JP2001522806A - Methods for treating tumors and tumor cells using ex vivo activated T cells - Google Patents

Abstract

  (57) [Summary] A novel method of modulating an immune response using treatment with ex vivo treated T cells is provided. The present invention provides a solution to the problem of treating various diseases, especially malignant diseases. The present invention also provides a method of treating a non-malignant disease by modulating the immune response of a mammal.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD [0001] The present invention relates generally to the fields of immunotherapy and oncology. For more information,
The present invention relates to a method for inducing activated T cells ex vivo and a method for treating tumors and tumor cells with activated T cells to stimulate an immune response.

BACKGROUND ART CD4 + T helper (T _H ) cells play a very important role in regulating the immune response and may be involved in regulating the immune response against tumor cells. T _H cells, they are based on the pattern of cytokine expression and immune responses mediated, it can be divided into two major subclasses T _H 1 and T _H 2. T _H 1 cells produce interleukin -2 (IL-2), interferon - gamma (IFN-γ), tumor necrosis factor -
Produces alpha (TNF-α) and lymphokines (LT), but not IL-4 or IL-5. T _H 2 cells produce IL-4 and IL-5, but do not produce IL-2, IFN-γ, TNF-α or LT. Third class T _H of T _H cells 0 indicates cytokine expression are not restricted pattern, produce both T _H 1 and T _H 2 cytokines. In experimental animal models of infection, T _H 1
Cells modulate cellular arm of the immune response, macrophages and monocytes and delayed over the sensitivity activated, while, T _H 2 cells turned into active humoral arm of the immune response, provided the B cell donor, Stimulates IgG and IgE production. T _H 0 cells can function to activate both arms of the immune response.

[0003] IFN-α, IFN-γ, IL-4 and IL-12 are T_HCell subsets can be differentially regulated. IFN-α, IFN-γ, and IL-12 are native T _H Cell T_H1 induces differentiation into one cell, whereas IL-4 induces T cells in na ー ブ ve cells._HIt is the only cytokine that promotes differentiation into two cells. In addition, these
Differentiated T_HCell responsiveness changes. For example, IFN-γ is T_HTwo
Inhibits growth, but_HOne cell growth is not inhibited, while T_HNot two cells
Iga T_HOne cell retains IL-12 responsiveness.

[0004] While there is data supporting a role in both T _H 1 and T _H 2 cells in anti-tumor responses, most preclinical animal studies suggest that the T _H 1 response plays a key role. ing. The ability of mice to reject P815 tumor cell line has been shown to be due largely to the presence of CD4 + cells producing T _H 1 cytokines (Fa
llarino et al., 1996). In these experiments, high IFN-γT _H 1 can not produce a response mouse can not reject tumor, while mice which develop such responses rejected their tumors more effectively. IL-12 was required for the generation of T _H 1 responses. Evidence made Sara for the role of T _H 1 responses in anti-tumor immunity is obtained from experiments in mice with established sarcomas (Zitv
ogel et al., 1995). Established tumors were rejected with fibroblasts created to secrete IL-13 or with control fibroblasts. IL-12 secreting fibroblasts effectively induced a CD4T _H1 anti-tumor response, and were thus successful in controlling the tumor.

[0005] Aggressive non-Hodgkin's lymphoma (NHL) can often be cured with intense combination chemotherapy, with 40-50% of patients alive and disease free in 5 years (Armi)
stage, 1993; Salles et al., 1994). However, patients who relapse or have major intractable illness have a poor prognosis. The use of intensive therapy with similar bone marrow or peripheral blood progenitor cells (PBPC) has become the standard treatment approach for these patients. However, even with this aggressive therapy, only 30-40% of patients with relapsed or refractory NHL survive and are ill at 5 years (Gribben et al., 1989; Philip et al., 1995).
).

[0006] Patients with relapsed or refractory NHL with incomplete or short lasting response to dose-intensive therapy have an extremely poor prognosis with a survival rate of 5% or less after 5 years . Experiments have also shown that patients with molecular evidence of residual NHL following intensive therapy and patients with evidence of bone marrow or stem cell involvement with NHL at reinfusion are at risk of failing intensive therapy. (Sharp et al., 1992; Gribben et al., 1994). Thus, patients who do not show chemosensitivity to the method of induction, or who have molecular evidence of residual disease, do not appear to achieve long-term disease-free survival with the intensive dose regimen. These "high-risk" patients are thus candidates for further supplemental therapies with intensive therapy that improves long-term event-free survival.

SUMMARY OF THE INVENTION The present invention generally relates to methods of modulating an immune response comprising treating a mammal with a T cell or population of T cells. In some currently preferred embodiments, the invention generally relates to a method of inducing an immune response comprising treating a mammal with a T cell or population of T cells. The present invention provides a solution to the previously discussed problems of treating a variety of diseases, especially those that are malignant. The methods taught herein make it possible to induce an immune response directed against a malignancy.

As used herein, the phrase “inducing an immune response” refers to any measurable increase in an indicator of an immune response. Induction of an immune response involves the onset of an immune response in a mammal that has not laid an immune response at the time treatment is initiated. Similarly, increasing or enhancing an immune response that is already in progress is induction of an immune response. The present invention also provides a method of treating a non-malignant disease by modulating the immune response of a mammal. "Modulation of an immune response" is defined as any measurable change in an indicator of an immune response in response to T cell administration.

In many cases, mammals have a disease, such as a malignant disease, and the treatment is expected to benefit from induction of an immune response. In some cases, the mammal is a human. The method of the present invention is expected to have broad applications for immunoreactive and systemic cancers.

[0010] The present invention provides for obtaining a population of peripheral blood mononuclear cells from a mammal and activating the population of peripheral blood mononuclear cells outside the mammal or ex vivo to produce a population of peripheral blood mononuclear cells outside the mammal or ex vivo. Obtaining a population of T cells already activated in vivo and administering to the mammal a therapeutically effective amount of the population of T cells, thereby inducing an immune response and treating a mammal with an immune responsive cancer Providing a method of treating a mammal having an immune responsive cancer, comprising: As used herein, a "therapeutically effective amount" refers to a substance that specifically delays the progression of a tumor, induces necrosis in at least a portion of the tumor, and / or regresses tumors upon administration to a selected mammal or patient. The amount of activated T cells effective to induce remission.

[0011] Immune responsive cancers contemplated for treatment using the methods of the invention include, but are not limited to, non-Hodgkin's lymphoma, multiple myeloma, kidney cancer, ovarian cancer, prostate cancer, low grade lymphoma, chronic Lymphocytic leukemia (CLL), chronic myeloid leukemia (CML)
Lymphomas such as acute myeloid leukemia (AML), osteosarcomas, sarcomas such as lung cancer,
Includes opportunistic malignancies, such as Kaposi's sarcoma, and cancers associated with other viruses, such as cervical and rectal cancer, or melanoma.

[0012] In a most general sense, any cancer that responds to an immunological intervention in which manipulation of the immune system produces a response to the disease is considered to be responsive to the treatment provided herein. Many different techniques are available for measuring the immunological responsiveness of a particular cancer and will be understood by those skilled in the art. For example, leukemias and lymphomas have been shown to respond immunologically through the use of allogeneic transplantation, and melanomas have been shown to respond to LAK and TIL.
It has been determined that cell technology, as well as immunological response to interferon, kidney cancer has been shown to respond immunologically to interferon and IL-2, and prostate cancer is CTLA-4 mouse It has been determined to respond immunologically using the model.

In a particular embodiment of the invention, activating the population of peripheral blood mononuclear cells comprises contacting the peripheral blood mononuclear cells with at least a first antibody and at least a second antibody. In certain embodiments, the at least first and at least second antibodies are linked to a particle, such as a bead. Beads that could be used are
Manufactured from a variety of different materials, for example, plastic or magnetic beads, such as polystyrene. In a preferred embodiment, the antibodies are immobilized on a structure (ie, a bead) such that distinct point contacts are made between the multiple beads and each individual T cell that is the target of costimulation. The immobilized antibody stimulates individual T cells from multiple contact points. In particular embodiments, the optimal number of contact points is greater than or equal to three. However, in certain embodiments of the invention, the use of superantigens in combination with calcium ionophores, such as ionomycin, or other forms of activation, such as the use of protein kinase C activators, such as phorbol, are contemplated. Can be

In any of the methods described above, “antibodies, naked antibodies and non-conjugated antibodies” as used herein include polyclonal and monoclonal IgG,
Any immunological binder such as IgM, IgA, IgD and IgE antibodies is broadly referred to. Generally, IgG and / or IgM are preferred. Because they are the most common antibodies in physiological situations, and because they are most easily produced in laboratory settings.

[0015] Polyclonal antibodies obtained from antisera can be used in the present invention.
However, the use of monoclonal antibodies (MAbs) will generally be preferred. It has been recognized that MAbs have certain advantages, such as reproducibility and large-scale production, which makes them suitable for clinical treatment. The invention thus provides a monoclonal antibody of murine, human, monkey, rat, hamster, rabbit or frog or even chicken origin.

As will be appreciated by those skilled in the art, immunological binding agents encompassed by the term “antibody” include dimeric, trimeric and multimeric antibodies, bispecific antibodies, chimeric antibodies,
It also includes all naked and non-conjugated antibodies and antigen-binding fragments thereof from any species, including human and humanized antibodies, recombinant and genetically engineered antibodies, and fragments thereof.

The term “antibody” thus refers to any antibody-like molecule having an antigen-binding region, including Fab ′, Fab, F (ab ′) ₂ , single domain antibody (DAB), Fv, scFv (Single-chain Fv) and the like. Techniques for preparing and using various antibody-based constructs and fragments are well known in the art.

In certain embodiments, the antibodies used are “humanized” or human antibodies. "Humanized" antibodies are generally chimeric monoclonal antibodies from mouse, rat or other non-human species that carry human constant and / or variable region domains ("partially human chimeric antibodies"). For the most part, the humanized monoclonal antibodies used in the present invention are chimeric antibodies, in which the complementarity determining regions (CDRs) of a mouse, rat or other human monoclonal antibody operate on a human antibody constant region or "framework". Possibly linked or "grafted" to it.

Also, as used herein, a “humanized” antibody is a monoclonal antibody from a non-human species, wherein one or more selected amino acids are exchanged for those more commonly found in human antibodies. ing. This can be easily achieved through the use of routine recombination techniques, especially site-directed mutagenesis.

In some embodiments of the invention, at least the first antibody and at least the second antibody
Antibodies are distinct antibodies. In certain preferred embodiments, at least the first antibody is an anti-CD3 antibody, such as an OKT3 or G19-4 antibody. In another embodiment, at least the second antibody is 9.3, KOLT-2, 15E8, 248.23.
Anti-CD28 antibody, such as 2 or EX5.3D10 antibody. In a particularly preferred embodiment of the invention, at least the first antibody is an anti-CD3 antibody and at least the second antibody is an anti-CD28 antibody. In a further embodiment, the anti-CD2 antibody is utilized in combination with an anti-CD28 antibody, and the anti-CD3 antibody is IL-2 or I-2.
Available in combination with L-15 or anti-B7- (CD80) or anti-B
The 7-2 (CD86) antibody can be used in combination with an anti-CD3 antibody.

In a particular embodiment of the invention, the population of T cells comprises CD4 + T cells. In certain embodiments, the population of T cells predominantly comprises CD4 + T cells, while in other embodiments, the population of T cells comprises CD4 + T cells and CD8 + T cells. In some embodiments of the invention, the population of T cells is not activated upon administration to a mammal. Thus, in some preferred embodiments, the T cells are inactive or inactivated upon administration to the mammal.

[0022] CD4 + and / or CE8 + T cells can be expanded or derived from a population of CD4 + and CD8 + T cells that have been expanded by treatment with the antibody.
In many cases, growth due to treatment with the antibody occurs ex vivo. In a preferred embodiment, expansion by treatment with the antibody comprises costimulation with two distinct populations of the antibody. Preferred of the distinct population of antibodies are anti-CD3 antibodies, such as O
KT3 population. Preferably, one of the distinct populations of antibodies is a population of anti-28 antibodies. In a currently preferred embodiment, expansion comprises costimulation with a population of anti-CD3 antibodies and a population of anti-CD28 antibodies via the methods described elsewhere in this application.

In a particular embodiment of the invention, the immune response comprises the production of T cells in the mammal. In another embodiment, the immune response comprises a population of CD8 + T cells in the mammal. In a preferred embodiment, the population of T cells administered to the mammal is predominantly or completely CD4 + T cells. In another preferred embodiment, the population of T cells is predominantly CD4 + T cells, and the immune response comprises a population of CD8 + T cells directed against immune responsiveness in a mammal.

In addition to malignant diseases, CD4 + T cells can be used in patients with other disorders of the immune system, for example,
It can be used to treat patients with a hyperimmune condition or immunodeficiency.
In these cases, the invention involves modulating the immune response mediated by CD4 + cells, in some cases by switching off or switching on the immune response, or by recognizing the immune response. . For example, C
D4 + cells can be used to treat diseases such as diabetes, lupus and rheumatoid arthritis. Similarly, T cells can be used to prevent organ rejection after transplantation. In the case of implant rejection prevention, it will often benefit from T cell treatment prior to the transplantation procedure.

In some cases, T cells will be administered in combination with other treatment modalities, such as chemotherapy, antibiotic therapy, antibody therapy, radiation therapy, transplantation, and the like. In some preferred methods of practicing the present invention, stem cell transplantation can be provided in combination with T cell treatment. In the treatment of malignancies, administration of CD4 + T cells can serve to modify the proper activation of the immune system. In other diseases, there are also problems associated with a defective immune response. Therefore, it would be advantageous to immunosuppress the patient prior to T cell therapy. By immunosuppressing the patient, the inappropriate immune response is suppressed and T cell therapy can thus influence the proper activation of the immune system. If a stem cell transplant is given, the chemotherapy associated with the transplant procedure is CD4 + T
Immunosuppress the patient prior to cell administration.

Thus, in a further embodiment of the invention, the method further comprises immunosuppressing the mammal prior to administering the T cells to the patient. Corticosteroids such as, but not limited to, chemotherapy to mammals, radiation therapy, azathioprine, cyclophosphamide, rapamycin, prednisone, cyclosporin A
, FK506, purine analogs and alderylsen, 2-chloro-deoxyadenosine, mercaptopurine (6-mercaptopurine), thioguanine (6-thioguanine) and related inhibitors such as pentostatin (2-deoxyformycin), alkylating agents, For example, nitrogen mustards such as mechloresamine (NH ₂ ), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) and chlorambucil, ethylmelenimen and methylmelamines such as hexamethylmelamine and thiotepa, alkylsulfonates such as dusulfan,
Carmustine (BCNU), lomustine (CCNU), semustine (methyl-C
Nitrosoureas such as CNU) and streptozocin (streptozotocin), and triazines such as decarbazine (DTIC; dimethyltriazenoimidazolecarboxamide), or any of a number of techniques for immunosuppression, including anti-T cell monoclonal antibodies. It is contemplated for use in the present invention and is known to those skilled in the art.

[0027] In yet another aspect of the invention, the method comprises administering the stem cell transplant to a mammal. In a preferred embodiment, the stem cells include CD34 + cells.
The present invention involves co-administering CD4 + T cells with cytokines or other biological compounds capable of modulating or specifying the phenotype of CD4 + T cells or CD8 + T cells. For example, IL-2, IL-12, IL-4, IL-10, or IL
The function of the cell product produced using the treatment at -6 can be modified.

[0028] The present invention also provides a T cell that is obtained from a mammal and obtains a population of peripheral blood mononuclear cells, activates the population of peripheral blood mononuclear cells outside the mammal, and is activated outside the mammal. And administering the population of T cells to a mammal, thereby inducing an immune response and treating the mammal with non-Hodgkin's lymphoma. I will provide a.

In a preferred embodiment of the present invention, the activation of the population of peripheral blood mononuclear cells comprises:
8 with the antibody. In a particularly preferred embodiment, at least the first anti-CD3 antibody and at least the first anti-CD28 antibody are linked to magnetic beads. In another preferred embodiment, the population of T cells predominantly comprises CD4 + T cells.

[0030] The present invention further provides a population of peripheral blood mononuclear cells from a mammal and activates the population of peripheral blood mononuclear cells outside of the mammal to activate T cells that are activated outside of the mammal.
Obtaining a population of cells and administering the population of T cells to a mammal, thereby treating the mammal with T cells having a defective cytokine profile, comprising treating the mammal with T cells having a defective cytokine profile Provide a way to In a preferred embodiment, activating the population of peripheral blood mononuclear cells comprises co-stimulating a population of anti-CD3 antibodies and an anti-CD28 antibody.

Obtaining a population of peripheral blood mononuclear cells from the mammal, activating the population of peripheral blood mononuclear cells outside of the mammal to obtain a population of T cells activated outside of the mammal,
There is provided a method of inducing lymphocytosis in a mammal, comprising administering the population of cells to a mammal, thereby inducing lymphocytosis in the mammal.

According to longstanding patent law, as used herein, including the claims, the term “
“(A) or (an)” indicates “one or more”. Specifically, "Target"
The word “comprising,” “having,” or other open terms in the claims claiming a combination or method of use indicates that “one or more of the objects” can be used in the claimed method or combination.

BEST MODE FOR CARRYING OUT THE INVENTION In designing a novel biological therapy for immunoreactive cancers, we developed a method for ex vivo expansion of activated CD4 + and CD8 + T cells. And
Polished. An immune-responsive cancer is herein any cancer that responds to an immunological intervention, where manipulation of the immune system results in a response to the disease. In general, cancer has been shown to be immunoresponsive using any of a number of different technologies. For example, certain leukemias and lymphomas have been shown to be immunologically responsive through the use of syngeneic xenografts and immunologically responsive to LAK and TIL and TIL cell technology, and to interferons. Renal cancer has been determined to be immunologically responsive to interferon and IL-2, and ovarian cancer has been shown to be immunologically responsive to IL-2; Cancer is CTLA
-4 has been determined to be immunologically responsive using a mouse model (e.g., Cancer principal, which is incorporated herein by reference).
ples and practice of oncology, Lippi
cot-Raven, Philadelphia, 1997).

An exemplary use of this therapy is as an adjunct therapy in patients undergoing dose-intensive therapy for relapsed non-Hodgkin's lymphoma (NHL). The antitumor activity of these activated cells was measured and is presented here.
T helper subset profiles were examined in patients when NHL was first present. In vitro cell cultures, which studied identifying cytokines that modulate T helper responses in patients, were also performed.

In one embodiment of the invention, a novel system is used to drive the ex vivo expansion of CD4 + and CD8 + T cells. In this system, immobilized on magnetic beads,
Antibodies to the T cell accessory molecules CD3 and CD28 were used to provide a costimulatory signal to drive logarithmic growth and proliferation of CD3 + CD28 + human T cells. The previous T cell culture system used in the human immunotherapy trial (ie, LAK
Or TIL cell technology) resulted in predominantly CD8 + effector T cell growth and proliferation. This CD3 + 28 co-stimulatory system is the first available method to enable large-scale expansion and activation of polyclonal human CD4 + T cells ex vivo with CD8 + T cells, specifically the relapsed NHL (BBIND)
These ex vivo expanded CD3 + cells can be administered to patients receiving intensive therapy on autologous CD34 selected peripheral blood progenitor cell (PBPC) support for 6914).
A clinical trial was performed in which increasing doses of 28 costimulatory T cells were administered. The ability to eliminate tumors and tumor cells as measured by both conventional and newly developed assays specific for this protocol was demonstrated. However, other methods of activating T cells are also contemplated for use in certain aspects of the invention (June et al., PCT Application WO 94, each of which is specifically incorporated herein by reference).
/ 29436 and WO 95/33823).

One approach to improving the outcome of treatment for patients with relapsed / refractory NHL is to combine another treatment modality, such as biological therapy with intensive dose therapy or immunomodulation. As a result, minimal residual disease that fails to fully respond to the intensive dosing regime may be eradicated. The study was based on intensive dose therapy followed by residual N
Patients with molecular evidence of HL and those with evidence of bone marrow or stem cell involvement with NHL at reinfusion show an increased risk of failing the intensive dosing regimen (Zw
icky et al., 1996). Thus, it appears to be useful in identifying patients who are unable to achieve long-term disease survival with the intensive dosing method alone, and is thus a candidate for additional supplemental treatment modalities with intensive therapy.

A. Antibodies 1. Polyclonal Antibodies Means for preparing and characterizing antibodies are well known in the art (eg, Antibodies: A, which is incorporated herein by reference).
Laboratory Manual, Cold Spring Harbo
r Laboratory, 1988). To prepare a polyclonal antiserum, an animal is immunized with an immunogenic composition, and the antiserum is collected from the immunized animal. A wide range of animal species can be used in the production of antisera. Typically, the animals used in the production of anti-antisera are rabbits, mice, rats, hamsters, guinea pigs or goats. Rabbits are a preferred choice for the production of polyclonal antibodies because of their relatively large blood volume.

[0038] The amount of the immunogen composition used in the production of polyclonal antibodies will vary depending on the nature of the immunogen as well as the animal used in the immunization. Various routes can be used for administering the immunogen. Subcutaneous, intramuscular, intradermal, intravenous, intraperitoneal and intrasplenic administration. Production of polyclonal antibodies is monitored by sampling blood from the immunized animal at various times after immunization. A second booster injection can also be given. The process of boosting and titration is repeated until the appropriate titer is achieved. When the desired titer level is obtained, blood can be collected from the immunized animal and the serum can be isolated and stored. Alternatively, monoclonal antibodies can be produced using animals.

As is well known in the art, the immunogenicity of a particular composition can be enhanced by the use of a non-specific stimulator of the immune response known as an adjuvant. An exemplary adjuvant is Freund's complete adjuvant, killed Mycoba
a non-specific stimulator of the immune response containing terium tunerculosis; incomplete Freund's adjuvant; and aluminum hydroxide adjuvant.

It may be desirable to boost the host immune system, as can be achieved by coupling the immunogen to a carrier. Exemplary carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.

[0041] 2. Monoclonal Antibodies Various methods for producing monoclonal antibodies (MAbs) are also well known in the art. The most standard monoclonal antibody generation techniques generally begin along the same system as that for preparing polyclonal antibodies (Antibod).
ies: A Laboratory Manual, Cold Spring
g Harbor Laboratory, 1988; incorporated herein by reference). A polyclonal antibody response is initiated by immunizing an animal with the immunogenic composition, and once the desired titer level has been obtained, the immunized animal can be used to generate a MAb.

MAbs are incorporated by reference in US Pat.
It can be readily prepared by the use of well-known techniques, such as those exemplified in 196,265. Typically, this technique involves immunizing a suitable animal with a selected immunogenic composition. The immunizing composition is administered to be effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, but the use of rabbit, sheep and frog cells is also possible.
Although the use of rats can provide certain advantages (Goding, 1986, pp. 60-61; incorporated herein by reference), mice are preferred and BALB / c mice are the most preferred. preferable. This is because it is most routinely used and generally gives a higher percentage of stable fusion.

Following immunization, antibodies, specifically somatic cells capable of producing B lymphocytes (B cells), are selected for use in the MAb production protocol. These cells can be obtained from biopsied spleens, tonsils or lymph nodes, or from peripheral blood samples. Spleen cells and peripheral blood cells are preferred, because the former is a rich source of antibody-producing cells at the dividing plasmablast stage, and the latter is easily accessible by peripheral blood. Frequently, a panel of animals is immunized, the spleen of the animal with the highest antibody titer is removed, and spleen lymphocytes are obtained by homogenizing the spleen with a syringe. Typically, spleens from immunized mice contain approximately 5 × 10 ⁷ to 2 × 10 ⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortalized myeloma cell (generally of the same species as the immunized animal). Myeloma cell lines suitable for use in the hybridoma production fusion procedure are preferably non-antibody-producing, have high fusion efficiency, and are in certain selection media that support growth of only the desired fusion cells (hybridomas). Has an enzyme deficiency that prevents growth.

[0045] Any of a number of myeloid cells can be used as known to those skilled in the art (Goding, pages 65-66, 1986; Campbel).
1, pages 75-83, 1984; each of which is incorporated herein by reference). For example, if the immunized animal is a mouse, P3-X63 / A
g8, X63-Ag8.653, NS1 / 1. Ag41, Sp210-Ag
14, FO, NSO / U, MPC-11, MPC-X45-GTG1.7 and S194 / 5XXO Bul can be used; in rats, R210
. RCY3, Y3-Ag 1.2.3. , IR983F, 4B210 or one of the mouse cell lines listed above; U-266, GM1500-
GRG2, LICR-LON-NMy2 and UC729-6 are all useful in the context of human cell fusion.

Methods for producing hybrids of antibody producing spleen or lymph node cells and myeloma cells generally involve somatic cells in the presence of an agent that promotes cell membrane fusion or multiple agents (chemical or warm). Including mixing at a 4: 1 ratio with myeloma cells (
However, the proportions can each vary from about 20: 1 to about 1: 1)
. Fusion methods using Sendai virus are described by Kohler and Milstein.
(1975; 1976; incorporated herein by reference) and methods using polyethylene glycol (PEG), such as 37% (v / v) PEG, are described in Getter (1977). ; Incorporated herein by reference). Electrically induced fusion methods are also suitable (Goding, 71-74, 1986, which is incorporated herein by reference).

The fusion approach usually produces live hybrids at low frequency, about 1 × 10 ⁻⁶ to 1 × 10 ⁻⁸ . However, this does not pose a problem. This is because live fusion hybrids are differentiated from parental non-fusion cells, especially non-fusion myeloma cells, which usually divide indefinitely, by culturing the selection medium. The selection medium generally contains an agent that blocks de novo synthesis of nucleotides in the tissue culture medium. Exemplary and preferred agents are aminopterin, methotrexate and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, while azaserine blocks only purine synthesis. If aminopterin and methotrexate are used, the medium is supplemented with hypoxanthine and thymidine as nucleotide sources (HAT medium). When using azaserine, supplement the medium with hypoxanthine.

A preferred selection medium is HAT. Only cells capable of operating the nucleotide salvage pathway can survive in HAT medium. Myeloma cells
Lacking key enzymes of the salvage pathway, such as hypoxanthine phosphatidyltransferase (HPRT), they cannot survive. B cells can operate this pathway but have a limited lifespan in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selection medium are hybrids formed from myeloma and B cells.

This culture provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas involves culturing the cells by single-clone dilution into microtiter plates, followed by the desired reactivity (for
This is done by testing individual clonal supernatants (after about two to three weeks). The assay should be sensitive, simple and rapid, such as a radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunoblotting assay, and the like.

[0050] The selected hybridomas can then be serially diluted and cloned into individual hybridoma producing cell lines, and the clones can then be grown indefinitely to obtain MAbs. Cell lines can be developed for MAb production in two basic ways. The hybridoma sample can be injected (often intraperitoneally) into a type of histocompatible animal that can be used to provide the somatic and myeloma cells for the original fusion. The injected animals develop tumors that secrete specific monoclonal antibodies produced by the fused cell hybrids. The body fluid of the animal, such as serum or ascites fluid, can then be collected to obtain a high concentration of MAb. Alternatively, if the MAb is naturally secreted into a medium from which high concentrations can be obtained, individual cell lines can be cultured in vitro.

MAbs produced by either means are generally, for example, filtered, centrifuged,
And will be further purified using various chromatographic methods such as HPLC or affinity chromatography, all of which are well known to those skilled in the art. Each of these purification techniques involves fractionation, which separates the desired antibody from other components of the mixture. Analytical methods particularly suitable for preparing antibodies include, for example,
Protein A-Sepharose and / or Protein G-Sepharose
Including ose chromatography.

[0052] 3. Antibodies from Phagemid Libraries Recombinant technology today allows the preparation of antibodies with the desired specificity from recombinant genes encoding a range of antibodies (Van Dijk et al., 1989; Of the present specification). Certain recombinant technologies include:
Involves isolation of antibody genes by immunological screening of a combined immunoglobulin phage expression library prepared from RNA isolated from the spleen of the immunized animal (Morrison et al., 1986; Winter and Milstein,
1991; each of which is incorporated herein by reference).

