EP1030674A1 - Verfahren und behandlung von tumoren und tumorzellen mit ex vivo aktivierten t-zellen - Google Patents

Verfahren und behandlung von tumoren und tumorzellen mit ex vivo aktivierten t-zellen

Info

Publication number
EP1030674A1
EP1030674A1 EP98957775A EP98957775A EP1030674A1 EP 1030674 A1 EP1030674 A1 EP 1030674A1 EP 98957775 A EP98957775 A EP 98957775A EP 98957775 A EP98957775 A EP 98957775A EP 1030674 A1 EP1030674 A1 EP 1030674A1
Authority
EP
European Patent Office
Prior art keywords
cells
mammal
population
antibody
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98957775A
Other languages
English (en)
French (fr)
Inventor
David N. Liebowitz
Carl June
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Arch Development Corp
Henry M Jackson Foundation for Advancedment of Military Medicine Inc
Original Assignee
Arch Development Corp
Henry M Jackson Foundation for Advancedment of Military Medicine Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Arch Development Corp, Henry M Jackson Foundation for Advancedment of Military Medicine Inc filed Critical Arch Development Corp
Publication of EP1030674A1 publication Critical patent/EP1030674A1/de
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/05—Immunological preparations stimulating the reticulo-endothelial system, e.g. against cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex

