and equivalent translation Amendment Contents (1) Submit an application stating the representative pitch name of the patent applicant company. (2) Submit an engraving of the drawings (no changes in content) (3) Submit a power of attorney and a translation thereof. 7 List of attached documents (1) Application form 1 copy (2
) drawing
1 copy (3) Power of attorney and its translation
1 copy each 2-

Claims (1)

[Claims] 1. Contains plasma of a normal or tumor-bearing host, and has the following characteristics (a) to (4I) (&) Mainly I
Contains gG and IgM and has a small amount of albumin; (bJ) has C1q binding in fractions 78 or higher on a sucrose density gradient; (c) produces a tumoricidal response when injected into a tumor-bearing host; (d) ) reduced IgG, Ig compared to unprocessed enriched immunoglobulin derived from forced flow electrophoresis of plasma.
(s) when the plasma is from a tumor-bearing host; Tumor-related antibody titer 20,000
A tumor immunopreparation characterized by having the following. 2. Derived from a normal or tumor-bearing host, with the following characteristics (&) to (f), containing (&) immunoglobules +)yG and a small amount of IgA,
Substantially free of IgM and complement, (b>° shows an increase in ctq-bound IgG in fractions 7 or more sedimented by sucrose density gradients, (0) has very little anti-complement activity, (a) (e) non-acidified corn-fractionated gamma which produces a tumoricidal response when administered to a tumor-bearing host;
(f) have lower levels of IgG and IgA compared to globulin and show increased C1q binding in plaques of 78 and above; A tumor immunoglobulin preparation characterized in that it has an increased level and low anti-complement activity. 3. Characteristics of the following (a) to (k) (a) By polyacrylamide gel electrophoresis::
redundancy:g: :H(a and L chain and dip(bl)
7 on a sucrose density gradient with increased C14 binding activity.
(c) Dissociates into lower molecular weight C1q-binding fragments under acidic conditions; (4) Dissociates in 5% polyethylene glycol or other known immunoglobulin fractions; (e) Has anti-complement activity; (f) Suppresses Fc-dependent lymphocyte rosette formation; (g) neutrophils aggregate, myeloperoxidase,
Promote the release of catezin and peroxide anions; (h) Cause aggregation of dog blood red cells; (1)
(j) causing non-specific complement-dependent cytotoxicity of canine thymic carcinoma cells, murine L cells and human red blood cells; (k) complement non-activation with the production of anaphylatoxins; Protein A is produced by an interaction between plasma and free or non-covalently bound Protein A by a dependent mechanism and is not present in plasma before treatment. IgG formulation. 4. Characteristics of (al to (j)) (a) Contains primarily IgM H and L chains and protein molecules as determined by polyacrylamide gel electrophoresis (b) With increased C14 binding activity (e) dissociates into lower molecular weight C1q-binding fragments under acidic conditions, (d) oxidation with 5% polyethylene glycol, or other It can be precipitated by a known immunoglobulin precipitation method. (e) It has anti-complement activity. (f) It suppresses Fc-dependent lymphocyte pore and zet formation. (g) It inhibits neutrophil myelopathy. (h) causes aggregation of dog blood red cells, (i) maintains its antigen affinity in the presence of complement, and characterized by having a relatively increased cytotoxic ability, (jl) producing a tumoricidal response and tumor singation when injected into a tumor-bearing host of protein-, IgG complexes at various ratios. 5. The following properties (a) to (f): (a) The titer of tumor-associated antibodies is 10 by indirect immunofluorescence.
．． 000 to 50,000, (bl radioimmunoassay, tumor-associated binding rep 5
- increase in C14 binding by more than 85% compared to untreated plasma; (a) increase in C14 binding in sedimentation fractions larger than 78 in the sucrose density gradient; -200, the protein content decreases in the fraction contained. (e) the tumor-immune plasma produces a tumoricidal response when injected into a tumor-bearing host, (fl) reduced IgG, IgM, Ig&, and fibrinogen levels in the tumor-immune plasma compared to live plasma; A tumor characterized in that it shows an increase in C14 binding in the 9-lactone (heavily precipitated), and in G-200 chromatography it shows an increased protein level in the void volume and a decreased level in the containing fraction. Immune plasma preparation. 6. Applying an electric field to the plasma, activating the current to obtain an effluent from the plasma having enriched 7-lactones containing immunoglobulins, and 6- acidifying or acidifying the plasma sufficiently to dissociate the antigen-antibody complexes. Claim 1, characterized in that alkalization is applied to the effluent to liberate tumor-specific antibodies, neutralize the effluent, and then filter the neutralized effluent containing precipitate. Method of manufacturing the described formulation. 7. Acidifying gamma globulin from normal or tumor-bearing serum. 3. The method for producing a preparation according to claim 2, which comprises neutralizing the acidified gamma globulin and removing a precipitate from the neutralized gamma globulin. 8. Precipitating protein A-IgG complexes from tumor-bearing or normal plasma after perfusion with immobilized Staphylococcus aurecus protein A, and separating the protein A-IgG complexes. A method for producing a preparation according to claim 3. 9. The method for producing the preparation according to claim 3, which comprises keeping protein A and IgG warm. 10. The method for producing the preparation according to claim 3, which comprises keeping protein A and IgG warm and then isolating the obtained protein A-IgG complex. 11. The production method according to claim 10, wherein the IgG has tumor specificity. 12. A method for producing a preparation, characterized in that serum from a normal or tumor-bearing host is incubated in demosan and then thymosan is removed. (Margin below)