In such a method, a combinatorial immunoglobulin phagemid library is prepared from RNA isolated from the spleen of an immunized animal, and phagemid expressing the appropriate antibody is isolated from cells expressing the antigen and control cells. Select by dividing. Advantages of excellent this approach than conventional hybrid technology, produced almost ¹⁰⁴ times more antibodies in a single round, to be secreted, and new specificities are created by H and L combinations, which It is also to increase the percentage of suitable antibodies created.

One method for creating a large repertoire of diverse antibody molecules in bacteria is
Utilizing bacteriophage lambda as a vector (Huse et al., 1989; incorporated herein by reference). Production of antibodies using lambda vectors involves the cloning of heavy and light chain populations of DNA sequences into separate starting vectors. The vectors are subsequently randomly combined to form a single vector that directs co-expression of the heavy and light chains to yield the antibody fragment. The heavy and light chain DNA sequences are, for example, those of mRNA isolated from spleen cells (or hybridomas thereof) from an animal immunized with the selected antigen, preferably PC
Obtained by amplification with ^RTM or related amplification techniques. The heavy and light chain sequences are:
Typically, amplification is performed using primers that incorporate restriction sites at the ends of the amplified DNA segment to facilitate cloning of the heavy and light chain segments into the starting vector.

Another method of creating and screening large libraries of totally or partially synthetic antibodies combining sites or paratopes utilizes a display vector derived from a filamentous phage such as M13, fl or fd. "
These filamentous phage display vectors, "phagemids," yield large libraries of monoclonal antibodies with diverse and novel immunospecificities. The technology uses the filamentous phage coat protein membrane anchor domain as a means of linking gene products and genes during the assembly phase of filamentous phage replication, which is used in the cloning and expression of antibodies from combinatorial libraries. (Kang et al., 1991; Barbas et al., 1991; each incorporated herein by reference).

This general technique for filamentous phage display is described in US Pat. No. 5,658,727, which is incorporated herein by reference. In its most general sense, the method provides a system for co-cloning and screening for pre-selected ligand binding specificities from antibody genes using a single vector system. Screening of isolated members of the library for preselected ligand binding capacity correlates the binding capacity of the expressed molecule with a convenient means for isolating the gene encoding the member from the library. Is possible.

The coupling of expression and screening involves targeting of the fusion polypeptide to the periplasm of bacterial cells, which allows for the assembly of functional antibodies, and phage assembly, which allows for convenient screening of library members of interest. By targeting the fusion polypeptide to the coat of the filamentous phage particles. Periplasmic targeting is provided by the presence of a secretory signal domain in the fusion polypeptide. Targeting to phage particles is accomplished by binding the filamentous phage coat protein membrane anchor domain (ie, cpIII-
Or cpVIII-derived membrane anchor domain).

The diversity of a filamentous phage-based combinatorial antibody library is determined by shuffling the heavy and light chain genes, thereby altering one or more complementary determining regions of the library's cloned heavy chain genes. Alternatively, it can be increased by introducing random mutations into the library by an error-prone polymerase chain reaction. Additional methods of screening phagemid libraries are described in US Pat. Nos. 5,580,717, 5,427,908, 5,403,484, which are incorporated herein by reference. No. 5,223,409.

Another method has been developed to screen large combinatorial antibody libraries utilizing the expression of diverse heavy and light chain populations on the surface of filamentous bacteriophages such as M13, fl or fd ( U.S. Patent No. 5,698,426, which is hereby incorporated by reference. Two populations of diverse heavy (Hc) and light (Lc) sequences are synthesized by the polymerase chain reaction (PCR ^™ ). These populations are cloned into another M13-based vector containing the necessary elements for expression. The heavy chain vector has a heavy chain sequence translation of gVII.
Contains the gene VIII (gVIII) coat protein sequence to generate an I-Hc fusion protein. The two vector populations are randomly combined such that only the vector portion containing the Hc and Lc sequences is ligated into a single circular vector.

The combined vector directs the assembly of the two popeptides and the co-expression of the Hc and Lc sequences for surface expression at M13 (US Pat. Patent No. 5,698,426). The combining step randomly places the different Hc and Lc coding sequences in the two diverse populations together into a single vector. Vector sequences provided from each independent vector are required for live phage production. Also, since the pseudogVIII sequence is contained in only one of the two starting vectors, the Lc-associated gVIII-H
Co-expression of a functional antibody fragment as a c-fusion protein can be achieved on the phage surface until vector expression is ligated in a single vector.

[0061] Surface expression of the antibody library is performed in an Amber suppressor strain. The amber stop codon between the Hc sequence and the gVIII sequence releases two components in the non-suppressor strain. Isolation of the phage produced from the non-suppressor strain and infection of the suppressor strain will link the Hc sequence to the gVIII sequence during expression. Culture of the suppressor strain after infection was performed using the gVIII fusion protein (gVIII).
-Fab fusion protein) to allow coexpression of all antibody species in the library on the surface of M13. Alternatively, DNA can be isolated from a non-suppressor strain and then introduced into a suppressor strain to achieve the same effect.

Surface expression libraries are screened for specific Fab fragments that bind to the preselected molecule by standard affinity isolation techniques. Such methods include, for example, panning (Parmley and Smith, 1988; incorporated herein by reference), affinity chromatography, and solid phase blotting techniques. Panning is preferred. This is because high titer phage can be easily, rapidly and screened in a small volume. In addition, this approach can select for small Fab fragment species within a population that would otherwise be undetectable and would be amplified to a substantially homogeneous population. Selected Fab fragments can be characterized by sequencing the nucleic acid encoding the popeptide after amplification of the phage population.

Another method for producing a diverse library of antibodies and screening for the desired binding specificity is described in US Pat. No. 5,667,988, which is hereby incorporated by reference. And U.S. Pat. No. 5,795,817. The method comprises CDRs of immunoglobulin variable heavy and light chain variable domains.
The preparation of a library of heterodimeric immunoglobulin molecules in the form of a phagemid library using degenerate oligonucleotides and primer extension reactions to incorporate degeneracy into the display of the mutated polypeptide on the region and phagemid surface. Including. Thereafter, the display proteins are screened for the ability to bind to the preselected antigen.

A method for producing a homodimer immunoglobulin molecule generally includes (1) introducing a heavy or light chain V region coding gene of interest into a phagemid display vector, and (2) applying a CDR to an antibody V region gene. A randomized binding site is introduced into the phagemid display protein vector by primer extension with an oligonucleotide containing a region of homology and containing a region of degeneracy for the randomized coding sequence, each of which has a phagemid surface display. Forming a large population of display vectors capable of expressing different putative binding sites displayed on the protein, (3) expressing the display protein and binding sites on the surface of the filamentous phage particles, and (4) Such as panning of phage particles against a selected antigen The surface-expressed phage particles are isolated (screened) using affinity technology, thereby isolating one or more species of phagemid containing a display protein containing a binding site that binds to a preselected antigen. Including doing.

A further variation of this method of producing a diverse library of antibodies and screening for a desired binding specificity is described in US Pat. No. 5,702, incorporated herein by reference. No. 892. In this method, only the heavy chain is used, the light chain sequence is randomized at all nucleotide sites that encode either the CDRI or CDRIII hypervariable region, and the genetic variability in the CDRs is reduced by any biological process. Created independently.

In the method, two libraries are created to genetically shuffle oligonucleotide motifs within the framework of the heavy chain gene structure. CD
Reconstruction of the hypervariable region of the heavy chain gene via random mutation of either RI or CDRIII results in a collection of highly diverse sequences. The heavy chain protein encoded by the collection of mutated genes retained the ability to have all of the binding characteristics of the immunoglobulin, while requiring only one of the two immunoglobulin chains.

Specifically, the method is performed in the absence of an immunoglobulin light chain protein. The library of phage displaying modified heavy chain proteins is incubated with the immobilized ligand to select a clone encoding a recombinant protein that specifically binds to the immobilized ligand. The bound phage is then dissociated from the immobilized ligand and amplified by growth in bacterial host cells. Individual viral phages, each expressing a different recombinant protein, can be grown and individual clones can then be assayed for binding activity.

[0068] 4. Antibodies from Human Lymphocytes Human antibodies can also be created using in vitro immunization or antigen stimulation. Such techniques can be used to stimulate peripheral blood lymphocytes from normal healthy subjects. Such “in vitro immunization” generally involves antigen-specific activation of non-immunized B lymphocytes within a mixed population of lymphocytes (mixed lymphocyte culture, MLC)
. In vitro immunization assays can also be supported by B cell proliferation and differentiation factors and lymphokines. The antibodies produced by these methods are often IgM antibodies (Bo, which is incorporated herein by reference).
rrbaeck et al., 1986).

Another method has been described (US Pat. No. 5,681,729, which is hereby incorporated by reference), in which IgG (or IgA) antibodies are primarily Human lymphocytes to be produced can be obtained. The method generally involves transplanting human lymphocytes into an immunodeficient animal so that the human lymphocytes "take" in the animal body; immunizing the animal with the desired antigen to produce antibodies specific for the antigen Creating antibody-producing human lymphocytes; and then collecting antibody-producing human lymphocytes from the animal. Using the human lymphocytes thus produced, human lymphocytes producing antibodies are immortalized, the resulting immortalized human-derived lymphocytes producing antibodies are cloned, and then cloned from immortalized human-derived lymphocytes. Monoclonal antibodies can be produced by collecting monoclonal antibodies specific to the desired antigen.

[0070] Immunodeficient animals that can be used in this technique are those that do not exhibit rejection when human lymphocytes are transplanted into the animal. Such animals can be prepared artificially by physical, chemical or biological treatment. Any immunodeficient animal can be used. Human lymphocytes can be obtained from human peripheral blood, spleen, lymph nodes, tonsils and the like.

“Taking” transplanted human lymphocytes in an animal can be accomplished by simply administering the human lymphocytes to the animal. The route of administration is not limited and can be, for example, subcutaneous, intravenous or parenteral. The dose of human lymphocytes is not limited and can usually be 10 ⁶ to 10 ⁸ lymphocytes per animal. The immunodeficient animal is then immunized with the desired antigen.

After immunization, human lymphocytes are collected from blood, spleen, lymph nodes or other lymphoid tissues by any conventional method. For example, monoclonal antibodies can be separated by Ficoll-Hypaque (specific gravity: 1.077) centrifugation, and monocytes can be separated by the plastic dish antibody method. Contaminating cells from immunodeficient animals can be removed by using an antiserum specific for the animal cells. Antisera can be obtained, for example, by immunizing a second distinguished animal with spleen cells of an immunodeficient animal and then collecting serum from the distinguished immunized animal. The treatment with the antiserum can be performed at any stage. Human lymphocytes can also be recovered by immunological methods using human immunoglobulins expressed on the cell surface as markers.

[0073] By these methods, human lymphocytes that primarily produce IgG and IgA antibodies specific for one or more selected antigens can be obtained. Monoclonal antibodies can then be obtained from human lymphocytes by immortalization, selection, cell proliferation and antibody production.

[0074] 5. Transgenic mice containing human antibody libraries Recombinant technology is available today for the preparation of antibodies. In addition to the combined immunoglobulin phage expression library disclosed above, another molecular cloning approach is to prepare antibodies from transgenic mice containing a human antibody library. Such techniques are disclosed in U.S. Patent No. 5,545,807, which is incorporated herein by reference.

In the most general sense, these methods involve encoding at least some of the immunoglobulins of human origin, or their germline genetic material, which can be rearranged to encode a repertoire of immunoglobulins. Includes production of inserted transgenic animals. The inserted genetic material can be produced from a human source or can be produced synthetically. The agent can encode at least a portion of a known immunoglobulin, or can be modified to encode at least a portion of a modified immunoglobulin.

[0076] The inserted genetic material is expressed in the transgenic animal, resulting in the production of immunoglobulins derived at least in part from human immunoglobulin genetic material. The genetic material is such that, if the inserted genetic material is incorporated into the reproductive system at the wrong location or in the wrong geometry, some or more of the derived location material can be produced. , Was found to be rearranged in transgenic animals.

The inserted genetic material may be in the form of DNA cloned into a prokaryotic vector such as a plasmid and / or cosmid. A yeast artificial chromosome vector (Burke et al., 1987, which is hereby incorporated by reference) or by introduction of a chromosomal fragment (which is incorporated herein by reference). Richer and Lo, 1989), a larger DNA fragment is inserted. The inserted genetic material can be introduced into a host by conventional methods, for example, by injection into fertilized eggs or embryonic stem cells or other techniques.

In a preferred embodiment, the host animal initially carries no genetic material encoding the immunoglobulin constant region, such that the resulting transgenic animal utilizes only the inserted human genetic material when expressing the immunoglobulin. Use this is,
Achieved by using a mutant mutant host lacking the relevant genetic material, for example, or by artificially creating the mutant in a cell line that will eventually create a host from which the relevant genetic material has been removed. can do.

When the host animal carries an immunoglobulin constant region, the transgenic animal carries the naturally occurring genetic material and the inserted genetic material, and the naturally occurring genetic material,
It will produce immunoglobulins from the inserted genetic material and from a mixture of both types of genetic material. In this case, the desired immunoglobulin can be obtained by screening hybridomas from transgenic animals, for example, by developing the phenomenon of allelic exclusion of antibody gene expression or differential chromosome loss.

Once the appropriate transgenic animal has been prepared, the animal is simply immunized with the desired immunogen. Depending on the nature of the inserted material, the animal can, for example, produce chimeric immunoglobulins of mixed mouse / human origin, wherein the genetic material of foreign origin encodes only a portion of the immunoglobulin; Alternatively, the animal can, for example, produce entirely foreign immunoglobulins of all human origin, wherein the genetic material of foreign origin encodes whole immunoglobulins.

[0081] Polyclonal antisera can be produced from transgenic animals by immunization. Immunoglobulin producing cells can be removed from the animal to produce the immunoglobulin of interest. Preferably, monoclonal antibodies are produced from transgenic animals, for example, by fusing spleen cells from the animal with myeloma cells and screening the resulting hybridomas to select those that produce the desired antibody. Techniques suitable for such a process are described herein.

In another approach, the genetic material can be incorporated into the animal such that the desired antibody is produced in a body fluid, such as serum, or in the animal's exocrine secretions, such as milk, colostrum, or saliva. For example, by injecting in vitro the in vitro genetic material encoding for at least a portion of human immunoglobulin into a mammalian gene encoding for milk protein by injection, the eggs can be inserted into the inserted human immunoglobulin genetic material. Adult female mammals containing immunoglobulins derived at least in part can be grown. The desired antibody can then be harvested from the milk. Suitable techniques for performing such a process are known to those skilled in the art.

The transgenic animals are typically used to generate human antibodies of a single isotype, more specifically an isotype essential for B cell maturation, such as IgM and possibly IgD. Another preferred method of producing human antibodies is described in US Pat. No. 5,545,80, each of which is incorporated herein by reference.
No. 6, No. 5,569,825; No. 5,625,126; No. 5,633, 42
No. 5,661,016; and 5,770,429, which describe transgenic animals capable of switching from an isotype required for B cell development to another isotype. .

In developing B lymphocytes, cells initially produce IgM with a binding specificity determined by the productively rearranged V _H and V _L regions. Subsequently, each B
Cells and their progeny synthesize antibodies with identical light and heavy chain regions, but they are able to switch heavy chain isotypes. The use of the mu and delta constant regions is largely determined by alternative splicing, with IgM and IgD
Can be co-expressed in a single cell. Other heavy chain isotypes (gamma, alpha and epsilon) are only naturally expressed after a gene rearrangement event has deleted the Cmu and Cdelta exons. This gene rearrangement process (referred to as isotype switching) typically occurs by recombination between so-called switch segments located immediately upstream of each heavy chain gene (excluding delta). The individual switch segments are between 2 and 10 kb in length and consist mainly of short repeating sequences.

For these reasons, it is preferred that the transgene incorporate transcriptional regulatory sequences within about 1-2 kb upstream from each switch region to be used in isotype switching. These transcription control sequences preferably include a promoter and enhancer element, and more preferably include a 5 'flanking (ie, upstream) region that naturally associates with the switch region (ie, occurs in the germline conformation). Including.
While the 5 'flanking sequences from one switch region can be operably linked to different switch regions for transgene construction, in certain embodiments, each switch region incorporated into the transgene construct has It is preferred to have a 5 'flanking region that occurs immediately upstream in the naturally occurring germline conformation. Sequence information relating to the immunoglobulin switch region is known (Mil
ls et al., 1991; Sideras et al., 1989; each is hereby incorporated by reference).

US Pat. Nos. 5,545,806; 5,569,825; 5,625,
No. 126; 5,633,425; 5,661,016; and 5,7.
In the method described in US Pat. No. 70,429, the human immunoglobulin transgene contained in the transgene animal functions properly through the B cell development pathway and leads to isotype switching. Thus, in this method, these transgenes are isotype switched and have the following: (1) high levels and cell type-specific expression, (2) functional electronic rearrangement, (3) activation of allelic exclusion and Response to allele exclusion, (4) expression of sufficient primary repertoire, (5
) Are constructed to produce one or more of the dominance of the transgene antibody locus during signal transduction, (6) somatic hypermutation, and (7) the immune response.

A key requirement for transgene function is the creation of a primary antibody repertoire that is sufficiently diverse to trigger a secondary immune response to a range of antigens. The rearranged heavy chain gene consists of a tandem array of signal peptide exons, variable region exons and multidomain constant regions, each of which is encoded by several exons. Each of the constant region genes encodes a constant portion of a different claka of immunoglobulin. During B cell development, the V region proximal constant region is deleted, leading to the expression of a new heavy chain class. For each heavy chain class, RN
Another pattern of A splicing gives rise to both transmembrane and secreted immunoglobulins.

The human heavy chain locus is a gene segment of approximately 200 V over 3 Mb, approximately 4
30D gene segment spanning 0 kb, 6 clustered within 3 kb span
Consists of one J segment and nine constant region gene segments expanded over approximately 300 kb. All loci span approximately 2.5 Mb of the distal portion of the long arm of chromosome 14. All six known _VH families, D and J gene segments, and mu, delta, gamma 3, gamma 1 and alpha 1
Heavy chain transgene fragments containing constant region members are known (Berm
an et al., 1988, which is incorporated herein by reference). A genomic fragment containing all the necessary gene segments and regulatory sequences from the human light chain locus is similarly constructed.

The expression of successfully rearranged immunoglobulin heavy and light chain transgenes has a profound effect by suppressing the rearrangement of endogenous immunoglobulin genes in transgenic non-human animals. However, in certain embodiments, such that a hybrid immunoglobulin chain comprising a human variable region and a non-human (eg, murine) constant region cannot be formed, for example, by transswitching between the transgene and the endogenous Ig sequence. It is desirable to have complete inactivation of the endogenous Ig locus. Using embryonic stem cell technology and homologous recombination, the endogenous immunoglobulin repertoire can be easily eliminated. In addition, endogenous I
Suppression of the g gene can be achieved using various techniques such as antisense technology.

In another embodiment of the present invention, it may be desirable to produce transswitched immunoglobulins. Antibodies comprising such chimeric transswitched immunoglobulins can be used in a variety of applications where it is desirable to have a non-human (eg, murine) constant region, eg, for retention of effector function in a host. The presence of murine constant regions provides advantages over human constant regions to provide murine effector functions (eg, ADCC, murine complement fixation), such that such chimeric antibodies can be detected in mouse disease models. be able to.
Following animal testing, the human variable region coding sequence is isolated, for example, by PCR amplification or a cDNA clone from a source (hybridoma clone) and spliced into a sequence encoding the desired human constant region, making it more suitable for human therapeutic applications. It can encode a human sequence antibody.

[0091] 6. Humanized antibodies Humanized antibodies generally have at least three potential advantages for use in human therapy. First, because the effector moiety is human, it interacts well with other parts of the human immune system, for example, by complement-independent cytotoxicity (CDC) or antibody-independent cytotoxicity (ADCC). The target cells can be destroyed more effectively. Second, the human immune system should not recognize antibodies as foreign. Third, the half-life in human circulation is similar to natural human antibodies, allowing smaller and less frequent doses to be given.

[0092] Various methods for preparing human antibodies are provided herein. In addition to human antibodies, "humanized" antibodies have many advantages. "Humanized" antibodies are generally chimeric or mutant monoclonal antibodies from mouse, rat, hamster, rabbit or other species, which carry human constant and / or variable region domains or specific changes. Techniques for creating so-called "humanized" antibodies are well known to those skilled in the art.

[0093] Humanized antibodies also have the above advantages. First, the effector portion is human. Second
In addition, the human immune system should not recognize the framework or constant region as foreign, and therefore the antibody response to such injected antibodies should be lower for whole foreign mouse antibodies. Third, the injected humanized antibody, unlike the injected mouse antibody, probably has a half-life that closely resembles the naturally occurring human antibody, also allowing for smaller and less frequent doses Would be.

A number of methods for producing humanized antibodies have been described. The controlled rearrangement of antibody domains joined via protein disulfide bonds to form new artificial protein molecules or "chimeric" antibodies can be utilized (Bobrez)
ekka et al., 1980, Konieczny et al., 1981, each of which is incorporated herein by reference). Recombinant DNA techniques can also be used to construct gene fusions between DNA sequences encoding the murine antibody variable light and heavy chain domains and the human antibody light and heavy chain constant domains (Morris
on et al., 1984, which is hereby incorporated by reference).

The DNA sequence encoding the antigen binding portion or complementarity determining region (CRD) of a murine monoclonal antibody can be implanted by molecular means into a DNA sequence encoding the framework of the human antibody heavy and light chains. (Jones et al., 19
86, Riechmann et al., 1988, each of which is incorporated herein by reference). The expressed recombinant product is a so-called "remodeled" or humanized antibody, comprising the framework of a human antibody light or heavy chain and the antigen recognition site CDR of a murine monoclonal antibody.

[0096] Another method of producing humanized antibodies is described in US Patent No. 5,639,461, which is incorporated herein by reference. The method provides a humanized rodent antibody with improved therapeutic effect by displaying a human surface in the variable region, via applying a new surface. In the method, (1)
A pool of antibody heavy and light chain variable regions is aligned to provide a set of heavy and light chain variable region framework surface exposed positions, wherein the aligned positions for all variable regions are at least about 98% (2) a set of heavy and light chain variable region framework surface exposed amino acid residues is defined for a rodent antibody (or fragment thereof); (3) a set of rodent surface exposed amino acid residues A set of heavy and light chain variable region framework surface exposed amino acid residues is identified that is almost identical to the set; (4) the heavy and light chain variable region framework surface exposed amino acid residues defined in step (2) are identified. The set of groups is heavy chain and light chain identified in step (3), except for those amino acid residues that are within 5% of any atom of any residue in the complementarity determining region of the rodent antibody. It is replaced by a set of variable region framework surface exposed amino acid residues; then, (5) a humanized rodent antibody having binding specificity is produced.

[0097] Similar methods for the production of humanized antibodies are described in US Pat. Nos. 5,693,762, 5,693,76, each of which is incorporated herein by reference.
No. 1, No. 5,585,089, and No. 5,530,101. These methods involve producing a humanized immunoglobulin having one or more complementarity determining regions (CDRs) and possibly additional amino acids from a donor immunoglobulin and framework regions from a recipient human immunoglobulin. Each humanized immunoglobulin chain typically interacts with CDRs, such as one or more amino acids immediately adjacent to the CDRs in the donor immunoglobulin and within about 3 mm, as predicted by molecular modeling, in addition to the CDRs. Amino acids from a donor immunoglobulin framework that can perform binding affinity. The heavy and light chains are each described in U.S. Patent Nos. 5,693,762, 5,693,761, 5,585,089, each of which is incorporated herein by reference. And any of the various position references described in US Pat. No. 5,530,101,
It can be designed by using any combination or all.
When combined with an intact antibody, the humanized immunoglobulin is substantially non-immunogenic in humans and retains substantially the same affinity for the original antigen as the donor immunoglobulin.

[0098] Further methods of producing humanized antibodies are described in US Patent Nos. 5,565,332 and 5,733,743, which are incorporated herein by reference. . The method combines the concept of humanizing antibodies with a phagemid library, also described herein. In a general sense, the method comprises:
Utilize sequences from the antigen binding site of an antibody or population of antibodies directed to the antigen of interest. Thus, in a single rodent antibody, sequences that include a portion of the antibody's antigen-binding site can be combined with a diverse repertoire of human antibody sequences that can produce a complete antigen-binding site.

The antigen binding site created by this process differs from that created by CDR grafting in that only a portion of the sequence of the original rodent antibody appears to contact the antigen in a similar manner. The selected human sequences differ in sequence and appear to make additional contacts with antigen from that of the original binding site. However, the constraints imposed by the binding of a portion of the original sequence to the antigen and the shape of the antigen and its antigen binding site appear to drive new contacts of the human sequence to the same region or epitope of the antigen. Therefore, this process has been called "epitope imprinted selection" (EIS).

Starting with animal antibodies, one process results in the selection of antibodies that are partially human. Such antibodies are sufficiently similar in sequence to human antibodies and can be used directly in therapy or after modification of a few key residues. Sequence differences between the rodent components of selected antibodies with human sequences can be attributed to different residues, e.g., by site-directed mutagenesis of individual residues or by CDR grafting of the entire loop. Can be minimized by replacing Thus, EIS provides a method of making partially human or fully human antibodies that bind to the same epitope as an animal or partially human antibody, respectively. In the EIS, a repertoire of antibody fragments is displayed on the surface of the fibrous phase, and genes encoding fragments having antigen-binding activity can be selected by phage binding to the antigen.

Additional methods of humanizing antibodies contemplated for use in the present invention are described in US Pat. No. 5,502,598, each of which is incorporated herein by reference.
No. 078, 5,502,167; 5,705,154, 5,770,
No. 403, 5,698,417, 5,693,493, 5,558,
Nos. 864, 4,935,496, and 4,816,567.

7. Antibody Fragments Regardless of the source of the original antibody, any one of the intact antibody, the antibody multimer, or any of the various functional antigen binding regions of the antibody can be used in the present invention. Examples of functional regions include scFv, Fv, Fab ', Fab and F (ab') ₂ fragments of an antibody. Techniques for preparing such constructs are well known to those of skill in the art and are further exemplified herein.

[0103] The choice of antibody construct can be influenced by a variety of factors. For example, an extended half-life can result from active re-adsorption of intact antibodies in the kidney, which is characteristic of immunoglobulin Fc pieces. Thus, an IgG-based antibody is labeled with its Fab ′
It is expected to exhibit slower blood clearance than the counterpart. However, Fab ′ fragment-based compositions will generally exhibit good tissue penetration capacity.

[0104] Fab fragments can be obtained by proteolysis of whole immunoglobulins with a non-specific thiol protease, papain. Papain, first, cysteine,
Activation can be achieved by reducing the sulfhydryl groups at the active site with 2-mercaptoethanol or dithiothreitol. Heavy metals in the stock enzyme should be removed by chelation with EDTA (2 mM) to ensure maximum enzyme activity. The enzyme and the substrate are usually mixed together in a 1: 100 weight ratio. After incubation, the reaction can be stopped by irreversible alkylation of the thiol group with iodoacetamide or simply by dialysis. Digestion integrity should be monitored by SDS-PAGE and the various fractions separated by protein A-sepharose or ion exchange chromatography.

A common procedure for the preparation of F (ab ′) ₂ fragments from IgG of rabbit and human origin is limited proteolysis with the enzyme pepsin. Conditions, pH 4.5, 3
A 100 × antibody excess in acetate buffer at 7 ° C. indicates that the antibody is cleaved C-terminal to the heavy chain disulfide bond. The digestion rate of mouse IgG can vary with the substrate,
It may be difficult to obtain high yields of active F (ab ') ₂ fragments without some undigested or completely degraded IgG. In particular, IgG _2b is highly sensitive to complete degradation. Other subclasses require different incubation conditions for optimal results, all of which are known in the art.