Definitions

  • the present invention relates generally to the fields of immunotherapy and oncology. More particularly, it concerns methods of inducing activated T cells ex vivo and methods of treating tumors and tumor cells with activated T cells in order to stimulate an immune response.
  • T helper (T H ) cells play a critical role in regulating immune responses and may be involved in regulating immune responses against tumor cells.
  • T H cells can be divided into two major subclasses, T H 1 and T H 2, based on their pattern of cytokine expression and the immune responses they mediate.
  • T H 1 cells produce interleukin-2 (IL-2), interferon-gamma (IFN- ⁇ ), tumor necrosis factor-alpha (TNF- ⁇ ) and lymphotoxin (LT), but not IL-4 or IL-5.
  • T H 2 cells produce IL-4 and IL-5, but not IL-2, IFN- ⁇ , TNF- ⁇ or LT.
  • T H 1 cells regulate the cellular arm of the immune response, activating macrophages and monocytes and delayed type hypersensitivity, while T H 2 cells activate the humoral arm of the immune system, providing B cell help and stimulating IgG and IgE production.
  • T H 0 cells can function to activate both arms of the immune response.
  • IFN- ⁇ , IFN- ⁇ , IL-4 and IL-12 are capable of differentially regulating T H cell subsets. IFN- ⁇ , IFN- ⁇ , and IL-12 induce the differentiation of naive T H cells into
  • T H 1 cells while IL-4 is the only cytokine known to promote the differentiation of naive cells into T H 2 cells. Furthermore, the responsiveness of differentiated T H cells to these cytokines also varies. For instance, IFN- ⁇ inhibits the proliferation of T H 2 but not T H 1 cells, while T H 1, but not T H 2 cells retain IL-12 responsiveness.
  • T H 1 responses play a key role.
  • the ability of mice to reject the P815 tumor cell line has been shown to be largely due to the presence of CD4+ cells producing T H 1 cytokines (Fallarino et al, 1996). In these studies, mice who failed to generate a high IFN- ⁇ T H 1 response failed to reject tumors, while those that did generate such a response rejected their tumors more efficiently. IL-12 was required for the generation of the T H 1 response.
  • T H 1 responses in anti-tumor immunity come from studies in mice with established experimental sarcomas (Zitvogel et al, 1995). Established tumors were injected with fibroblasts engineered to secrete IL-12 or with control fibroblasts. The IL-12 secreting fibroblasts efficiently elicited a CD4 T H 1 anti-tumor response that resulted in successful control of the tumor.
  • the present invention relates generally to methods for the modulation of an immune response involving the treatment of a mammal with a T cell, or population of T cells.
  • the invention relates generally to methods for the induction of an immune response involving the treatment of a mammal with a T cell, or population of T cells.
  • the invention provides a solution to the previously-discussed problems of treating various diseases, especially those diseases that are of a malignant nature.
  • the methods taught herein allow one to induce an immune response directed against a malignant disease.
  • induction of an immune response denotes any measurable increase in a indicator of immune responses. Induction of an immune response includes both initiation of an immune response in a mammal that is not, at the time treatment begins, mounting an immune response. Likewise, increasing or potentiating an already ongoing immune response is induction of an immune response.
  • the invention also provides methods of treating diseases of a non- malignant nature, by modulating a mammal's immune response. "Modulation of an immune response” is defined as any measurable change in an indicator of immune responses, in response to T cell administration.
  • the mammal will have a disease, such as a malignancy, the treatment of which is expected to benefit from the induction of an immune response.
  • the mammal is a human.
  • the present methods are anticipated to be of broad application to immunoresponsive and systemic cancers.
  • the present invention provides methods of treating a mammal with an immunoresponsive cancer, comprising obtaining a population of peripheral blood mononuclear cells from the mammal, activating the population of peripheral blood mononuclear cells outside of the mammal, or ex vivo, to obtain a population of T cells that have been activated outside of the mammal, or ex vivo, and administering a therapeutically effective amount of the population of T cells to the mammal, thereby inducing an immune response and treating a mammal with an immunoresponsive cancer.
  • the "therapeutically effective amounts" for use in the invention are amounts of activated T cells effective to specifically slow progression of a tumor, to induce necrosis in at least a portion of a tumor and/or to induce tumor regression or remission upon administration to selected mammals or patients.
  • Immunoresponsive cancers contemplated for treatment using the methods of the present invention include, but are not limited to, lymphomas, such as non- Hodgkin's lymphoma, multiple myeloma, renal cancer, ovarian cancer, prostate cancer, low-grade lymphoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), sarcomas, such as osteosarcomas, lung cancer, opportunistic malignancies, such as Kaposi's sarcoma and other virally related cancers such as cervical cancer and rectal cancer, or melanoma.
  • lymphomas such as non- Hodgkin's lymphoma, multiple myeloma, renal cancer, ovarian cancer, prostate cancer, low-grade lymphoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), acute myelogenous leuk
  • any cancer that responds to immunologic intervention wherein the manipulation of the immune system yields a response to the disease, is contemplated to respond to the treatments provided herein.
  • a number of different techniques for determination of the immunologic responsiveness of a particular cancer are available and understood by those of skill in the art.
  • leukemias and lymphomas have been shown to be immunologically responsive through the use of allogenic transplantation
  • melanoma has been determined to be immunologically responsive to LAK and TIL cell technology, as well as interferons
  • renal cancer has been shown to be immunologically responsive to interferons and IL-2
  • ovarian cancer has been shown to be immunologically responsive to IL-2
  • prostate cancer has been determined to be immunologically responsive using the CTLA-4 mouse model.
  • the activation of the population of peripheral blood mononuclear cells comprises contacting the population of peripheral blood mononuclear cells with at least a first antibody and at least a second antibody.
  • the at least a first antibody and the at least a second antibody are linked to a particle, such as a bead.
  • Beads contemplated for use can be fabricated from a variety of different materials, for example a plastic bead such as polystyrene, or a magnetic bead.
  • the antibodies are immobilized on structures (i.e., beads), such that discreet points of contact are made between multiple beads and each individual T cell that is the target of costimulation.
  • the antibodies thus immobilized costimulate individual T cells from multiple contact points.
  • the optimal number of contact points may be greater than or equal to 3.
  • other forms of activation such as the use of superantigens, or the use of a protein kinase C activator, such as a phorbol ester, in combination with a calcium ionophore, such as ionomycin, are contemplated for use.
  • antibody naked antibody and unconjugated antibody
  • immunologic binding agent such as polyclonal and monoclonal IgG, IgM, IgA, IgD and IgE antibodies.
  • IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting.
  • MAbs monoclonal antibodies
  • the invention thus provides monoclonal antibodies of the murine, human, monkey, rat, hamster, rabbit and even frog or chicken origin.
  • the immunologic binding reagents encompassed by the term "antibody” extend to all naked and unconjugated antibodies from all species, and antigen binding fragments thereof, including dimeric, trimeric and multimeric antibodies; bispecific antibodies; chimeric antibodies; human and humanized antibodies; recombinant and engineered antibodies, and fragments thereof.
  • antibody is thus used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab,
  • F(ab') 2 single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like.
  • the antibodies employed will be “humanized” or human antibodies.
  • “Humanized” antibodies are generally chimeric monoclonal antibodies from mouse, rat, or other non-human species, bearing human constant and/or variable region domains ("part-human chimeric antibodies").
  • humanized monoclonal antibodies for use in the present invention will be chimeric antibodies wherein at least a first antigen binding region, or complementarity determining region (CDR), of a mouse, rat or other non-human monoclonal antibody is operatively attached to, or "grafted” onto, a human antibody constant region or "framework”.
  • CDR complementarity determining region
  • “Humanized” monoclonal antibodies for use herein may also be monoclonal antibodies from non-human species wherein one or more selected amino acids have been exchanged for amino acids more commonly observed in human antibodies. This can be readily achieved through the use of routine recombinant technology, particularly site-specific mutagenesis.
  • the at least a first antibody and the at least a second antibody are distinct antibodies.
  • the at least a first antibody is an anti-CD3 antibody, such as the OKT3 or G19-4 antibody.
  • the at least a second antibody is an anti-CD28 antibody, such as the 9.3, KOLT-2, 15E8, 248.23.2 or EX5.3D10 antibody.
  • the at least a first antibody is an anti-CD3 antibody and the at least a second antibody is an anti-CD28 antibody.
  • an anti-CD2 antibody can be utilized in combination with an anti-CD28 antibody
  • an anti-CD3 antibody can be utilized in combination with IL-2 or IL-15
  • an anti-B7-l (CD80) or anti-B7-2 (CD86) antibody can be used in conjunction with an anti-CD3 antibody.
  • the population of T cells comprises CD4+ T cells.
  • the population of T cells comprises predominantly CD4+ T cells, while in other embodiments, the population of T cells are only CD4+ T cells.
  • the population of T cells comprises CD4+ T cells and CD8+ T cells.
  • the population of T cells are not activated upon administration to the mammal.
  • the T cell is in a quiescent or non-activated state upon administration to the mammal.
  • the CD4+ and/or the CD8+ T cells can be expanded or derived from a population of CD4+ and CD8+ T cells that have been expanded by treatment with antibodies.
  • the expansion by treatment with antibodies has occurred ex vivo.
  • the expansion by treatment with antibodies involves costimulation with two distinct populations of antibodies.
  • one of the distinct populations of antibodies is a population of anti-CD3 antibodies, for example OKT3.
  • one of the distinct populations of antibodies is a population of anti-CD28 antibodies.
  • the expansion involves costimulation with a population of anti-CD3 antibodies and a population of anti-CD28 antibodies, via methods described elsewhere in this application.
  • the immune response comprises the production of T cells in the mammal.
  • the immune response comprises the production of CD8+ T cells in the mammal.
  • the population of T cells administered to the mammal are predominantly or totally CD4+ T cells, and the immune response comprises the production of CD8+ T cells in the mammal.
  • the population of T cells are predominantly CD4+ T cells, and the immune response comprises the production of CD8+ T cells directed against the immunoresponsive cancer in the mammal.
  • CD4+ T cells may be used to treat patients with other disorders of the immune system, for example, those patients with hyper- immune states or immunodeficiencies.
  • the invention involves modulating the immune response that is mediated by CD4+ cells, in some cases by turning off or turning down an immune response or by reorganizing the immune response.
  • CD4+ T cells may be employed to treat diseases such as diabetes, lupus, or rheumatoid arthritis.
  • T cells may be employed to prevent organ rejection after transplantation. In cases of implant rejection prevention, there will often be benefits derived from T cell treatment prior to the transplantation procedure.
  • the T cells will be administered in combination with other treatment modalities, for example, chemotherapy, antibiotic therapy, antibody therapy, radiation therapy, transplants, etc.
  • the patient may be given a stem cell transplant in conjunction with the T cell treatment.
  • administration of CD4+ T cells may serve to correct inappropriate activation of the immune system.
  • the chemotherapy associated with the transplant procedure immunosuppresses the patient prior to the CD4+ T cell administration.
  • the methods further comprise immunosuppression of the mammal prior to administration of the population of the T cells to the mammal.
  • immunosuppression Any of a number of techniques for immunosuppression are contemplated for use in the present methods and are known to those of skill in the art, including, but not limited to, administration of chemotherapy, radiation therapy, azathioprine, cyclophosphamide, rapamycin, corticosteroids, such as prednisone, cyclosporine A, FK506, purine analogs and related inhibitors, such as fluderilzene, 2- chloro-deoxyadenosine, mercaptopurine (6-mercaptopurine), thioguanine (6-thioguanine) and pentostatin (2-deoxycoformycin), alkylating agents, for example nitrogen mustards such as mechlorethamine (HN 2 ), cyclophosphamide, ifosfamide, melphalan (L-sarcolys
  • the methods further comprise administering a stem cell transplant to the mammal.
  • the stem cells comprise CD34+ cells.
  • the invention also encompasses a combination therapy comprising the co-administration of CD4+ T cells with cytokines or other biological compounds that can modulate or specify the phenotype of the CD4+ T cells or the CD8+ T cells. For example treatment with IL-2, IL-12, IL-4, IL-10, or IL-6 can be used to modify function of cell product produced.
  • the present invention also provides methods of treating a mammal with non- Hodgkin's lymphoma, comprising 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 that have been activated outside of the mammal, and administering the population of T cells to the mammal, thereby inducing an immune response and treating a mammal with non- Hodgkin's lymphoma.
  • 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.
  • the at least a first anti-CD3 antibody and the at least a first anti-CD28 antibody are linked to a magnetic bead.
  • the population of T cells comprises predominantly CD4+ T cells.
  • the present invention further provides methods of treating a mammal with T cells that have a defective cytokine profile, comprising 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 that have been activated outside of the mammal, and administering the population of T cells to the mammal, thereby treating a mammal with T cells that have a defective cytokine profile.
  • the activation of the population of peripheral blood mononuclear cells comprises costimulation with a population of anti-CD3 antibodies and a population of anti-CD28 antibodies.
  • methods of inducing lymphocytosis in a mammal comprising 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 that have been activated outside of the mammal, and administering the population of T cells to the mammal, thereby inducing lymphocytosis in the mammal.
  • FIG. 1A and FIG. IB CD3+28 T cell expansion from PBMCs. A typical pre-clinical expansion of CD3+28+ T cells is shown.
  • FIG. 1A shows an expansion in X-VIVO 15® with 5% autologous human serum
  • FIG. IB is an expansion in the same medium with 100 U/ml interleukin-2 (Cetus) added.
  • the method supports the logarithmic growth and expansion of CD3+ T cells, and that CD4+ T cells kinetically expand greater than do CD8+ T cells.
  • FIG. 2 Clinical CD3+28 T cell expansion (Patient SE 8558-06). A clinical expansion of CD3+28 T cells is shown. Similar results are obtained to the preclinical scale expansions such as that shown in FIG. 1A and FIG. IB. Specifically, CD4+ T cells are expanded preferentially over CD8+ T cells.
  • FIG. 3 Immunophenotypic analysis of CD3+28 costimulated T cell expansion culture.
  • the time points SI DO, S1D4, S1D9 and S1D13 correspond to days 1, 5, 10 and 14 of culture, respectively.
  • the starting population of cells shown here is typical for these patients.
  • 40-60% of the cells are monocytes (CD 14+)
  • 10-40% are T cells (CD3+)
  • 5-15% are B cells (CD 19+)
  • 5-30% are CD4+ T cells (CD3+CD4+)
  • 1-15% CD8+ T cells
  • S1D4 nearly all of the cells in the culture are T cells, with a few monocytes persisting.
  • all of the cells are either CD4+ or CD8+ T cells.
  • >99% of all of the cells in the culture are CD3+ T cells and 90% are CD4+.
  • B cells are not expanded and are rapidly lost from the cultures.
  • FIG. 4 Recovery of lymphoid and myeloid cell compartments of patient (AS 8558-03) following dose-intensive chemotherapy with CD34-detected PBPC support and CD3+28 costimulated T cells. Blood cell counts were measured during the recovery phase from dose-intensive therapy and CD34-selected PBPC support and CD3+28 costimulated T cell reinfusion. The costimulated T cells were reinfused on day 13 on the x-axis. Note that neutrophils (POLY + BAND COUNT) recovered by day 15 and have remained relatively constant since that time. In contrast, the lymphocytes had an initial phase of recovery beginning at day 20 and then a steep second phase beginning at day 30. This second phase is an absolute and relative lymphocytosis, which is currently still sustained.
  • neutrophils POLY + BAND COUNT
  • these cells are atypical, predominantly CD8+ with an activated phenotype and show monoclonal or oligoclonal V ⁇ repertoire usage (FIG. 10A, FIG. 10B, FIG. 10C, FIG. 10D, FIG. 11A, FIG. 1 IB, FIG. 1 IC and FIG. 1 ID).
  • this second phase correlates with a specific immune response that is likely initiated and maintained by the reinfused CD3+28 costimulated T cells.
  • FIG. 5 Differential of Patient (AS 8558-03) following dose-intensive chemotherapy with CD34-detected PBPC support and CD3+28 costimulated T cells. Shown is the differential of patient AS 8558-03's blood counts over the same period as FIG. 4. Note the relative lymphocytosis with the first (days 21-27) and second (days 30-72) waves of lymphocytes recovery. Another interesting feature found in all 4 evaluable patients is that eosinophilia (days 21-30) precedes the second wave of lymphocytosis. This is useful to predict when this second wave of lymphocytes will appear.
  • this patient's second phase has a relative, although not absolute lymphocytosis. Like AS 8558-03, this patient's relative lymphocytosis is currently still sustained. Notably, as with AS 8558-03, this patent's lymphocytes are atypical, predominantly CD8+ with an activated phenotype and show monoclonal or oligoclonal V ⁇ repertoire usage.
  • FIG. 7 Differential of Patient (CR 8558-05) following dose-intensive chemotherapy with CDE34-detected PBPC support and CD3+28 costimulated T cells. Shown is the differential of patient AS 8558-05's blood counts over the same period as FIG. 6. Note the relative lymphocytosis with the first (days 20-29) and second (days 35-57) waves of lymphocytes recovery. Again, as noted in FIG. 5, eosinophilia (days 21-30) predicts the second wave of lymphocytosis.
  • FIG. 8 Recovery of lymphoid and myeloid cell compartments of patient (SE/8558-06) following dose-intensive chemotherapy with CD34-selected PBPC support and CD3+28 costimulated T cells. Blood cell counts were measured during the recovery phase from dose-intensive therapy with CD34-selected PBPC support and CD3+28 costimulated T cell reinfusion. The costimulated T cells were reinfused on day 13. The neutrophils (POLY + BAND COUNT) recovered by day 14 and, after peaking at day 32, leveled off at approximately 4000/ml. In contrast to the previous patients AS/8558-03 and CR/8558-05 (FIG. 4, FIG. 6), the lymphocytes remained relatively constant.
  • FIG. 9 Differential of Patient SE/8558-06 following dose-intensive chemotherapy with CD34-seIected PBPC support and CD3+28 costimulated T cells. Shown is the differential of patient SE/8558-06's blood counts over the same period as FIG. 8. Note that although the total lymphocytes appear relatively constant as plotted in FIG. 5, there is a gradual increase in the % lymphocytes in the differential (days 48-64) which, as noted in FIG. 5 and FIG. 7 is preceded by eosinophilia (days 22-32).
  • FIG. 10A, FIG. 10B, FIG. IOC and FIG. 10D Analysis of V ⁇ 20 usage in CD3+28 costimulated T cells after 14 days of culture (day of reinfusion) and in peripheral blood T cells 30 days after reinfusion.
  • V ⁇ T cell antigen receptor usage was determined by PCR.
  • FIG. 1 OA and FIG. 1 OB show the V ⁇ 20 usage for the CD3+ 28 costimulated T cells after 14 days of stimulation (at the time of reinfusion into patient AS 8558-03).
  • the analysis shows the expected gaussian distribution for a polyclonal population of T cells.
  • FIG. IOC and FIG. 10D show the V ⁇ 20 usage for patient AS 8558-03's peripheral blood T cells that were collected 30 days after CD3+28 costimulated T cell reinfusion.
  • FIG. 10C and FIG. 10D show a single peak representing a monoclonal population of T cells.
  • FIG. 11A, FIG. 11B, FIG. 11C and FIG. 11D Analysis of V ⁇ l8 usage in CD3+28 costimulated T cells after 14 days of culture (day of reinfusion) and in peripheral blood T cells 30 days after reinfusion. V ⁇ T cell antigen receptor usage was determined by PCR.
  • FIG. 11A and FIG. 1 IB show the V ⁇ l8 usage for the CD3+ 28 costimulated T cells after 14 days of stimulation (at the time of reinfusion into patient AS 8558-03). The analysis shows the expected gaussian distribution for a polyclonal population of T cells.
  • FIG. 11D show the V ⁇ l8 usage for patient AS 8558-03's peripheral blood T cells that were collected 30 days after CD3+28 costimulated T cell reinfusion.
  • FIG. 11C and FIG. 1 ID show two peaks representing an oligoclonal population of T cells.
  • Immunoresponsive cancers are defined herein as any cancer that responds to immunologic intervention, wherein the manipulation of the immune system yields a response to the disease. In general, cancers are shown to be immunoresponsive using any of a number of different techniques.
  • leukemias and lymphomas have been shown to be immunologically responsive through the use of allogenic transplantation
  • melanoma has been demonstrated to be immunologically responsive to LAK and TIL cell technology, as well as interferons
  • renal cancer has been determined to be immunologically responsive to interferons and IL-2
  • ovarian cancer has been shown to be immunologically responsive to IL-2
  • prostate cancer has been determined to be immunologically responsive using the CTLA-4 mouse model (see, e.g., Cancer principles and practice of oncology. Lippicott-Raven, Philadelphia, 1997, incorporated herein by reference).
  • An exemplary use of this therapy is as an adjunct therapy in patients undergoing dose-intensive therapy for relapsed non-Hodgkin's lymphoma (NHL).
  • NHL non-Hodgkin's lymphoma
  • the anti-tumor activity of these expanded activated cells is measured, and presented herein.
  • the T helper subset profile was examined in patients at the time they initially present with NHL. In vitro cell culture studies to identify cytokines which modulate T helper responses in these patients was also performed.
  • a novel system is used to drive the ex vivo expansion of CD4+ and CD8+ T cells.
  • Previous T cell culturing systems i.e., LAK or TIL cell technology
  • This CD3+28 costimulation system is the first available method to allow the large scale expansion and activation of polyclonal human CD4+ T cells ex vivo, along with CD8+ T cells.
  • the amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization.
  • a variety of routes can be used to administer the immunogen; subcutaneous, intramuscular, intradermal, intravenous, intraperitoneal and intrasplenic.
  • the production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired titer level is obtained, the immunized animal can be bled and the serum isolated and stored. The animal can also be used to generate monoclonal antibodies.
  • the immunogenicity of a particular composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants.
  • adjuvants include complete Freund's adjuvant, a nonspecific stimulator of the immune response containing killed Mycobacterium tuberculosis; incomplete Freund's adjuvant; and aluminum hydroxide adjuvant.
  • exemplary carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA).
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
  • MAbs monoclonal antibodies
  • the most standard monoclonal antibody generation techniques generally begin along the same lines as those for preparing polyclonal antibodies (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference).
  • a polyclonal antibody response is initiated by immunizing an animal with an immunogenic composition and, when a desired titer level is obtained, the immunized animal can be used to generate MAbs.
  • MAbs may be readily prepared through use of well-known techniques, such as those exemplified in US. Patent 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with the selected immunogen composition. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep and frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, pp. 60-61 ; incorporated herein by reference), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
  • somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol.
  • B cells B lymphocytes
  • These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible.
  • a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe.
  • the antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized.
  • Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
  • any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984; each incorporated herein by reference).
  • the immunized animal is a mouse
  • rats one may use R210.RCY3, Y3-Ag 1.2.3, IR983F, 4B210 or one of the above listed mouse cell lines; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6, are all useful in connection with human cell fusions.
  • Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 4:1 proportion, though the proportion may vary from about 20:1 to about 1 :1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes.
  • Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976; each incorporated herein by reference), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977; incorporated herein by reference).
  • PEG polyethylene glycol
  • the use of electrically induced fusion methods is also appropriate (Goding pp.
  • Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10 " to 1 x 10 " .
  • the selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.
  • agents are aminopterin, methotrexate, and azaserine.
  • Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis.
  • aminopterin or methotrexate the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
  • HAT medium hypoxanthine and thymidine as a source of nucleotides
  • the preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium.
  • the myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.
  • HPRT hypoxanthine phosphoribosyl transferase
  • the B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells.
  • This culturing provides a population of hybridomas from which specific hybridomas are selected.
  • selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity.
  • the assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
  • the selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide MAbs.
  • the cell lines may be exploited for MAb production in two basic ways.
  • a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion.
  • the injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid.
  • the body fluids of the animal such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration.
  • the individual cell lines could also be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.
  • MAbs produced by either means will generally be further purified, e.g., using filtration, centrifugation and various chromatographic methods, such as HPLC or affinity chromatography, all of which purification techniques are well known to those of skill in the art. These purification techniques each involve fractionation to separate the desired antibody from other components of a mixture. Analytical methods particularly suited to the preparation of antibodies include, for example, protein A- Sepharose and/or protein G-Sepharose chromatography.
  • combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the spleen of the immunized animal, and phagemids expressing appropriate antibodies are selected by panning using cells expressing the antigen and control cells.
  • the advantages of this approach over conventional hybridoma techniques are that approximately 10 times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination, which further increases the percentage of appropriate antibodies generated.
  • the bacteriophage lambda vector involves the cloning of heavy and light chain populations of DNA sequences into separate starting vectors. The vectors are subsequently combined randomly to form a single vector that directs the co-expression of heavy and light chains to form antibody fragments.
  • the heavy and light chain DNA sequences are obtained by amplification, preferably by PCRTM or a related amplification technique, of mRNA isolated from spleen cells (or hybridomas thereof) from an animal that has been immunized with a selected antigen.
  • the heavy and light chain sequences are typically amplified using primers that incorporate restriction sites into the ends of the amplified DNA segment to facilitate cloning of the heavy and light chain segments into the starting vectors.
  • filamentous phage display vectors such as M13, fl or fd. These filamentous phage display vectors, referred to as "phagemids", yield large libraries of monoclonal antibodies having diverse and novel immunospecificities.
  • the technology uses a filamentous phage coat protein membrane anchor domain as a means for linking gene- product and gene during the assembly stage of filamentous phage replication, and has been used for the cloning and expression of antibodies from combinatorial libraries (Kang et al., 1991 ; Barbas et al., 1991 ; each incorporated herein by reference).
  • Patent 5,658,727 incorporated herein by reference.
  • the method provides a system for the simultaneous cloning and screening of pre-selected ligand-binding specificities from antibody gene repertoires using a single vector system. Screening of isolated members of the library for a pre-selected ligand- binding capacity allows the correlation of the binding capacity of an expressed antibody molecule with a convenient means to isolate the gene that encodes the member from the library.
  • Linkage of expression and screening is accomplished by the combination of targeting of a fusion polypeptide into the periplasm of a bacterial cell to allow assembly of a functional antibody, and the targeting of a fusion polypeptide onto the coat of a filamentous phage particle during phage assembly to allow for convenient screening of the library member of interest.
  • Periplasmic targeting is provided by the presence of a secretion signal domain in a fusion polypeptide.
  • Targeting to a phage particle is provided by the presence of a filamentous phage coat protein membrane anchor domain (i.e., a cpIII- or cpVIII-derived membrane anchor domain) in a fusion polypeptide.
  • the diversity of a filamentous phage-based combinatorial antibody library can be increased by shuffling of the heavy and light chain genes, by altering one or more of the complementarity determining regions of the cloned heavy chain genes of the library, or by introducing random mutations into the library by error-prone polymerase chain reactions. Additional methods for screening phagemid libraries are described in U.S. Patent No. 5,580,717; 5,427,908; 5,403,484; and 5,223,409, each incorporated herein by reference.
  • Another method for the screening of large combinatorial antibody libraries has been developed, utilizing expression of populations of diverse heavy and light chain sequences on the surface of a filamentous bacteriophage, such as Ml 3, fl or fd (U.S. Patent No. 5,698,426; incorporated herein by reference).
  • Two populations of diverse heavy (He) and light (Lc) chain sequences are synthesized by polymerase chain reaction (PCRTM). These populations are cloned into separate M13-based vector containing elements necessary for expression.
  • the heavy chain vector contains a gene VIII (gVIII) coat protein sequence so that translation of the heavy chain sequences produces gVIII-Hc fusion proteins.
  • the populations of two vectors are randomly combined such that only the vector portions containing the He and Lc sequences are joined into a single circular vector.
  • the combined vector directs the co-expression of both He and Lc sequences for assembly of the two polypeptides and surface expression on Ml 3 (U.S. Patent No. 5,698,426; incorporated herein by reference).
  • the combining step randomly brings together different He and Lc encoding sequences within two diverse populations into a single vector.
  • the vector sequences donated from each independent vector are necessary for production of viable phage.
  • the pseudo gVIII sequences are contained in only one of the two starting vectors, co-expression of functional antibody fragments as Lc associated gVIII-Hc fusion proteins cannot be accomplished on the phage surface until the vector sequences are linked in the single vector.
  • the surface expression library is screened for specific Fab fragments that bind preselected molecules by standard affinity isolation procedures. Such methods include, for example, panning (Parmley and Smith, 1988; incorporated herein by reference), affinity chromatography and solid phase blotting procedures. Panning is preferred, because high titers of phage can be screened easily, quickly and in small volumes. Furthermore, this procedure can select minor Fab fragments species within the population, which otherwise would have been undetectable, and amplified to substantially homogenous populations. The selected Fab fragments can be characterized by sequencing the nucleic acids encoding the polypeptides after amplification of the phage population.
  • the method for producing a heterodimeric immunoglobulin molecule generally involves (1) introducing a heavy or light chain V region-coding gene of interest into the phagemid display vector; (2) introducing a randomized binding site into the phagemid display protein vector by primer extension with an oligonucleotide containing regions of homology to a CDR of the antibody V region gene and containing regions of degeneracy for producing randomized coding sequences to form a large population of display vectors each capable of expressing different putative binding sites displayed on a phagemid surface display protein; (3) expressing the display protein and binding site on the surface of a filamentous phage particle; and (4) isolating (screening) the surface-expressed phage particle using affinity techniques such as panning of phage particles against a preselected antigen, thereby isolating one or more species of phagemid containing a display protein containing a binding site that binds a preselected antigen.
  • two libraries are engineered to genetically shuffle oligonucleotide motifs within the framework of the heavy chain gene structure.
  • CDRI or CDRIII the hypervariable regions of the heavy chain gene were reconstructed to result in a collection of highly diverse sequences.
  • the heavy chain proteins encoded by the collection of mutated gene sequences possessed the potential to have all of the binding characteristics of an immunoglobulin while requiring only one of the two immunoglobulin chains.
  • the method is practiced in the absence of the immunoglobulin light chain protein.
  • a library of phage displaying modified heavy chain proteins is incubated with an immobilized ligand to select clones encoding recombinant proteins that specifically bind the immobilized ligand.
  • the bound phage are then dissociated from the immobilized ligand and amplified by growth in bacterial host cells.
  • Individual viral plaques, each expressing a different recombinant protein, are expanded, and individual clones can then be assayed for binding activity.
  • Antibodies from Human Lymphocytes may also be used to generate a human antibody. Such techniques can be used to stimulate peripheral blood lymphocytes from normal, healthy subjects. Such "in vitro immunization” involves antigen-specific activation of non-immunized B lymphocytes, generally within a mixed population of lymphocytes (mixed lymphocyte cultures, MLC). In vitro immunizations may also be supported by B cell growth and differentiation factors and lymphokines. The antibodies produced by these methods are often IgM antibodies (Borrebaeck et al, 1986; incorporated herein by reference).
  • Another method has been described (U.S. Patent No. 5,681,729, incorporated herein by reference) wherein human lymphocytes that mainly produce IgG (or IgA) antibodies can be obtained.
  • the method involves, in a general sense, transplanting human lymphocytes to an immunodeficient animal so that the human lymphocytes "take” in the animal body; immunizing the animal with a desired antigen, so as to generate human lymphocytes producing an antibody specific to the antigen; and recovering the human lymphocytes producing the antibody from the animal.
  • the human lymphocytes thus produced can be used to produce a monoclonal antibody by immortalizing the human lymphocytes producing the antibody, cloning the obtained immortalized human-originated lymphocytes producing the antibody, and recovering a monoclonal antibody specific to the desired antigen from the cloned immortalized human-originated lymphocytes.
  • the immunodeficient animals that may be employed in this technique are those that do not exhibit rejection when human lymphocytes are transplanted to the animals. Such animals may be artificially prepared by physical, chemical or biological treatments. Any immunodeficient animal may be employed.
  • the human lymphocytes may be obtained from human peripheral blood, spleen, lymph nodes, tonsils or the like.
  • the "taking" of the transplanted human lymphocytes in the animals can be attained by merely administering the human lymphocytes to the animals.
  • the administration route is not restricted and may be, for example, subcutaneous, intravenous or intraperitoneal.
  • the dose of the human lymphocytes is not restricted, and can usually be 10 to 10 lymphocytes per animal.
  • the immunodeficient animal is then immunized with the desired antigen.
  • human lymphocytes are recovered from the blood, spleen, lymph nodes or other lymphatic tissues by any conventional method.
  • mononuclear cells can be separated by the Ficoll-Hypaque (specific gravity: 1.077) centrifugation method, and the monocytes removed by the plastic dish adsorption method.
  • the contaminating cells originating from the immunodeficient animal may be removed by using an antiserum specific to the animal cells.
  • the antiserum may be obtained by, for example, immunizing a second, distinct animal with the spleen cells of the immunodeficient animal, and recovering serum from the distinct immunized animal.
  • the treatment with the antiserum may be carried out at any stage.
  • the human lymphocytes may also be recovered by an immunological method employing a human immunoglobulin expressed on the cell surface as a marker.
  • human lymphocytes mainly producing IgG and IgA antibodies specific to one or more selected antigen(s) can be obtained.
  • Monoclonal antibodies are then obtained from the human lymphocytes by immortalization, selection, cell growth and antibody production.
  • these methods involve the production of a transgenic animal that has inserted into its germline genetic material that encodes for at least part of an immunoglobulin of human origin or that can rearrange to encode a repertoire of immunoglobulins.
  • the inserted genetic material may be produced from a human source, or may be produced synthetically.
  • the material may code for at least part of a known immunoglobulin or may be modified to code for at least part of an altered immunoglobulin.
  • the inserted genetic material is expressed in the transgenic animal, resulting in production of an immunoglobulin derived at least in part from the inserted human immunoglobulin genetic material. It is found the genetic material is rearranged in the transgenic animal, so that a repertoire of immunoglobulins with part or parts derived from inserted genetic material may be produced, even if the inserted genetic material is incorporated in the germline in the wrong position or with the wrong geometry.
  • the inserted genetic material may be in the form of DNA cloned into prokaryotic vectors such as plasmids and/or cosmids. Larger DNA fragments are inserted using yeast artificial chromosome vectors (Burke et al., 1987; incorporated herein by reference), or by introduction of chromosome fragments (Richer and Lo, 1989; incorporated herein by reference).
  • the inserted genetic material may be introduced to the host in conventional manner, for example by injection or other procedures into fertilized eggs or embryonic stem cells.
  • a host animal that initially does not carry genetic material encoding immunoglobulin constant regions is utilized, so that the resulting transgenic animal will use only the inserted human genetic material when producing immunoglobulins. This can be achieved either by using a naturally occurring mutant host lacking the relevant genetic material, or by artificially making mutants e.g., in cell lines ultimately to create a host from which the relevant genetic material has been removed.
  • the transgenic animal will carry the naturally occurring genetic material and the inserted genetic material and will produce immunoglobulins derived from the naturally occurring genetic material, the inserted genetic material, and mixtures of both types of genetic material.
  • the desired immunoglobulin can be obtained by screening hybridomas derived from the transgenic animal, e.g., by exploiting the phenomenon of allelic exclusion of antibody gene expression or differential chromosome loss.
  • the animal is simply immunized with the desired immunogen.
  • the animal may produce a chimeric immunoglobulin, e.g. of mixed mouse/human origin, where the genetic material of foreign origin encodes only part of the immunoglobulin; or the animal may produce an entirely foreign immunoglobulin, e.g. of wholly human origin, where the genetic material of foreign origin encodes an entire immunoglobulin.
  • Polyclonal antisera may be produced from the transgenic animal following immunization. Immunoglobulin-producing cells may be removed from the animal to produce the immunoglobulin of interest. Preferably, monoclonal antibodies are produced from the transgenic animal, e.g., by fusing spleen cells from the animal with myeloma cells and screening the resulting hybridomas to select those producing the desired antibody. Suitable techniques for such processes are described herein.
  • the genetic material may be incorporated in the animal in such a way that the desired antibody is produced in body fluids such as serum or external secretions of the animal, such as milk, colostrum or saliva.
  • body fluids such as serum or external secretions of the animal, such as milk, colostrum or saliva.
  • the desired antibody can then be harvested from the milk. Suitable techniques for carrying out such processes are known to those skilled in the art.
  • transgenic animals are usually employed to produce human antibodies of a single isotype, more specifically an isotype that is essential for B cell maturation, such as IgM and possibly IgD.
  • Another preferred method for producing human antibodies is described in U.S. Patent No. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,770,429; each incorporated by reference, wherein transgenic animals are described that are capable of switching from an isotype needed for B cell development to other isotypes.
  • the cell In the development of a B lymphocyte, the cell initially produces IgM with a binding specificity determined by the productively rearranged V H and V L regions.
  • each B cell and its progeny cells synthesize antibodies with the same L and H chain V regions, but they may switch the isotype of the H chain.
  • mu or delta constant regions is largely determined by alternate splicing, permitting IgM and IgD to be coexpressed in a single cell.
  • the other heavy chain isotypes (gamma, alpha, and epsilon) are only expressed natively after a gene rearrangement event deletes the C mu and C delta exons.
  • This gene rearrangement process termed isotype switching, typically occurs by recombination between so called switch segments located immediately upstream of each heavy chain gene (except delta).
  • the individual switch segments are between 2 and 10 kb in length, and consist primarily of short repeated sequences.
  • transgenes incorporate transcriptional regulatory sequences within about 1 -2 kb upstream of each switch region that is to be utilized for isotype switching.
  • These transcriptional regulatory sequences preferably include a promoter and an enhancer element, and more preferably include the 5' flanking (i.e., upstream) region that is naturally associated (i.e., occurs in germline configuration) with a switch region.
  • a 5' flanking sequence from one switch region can be operably linked to a different switch region for transgene construction, in some embodiments it is preferred that each switch region incorporated in the transgene construct have the 5' flanking region that occurs immediately upstream in the naturally occurring germline configuration. Sequence information relating to immunoglobulin switch region sequences is known (Mills et al., 1991 ; Sideras et al., 1989; each incorporated herein by reference).
  • the rearranged heavy chain gene consists of a signal peptide exon, a variable region exon and a tandem array of multi-domain constant region regions, each of which is encoded by several exons.
  • Each of the constant region genes encode the constant portion of a different class of immunoglobulins.
  • V region proximal constant regions are deleted leading to the expression of new heavy chain classes.
  • alternative patterns of RNA splicing give rise to both transmembrane and secreted immunoglobulins .
  • the human heavy chain locus consists of approximately 200 V gene segments spanning 2 Mb, approximately 30 D gene segments spanning about 40 kb, six J segments clustered within a 3 kb span, and nine constant region gene segments spread out over approximately 300 kb. The entire locus spans approximately 2.5 Mb of the distal portion of the long arm of chromosome 14. Heavy chain transgene fragments containing members of all six of the known V H families, the D and J gene segments, as well as the mu, delta, gamma 3, gamma 1 and alpha 1 constant regions are known (Berman et al., 1988; incorporated herein by reference). Genomic fragments containing all of the necessary gene segments and regulatory sequences from a human light chain locus is similarly constructed.
  • the expression of successfully rearranged immunoglobulin heavy and light transgenes usually has a dominant effect by suppressing the rearrangement of the endogenous immunoglobulin genes in the transgenic nonhuman animal.
  • it is desirable to effect complete inactivation of the endogenous Ig loci so that hybrid immunoglobulin chains comprising a human variable region and a non-human (e.g., murine) constant region cannot be formed, for example by trans- switching between the transgene and endogenous Ig sequences.
  • a non-human (e.g., murine) constant region cannot be formed, for example by trans- switching between the transgene and endogenous Ig sequences.