Digestion of rat IgG with pepsin requires conditions including dialysis in 0.1 M acetate buffer, H4.5, and then incubation for 4 hours with 1% w / w pepsin; IgG ₁ and IgG _2a digestion is improved if it is first dialyzed at 4 ° C. against 0.1 M formate buffer, pH 2.8 for 16 hours, followed by acetate buffer. IgG _2b was prepared from 0.1M phosphate buffer, pH, 4 hours at 37 ° C.
It gives results more consistent with the results of incubation in Staphylococcus V8 protease (3% w / w) in 7.8.

8. In certain methods of the invention, bispecific antibodies are preferred for use. Preparation of bispecific antibodies is also well known in the art. One method of preparation involves, on the one hand, another preparation of an antibody having specificity for a first particular component, and on the other hand, another preparation of an antibody having specificity for a second particular component. Then
A pepsin F (ab'γ) ₂ fragment is generated from the two selected antibodies, and each is subsequently reduced to give separate Fab'γ fragments. The SH group on one of the two partners to be coupled is then alkylated with a crosslinker, such as o-phenylenedimaleimide, to give a free arrayamide group on one partner. This partner is then conjugated to the other by a thioether bond to give the desired F (ab'γ) ₂ heteroconjugate (Glennie et al., 1987; incorporated herein by reference). ). Other approaches, such as cross-linking with SPDP or Protein A, can also be performed.

[0108] Another method for producing bispecific antibodies is to use a 2
Of two hybridomas. As used herein, "quadroma"
The term is used to describe a productive fusion of two B cell hybridomas. This time, using standard techniques, two antibody-producing hybridomas are fused to obtain daughter cells, and then cells that maintain expression of both sets of clonal-type immunoglobulin genes are selected.

A preferred method of generating a clonal type involves the selection of at least one enzyme deficient mutant of the parent hybridoma. This first mutant hybridoma cell line is then fused to cells of a second hybridoma, for example, that have been lethally exposed to iodoacetamide. Cell fusion allows the rescue of a first hybridoma by obtaining the gene for its enzyme deficiency from a lethally processed hybridoma, and the rescue of a second hybridoma via fusion to the first hybridoma. . Preferred, but not required, are fusions of immunoglobulins of the same isotype rather than different subclasses. Mixed subclass antibodies allow the use of alternative assays for the isolation of the preferred quadromas.

[0110] Preferred quadromas can be identified using microtiter identification embodiments, FACS, immunofluorescent staining, idiotype-specific antibodies, antigen binding competition assays, and other methods routine in the art of antibody characterization. Following isolation of the quadroma, the bispecific antibody is purified from other cell products. This can be achieved by a variety of antibody isolation techniques known to those skilled in the art of immunoglobulin purification (eg, Antibodies: A Laboratory Manu).
al, 1988; incorporated herein by reference). Protein A or protein G Sepharose columns are preferred.

The following examples are included to illustrate preferred embodiments of the present invention. The techniques disclosed in the following examples represent techniques discovered by the present inventors that work well in the practice of the present invention, and thus can be considered to constitute a preferred mode for its practice. Should be understood by the trader. However, one of ordinary skill in the art can, in light of the present disclosure, make many variations in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention. Will recognize that

Example 1 CD3 + 28 costimulation for ex vivo expansion and activation of CD4 + T cells The present inventors have developed a novel system that drives the expansion of CD4 + T cells (Levi).
ne et al., 1996; Caroll et al., 1997). This system is used to drive the polyclonal logarithmic expansion of human T cells in vitro to achieve the T cell accessory molecule C
Antibodies against D3 and CD28 are used. In addition, the present inventors have developed expanded autologous human CD4 + T cells as an immunotherapy adjunct for does-intensive therapy for patients with high-risk relapsed / refractory NHL ex vivo. A clinical protocol for use has been developed (described below). In this protocol, patients with relapsed / refractory NHL, a high-risk candidate for intensive therapy, defined as a refractory or chemically insensitive disease in the first refractory disease, have PBMC as a source of T cells. Undergoes steady-state leukocyte export to collect,
Then receive PBMC mobilization with chemotherapy plus cytokines. Minimum number of CD34
+ After collection of progenitor cells, the harvested product undergoes a CD34 positive selection step. Selected CD34 + cells are used in hematological rescue after the patient receives intensive dose chemotherapy. The positive selection step depletes autologous T cells in PBMCs that exhibit recovery of endogenous T cell compartments after autologous stem cell transplant. Peripheral blood mononuclear cells (PBMC) from cryopreserved steady state pheresis products were converted to CD4
Culture for 14 days in a CD3 + 28 costimulatory system that promotes preferential proliferation of + T cells.

[0113] Preclinical data indicate that 2.5-3 log ₁₀ T cell expansion was achieved with 14 days of culture in this system (FIGS. 1A, 1B, 2 and 3). The T cell expansion system used in this application drives polyclonal expansion of CD3 + CD28 + T cells. CD4 and CD8 positive T cells were expanded together, while CD4 + cells
Proliferate better than + cells (FIGS. 1A, 1B, 2 and 3). Cytokine secretion phenotype of proliferated Cd4 + cells generally have a T _H pattern. T cell doses were escalated in patients using a Phase I experimental design (described in detail below).

Example 2 Immunological Profile of Patients Receiving CD3 + 28 Co-Stimulatory T Cells Eight patients were treated with CD3 + 28 co-stimulatory T cells following intensive dose chemotherapy with protocol 8558 [BB IND 6914] and PBMC reinfusion. Processed.
Toxicity was associated with No. 3 associated with reinfusion of CD3 + 28 T cells up to 2 × 10 ⁹ total cells. Grade 2 or higher initial (24 h) toxicity and mild (see protocol). The immunological results of the re-injected T cells were higher than expected. Cell production and release data are summarized in Table 1 below. The reverse events are summarized in Table 2 below.

[0115]

[Table 1]

[0116]

[Table 2]

In the eight evaluable patients, the following observations were made. First, one patient is C
Absolute or relative lymphocytosis was shown within 2-4 weeks after reinfusion of D3 + 28 T cells (FIGS. 4, 5, 6 and 7). In addition, the predominant lymphocyte phenotype in these patients was due to the CD3 + 28 costimulatory T cells reinfused in each of these patients.
Not the same as the cell phenotype. Re-injected T cells are overwhelmingly (60-90%
) Lymphocytes that were CD4 + (FIGS. 1A, 1B, 2 and 3) but predominate in these patients 2-4 weeks after reinfusion of CD3 + 28 T cells were predominantly (85-955) CD8 + Met.

The cytokine expression profile of CD4 + and CD8 + T cells that were present during the period of lymphocytosis was measured in the four patients evaluated. For CD4 + T cell profiles, T cells (CD3 cells perP + cells) were gated and then analyzed for CD4 + surface staining and intracellular cytokine expression as described herein. The results show that 68% of CD4 + T cells express TNF-α and 25%
Expressed IL-4 and 85% expressed IFN-γ. Quadrants were set with isotype matched negative control antibodies. Thus, the majority of activated cells express T _H 1 cytokines TNF-alpha and IFN-gamma. For CD8 + T cell profiles, see T cells (CD3 perCP + cells)
Were gated and then analyzed for CD8 + surface staining and intracellular cytokine expression as described herein. These results indicate that 48% of CD8 + T cells
It showed that NF-α was expressed, 4.6% expressed IL-4, and 70% expressed IFN-γ. Quadrants were set with isotype matched negative control antibodies. These data indicate that a significant proportion of circulating CD8 + T cells resulting in T _c I cytokines is activated. CD8 + clonality data (FIGS. 10A, 10B, 10C, 10D, 11A, 11B, 11C and 1C)
This in combination with 1D) suggests that these cells represent specifically activated effector cells. A lower total number of activated CD4 + T cells compared to that of CD8 + T cells suggests that most of the activated CD4 + T cells do not circulate and can be sequestered in the lymphatic compartment or at the site of the tumor.

Although the importance of the cytokine production profile by human CD8 + T cells is not well characterized, cells with a type 1 cytokine secretion profile (T _c I) are more cytolytically active than those with a T _c 2 profile. It is possible (Maggi et al., 1997). T cell repertoire analysis using PCR ^™ for the Vβ T cell antigen receptor revealed that the Vβ usage of the CD3 + 28 costimulatory product had a Gaussian (polyclonal) distribution, indicating a relative non-specificity of the expanded cell product. Shows polyclonal properties. This is expected because costimulation through CD3 + 28 acts globally, proliferates T cells, and hopefully overcomes activation defects that may exist in these patients (Ochoa and Longo, 1995). In contrast, the Vβ usage of CD8 + T cells that appear in the wave of lymphocytosis is not Gaussian and is often monoclonal or oligoclonal. Taken together, these data indicate that CD8 + lymphocytes that emerge in patients 2-4 weeks after CD3 + 28 costimulatory T cell reinfusion are antigen or tumor-specific effector cells driven by CD3 + 28 costimulatory CD4 + T cells. It is within our hypothesis. We show that the specificity of these CD8 + clonal T cell populations is that if these same cells were present in the biopsy of the remaining tumors of the selected patients, then their killing of the autologous transformed B cell line It can be determined by testing the ability of the cells.

These data are strikingly similar to animal experiments in which tumor-bearing mice have alterations in T-cell signaling pathways that prevent normal immunological recognition and elimination of tumor cells (Mizoguchi et al., 1992). In mice with tumors, T
There is sequential development proceeds functional alterations in cells, also have progressive loss of T _H 1 cell population (Correa et al., 1997; Ghosh et al., 1995).
Spleen cells or CD4 + T from these tumor-bearing cells with anti-CD3 + IL-2
Cell activation overcomes these T cell activation defects and results in a tumor-specific response when the activated cells are adoptively transferred to mice following bone marrow transplantation or treatment with cyclophosphamide (Katsanis et al., 1994). Saxton et al., 1997).

Example 3 CD3 + 28 costimulatory CD4 + and CD8 + grown and activated ex vivo
T Cell Immunological Results and Anti-Tumor Efficacy A clinical protocol was developed to examine growing CD4 + T cells for human immunotherapeutic applications. The safety of these cells when delivered to the post-transplant setting at increasing doses, the dose tolerance of these cells in these patient populations, and these cells in reducing the presence of residual molecularly detectable disease Was evaluated and the recurrence rate in these patients was evaluated. The clinical protocol is CD3 + 2 after intensive dose therapy
Developed using 8 costimulatory T cells. Specific methods for isolating and growing these cells are provided below.

Patients were selected according to the eligibility criteria outlined here. After undergoing the leukocyte export method,
PBPC was separated into CD34 positive and CD34 negative fractions. The CD34 positive fraction was used in hematopoietic rescue of patients after intensive chemotherapy. CD3 + 28 costimulation cultures were started using the CD34 negative fraction as described more fully below. CD
The 3 + 28 costimulatory culture started on the same day as patients returned and received their CD34 positive PBPC. Fourteen days later, CD3 + 28 costimulatory T cells were harvested, counted, and the patient was reinjected with a specific dose. The experimental design was a Phase I experiment, in which a cohort of these patients received increasing doses of autologous CD3 + 28 costimulatory T cells.

This study evaluated the administration of ex vivo expanded CD3 + 28 costimulatory T cells and CD34 selected peripheral blood progenitor cell support after intensive dose therapy in patients with relapsed or refractory B cell NHL. Increasing dose of C in population
The toxicity of D3 + 28 costimulatory T cells was evaluated. Also, this experiment was performed by Ex Vivo C
The immunoreactivity of the patient's T cells in response to D3 + 28 costimulation, prior to therapy, and after adoptive therapy with costimulatory T cells, was also evaluated.

[0124] In this experiment, collect a sample from peripheral blood, bone marrow and cell products were measured T _H subsets by flow cytometry using intracellular cytokine staining. Measurement of pre- and post-injected _TH subsets helped measure the immunophysiological consequences of delivering these ex vivo-derived CD4 + T cells to patients with recurrent NHL. Finally, correction of molecular response with T cell injection or _TH subset polarization helped identify useful surrogate immunological markers for antitumor activity.

A. Peripheral Blood Collection Peripheral blood clinical specimens were collected at the time of phlebotomy for standard clinical laboratory tests. All patients provided written informational consent.

B. In vitro isolation of PBPC and isolation of B and T cells Mononuclear cells were Ficoll-Hypaque (BioWhittaler, Inc.).
) Was isolated from peripheral blood by density gradient centrifugation. It is then washed by purification on human T-cell enriched columns (R & D Systems) according to the manufacturer's instructions or by E-reset on luminoethylisothiouronium hydrobromide (AET) treated sheep red blood cells. Monocytes in T cell rich and B
Separated into cell-rich fractions. B cells and T cells were used as described below.

C. Intracellular cytokine staining and analysis by flow cytometry T cells were analyzed for phorbol 12-myristate 13-acetate (PMA; Sig
ma Chemical Co. ) And ionophore ionomycin (I; Sigma Chemical Co.)
(Ie, T_H1 or T_H2) was measured. T cells (0.5 to 1.0 × 10 ⁶ Cells / ml) with 10% heat-inactivated fetal bovine serum (BioWhittaler, Inc.), PMA + I (25 ng / ml and 1 mg / ml, respectively) and
RPMI-1640 supplemented with 2 mM monensin (Calbiochem)
BioWhittaler, Inc. ), 37 ℃ / 5% CO_TwoAnd cultured. Control cultures were identical except that PMA + I was omitted. Monensin is cytoca
Added to the culture to inhibit in secretion, resulting in cytokines being stored in the cell.
Piled up.

At the end of 4 hours, a surface antigen-specific fluorescent conjugated monoclonal antibody was incubated with the cells for 15-30 minutes at room temperature. FACS lysis solution (Becton Dickinson) was added to lyse the remaining red blood cells and partially fix the T cells. The cells are removed by washing the solution and the cells are then allowed to stand at room temperature for 10 minutes in a FACS infiltration solution (Becton Dickinson).
) To infiltrate the lymphocyte membrane prior to intracellular immunofluorescence staining. The cells were washed and then incubated for 30 minutes at room temperature with a fluorescently conjugated intracellular monoclonal antibody. After the second wash, cells are removed for 1
% BSA, FACScan (Becton Dickinson)
Analysis was performed by three-color flow cytometry on a flow cytometer. The surface antigen-specific monoclonal antibody used was perCP-anti-CD3 (Becton
Dickinson), FITC-anti-CD4, and FITC-anti-CD8 (DA
Both from KO). The intracellular monoclonal antibody used was PE anti-IL-
2, PE anti-IL-4, PE anti-IFN-γ, and PE anti-TNF-α (all Bec
ton Dickinson).

Example 4 CD34 + selective intensive dose therapy was supported by mobilized PBPC. The use of this stem cell source has been associated with more rapid hematologic recovery (Zimmerman et al., 1995).
). In addition, patients transplanted with PBPC can have better survival benefits than those who consume harvested bone marrow (Vose et al., 1993). PBPC was recruited with cyclophosphamide and GM-CSF + G-CSF. The use of high doses of cyclophosphamide to recruit PBPC provides a therapeutic benefit by using non-replicating active agents in transplant preparation procedures. In some embodiments, G
The combination of M-CSF and G-CSF is more effective than growth factors in recruiting progenitor cells (Winter et al., 1996).

Improving Treatment Outcomes for Patients with “High Risk” Relapsed / Refractory NHL 1
One approach is to combine it with another treatment modality, such as intensive therapy immunotherapy. As a result, diseases that did not completely respond to the intensive administration method are eradicated. In this study, increasing doses of ex vivo expanded CD4 + CD2
8+ T cells are used as immunotherapeutic adjuncts for intensive administration. Then C
The product of the D34 positive selection is used as a source of progenitor cells for rescue from intensive dosing, and the steady state leukocyte export product is CD4 + C against a target for ex vivo expansion.
Used as a source of D28 + T cells.

The protocol expands a population of ex vivo CD4 + T cells from patients with relapsed / refractory non-Hodgkin's lymphoma (NHL) and uses these as replacement therapy for patients whose tumors did not respond to induced chemotherapy. Of the expanded cells.
This protocol is also contemplated for use in patients targeting other immunotherapy protocols whose immunoresponsive tumors do not respond to chemotherapy alone.

All of the published adoptive immunotherapy trials for treating patients with malignancies used T cells that are predominantly CD8 +. This is, in part, C
This was due to the large scale expansion of D4 + T cells and the absence of available systems for expansion.

Preclinical Data for CD4 + T Cell Proliferation The proliferative capacity of in vitro expanded T cells is a major consideration for adoptive immunotherapy. Antigen-specific and polyclonal CD8 + T cells successfully expanded in vitro by the addition of IL-2 or anti-CD3 Ab + IL-2. However, a mixed population of CD4 ⁺ and CD8 ⁺ T cells stimulated in this way eventually results in a population that is all or almost CD8 +. Furthermore, CD4 + T
Long-term expansion of cells requires the addition of exogenous lymphokines and syngeneic xenogeneic feeder cells, which excludes large-scale expansion of CD4 + T cells for the treatment of disease.

A two-signal model of lymphocyte activation states that lymphocytes require the delivery of both antigen-specific signals as well as simultaneous costimulatory signals. In the absence of a costimulatory signal, interaction of the T cell receptor with the antigen MHC complex can cause T cell clonal anergy or deletion. Data from many laboratories indicate that CD28 can provide important costimulatory signals. The present inventors have developed a novel method of expanding CD4 + T cells preferentially independently of exogenous cytokines or feeder cells using anti-CD3 Ab + anti-CD28 Ab conjugated to magnetic beads.

Autocrine growth in normal donors is maintained at 4-6 log _10- fold growth.
These cells remain polyclonal and are predominantly CD4 ⁺ . In some patients, the addition of IL-2 enhanced lymphocyte proliferation, which
In certain embodiments, this is an optional step. Activated cells are T helper type 1
Overwhelming secretion of function-related cytokines.

We have recently used this system to develop CD4 + CD28 from CD34-negative selection products from normal peripheral syngeneic xenogeneic donors as well as autologous patient donors with malignant disease.
+ T cells were preferentially expanded. The CD34 negative selection product is used to inoculate the first culture without further selection or manipulation. The culture set up in this way is 1
Showing 2-3 log ₁₀ growth over 4 days, 85-90% CD4 + and 1
The final cell culture product is 0-15% CD8 + T cells. Thus, targeted numbers of reinjected CD4 + T cells were expanded from 1 × 10 ⁸ to 1 × 10 ⁹ CD34 negative selection products without any additional processing.

B. Clinical data for the safety of ex vivo expanded CD4 + T cells Thus, two additional TIV infected patients had a total of 3 infusions of ex vivo expanded C
D4 + T cells were ingested. The first patient received 2 × 10 ⁹ expanded CD4 + T cells in case 1, followed by 1.1 × 10 ¹⁰ cells during the second reinfusion. The second patient received 3 × 10 ¹⁰ cells. Toxicity was mild with night sweats (without fever), headache was only a side effect reported in patient 1 12 hours after infusion of a 1.1 × 10 ¹⁰ cell dose, and fever and Redman syndrome were 3 × 10 ^{The 10} cell dose was reported in patient 2.

C. Experimental Design This is a single arm open labeling possibility experiment. High-risk patients (as defined below) were treated with CD34-selected PBPC support (BCNU, VP-16, Ara-
C and cyclophosphamide (BEAC)), receiving standard intensive chemotherapy for relapsed / refractory NHL. 14 days after reinfusion of CD34 selected PBPC,
Patients will receive a reinfusion of ex vivo expanded CD4 + T cells. T cell dose is 2 ×
It ranges from 10 ⁹ to 5 × 10 ¹⁰ cells.

[0139] 1. Eligibility Criteria Recurrent NHL is defined as a patient with a recurrent disease within 5 years of achieving the CR if the tumor cannot be shown to be identical to the original clone, even after the CR (defined below). Intractable refers to a major intractable disease.

“High risk” disease is defined by the standard pre-transplant induction method, creatinine <1.5 or>
Calculated creatinine clearance, 60 ml / min, total bilirubin, AST and ALT <1.5 x defined as less than the PR to the upper limit of normal (unless by Gilbert for bilirubin) (ie, SD or PD).

[0141] 2. Pretreatment evaluation Review the pathological substance to make it suitable for diagnosis. Plaffin blocks or frozen material from early biopsies and relapses should be provided for entry within 30 days. A small percentage is used to develop a tumor-specific oligonucleotide probe for the CDR region of the immunoglobulin heavy chain gene. A CT scan of the chest / abdomen / pelvis and a SPECT-gallium scan are performed with bone marrow aspirate and biopsy. Using a PCR-based assay with a tumor-specific probe, 5-10 cm ³ (cc) of bone marrow aspirate per bone marrow mononuclear cell fraction and at the stage of measuring bone marrow mononuclear cell culture on day 21 can get. 20 cc of heparinized peripheral blood is collected at the time of pre-transplant evaluation and measured on the peripheral blood mononuclear cell fraction using a PCR-based assay with a tumor-specific probe.

[0142] 3. PBPC recruitment and leukapheresis (PBPC collection) Stem cells are recruited to peripheral blood using standard methods. Apheresis uses a large-bore dual lumen catheter for venous access and uses a Fenwal CS30
Performed with a 00 Plus cell clector, or equivalent, at approximately 1 per session.
0-12 liters of total processing. A small volume collection chamber (SVCC) of CS3000Plus is used for collection.

Recruitment is assessed by monitoring the levels of circulating CD34 + cells in peripheral blood, and a circulating CD34 + cell of ≧ 5 / μl is considered successful. After a successful recruitment, if WBC ≧ 1000 / μl, the patient will undergo leukapheresis daily. The endpoint of a successful PBPC harvest involves harvesting at least 5 × 10 ⁶ CD34 + cells / kg. From this time, 1.0 × 10 ⁶ CD34 + cells / kg are cryopreserved without further manipulation and stored for patient rescue in case of graft failure. Failure to meet the backup collection 6 days after leukapheresis or the collection minimum within 12 days of apheresis is not eligible for the patient for the CD34 positive selection procedure and T cell expansion. Standard procedures for CS3000 operation and cell collection are provided below.

A backup aliquot of the mononuclear cell suspension obtained by apheresis is cryopreserved without further manipulation. Peripheral stem cells are stored according to Stem Cell Cryolab standards. Details of the low-temperature storage method will be outlined later.

[0145] 4. CD34 + Selection from PBSC Apheresis Collection Selection is performed using the Isolex 300 system for positive selection of CD34 cells. The system comprises the following components: a monoclonal antibody directed against the CD34 antigen expressed on hematopoietic progenitor / stem cells, which antigen is selectively expressed by a small percentage of bone marrow and peripheral blood mononuclear cells; Isolex ^™ 300i Magnetic Cell Separator; a paramagnetic microsphere coated with sheep anti-mouse antibody; and a non-enzymatic stem cell releasing agent to separate targeted CD34 + cells from the paramagnetic microsphere. The Isole
The x ^™ 300i Magnetic Cell Separator provides for separation, including washing and sensitization of target cells, separation of paramagnetic microspheres / target cells from mononuclear cell suspension, and release of target cells from paramagnetic microspheres. A device that controls all of the steps in the process.

The components of the Magnetic Cell Separator are: a primary magnet whose adjustable position is automatically controlled; a stationary secondary magnet; a series of clamps and pumps; a support module for cleaning the chamber; used as an operator interface. Including a graphic LCD display with a touch screen. Connected Isolex ^™ 300i Disposable Set
The (disposable set) includes a sterile biocompatible fluid channel for the cells. The main components of the set include a wash chamber, a mixing / separation chamber (which connects to a primary magnet), and a secondary chamber (which connects to a secondary magnet). Sheep anti-mouse antibody-coated microspheres (Dynabeads (M-450 sheep anti-mouse)) bind to murine immunoglobulins and provide a mechanism for targeting antibody-coated cells for selection. Displacing the cells from the bead cell reset, the target cells are won, allowing the beads to be retained by the magnet.

CD34 + cells are isolated from a PBSC collection of test groups using a Baxter Isolex 300 System. All procedures are performed using aseptic techniques. Selection procedures include sterile steps for PBSC preparation, sensitization of mononuclear cells with 9069 anti-CD34 monoclonal antibody, resetting of target cells and magnetic beads, release of target cells from beads, and isolation of isolated CD34 + cells. Including washing. Details of the selection method will be outlined later.

In preparing PBSC, the apheresis product is washed with platelets prior to the isolation procedure. This occurs using a spinning membrane assembly that is part of a disposable set (wash chamber). Sensitization of mononuclear cells, total volume 100m
Performed with 2.5 mg of L anti-CD34 antibody. The sensitization is performed at room temperature for 15 minutes,
Thereafter, the cells are washed to remove excess / unbound antibody. The freshly prepared Dynal paramagnetic microspheres (SAM IgG) are then added to the washed and sensitized cells. After incubation at room temperature, the bead cell complexes are separated from unbound cells using a primary magnet. The Biz / target cell complex is retained in the separation chamber and non-target cells are washed away. Non-target fractions are collected using aseptic techniques for T cell expansion. A stem cell releasing agent is used to displace CD34 + cells and release them from the beads. This requires incubation at room temperature, after which the target cells are collected using a primary magnet and the beads are retained in the chamber.
The final CD34 + product is assayed for bacterial culture and sensitivity, sterility in mycoplasma and fungal cultures.

D. Ex Vivo Expansion of CD4 + CD + 28T Lymphocytes 1: Cell Culture of PBMC Steady-state pheresis obtained prior to stem cell recruitment was performed using CD4 + CD28 +
Used as a source of PBMC that is cultured in a system that facilitates preferential proliferation of T lymphocytes. The cells are inoculated into 300 ml Baxter Lifecell Flask, or a Baxter 2417 flask if the starting volume of cells is at least 100 × 10 ⁶ cells. Baxter Lifecell for cultured cells
Flash and Solution Introducing Pump (Solution Transfer Pu)
Details of the use of mp) are provided below.

X-VIV supplemented with 5% autologous serum collected and prepared during apheresis
Proliferate cells with O 15 (). Wash magnetic beads (BB IND 6675) containing immobilized anti-CD3 (OKT3) and anti-CD28 (9.3) antibodies,
Add at a 3: 1 bead: CD3 + cell ratio. Cultures are maintained for 14 days prior to preparative harvest for reinfusion. Cells were counted daily, it keeps the cells at a density of about 0.75 to 2 × 10 ⁶ per 1ml as outlined later with the addition of fresh medium.

The phenotype of ex vivo expanded cells is measured at the time of initial inoculation, day 7 of expansion, and then at the time of harvest at section 14 of expansion. Cells are treated with anti-CD3, anti-CD4 and anti-CD
8, and analyzed by flow cytometry to determine total T cells (CD3 +),
And determine the total number and relative frequency of CD4 + and CD8 + T cells.

[0152] 2. Preparation of Cultured Cells for Injection Cells are grown ex vivo for 14 days and then processed for reinfusion on day 14. Magnetic beads are removed using a Baxter Fenwal Maxsep magnetic cell separation system. Preferably, the microbeads are effectively removed prior to reinfusion into the patient to prevent or reduce the potential for unwanted side effects that may be caused by the injection of the antibody-coated microbeads. Details of the bead removal process are provided below.

Once the culture has undergone a magnetic cell separation process for removal of microbeads used in cell stimulation, cell culture harvest is performed on the day of the planned cell reinfusion. The cells are harvested, washed, and resuspended in Plaxamlyte A containing 1% human serum albumin together in a Baxter Fenwal cell harvester, as described below.

E. Reinfusion of expanded T cells The total dose of reinjected T cells is based on the total cell count and the number of CD3 + cells as measured by flow cytometry with anti-CD3. 14 of proliferation
After a day, this constitutes> 99% of the total cells in culture.

The starting CD3 + cell dose is 100-250 containing 1% human serum albumin.
2 × 10 ⁹ cells in ml of Plasmalyte A. The dose of cells is infused over 20-30 minutes. Do not use leukocyte filters. Details of the re-injection technique are provided below.

CD3 + cell dose is increased as follows: dose level 2,5 × 10 ⁹ CD 3+ cells; dose level 3,1 × 10 ¹⁰ CD3 + cells; dose level 4,2.5 × 1
0 ¹⁰ CD3 + cells; and dose level 5,5 × 10 ¹⁰ CD3 + cells. At all dose levels, cells are re-infused for 20-30 minutes.

The following minimum criteria are required for the release of expanded CD3 + 28 T cells for reinfusion: a) 80% minimal cell viability, b) less than 100 residual Cd3 + 28 beads / 3
× 10 ⁶ reinfused cells, c) - bacteria from 2 days Maikopurazumu and fungal culture reports must be read as no growth, d) grams strains harvested products must be reported as not organisms observed, e) Endotoxin assay from -2 days must be <1.

Dose limiting toxicity (DLT) is a toxic event in a single patient that limits further dose escalation. For the purpose of this study, DLT was a CD3 + other than nausea, vomiting or fatigue
It is defined as any grade 3 or higher non-hematological toxicity that is probably or clearly associated with T cells. For the purposes of this study, the minimum tolerated dose (MTD) is equal to the dose at which at least 33% of 6 patients experience DLT. For the purpose of this study, recommended phase II
The dose (RPTD) is equal to one dose level below the MTD.

Toxicity is based on CALGB proliferation normal toxicity criteria (Expanded Common To
xicity Criteria) (Table 3). Reverse reactions not included in these criteria are graded as follows: mild, grade 1
Moderate, grade 2; severe, grade 3; life threatening, grade 4; and fatal, grade 5. Each toxic event can be considered for a relationship to proliferating T cells as follows: Irrelevant : This category is due, after careful medical consideration, to an extrinsic cause (disease, environment, etc.) unrelated to the administration of proliferating T cells. Applies to adverse events that are clearly felt to be; it does not seem to be : this category
After careful medical considerations, it applies to adverse experiences that may not be relevant to the administration of proliferating T cells. The reverse experience is if 1) it does not follow a reasonable temporal sequence from the administration of proliferating T cells; and 2) it is the patient's clinical condition, environmental or toxic factors, or other therapies administered to the patient. It is likely that this was not the case if not easily possible; perhaps : after careful medical considerations, this category does not seem to be related to the administration of proliferating T cells, Possibilities apply to reverse experiences that cannot be ruled out with certainty. If the reverse experience is 1
And / or 2) it may be the result of the patient's clinical condition, environmental or toxic factors, or other therapies administered to the patient. Perhaps it can be considered relevant; maybe : after careful medical considerations, this category applies to adverse experiences that are considered to be relevant with a high degree of certainty to the administration of proliferating T cells. The reverse experience is, if 1) it is a proliferation T
Follow a rational temporal sequence from administration of the cells; and 2) consider it probably relevant if it cannot be reasonably explained by the patient's clinical condition, by environmental or toxic factors or by other therapies administered to the patient can; clear: this category, after careful medical consideration, are applied in the opposite experience felt to be related to the administration of proliferating T cells. The reverse experience is that if 1) it follows a reasonable temporal sequence from the administration of the expanded T cells or while the expanded T cells are established in a body fluid or tissue; It may clearly be considered relevant if it cannot be reasonably explained by the patient's clinical condition, by environmental or toxic factors, or by other therapies administered to the patient.

A minimum of one patient is evaluated at each dose level that is at most grade 0-1 toxicity. After at least one episode of Grade 2 toxicity is probably or apparently related to treatment (except alopecia, nausea / vomiting), subsequent dose levels entail a minimum of 3 patients. No more than one new patient is treated per week. A third patient entering a dose level must be observed for at least one week before the first patient enters the next dose level. If dose limiting toxicity occurs in one of the first three patients at a dose level, at least three additional patients are treated at that dose level where the MTD is determined to have been exceeded (if MT
D less than 3 if 6 patients are clearly exceeded before being treated). If none of the first three patients at a dose level experiences dose-limiting toxicity, the dose escalation proceeds to the next level. If dose limiting toxicity occurs in more than 2 out of 6 patients, the MTD has been reached and the dose is reduced to define a recommended Phase II dose.

The toxicity associated with cell therapy on T cells is generally thought to be due to the release of cytokines from the cells. Experience with LAK cells and marginal experience with CD4 + T cells suggests minimal toxicity from these cells. The most common side effects are fatigue, headache, fever, nausea and chills. The most severe side effects associated with cytokines such as hypertension, capillary leaky syndrome and cardiovascular collapse have not been encountered with T cell infusion therapy.

F. Bone marrow aspirate at biopsy for pathological review after transplant recovery
Obtained within 60 days after reinfusion of BPC. Using a PCR-based assay with a tumor-specific probe, 5-10 cm ³ of bone marrow aspirate is obtained to measure tumor cell contamination in the bone marrow mononuclear cell fraction. CBC, differential and platelet counts are performed every other week. 20 cm ³ of heparinized peripheral blood was re-infused the same day as the post-transplanted bone marrow specimen, then every 30 days for the first 3 months, every 60 days for the next 3 months, and then for 6 months or for CD34 + cells 90 for at least 18 months
Collect daily. Tumor cell contamination is measured on the peripheral blood mononuclear cell fraction using a PCR-based assay with a tumor-specific probe. Tumor re-evaluation is post-treatment 1
Performed annually on CT and SPECT gallium scans every three months.

A complete response (CR) is defined as no clinical, radiological or histopathological evidence (bone marrow assessment) of the tumor, lasting more than 6 months. Partial response (PR) is defined as a minimum 50% contraction in the tumor with no evidence of progression at any site and no site of new disease. Patients who do not meet the criteria for CR or PR are considered no response (NR). Progressive disease (PD) is defined as a> 5% increase in the size of the appearance of an existing lesion or any new lesion. Survival time is calculated from CD34 selective PBPC reinfusion (day 0) to death or last patient contact. Time-to-progress (TTP) and time-to-treatment failure (TTF) are the re-infusion to disease progression or last patient contact, and any event (PD, any cause) defining re-infusion to treatment failure, respectively. , Death from a second malignant disease, etc.).

[0164]

[Table 3]

Example 5 Collection of Mononuclear Cells Mononuclear cells were collected from Baxter Fenwal CS3000 Plus Blood.
d Harvest using the 1-120 stem cell program on the Cell Separator. The CS3000 Plus Blood Cell Separato
r is a self-contained continuous flow centrifugal device that separates whole blood into some of its components. Collection of specific blood components is performed automatically and monitored. The programs for each type of acquisition are stored in the solid state memory of the separator. Two modes of operation are provided: automatic and manual. During automatic operation, all functions are controlled by the microcomputer, except during situations where operator intervention is required. During manual operation, all functions are under the control of the operator. The message center on the operator panel provides help and status messages to the operator and allows the operator to program the separator and perform special techniques. Blood components are collected with a sterile disposable apheresis kit. The components are separated by centrifugation in an apheresis kit due to differences in density. A two-stage centrifugation process is used in most approaches.

Various monitoring systems are continuously checked for abnormalities or potential adverse conditions. Digital readouts, indicators and audible alarms keep operators informed of conditions and abnormal conditions. If the appropriate action is not taken to correct the abnormal condition within a certain amount of time, the separator stops the procedure and removes the donor from the system. The alphanumeric message center provides status messages, alarm information and help messages during the separation procedure.

The centrifuge consists of two main mechanical components: a motor-driven rotor and a protection assembly. The rotor consists of a yoke that supports two radially opposed clamp assemblies. Each clamp assembly supports a container holder that secures one of the flexible plastic containers (separation or collection) of the apheresis kit. Each container holder consists of a plastic insert and a metal plate that together form a cavity of a particular shape. When the combined plastic container of the kit is clamped between the insert and the plate, the container takes the shape of a cavity. Separation container holders are used to harvest leukocytes. A small collection vessel (SVCC) is used to collect mononuclear cells.

During operation, whole blood subjected to anticoagulation is pumped by a whole blood pump (WA / ACD pump) to a rotary separation vessel. The main stage of separation occurs in the separation vessel, which is similar to the "first spin" operation of the blood component collection process. As whole blood enters the separation container, higher blood components, mainly RBCs, are filled into the outer edge of the container by centrifugal force. These heavier components then exit the separation vessel via a PRBC (filled red blood cell) line. The lower density components, such as plasma, platelets and white blood cells, are then extracted by a plasma pump via a CRP (component rich plasma) line. The CRP is pumped into a collection container where a second stage of component separation occurs, similar to the "second rotation" of the blood component collection process. As the CRP enters the bottom of the collection container, heavier components (white blood cells) are filled against the back wall of the container by centrifugal force. The separated components remain filled in the container, while the lower density components (plasma and platelets) exit the container via the CPP (poor component plasma) line. The CPP is then recombined with the PRBC and returned to the donor or collected in one of the Transfer Pack containers of the apheresis kit.

In this procedure for collection of mononuclear cells (stored under Special Procedure I (separate procedure I) in the memory of the separator), 1-120 S
The tem Cell Program (stem cell program) is used. Mononuclear cells are cells with one nucleus and are collected because target cells are found in this fraction of leukocytes. The 1-120 program is slightly different from the original 1-100 program. It allows plasma flow rate setting according to donor hematocrit,
Any setting according to the absolute monocyte count of the donor is possible. This program can be used at draw speeds between 20 and 80 ml / min by changing the draw speed using the front panel keys in the same way used in platelet apheresis. If the draw speed is changed, no program change is made.

A. Switch on CS2000 power by pressing down the power button on the front panel to ensure successful power on self test. The master reset control knob is then closed by rotating it clockwise until "pre-prime" is displayed in the message center. Select procedure 8 (baseline cell collection) using the procedure selection keys and press the edit key on the manual control panel. The table editing feature allows the operator to change the preset procedure stored as a table in the memory of the separator and store a modified version of the table for future use. These tables govern the method used to control the pump speed, centrifugal speed, endpoint, interface detector offset, clamp settings and the algorithm used to calculate the plasma pump speed during operation in automatic mode. I do.

By using either the position or the contents key, a desired table for editing the (run table) is selected. The main table remains unchanged. The main cycle is the same for all procedures. Press the edit key to edit the run table: change the position using the up / down position arrow keys and change the content using the up / down content arrow keys. New values appear. Enter the content values on Table 4 shown below for each location identified. Repeat using the up / down arrow keys until all desired position changes have been entered.

Then, using the up / down arrow keys, a special procedure 1 for storage
Select Press the store key to store the new content value in the special procedure 1 permanent memory. The special procedure 1 is selected using the procedure selection key. Repeat the above procedure to check the location for correct content. This step is to confirm that the values from Table 4 previously entered were properly stored in the permanent memory of Special Procedure 1. If any errors are found, use the up / down content arrows to reprogram the correct content and store the correct procedure during special one. If everything is correct, press Edit to exit.

[0173]

[Table 4]

[0174]

[Table 5]

[0175]

[Table 6]

MNC (monocyte) counts are absolute lymphocyte counts per microliter + absolute monocyte counts. It is calculated by multiplying the donor's WBC count by the sum of the percentages of lymphocytes and monocytes. For example, the donor CBC has 35% lymphocytes, 4% monocytes, 63% granulocytes and 39% HCT WCT.
If the BC count is reflected, the MNC count is 7400 × (33 + 0.04)
= 2738. Thus, the absolute MNC count of the donor is 2738 / μ
L. The interface setting (position 71) is 120.

It is important to accurately calculate the MNC count of an immunocompromised donor and adjust the position 71 value appropriately. During the MNC collection process described here, location 71
Is routinely programmed to 90 to reduce the amount of red blood cell and granulocyte contamination in the final product. In general, position 71 should always have a value of 90. Table 6 is provided to set guidelines for programming position 71, assuming the Interface Optical Detector was set according to the donor's MNC count.

Next, the special procedure 1 is selected using the procedure selection key. Press the edit key on the manual control panel. Use the position or content keys to select a reinjection table.
Program the position values in Table 7 into the refill table.

[0179]

[Table 7]

The verification process described above is repeated to ensure that the values from Table 7 programmed into the special procedure 1 reinfusion table are properly stored in permanent memory.

Next, the apheresis kit of the closed system was prepared using CS3000 Plus Sep.
Install on arrator. Separation chamber (Separation C
hamber) is installed on the separation vessel holder clamp assembly,
Verify that SVCC is installed in the collection vessel holder clamp assembly. Apart from the specific changes, all other steps for priming the closed system apheresis kit are unchanged.

B. Mononuclear Cell Collection Set Auto / Manual Run to Auto (Auto On) and confirm Special 1 is displayed in the Message Center. Press the display / edit key on the front panel.
Pressing the Enter key reduces the endpoint volume to 5000 ml. The message center displays the current settings (10000) and the new settings. 50
Press the double down arrow key until 00 is displayed under "New." Press the Enter key again to enter the new settings. The end point is routinely 5000m
to 1.

Press the single up arrow key to move to the next parameter, which is the whole blood flow rate. The default of the blood flow rate is 50 ml / min. The first overflow (Spillover
) Occurs and it is recommended that this parameter not be changed until the blood flow rate from the Inlet Needle (inflow needle) is found to be adequate. Press the single up key to move to the next parameter. The plasma flow rate remains unchanged. This is because the separator computer determines this parameter according to the location programmed in memory. The centrifugation speed parameter should be 1600. The interface detector offset should be set to 90. If a parameter other than 90 is displayed, press the Enter key, use the up or down arrow keys to select 90 and press the Enter key again.

The interface detector baseline parameters are set automatically by the computer's memory and should remain unchanged. The plasma volume (Col / Exch) is set to 200. Press the Enter key once, press the double up arrow key twice to select 200 and press the Enter key again. By setting this parameter to 200, CS3000 Plus Se
The parator will use the first 200 ml of the apheresis kit at the beginning of the run.
Pump into 0 ml Transfer Pack. During the first cycle,
Apheresis kit is filled with approximately 300 ml of the initial solution and 10
0 ml is ACDA anticoagulant. Converting the initial volume of 200 ml to Transfer Pack reduces the likelihood of causing early signs of citrate toxicity in the donor. If the extracorporeal volume is not of interest, the default setting for the plasma volume parameter is 200.

The single access cycle capacity and Transfer Pack capacity parameters are not changed. Both defaults are zero. These parameters are only used when performing the one-arm approach. Press View / Edit Kit to allow the parameters to enter and return to the pre-run screen. The dip chambers for saline and anticoagulant containers should be at least 1/2 full volume and not more than 3/4 full volume to facilitate drip rate measurements and prevent air from entering the system. is there.

Activate the return line to the “Y” junction, open the Roberts clamp and fill the Sampling Pack with sufficient blood to collect the required sample. Close Roberts pump, Sampling Pac
Heat seal k twice. Open the return line roller clamp and flush the line. The saline flow rate is controlled with a return line roller clamp to maintain the site at the KVO rate. The required blood sample is collected from the Sampling Pack using a Vacutainer needle and holder. Sa
The mpling pack can be detached from the return line or left coupled. Activate the inflow line just above the needle. Press the Mode key to select a run. With the run indicated in the message center,
Press the Start / Resume key twice to start the automatic run. Immediately open the inflow and return line roller clamps. The centrifuge and pump start immediately.

Measure the anticoagulant flow rate by monitoring the drip rate in the ACD drip chamber of the apheresis kit and record the flow rate. To achieve the best possible separation, a 10: 1 WB: A, unless the donor is very sensitive to the anticoagulant or no signs of hypocalcemia are reported by the donor.
CD speed is recommended. See Table 8 below to determine the WA: ACD ratio according to the drip speed.

[0188]

[Table 8]

Ensure that the saline and vent line roller clamps are fully open. This allows free flow of saline solution whenever the stop / wash key is depressed. Within five to ten minutes, the first spill occurs and message 80 (the spill step in the program) is displayed in the message center. Once the overflow is over, message 89 (a run in the program) reappears. At this point, if the blood flow rate from the donor is appropriate, increase the whole blood flow rate and press the display / edit key. Use the single up / down arrow keys to select the whole blood flow screen. Press the Enter key and use the double or single up arrow keys to increase the value to 60. Press the Enter key again to enter the change. The single up / down key changes the value by one, and the down up / down arrow key changes the value by ten.

The WBRF value is now 60. Press the View / Edit key to allow and execute the change. The blood (ml / min) display on the front panel is W
Changes in BRF should reflect. After the blood flow rate has stabilized, spillover occurs approximately every 3.5 minutes throughout the procedure. Every 15 minutes, process the volume, the whole blood pump flow rate,
Check and record the plasma pump flow rate, anticoagulant drip rate, centrifuge speed and return plasma clarity. Separated plasma in the plasma return line is visually observed for hemolysis. The red coloration of the plasma in the return line is an assessment to determine if this is from red blood cell contamination of the hemolyzed or separated plasma (
(Before re-injection).

After 4100 ml of blood has been processed, message 61 (anticoagulant container check) alerts the operator to confirm that sufficient anticoagulant remains in the container to complete the procedure. . Clear the alarm, clear the message, breed the apheresis technique, and press the start / resume key once. Continue the automatic run until the end point is reached. If it is desired to stop before reaching the end point, press the stop / wash key once. At this point, the mode and start / resume LEDs are flashed. Press the mode key to select reinfusion. Then, the start / restart key is pressed to start the re-injection operation. If for some reason it is desired to interrupt the procedure and restart at the same point where it was stopped, press the Stop / Wash key. Once the stop reason is resolved, press the Start / Resume key to continue the technique.

If the end point is reached (blood volume is equal to or greater than the end point indication), a message 60 (End Point Reach) is displayed, a single attention chime sounds, and the mode and start / restart LED flash. To stop the run procedure and start the reinfusion operation, press the mode key and select reinfusion. Then, the start / restart key is pressed to start the re-injection operation. To continue the procedure, select a new end point using the display / edit function, then press the start / resume key. If message 80 (the overflow step in the program) is displayed in the message center when the end point is reached, message 60 will not be displayed until the overflow step is completed. The stop / wash key must not be pressed until message 60 has appeared to prevent the purity of the end product from being reduced.

While the re-injection operation is in progress, close the inflow line roller clamp, heat seal the line twice and separate from the apheresis kit. Remove the inflow needle from the donor and place in a Sharps container. After approximately 3 minutes of pressure is applied to the phlebotomy site, the donor arm is wrapped using a sterile 2x2 adhesive bandage. Instruct the donor not to move the arm with the return needle. This is because fluid is being injected into the arm and invasion can occur. Once the re-injection operation is complete,
Message 25 (method complete) is displayed and one caution chime sounds. Press the mute key to turn off the chime, close the return line roller clamp, heat seal the line twice and separate from the apheresis kit. Remove the return needle from the donor's arm and place it in the Sharps container. As described above, pressure is applied to the phlebotomy site and a bandage is applied to the arm. Donors will be provided with a cooling agent and given oral and written post-donation instructions. Check the donor for vital signs,
Record. Close all roller clamps on the apheresis kit. The end time, the total volume of blood processed, the donor's post-donation vital signs, the amount of saline and anticoagulant used, and how much the procedure was tolerated by the donor are recorded.

C. Introduction of Harvested Cells Once the roller clamp on the apheresis kit is closed, the door to the centrifuge compartment of the separator is opened and multiple lumen tubing is removed from the upper support bar and the lower restriction collar. Open the separation and collection container holder and remove the collection and separation container. The cells are resuspended in the collection vessel by gentle agitation for 3-5 minutes or until the aggregates are no longer visible. Press the Start / Resume key to open the plasma collection clamp and remove the tubing from the clamp. 600ml transfer pack and P
Remove the L732 container from its hook. Transfer even air to the collection container by temporarily inverting the PL732 container, opening its roller clamps and squeezing out the air. Before discharging the PL732 container, close the roller clamp. Place PL732 container and 600 ml inlet pack in centrifuge compartment.
Hang the collection container from the WBIACD clamp handle. 1 of PL732 container
Open the upper roller clamp and drain all cells in the collection container to this container.
Once all cells have been expelled into the PL732 container, the empty collection container is squeezed to expel air into the PL732 container. Close the roller clamp to the PL732 container now containing the cells. This action wipes out the remaining cells from the line and facilitates the collection and separation vessel through the guide holes of the roller assembly during removal of the apheresis kit from the separator. Shake the PL732 container containing the cells to push excess air towards the container port. Open the roller clamp on the container and purge excess air into the component poor plasma line, leaving some air in the container to facilitate proper evacuation of the container during further processing. Close the roller clamp to this container as close as possible to the port.

The tubing derived from the PL732 container containing cells was added to the manifold by 3
Heat seal once. Cut between the seals, leaving two seals on the tubing of the PL732 container containing the cells. Weigh the cell container and record the weight. Any fluid leaks or apheresis kit breaks constitute a sterility disruption and the set must be discarded and reinstalled. If this happens during the priming procedure, simply discard the defective apheresis kit and install a new one. If the sterility disruption occurs during the actual collection process, but not inside the centrifuge compartment of the cell separator, press the stop / wash key and immediately close the inflow and return line roller clamps to prevent loss of sterility. Investigate the cause, include any fluid spills, explain the situation to the donor, and do not perform the reinfusion operation. Remove the apheresis kit and process appropriately.

Fluid spill triggers a code 42 alarm if sterility disruption occurs inside the centrifuge compartment. This alarm condition shuts down all separator functions. Press the mute key to silence the audible alarm and close the inlet and return line roller clamps. Again, investigate the cause of the loss of sterility,
No re-injection operation is performed.

If the starting plasma flow rate is set according to the donor's hematocrit, CS30
00 will soon start collecting MNCs and, as a result, will have higher overall collection efficiency. The computer selects the appropriate settings during the first 1 liter process, but pre-setting the plasma flow rate speeds up the process and improves the yield by a few percent.

The use of SVCC (small volume collection chamber) reduces the amount of end product platelet contamination and reduces extracorporeal volume. Although the final volume of SVCC is approximately 50 ml, it contains about 25 ml of cells and requires about 25 ml of space for plasma.
Approximately 2 ml of red and white blood cells are enriched from the donor, up to 3000 MNC / μL per liter of processed whole blood. The 1-120 program collects an average of 60% +/- 25% of processed MNCs, but collection efficiency is very donor dependent. ACD is important for cell collection. The ideal ratio of WA: ACD is 10:
1, which means that 100 ml of ACD is used per 1000 ml of processed whole blood. If not enough ACD is used, it may cause cells to clump and not separate well for pure collection.

Example 6 Autologous stem cell rescue Autologous stem cell rescue (ASCR) is used to restore hematopoietic function following high-dose chemotherapy radiation therapy. There are many sources of stem cells, including bone marrow, peripheral blood, and umbilical cord blood. During the interval between cell collection and reinfusion, the cell's metabolic "clock" should be completely stopped. Successful "freezing" or cryopreservation of stem cells inhibits further metabolism while maximizing viability. A key aspect is dimethyl sulfoxide (SMDO)
)), Including the use of cryoprotectants and controlled rates of cooling.

Cells are easily damaged by rapid or slow cooling rates. Dimethyl sulfoxide protects cells from damage due to slow cooling, while a controlled rate protects against rapid cooling. This is especially true at the most critical point when cells undergo a phase change from liquid to solid. In this heating of the melting point, the cells release energy in the form of destructive heat for the cells, unless the heat is compensated by further cooling. However, overcooling is also lethal to these sensitive cells.
The following protocol was developed for variable melting point heat, using real samples rather than "dummy" bags to monitor the rate of cooling. Following cryopreservation, the cells are immediately stored in a liquid phase of nitrogen, where they can stop for days, months, or years without significant denaturation.

The DMSO vehicle solution should be made on the same day as the cryopreservation. If the DMSO vehicle is mixed with the cells at a 1: 1 ratio, the final concentration of DMSO is 10%. Therefore,
The concentration of DMSO in the medium must be twice that, or 20%. DMS
The sample is weighed to determine the total amount of O and required media. The weight approximates the volume of the analyte that requires an equal volume of DMSO media solution. In addition, an extra few milliliters (ml) for the cold vian are required.

Tissue culture medium, dimethyl sulfoxide (Cryoserv; Research)
h Industries Corporation, Salt Lake
City, UT), two sterile 50 cc conical tubes (any container is suitable), one 18G 1.5 "sterile hypodermic needle, GIBSO medium 199 containing Hanks' salt and L-glutamine (GIBSO Laboratories,
Grand Island, NY), 1 sterile 25 or 50 ml pipette, pipetting aid (Drummond Sci. Co., Bloomall,
PA), place a metal or plastic rack for conical tubes, a sterile alcohol prep pad, and one sterile syringe under a laminar flow hood. Remove the plastic wrap from the bottom of the medium and loosen the cap. Carefully pour the medium into two sterile 500 cc tubes. Pipette the appropriate volume into the plastic bottle and cap again loosely. If it adheres, put the metal cap on the alcohol prep pad with C
Take out from ryoserv. Wipe the rubber injection site with alcohol. 18G1.5
Attach needle to syringe, insert needle, aspirate DMSO. Inject DMSO into media, cap tightly, shake well (outside hood) and cool in refrigerator at 4 ° C.

Turn on the computer and recorder. Accordingly, remove the protective cap from the recorder pen and "zero". Use "adv" to look at pen location. On the computer, press the "Cool", "Cool +", "Fan", "Alarm" and "Run" keys in sequence, then press number 1 if program 1 is used last. Close the chamber door and place the Styrofoam blood tube box near the freezer.

A. Specimen preparation The metal holder should be marked with a unique number, name, specimen type, date of birth, cryopreservation date and other relevant information such as the etiology chart number. "holder"
, "Cassettes" or "canisters" are all metal plates used to push analytes into even layers and protect frozen cryobags. The Fenwal cryogenic bag is incomplete with a bespoke metal holder from Stealcon, which fits into a frame created by Cut Biogenic Systems for storage in liquid nitrogen (Stericon, Broadview, IL). Fits properly. Cryogenic bags require similar information and equipment. Do not write directly on cryogenic bag plastic. This is because the ink can diffuse into the bag. Instead, write on the left and right corners above the cryobag, or use a cryobag with pockets. To ensure the same freezing rate, each bag should contain the same volume.
Cool the holder while waiting for the sample.

Next to the hood, switch on the Sebra sealer. Place the following with your specimen under the hood: n cryobags (n is the final number of cryobags); 1 blood component infusion set (bcis); (n + 1) 60 cc syringe; 10 cc syringe; sterile conical tube containing one label "immix"; metal or plastic tube rack; pipette aid; sterile 2 in 1
ml pipette; and 2 sterile 1 or 2 ml pipettes (depending on the number and volume of cryovials).

Close all clamps on the cryogenic bag. take bcis out of the box,
Close all clamps. Remove the cover from the sample bag outer port and hold or set the bag aside without touching the port. Insert the bcis spike into the outflow pod of the sample bag. Open the 100cc syringe and set it sideways. Remove the cover from the bcis luer female connelter and attach it to a 10 cc syringe. Open the clamp in the sample bag, dispense a predetermined amount for cold vials, CD34 + tests, etc., then close and clear the clamp. Open the 60 cc syringe, set it sideways, then remove and secure the 10 cc syringe between the thumb and the remaining finger at the tip of the hand, and attach the 60 cc syringe to the bcis female luer.

There are four different sized cryogenic bags called Cryocyte Freezing Cotains. The plastic is PL269. 500ml
Total capacity in size bag, code 4R5462 (changed to 4R9594) should not exceed 110ml / because the bag is too large and 4m
This is because it is wrapped in a Stericon holder having a depth of m. Similarly, the total volume in a 250 ml bag, code 4R5461 (changed to 4R9952) should not exceed 60 ml. Each cryobag has two female leads ("pig tail") for introducing DMSO media and analyte. The plastic tube adjacent to the bag is easily sealed with a Sebra sealer. Two sterile pods are located at the top center of the cryobag for use after thawing. Fenwal
Cryogenic Bag (Baxter Health Care Corporation)
, Deerfield, IL) have been used with exceptional results (<0.06% destruction). Fenwal blood component infusion set (bcis), code 4
C2223 constitutes a closed system, thereby acting as an added measure for contamination, which further contains a filter (pore size of 170-210 microns).

Inject the contents of the 10 cc syringe into a conical tube. Loosen the cap,
Pipette 1.1 or 2.1 ml of sample into the "immix" tube.
Process the pipette. Remove the sample using a 60 cc pipette (the volume should be half of the total volume calculated for each cryogenic bag, eg, 50 ml total volume = 25 m
(1 sample + 25 ml DMSO medium), then close the clamp and close the sample bag. Remove the luer adapter cover from one cryogenic bag lead and bci
Attach to s coupler. Open the clamp on the cryogenic bag bcis and inject the sample.