  • the endogenous immunoglobulin repertoire can be readily eliminated.
  • suppression of endogenous Ig genes may be accomplished using a variety of techniques, such as antisense technology.
  • trans- switched immunoglobulin it may be desirable to produce a trans- switched immunoglobulin.
  • Antibodies comprising such chimeric trans-switched immunoglobulins can be used for a variety of applications where it is desirable to have a non-human (e.g., murine) constant region, e.g., for retention of effector functions in the host.
  • a murine constant region can afford advantages over a human constant region, for example, to provide murine effector functions (e.g., ADCC, murine complement fixation) so that such a chimeric antibody may be tested in a mouse disease model.
  • the human variable region encoding sequence may be isolated, e.g., by PCR amplification or cDNA cloning from the source (hybridoma clone), and spliced to a sequence encoding a desired human constant region to encode a human sequence antibody more suitable for human therapeutic use.
  • Human antibodies generally have at least three potential advantages for use in human therapy.
  • humanized antibodies have many advantages.
  • “Humanized” antibodies are generally chimeric or mutant monoclonal antibodies from mouse, rat, hamster, rabbit or other species, bearing human constant and/or variable region domains or specific changes. Techniques for generating a so-called “humanized” antibody are well known to those of skill in the art.
  • Humanized antibodies also share the foregoing advantages.
  • DNA sequences encoding the antigen binding portions or complementarity determining regions (CDR's) of murine monoclonal antibodies can be grafted by molecular means into the DNA sequences encoding the frameworks of human antibody heavy and light chains (Jones et al, 1986; Riechmann et al., 1988; each incorporated herein by reference).
  • the expressed recombinant products are called "reshaped" or humanized antibodies, and comprise the framework of a human antibody light or heavy chain and the antigen recognition portions, CDR's, of a murine monoclonal antibody.
  • position alignments of a pool of antibody heavy and light chain variable regions is generated to give a set of heavy and light chain variable region framework surface exposed positions, wherein the alignment positions for all variable regions are at least about 98%o identical;
  • a set of heavy and light chain variable region framework surface exposed amino acid residues is defined for a rodent antibody (or fragment thereof);
  • a set of heavy and light chain variable region framework surface exposed amino acid residues that is most closely identical to the set of rodent surface exposed amino acid residues is identified;
  • the set of heavy and light chain variable region framework surface exposed amino acid residues defined in step (2) is substituted with the set of heavy and light chain variable region framework surface exposed amino acid residues identified in step (3), except for those amino acid residues that are within 5A of any atom of any residue of the complementarity determining regions of the rodent antibody; and
  • the humanized rodent antibody having binding specificity is produced.
  • the heavy and light chains may each be designed by using any one, any combination, or all of the various position criteria described in U.S. Patent No. 5,693,762; 5,693,761; 5,585,089; and 5,530,101, each incorporated herein by reference.
  • the humanized immunoglobulins When combined into an intact antibody, are substantially non- immunogenic in humans and retain substantially the same affinity as the donor immunoglobulin to the original antigen.
  • the antigen binding sites created by this process differ from those created by
  • EIS epitope imprinted selection
  • an animal antibody Starting with an animal antibody, one process results in the selection of antibodies that are partly human antibodies. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or after alteration of a few key residues. Sequence differences between the rodent component of the selected antibody with human sequences could be minimized by replacing those residues that differ with the residues of human sequences, for example, by site directed mutagenesis of individual residues, or by CDR grafting of entire loops. However, antibodies with entirely human sequences can also be created. EIS therefore offers a method for making partly human or entirely human antibodies that bind to the same epitope as animal or partly human antibodies respectively. In EIS, repertoires of antibody fragments can be displayed on the surface of filamentous phase and the genes encoding fragments with antigen binding activities selected by binding of the phage to antigen.
  • exemplary functional regions include scFv, Fv, Fab', Fab and F(ab') 2 fragments of the antibodies. Techniques for preparing such constructs are well known to those in the art and are further exemplified herein.
  • antibody construct may be influenced by various factors. For example, prolonged half-life can result from the active readsorption of intact antibodies within the kidney, a property of the Fc piece of immunoglobulin. IgG based antibodies, therefore, are expected to exhibit slower blood clearance than their Fab' counterparts. However, Fab' fragment-based compositions will generally exhibit better tissue penetrating capability.
  • Fab fragments can be obtained by proteolysis of the whole immunoglobulin by the non-specific thiol protease, papain.
  • Papain must first be activated by reducing the sulphydryl group in the active site with cysteine, 2-mercaptoethanol or dithiothreitol.
  • Heavy metals in the stock enzyme should be removed by chelation with EDTA (2 mM) to ensure maximum enzyme activity.
  • Enzyme and substrate are normally mixed together in the ratio of 1 : 100 by weight. After incubation, the reaction can be stopped by irreversible alkylation of the thiol group with iodoacetamide or simply by dialysis. The completeness of the digestion should be monitored by SDS-PAGE and the various fractions separated by protein A-Sepharose or ion exchange chromatography.
  • Digestion of rat IgG by pepsin requires conditions including dialysis in 0.1 M acetate buffer, pH 4.5, and then incubation for four hours with 1% w/w pepsin; IgGj and IgG 2a digestion is improved if first dialyzed against 0.1 M formate buffer, pH 2.8, at 4°C, for 16 hours followed by acetate buffer. IgG 2b gives more consistent results with incubation in staphylococcal V8 protease (3% w/w) in 0.1 M sodium phosphate buffer, pH 7.8, for four hours at 37°C.
  • bispecific antibodies are preferred for use.
  • the preparation of bispecific antibodies is also well known in the art.
  • One preparative method involves the separate preparation of antibodies having specificity for a first particular component, on the one hand, and a second particular component on the other.
  • Peptic F(ab' ⁇ ) 2 fragments from the two chosen antibodies are then generated, followed by reduction of each to provide separate Fab' ⁇ sH fragments.
  • the SH groups on one of the two partners to be coupled are then alkylated with a cross- linking reagent, such as o-phenylenedimaleimide, to provide free maleimide groups on one partner.
  • This partner may then be conjugated to the other by means of a thioether linkage, to give the desired F(ab' ⁇ ) 2 heteroconjugate (Glennie et al, 1987; incorporated herein by reference).
  • Other approaches, such as cross-linking with SPDP or protein A may also be carried out.
  • quadroma Another method for producing bispecific antibodies is by the fusion of two hybridomas to form a quadroma.
  • quadroma is used to describe the productive fusion of two B cell hybridomas.
  • two antibody producing hybridomas are fused to give daughter cells, and those cells that have maintained the expression of both sets of clonotype immunoglobulin genes are then selected.
  • a preferred method of generating a quadroma involves the selection of an enzyme deficient mutant of at least one of the parental hybridomas. This first mutant hybridoma cell line is then fused to cells of a second hybridoma that had been lethally exposed, e.g., to iodoacetamide, precluding its continued survival. Cell fusion allows for the rescue of the first hybridoma by acquiring the gene for its enzyme deficiency from the lethally treated hybridoma, and the rescue of the second hybridoma through fusion to the first hybridoma.
  • Preferred, but not required is the fusion of immunoglobulins of the same isotype, but of a different subclass.
  • a mixed subclass antibody permits the use if an alternative assay for the isolation of a preferred quadroma.
  • Microtiter identification embodiments FACS, immunofluorescence staining, idiotype specific antibodies, antigen binding competition assays, and other methods common in the art of antibody characterization may be used to identify preferred quadromas.
  • the bispecific antibodies are purified away from other cell products. This may be accomplished by a variety of antibody isolation procedures, known to those skilled in the art of immunoglobulin purification (see, e.g., Antibodies: A Laboratory Manual, 1988; incorporated herein by reference). Protein A or protein G sepharose columns are preferred.
  • the inventors developed a novel system to drive the expansion of CD4+ T cells (Levine et al, 1996; Carroll et al, 1997). This system uses antibodies to the T cell accessory molecules CD3 and CD28 to drive polyclonal logarithmic growth of human T cells in vitro. The inventors also have developed a clinical protocol to use ex vivo expanded autologous human CD4+ T cells as an immunotherapeutic adjunct to dose-intensive therapy for patients with high risk relapsed refractory NHL (described below).
  • relapsed/refractory NHL who are high risk candidates for dose-intensive therapy, defined as chemo-insensitive disease in relapse or primary refractory disease, undergo a steady-state leukapheresis to collect PBMCs as a source of T cells, and then undergo PBPC mobilization with chemotherapy plus cytokines.
  • the product of the harvest undergoes a CD34 positive selection step.
  • the selected CD34+ cells are used for hematologic rescue after the patient has received a dose-intensive chemotherapy regimen.
  • the positive selection step depletes the autologous T cells in the PBPC thus delaying recovery of the endogenous T cell compartment following the autologous stem cell transplant.
  • PBMCs Peripheral blood mononuclear cells
  • T cell expansion system used in this application drives the polyclonal expansion of CD3+CD28+T cells. Although both CD4- and CD8-positive T cells are expanded, the CD4+ cells are expanded to a greater extent than the CD8+ cells (FIG. 1A, FIG. IB, FIG. 2 and FIG. 3).
  • the cytokine-secretion phenotype of the expanded CD4+ cells generally has a T H 1 pattern. The doses of T cells were escalated in patients using a phase I study design (described in detail below).
  • EXAMPLE 2 Immunological profile of patients receiving CD3+28 costimulated T cells
  • Toxicity was mild with no grade 2 or higher early (24 hr.) toxicities (see protocol) associated with CD3+28 T cell reinfusion of up to 2 ⁇ l0 9 total cells.
  • the immunologic consequences of reinfused T cells were greater than expected.
  • the cell manufacturing and release data are summarized in Table 1 below.
  • the adverse events are summarized in Table 2, below.
  • the cytokine expression profile of the CD4+ and CD8+ T cells that were present during the period of lymphocytosis was determined in the 4 patients evaluated.
  • T cells CD3 perCP+ cells
  • CD4+ surface staining and intracellular cytokine expression as described herein.
  • the results showed that 68% of the CD4+ T cells expressed TNF- ⁇ , 25%o expressed IL-4 and 85% expressed IFN- ⁇ .
  • Isotype-matched negative control antibodies were used to set quadrants.
  • the majority of activated cells express the T H 1 cytokines TNF- ⁇ and IFN- ⁇ .
  • T cells (CD3 perCP+ cells) were gated and then analyzed for CD8+ surface staining and intracellular cytokine expression as described herein. These results showed that 48%> of the CD8+ T cells expressed TNF- ⁇ , 4.6% expressed IL-4 and 70% expressed IFN- ⁇ . Isotype-matched negative control antibodies were used to set quadrants. These data indicate that a significant fraction of the circulating CD8+ T cells are activated to produce T c l cytokines. This, combined with the CD8+ clonality data (FIG. 10A, FIG, 10B, FIG. 10C, FIG. 10D, FIG. 11 A, FIG. 1 IB, FIG. 11C and FIG.
  • V ⁇ usage of the CD3+28 costimulated product has a gaussian (polyclonal) distribution, indicating the relative nonspecific polyclonal nature of the expanded cell product. This is expected since costimulation through CD3+28 should globally act and expand T cells and hopefully overcome activation defects that may be present in these patients (Ochoa and Longo, 1995).
  • the V ⁇ usage of the CD8+ T cells that emerge in the wave of lymphocytosis are not gaussian and are often monoclonal or oligoclonal.
  • CD8+ lymphocytes which emerge in the patient 2-4 weeks following CD3+28 costimulated T cell reinfusion are antigen or tumor specific effector cells that are being driven by the CD3+28 costimulated CD4+ T cells.
  • the inventors expect that the specificity of these CD8+ clonal T cell populations can be determined if these same cells are present in biopsies of residual tumor in select patients and then by testing the ability of these cells to kill autologous transformed B cell lines.
  • mice with tumors have alterations in T cell signaling pathways that prevent normal immunologic recognition and elimination of tumor cells (Mizoguchi et al, 1992).
  • mice with tumors there is a progressive, sequential development of functional alterations in T cells, and also a progressive loss of T H 1 cell populations (Correa et al, 1997; Ghosh et al, 1995).
  • a clinical protocol was developed to examine expanding CD4+ T cells for human immunotherapeutic use. The safety of these cells when delivered in escalating doses in the post-transplant setting, the dose tolerance of these cells in this patient population, and the efficacy of these cells in reducing the presence of molecularly detectable residual disease and relapse rates in these patients were evaluated.
  • the clinical protocol was developed using CD3+28 costimulated T cells following dose-intensive therapy. The specific methods for the isolation and growth of these cells are provided below.
  • the PBPC were separated into CD34-positive and CD34-negative fractions.
  • the CD34-positive fraction was used for hematopoietic rescue of patients following dose-intensive chemotherapy.
  • the CD34-negative fraction was used to start the CD3+28 costimulation cultures as described fully below.
  • the CD3+28 costimulation cultures were started on the same day that patients received back their CD34-positive PBPCs.
  • the CD3+28 costimulated T cells were harvested, counted, and a specific dose was reinfused into the patient.
  • the study design is a phase I study in which cohorts of three patients received escalating doses of autologous CD3+28 costimulated T cells.
  • T cells following dose-intensive therapy and CD34-selected peripheral blood progenitor cell support in patients with relapsed or refractory B cell NHL, and assessed the toxicity of escalating doses of CD3+28 costimulated T cells in this patient population. This study also assessed the immune responsiveness of patient's T cells prior to therapy, in response to ex vivo CD3+28 costimulation, and following adoptive therapy with costimulated T cells. Specimens were collected from peripheral blood, bone marrow and cell products in the study to measure T H subsets by flow cytometry using intracellular cytokine staining.
  • T H subsets helped to determine the immunophysiologic consequences of delivering these ex vivo derived CD4+ T cells to patients with relapsed NHL.
  • correlation of molecular responses with T cell infusion or with T H subset polarization helped to identify useful surrogate immunologic markers for anti-tumor activity.
  • Mononuclear cells were isolated from peripheral whole blood by density gradient centrifugation on Ficoll-Hypaque (BioWhittaker, Inc.). The washed mononuclear cells then were separated into T cell-rich and B cell-rich fractions either by purification on a human T cell enrichment column (R&D Systems) according to the manufacturer's directions, or by E rosetting with aminoethylisothiouronium hydrobromide (AET) treated sheep red blood cells. The B cells and T cells were used as described below.
  • T H 1 or T H 2 T cells (0.5-1.0 x 10 cells/ml) were cultured at 37°C/5%CO 2 in RPMI-1640 (BioWhittaker, Inc.) supplemented with 10%) heat-inactivated fetal bovine serum (BioWhittaker, Inc.), PMA+I (25 ng/ml and 1 mg/ml, respectively) and 2 mM monensin (Calbiochem). Control cultures were set up identically with the exception that the PMA+I was omitted. Monensin was added to the culture to inhibit cytokine secretion, resulting in the intracellular accumulation of cytokines.
  • FACS lysing solution (Becton Dickinson) was added to lyse any remaining red cells and to partially fix the T cells. The solution was washed off the cells, and then the cells were incubated with FACS permeabilizing solution (Becton Dickinson) at room temperature for 10 minutes in order to permeabilize the lymphocyte membranes prior to intracellular immunofluorescence staining. The cells were washed, and then incubated with fluorscent-conjugated intracellular monoclonal antibodies at room temperature for 30 minutes.
  • the cells were resuspended in PBS with 1% BSA and analyzed by three color flow cytometry on a FACScan (Becton Dickinson) flow cytometer.
  • Surface antigen specific monoclonal antibodies that were used included perCP-anti-CD3 (Becton Dickinson), FITC-anti-CD4, and FITC-anti- CD8 (both from DAKO).
  • Intracellular monoclonal antibodies that were used included PE-anti-IL-2, PE-anti-IL-4, PE-anti-IFN- ⁇ , and PE-anti-TNF- ⁇ (all from Becton Dickinson).
  • PBPC are mobilized with cyclophosphamide and GM-CSF plus G-CSF.
  • the use of high dose cyclophosphamide to mobilize PBPC provides therapeutic effect by using an active agent which is not duplicated in the transplant preparative regimen.
  • the combination of GM-CSF and G-CSF is more effective than either growth factor alone in mobilizing progenitor cells (Winter et al, 1996).
  • One approach to improving the results of therapy for patients with "high risk" relapsed/refractory NHL is to combine another therapeutic modality such as immunotherapy with dose-intensive therapy. This results in the eradication of disease that has failed to respond completely to the dose-intensive regimen.
  • escalating doses of ex vivo expanded CD4+CD28+ T cells are used as an immunotherapeutic adjunct to the dose-intensive regimen.
  • the product of CD34 positive selection is then used as a source of progenitor cells for rescue from the dose- intensive regimen and the steady-state leukapheresis product as the source of CD4+CD28+ T cells to target for ex vivo expansion.
  • the protocol expands a population of CD4+ T cells ex vivo from patients with relapsed refractory non-Hodgkin's lymphoma (NHL), and delivers these expanded cells as complimentary therapy for patients whose tumors failed to respond to induction chemotherapy.
  • This protocol is also contemplated for use in other immunotherapeutic protocols targeting patients whose immunoresponsive tumors are not responsive to chemotherapy alone.
  • T cells which are predominantly CD8+. This was due, in part, to the absence of a system available for the large scale growth and expansion of CD4+ T cells.
  • CD4+ T Cell Expansion The proliferative potential of T cells expanded in vitro is a major consideration for 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.
  • mixed populations of CD4 + and CD8 + T cells stimulated in this manner will eventually result in a population that is all or mostly CD8 + .
  • the long-term growth of CD4 + T cells has necessitated the addition of exogenous lymphokines and allogeneic feeder cells, which precludes a large-scale expansion of CD4+ T cells for the treatment of disease.
  • lymphocytes require the delivery of both an antigen-specific signal as well as a simultaneous co- stimulatory signal.
  • interaction of the T cell receptor with the antigen-MHC complex may cause T cell clonal anergy or deletion.
  • CD28 can provide an important co- stimulatory signal.
  • the inventors developed a novel method for preferentially expanding CD4+ T cells independent of exogenous cytokines or feeder cells using anti-CD3 Ab plus anti-CD28 Ab conjugated to magnetic beads.
  • the inventors have recently used this system to preferentially expand CD4+CD28+ T cells from the CD34 negative selection product from normal peripheral allogeneic donors as well as autologous patient-donors with malignancy.
  • the CD34 negative selection product is used to seed the initial cultures without further selection or manipulation. Cultures set up in this fashion show a 2-3 log 10 expansion over 14 days with a final cell culture product that is 85-90% CD4+ and 10- 15% CD8+ T cells.
  • the targeted numbers of CD4+ T cells for reinfusion are expanded from 1 x 10 to 1 x 10 CD34 negative selection product cells without any further processing.
  • Relapsed NHL is defined as patients with recurrent disease after CR (defined below), but not more than 5 years after achieving CR, unless tumor can be shown to be identical to the original clone.
  • Refractory refers to primary refractory disease.
  • High risk disease is defined as less than a PR (i.e., SD or PD) to a standard pre-transplant induction regimen, creatinine ⁇ 1.5 or calculated creatinine clearance of > 60 ml min, total bilirubin, AST and ALT ⁇ 1.5 ⁇ upper limit of normal (unless due to Gilbert's for bilirubin).
  • PR i.e., SD or PD
  • Pathology material is reviewed to confirm diagnosis. Paraffin blocks or frozen material from initial biopsy and relapse should be provided within 30 days of entry. A small portion is used to develop tumor-specific oligonucleotide probes to the CDR3 region of the immunoglobulin heavy chain gene.
  • a CT scan of chest/abdomen/pelvis and SPECT-gallium scan are performed, along with a bone marrow aspirate and biopsy. Five to ten cubic centimeters (cc) of bone marrow aspirate is obtained at the time of disease staging to measure tumor cells on the bone marrow mononuclear cell fraction and on 21 -day bone marrow mononuclear cell cultures using a PCR-based assay with tumor specific probes. Twenty cc of heparinized peripheral blood is collected at the time of pre-transplant evaluation, and tumor cell contamination is determined on the peripheral blood mononuclear cell fraction using a PCR-based assay with tumor specific probes.
  • Stem cells are mobilized into the peripheral blood using a standard regimen. Apheresis is performed with the Fenwal CS3000 Plus cell collector, or equivalent, using a large bore double lumen catheter as venous access with total processing of approximately 10-12 liters per session.
  • the small volume collection chamber
  • Mobilization is assessed by monitoring levels of circulating CD34+ cells in peripheral blood, and is considered successful when circulating CD34+ cells > 5/ ⁇ l. Following successful mobilization, and when WBC ⁇ 1000/ ⁇ l, patients undergo leukapheresis daily. Endpoints for successful PBPC harvest include harvest of at least 5 ⁇ l0 6 CD34+cells/kg. From this, 1.0 x lO 6 CD34+ cells/kg are cryopreserved without further manipulation, and stored for rescue of the patient in case of graft failure. Failure to meet the backup collection after 6 days of leukapheresis or the total collection minima in 12 or fewer days of apheresis disqualifies the patient for the CD34 positive selection procedure and T cell expansion. The standard procedure for operation of the CS3000 and collection of cells is provided below.
  • the backup aliquot of mononuclear cell suspensions obtained by apheresis is cryopreserved without further manipulation.
  • the peripheral stem cells are stored according to the Stem Cell Cryolab standard. Details on the cryopreservation procedure are outlined below. 4. CD34+ Selection from PBSC Apheresis Collection
  • the Isolex 300 System for positive selection of CD34 cells.
  • the system is comprised of the following components: a monoclonal antibody directed against the CD34 antigen expressed on hematopoietic progenitor/stem cells (this antigen is selectively expressed by a small percentage of bone marrow and a peripheral blood mononuclear cells); the Isolex 300i Magnetic Cell Separator; paramagnetic microspheres coated with sheep anti-murine antibody; and a non- enzymatic stem cell releasing agent to separate the targeted CD34+ cells from the paramagnetic microspheres.
  • the Isolex 300i Magnetic Cell Separator is a device which controls all of the steps in the separation process, including washing and sensitization of the target cells, separation of the paramagnetic microspheres/target cells from the mononuclear cell suspensions, and release of the target cells form the paramagnetic microspheres.
  • Components of the Magnetic Cell Separator include: a primary magnet whose adjustable position is controlled automatically; a stationary secondary magnet; a series of clamps and pumps; a support module for the washing chamber; and a graphic LCD display with a touch screen used as the operator interface.
  • the associated Isolex 300i Disposable Set comprises a sterile biocompatible fluid path for the cells.
  • the main components of the set include the washing chamber, the mixing/separation chamber (which interfaces with the primary magnet), and the secondary chamber, which interfaces with the secondary magnet.
  • the sheep anti-murine antibody coated microspheres (Dynabeads M-450 sheep anti-mouse) bind murine immunoglobulin and provide the mechanism for targeting the antibody coated cells for selection.
  • the non- enzymatic stem cell releasing agent displaces the cells from the bead-cell rosettes, allowing the target cells to be collected and the beads to be retained by the magnet.
  • CD34+ cells are isolated from the PBPC collections of the test group using the
  • Baxter Isolex 300 System All procedures are performed using aseptic technique.
  • the selection procedure includes specific steps for PBPC preparation, sensitization of the mononuclear cells with the 9069 anti-CD34 monoclonal antibody, rosetting of target cells and paramagnetic beads, release of target cells from the beads, and washing of isolated CD34+ cells. Details of the selection procedure are outlined below.
  • a platelet wash is performed on the apheresis product prior to the isolation procedure. This occurs using the spinning membrane assembly, which is part of the disposable set (wash chamber).
  • Sensitization of the mononuclear cells is performed with 2.5 mg of anti-CD34 antibody in a total volume of 100 mL. Sensitization is performed for fifteen minutes at room temperature, after which the cells are washed to remove excess/unbound antibody.
  • Freshly prepared Dynal® paramagnetic microspheres (SAM IgG) are then added to washed, sensitized cells. After incubation at room temperature, the bead-cell complexes are separated from the unbound cells using the primary magnet.
  • the bead/target cell complexes are retained in the separation chamber and the non-target cells are washed away.
  • the non-targeted fraction is collected using aseptic technique for T cell expansion.
  • the stem cell releasing agent the CD34+ cells are displaced, releasing them from the beads. This requires incubation at room temperature, after which the target cells are collected using the primary magnet to hold the beads in the chamber.
  • the final CD34+ product is assayed for sterility with bacterial cultures and sensitivity, mycoplasma and fungal cultures.
  • a steady-state pheresis product obtained prior to stem cell mobilization is used as the source of PBMCs that are cultured in a system which facilitates the preferential expansion of CD4+CD28+ T lymphocytes.
  • Cells are seeded into a 300 ml Baxter Lifecell Flask, or Baxter 2417 flask if the starting amount of cells is at least 100 x 10 cells. Details on the use of Baxter Lifecell® Flask and Solution Transfer Pump to culture cells are provided below.
  • the cells are grown in X-VIVO 15® supplemented with 5% autologous serum collected and prepared during apheresis. Magnetic beads (BB IND 6675) containing immobilized anti-CD3 (OKT3) and anti-CD28 (9.3) antibodies are washed and added at a 3:1 Bead: CD3+ cell ratio. The cultures are maintained for 14 days prior to harvesting for preparation for reinfusion. The cells are counted daily and fresh medium is added to maintain the cells at a density of about 0.75-2 x 10 6 per ml as outlined below.
  • the phenotype of the ex vivo expanded cells is determined at the time of initial seeding, at 7 days of expansion, and then on day 14 of expansion at the time of harvesting.
  • the cells are stained with anti-CD3, anti-CD4 and anti-CD8, and analyzed by flow cytometry to determine the total number and relative frequency of total T cells (CD3+), as well as CD4+ and CD8+ T cells.
  • the cells are expanded ex vivo for 14 days and then processed for reinfusion on day 14.
  • the magnetic beads are removed using a Baxter Fenwal Maxsep® magnetic cell separation system. It is preferable to efficiently remove the microbeads prior to reinfusion into the patient to prevent or reduce the possibility of undesirable side effects that may be caused by the infusion of the antibody-coated microbeads. Details of the bead removal process are provided below.
  • Cell culture harvest is performed the day of scheduled cell reinfusion and once the cultures have undergone the magnetic cell separation process for removal of the microbeads used for cell stimulation.
  • the cells are harvested, washed and resuspended in Plasmalyte A containing 1% human serum albumin with the Baxter Fenwal cell harvester, as described below.
  • the total dose of reinfused T cells is based on the number of CD3+ cells as determined by the total cell count and flow cytometry with anti-CD3. After fourteen days of expansion this constitutes >99% of the total cells in the culture.
  • the starting CD3+ cell dose is 2 x 10 9 cells in 100-250 ml Plasmalyte A containing 1% human serum albumin. The dose of cells is infused over 20-30 minutes. No leukocyte filter is used. Details of the reinfusion procedure is provided below.
  • the CD3+ cell dose escalates as follows: Dose level 2, 5 x 10 9 CD3+ cells; Dose level 3, 1 x 10 10 CD3+ cells; Dose level 4, 2.5 x 10 10 CD3+ cells; and Dose level 5, 5 x 10 CD3+ cells. At all dose levels, the cells are reinfused over 20-30 minutes.
  • the following minimum criteria are required for release of the expanded CD3+28 T cells for reinfusion: a) Minimum cell viability of 80%; b) Less than 100 residual CD3+28 beads/3 x 10 cells reinfused; c) The bacterial, mycoplasma and fungal culture reports from day -2 must read no growth; d) The gram stain of the harvest product must report no organisms seen; and e) The endotoxin assay from day - 2 must be ⁇ 1.
  • DLT Dose Limiting Toxicity
  • MTD Miximum Tolerated Dose
  • RPTD Recommended Phase II Dose
  • Toxicity is graded according to the CALGB Expanded Common Toxicity Criteria (Table 3). Adverse reactions not included in these Criteria are graded as follows: Mild, Grade 1 ; Moderate, Grade 2; Severe, Grade 3; Life-Threatening, Grade 4; and Lethal, Grade 5. Each toxic event is considered for relationship to the expanded T cells as follows: Unrelated: This category applies to those adverse events which, after careful medical consideration, are clearly felt to be due to extraneous causes (disease, environment, etc.) unrelated to the administration of the expanded T cells; Unlikely: This category applies to those adverse experiences which, after careful medical consideration, are felt unlikely to be related to the administration of the expanded T cells.
  • An adverse experience can be considered possibly related if: 1) It follows a reasonable temporal sequence from administration of the expanded T cells; and 2) It could have been a result of the patient's clinical condition, environmental or toxic factors, or other therapies administered to the patient; Probable: This category applies to those adverse experiences which, after careful medical consideration, are felt with a high degree of certainty to be related to administration of the expanded T cells.
  • An adverse experience can be considered probably related if: 1) It follows a reasonable temporal sequence from administration of the expanded T cells; and 2) It could not be reasonably explained by the patient's clinical condition, by environmental or toxic factors or by other therapies administered to the patient; Definite: This category applies to those adverse experiences which, after careful medical consideration, are felt to be related to the administration of the expanded T cells.
  • An adverse experience can be considered definitely related if: 1) It follows a reasonable temporal sequence from administration of the expanded T cells or is associated within a period during which the expanded T cells have been established in body fluids or tissues; and 2) It could not 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 at which there is at most grade 0-1 toxicity. After at least one episode of grade 2 toxicity probably or definitely related to treatment (excluding alopecia, nausea/vomiting), subsequent dose levels enroll a minimum of three patients. No more than one new patient is treated per week. The third patient entered at a dose level must be observed for at least one week prior to enrolling the first patient at the next dose level. If dose limiting toxicity occurs in one of the first three patients at a dose level, then at least three additional patients are treated at that dose level to determine if the MTD has been exceeded (fewer than three if MTD is clearly exceeded before six patients have been treated).
  • dose escalation proceeds to the next level. If dose limiting toxicity occurs in more than 2 of the 6 patients, then the MTD has been reached, and the dose is decreased to define recommended phase II dose.
  • the toxicities related to cellular therapy with T cells are generally thought to be due to the release of cytokines from the cells.
  • the experience with LAK cells and limited experience with CD4+ T cells suggest that the toxicity from these cells is minimal.
  • the most common side effects are fatigue, headache, fever, nausea and chills.
  • the more serious side effects associated with cytokines such as hypotension, capillary leak syndromes and cardiovascular collapse have not been encountered with T cell infusion therapy.
  • Bone marrow aspirate with biopsy for pathology review is obtained within 60 days post-reinfusion of CD34 selected PBPCs.
  • Five to ten cubic centimeters of bone marrow aspirate is obtained in order to measure tumor cell contamination on the bone marrow mononuclear cell fraction using a PCR-based assay with tumor specific probes.
  • CBC Differential and platelet count are performed every other week.
  • Chem- 17 is performed every other week.
  • Twenty cubic centimeters of heparinized peripheral blood is collected on the same day as the post-transplant bone marrow specimen, then every 30 days for the first three months, every 60 days for the next three months, followed by every 90 days for six months or for a minimum of 18 months after reinfusion of CD34+ cells.
  • Tumor cell contamination is determined on the peripheral blood mononuclear cell fraction using a PCR-based assay with tumor specific probes.
  • Tumor reevaluation is performed every three months with CT and SPECT-gallium
  • Complete Response is defined as no clinical, radiologic, or histopathologic evidence (marrow evaluation) of tumor for 6 or more months duration.
  • Partial Response is defined as a minimum of 50% shrinkage in tumor with no evidence of progression at any site and no new sites of disease. Patients not meeting the criteria for CR or PR are considered to have No Response (NR).
  • Progressive disease is defined as > 25%. increase in size of an existing lesion or the appearance of any new lesion.
  • Survival time is calculated from CD34 selected PBPC reinfusion (Day 0) until death or last patient contact.
  • Time to Progression (TTP) and Time to Treatment Failure (TTF) are calculated from reinfusion until disease progression or last patient contact, and from reinfusion until any event defining treatment failure (PD, death from any cause, second malignancy, etc.) respectively.
  • Hgb WNL 10. 0-normal 8.0-10.0 6.5-7.9 ⁇ 6.5
  • Diarrhea none increase of 2-3 increase of 4-6 increase of 7-9 increase of>10 stools/day over pre Rx stools/day, or stools/day, or stools/day or nocturnal stools, or incontinence, or grossly bloody moderate cramping severe cramping diarrhea, or need for parenteral support
  • Stomatitis none painless ulcers, painful erythema, painful erythema, requires parenteral erythema, or mild edema, or ulcers, edema, ulcers, and or enteral support soreness but can eat cannot eat
  • Esophagitis/Dysphagia none painless ulcers, painful erythema, cannot eat solids or requires parenteral erythema, mild edema, or ulcers or requires narcotics or enteral support soreness, or mild moderate dysphagia to eat or complete dysphagia but can eat without obstruction or narcotics perforation
  • Gastritis/Ulcer no antacid requires vigorous uncontrolled by perforation or medical medical bleeding management or management; non-surgical requires surgery for treatment GI ulceration
  • Alk Phos or WNL >2.5xN 2.6-5.0xN 5.1-20.0xN >20.0xN 5'nucleotidase
  • Urinary Retention none urinary residual self catheterization surgical procedure >100 cc or always required for required (TUR or occasionally requires voiding dilation) catheter or difficulty initiating urinary stream
  • Cardiac Function none asymptomatic, asymptomatic, decline mild CHF, severe or decline of resting of resting ejection responsive to refractory CHF ejection fraction by fraction by more than therapy less than 20% of 20% of baseline value baseline value
  • Cardiac Ischemia none asymptomatic, asymptomatic, decline mild CHF, sever or refractory decline of resting of resting ejection responsive to CHF ejection fraction by fraction by more than therapy less than 20% of 20% of baseline value baseline value
  • Hypertension none or no asymptomatic, recurrent or persistent requires therapy hypertensive crisis change transient increase increase by greater by greater than 20 than 20 mm Hg (D) or mm Hg(D) or to to > 150/100 if > 150/100 if previously WNL. previously WNL. No treatment required No treatment required.
  • Edema none 1+ or dependent in 2+ or dependent 3+ 4+, generalized evening only throughout day anasarca
  • Neuro-headache none mild moderate or severe but unrelenting and transient severe Neuro-constipation none or no mild moderate severe ileus >96 hrs change
  • Mononuclear cells are collected using the 1-120 stem cell program on the Baxter Fenwal CS3000 Plus Blood Cell Separator.
  • the CS3000 Plus Blood Cell Separator is a self-contained, continuous flow, centrifugal device that separates whole blood into some of its components. Collection of specific blood components can be automatically implemented and monitored. A program for each type of collection procedure is stored in the solid state memory of the separator. Two operating modes are provided: automatic and manual. During automatic operation, all functions are controlled by a microcomputer except during situations when operator intervention is required. During manual operation, all functions are under control of the operator. A message center on the operator panel provides operator help messages and status messages, and also allows the operator to program the separator to perform special procedures. Blood components are collected in a sterile, disposable Apheresis Kit. The components are centrifugally separated within the Apheresis Kit by density differences. A two-stage centrifugation process is used in most procedures.
  • the centrifuge consists of two primary mechanical components: the motor driven rotor and the shield assembly.
  • the rotor consists of a yoke which supports two diametrically 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 metal plate which together form a cavity of a specific configuration. When the associated plastic container of the kit is clamped between the insert and plate, the container takes the shape of the cavity.
  • the separation container holder is used to harvest white cells.
  • the small volume collection container (SVCC) is used to collect the mononuclear cells.
  • anticoagulated whole blood is pumped into the spinning separation container by the whole blood pump (WB/ACD pump).
  • the primary stage of separation takes place in the separation container, which is analogous to the "first spin" operation of a blood component collection process.
  • the higher density blood components primarily RBC's
  • PRBC packed red blood cells
  • the lower density components such as plasma, platelets and white cells can then be extracted by the plasma pump via the CRP (component-rich plasma) line.
  • the CRP is pumped into the collection container where the secondary stage of component separation takes place, which is analogous to the "second spin" of a blood component collection process.
  • the heavier component (white cells) is packed by centrifugal force against the back wall of the container.
  • the separated components remain packed within the container while the lower density components (plasma and platelets) exit the container via the CPP (component-poor plasma) line.
  • the CPP can then be recombined with the PRBC's and returned to the donor or collected in one of the Transfer Pack containers of the Apheresis Kit.
  • the 1-120 Stem Cell Program is used in this procedure for the collection of mononuclear cells (stored under Special Procedure I in the separator's memory). Mononuclear cells are those cells with one nucleus and are collected because the targeted cells are found in this fraction of leukocytes.
  • the 1-120 Program is slightly different from the original 1-100 Program.
  • the 1-120 Program includes several changes to reduce the blocked line alarms at spillover. It permits plasma flow rate settings according to the donor's hematocrit and permits optical settings according to the absolute mononuclear cell count of the donor.
  • This program can be used at draw rates between 20 and 80 ml/min by changing the draw rate using the front panel keys in the same method used in platelet apheresis. No program changes are made when changing draw rates.
  • Procedure 8 Baseline Cell Collection
  • the table edit feature allows the operator to alter the preset procedures which are stored as tables in the separator's memory and to store the altered version of the tables for future use. These tables govern the method used to control the pumps speed, centrifuge speed, end point, interface detector offset, clamps settings and algorithms used to compute the plasma pump speed during the automatic mode of operation.
  • the desired table to edit (the run table) is selected using either the location or contents keys.
  • the prime table is left unaltered as the prime cycle is identical for all procedures.
  • the interface setting (Location 71) is 120.
  • Location 71 It is important to accurately calculate the MNC count of the immunocompromised donor and adjust the value of Location 71 as appropriate.
  • the value of Location 71 is routinely programmed as 90 to reduce the amount of erythrocyte and granulocyte contamination in the final product. In general, Location 71 should always have a value of 90. Table 6 is provided to set guidelines for the programming of Location 71 should the Interface Optic Detector be set according to the donor's MNC count.
  • the Closed System Apheresis Kit is installed on the CS3000 Plus Separator. Ensure that the Separation Chamber is installed in the separation container holder clamp assembly and that the SVCC is installed in the collection container holder clamp assembly. Aside from the changes specified above, all other steps for priming the Closed System Apheresis Kit are unaltered.
  • the single up arrow key Press the single up arrow key to move to the next parameter which is whole blood flow rate.
  • the default for the blood flow rate is 50 ml/min. It is advisable to leave this parameter unchanged until the first Spillover occurs and the blood flow from the Inlet Needle is proven to be appropriate.
  • the plasma flow rate is left unchanged as the separator's computer determines this parameter according to the location contents programmed into the memory.
  • the centrifuge 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, and using the up or down arrow keys, select 90 and press the enter key again.
  • the interface detector baseline parameter is set by the computer's memory automatically, and should be left unaltered.
  • the plasma volume (Coll/Exch) is set to 200. Press the enter key once, the double up arrow key twice to select 200 and the enter key again.
  • the CS3000 Plus Separator pumps 200 ml of the prime into the 600 ml Transfer Pack of the Apheresis Kit at the beginning of the run.
  • the Apheresis Kit is filled with approximately 300 ml of priming solution, about 100 ml of this volume is ACDA anticoagulant.
  • the standard setting for the plasma volume parameter is 200.
  • the single access cycle volume and the transfer pack volume parameters are unaltered. The default for both is 0. These parameters are used only when performing a one-arm procedure.
  • press the display/edit key To accept the parameters entered and return to the prerun screen, press the display/edit key. Make sure that the drip chambers of the Saline and Anticoagulant containers are at least 1/2 full but no more than 3/4 full to facilitate determination of drip rates and to prevent air from entering the system.
  • Roberts clamp and heat seal the Sampling pack twice. Open the Return Line roller clamp to flush the line. Control the Saline flow rate with the Return Line roller clamp and maintain the site at KVO rate. Collect the required blood samples from the Sampling Pack using a Vacutainer needle and holder. The Sampling Pack may be detached from the Return Line or left connected. Prime the Inlet Line just to the tip of the needle. Press the mode key to select run. With run indicated in the message center, press the start/resume key twice to initiate the Auto Run. Immediately open the Inlet Line roller clamp and the Return Line roller clamp. The centrifuge and pumps start immediately.
  • the WBFR value is now 60. Press the display/edit key to accept and implement the change entered.
  • the blood (ml/min) display on the front panel should reflect the change in WBFR.
  • spillovers occur approximately every 3.5 minutes throughout the procedure. Every 15 minutes, the volume processed, the whole blood pump flow rate, the plasma pump flow rate, the anticoagulant drip rate, the centrifuge speed and the return plasma clarity are checked and recorded.
  • the separated plasma in the Plasma Return Line is visually inspected for hemolysis. A red tinge to the plasma in the Return Line is cause for evaluation (prior to reinfusion) to determine if this is from hemolysis or red cell contamination of the separated plasma.
  • message 61 Check Anticoagulant Container
  • message 61 alerts the operator to ensure that sufficient anticoagulant remains in the container to complete the procedure.
  • press the start resume key once.
  • the Auto Run continues until the end point is reached. If it is desired to terminate the procedure before the end point is reached, press the halt/irrigate key once. At this time, the mode and start/resume LEDs flash. Press the mode key to select Reinfuse. Then press the start resume key to initiate the Reinfuse operation. If it is desired to pause the procedure for any reason and later restart at the same point where paused, press the halt/irrigate key. Once the reason for pausing has been resolved, press the start/resume key to continue with the procedure.