[0209] Approximately 5 ml of air is pushed back into the syringe, which is injected and passed through the sample captured in the tube. Close all clamps on cryobag bcis. If more bags are needed, attach a small syringe to the cryobag lead, attach the next cryobag to the bcis, and repeat the procedure. Under a hood, use a Sebra sealer to seal, pull, and discard the cryogenic bag tube (between the clamp and the base of the Y-tube) for the sample to be injected. If the sample bag is to be sent for microbiological testing, any sample captured in the tube is injected back into the sample bag, the sample bag is sealed near the base of the tube, the tube is pulled, and the bag is sent. Finish labeling the cryogenic bag and holder for the final volume. Cool the cryogenic bag and the "immix" tube.

DMSO media (shook vigorously before placing under hood) and n 15G
Place a 1.5 "needle (where n is the number of cold bags) under the hood. DMSO
Loosen the cap on the media. Open the syringe and set it aside. Attach the needle to the syringe (be careful not to touch the tapered end of the syringe as it can be at the bottom of the sterile tube or DMSO media). For each volume of sample, dispense an equal volume of DMSO medium. If more than one different volume of syringe is cooled together, label the syringe. Cool in refrigerator for 15 minutes. D
The temperature of the analyte mixed with the MSO medium must not reach room temperature before the “freeze” begins.

B. Cryogenic vial This step is performed just before the start of the freezing program. The cryovial is labeled with the patient's initials, specimen type and unique number, hospital number and date, and is pre-cooled with the cryogenic bag and holder.

Place 4 to 8 labeled cryovials into cryovials and place “immix” tubes and DMSO media into hood in 0 ° C. wet ice. Immediately before pumping it into the hood, shake the DMSO medium and swirl the "immix" tube. Under the hood, loosen each cap. The pipette volume of the DMSO medium is equal to the volume of the sample in the "immix" tube, for example, 2.1 ml, and added to the "immix" tube of the sample. Clean the pipette, close the cap and swirl the mixture. Loosen the "immix" tube cap. Samples are pipetted into cryogenic vials, capped and submerged in wet ice. Repeat for each cryovial. Remove from hood, place in Styrofoam box and partially submerge in wet ice near freezer. Due to its small volume, the cryogenic vial of the specimen does not have the same eutectic point (melting heat phase change) as its cryogenic vial counterpart. Thus, the cryovial is placed in a Styrofoam blood tube box to slow the rate at which it freezes.

C. Controlled Speed Freezer Program This program was written specifically for the Custom Biogenic Systems controlled speed freezer, but the principles for freezing stem cells are based on any controlled speed freezer. Is also applicable. For stem cell cryobag volumes between 40 and 110 ml, the heat of fusion is typically-
Occurs between 8 ° C and -22 ° C. Ideally, in the heat of fusion, the specimen should be heated to a minimum (between 2 and 8 ° C.) and warmed to a temperature no higher than −6 ° C., with a heat of fusion within 4 minutes, and certainly 8 minutes. Return to the temperature occurring within, then cool at -1 to -2 ° C per minute.

The Custom Biogenic Systems (CBS) program 1 has two functions: 1) cool the specimen or chamber to the specified temperature, maintain that temperature until the operator proceeds with the program, and 2) single button Note that it includes several "waiting" functions that allow the operator to proceed to the next step by pressing. The timely seconds saved by pressing one button instead of several are very important in heat at the melting point. If necessary, additional coolant can be added by using the "Cool +" button.

[0215]

[Table 9]

Switch on liquid nitrogen. On the computer, the program is executed in process “1”.
Proceed until If program "1" is used, the programs and sections show "1.1" in green. The control states highlighted in green are:
It looks "cool +" and "wait". The chamber is cooled to 0 ° C. and maintains that temperature according to its waiting function.

The DMSO medium and the analyte are mixed just prior to cryopreservation of the analyte to limit the extent to which DMSO, which is lethal to cells at room temperature, is warmed. Mixing should be as fast as possible without spoiling sterile techniques. All labels should be done before mixing. The controlled speed freezer should be as close as possible to the refrigerator (4 ° C.) directly below or several steps away from the hood and freezer chamber where mixing takes place, to prevent as much warming as possible. If a liquid nitrogen storage refrigerator is not located next to the freezer, a dry nitrogen containing liquid nitrogen or Styr
An foam box can be used to transport frozen samples to a liquid nitrogen refrigerator.

Following the hood, a plasma extractor should be available and the Sebra sealer should still be on. Select an equal volume of sample bag and syringe. Quickly place them under the hood, remove the protective cover from the cold bag lead, remove the needle from the syringe, and attach the syringe to the bag. Remove the coupled bag syringe from the hood, open the clamp and inject DMSO media into the cryogenic bag. The syringe does not desorb, is mixed and placed in the plasma extractor. Extract excess air into syringe + small sample. If there is more than 60 cc of air, return the coupled cryogenic bag syringe under the hood, separate the syringe, squirt some air out of the syringe, reconnect it, put it in the plasma extractor and remove it from the bag. Continue to vent. Inject sample and close clamp. Under the hood, seal, pull apart and discard the syringe and tube. Put the cryogenic bag in the holder. If there is more than one bag, refrigerate and repeat the process starting with bag and sample selection.

D. The following steps should be performed quickly. The chamber temperature should be 0 ° C,
maintain. Open the chamber door and place the ribbon thermocouple in the center of the bag (referred to as reference bag). Choose the holder carefully so as not to cut the thermocouple. Insert the holder horizontally and insert the ports inward into the horizontal rack.

Press “Scan” on the computer and press “Start” on the recorder. If you have another holder, quickly insert it into the rack. Place the cryovial box upright into the chamber and close the chamber door. Chamber at 0 ° C
As soon as returning to step 1, press "Run" once and run the program at -1 ° / min (step 1.2)
Proceed to The analyte temperature should now drop at an appropriate rate and need not be monitored until it is about -8 ° C. This time is used for recording. Step 1.3 Just before and clearly after engagement, look carefully at the recorder pen to detect warming of the specimen. Be prepared to press "Run" as soon as heat of fusion occurs (sudden pen movement). After the heat of fusion has occurred, the specimen temperature is quickly returned to the same temperature at which the heat of fusion occurs without receiving a subsequent rapid cooling rate. If the sample begins to cool quickly, engage the chamber warming step 1.6 or open the chamber door, but if the sample cools at less than 1 ° / min or plateau, -1 °
/ Min cooling (step 1.7), stopping between shutdown (no coolant added) and steady cooling rate (step 1.7). Care should be taken. This is because any process that engages too long will cause excessive heating or cooling.
Once the specimen has returned to its melting point, monitor it for about 10 minutes to make sure it cools below -2 ° C / min. The program is then reliable and does not need to be monitored. However, it is not recommended to leave the room if liquid nitrogen runs out fast or there is no electricity supply or the like.

The computer warns visually or audibly when finished. Press "Run" and switch off liquid nitrogen. Wear cryogenic gloves, open the chamber door, and place the holder in a shipper or Styrofoam box except for the reference bag. Mild but quick and try not to drop it. The thermocouple is removed by the reference bag, the pen is raised, the holder on the reference bag is closed again, and placed in a shipper or Styrofoam box. Place the box of the cryovial in a shipper or Styrofoam box. If already done, mark the frame on the left with the patient's last name and on the right with the date and record the placement of the cryobag in the liquid nitrogen refrigerator and the location in the cryobag box. Transport the holder with the cryovial box and store in a liquid nitrogen refrigerator. Place the holder in the frame, facing the hinge, and submerge in a liquid nitrogen refrigerator.

If the stem cell follows an ideal freezing curve, it cools at -1 ° C / min to reach -45 ° C, and then at -10 ° C / min until it reaches -90 ° C. . If visible, the heat of fusion is a small round hill on the graph, which is successfully predicted and automatically accounted for by a controlled speed freezer program. However, the analytes are derived from different sources using different means, do not undergo the Ficoll procedure, vary widely in volume, cell count and cell composition, and thus have a variable melting point heat that requires manual intervention. give. Optimally, the intervention minimizes the warming and number of minutes of analyte temperature and returns to the heat of fusion. Flexibility of the program is required, and care must be taken to manually control the freezing curve of the specimen through this critical point, the heat of thawing. The most difficult aspect is to determine if the analyte temperature has been properly reduced just after the heat of fusion has occurred and to know when to proceed to the next step. For example, if the sample receives a heat of fusion at -16 ° C and is warmed to -11 ° C, the rate at which the sample returns at -16 ° C will be at -5 ° C /
Minutes or faster. However, after returning to the heat of fusion,
The rate is not critical and should not exceed -2 ° C / min. Any operation of cooling or warming the chamber has a potential effect on analyte velocity after it has returned to its heat of fusion. The effects of these actions must be predicted. Clues to manual freezing via heat of thawing are visible.

As soon as the heat of fusion is detected, the chamber is cooled. If the specimen is cooled immediately after the extreme cooling of the chamber is started, overcooling is prevented by proceeding with a step of cooling the chamber to a cooling once per minute of the chamber. If the sample continues to cool excessively, stop the program or open the chamber door. If the specimen is heated after the heat of fusion, a short "burst"
Prevent plateau by adding coolant through cooling + 2
Avoid keeping the chamber 20 degrees below the sample for more than a minute. If the chamber temperature reaches its maximum cooling temperature and the specimen starts to warm slightly, or if it reaches a plateau, proceed to cooling at -1 ° C / min (step 1.7) to increase the heating tendency. Allows the specimen to start cooling until is reversed. Alternatively, a "burst" of cooling + is used.

In general, the higher the heat of fusion (−12 ° C. and above), the lower the warming of the specimen. In other words, the heat of fusion occurring at -10 ° C only warms twice to -8 ° C and returns to -10 ° C within 60 seconds without 12 with little room to make a decision. If the chamber is overcooled and the heating step is severely lacking, open the chamber door and warm quickly, keeping the eye at the sample temperature rather than the chamber temperature. In the Linsei graph recorder, each specimen pen marking represents 10 seconds, while the recording of the chamber "spike" represents 2 seconds. Thus, five of these "boxes" are equivalent to minutes. Ideally, the analyte temperature would require 5 "boxes" to decrease by 1 (or 2) degrees.

Specimens are clamped between steady-state cooling (step 1.7) and warming (step 1.6 or opening the door), with subsequent efforts to ensure a rate of -1 ° C (or -2 ° C). To the heat of fusion. The danger of bringing the formulation too close (minimizing the warming of the sample temperature and the number of minutes to return to the heat of fusion) is that the chamber cools too or too long, and thus later than -2 ° C / min. It is to jeopardize the large sample cooling rate. Rates greater than 10 ° C. per minute are detrimental to the analyte.

Example 7 Selection of CD34 + Cells The following procedure is for the Isomagnetic 300 for immunomagnetic selection of CD34 + cells.
Used when i is used. Reagents were RP34 + releaser, Dulbecco's phosphate buffered saline (Ca ⁺⁺ and Mg ⁺⁺ free), immunoglobulin, intravenous (supplemented with 1% HSA and 12% sodium citrate (v / v)). Human), one vial of human serum albumin (25% solution), sodium citrate (4% solution), and finishing buffer (DPBS; Ca and Mg ⁺⁺ free). Finishing buffer was 3000 ml DPBS, 360 ml sodium citrate and 80 ml
By mixing 25% HSA. The buffer can be stored in a refrigerator, but should be warmed to room temperature prior to use. The buffer should be used within 24 hours of preparation.

Prepare 5% immunoglobulin intravenous (human) according to manufacturer guidelines. Withdraw the contents of the antibody vial (approximately 2.5 ml) aseptically into a syringe. Withdraw (approximately 20 ml) RP34 + stem cell release agent vial and aseptically place into syringe.

Dynabeads ((should be freshly prepared daily. Remove the contents of one vial of the tube for a total of 20 ml. Expose Dynabeads (to the MPC-1 magnet for 2 minutes. Aspirate the supernatant. Remove the magnet and add 10ml beads
Resuspend in finishing buffer. Avoid foaming and minimize bead loss. Transfer beads to syringe.

Using a peripheral blood stem cell collection, an enriched mononuclear cell product containing up to 4.5 × 10 ¹⁰ cells is collected from the mobilized peripheral blood. Spike the apheresis product bag with the sampling site coupler and weigh the bag. Clean the septum with an alcohol wipe and remove the well-suspended sample with a needle and syringe (2 ml). Measure nucleated cell count and viability. Samples are stored for hematology / differential, immunophenotyping, tumor cell detection (autologous transplantation) and microbiology.

The 300 ml plasma infusion set is aseptically coupled to the collection tube of the Y site blood infusion set (including the filter) and the roller clamp on the collection tube is closed. The 300 ml inlet bag filled with finishing buffer is aseptically attached to the blood infusion set and the roller clamp is closed. Attach the mononuclear cell product to another inflow tube and close the roller clamp. Close the roller clamp on the mononuclear cell product tube and the collection bag tube and drain the mononuclear cells into the collection bag. After draining the cells,
Close the roller clamp on the collection bag tube. Close the roller clamp on the buffer bag and allow the buffer to flow into the product cell bag. Close the roller clamp on the buffer bag, open the clamp on the collection bag and allow the buffer rinse to flow into the collection bag.

The above steps are repeated until approximately 200-250 ml of cell product / buffer has been added to the collection bag. Weigh the collection bag. When you are ready to install the set, clean the rubber septum on the apheresis bag with an alcohol wipe,
10 ml of the prepared immunoglobulin vein is inserted with a needle and a syringe.

Turn on the main power switch of the Isolex TM300i magnetic cell separator located on the back panel of the device. The light next to the "error" and "start" keys on the keypad glows instantly. An identification screen appears. This screen is
Displayed while the instrument is undergoing a self-test. During the last part of the self-test, text indicating the Isolex TM300i software version number is displayed in the lower right corner of the screen. Without any further intervention by the user, the System Stop Verification
(System stop confirmation) display should appear. This is an internal test of the system. At this point, the user should press "Stop" on the keypad. Sy
The stem Initialization Preparation display appears. The operator should follow the instructions to clear the machine and then press "OK". System Initialization display is completed S
will display the percentage of the system initialization test. While the System Initialization screen is displayed, scale and pressure references are obtained and the mechanical parts of the instrument are tested.

Once the test is completed, “Select a Procedure (select a procedure)” appears. Press the box on the “Positive Selection” side, then press the “OK” box to make Pos
Select the active Selection application. "Install Set (
Install the set)] appears. Remove the tray containing the disposable set from the bag. Open pump door. If possible, hold the tray longitudinally with the next chamber of the weight scale arm. Hang the two large waste bags on the holder to the right of the machine. Install the chamber on the rocket module. Both recirculation wash bags are hung on the weight scale 3.
Hang the release agent bag on the weight scale 2.

[0234] Loosen all the disposable set parts remaining on the tray, and take out the tray. The rotating membrane assembly is installed on the spinner module to ensure that the rotating device is correctly positioned after the support arm is in place. Lock the upper two clamp manifolds in place. Snap the pump organizer into place. Ensure that all tubes in the organizer are located on the pump rollers or on the grooved surface of the pump module cover. Open the pump door and ensure that the PI tube and rotating device tube are not pinched by the door. Lock the lower three clamp manifolds in place.

Using a tube guide, install a second magnet pouch on the second magnet. Match the blue dots, install the tubing on fluid detectors 1,2, and attach the tubing firmly to pressure transducers 1 and 2. Match the blue dots and install the tubing on the fluid detector 3. Install the tube in the rocket tube guide. Hang the cell source bag and buffer bag tubes so that they do not interfere with the tubes from the bag on the scale. Check to make sure the bag is hanging upright on the weight scale. Check the disposable set for type and pinch.

After properly installing the disposable set, the “Installation S
"OK" on the "et" screen. “Install Check” appears. P1 and P2 pressures are displayed in the bottom window of the screen.

Once the installation check is completed, “Connect Buff
er (connect buffer) "appears. The user spikes the port of the finishing buffer bag with the disposable set buffer line spike, hangs the bag on the weight scale 6, confirms that the clamp on the finishing buffer bag is open, and holds the finishing buffer bag stationary. If so, you should press "OK".
The Prime Set display appears. Prime Set
During display, the disposable set is automatically activated using the sequence of steps.
The display is updated to reflect the steps in the program. The weight scale value is also displayed.

When the Prime Set state is completed, the Add Release Agen
t (Add release agent) screen is displayed. The user is the weight screen 2
The septum of the release agent bag above should be cleaned with an alcohol wipe, the contents of the syringe containing the release agent should be added, check that the weight scale bag is hanging upright on the scale and press "OK" It is. "Add Antibody (
Add antibody) "appears. The user cleans the antibody bag on the weight scale 3 with an alcohol wipe, adds the contents of the syringe containing the monoclonal antibody, and checks that the weight scale bag is hanging upright on the scale,
You should press "OK". The "Add Beads" indication appears. The user should clean the chamber septum with an alcohol wipe and inject the contents of one vial of Dynabeads () that has been washed.

Aiming at the center of the chamber, avoiding the deposition of beads on the chamber wall, "OK
"push. “Connect Cell Source Bag” appears. The user spikes the cell bag with the C1 line spike provided on the disposable set, hangs the bag on the weight scale 1, checks that the cell bag tube is not in the passage of the rocket module,
Check that the weight scale bag is hanging upright on the scale.

After attaching the cell source bag, press OK. In this part, the instrument automatically initiates the selection procedure and runs through the following steps; adding buffer to reagents; washing platelets; introducing antibodies; incubating cells / antibodies And;
Wash antibodies; introduce cells into chamber; reset; chamber cell wash 1-3
The release agent introduces a cell release incubation; rinsing the cell release; washing the released cells; washing the release agent; and introducing the cells into the product bag.

After completion of the procedure, a Procedure Complete (procedure complete) display appears. The user should perform the following steps: 1) close the clamp in the target product bag (weight scale 4); 2) heat seal the target product bag tube; 3) heat seal both pressure transducer tubes. 4) heat seal the buffer bag (weight scale 6) tube; 5) heat seal the first chamber tube; 6) heat seal the cell source bag (weight scale 1) tube; 7) desired product Remove bag from weight scale 4 (ensure that target product bag is properly labeled); 8) Remove disposable set from instrument and process according to all applicable regulations for biohazard disposal; 9)
Press "OK". If “OK” is selected, End of Procedure
Selection (end of procedure selection) appears.

Example 8 Inoculation of Tissue Culture Flasks, Addition of Fresh Media to Flasks, and Splitting of Cell Cultures This is a positive displacement fluid pumping system intended for simple introduction.
answer Pump, Lifecell Transfer Set (life cell introduction set) and Lifecell Tissue Culture Fl
Ask (live cell tissue culture flask) is used to provide safe, fast and accurate pumping of a wide variety of fluids.

[0243] The system has three main elements. The first one is Solutio
n Tissue Pump, an electromechanical device that pumps opaque and clear solutions to operator programmed volumes. The pump weighs the pumped solution by gravity and monitors the exact actual volume of the pumped solution. The pumping unit is designed to perform two types of laboratory solution delivery: large volume (red and green pumps) and small volume (orange pumps). The red and green pumps are electronically coupled to the run and operate simultaneously to introduce a large volume of solution. In this procedure, the red and green pumps will be used only for the delivery of fresh culture medium to the culture flask. An orange pump is used to introduce the existing cell culture from the flask to one or more flasks, and in some cases it is not utilized at all. Second
The third is the Lifecell Tissue Set, a sterilized multi-use fluid introduction set, and the third is the Lifecell Tissue Cultu.
re Flask, a sterile, single-use container.

In certain embodiments, the cell culture being treated and processed is from cells collected from an HIV seropositive individual. During tissue culture flask inoculation, addition of fresh medium to the cell culture flask, and splitting of the cell culture, an effective and safe method to safeguard the sterility of the culture is essential. In addition, the development of methods to provide a closed system, the reduction of the need for unnecessary exposure to biohazard fluids, and the use of sharps are highly empirical for the protection of treated individuals.

The supply and equipment are provided by Fenwal Solution Tissue Pum
p (Baxter Cat. # 4R4345), Fenwal Lifec
cell Tissue Set (Baxter Cat. # 4C2474)
, Fenwal Lifecell Adapter Set (Baxter Cat. # 4C2476), laminar flow hood, and Sebra dielectric heat sealer. These are standard supplies and equipment used in all the procedures described below. Any additional supplies and equipment required for the specific task are listed individually under each procedure.

A. First start First, initialize the introduction pump. The cable connection from the control module to the pump module and the power cord connection to the pump module must be tightly coupled. Switch on the pump module near the power switch. The membrane panel power on the indicator should be lit and stay on. The power-up self-test should occur as specified in the operator manual. The self-test ends with all displays showing a just-adjusted zero with the "g" indicator lit. If a problem occurs,
Without pump, section 6 of the operator's manual (trouble guide)
Was referred to. Allow the pump to warm up for approximately 5 minutes before proceeding. A Load Cell Performance Check (load cell efficiency check) is performed. See section 7.2 of the operator's manual for procedures.

Next, introduction and an adapter set are set. Remove the introductory set from the pouch. Make sure the Inlet Set Coupling Cover is tightly closed over the junction port opening. The introduction set is placed on the introduction pump with each of the silicone segments pre-stretched before starting. Grab the segment with the end connector and slowly extend it approximately 2 inches to release the segment. Load the introductory set on the pump module as follows: a) open the safety door on the pump module; b) insert the introductory set junction into the junction holder on the pump module; c) check the color code of the pump segment for the pump Check that it matches the color code of the head. Starting at the right (green) pump station, insert the white connector into the white outlet tube guide (right guide of the pump section) so that the connector is on the top surface of the guide; d) Insert the pump tube into the roller of the pump rotor E) position the tube in the tube guide slot without twisting and retract the green connector into the green tube guide; f) turn the pump rotor counterclockwise G) Repeat steps (c)-(f) for the center (orange) and left (red) pump stations, making at least one revolution of the tube around the rotor. All tube segments must be installed on the pump rotor for proper instrument operation; h) Close the safety door and do not twist, entangle, or tighten the introducer set tube. Remove the Lifecell Adapter Set from the pouch. The red and green pump segment couplers are coupled to the ends of an adapter set having two ports. A single port on the adapter set is used to connect cell culture media containers.

Next, the solution introduction pump is programmed. Press the volume control pad for the display to be programmed. One audible signal sounds and the display flashes. Using the numeric keyboard, enter the volume for the corresponding solution. As the data is entered, each new order shifts from right to left sequentially in the display. Ensure that the volume entered for each pump corresponds to the solution and volume associated with each pump station. It is recommended that half of the total volume of fresh culture medium to be introduced be entered into each of the red and green pumps. This both allows the extension tube on the final container to be activated and rinsed with the culture medium. Press the display specific gravity control pad to program. One audible signal sounds and the display flashes. Using the numeric keyboard, enter the specific gravity for the corresponding solution. As the data is entered, each new order is shifted sequentially from right to left in the display. A specific gravity of 1.00 can be used for most cell culture solutions.

B. Inoculation of tissue culture flask Lifecell tissue culture flask of desired size, cell culture medium container, plasma transduction set (Baxter Cat. # 4C243), Terumo SCD3
Containers containing 12 devices / welding wafer and cells to be inoculated should be collected inside a laminar flow hood. Using SCD312, the plasma transfer set is coupled to a tube already attached to the cell culture media container. Make sure the roller clamp on this line is closed. Connect the media container to the adapter set already installed on the introduction pump. Use a coupler on a single port of the plasma transfer set and adapter set. Open the roller clamp to the culture vessel. Carefully remove the joint cap to prevent contamination of the joint port.
The Lifecell flask is coupled to the introductory set junction using the coupling coupler on the flask. Close all roller clamps on the flask except for the mating coupler. Hang the Lifecell flask from the final container hook into the load cell. Make sure the flask is freely suspended without restrictions that can alter the operation of the load cell.

Connect the cell container to the orange pump tube coupler and hang it from one of the container hooks of the pump module. The solution introduction pump is designed to contain the solution in a collapsible container. If the cells to be inoculated are in vials or other non-collapsible containers, the containers must be ventilated to allow the solution to flow properly. Final Vessel (Lifecell Flask coupled to pump joint
Enter the volume of the culture medium to be delivered. This volume must be split between the red and green pumps. For example, if 500 ml of medium is to be introduced into the final container, the red pump is programmed to 250 ml and the green pump to 250 ml. Enter the volume of cell suspension to be inoculated by programming the orange pump. For example, if the total volume of the cell suspension is 200 ml, if you want all the cell suspension to be introduced into the final container, program the orange pump to deliver 200 ml. Enter the specific gravity of the solution to be introduced into each pump. Head, Cl
Unless dictated by the internal Cell Production Facility, the specific gravity is typically 1.00 programmed for each pump.

Before proceeding any further, the following conditions should exist: The final container must be properly coupled to the introductory set junction, the junction coupler clamp of the final container should be open while all others are closed, and the culture medium container clamp should be open. Should be, there should be no twisting or tightening of the introducer set tube or final container extension tube, and the total programmed capacity should not exceed the capacity capacity of the final container.

Press the start pad on the control module to start the pumping cycle.
All volume displays show four zero and ml indicator emissions. As the introduction from each pump station occurs, the capacity display shows the actual installed capacity, with zero leading for each station. In addition, the cumulative delivered volume in ml is shown in the total deliver display. The pumping cycles are as follows: a) red and green pumps simultaneously introduce the programmed volume to the red pump simultaneously; b) orange pumps individually introduce the programmed volumes individually to the orange pump. C) The red and green pumps first introduce the programmed volume into the green pump at the same time.

If the pumping cycle needs to be stopped, press the stop / mute pad. The active pumping cycle is stopped and cannot be restarted. Remove the final container and start a new cycle with a new final container. If the pumping cycle needs to be stopped for any reason, but stopping the active cycle is not desired, simply open the pumping module cover. This causes the pumping cycle to temporarily stop without having to reprogram the pumping cycle. To restart the pumping cycle, close the pump module cover and press the start pad. The pumping cycle that is running first. It will be resumed when it is stopped. At the completion of the pumping cycle, the green light has finished emitting the LED indicator, an audible signal sounds and the pumping operation automatically stops. The volume displayed on the total deliver display should be equal to the sum of the volumes programmed for each pump. For example, using the volume used as an example in steps 7 and 8, the total deliver reading should read 700 ml. A positive or negative deviation of 1-2 ml in the total delivery display is acceptable.

Remove the final container from the hook on the load cell without disconnecting the final container connector from the junction and place it next to the junction. Using a Sebra heat sealer,
Seal the mating coupler tube of the final container as close as possible to the mating coupler and three times in the direction of the container. Close the roller clamp on the mating coupler tube. The final container is separated from the mating coupler by detaching the tube from the heat seal closest to the mating coupler. In this method, 21/2
Heat seal remains on the tube attached to the final container, and half the heat seal remains with the mating coupler. Suitably label the final container and use the volume indicated on the total delivery label as the volume of the final container. Cell culture vessel (Lifec
(Fall flask / final container) in a 37 ° C. humidified CO ₂ incubator. Heat seal the tube attached to the cell culture medium container twice to obtain sufficient tube length for future SCD312 attachment. The media container is detached from the solution introduction set and stored in its box inside the refrigerator at 4 ° C. Remove all disposables from the solution introduction pump and process them according to the infection control manual. Disinfect the inlet pump with a 50% solution of sodium hypochlorite and water. Wash laminar flow hood with 70% ethanol solution.

C. Addition of Medium to Existing Cell Culture Flasks See the previous section for preliminary steps before adding medium to existing cell culture flasks. Introductory pump start and introductory pump maintenance are performed and programmed as specified in Section A above. The inlet pump is programmed as specified in Section A above, except that the orange pump is not programmed with a container. In this procedure, fresh medium is added to the existing cell culture flask, and only red and green pumps are used to introduce the solution.