  • the doors to the centrifuge compartment of the separator are opened and the multiple lumen tubing is removed from the upper support bar and the lower restraining collar.
  • the starting plasma flow rate is set according to the donor's Hematocrit
  • CS3000 begins collecting MNCs sooner and consequently has an overall higher efficiency of collection. While the computer selects the appropriate setting during the first liter processed, pre-setting the plasma flow rate speeds the process and improves yield by a few percent.
  • SVCC Small Volume Collection Chamber
  • the final volume of the SVCC is approximately 50 ml, it contains about 25 ml of cells and requires about 25 ml of space for plasma. About 2 ml of red and white cells are concentrated from donors with up to 3000 MNCs/ ⁇ L for each liter of whole blood processed.
  • the 1-120 Program collects an average of 60% +/- 25% of the MNCs that are processed, although the collection efficiency is very donor dependent.
  • ACD is important for cell collection.
  • the ideal ratio of WB:ACD is 10:1, which means using 100 ml of ACD per 1000 ml of whole blood processed. If not enough
  • ACD is used, it may cause the cells to clump and not separate finely for a pure collection.
  • Autologous stem cell rescue is used to restore hematopoietic function following high-dose chemotherapy-radiotherapy.
  • stem cells including bone marrow, peripheral blood, and umbilical cord blood.
  • DMSO dimethyl sulfoxide
  • the DMSO-Medium solution should be made on the same day as the cryopreservation.
  • the final concentration of DMSO when DMSO-medium is mixed with cells in a 1 :1 proportion is 10%.
  • the initial concentration of DMSO in medium must be twice that, or 20%.
  • the metal holders should be labeled with unique number, name, specimen type, date of birth, cryopreservation date and any other pertinent information such as hospital chart number.
  • a "holder”, "cassette” or “canister” are all metal plates used to press the specimen into an even layer and protect the frozen cryobag.
  • the Fenwal cryobags fit imperfectly but adequately in the metal holders custom-made by Stealcon, which in turn, fit into frames made by Custom Biogenic Systems, for storage in liquid nitrogen (Stericon, Broadview, IL).
  • Cryobags require similar information plus institution. Do not write directly on the cryobag plastic because the ink may diffuse into the bag. Instead write on the upper left and right corners of cryobags, or use cryobags with pockets. To ascertain the same freeze rate, each bag should contain the same volume. Chill holders while awaiting the specimen.
  • cryobags There are four different sizes of cryobags called Cryocyte Freezing Containers.
  • the plastic is PL269.
  • total volume in the 250 ml bag, Code 4R5461 (Changed to 4R9952), should not exceed 60 ml.
  • Each cryobag has two female leads ('pigtails') for transferring DMSO-medium and specimen. The plastic tubing adjacent to the bag seals easily in the Sebra sealer.
  • Fenwal cryobags (Baxter Health Care Corporation, Deerfield, IL) have been used with exceptional results ( ⁇ 0.06% breakage).
  • Cryovials This step is performed just before starting the freeze program. Cryovials are labeled with the patient's initals, specimen type and unique number, hospital number, and date, and are pre-chilled with the cryobags and holders.
  • DMSO-medium and vortex the "immix” tube Under the hood, loosen the caps on each.
  • Pipet volume of DMSO-medium equal to volume of specimen in "immix” tube, e.g., 2.1 ml, and add to "immix” tube of specimen. Dispose of pipet, tighten cap and vortex mixture. Loosen "immix” tube cap. Pipet specimen, put in cryovial, tighten cap, and submerge in wet ice. Repeat for each cryovial. Remove from hood, put in Styrofoam box and submerge partially in wet ice near freezer.
  • cryovials of specimen do not have the same eutectic point (Heat of Fusion ⁇ Phase Change) as their cryobag counterparts. Hence, the cryovials are placed in a Styrofoam blood tube box to stagger the rate at which they freeze.
  • Custom Biogenic Systems (CBS) program 1 includes several components
  • the DMSO-medium and specimen are mixed immediately before cryopreserving the specimen in order to limit the degree to which the DMSO, which is lethal to cells at room temperature, warms.
  • the mixing should be done as quickly as possible without compromising aseptic technique. All labeling should be done prior to mixing.
  • the controlled rate freezer should be close to the hood where the mixing is done and a refrigerator (4°C) directly beneath the freezer chamber or a few steps away, to prevent as much warming as possible. If the liquid nitrogen storage refrigerator is not located next to the freezer, then a dry shipper or a Styrofoam box containing liquid nitrogen can be used to transport the frozen specimen to the liquid nitrogen refrigerator.
  • cryobag- syringe If there is more than 60 cc of air, pat the coupled cryobag- syringe back under the hood, disconnect the syringe, eject some of the air from the syringe, reconnect it and place it in the plasma extractor to continue withdrawing air from the bag. Inject specimen, close clamp. Under hood, seal, pull apart and discard syringe and tubing. Put cryobag in holder. If there is more than one bag, refrigerate and repeat steps starting with selecting a bag a specimen.
  • the chamber temperature should be 0°C and holding. Open the chamber door and place the ribbon thermocouple on the center of the bag (now referred to as the reference bag). Close the holder carefully so as to not cut the thermocouple. Insert the holder horizontally, ports inward, in a horizontal rack.
  • Step 1.2 push "scan” on the computer, and on recorder push "start”. If there are other holders insert them quickly in the rack. Insert the box of cryovials upright in the chamber and close chamber door. As soon as the chamber returns to 0°C, push "run” once to advance the program to -1 min (Step 1.2). The specimen temperature should now drop at an appropriate rate and does not need to be monitored until it is about - 8°C. Use this time for documentation.
  • Step 1.3 engages, watch the recorder pen very carefully in order to detect specimen warming. Be prepared to push "run” as soon as heat of fusion occurs (a sudden rightward pen movement). After heat of fusion occurs, quickly return the specimen temperature to the same temperature at which heat of fusion occurred without incurring too rapid a cooling rate thereafter.
  • Step 1.7 advance to -17min cooling
  • Step 1.7 toggle between pausing (no coolant added) and a steady cool rate
  • Step 1.7 steady cool rate
  • the computer will alarm visibly and audibly when finished.
  • stem cells could follow the ideal freeze curve, they would cool at - l°C/minute until reaching " 45°C, thereafter cooling at -10°C/min until reaching
  • the rate at which the specimen returns to -16°C can be -5°C/minute or faster without compromising the specimen.
  • the rate does matter and should not exceed -2°C/minute. Any manipulations cooling or warming the chamber has a latent effect on the specimen rate after it returns to heat of fusion. The effect those actions have must be anticipated.
  • the cues to a successful freeze manually through heat of fusion are visual.
  • the specimen temperature will take 5 'boxes' to lower one (or two) degrees. Toggle between steady cooling (Step 1.7) and warming (Step 1.6 or opening the door), nursing the specimen back to the heat of fusion temperature while also endeavoring to ensure a rate of -1°C (or -2°C) thereafter.
  • the danger in adhering too closely to the formula above is that the chamber will cool too much or for too long, thus engendering a specimen cooling rate of much greater than -2°C /minute hereafter. Rates of more than 10°C per minute are detrimental to the specimen.
  • the following procedure is used when using the Isolex 300i for the immunomagnetic selection of CD34+ cells.
  • the reagents include one vial of PR34+ Release Agent Dulbecco's Phosphate Buffered Saline (Ca ++ and Mg ⁇ free), Immune Globulin; Intravenous (Human), Human Serum Albumin (25% Solution), Sodium Citrate (4% Solution), and Working Buffer (DPBS; Ca ++ and Mg " ⁇ free), supplemented with 1% HSA and 12% Sodium Citrate (v/v).
  • the working buffer should be prepared by mixing 3000 ml of DPBS, 360 ml of sodium citrate and 80 ml of 25% HSA.
  • the buffer may be stored in the refrigerator, but should be warmed to room temperature prior to use.
  • the buffer should be used within 24 hours of preparation.
  • the Dynabeads should be prepared fresh each day. Remove the contents of one vial of tube for a total of 20 ml. Expose the Dynabeads to the MPC-1 magnet for
  • the identification screen will appear. This screen is displayed while the instrument is undergoing self test. During the last portion of self test, text indicating the IsolexTM 300i software version number is displayed in the lower right corner of the screen. Without any further intervention by the user, the System Stop Verification display should appear. This is an internal test for the system. At this time the user should press "stop” on the keypad. The System Initialization Preparation display will appear. The operator should follow the instructions to clear the machine and then press “OK.” The System Initialization display will indicate the percentage of the System Initialization Tests completed. While the System Initialization screen is displayed, the scale and pressure reference values are obtained and the mechanical parts of the instrument are tested.
  • the "Select a Procedure” display will appear. Select the Positive Selection application by pressing the box beside “Positive Selection” and then pressing the "OK” box.
  • the "Install Set” display will appear. Remove the tray containing the disposable set from bag. Open the pump door. If possible, hold the tray lengthwise with the chamber next to weight scale arm. Hang the two large waste bags on the holder on the right side of the machine. Install the chamber in the rocker module. Hang both recirculation wash bags on Weight Scale 5. Hang the end product bag on Weight Scale 4. Hang the antibody bag on Weight Scale 3. Hang the release agent bag on Weight Scale 2.
  • the "Connect Buffer" display will appear.
  • the user should spike port of working buffer bag with disposable set buffer line spike and hang bag on Weight Scale 6, verify that clamps on the working buffer bag are open, and when the working buffer bag is stationary, press "OK.”
  • the Prime Set Display will appear.
  • the disposable set is automatically primed using a sequence of steps.
  • the display is updated to reflect the step in progress.
  • the weight scale values are also displayed.
  • the Add Release Agent screen is displayed.
  • the user should cleanse septum of the release agent bag on Weight Scale 2 with an alcohol wipe, add the contents of the syringe containing the release agent, check that the weight scale bags are hanging straight on the scales, and press "OK.”
  • the 'Add Antibody' display will appear.
  • the user should cleanse septum of the antibody bag on Weight Scale 3 with an alcohol wipe, add the contents of the syringe containing the monoclonal antibody, check that the weight scale bags are hanging straight on the scales, and press “OK.”
  • the 'Add Beads' display will appear.
  • the user should cleanse septum of the chamber with an alcohol wipe and inject contents of one vial of washed Dynabeads .
  • the instrument After connecting cell source bag, press OK. At this part, the instrument will automatically begin the selection procedure, running through the following steps: add buffer to reagent; platelet wash; antibody transfer; cells/antibody incubation; antibody wash; transfer cells to chamber; rosetting; chamber cell wash 1-3 release agent transfer cell release incubation; cell release rinse; clear released cells; release agent wash; and transfer cells to and product bag.
  • the Procedure Complete display will appear.
  • the user should perform the following steps: 1) Close clamp to end product bag (Weight Scale 4); 2) Heat seal the end product bag tubing; 3) Heat seal both pressure transducer tubing; 4) Heat seal buffer bag (Weight Scale 6) tubing; 5) Heat seal primary chamber tubing; 6) Heat seal cell source bag (Weight Scale 1) tubing; 7) Remove end product bag from Weight Scale 4 (ensure end product bag is properly labeled); 8) Remove disposable set from instrument and dispose of in accordance with all applicable regulations regarding biohazardous waste; and 9) Press “OK.” When "OK" is selected, the End of Procedure Selection display will appear.
  • the Fenwal Solution Transfer Pump is a positive displacement fluid pumping system intended for the accurate transfer of large and small volume laboratory solutions. It employs the Solution Transfer Pump, a Lifecell Transfer Set and Lifecell Tissue Culture Flasks to provide safe, fast and accurate pumping of a wide variety of fluids.
  • the system has three principal elements.
  • First is the Solution Transfer Pump, an electromechanical device which pumps opaque and transparent solutions to operator programmed volumes.
  • the pump features gravimetric weighing of pumped solutions to monitor the actual volume of solutions pumped.
  • the pumping unit is designed to perform two types of solution delivery for the laboratory; large volume (red and green pumps) and small volume (orange pump).
  • the red and green pumps are electronically connected to run simultaneously and transfer large volumes of solution.
  • the red and green pumps will be used solely for the delivery of fresh culture media to the culture flasks.
  • the orange pump operates individually and transfers small volumes of solution more accurately.
  • the orange pump will be used to transfer existing cell cultures from a flask to one or more flasks or in some instances it is not utilized at all.
  • Second is the Lifecell Transfer Set, a sterile, multiple use fluid transfer set
  • third is the Lifecell Tissue Culture Flask, a sterile, single use container.
  • the cell cultures being handled and processed are of cells collected from HIV seropositive individuals.
  • tissue culture flask seeding the addition of fresh media to cell culture flasks and the splitting of cell cultures, an efficient and safe method that safeguards the sterility of the cultures is essential.
  • developing a method that provides a closed system reducing the need for unnecessary exposure to biohazardous fluids and the use of sharps is of high priority.
  • the supplies and equipment include the Fenwal Solution Transfer Pump
  • the transfer and adapter sets are set up. Remove the Transfer Set from the pouch. Ensure that the Transfer Set Junction Cover is securely closed over the Junction port opening. Pre stretch each of the silicone segments before starting to place the Transfer Set on the Transfer Pump. Grasp the segment by the end connectors and slowly stretch approximately two inches and relax the segment. Load Transfer Set onto the Pump Module as follows: a) Open safety door on pump module; b) Insert transfer set junction into junction holder on pump module; c) Ensure the color coding of the pump segments matches the color coding of the pump heads.
  • the solution transfer pump is programmed. Press the volume control pad for the display to be programmed. The audible signal sounds once and the display flashes. Using the numeric keyboard, enter the volume for the corresponding solution. As data is entered, each new digit shifts sequentially from right to left in the display. Ensure that the volumes entered for each pump correspond to the solutions and amounts that are connected to each pump station. It is recommended that one half of the total amount of fresh Culture Media to be transferred be entered on each of the red and green pumps. This allows for the extension tube on the final container to be both primed and rinsed with Culture Media. Press specific gravity control pad for the display to be programmed. The audible signal sounds once and the display flashes. Using the numeric keyboard, enter the specific gravity for the corresponding solution. As data is entered, each new digit shifts sequentially from right to left in the display. A specific gravity of 1.00 may be used for most cell culture solutions.
  • the Lifecell Tissue Culture Flask of the desired size, the Cell Culture Media Container, the Plasma Transfer Set (Baxter Cat # 4C2243), the Terumo SCD312 Device/Welding wafers and the container with the cells to be seeded should be gathered inside the laminar flow hood.
  • the SCD312 connect the Plasma Transfer Set to the tubing already attached to the Cell Culture Media Container. Ensure that the roller clamp on this line is closed.
  • the Solution Transfer Pump is designed to accommodate solutions in collapsible containers. If the cells to be seeded are in a glass bottle or other noncollapsible container, the container must be vented to permit the solution to flow properly. Enter the volume of Culture Media to be delivered to the final container (Lifecell Flask connected to the pump junction). This volume must be divided between the red and green pumps. For example, if 500 ml of media are to be transferred into the final container, program the red pump for 250 ml and the green pump for 250 ml. Enter the volume of the cell suspension to be seeded by programming the orange pump.
  • the orange pump For example, if the total volume of the cell suspension is 200 ml, program the orange pump to deliver 200 ml if you want all the cell suspension to be transferred to the final container. Enter the specific gravity of the solutions being transferred on each pump. A specific gravity of 1.00 is normally programmed for each pump unless otherwise directed by the Head, Clinical Cell Production Facility.
  • the final container should be properly connected to the transfer set junction, the junction coupler clamp of the final container should be open while all the others are closed, the culture media container clamp should be open, there should be no kinking or clamping of the transfer set tubing or final container extension tube, and the total programmed volume should not exceed the volume capacity of the final container.
  • the pumping cycle is as follows: a) The red and green pumps simultaneously transfer the volume initially programmed into the red pump; b) The orange pump individually transfers the volume initially programmed into the orange pump; and c) The red and green pumps simultaneously transfer the volume initially programmed into the green pump.
  • the pumping cycle needs to be stopped, press the stop/mute pad.
  • the pumping cycle underway is aborted 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 aborting the cycle underway is not desired, simply open the pump module cover. This will cause the pumping cycle to be halted temporarily without the need for reprogramming the pumping cycle.
  • To restart the pumping cycle close the pump module cover and press the start pad. The pumping cycle previously underway will resume at the point where it was halted.
  • the green complete LED indicator illuminates and an audible signal sounds and pumping action automatically stops.
  • the volume displayed in the total delivered display should equal the sum of the volumes programmed into each pump. For example, using the volumes used as examples on Steps 7 and 8, the total delivered display should read 700 ml. A positive or negative deviation of one to two ml in the total delivered display is acceptable.
  • the volume in the culture container is now the sum of the initial culture volume plus the volume displayed in the total delivered display. For example, if the volume of the cell culture container before fresh media was added was 1000 ml and the total delivered display shows a volume of 500 ml (this volume should be equal to the sum of the programmed volumes of the red and green pumps +/- 2 ml), the cell culture container now holds 1500 ml. Store the cell culture and seal off the cell culture media container as specified in Section B above. Remove all the disposables from the Solution Transfer Pump and dispose of them in accordance to the Infection Control Manual. Disinfect the Transfer Pump with a 50% solution of Sodium Hypochlorite and water. Clean the Laminar Flow Hood with 70% Ethanol solution.
  • the culture is to be split into two new Lifecell Flasks with a total volume of 1000 ml each. 750 ml of the existing cell culture will be transferred into each individual Lifecell Flask. The total volume of fresh media required is 500 ml which will be equally divided (250 ml each) between the two new Lifecell Flasks. Therefore, program the pumps as follows: Red Pump (media) - 125 ml / Green Pump (media) - 125 ml / Orange Pump (cell culture) - 750 ml. The total volume transferred to each new flask will be 1000 ml.
  • the final container should be properly connected to the transfer set junction, the junction coupler clamp of the final container should be open while all the others are closed, the culture media container clamp should be open, there should be no kinking or clamping of the transfer set tubing or final container extension tube, and the total programmed volume should not exceed the volume capacity of the final container.
  • a positive or negative deviation of one to two ml in the total delivered display is acceptable. Close the roller clamp on the tube extension of the flask hanging from the hook of the Load Cell. Remove the flask from the hook and place next to the junction. The cell culture split is now complete. Using the Sebra Heat Sealer, seal the Lifecell Flasks' tubing lines connected to the "Y" juncture three times. Ensure that the roller clamps remain with the flasks. Disconnect the flasks from the "Y" juncture by separating the heat seal closest to the "Y" juncture. This way the flasks have two and a half seals in their lines and a half seal remains on each leg of the "Y" juncture.
  • the 2:4 split of cell cultures is identical to the 1 :2 split except for the following. Instead of two Lifecell Flasks, four are required, a double "Y" juncture from a 600 ml Transfer Pack with 8 Couplers is required instead of a single "Y" juncture (Baxter Cat # 4R2027), and a 5-Prong Manifold is added to the required supplies (Baxter Cat # 5C4446).
  • the junction couplers of the Lifecell Flasks are still sealed off but in this procedure it is done four times since there are four flasks.
  • the SCD312 is used to connect the flasks to the double "Y" juncture. The only difference is that a double "Y" juncture has four available legs which correspond with the number of Lifecell Flasks used in this procedure. This repeated for each flask.
  • the 5-Prong Manifold is used to convert the orange pump coupler from single to double - this eliminates the need to disconnect a culture container from the orange coupler to connect another. To do this, using the Sebra Heat Sealer, seal off three of the prongs on the 5-Prong Manifold.
  • the 5-Prong Manifold now has only two couplers at one end which will be used to connect the cell culture containers to the orange pump. On the other end of the manifold there is a coupler port. Connect the orange pump tubing coupler to this port of the manifold. Ensure that the clamps on the two remaining prongs of the manifold are closed. Using the couplers at the end of the prongs, connect the two culture containers to be split to the orange pump and hang the containers from the hooks on the pump module.
  • the volume on each cell culture container to be split will be the same. This simplifies the splitting process since once the transfer of cell culture and fresh media into a Lifecell Flask connected to the Transfer Set junction is complete, all that is required is that the roller clamp to that flask be closed, the flask removed from the Load Cell hook and replaced with an empty flask whose clamp is opened and the start pad pressed to start a new pumping cycle. In cases that the volumes of the two cell cultures being split are different, reprogramming of all the pumps would be necessary once the first culture container has been split. This is determined prior to starting the splitting process but does not require any other alteration in the procedure besides the reprogramming of the pumps between cultures.
  • lymphocytes require the delivery of both an antigen-specific signal as well as a simultaneous costimulatory signal.
  • interaction of the T cell receptor with the antigen-MHC complex may cause T cell clonal anergy or deletion.
  • CD28 can provide an important costimulatory signal.
  • CD8 + T cells The proliferative potential of T cells expanded in vitro is a major consideration for 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.
  • mixed populations of CD4 + and CD8 T cells stimulated in this manner will eventually result in a population that is all or mostly CD8 + .
  • the long-term growth of CD4 T cells has necessitated the addition of exogenous lymphokines and allogeneic feeder cells, which precludes a large-scale expansion of CD4 + T cells for the treatment of disease.
  • the procedure described here is a method for expanding purified CD4 + T cells independent of exogenous cytokines or feeder cells using anti-CD3 antibody (Ab) plus anti-CD28 Ab conjugated to magnetic beads.
  • Autocrine growth in normal donors is maintained for a 4-6 log 10 fold expansion, and in HIV + donors for a 3-5 log 10 fold expansion. These cells remain polyclonal and >97% CD4 + .
  • the addition of IL-2 enhances growth of lymphocytes, and thus this is an optional step in certain embodiments.
  • Activated cells secrete predominantly cytokines associated with T helper type I function.
  • MPC-1 Magnetic Particle Concentrator
  • the primary stimulation is referred to as S 1
  • the first restimulation is referred to as S2
  • the second restimulation is referred to as S3
  • the third restimulation is referred to as S4, etc.
  • the day of stimulation is referred to as DO
  • 24 hr after stimulation is referred to as Dl
  • 48 hr after stimulation is referred to as D2, etc.
  • S4D3 72 hr after the fourth stimulation (third restimulation).
  • the operation of the counter is based on the principle that particles (cells suspended in an electrolyte) passing an aperture through which electric current is flowing alter the electrical resistance of the electrolyte and give rise to changes in the current flow and voltage. The magnitude of these changes is directly proportional to the particle size and can be electronically converted to a particle count. Up to 500 particles are individually counted and sized per second, and particle size is determined independently of shape or orientation in solution. In most instruments a lower threshold control is available to select minimum particle size so as to eliminate counting of particles too small to be of interest.
  • the electronic counter utilizes side scatter to distinguish live from dead cells. The operation of the electronic counter is fully explained in the manufacturer's manual.
  • the sampling stand should be equipped with a 70 micron "long-bore" orifice tube for more accurate counting and sizing.
  • This long-bore aperture predisposes to clogging of the orifice but enhances size discrimination. This ensures that only live cells are counted by eliminating debris.
  • lymphocytes especially after beads have been added, the cells should be well dispersed to break up any clumps.
  • the lower gate for sizing should be set at 100 fl, and sample volume is set for 0.5 ml. For sizing stimulated cells, set the sizing (analysis) gate at 200 fl and maintain this gate throughout the culture period. This ensures that beads (4.5 micron diameter) are not counted and are electronically gated out.
  • CD34-fraction cells to X-VIVO 15® with 5% autologous human serum at a concentration of 1 x 10 per ml.
  • the culture may be started in a 300 ml Baxter Lifecell Flask if the starting amount of cells is at least 50 x 10 6 cells.
  • Baxter Lifecell® Flask and Solution Transfer Pump to culture cells see Example 8. Wash appropriate amount of tosylactivated beads coated with anti-CD3 plus anti-CD28 Ab (BB IND 6675) 3 ⁇ with X-VIVO 15® and add to cells, maintaining a concentration of 1 x 10 cells per ml.
  • Standard cell culture technique uses a microscope with a practiced eye to determine the optimal time of culture restimulation.
  • cell volume is used to determine the time of cell restimulation.
  • the use of an electronic cell volume determination can reliably discriminate activated from resting cells, and is quicker and easier than counting cells using a hemacytometer.
  • electronic cell volume determinations are subject to error due to the presence of cell clumps or beads.
  • electronic counting requires precise resetting of the instrument for cell populations of different sizes, possibly leading to inaccurate counts for heterogeneous cell populations and necessitating separate settings for counting of resting-and activated cells.
  • cell populations derived from tissues that may contain significant numbers of dead cells and cell clumps are difficult to size with an electronic counter. Overall, the electronic cell counter is best reserved for repetitive and rapid sizing of peripheral blood cells in suspension culture.
  • Resting T cells have a mean volume of -170 fl, with only a small deviation from the mean (less than 40 fl). Resting T cells from HIV donors have a slightly larger volume of -200 fl. The activated T cells will reach a maximum mean volume of 780-900 fl by Day 5-8 of stimulation. Also, the deviation from the mean will be larger (250-350 fl). Around Day 7-10, the mean cell volume and the deviation from the mean will begin to fall. Eventually the cells will return to a resting T cell volume. Restimulate the culture when the mean cell volume falls below 400 fl (usually between S1D12 and S1D20). The cells should not be allowed return all the way to a resting cell volume (this will be too late and the cells die).
  • CD28-stimulated cells from normal blood donors do not require addition of exogenous IL-2 to the medium for cell growth.
  • cells from some donors may have deficient IL-2 secretion, and in these cases, optimal cell growth is achieved by addition of rIL-2.
  • rIL-2 Chiron
  • rIL-2 Chiron
  • the magnetic beads may be removed from cell culture media using the Baxter Fenwal MaxSep® Magnetic Cell Separation System.
  • the Baxter Fenwal MaxSep® Magnetic Cell Separation System consists of the MaxSep Magnetic Cell Separator and the MaxSep Magnetic Cell Separator Disposable Set.
  • the larger Primary Magnet is a strong permanent magnet constructed of Neodymium-Iron-Boron Bars and engineered to provide optimal separation characteristics. It is used to attract the microbeads to the magnet surface allowing the remaining cells and suspension to flow out of the Primary Container.
  • the Secondary Magnet is designed to capture microbeads which may escape the Primary Magnet.
  • the residual cell/fluid mixture is collected in a container (Cell Recovery Container) and then further processed on the Baxter Fenwal Cell Harvester if desired.
  • the MaxSep System incorporates a peristaltic pump with a flow rate range of
  • the pump may be bypassed to allow processing of the cell suspension at a faster flow rate. This reduces the total processing time and the time the cells are maintained at room temperature out of the incubator which could affect cell viability.
  • other modifications are made on the MaxSep Disposable Set to both accelerate the separation process and to create a semi-closed system. These modifications ensure the sterility of the cell suspension and add protection to the processing staff.
  • antibody-coated paramagnetic microbeads are used to stimulate cell growth and expansion. Once the cell expansion goal is achieved, it is necessary to efficiently remove the microbeads prior to reinfusion into the patient-donor to prevent or reduce the possibility of undesirable side effects which may be caused by the infusion of these antibody-coated microbeads.
  • the magnetic cell separation procedure is performed the day of scheduled cell reinfusion and prior to cell harvesting or as desired.
  • Sebra Heat Sealer Carefully remove from the rest of the set and save. Store unused Plasmacell-C Set in a sealed plastic bag for future use; 2) Using the Sebra Heat Sealer, seal off the Secondary Chamber Line, the Priming Line and the Cell Recovery Line from the unmodified MaxSep Disposable Set. Seal the Secondary Chamber Line and the Priming Line as close to the "Y" connection as possible. The Cell Recovery Line is sealed off approximately 1 inch away from the Recovery Line Bushing eliminating the Pump Segment altogether. Carefully detach these tubing lines from the "Y” and save; and 3) Using the SCD312, weld the single leg below the "Y" juncture from the Plasmacell-C Disposable Set to the Cell Recovery Line - this line will become the Reservoir Bag Line. Weld one of the legs above the "Y" to the Priming Line - this line is the Priming Solution Container Line. The leg that remains is welded to the Secondary Chamber Line. Ensure that all clamps are closed and remain part of the setup.
  • Transfer Packs become the Cell Recovery Containers. Discard the spike connectors.
  • MaxSep Disposable Set is fully primed with the exception of the Cell Suspension Line and the 8-Couplers Manifold that will connect to the Cell Suspension Containers. Also, the Primary Separation Chamber contains air that will need to be expressed into one of the Cell Suspension Containers once they are connected to the 8-Couplers Manifold. Inspect the entire MaxSep Disposable Set for leaks, specially around SCD312 welds. If any leaks are found, consider the set contaminated and discard. Go back and set up a new disposable set.
  • Restraining Lock 90° so that the Port Restraining Arm can be lifted to the open position.
  • the flask must be at a lower level than the Primary Container for fluid to flow in the direction of the flask. Close both clamps once door closure is achieved. Position the Primary Magnet at a 45° angle and gently tap the Primary Magnet Door to dislodge any air bubbles that may have formed during manipulation of the container. Any bubbles present must be at the end of the Primary Container that has no ports and as far away from the Primary Line Coupler as possible. Place the Primary Magnet at a 15° angle. Ensure all clamps are closed.
  • the maximum flow rate using the peristaltic pump built into the MaxSep is 30 ml/min.
  • the MaxSep's pump is bypassed and gravitational force is used to feed the fluid through the primary and secondary separation chambers and into the recovery containers.
  • a semi-closed system has been developed to reduce the risk of exposure to biohazardous fluids. All other manufacturer's guidelines have been followed to the fullest extent possible.
  • Suspension Container currently being processed to empty, and close the clamp to the container. Close the Cell Suspension Line Clamp. Do not close the Primary Line Clamp. Allow the Primary Separation Chamber to partially empty. Close the Primary Line Clamp, the Secondary Chamber Line Clamp and the Reservoir Bag Line Clamp. Open the Secondary Magnet Door and remove the Secondary Separation Chamber. Open the Priming Solution Line Clamp and the Secondary Chamber lamp. Allow the Secondary Chamber to fill with Priming Solution and close the Secondary Chamber Line Clamp. With a massaging action resuspend the microbeads inside the Secondary Chamber. Elevate the Secondary Chamber so that it is higher than the Primary Separation Chamber. Open the Primary Chamber Line Clamp and allow the fluid in the Secondary Chamber to flow into the Primary Chamber. Close the Primary Chamber Line Clamp.
  • the Fenwal Harvester® System is a system for performing rapid, continuous, centrifugal separation of large volume suspensions.
  • the system consists of the Harvester and various disposable sets.
  • the Harvester is manually operated by means of a control panel.
  • Suspensions are processed within sterile disposable sets.
  • the system is designed to collect either particulate concentrate, supernatant or both.
  • the particulate phase of the suspension is centrifugally separated and collected within a belt-type centrifuge chamber while the liquid phase or supernatant is removed via a line for aseptic collection or disposal.
  • various monitors continuously check for abnormal conditions. A combination of visual and audible alarms keep the operator informed of the operating status and any abnormal conditions.
  • Cell culture volumes average approximately twelve liters per individual culture once the cell expansion goal is reached. It is advantageous to efficiently reduce the cell containing media to a manageable volume both for handling and for transfusion purposes.
  • the final component that is used for transfusion into the donor- recipient should be of a volume that will not cause fluid overload. At the same time, it is advantageous to achieve this volume reduction without incurring a significant loss of cells.
  • the Baxter Fenwal Harvester efficiently accomplishes these goals.
  • Plasmalyte-A is also the final cell suspension solution.
  • Cells collected from HIV seropositive donors are contained in the Cell Suspension Containers that are to be processed.
  • the Fenwal Harvester System and its sterile Disposable Sets provide a semi-closed system that protects the sterility of the cell cultures being harvested and reduces the risk of exposure to biohazardous fluids.
  • Cell culture harvest is performed the day of scheduled cell reinfusion and once the cultures have undergone the magnetic cell separation process for removal of the microbeads used for cell stimulation.
  • the tubes as follows: the Inlet Line (it has two small tubes with a Pump Segment and a Coupler) lays across the top of the Harvester; the Harvest Line (one small tube) lays across the top/back of the machine and the Waste Line is off to the left side.
  • the complete belt After the complete belt has been started into the slot, it can be easily pushed down until it is flush with the top surface of the Belt Support outer ring. Temporarily align the small tubing lines over the top of the Belt Support following their natural positioning.
  • the three Outlet Tubing Lines are on the left and the two Inlet Tubing Lines are on the right. Ensure that the tubing lines are not kinked or crossed as they go down into the Belt Slot. Also, confirm that the tubing lines are not twisted or kinked where they come out of the Restraining Collar. Tape the five tubing lines to the Rotor so as to avoid any twisting or kinking during the operation of the Harvester.
  • the tubing lines should lay flat against the Belt Support once they are taped down.
  • Preassemble the Waste/Harvest Containers as follows. Gather the Terumo SCD312 Sterile Connecting Device/Wafers (as needed), 1000 ml Lifecell Flask (1), 600 ml Transfer Pack (1), "Y" juncture from a 600 ml Transfer Pack with Eight Couplers (1), Plasma Transfer Set (1) and Sebra Dielectric Heat Sealer (1) inside a Laminar Flow Hood. Using the SCD312, attach the 1000 ml Lifecell Flask to one of the legs above the "Y" juncture. Close the clamp. Using the SCD312, attach the 600 ml Transfer Pack to the other leg above the "Y" juncture. Close the clamp. To the single leg below the "Y" juncture, connect a section of the Plasma Transfer Set with a spike connector at the opposite end using the SCD312 and close the clamp.
  • the Multiple Lumen Tubing should be straight and not twisted; b) the belt, associated tubing and cover should be in proper position and Cover Locking Handles should be in the locked position (red knobs out); c) Hex Strain Reliefs should be properly positioned in their respective Restraining Collars; d) the white Restraining Collar should be locked into place; e) the Multiple Lumen Tubing should be positioned completely inside the length of the Restraining Arm; f) the Multiple Lumen Tubing Retainer should be properly positioned; g) the front and top doors to the Centrifuge Compartment should be completely closed; h) Hemostats should be placed as specified - the Waste Line on the large tubing area close to the connection site of the three small tubes, and Inlet Lines of the Reservoir Bag; and i) All the clamps on the disposable sets should be closed except for the clamp between the Reservoir Bag and the Processing pump which remains open.
  • the LED display should indicate the rate at which the fluid is being pumped into the bag.
  • the Supply Pump flow rate should be set at 700 ml/min. If the flow rate is other than 700 ml/min, adjust the pump speed.
  • the LED display should indicate a flow rate of approximately 600 ml/min.
  • Affix an Identification Label to the Lifecell Flask that contains the cells. This label should include the date, donor information, recipient information, the phrase "for autologous use only", the processes the cells have undergone to this point, the suspension solution used, and a biohazard symbol. Place the final component container inside a leak-proof transporting container. Remove Disposables from the Harvester and Disinfect Harvester with a 50% Sodium Hypochlorite/water solution.
  • any fluid leakage or disposable set disconnection constitutes a break in sterility and the set must be discarded and replaced. If this happens during the priming procedure, simply discard the faulty disposable sets and install new sets. Should the break in sterility occur during the actual harvest process and not inside the Centrifuge Compartment of the Harvester, stop the Harvester pumps, clamp and Hemostat the Waste Line, stop the centrifuge, close all the clamps on the disposable sets and contain the fluid spill. Should a break in sterility occur inside the Centrifuge Compartment during the harvest process, the spill of fluid should trigger a Code 7 alarm. This alarm condition shuts down all the Harvester's operations until the condition is corrected and the mute button is depressed. All personnel in the processing room should leave the premises immediately to prevent exposure to any aerosol created by the spill.
  • This procedure may be used for the removal of magnetic beads from cultured CD3+28 T cells and concentration of cells on the Fenwal CS3000+, release criteria for the final cell product must be established before cell infusion.
  • the following materials were used: Coulter Multisizer lie; Trypan Blue 0.4% Solution, (Sigma Chemical, St. Louis, MO); Plasmalyte A with 1% Human Serum Albumin; Albumin 25% solution; and Gram Stain Kit, (Sigma).
  • Adequacy of magnetic bead removal may be assessed by microscopy according to the following protocol: 1) 1 x 10 cells are added to a 1.5 ml microfuge tube in 3 replicates. Thus, the residual beads contained in 3 x 10 cells are assessed; 2) The cells are lysed by addition of sufficient Triton X-100 to bring the final concentration to 1%; 3) The beads are pelleted by microfuge centrifugation, and the supernatant aspirated, being careful to leave a nearly dry pellet containing about 50 ⁇ l residual volume in each tube; 4) Use a pipet to gently resuspend the 50 ⁇ l, being careful not to create bubbles. Add the entire 50 ⁇ l to a microscope slide. Add a coverslip and scan the entire field for beads.
  • the bead removal release criterion for the final product should be less than 100 beads per
  • the cells are infused. Depending on the number of cells harvested and the target infusion number for a particular protocol, excess cells may be removed from the final bag and cryopreserved. The cells are thoroughly suspended in Plasmalyte A containing 1% human serum albumin in a final volume of 100 to 200 ml. The cells are reinfused unfiltered to the patient over 20 to 30 min and the patient should be monitored for adverse reactions as described herein. A leukocyte-reduction filter should not be used during administration of the final product. The cells should not receive gamma irradiation or be passed through x-ray irradiation devices designed to detect metal objects. Record the final disposition of the final product and any occurrence of adverse reaction.
  • infusions of 9.6 x 10 beads/kg were not toxic and the beads could not be detected in tissue sections.
  • in rodents given infusions of 8.3 x 10 beads/kg no toxicity was observed and beads were observed phagocytosed in cells of the reticuloendothelial system.
  • the residual beads in the final product may be quantified by measuring the beads in 3 x 10 cells. If ⁇ 100 beads/3 x 10 cells are observed, then infusions of 3 x 10 cells in a 70 kg patient would represent ⁇ 1 x 10 total beads or ⁇ 1.43 x 10 beads/kg. Proper coordination between the clinical facilities and the laboratory is advantageous throughout the harvest and infusion process.
  • Controlled haste is advantageous once cells have been harvested and removed from the cytokine containing culture medium.
  • the time from cell concentration to beginning cell infusion should not exceed 1 hour.
  • the final product should not be infused if more than 4 hours elapses after harvest.
  • the equipment and materials include the sample (pH 7-8), culture sample, 1 :10 dilution of culture sample heated at 70°C for five minutes, Stop Reagent (25% v/v glacial acetic acid in water), Limulus Amebocyte Lysate Test Kit, Chromogenic Substrate and Chromogenic Limulus Amebocyte Lysate (LAL).
  • This procedure describes a method of using the SpectraMax 340 and SOFTmax PRO software to determine the amount of endotoxin present in the cell culture.
  • the equipment and materials include: SpectraMax 340 Microplate Reader; SOFTmax PRO software; and Blank, Standards and Samples prepared in microplate.
  • the temperature may be adjusted and/or turned on or off by clicking the Incubator button located to the right of the READ icon. Open the drawer of the SpectraMax 340 by clicking on the Drawer button, the right button in the SpectraMax Status Bar. Place the microplate containing the Blank, Standards and Samples in the drawer. Close the drawer by clicking the Drawer button again.
  • the Blank, Standards, and first 10 Bags and their 1 :10 dilutions have been assigned wells. Delete or add wells if necessary. Delete wells by highlighting all undesired wells. Under the Group Pop-Up Menu in the Template Editor Tool Bar, click Clear. The SpectraMax 340 will no longer read these wells. Repeat if necessary. Add wells by highlighting the well desired. Under the Group Pop-Up Menu in the Template Editor Tool bar, click New. In the Group Settings window that appears, add the Name of the sample and, if necessary, select Unknown(Dilutions) under the Column Format section for Bag dilutions of 1 :10. Click OK when finished in the Group Settings window. Repeat if necessary.
  • PCR buffer 10X PCR buffer, 5 ml; 1st stage primer mixture, 1 ml; dNTP, 1 ml; MgCl 2 , 1 ml; Taq DNA polymerase, 0.2 ml; Distilled water to 45 ml.
  • a minimum of two positive DNA controls and one negative control should be included.
  • pipette 5 ml sample to each sample tube, making a final volume of 50 ml.
  • positive controls pipette 5 ml of each positive control DNA into separate positive control tubes.
  • DNA polymerase 0.2 ml, Distilled water 49 ml. Add the sample by carefully pipetting 1 ml from the first stage PCR reaction tube into the second stage reaction tube. Repeat the amplification reaction of this protocol as shown above.
  • PBMC peripheral blood mononuclear cell
  • Phenotype product to determine the degree of monocyte depletion and the composition of the final PBMC product.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Cell Biology (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Mycology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Hematology (AREA)
  • Oncology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Developmental Biology & Embryology (AREA)
  • Virology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
EP98957775A 1997-11-10 1998-11-10 Verfahren und behandlung von tumoren und tumorzellen mit ex vivo aktivierten t-zellen Withdrawn EP1030674A1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US6503197P 1997-11-10 1997-11-10
US65031P 1997-11-10
PCT/US1998/023954 WO1999024045A1 (en) 1997-11-10 1998-11-10 Methods for treatment of tumors and tumor cells using ex vivo activated t cells