The plasma transfer set is coupled to a media container as described in section G above. Connect the media container to the adapter set as described in section A above. Life containing Terumo SCD312 device, sterile mating coupler from Lifecell Flask, and cell culture inside laminar flow hood
Collect 11 Flash. Using SCD312, the mating coupler is connected to one of the tube extensions attached to the cell culture vessel. As described in Section B above, the joining coupler on the culture vessel is connected to the joining port of the introduction set. As described in Section B above, the cell culture vessel from the hook is hung on a load cell.

Program the red and green pumps with the desired capacity and program the specific gravity as described in section B above. Before proceeding, a check is performed as described in section B above. Press the start pad to start the pumping cycle. See Section B for details and guidance. The following exceptions apply: a) The orange pump is not programmed, so references made to the orange pump in the steps of the above list do not apply to this particular approach; b) displayed in full Dalibury display Displacement is the sum of the programmed displacements of the red and green pumps.

[0258] Suitably, the cell culture vessels are labeled. Check that the correct volume is recorded on the culture label. Now, the volume in the culture vessel is the sum of the initial culture volume plus the volume displayed on the total delivery display. For example, if the volume of the cell culture vessel before fresh medium is added is 1000 ml and the total delivery label is 500 ml (this volume is equal to the sum of the programmed volumes of the red and green pumps) +/- 2 ml should be), the cell culture vessel now holds 1500 ml.
Store the cell culture and seal off the cell culture vessel as described in section B above. Remove all disposables from the solution introduction pump and process them according to the infection control manual. Introduce pump to sodium hypochlorite 50
Disinfect with% solution and water. Wash laminar flow hood with 70% ethanol solution.

D. Split Cell Culture (1: 2 split) Prior to starting the set up, determine the ratio of cell culture to fresh medium needed to achieve the desired cell concentration. See previous sections for preliminary steps before adding media to existing cell culture flasks. The introduction pump is started as described in the section A, and the introduction pump is set. Program the inlet pump as follows (see Section A above)
. a) red pump-half of the total volume of fresh medium desired in the final container; b) green pump-half of the total volume of fresh media desired in the final container; and c) orange pump cell culture container to be split. Half of the total capacity at For example, the cell culture vessel to be split has a total volume of 1500 ml. The culture should be broken down into two new Lifecell Flasks each having a total volume of 1000 ml. 7
50 ml of the existing cell culture will be introduced into each individual Lifecell flask. The total volume of fresh medium required is 500 ml, which is equally divided between two new Lifecell Flasks (250 ml each)
. Therefore, program the pump as follows: red pump (medium) -12
5 ml / green pump (medium) -125 ml / orange pump (cell culture) -7
50 ml. The total volume introduced into each new flask will be 1000 ml.

As described in section B above, the plasma introduction set is connected to the medium container, and the medium container is connected to the adapter set. Temuro SCD312 device /
Wafer, Lifecell Flask (1), Lifecell Flash
k (2)-8 inside sterile mating coupler and laminar flow hood from required size
Collect the “Y” junction from a 600 ml introduction pack with two couplers (1) (part of Baxter Cat # 4R2027). Using SCD312, the joining coupler is joined to a single end of the “Y” joint. Using a Sebra heat sealer, seal off the coupler with each of the new Lifecell Flashes. Detach the coupler and store in a plastic bag for future use. Make sure the roller clamp on the mating coupler tube remains attached to the flask. The SCD312 is used to join a Lifecell Flask tube extension, which previously had a mating coupler at the end, to one of the available legs of a "Y" joint. Close all roller clamps. Using SCD312, connect the other Lifecell Flask tube extension to one of the available legs of the "Y" joint and close the roller clamp

The joining coupler attached to the two Lifecell Flashes is joined to the joining port of the introduction set as described in section B above. One of the Lifecell Flashes is hung on the hook of the load cell as described in section B above. Open the roller clamp on the tube line joining the "Y" joint. The red, green and orange pumps should already be programmed as described earlier in this section. If not, program the pump this time. The specific gravity is programmed for each solution as described in section B above. A specific gravity of 1.00 should normally be entered for each solution.

Before proceeding any further, the following conditions must exist: The final container is properly connected to the introductory set junction, the junction coupler clamp of the final container should be open while all others are closed, the culture medium container clamp should be open, There should be no twisting or squeezing of the introductory set tube or final container extension tube and the total program volume should not exceed the capacity capacity of the final container.

Remove the cell culture vessel to be split from the incubator and place inside a laminar flow hood. Resuspend the cells by shaking the container. An orange pump tube coupler is connected to the port furthest from the tube extension attached to the container. Hang the cell culture container from one of the container hooks of the pump module. Press the start pad to start the pumping cycle. See Section B above for details and guidance. Upon completion of the pumping cycle,
Green completes the LED indicator emission, emits an audible signal, and the pumping operation stops automatically. The volume displayed on the total delivery display should be equal to the combined volume programmed for each pump. A positive or negative deviation of 1-2 ml in the total delivery display is acceptable.

The roller clamp on the tube extension of the flask suspended from the hook of the load cell is closed. Remove the flask from the hook and place next to the joint. Hang another Lifecell Flask from the hook of the load cell and open the roller clamp on the extension tube connected to the "Y" junction. The tubing lines are not twisted or tangled so as to affect fluid flow or operation of the load cell. Press the pad to start the pumping cycle. See Section B above for details and guidelines. At the completion of the pumping cycle, green is LE
D-indicator emission is completed, an audible signal sounds, and the pumping operation automatically stops. The volume displayed on the full delivery display is the sum of the volumes programmed for each pump. A positive or negative deviation of 1-2 ml in the total delivery display is acceptable. Close the roller clamp on the tube extension of the flask suspended from the load cell hook. Remove the flask hook and place it next to the joint.
The cell culture split is now complete.

Lifecell bonded to “Y” junction using Sebra heat sealer
Seal the Flask tubing line three times. Make sure the roller clamp is still with the flask. The flask is cut from the “Y” joint by separating the heat seal closest to the “Y” joint. In this manner, the flask has a 21/2 seal in its line, with half the seals remaining on each leg of the "Y" joint. Suitably, each new culture vessel is labeled and stored as described in Section B above. Seal off the cell culture medium and save it as described in section B above. Remove all disposables from the solution introduction pump and process them according to the infection control manual. Disinfect the inlet pump with a 50% solution of sodium hypochlorite and water. Wash laminar flow hood with 70% ethanol solution.

E. Division of cell culture (2: 4 division) The 2: 4 division of cell culture is identical to the 1: 2 division except for the following. Two rifs
Instead of ecell Flash, 4 is required and a single “Y” junction (Bax
Instead of ter Cat # 4R2027), a double "Y" splice from a 600 ml introductory pack at 8 couplers is required and a five-pronged manifold is loaded into the required feed (Baxter Cat # 5C4446). The Lifecell Flask mating coupler is still sealed off, but in this approach it is done four times since there are four flasks. Using SCD312, connect the flask to the double "Y" junction. The only difference is the double “Y”
The junction is to have four available legs corresponding to the Lifecell Flask members used in this approach. This is repeated for each flask.

This is a 2: 4 split, where two existing cell cultures have four new Lifec
As the cells are split into cell flashes, the two cell culture vessels are removed from the incubator. Using a five-pronged manifold, convert the orange pump coupler from single to double-this eliminates the need to cut the culture vessel from the orange coupler that connects to the other. To do this, use Sebr
a Using heat sealing, seal off three of the crotches on the five-legged manifold. Now, the five-way manifold has only two couplers at one end used to couple the cell culture vessel to the orange pump. At the other end of the manifold is a coupler port. Connect the orange pump tube coupler to this port on the manifold. The two remaining riser clamps on the manifold are closed. Using a coupler at the crotch end, coupling the two culture vessels to be split to an orange pump hangs the vessels on hooks on the pump module.

Open the clamp on one of the culture vessels and press the start pad to start the pumping cycle. The cell culture on the now open container is introduced in equal amounts (half of the total volume) into two of the four Lifecell Flashes coupled to the introduction set junction. Once the culture vessel is empty, close the clamp and open the clamp to another culture vessel. Press the start pad to start the pumping cycle. See section B above for details and guidance.
At the completion of the pumping cycle, the green color has finished emitting the LED indicator, an audible signal sounds and the pumping operation automatically stops. The volume displayed on the total delivery display should be equal to the sum of the volumes programmed for each pump. A positive or negative deviation of 1-2 ml in the total delivery display is acceptable. Close the roller clamp on the tube extension of the flask suspended from the load cell hook. Remove the flask from the hook and place next to the joint. Other Life
Hang the cell flash from the load cell hook and open the roller clamp on the extension tube connected to the "Y" joint. The tubing lines are not twisted or tangled to affect fluid flow or load cell operation.

Lifecell bonded to a “Y” junction using a Sebra heat sealer
Seal the Flask tube line three times. Make sure the roller clamp stays on the flask. The flask is cut from the “Y” joint by separating the heat seal closest to the “Y” joint. Thus, the flasks have 21/2 seals in their lines, with half the seals remaining on each leg of the "Y" joint. This is repeated for each Lifecell Flash.

In most cases, the volume of each cell culture vessel to be split is the same. This simplifies the splitting process. Because Lifec once bound to the introductory set junction
Once cell culture and introduction of fresh media into the cell flask is complete, all that is required is to close the roller clamp on the flask, remove the flask from the loading cell hook, replace the empty flask with the clamp open, and start. Pressing a pad starts a new pumping cycle. If the volumes of the two cell cultures to be split are different, all pumps will need to be reprogrammed once the first culture vessel is split. This is determined prior to starting the splitting process, but does not require any other changes in the procedure other than reprogramming the pump between cultures.

[0271] Any leakage of fluid or cutting of the disposable set constitutes a sterile destruction, which must be discarded and replaced. The cell culture to be treated should be considered contaminated. If sterility disruption occurs during the actual solution introduction process, switch off the introduction pump and close all clamps on the introduction set and all associated tubing lines to allow for fluid overflow per standard guideline. Although this procedure is performed inside a laminar flow hood, it is recommended that all individuals in the treatment room leave immediately to prevent any aerosol exposure resulting from the disruption of the tubing line.

Example 9 Ex Vivo Proliferation of CD28 ⁺ / CD4 ⁺ T Lymphocytes via Bead-Immobilized Anti-CD3 Antibody (OKT3) + Anti-CD28 Antibody (9.3) The two-signal model of lymphocyte activation was It states that lymphocytes require delivery of both antigen-specific and costimulatory signals. In the absence of a costimulatory signal, interaction of the T cell receptor with the antigen MHC complex can cause T cell clonal anergy and deletion. Data from many laboratories is on CD2
8 shows that 8 can provide an important costimulatory signal.

The proliferative capacity of T cells grown in vitro is a major consideration in adoptive immunotherapy. Antigen-specific and polyclonal CD8 ⁺ T cells have been successfully expanded in vitro by the addition of IL-2 or anti-CD3 Ab + IL-2. However, a mixed population of CD4 ⁺ and CD8 ⁺ T cells stimulated in this way
Eventually, the population will be all or mostly CD8 ⁺ . Furthermore, long-term growth of C D4 ⁺ T cells requires the addition of exogenous lymphokines and allogeneic feeder cells, which eliminates the extensive proliferation of CD4 ⁺ T cells for treatment of disease.

The procedure described here uses an anti-CD3 antibody (A
b) Using + anti-CD28 Ab to expand CD4 ⁺ T cells purified independently of exogenous cytokines or feeder cells. Autocrine growth in normal donors is maintained for 4-6 log _10- fold expansions and for HIV ⁺ donors for 3-5 log _10- fold expansions. These cells are polyclonal and> 97%
It remains CD4 ⁺ . In some patients, the addition of IL-2 enhances lymphocyte proliferation, and thus is an optional step in certain embodiments.
Activated cells predominantly secrete cytokines associated with T helper type I function.

AccuComp software, magnetic particle concentrator (MPC-1;
P & B Biochemicals, Gaithersburg, MD)
Coulter Multisizer equipped with a channelizer connected to a PC with a
Magnetic separator for -50 mm tubes (Collaborative Bi)
chemical Products, Bedford, MA); magnetic separators for flasks (Collaborative Biochemical).
Products, Bedford, MA); and rare earth cobalt magnets (
Edmund Scientific Co. , Barrington; NJ
)It was used.

The reagents used were anti-CD3 (OKT3) + anti-CD28 (9.3) coated Dynal M-450 tosyl activated beads; X-VIVO 15 (BioWhittaker Walkersville, MD) containing 5% autologous human serum; L
Glutamine (BioWhittaker Walkersville, MD)
); Recombinant IL-2 (Chiron Corporation, Emeryv)
(Ille, CA).

The first stimulus is called S1, the first restimulation is called S2, the second restimulation is called S3, the third restimulation is called S4, and so on. The day of stimulation is called D0, the stimulation after 24 hours is called D1, the stimulation after 48 hours is called S2, and so on. For example: S4D3 = fourth stimulation after 72 hours (third restimulation).

A. Cell counting using Coulter Multisizer IIe Electronic cell counting and volume measurement are more accurate and more accurate than hemocytometer-based counting protocols, and are therefore the preferred method. The operation of the counter is
Particles (cells suspended in an electrolyte) passing through an opening through which current flows through are based on the principle that they change the electrical resistance of the electrolyte, causing a change in current and voltage. The magnitude of these changes is directly proportional to particle size and can be converted electronically to particle counting. Up to 500 particles are individually counted and sized per second, and particle size is measured independent of shape or orientation in solution. For most instruments, lower threshold controls are available to select the minimum particle size so as to exclude too small particle counts from being noticed. Electronic counters use side scatter to distinguish live cells from dead cells. The operation of the electronic counter is fully described in the manufacturer's manual.

[0279] The sunprint stand should be equipped with a 70 micron "long hole" orifice tube for more accurate counting and sizing. The opening of this long hole tends to clog the orifice, but enhances the size discrimination. this is,
Eliminating contaminants ensures that only viable cells are counted. To count lymphocytes, the cells should be well dispersed and any clumps destroyed, especially after the beads have been added. The lower gate for sizing should be set to 100 fl and the sample volume is set to 0.5 ml. To size the stimulated cells, set the sizing (analysis) gate to 200 fl and maintain this gate throughout the culture period. This ensures that beads (4.5 micron diameter) are not counted and are electronically exited from the gate.

To count cells, add 19.96 ml of PBS or Coulter diluent to a SunPrind vial. Add 160: 1 resuspended cells to vial and add 2: 1
Count twice. Record the next value of "Simultaneous Correction Count" and divide that number by 8 (2x for 2 counts and 4x for dilution factor), where ##### per ml is #. #### × 1
Obtain a concentration of cells per 1ml as 0 ⁶ cells. PBS background counts should only read below 100. Experience is CD3 +
28T cells were shown to be best restimulated at 350-400 fl. When complete, clean and disinfect the device using a 10% decolorizing solution.

B. Cell culture On day 0, CD34 fraction cells are added to X-VIVO 15 containing 5% autologous human serum at a concentration of 1 × 10 ⁶ per ml. If the starting volume of cells is at least 50 × 10 ⁶ cells, the culture can be started in 300 ml Baxter Lifecell Flash. Baxter for cultured cells
See Example 8 for details on the use of Lifecell Flash and a solution introduction pump. Anti-CD3 + anti-CD28 Ab (BB IND 6675
An appropriate amount of the coated tosyl-activated beads) were washed 3 times with X-VIVO 15, it was added to the cells, maintaining the 1 × 10 ⁶ cells per 1 ml.

On day three, by shaking / stirring the bag until the cell clumps were dispersed,
Detach cells from antibody-coated beads. Cells were counted and cells were 0.75-2 × 1
0 ⁶ / ml in so as to maintain the concentration, the addition of fresh medium. Cultures should not be diluted more than 1: 1 in fresh medium. On days 4, 5, 6, etc., cells are detached from the antibody-coated beads by shaking / stirring the bag until cell clumps are dispersed. Cells are maintained as described for day 3. Monitor the cell size as described above and access the correct time for restimulation of the culture.

Standard cell culture techniques employ a microscope with the practice of eye to determine the optimal time for culture restimulation. In this protocol, the cell volume is used to determine the optimal cell time. The use of electronic cell volume measurement reliably distinguishes activated cells from resting cells and is faster and easier than counting cells using a hemocytometer. However, if not properly calibrated, electronic cell volume measurements will be erroneous due to cell clumps or beads. In addition, electronic counting requires an accurate reset of the instrument for the percentage of cells of different sizes, possibly leading to inaccurate counting for heterogeneous cell populations, and for counting resting and activated cells. Requires different settings.
Thus, cell populations from tissues that can contain a significant number of dead cells and cell clumps are difficult to size on an electronic counter. Overall, electronic cell counters are best reserved for repetitive and rapid sizing of peripheral blood cells in suspension culture.

Resting T cells have a mean volume of １７０170 fl with only small deviations from the mean (less than 40 fl). Resting T cells from HIV ⁺ donors have a slightly larger volume of ~ 200 fl. Activated T cells are 7
A maximum average capacity of 80-900 fl is reached. Also, the deviation from the average is larger (250-350 fl). Around 7-10 days, the average cell volume and deviation from the average begin to decrease. Eventually, the cells return to the resting T cell volume. When the average cell volume drops below 400 fl (usually between S1D12 and S1D20), the culture is restimulated. Cells should not be returned to resting cell volume prematurely (this is because
Too late, the cells die). AccuComp software (Cult
er) to analyze the cell sizing data. A cell size histogram is obtained by the software. Digitally save data to a backup disk. Different donors will vary until the time of the first restimulation. The more restimulation of the culture, the shorter the time interval between restimulations.

C. Addition of IL-2 CD28-stimulated cells from a normal blood donor are treated with exogenous IL
No addition of -2 is required. However, cells from some donors have defective IL-
May have two secretions, in which case optimal cell growth is achieved by the addition of rIL-2. rIL-2 (Chiron) is added from a single use vial, 22 × 10 ⁶ IU / vial. Sufficient IL-2 was added to bring the culture medium to 100 IU /
up to ml.

At S1D11 (Day-2 re-injection), aliquots of each culture are removed, pooled, and sent for bacterial and sensitivity tests, fungal and mycoplasma cultures. In S1D11 (Day-2 re-infusion), follow the manufacturer's instructions for QCL
Test for endotoxin using a -1000 chromogen LAL assay (BioWhittaker, Inc.) and a SpectraMax 340 microplate reader. Each test is performed in triplicate and quantified using a proven endotoxin standard (BioWhittaker, Inc.). S1D11
At (Day-2 re-injection), aliquots of each culture were removed, pooled and ATCC
Test for mycoplasma using the mycoplasma detection kit. PCR reactions were performed on a Perkin-Elmer 9600 or Ida according to the manufacturer's instructions.
Run on either the Ho Technology heating cycler. Each test is performed in triplicate and confirmed against two positive standards (ATCC).

Example 10 (Removal of Magnetic Beads from Cell Culture Medium) In one embodiment of the present invention, the magnetic beads are a Baxter Fenwal M
Remove from cell culture media using axSep ™ magnetic cell separation system.
The Baxter Fenwal MaxSep magnetic cell separation system is based on MaxS
It consists of an ep magnetic cell separator and a MaxSep magnetic cell separator disposable set. The larger first magnet is a strong permanent magnet constructed from a neodymium iron boron bar and is designed to provide optimal separation characteristics. It attracts the microbeads to the magnet surface and removes the remaining cells and suspension to the first
Used to drain from containers. The second magnet is designed to capture microbeads that can escape from the first magnet. The remaining cell / fluid mixture is collected in a container (cell collection container) and then, if desired, the Baxter Fen
Further processing in a wal cell harvester.

The MaxSep system incorporates a peristaltic pump with a flow rate range of 1-30 ml / min. On average, approximately 12 liters of cell suspension are processed. In some procedures, the pump allows for processing of a higher flow rate of cell suspension through the bypass. This reduces the time the cells are maintained at room temperature outside the incubator, which can affect the overall processing time and cell viability. In addition to the pump passing through the bypass, other modifications to the MaxSep disposable set make the separation process faster and create a semi-closed system. These modifications ensure the sterility of the cell suspension and provide protection to the processing staff.

During cell culture, antibody-coated paramagnetic microbeads are used to stimulate cell growth and proliferation. Once the cell growth goals have been achieved, the microbeads must be sufficiently deficient prior to reinfusion into the patient donor to prevent or reduce the potential for unwanted side effects that can be caused by the injection of these antibody-coated microbeads. Need to be removed. The magnetic cell separation procedure is performed prior to the day of the planned cell reinfusion and cell harvest or when desired.

The following equipment and supplies are used: Baxter Fenwal Ma
xSep Magnetic Cell Separation System (1); Terumo SCD312 Sterile Tube Welder (1) / Welding Wafer (if needed); Sebra Dielectric Heated Tibe Sealer-Model 2100 (1); IV Pole (1); Max
Sep Disposable Set (1) (Baxter Cat # 4R540
1); 600 ml introduction pack with 8 couplers (2) (Baxter C
at # 4R2027); 1000 ml Lifecell Flask (1
) (Baxter Cat # 4R2111); 2000 ml introductory pack (
8) (Baxter Cat # 4R2041); 1000 ml bag Pl
asmalyte-A (1) (Baxter Cat # 2B2544) -priming solution; cell culture medium contained in 3 liters of Lifecell Flash (8); "Y" conjugation (Baxter Cat ##) from Plasmalyte-C disposable set (1) 4R2252); laminar flow hood (1);
And a plasma extractor (1).

A. Modification of MaxSep Disposable Set The following procedure should be performed before the cell culture flask is entered and the line is exposed to fluid. Maintain the sterility of the system according to the standard operating procedure for Terumo SCD312.

A cell suspension sample from each flask to be processed is obtained. Measure total cell count and cell viability as per protocol guidelines. The MaxSep set is prepared as follows: 1) Using a Sebra heat sealer, heat seal a tube approximately 3 inches away from the "Y" on each leg.
Detach the “Y” junction from the small-C disposable set. Carefully remove from the rest of the set and save. Unused Plasmacell-C
Store the set in a sealed plastic bag for future use; 2)
Using a Sebra heat sealer, seal off the second chamber line, the first line and the cell collection line from the unmodified MaxSep disposable set. Seal the second chamber line and the first line as close as possible from the "Y" bond. The cell collection line seals off approximately one inch from the collection line bushing, eliminating all pump segments. Carefully detach and save these tubing lines from “Y”; and 3) Use SCD312 to generate
Weld a single leg below the "Y" junction from the 11-C disposable set to the cell collection line-this line will be the reservoir bag line. "Y
Weld one of the upper legs to the priming line-this line is the priming solution container line. The remaining legs are welded to the second chamber line. Make sure all clamps are closed and remain part of the setting.

Prepare the effluent line as follows. Eight 2000 using SCD312
A single tube lead in a ml introduction pack, 600 with 8 coupler sets
Individually bind to eight tube leads of a ml introduction pack. After bonding, close all roller clamps. The 600 ml transfer pack serves as a reservoir bag, and the 2 liter transfer pack serves as a cell collection container. Discard the spike connector.

The inflow set is prepared as follows. First, using SCD312, the pre-attached lead on the 1000 ml Lifecell Flash above the "Y" junction is welded to the single tube lead of another 600 ml introduction pack with eight coupler sets. This line is the cell suspension line. 1000ml Life
cell Flash is a first separation container. Discard the 600 ml introduction pack.
Make sure the roller clamp is closed and remains part of the new setting. Second, the eight couplers attached to the first separation vessel were eventually spiked using a 3000 ml Lifecell Flass containing cell culture suspension.
k. Now make sure that all roller clamps are closed.

Combine the MaxSep, outflow and inflow sets as follows. MaxSe
Insert the first line coupler of the p-disposable set into the spike port furthest from the pre-attached lead (cell suspension line) on the 1000 ml Lifecell Flask that will be used as the first separation vessel. The reservoir bag line is connected to the spike port furthest from the reed coming out of the 600 ml introduction pack (reservoir bag) with eight couplers (cell collection containers). Close all clamps. The single line exiting the reservoir bag to the collection container is the cell collection line. Using a spike port, connect the priming line to a primin solution container (Plasmacell-A). Make sure the priming line clamp is closed before joining. Suspend the priming solution container vertically from the ring clamp stand located inside the laminar flow hood. Once all couplings are completed and all clamps on the disposable set are closed, proceed with the priming technique described below.

B. Priming Procedure It is very important to remove all air bubbles from the second separation chamber and the first line. The microbeads adhere strongly to the foam and can be carried downstream despite the presence of a strong magnet.

Open the priming line clamp and the reservoir bag line clamp.
Do not open the eight clamps (collection vessel clamps) for the 2000 ml cell collection vessel. Allow the primin solution to flow into the reservoir bag and make sure that all air has been purged from the tubes. Once approximately 200 ml has flowed into the reservoir bag, close the priming line clamp and the reservoir bag line clamp. Air in the reservoir bag emerges toward the cell collection container. Roll up the reservoir bag and make sure that the air in the bag is at the top (the end of the bag where the spike port is located) or put the reservoir bag into the plasma extractor. Open one of the cell collection line clamp and the cell collection container clamp. The air in the reservoir bag is removed by squeezing it or using a plasma extractor. Once all the air has been removed from the reservoir bag, continue to squeeze the bag and allow the priming solution to flow into the collection container to purge all air in the line. Close the collection vessel clamp.

Continue to apply light pressure to the reservoir bag. Open the clamp on the next collection container. Purging all air from the line by flushing with priming solution in the reservoir bag. Close the collection vessel clamp and maintain pressure on the reservoir bag. Repeat these steps for the rest of the collection vessel until all air in the tube has been purged. Close the reservoir bag clamp. If applicable, remove the reservoir bag from the plasma extractor. Fill the second separation chamber by opening the second chamber line clamp and the priming line clamp. Hold the second chamber upright so that all air is directed to the first line coupler. When the second chamber is filled with the first solution, close the second chamber line clamp and the priming line clamp. The massaging action removes any air that may be trapped at the corner of the second separation chamber towards the first line coupler. Tapping the chamber with your finger will help move the bubbles.

Open the priming line clamp, the second chamber line clamp and the first line clamp. While confirming that all air bubbles have been removed from the second separation chamber, the first line, and the second chamber line, approximately 200 ml of the priming solution flows into the first separation container. Close all clamps. At this point, Ma
The xSep disposable set is fully activated, with the exception of the 8 capulla manifold, which connects to the cell suspension line and cell suspension container. Also, the first separation chamber contains air that needs to be sent to one of the cell suspension containers once they are coupled to the 8-coupler manifold. The entire MaxSep disposable set is examined for leaks, especially around the SCD312 weld. If any leaks are found, the set is considered contaminated and discard it. Go back and set up a new disposable set.

On average, there will be eight 3 liter Lifecell Flasks inside a CO ₂ incubator containing the cell suspension to be treated. At some point remove two of them and place them inside a laminar flow hood. Confirm cell culture identification numbers against those previously recorded. Once the verification process is complete,
Remove the blue protective cover of the spike port of the flask from one of the cell suspension containers and remove the lid of one of the spike connectors from the 8-coupler manifold. The two are aseptically combined. Repeat this procedure for other cell suspension containers inside the hood. Remove two more flasks from the incubator and repeat the cell culture identification process to bind to the 8-coupler manifold.
These steps are repeated until all cell suspension containers to be treated have been connected to the first separation chamber via the 8-coupler manifold.

An IV pole is located near the opening of the laminar flow hood. Carefully move the cell suspension container from the hood to the IV pole and make sure that there is no tension in the tube line. The rest of the MaxSep disposable set is organized and organized inside a laminar flow hood to prevent the tubes from tangling. Move the IV pole and disposable set to the area where the MaxSep separation system is located. The IV pole is "heavy on top", so be careful when moving it to prevent the pole from tipping. The disposable set is placed on top of the counter where the MaxSep separation system is located. Suspend the priming solution container vertically from the mini IV pole created in the MaxSep system. Position the first separation container such that the air inside the container is on the side where the spike port is located. Roll up the container or place it in a plasma extractor.