Publications (1)

Publication Number Publication Date
EP1030674A1 true EP1030674A1 (de) 2000-08-30

Family

ID=22059894

Family Applications (1)

Application Number Title Priority Date Filing Date
EP98957775A Withdrawn EP1030674A1 (de) 1997-11-10 1998-11-10 Verfahren und behandlung von tumoren und tumorzellen mit ex vivo aktivierten t-zellen

Country Status (6)

Country Link
EP (1) EP1030674A1 (de)
JP (1) JP2001522806A (de)
AU (1) AU1395499A (de)
CA (1) CA2309206A1 (de)
NO (1) NO20002412L (de)
WO (1) WO1999024045A1 (de)

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7718196B2 (en) 2001-07-02 2010-05-18 The United States Of America, As Represented By The Department Of Health And Human Services Rapamycin-resistant T cells and therapeutic uses thereof
US8034334B2 (en) 2002-09-06 2011-10-11 The United States Of America As Represented By The Secretary, Department Of Health And Human Services Immunotherapy with in vitro-selected antigen-specific lymphocytes after non-myeloablative lymphodepleting chemotherapy
WO2004104185A1 (en) 2003-05-08 2004-12-02 Xcyte Therapies, Inc. Generation and isolation of antigen-specific t cells
US7435592B2 (en) 2003-05-13 2008-10-14 Immunovative Therapies, Ltd. Compositions for allogeneic cell therapy
PT2573166T (pt) 2004-02-26 2016-07-07 Immunovative Therapies Ltd Métodos para preparar células-t para terapia celular
US7592431B2 (en) 2004-02-26 2009-09-22 Immunovative Therapies, Ltd. Biodegradable T-cell Activation device
JP2010032444A (ja) * 2008-07-30 2010-02-12 Olympus Corp 生体組織処理装置
PL2704741T3 (pl) 2011-05-03 2018-02-28 Immunovative Therapies, Ltd. Sposoby postępowania z lekami biologicznymi zawierającymi żywe komórki
SG10201901391RA (en) 2011-05-03 2019-03-28 Immunovative Therapies Ltd Induction of il-12 using immunotherapy
WO2018039637A1 (en) 2016-08-26 2018-03-01 Juno Therapeutics, Inc. Methods of enumerating particles present in a cell composition
DK3644728T3 (da) * 2017-06-28 2022-10-31 Sci Group As Frysning af biologisk materiale
MA52114A (fr) * 2018-02-28 2021-01-06 Juno Therapeutics Inc Procédés de détection de particules présentes dans une composition cellulaire

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU617295B2 (en) * 1986-11-13 1991-11-28 Sloan-Kettering Institute For Cancer Research Compositions and method for treatment of cancer using monoclonal antibody against gd3 ganglioside together with il-2
CA2133075A1 (en) * 1992-04-07 1993-10-14 Craig B. Thompson CD28 Pathway Immunoregulation
ES2240962T3 (es) * 1993-06-04 2005-10-16 The United States Of America As Represented By The Secretary Of The Navy Metodo para estimular selectivamente la proliferacion de celulas t.
CA2227327A1 (en) * 1995-07-25 1997-02-13 Celltherapy, Inc. Autologous immune cell therapy: cell compositions, methods and applications to treatment of human disease

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9924045A1 *

Also Published As

Publication number Publication date
CA2309206A1 (en) 1999-05-20
WO1999024045A1 (en) 1999-05-20
AU1395499A (en) 1999-05-31
NO20002412L (no) 2000-07-07
JP2001522806A (ja) 2001-11-20
NO20002412D0 (no) 2000-05-09

Similar Documents

Publication Publication Date Title
Zheng et al. CTLA4 signals are required to optimally induce allograft tolerance with combined donor-specific transfusion and anti-CD154 monoclonal antibody treatment
Trial to Reduce Alloimmunization to Platelets Study Group Leukocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusions
Brett et al. Repopulation of blood lymphocyte sub‐populations in rheumatoid arthritis patients treated with the depleting humanized monoclonal antibody, CAMPATH‐1H
US7638121B2 (en) Methods for human allografting
US7718196B2 (en) Rapamycin-resistant T cells and therapeutic uses thereof
US5658564A (en) Xenograft thymus
US20050118142A1 (en) Cellular compositions which facilitate engraftment of hematopoietic stem cells while minimizing the risk of gvhd
JP2002502823A (ja) 移植における補刺激遮断および混合キメラ現象
EP1030674A1 (de) Verfahren und behandlung von tumoren und tumorzellen mit ex vivo aktivierten t-zellen
JP7262568B2 (ja) 免疫エフェクター細胞を使用して腫瘍を治療する方法
AU678179B2 (en) Methods for positive immunoselection of stem cells
Natarajan et al. CD4 depletion or CD40L blockade results in antigen-specific tolerance in a red blood cell alloimmunization model
EP1285577A1 (de) Chimäre maus, deren immunsystem mithilfe menschlicher cd34-positiver zellen erzeugt ist, und ihre verwendung(26.11.01)
JP2006528627A (ja) アロ反応性ナチュラルキラー細胞を使用する治療用抗体の有効性を増加するための方法および組成物
US8394368B2 (en) Method for producing a composition for promoting survival of transplanted hematopoietic stem cell
Van Gool et al. Expression of B7-2 (CD86) molecules by Reed–Sternberg cells of Hodgkin’s disease
Zhu et al. Simultaneous blockade of costimulatory signals CD28-CD80 and CD40-CD154 combined with monoclonal antibody against CD25 induced a stable chimerism and tolerance without graft-versus-host disease in rat
Keever-Taylor et al. Complement-mediated T-cell depletion of bone marrow: comparison of T10B9. 1A-31 and Muromonab-Orthoclone OKT3
Cowan et al. Tolerance induction post in utero stem cell transplantation
AU694120B2 (en) Xenograft thymus
Petrányi et al. Tolerance Induction: Historical and Scientific Backround and Recent Development in Clinical Practice.
Yuan et al. Tolerance is the achievable ‘Holy Grail’in transplantation
BOLEA BAILO et al. Mixed hematopoietic chimerism and immune tolerance through bone marrow transplantation and infusion of regulatory T cells in a preclinical large animal model
Alonso Guallart et al. Mixed hematopoietic chimerism and immune tolerance through bone marrow transplantation and infusion of regulatory T cells in a preclinical large animal model
Quinones Cellular Engineering of the Hematopoietic Graft

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20000516

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

17Q First examination report despatched

Effective date: 20020711

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20021122