Open the cell suspension line clamp and one of the individual Lifecell Flash clamps while maintaining pressure on the first container. Bleed air from the first vessel and send to the flask. Continue to flush fluid from the first container until the air is purged and the line is filled with the priming solution. Close the Lifecell Flash clamp. Repeat these steps until all Lifecell Flash lines are depleted of air and filled with priming solution. Make sure all clamps on the disposable set are closed. Cell collection container,
Place approximately 6 inches below the level of the surface where the MaxSep separator is located.

C. Installation of Disposable Set in MaxSep Separation System Shrink the locking pin and open the first magnet door. Rotate the port restraint lock 90 ° so that the port restraint arm is lifted to the open position. Horizontal (
Place the first separation vessel at a right angle above the first magnet in the 0 °) position. At this point the first
Do not close the magnetic door. Open the second magnet door and place the second separation chamber on the second magnet. The second chamber line, facing the magnetic cell separator,
Orient the line so that it faces towards the back of the magnetic cell separator.

A second chamber line bushing is placed in a recessed area provided in the second magnet facing the front of the separator. Next, the first container line bushing and tube are
Place in the recessed area provided facing the back of the separator. The second chamber should fit in the provided recessed area. Lower the second magnetic door above or below the second chamber. At this point the door does not close completely. Because the second
This is because the chamber contains excess priming solution. Close and lock the port restraining arms above the first container port to secure them. Rotate the port constraint lock 90 ° to secure the constraint arm. The notched area of the rocking block is for use in a 1000 ml container.

[0305] Excess priming solution is backflushed from the second separation chamber to the first container by opening the first line clamp. Magnetic force on the door on the second magnet causes the door to close and squeeze the fluid out of the second separation chamber.
When the backflush is completed, the first line clamp is closed. The first magnet door is closed by lifting from the hinge side and lowering the free end onto the first container. While supporting the door, two locking pins are slid over the door to restrain it. If this is not possible, the first container must be overfilled and fluid must be removed until the door can be closed.

[0306] Excess fluid from the first separation vessel is passed through a cell suspension line clamp and Life
It can be removed by opening one of the cell flash clamps. The flask must be below the first container so that fluid flows in the direction of the flask. Once the door closure is achieved, close both clamps. Position the first magnet at a 45 ° angle and tap the first magnet door to remove any air bubbles that may form during operation of the container. Any foam present must be as far as possible from the first line coupler at the end of the first container without ports. Position the first magnet at a 15 ° angle. Make sure all clamps are closed.

D. Separation Procedure This is a modified magnetic bead removal procedure using the Baxter Fenwal MaxSep magnetic cell separation system. The maximum flow rate using a peristaltic pump made in MaxSep is 30 ml / min. To achieve higher flow rates, the MaxSep pump is bypassed and gravity is used to supply fluid through the first and second separation chambers to the collection vessel. Semi-closed systems have been developed to reduce the risk of exposure to biohazardous fluids. All other manufacturers' guidelines have followed the maximum possible.

Make sure that all clamps on the MaxSep disposable set are closed. Adjust the height of the IV pole holding the cell suspension container so that the top of the fluid in the flask is 35 inches from the surface where the MaxSep separator is located. This height allows a flow rate of approximately 180 ml / min. Open each of the following clamps in a specific order: 1) one of the Lifecell Fenwal clamps; 2) the cell suspension line clamp; 3) the first line clamp; 4) the second.
Chamber line clamp; and 5) reservoir bag line clamp. The timing of the opening of the clamp is very important. Each must prevent pressure buildup in sequentially opened systems. Excessive pressure increases the likelihood that the beads will be washed downstream into the collection vessel.

Once the separation has begun, approximately 400 ml of the treated cell suspension is allowed to accumulate in the reservoir bag. The cell collection line clamp and cell collection container clamp are then opened. The cell collection containers can be filled individually or simultaneously. When filling the collection containers individually, care must be taken to prevent the formation of back pressure in the tube set. This increases the cell suspension flow rate. If the collection containers are filled at the same time, attempt to maintain an equal distribution of fluid in all containers. During the separation process, the condition of the first and second separation chambers is monitored for signs of improper magnetic separation. If too many microbeads are accumulating in the second separation chamber, backflush the first chamber with the priming solution. In some cases, backflushing of the first separation vessel may also be required.

To backflush the second separation chamber, empty the cell separation container currently to be processed and close the clamp to the container. Close the cell suspension line clamp. The first line clamp does not close. Partially empty the first separation chamber.
Close the first line clamp, the second chamber line clamp and the reservoir bag line clamp. Open the second magnet door and remove the second separation chamber. Open the priming solution line clamp and the second chamber clamp. Fill the second chamber with the priming solution and close the second chamber line clamp.
The microbeads are suspended inside the second chamber by a massage operation. Second
Raise the chamber so that it is higher than the first separation chamber. First
Open the chamber line clamp and allow the fluid in the second chamber to flow into the first chamber. Close the first chamber line clamp.

Open the second chamber line clamp, fill the second chamber with the priming solution, and close the second chamber line clamp. The microbeads are suspended inside the second chamber by the massage operation. The second chamber is the first
Raise so that it is higher than the separation chamber. Open the first chamber line clamp to allow the fluid in the second chamber to flow into the first chamber. Close the first chamber line clamp. Close the priming solution container clamp. Reposition the second chamber over the second magnet and close the second magnet door. If the door is not completely closed, the first line clamp is opened to allow excess fluid in the second chamber to exit to the first separation chamber. Restart the separation process.

[0312] Regularly agitate the Lifecell Flask to resuspend the cell suspension,
Maintain proper cell and bead distribution. This should be done lightly to avoid excessive foaming in the flask. As the Lifecell Flash to be processed nears emptying, it may be necessary to manipulate the flask to optimize cell recovery. To do this, tilt the Lifecell Flask so that the cell suspension is directed to the port connected to the cell suspension line. The first of the manifold
Close the empty Lifecell Flash clamp before the fluid level reaches the "Y" junction to prevent air from entering the first separation vessel. Repeat these steps until all flasks are empty. When all flasks are empty, close the cell suspension line clamp. Be careful to empty the first separation container. Several ml
When all fluids remain in the first container except for, or when foam begins to leave the first container, the first line clamp, the second chamber line clamp and the reservoir bag line clamp are closed. Empty the contents of the reservoir bag into the cell collection container.
Attempt to wash the cell suspension line for the cell suspension by applying pressure to the reservoir bag to maximize cell recovery. Close the cell collection line clamp and cell collection container clamp. The cell collection line is heat-sealed twice and detached from the disposable set. To detach the cell collection container from the 8-lead manifold, heat each individual tube lead twice (approximately 1 ") close to the container. The cell suspension is now ready for further processing as shown. Process the MaxSep disposable set according to the Infectious Disposal Control Plan.

[0313] Any fluid leakage or cutting of the disposable set constitutes a sterile destruction, which must be discarded and replaced. If this happens during the priming approach, simply discard the defective disposable set. If sterility disruption occurs during the actual magnetic cell separation process, all clamps on the disposable set must be closed and fluid spills must be contained according to standard guidelines.

Example 11 Harvesting and Washing of the CD3 + 28 Product The Fenwal Harvestor® system is a system for rapid and continuous centrifugation of large volume suspensions, the harvester and various systems. The harvester is manually operated by a control panel. The suspension is processed in a sterile disposable set.The system is designed to collect particulate concentrate, supernatant or both The particulate phase of the suspension is centrifuged and collected in a belt-type centrifugal chamber, while the liquid phase is collected via lines for aseptic collection or disposable. Monitor continuously checks for abnormal conditions, visible or audible Inform always the combination of operating conditions and any abnormal conditions of alarm to the operator.

Once the cell growth goals have been achieved, on average, the cell culture volume is approximately 12 liters per individual culture. For handling and transfusion purposes, it is advantageous to effectively reduce the cell-containing medium to a manageable volume. The final component used in transfusing the donor recipient should be of a volume that does not cause fluid overload. At the same time, it is advantageous to achieve this volume reduction without incurring significant cell loss. Baxter Fenwal harvesters achieve these goals effectively.

[0316] Injection of the culture medium into the recipient is undesirable. Because it is not a approved medium for intravenous use. Thus, a washing step is provided in this approach, using one liter of 0.9% sodium chloride followed by one liter of washing with Plasmalyte-A (electrolyte solution). Plasmaly-A is also the final cell suspension solution. Cells collected from an HIV seropositive donor are contained in a cell suspension container to be processed. The Fenwal harvester system and its sterile disposable set provide a semi-closed system that protects the sterility of the cell culture to be harvested and reduces the risk of exposure to biohazard fluids.
Cell culture harvest is performed on the day of the planned cell reinfusion once the culture has undergone a magnetic cell separation process to remove microbeads used in cell stimulation.

A. Harvester disposable set installation First, take out the storage set from the pouch. Close the two clamps on the reservoir set and place a hemostat on each inlet line. Hang the reservoir bag from the hook located on the metal support bar, open the feed pump cover and lift the rotor locking handle. Install the pump segment by placing the segment collar outside the pump inlet (lower groove) according to the arrow on the pump. Place the segment between the locator rods of the pump and rotate the locking handle counterclockwise to screw the segment through the pump. The installation is completed by placing the segment in the pump outlet groove and rotating the handle until it locks. Close the feed pump cover. Place the lower pump segment on the right side of the pressure monitor by extending the tubing and placing it in the groove.
Remove the harvester set from the pouch. Fold the belt in half and wind it up to exhaust any air. Close all clamps on the set.

Next, the door to the centrifuge compartment of the harvester is opened to access the belt support. Remove the cover by turning the locking handle so that the red rod knob faces toward the center of the belt support. Remove the white restraining collar from the center of the belt support. Release the three lines from the pack and insert them into the center of the belt support. Pull the line through the guide hole. The belt chamber is placed on top of a belt support with a belt inlet (two tube ends) on the right and a belt outlet (three tube ends) on the left. Place the restraining collar around the lower hex distortion relief. This assembly is inserted into the rotor and rotated until a distinct locking click is heard / feeled. Pull the line out of the guiding hole and over the top door.

[0319] The straight multiple lumen tube to the right of the restraining arm is then placed on the rotor shield. Rotate the rotor shield counterclockwise until the restraining arm and the multiple lumen tube align to the right of the centrifuge compartment. Align the upper hex strain relief on the support located above the centrifuge. Close the restraining color latch. Insert the multiple lumen tube inside the restraint arm along the entire length of the arm. Make sure the tube is straight (follow the blue line on the side of the tube). Slide the multiple lumen tube holder towards the open side of the restraining arm with the Baxter logo up.

[0320] Pull the multiple lumen tube to the left of the harvester and pull the tube
And hold it in a groove located towards the back of the top left side. Close the top door of the harvester far enough to hold the tube in place. Pull the excess length outside the centrifuge compartment. Separate the tubes as follows: inlet line, which has two small tubes with pumpment and coupler
Put across the top of the harvester; the harvest line (one small tube) is placed across the top / back of the machine, turning off the waste line to the left.

Open the processing pump cover and lift the rotor locking handle. Install the inlet line pump segment by placing the segment collar outside the pump inlet (upper groove) according to the arrow on the pump. Place the segment between the locator rods of the pump and rotate the locking handle counterclockwise to screw the segment through the pump. The installation is completed by placing the segment in the pump outlet channel and rotating the locking handle until it locks. Close the processing pump cover. Place the lower pump segment on the left side of the pressure monitor by extending the tubing and inserting it into the groove. Place a large tube hemostat on the waste line near the junction of the three small tubes.

The reservoir bag port protector and the processing pump inlet line spike protector are then removed. The two are aseptically combined. Open the clamp between the processing pump and the reservoir bag. Open the process pump cover and lift the rotor locking handle and slowly rotate it clockwise to deflate the belt into the reservoir bag. Rotate the handle until it locks and close the pump cover. Position the belt inlet (two tube ends) at the "inlet" marking on the belt support. Lift the plunger and insert the belt into the slot around the belt support. Starting at the belt inlet, the belt is inserted to a small depth and continues counterclockwise movement around the belt slot. After the complete belt has started into the slot, it can be easily depressed until it is flushed at the top surface of the belt support outer ring. After these natural positioning, a small tube line is temporarily aligned on top of the belt support. The three outlet tube lines are on the left and the two inlet tube lines are on the right. Make sure the tube lines do not twist or intersect as they descend into the belt slots. Again, make sure that the tube lines do not kink or tangle as they emerge from the restraining collar. Tape the five tube lines to the rotor to avoid twisting or tangling during harvester operation.

The belt support cover is repositioned over the alignment pins and plunger and lowered into position. Check the nearly parallel tube alignment of the five small tube lines in order. For they are fixed under the cover. The tube line can be manipulated for proper alignment by reaching through the central hole in the belt support cover. Make sure that the tubing lines are not crossed or pulled too tightly to cause twisting as they pass the belt support edge down to the belt slot. Lock the cover by rotating the locking handle to the locked position (outward facing red knob). If resistance is felt, check to see if the tube line and belt are properly positioned. Attach the pre-assembled waste / harvest container to the harvest line by removing the tip protector and spiking.

The disposal / harvest container is pre-assembled as follows. Terumo SCD312
Sterile binding device / wafer (if needed), 1000 ml Lifecell Flask (1), 600 ml introducer pack (1), “Y” junction from 600 ml introducer pack with eight couplers (1), plasma introducer set (1) And S
Collect the ebra dielectric heat sealer (1) inside the laminar flow hood. SCD312
Attach 1000 ml Lifecell Flask to one of the legs above the “Y” junction using Close the clamp. Using SCD312, attach a 600 ml introducer pack to the other leg above the “Y” junction. Close the clamp. "Y
On a single leg below the junction, use SCD312 to join the section of the plasma introduction set with spike connectors on opposite ends and close the clamp.

[0325] One of the spike protectors of the waste line and the plasma transfer set is removed and coupled. The ten-way manifold is coupled to another spike connector on the plasma delivery set. 2 tube lines attached to the 2000 ml introduction pack
Heat seal once and discard the tube. Attach a 2000 ml introducer pack to each of the spike connectors on the 10 crotch manifold. These would be waste containers. Close all clamps on the manifold. The top protector is removed from the other 10 crotch manifold and from one of the reservoir bag inlet line spike connectors and attached to each other. Close all clamps on the 10 crotch manifold. Hold the hemostat in place. Remove the top protectors from the five-pronged manifold and other reservoir bag inlet line spike connectors and attach to each other. Close all clamps on the 5-way manifold. Hold the hemostat in place.

Before proceeding, the following conditions should exist: a) Multiple lumen tubes should be straight and should not be twisted; b) Belts, associated tubes and covers should be in place The cover locking handles should be in the locked position (red knob out); c hex distortion reliefs should be properly positioned in their respective restraining collars; d) white restraining collars should be in place E) the multiple lumen tube should be located completely inside the length of the restraint arm; f) the multiple lumen tube holder should be properly located; g) the centrifuge The front and top doors to the compartment should be completely closed; h) the hemostat is in a specified position and is clear-three The waste line in the large tube area near the connection site of the small tube, and the inlet line of the reservoir bag; Should be closed except.

B. Harvester Disposable Set Activation Approach First, make sure that all clamps are closed except for the clamp between the reservoir bag and the processing pump. Three 1000m using spike connectors
One liter of 0.9% NaCl bag is attached to the five-leg manifold. Switch on the harvester by pressing the green power button on the control panel. The button presses the centrifuge "fast" button, which should emit light. 160
It should be programmed to 0 rpm and the display should include 1600 rpm. If the centrifuge does not spin at 1600 rpm, adjust the fast rotor knob on the back of the machine until the display reads 1600 rpm. Refer to the operator manual for further instructions on adjusting the back panel controls. Open the waste line clamp and the clamp to one of the 2000 ml waste containers. Do not remove the hemostat from the waste line. Na on 5-fold manifold
Open the Cl bag clamp, remove the hemostat and open the clamp to the reservoir inlet line connected to the five-way manifold. A Code 6 alarm should now be displayed; this indicates a low reservoir and is normal at this point. Press the mute button to silence the audible alarm.

Next, by pressing the F-JOG button of the feed pump, almost 500 ml of N
Pump aCl into the reservoir bag. The LED display should indicate the rate at which fluid is being pumped into the bag. The feed pump flow rate should be set at 700 ml / min. If the flow rate is other than 700 ml / min, adjust the pump speed. To adjust the feed pump flow rate, press the "fast" button on the feed pump on the back panel control of the harvester and position it on the "supply fast" knob to obtain a flow rate of 700 ml / min. adjust.

Next, the “fast” button is pressed for the processing pump. LED display is almost 60
It should show a flow rate of 0 ml / min. To adjust the process pump flow rate, press the "fast" button on the process pump (code 6 (low reservoir) condition should not exist or process pump will not operate), then "P
Position on the "processing fast" knob and adjust as needed to obtain a flow rate of 600 ml / min. The feed pump should be set to a higher flow rate than the process pump. NaCl from the reservoir bag should flow into the centrifuge via a manual lumen tube. Squeeze the processing line with a finger between the processing pump and the reservoir bag to eliminate any air bubbles that may form in that area.

[0330] Wait for a code 5 alarm indicating that the fluid being pumped into the centrifuge has filled the belt chamber and is ready to exit through the waste line. This alarm condition stops the processing pump and centrifuge. Remove the hemostat from the waste line. Since the build up of pressure has been resolved, the machine returns to normal operation once the hemostat has been removed (restarting the process pump and centrifuge). Alternatively, two of the three small tube lines exiting the centrifuge compartment together with the hemostat are pinched to remove any trapped air inside the belt chamber. Check fluid flow on waste line. It should be a smooth flow at approximately the programmed processing speed. Check if any bubbles have formed in the inlet line by pressing and holding the strobe button. Attempt to eliminate it, if any, by pinching one of the inlet multiple lumen tubes.

If there is no problem at this point, press the off buttons of the processing pump and the supply pump. Keep the centrifuge running and hemostat the waste line. Close the clamp to the 2000 ml waste container where the priming solution has been collected, as it should be almost full. Close the clamp to the NaCl bag and the clamp on the reservoir inlet line connected to the five-pronged manifold. Stop the inlet line.
The harvester is now ready to process the cell culture.

C. Cell Culture Harvest in Wash Close the clamp on the waste line and apply the hemostat to the line during alarm conditions or in any situation where the centrifuge slows, stops, or must stop. This prevents the cells from accidentally causing a sun horn phenomenon from the belt chamber to the waste container.

First, confirm the identification number on each of the cell culture vessels to ensure that the correct cell culture is being processed. In addition to confirming the cell culture identification number, confirm that the cell culture label indicates that the culture has undergone a magnetic separation process. Using a 10-fold manifold, spike one of the cell culture vessels. Open the clamp to the culture vessel and the clamp on the reservoir inlet line and remove the hemostat. Press the "fast" button for the feed pump. If the LED display does not illuminate and the pump stops once the button is released, it indicates that the reservoir bag has low capacity. Code 6 should not be displayed at this time. To resolve this condition, the LED display will hold the "fast" button down until the pump speeds up, then release.

Next, remove the hemostat from the waste line and open the clamp to one of the empty waste containers. The condition of the waste container is continuously monitored for overfilling. During the procedure, close and open the waste container, if necessary. Press the "fast" button on the processing pump. The cell culture in the reservoir bag has now been pumped into the belt chamber and harvesting has begun. Check that the feed pump flow rate is 700 ml / min and the processing pump is 600 ml / min. When the cell culture vessel being treated approaches empty, connect another vessel to the 10-fold manifold. When the container being processed is empty, close the clamp and open the clamp to the next culture container.

[0335] As the vessel being processed is emptied, the cell culture vessel continues to attach to the manifold. Whenever it is empty, tighten the appropriate cell culture vessel line clamp and then open the clamp to the full vessel to be processed. This confirms that there was a failure or problem during the harvest procedure that compromised the sterility of the cell culture, and all containers would not be affected. The cells are periodically resuspended by manually shaking the culture vessel. Monitor the progress of the harvesting process; in some cases, press the strobe button and check the centrifuge compartment for any signs of improper operation or failure.

After all cell culture vessels have been processed, a 1000 ml bag of 0.9% NaCl and a 1000 ml bag of Plasmalyte-A are attached to the quintuple manifold. The clamp does not open. These solutions will be used in the washing step. Once the cell culture vessel is empty and the end of the cells is in the reservoir bag, press the off button on the feed pump. Close the clamp on the reservoir bag inlet line that is connected to the 10-fold manifold and place the hemostat on the line. At this point, the reservoir bag is low and the processing pump automatically shuts down. Press the mute button to turn off the alarm. Press the F-JOG button for the processing pump until the reservoir bag is almost empty, then release. Do not allow air to enter the processing line (leave on centrifuge if left unchecked). 0.9% sufficient for three bags initially connected to the five-legged manifold used to activate the harvester
There should be NaCl. If less than 1000 ml of NaCl remains (
Combine the volumes in each bag) and combine into another bag. Remove the hemostat and open the clamp on the reservoir bag inlet line that connects to the 5-way manifold.

For the feed pump, press the F-JOG button to allow approximately 300 ml of NaCl to enter the reservoir bag and then release. Massage and rinse the reservoir bag. For the processing pump, keep the F-JO until the reservoir bag is almost empty.
Press G button. Again, do not allow air to enter the processing line. These steps are repeated twice. Once completed, the 0.9% N used in these steps
Close the clamp to the aCl bag. Open the clamp to the full 0.9% NaCl bag and press the "fast" button on the feed pump until the machine automatically takes over filling the reservoir bag. For the feed pump, once the bag is empty, press the off button and close the clamp.

Then, for the feed pump, press the “slow” button, where it runs at a flow rate of 300 ml / min. If the LED display indicates a different flow rate, adjust it by using the back panel control (using the processing delay knob to adjust the pump speed). When the low reservoir alarm sounds (code 6), press the process pump off button and mute button. 100 of Pharmalyte-A
Open clamp to 0 ml bag. For the feed pump, press the "fast" button until the machine takes over filling the reservoir bag. Once the bag is empty, press the off button for the feed pump. Close the clamp to the Plasmaly-A bag and the clamp on the reservoir bag inlet line. Place the hemostat on the line. Press the "slow" button for the feed pump, where it operates at a flow rate of 300 ml / min. Run the solution through the machine.
When the low reservoir alarm sounds (code 6), press the process pump off button and mute button. Place the hemostat on the waste line near the point where the three small tube lines join. Close the waste line clamp. Press the off button for centrifuges.

Open the feed pump compartment cover, lift the locking handle and remove the tubing by turning the door. (Follow the same steps used in installing the inlet line pump segment-see section A above) Install the harvest line on the feed pump. Open both small and large clamps on the harvest line. Open the roller clamp to the waste container (600 ml introduction pack) connected to the harvest line.

Open the door to the centrifuge compartment. Remove the belt support cover (turn the locking handle so that the red knob faces the center of the belt support). Make sure that all five small tube lines entering and exiting the belt chamber are visible. Press the Feed Pump F-JOG button while looking closely at the three outlets to ensure that only the Plasmalyte-A solution exits the belt. If cells are seen at the outlet line of the belt, this step of releasing the F-JOG button is only used to siphon excess of the Plasmalyte-A solution; at this point the cells Should not leave the belt chamber. Close the roller clamp to the waste bin connected to the harvest line. Harvest container (
Open roller clamp to 1000 ml Lifecell Flask).

Lift the belt support plunger and carefully remove the belt chamber from around the belt slot. Make sure the line is not pinched during the removal process. At this point, the restraining collar should not be removed. Using the back of one hand, scrape or lower the belt chamber until all cells are resuspended. Any other form of operation of the belt can be used to achieve the best possible resuspension of the cells, as long as the openness of the belt chamber is not compromised. Hold the belt chamber with one hand so that the three outlet tube lines are at the bottom. Press the feed pump F-JOG button so that cells flow from the belt to the harvest container. Once the vacuum is formed, release the F-JOG button. Processing pump FJ
Add some Plasmalyte-A solution from the reservoir bag to the belt chamber by pressing the OG button and then releasing it. The cell resuspension procedure is then repeated.

Next, as described above, the contents of the belt are emptied into harvest containers. Add the Plasmalyte-A solution from the reservoir bag to the belt chamber and allow the cells to flow from the belt chamber to the harvest container until all cells have been cleared from the belt chamber (two or three belt rinses are sufficient). Then, both press the feed pump F-JOG button is repeated. Remove the reservoir bag from the support and invert it. Press the Process Pump F-JOG button until the belt chamber starts filling with air. Hold the belt with the three outlet tube lines at the bottom. Press the feed pump F-JOG button, put air inside the belt,
Wash all lines on the way to the harvest container. Close the roller clamp to the harvest container. Heat seal the harvest vessel line twice, as close as possible to the "Y" junction, and detach. A cell culture identification label is attached to the harvest container.

Inside the laminar flow hood, couple another 1000 ml of Lifecell Flask to the harvest vessel. This flask is the final component container. Make sure the roller lamp remains part of the setting. Baxter Fenwal Ma
Place the harvest container flat (0 ° position) on the first magnet of the xSep magnetic cell separation system. Close the first magnet door and let the harvest container rest on the magnet for approximately 1 minute. This captures any microbeads that may have escaped the initial magnetic separation process. Position where the first magnet is placed in the vertical position (90 °). Open the roller clamp between the harvest container and the final component container (1000 ml Lifecell Flask). Empty the harvest container completely and close the roller clamp. The tube between the two containers is heat sealed three times and detached. At least 2
Make sure that one heat seal stays on the tubing line connected to the final component container.

The Lifecell Flask containing the cells is identified and labeled. This label should include the date, donor information, receptor information, the phrase "for autologous use only", the process the cell has undergone up to this point, the suspension used, and the biohazard symbol. Place the final component container inside the leak free shipping container. Remove the disposable from the harvester and disinfect the harvester with 50% sodium hypochlorite / water solution.

[0345] Any fluid leaks or disposable set cuts constitute a break in sterility, and the set must be discarded and replaced. If this happens during the boot procedure, simply discard the defective disposable set and install a new one.
If sterility disruption occurs during the actual harvesting process and not inside the harvester's centrifuge compartment, shut off the harvester pump and shut down the waste line,
Stop the centrifuge and close all clamps on the disposable set to contain fluid spills. If sterility breakage occurs inside the centrifuge compartment during the harvest process, fluid spillage should trigger a Code 7 alarm. This alarm condition shuts down all harvester activity until the condition is corrected and the mute button is depressed. All individuals in the treatment room should leave immediately to prevent any aerosol-borne exposure caused by the spill.

Cells harvested and grown from HIV seropositive individuals are contained in cell culture vessels; therefore, at all times, Universal Blood and Body.
Fluid Precautions (universal blood and fluid precautions) should be rigorously implemented. Gloves and disposable lab coats should be worn during all steps of the procedure where cell culture is handled. If splashes or aerosols are possible, goggles and / or face protection should be worn.

Example 12 Procedure for Injection of CD3 + 28 T Cells This procedure involves the removal of magnetic beads from cultured CD3 + 28 T cells, and F
It can be used for cell enrichment with enwal CS3000 + and release criteria for the final cell product must be established prior to cell injection. The following materials were used: Coulter Multisizer IIe; Trypan Blue 0
. 4% solution (Sigma Chemical, St. Louis, MO
); Plasmalyte-A containing 1% human serum albumin; albumin 25
% Solution; and Gram stain kit (Sigma).

An aliquot of cells is removed from the final product bag by harvest in cell culture and washing as detailed above. Cells are counted and Coulter Multisi
Electronic capacity measurements are performed on the Zer IIe. Cell viability can be assessed by trypan blue staining. Survival should be at least 80%. Culture sterility can be assessed by reference to microbiological reports from culture performed on day -2. To further confirm sterility, the Gram stain report should report the unobserved microorganisms, and bacterial, mycoplasma and fungal cultures from all previous culture reports, including day -2, show no growth. Should be reported. No.-2
Endotoxin assays from day should have a result of <1 EU / ml /.

The suitability of removing magnetic beads can be assessed by microscopic observation according to the following protocol: 1) Add 1 × 10 ⁶ cells in triplicate to a 1.5 ml microcentrifuge tube. Thus, evaluate the remaining beads contained in 3 × 10 ⁶ cells; 2) lyse the cells by adding enough Triton X-100 to a final concentration of 1%; 3) microcentrifuge tubes Pellet the beads by centrifugation, aspirate the supernatant and take care to leave a nearly dried pellet containing 50 μl residual volume in each tube; 4) Use a pipette to resuspend 50 μl gently Be careful not to form bubbles. Add a total of 50 μl to the microscope slide. Add a coverslip and scan the entire field of view of the beads. Record the total number of beads remaining; and 5) If more than 100 beads are observed on three slides, repeat the magnetic bead depletion by passing the final product through a magnetic field on the MaxSep device. In steps 1-4, the magnetic bead quantification protocol is repeated. The bead removal release criteria for the final product should be less than 100 beads per 3 × 10 ⁶ cells.

Inject cells with concomitant results from the release criteria. Depending on the number of cells harvested and the number of target injections for a particular protocol, excess cells can be removed from the final bag and cryopreserved. 100-200ml
Thoroughly lyse cells in Plasmalyte-A containing 1% human serum albumin in a final volume of. The cells should be reinfused into the patient without filtration for 20 to 30 minutes, and the patient should be monitored for adverse reactions as described herein. Leukopenia filters should not be used during the administration of the final product. Cells should not receive gamma rays or pass through an x-ray irradiation device designed to detect metal objects. The final trend of the end product and any appearance of adverse reactions are recorded.

In rodents, injection of 9.6 × 10 ⁴ beads / kg was not toxic and no beads could be detected in the tissue pieces. No toxicity was observed in rodents receiving 8.3 × 10 ⁸ beads / kg, and beads were observed phagocytosed in cells of the reticuloendothelial cell lineage.
The remaining beads in the final product can be quantified by measuring beads in 3 × 10 ⁶ cells. If <100 beads / 3 × 10 ⁶ cells are observed, injection of 3 × 10 ¹⁰ cells in a 70 kg patient represents <1 × 10 ⁶ total beads or <1.43 × 10 ⁴ beads / kg. Appropriate agreement between clinical facilities and laboratories is advantageous throughout the harvesting and infusion process. "Controlled rapid" is advantageous once the cells are harvested and removed from the cytokine-containing culture medium. The time between cell enrichment and the start of cell infusion should not exceed one hour. The final product should not be injected if more than 4 hours after harvest.

Example 13 Obtaining Cell Counts and Sizes To obtain cell counts and sizes using a Coulter Multisizer II counter, first obtain Isoton as outlined in the calibration procedure above.
(Blank) to achieve a background <100. Next, the sample is gently turned upside down three to four times to evenly distribute the cells. Then 10ml Isot
Add on to an empty sample vial. Next, add 0.05 ml of sample to the sample vial. The sample vial is then gently turned upside down three to four times to distribute the cells evenly. Next, make sure the probe is not clogged and place the sample vial under the probe and run to get cell count and size. Then open the Multisizer II software on the computer. Multi
Press the print button on the sizer II to transfer the data to the computer.
Analyze with software. Coulter Multisizer Acci
See the Comp Color Software manual. Finally,
After running the sample, rinse the orifice of the sampling stand with distilled water on a waste bucket.

Example 14 To calibrate a Coulter Multisizer II with a sampling stand, switch on the Coulter by turning on the power switch of the Multisizer II and the associated sampling stand. Iso
Check to make sure that the ton bin is properly sealed and full. If necessary, empty the waste bottle. Allow the Coulter to warm for approximately 10 minutes.

A Coulter control is then prepared. To set the control properly, C
See the Oulter Multisizer II reference manual.
Add 10 ml Isoton to the vial. Add one drop of the appropriate Coulter size standard to the vial. Mildly invert the sample vial 3-4 times to evenly distribute the cells. Make sure the probe is not clogged and place the sample vial under the probe. Make sure the instrument control is in siphon mode and first run a calibration vial by pressing the reset button on the Multisizer. Next, the upper stopcock is rotated by a quarter turn clockwise. Switch on the light of the sampling stand and increase the height of the manometer. Wait a few seconds for the green light screen to indicate the presence of cells, then
Start the upper stopcock a further 1/4 turn clockwise. The light goes out, Mu
The count is complete when the ltizer II beeps. If all expected value ranges match, the calibration is complete. Before starting sample analysis,
Run ton through the system.

Example 15 Measurement of the amount of endotoxin present in cell culture Equipment and materials were sample (pH 7-8), culture sample, 1:10 dilution of culture sample heated at 70 ° C for 5 minutes, stop reagent ( 25% v / v glacial acetic acid in water), Limu
luc Amebocyte Lysate Test Kit, Chromogenic Substrate and Chromogenic Li
mulus Amebocyte lysate (LAL).

[0356] 1.0 ml of Limulus Amebocyte lysate warmed to room temperature (
LAL) by adding reagent water. Restores E. coli endotoxin. The actual concentration is determined by the values stated in the enclosed certificate of analysis. Shake vigorously with a stirring mixer for at least 15 minutes. This stock solution should be
Monthly stable. Prior to use, the solution should be warmed to room temperature and mixed vigorously for 15 minutes. This is advantageous because endotoxin tends to adhere to glass.

To prepare four standard endotoxin solutions, add 0.1 ml of endotoxin stock solution to (x-1) / 10 ml of l in a 6 ml Falcon tube.
Prepare a solution containing 1.0 EU / ml endotoxin by diluting with AL reagent water, where x is equal to the concentration of endotoxin vial (found in the certificate of analysis). The solution should be stirred vigorously for at least 1 minute before proceeding.

Transfer 0.5 ml of this 1.0 EU / ml solution to 0.5 ml LAL reagent water in a 6 ml Falcon tube and label 0.5 EU / ml. This solution is
It should be stirred vigorously for at least 1 minute before use. Next, 6 ml of Falc
on 0.5 ml of 1.0 EU / m in 1.5 ml of LAL reagent water in a tube
Transfer the solution and label 0.25 EU / ml. This solution should be stirred vigorously for at least 1 minute before use. Finally, 0.9 ml LA in a suitable container
Transfer 0.1 ml of 1.0 EU / ml solution to L reagent water and label as 0.1 EU / ml. This solution should be stirred vigorously for at least 1 minute before use.

One vial of 7 mg of lyophilized chromogenic substrate is reconstituted by adding 6.5 ml of LAL reagent water to obtain a concentration of approximately 2 mm. Protect the substrate from prolonged exposure to light. Just before use, reconstitute the LAL with 1.4 ml LAL reagent water. Gentle stirring to avoid foaming.

The addition of all reagents in the Limulus assay should be consistent. All tubes or microplate wells should be treated in exactly the same way to determine the appropriate endotoxin concentration. Reagents are pipetted into wells in the same order and at the same speed. Place the microplate in a microplate reader at 37
Pre-equilibrate at ± 1.0 ° C. While the microplate is standing at 37 ° C. ± 1.0 °, carefully dispense 50 μl of the standard or sample into the appropriate microplate well. Each series of measurements should include four endotoxin standards performed in Planck + duplicate. Blank wells are replaced with 50 μl of LAL instead of sample
Contains reagent water. All reagent additions and incubation times are the same.

At T = 0, add 50 μl of LAL to the first microplate well. Timing starts when the LAL is added. Once the LAL has been dispensed into the microplate wells containing the samples or standards, mix in a microplate reader by remote control with SpectraMax software. T = 1
At 0 minutes, add 100 μl of substrate solution (pre-warmed to 37 ° C. ± 1.0 ° C.). Pipette the substrate solution as described above. Once the substrate solution has been dispensed into all microplate wells containing samples or standards, mix in a microplate reader by remote control with SpectraMax software. At T = 16 minutes, add 100 μl of stop reagent. Pipette the substrate solution as described above. Once the substrate solution has been dispensed into all microplate wells containing the sample or standard, the SpectraM
Mix in microplate reader by remote control with ax software. The absorbance of each microplate well at 405 nm is read, analyzed and graphed with SpectraMax software.

Example 16 Sample Analysis of Endotoxin Control This procedure was performed using SpectraMax 340 and SOFTmax PRO.
A method for measuring the amount of endotoxin present in cell culture using software is described. Equipment and materials include: SpectraMax 340 microplate reader; SOFTmax PRO software; and blanks, standards and samples prepared in microplates.

Open SOFTmax PRO in MS-DOS in the computer. (The computer will have to be restarted to enter MS-DOS). Under the FILE menu, open the file named "template.pda." This can serve as a template for all samples. Check to make sure that the SOFTmax icon at the top left corner of the status bar does not have a red "X" at its top. If an "X" appears on its top, the computer will not contact the instrument, and therefore
Check if SpectraMax is switched on. SpectraM
Check the current temperature of the ax chamber. The current chamber temperature display is located to the right of the SpectraMax icon in the status bar. RESD
By clicking the incubator button located to the right of the icon, the temperature can be adjusted and / or turned on or off.

Drawer Button, SpectraMax Open the SpectraMax 340 drawer by clicking the right button in the status bar. Place blanks, standards and samples in drawer. Close the drawer by clicking the drawer button again. SpectraMax 340
Automi whenever the SOFTmax PRO software is on
The x button can be used. During the endotoxin experiment, T = 0, T = 1
An Automix button can be used at 0 and T = 16. It is recommended to use Automix just before reading the microplate. In the introductory section, enter the patient number on the specified line. In the plate section,
Click the mold button located on the status bar. Blank, standard and first one
Zero bags and their 1:10 dilution were assigned to wells. If necessary, remove or add wells. Remove wells by highlighting all unwanted wells. Click Clear under the Group Pop-Up menu on the template editor toolbar. SpectraMax 340 no longer reads these wells. Repeat if necessary. Add wells by highlighting the desired wells. Group Pop- in the template editor toolbar
Click New under the Up menu. In the appearing group setting window, add the name of the sample and, if necessary, select Unknown (under dilution) under the column format section for 1:10 bag dilution. Click OK when finished with the group setting window. Repeat if necessary.

By clicking the print button at the bottom left corner of the mold window,
Print a copy of the mold. Exit the mold window by clicking OK at the bottom right corner. Remove all unnecessary active bag bars. First, highlight the unwanted active bag bar. Then, under the edit menu, "Delete
Click “e Bag #”. Repeat if necessary. Delete or add plots for bags. Pan to the graph section toolbar. Click the plot button in the graph section toolbar to delete plots for unnecessary bags. Highlight unwanted plots. Click the delete button. When finished, click the OK button. Add a plot for each new bag by clicking the plot button in the graph section toolbar. Click the New button in the right corner of the plot window. Identify the new bag number and X and Y axis labels by clicking on the Pop-Up menu located to the right of the label groups, X and Y, respectively. Click OK after each new bag and enter it into the computer. Repeat if necessary.

When the microplate is ready to be analyzed, start reading by clicking the read button in the SpectraMax status bar. If further analysis is needed, refer to the SOFTmax user's manual. Universal blood and fluid attention should be strictly enforced at all times.

Example 17 Mycoplasma Detection in Cell Culture First, a fresh cell culture is prepared by centrifuging a 0.5 ml sample at 12,000 × g for 10 minutes at 4 ° C. Discard 400 ml of supernatant and get 100 ml of medium and cell pellet in tube. Using a pipette, leave the remaining 100
Resuspend the cell pellet with ml of medium. Gentle pipetting to avoid foaming. 100 ml of the cell suspension is ready for PCR.

For the first step of the nested PCR, prepare the following reagents for each PCR tube: 10 × PCR buffer, 5 ml; first step primer mix, 1 ml; d
NTP, 1 ml: MgCl ₂ , 1 ml; Taq DNA polymerase, 0.2 ml; make up to 45 ml with distilled water. For each set of unknown samples to be tested, a minimum of two positive DNA controls and one negative control should be included. For samples, a 5 ml sample is pipetted into each sample tube to a final volume of 50 ml. For the positive controls, 5 ml of each positive control DNA is pipetted into a separate positive control tube.
For the negative control, 5 ml of sterile distilled water is pipetted into the sample tube. Place all tubes in a heated cycler and program as follows: denaturation: step 1:30
Denaturation: step 2: 94 ° C. for 30 seconds; annealing: step 3: 55 ° C. for 2 minutes; extension: step 4: 72 ° C. for 2 minutes; final extension: step 5: 72 for 5 minutes.
C .; Steps 2 to 4 are repeated for 30 cycles.

In the second stage of PCR, prepare the following reagents for each reaction: 10 × PCR buffer, 5 ml; first stage primer mix, 1 ml; dNTPs, 1 ml; MgC
l _2, 1ml; Taq DNA polymerase, 0.2 ml, distilled water 49 ml. The sample is added by carefully pipetting 1 ml from the first stage PCR reaction tube into the second stage reaction tube. The amplification reaction of this protocol is repeated as indicated above.

Next, gel analysis and interpretation of the PCR results are performed. 10 ml second stage PCR
The product is mixed with an appropriate amount of loading buffer and added to a sample well of a 1.2% agarose gel stained with ethidium bromide. A 100 bp DMA ladder is used as a size marker. Run the gel and then observe the results in a UV light box. (A
Mycoplasma species normally encountered as cell culture contaminants (according to the TCC Mycoplasma PCR detection kit) are the second stage PCs in the size range of 236-365 bp.
It should produce an R DNA amplicon. They are 426-219 bp
Generating two DNA amplicons of A. laidawii (positive control)
Producing a 323 bp DNA amplicon. pirum, and a negative control that should not contain any amplicons.

Example 18 Monocyte Depletion This procedure describes a method for depleting the amount of monocytes present in peripheral blood mononuclear cell (PBMC) products prior to cell culture. Equipment and materials include PBMC, Falcon Vented F in X-VIVO 15 containing 5% autologous human serum.
Includes lask, 175 cm ² , straight neck, 37 ° C. incubator, sterile gloves, and sterile individually wrapped pipette.

First, cells are counted using a Coulter Multisizer IIe as described above. The phenotype of the cells was then determined and CD3 / 4, CD3 in PBMC products
/ 8, CD19 and CD45 / 14 cell proportions are determined. Add 1 × 10 ⁸ cells of PBMC to each 175 cm ² Falcon flask. Cells should be in a volume of 20-40 ml. Place each Falcon flask in a 37 ° C. incubator containing 5% CO ₂ . Place each flask on its side to ensure optimal cell attachment to the plastic of the flask. Incubate for 2 hours at 37 ° C., 5% CO ₂ . The supernatant containing the desired cells from each flask is gently removed.
Monocytes can be observed macroscopically attached to the inner surface of the flask. The product phenotype is measured to determine the degree of monocyte depletion and the composition of the final PBMC product. Cells are counted using a Coulter Multisizer IIe as described above to determine recovery. Universal blood and fluid attention should be strictly enforced at all times.

All of the methods disclosed and claimed herein can be performed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described by way of preferred embodiments, without departing from the concept, spirit and scope of the invention,
It will be apparent to those skilled in the art that variations in the steps or sequence of steps of the methods described herein may be applied to the methods. More specifically, it is clear that certain agents, both chemically and physiologically, can be substituted for the agents described herein while achieving the same or similar results. There will be. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

The following references, to the extent that they provide supplemental to the exemplary method or other details described herein, are incorporated herein by reference.

[0375]

[Table 10]

[0376]

[Table 11]

[0377]

[Table 12]

[0378]

[Table 13]

[Brief description of the drawings]

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood with reference to one or more drawings, in combination with the detailed description of specific examples provided herein.

1A and 1B. Expansion of CD3 + 28 T cells from PMNC. 1 shows typical preclinical expansion of CD3 + 28 + T cells. FIG. 1A shows growth in X-VIVO15 with 5% autologous human serum, and FIG. 1B shows 100 U / ml interleukin-2 (Cetu).
s) shows growth in the same medium to which s) was added. In both cases, it can be seen that the method supports logarithmic expansion and expansion of CD3 + T cells, with CD4 + T cells expanding more dynamically than CD8 + T cells.

FIG. 2. Clinical CD3 + 28 T cell proliferation (patient SE8558-06). CD3 + 28T
The clinical proliferation of the cells is indicated. Similar results are obtained for preclinical scale growth, such as those shown in FIGS. 1A and 1B. Specifically, CD4 + T cells proliferate preferentially over CD8 + T cells.

FIG. 3. Immunophenotypic analysis of CD3 + 28 costimulatory T cell expansion cultures. Time point SID0, SID
4, SID9 and SID13 correspond to day 1, day 5, day 10 and day 14 of culture, respectively. The starting population of cells shown here is typical for these patients. Generally, 40-60% of the cells are monocytes (CD15 +) and 10-40
% Are T cells (CD3 +), 5-15% are B cells (CD19 +), 5%
-30% are CD4 + T cells (CD3 + CD4 +), 1-15% are CD8 +
(CD3 + CD8 +). By day 5 (SID4), almost all cells in culture are T cells and a few monocytes are persistently present. After this, all of the cells are CD4 + or CD8 + T cells. By day 14, all> 99% of the cells in culture are CD3 + T cells and 90% are CD4 +. B cells do not proliferate and are rapidly lost from culture.

FIG. 4. Recovery of lymph and bone marrow cell compartments in patients (AS8668-03) following dose-intensive therapy with CD34-detected PBPC support and CD3 + 28 costimulatory T cells. Blood cell counts were measured during intensive dose therapy and the recovery phase from CD34 selected PBPC support and CD3 + 28 co-stimulated T cell reinfusion. Co-stimulated T cells were re-infused on day 13 on the x-axis. Note that neutrophils recovered by day 15 (POLY + BAND COUNT) have remained relatively constant since then. In contrast, lymphocytes had an early phase of recovery beginning at day 20 and a steep second phase beginning at day 30. This second phase is absolute and relative lymphocytosis and is still maintained today. Of note, these cells are atypical, overwhelmingly CD8 + with an activated phenotype, demonstrating a monoclonal or oligoclonal Vβ repertoire usage (FIGS. 10A, 10B, 10C, 10C). 10D, 11A, 11B, 11C and 11D). Thus, this second phase correlates with a specific immune response that appears to be initiated and maintained by the reinjected CD3 + 28 costimulatory T cells.

FIG. 5. Differences in patients (AS 8558-03) following intensive dose chemotherapy with CD34 detected PBPC support and CD3 + 28 costimulatory T cells. Shown is the blood count difference for patient AS 8558-03 over the same period in FIG. The first of lymph recovery (
Note the relative lymphocytosis at wave 21-27) and wave 2 (day 30-72). Another interesting feature found in four evaluable patients is that eosinophils (days 21-30) precede the second wave of lymphocytosis. This is useful in predicting when a second wave of lymphocytosis will appear.

FIG. 6: Recovery of lymphoid and bone marrow cell compartments in patients (CR 8558-05) following intensive dose chemotherapy with CD34-detected PBPC support and CD3 + 28 costimulatory T cells. Intensive dose therapy and CD34 selective PBPC support and CD3 + 28 costimulatory T
Blood cell counts were measured during the recovery phase from cell reinfusion. Co-stimulated T cells were re-infused on day 13 on the x-axis. Neutrophils (POLY + BAND COUNT) recovered by day 14 and peaked on day 35 were nearly 1800
Had a level off at / μl. In contrast, lymphocytes had an early phase of recovery beginning at day 20 and a steep second phase beginning at day 29. Patient AS 8558
In contrast to -03 (FIG. 4), the second phase of this patient has a relatively, if not absolute, lymphocytosis. As with AS 8558-03, the patient's relative lymphocytosis is still maintained today. Note that AS 8558
As with -03, the lymphocytes of this patient are atypical, predominantly CD8 + with an activated phenotype, and monoclonal or oligoclonal Vβ
Show repertoire usage.

FIG. 7: Differences in patients (CR 8558-05) following intensive dose chemotherapy with CD34-detected PBPC support and CD3 + 28 costimulatory T cells. The difference in blood count of patient AS 8558-05 over the same period in FIG. 6 is shown. The first of lymph recovery (
Note the relative lymphocytosis in the second (day 20-29) and second (day 35-57) waves. Again, as shown in FIG. 5, eosinophils (days 21-30) predict a second wave of lymphocytosis.

FIG. 8. Recovery of lymph and bone marrow cell compartments in patients (SE / 8558-06) following intensive dose chemotherapy with CD34 selected PBPC support and CD3 + 28 costimulatory T cells. Blood cell counts were measured during the recovery phase from intensive therapy with CD34 selected PBPC support and CD3 + 28 co-stimulated T cell re-infusion. Co-stimulated T cells were re-infused on day 13 on the x-axis. Neutrophils (POLY + BAND COUNT) recovered by day 14 and peaked on day 32 had nearly 4000 /
It had level off in μl. Previous patient AS / 8558-03 and CR
In contrast to / 8558-05 (FIGS. 4, 6), the lymphocytes remained relatively constant.

FIG. 9: Differences in patient SE / 8558-06 following intensive dose chemotherapy with CD34 selected PBPC support and CD3 + 28 costimulatory T cells. Patient S over the same period as in FIG.
The blood count difference for E / 8558-06 is shown. Although all lymphocytes appear relatively constant as plotted in FIG. 5, the difference (days 48-64) preceded by eosinophils (days 22-32), as shown in FIGS. There is an increase in% lymphocytes.

FIGS. 10A, 10B, 10C and 10D Analysis of Vβ20 usage in CD3 + 28 co-stimulated T cells 14 days after culture (day of reinfusion) and in peripheral blood T cells 30 days after reinfusion. VβT cell antigen receptor usage was measured by PCR. 10A and 10B show one of the stimuli.
4 days later (at the time of reinfusion into patient AS 8558-03) CD3 + 28 costimulatory T
2 shows Vβ usage for cells. Analysis shows the expected Gaussian distribution for the polyclonal population of T cells. FIGS. 10C and 10D show Vβ20 usage on patient AS 8558-03 peripheral blood T cells collected 30 days after CD3 + 28 costimulatory T cell re-infusion. In contrast to FIGS. 10A and 10B, FIG.
C and FIG. 10D show a single peak representing a monoclonal population of T cells.

FIGS. 11A, 11B, 11C and 11D Analysis of Vβ18 usage in CD3 + 28 co-stimulated T cells 14 days after culture (day of reinfusion) and in peripheral blood T cells 30 days after reinfusion. VβT cell antigen receptor usage was measured by PCR. 11A and 11B show one of the stimuli.
4 days later (at the time of reinfusion into patient AS 8558-03) CD3 + 28 costimulatory T
Shows Vβ18 usage for cells. Analysis shows the expected Gaussian distribution for the polyclonal population of T cells. FIGS. 11C and 11D show CD3 + 28.
Patient AS 8558-03 peripheral blood T collected 30 days after costimulatory T cell reinfusion
Shows Vβ18 usage for cells. In contrast to FIGS. 11A and 11B, FIGS. 11C and 11D show two peaks representing an oligoclonal population of T cells.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 39/395 A61K 45:00) (A61K 35/14 C12N 5/00 E 45:00) A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, U, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR , LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventors Lebowitz, David N. Green Street, Philadelphia, 19130, Pennsylvania, USA 2146 (72) Inventors June, Carl 7850 F-term (Reference) 4850, AA91X AA93X AC20 BC50 BD50 CA44 4C084 AA02 BA04 CA17 MA66 ZB262 ZB272 4C085 AA40 BA99 BB33 BB34 BB35 BB36 BB37 BB50 GG10 4C087 AA01 AA02 BB34 CA13 DA19 NA14 ZB26 ZB27 4H045 AA11 BA09 CA42 DA76 DA86 EA28 FA74

Claims (34)

[Claims] 1. a) obtaining a population of peripheral blood mononuclear cells from a mammal; b) obtaining peripheral blood outside the mammal to obtain a population of T cells that are activated outside the mammal. Activating said population of mononuclear cells; c) administering said population of T cells to said mammal, thereby inducing an immune response;
A method of treating a mammal having an immune responsive cancer, comprising treating a mammal having an immune responsive cancer. 2. The method of claim 1, wherein the mammal is a lymphoma, multiple myeloma, kidney cancer, ovarian cancer, prostate cancer, low grade lymphoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), acute myeloid leukemia ( 2. The method according to claim 1, wherein the patient has AML), sarcoma, lung cancer, opportunistic malignancy or melanoma. 3. The method of claim 2, wherein said mammal has lymphoma. 4. The method of claim 3, wherein said mammal has non-Hodgkin's lymphoma. 5. The method of claim 1, wherein said activating said population of peripheral blood mononuclear cells comprises contacting said population of peripheral blood mononuclear cells with at least a first antibody and at least a second antibody. 6. The method of claim 5, wherein said at least first antibody and said at least second antibody are linked to magnetic beads. 7. The method of claim 5, wherein said at least first antibody and said at least second antibody are distinct antibodies. 8. The method according to claim 5, wherein said at least first antibody is an anti-CD3 antibody. 9. The method according to claim 8, wherein said at least first antibody is an OKT3 antibody. 10. The method according to claim 5, wherein said at least second antibody is an anti-CD28 antibody. 11. The method according to claim 5, wherein said at least first antibody is an anti-CD3 antibody and said at least second antibody is an anti-CD28 antibody. 12. The method of claim 1, wherein said population of T cells comprises CD4 + T cells. 13. The method of claim 11, wherein said population of T cells comprises CD4 + T cells. 14. The method of claim 13, wherein said population of T cells is CD4 + T cells. 15. The method of claim 1, wherein said population of T cells comprises CD4 + T cells and CD8 + T cells. 16. The method of claim 1, wherein said population of T cells has not been activated upon administration to said mammal. 17. The method of claim 1, wherein said immune response comprises production of T cells in said mammal. 18. The method of claim 17, wherein said immune response comprises production of CD8 + T cells in said mammal. 19. The method according to claim 19, wherein said population of T cells administered to said mammal is predominantly CD4.
2. The method of claim 1, wherein the immune response comprises the production of CD8 + T cells in the mammal. 20. The method of claim 19, wherein said population of T cells is predominantly CD4 + T cells, and wherein said immune response comprises production of CD8 + T cells directed to said immune responsive cancer in said mammal. 21. The method of claim 1, wherein said mammal is a human. 22. The method of claim 1, further comprising immunosuppressing said mammal prior to administering said T cells to said mammal. 23. The method of claim 22, wherein said immunosuppression comprises providing chemotherapy, azathioprine, cyclosporin A, FK506, a purine analog, an alkylating agent, or an anti-T cell monoclonal antibody to the mammal. 24. The method of claim 1, further comprising providing a stem cell transplant to said mammal. 25. The method of claim 24, wherein said stem cells comprise CD34 + cells. 26. Peripheral blood outside the mammal to obtain a population of peripheral blood mononuclear cells from the mammal, and b) obtaining a population of T cells that are activated outside the mammal. Activating said population of mononuclear cells; c) administering said population of T cells to said mammal, thereby inducing an immune response;
A method of treating a mammal having non-Hodgkin's lymphoma, comprising treating a mammal having non-Hodgkin's lymphoma. 27. The activation of the population of peripheral blood mononuclear cells comprises contacting the population of peripheral blood mononuclear cells with at least a first anti-CD3 antibody and at least a first anti-CD28 antibody. 26. The method of claim 26. 28. The method of claim 27, wherein said at least a first anti-CD3 antibody and said at least a first anti-CD28 antibody are linked to magnetic beads. 29. The T cell population is predominantly CD4 + T cells.
6. The method according to 6. 30. The method according to claim 26, wherein said mammal is a human. 31. A method for obtaining a population of peripheral blood mononuclear cells from a mammal, and b) for obtaining a population of T cells that are activated in vitro in a mammal. Activating said population of mononuclear cells; c) administering said population of T cells to said mammal, thereby treating a mammal with T cells having a defective cytokine profile. A method for treating a mammal having T cells. 32. The method of claim 31, wherein said activating said population of peripheral blood mononuclear cells comprises co-stimulating with a population of anti-CD3 antibodies and a population of anti-CD28 antibodies. 33. The method of claim 32, wherein said population of anti-CD3 antibodies and said population of anti-CD28 antibodies are linked to magnetic beads. 34. A method for obtaining a population of peripheral blood mononuclear cells from a mammal, and b) for obtaining a population of T cells that are activated in vitro in a mammal. Activating said population of mononuclear cells; c) administering said population of T cells to said mammal, thereby inducing lymphocytosis in said mammal; How to guide.