Multifunctional immunotherapeutic monoclonal antibody complexes and conjugates
12594341 ยท 2026-04-07
Inventors
Cpc classification
A61K35/17
HUMAN NECESSITIES
C07K16/2809
CHEMISTRY; METALLURGY
A61K40/11
HUMAN NECESSITIES
C07K2317/73
CHEMISTRY; METALLURGY
A61K40/15
HUMAN NECESSITIES
A61K39/3955
HUMAN NECESSITIES
A61K47/6849
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
A61K47/68
HUMAN NECESSITIES
A61K35/17
HUMAN NECESSITIES
A61K39/00
HUMAN NECESSITIES
A61K39/395
HUMAN NECESSITIES
A61K40/11
HUMAN NECESSITIES
A61K40/15
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
Immunotherapeutic Monoclonal Antibody Complexes or Conjugates (IMAC) comprising readily accessible antibodies designed and approved for clinical use are provided using a one-step method that combines killing of existing cancer cells in parallel with induction of long-lasting anti-cancer vaccination. Methods for their use, alone or in combination with cancer killer cells including intentionally mismatched donor T cells, NK cells concomitantly with additional anti-cancer or immune activating agents, or activation of patient's own immune system for personalized treatment of cancer and elimination of undesirable non-malignant cells are also provided. In addition, treatment method based on IMAC can be applied for in vivo vaccination against cancer using an existing malignant lesion as internal anti-cancer vaccine by engagement of patients antigen presenting cells for induction of long-lasting anti-cancer vaccination in situ against residual or recurrent disease.
Claims
1. A method for treating a subject having cancer, the method comprising administering to the subject: a) one or more Immunotherapeutic Monoclonal Antibody Complexes or Conjugates (IMAC) comprising at least two different monoclonal antibodies, wherein each antibody comprises two hypervariable regions (F(ab)2 domains) and a constant region (Fc domain), and wherein the at least two different antibodies are linked through an Avidin-Biotin connection between biotin moieties covalently coupled to the antibodies and an avidin moiety, wherein the IMAC comprises a first antibody capable of binding to a lymphocyte; and a second antibody capable of binding to at least one antigen on a tumor cell or tumor-specific blood vessel, and b) one or more preactivated mismatched allogeneic lymphocytes.
2. The method of claim 1 wherein the IMAC comprises an antibody capable of binding to a T cell, an NK cell or an NKT cell.
3. The method of claim 1, wherein the method further comprises administering a moiety capable of activating the immune system or an anti-cancer moiety.
4. The method of claim 1, wherein the IMAC comprises antibody binding regions to at least 2 different cell-surface antigens.
5. The method of claim 4, wherein the IMAC comprises a combination of antibodies to two cell-surface antigens of malignant cells selected from the group consisting of: CD19 and CD20; Her2/neu and EGFR; Her2/neu and GD2; Her2/neu and MUC-1; CD38 and CD138; and CD34 and CD133.
6. The method of claim 1, wherein the IMAC comprises at least one monoclonal antibody specific for regulatory T cells or for a cell that can suppress induction of anti-cancer immunity.
7. The method of claim 1, wherein the IMAC comprises at least one antibody is specific to a checkpoint inhibitor.
8. The method of claim 1, wherein the IMAC comprises at least one antibody capable of binding to T cells, NK cells, dendritic cells or macrophages through a domain selected from an F(ab)2 (hypervariable region) domain and an Fc domain (constant region).
9. The method of claim 1, wherein the IMAC comprises at least one antibody capable of binding through its F(ab)2 or Fc domain, to an Fc receptor on activated immune cells selected from the group consisting of NK cells, T cells, NKT cells and macrophages.
10. The method of claim 1, wherein the IMAC comprises: (1) at least one antibody specific to T cells or NK cells selected from the group consisting of: anti-CD2, anti-CD3, anti-CD4, anti-CD8, anti-CD28, anti-CD25, anti-CD80, anti-CD86, anti-CD45RA, anti-CD45RO, anti-CD 134, anti-CD196, anti-CD197, anti-CD62L, anti-CD69, anti-CTLA-4, anti-PD-1 and anti-PD-L1; or (2) at least one antibody specific to dendritic cells or macrophages, wherein said antibody is selected from the group consisting of: anti-HLA-DR, anti-CD16, anti-CD56, anti-NKG2D, anti-NKG2A, anti-CD94, anti-CD11b, anti-CD14, and anti-CD136; and/or (3) at least one antibody specific to mesenchymal stromal cell markers (MSCs) selected from the group consisting of: anti-CD73, anti-CD105, anti-CD90 and anti-CD200.
11. The method of claim 1, wherein the IMAC comprises an antibody selected from the group consisting of: anti-CD19, anti-CD20, anti-EGFR, anti-HER2, anti-GD2, anti-CD30, anti-CD37, anti-CD38 and anti-CD138.
12. The method of claim 1, wherein the IMAC comprise 3 or 4 antibodies.
13. The method of claim 1, wherein the method comprises selecting a combination of at least two different antibodies suitable for treatment of the subject, creating an IMAC by connecting the at least two antibodies, and administering to the subject a pharmaceutical composition comprising the IMAC.
14. The method of claim 1, wherein the cancer is hematologic malignancy.
15. The method of claim 1, further comprising administering a lymphocyte activating agent.
16. The method of claim 1, wherein the preactivated mismatched allogeneic lymphocytes were pre-activated by cytokines.
17. The method of claim 1, wherein the preactivated mismatched allogeneic lymphocytes were pre-activated by IL-2.
18. A pharmaceutical composition comprising a) at least one Immunotherapeutic Monoclonal Antibody Complexes or Conjugates (IMAC) comprising at least two different monoclonal antibodies, wherein each antibody comprises two hypervariable regions (F(ab)2 domains) and a constant region (Fc domain), and wherein the at least two different antibodies are linked through an Avidin-Biotin connection between biotin moieties covalently coupled to the antibodies and an avidin moiety, wherein the IMAC comprises a first antibody capable of binding to a lymphocyte; and a second antibody capable of binding to at least one antigen on a tumor cell or tumor-specific blood vessel, b) preactivated mismatched allogeneic lymphocytes; and c) a pharmaceutically acceptable carrier, diluent or excipient.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
DETAILED DESCRIPTION OF THE INVENTION
(17) The present invention provides molecules, compositions and methods for immunotherapy, in particular against all MHC incompatible cancer cells, including multi-drug resistant and cancer stem cells. The present invention provides methods and compositions for direct elimination of cancer cells combined with induction of anti-cancer vaccination, by a 2-stage procedure, first selective targeting of mismatched killer cells against cancer cells followed by induction of anti-cancer immunity by patient's own antigen presenting cells and T cells using multi-specific combinations of complexes or conjugates of antibodies termed IMAC. The IMAC may consist of avidin's high affinity binding of four biotinylated antibodies and/or other potential biotinylated anti-cancer agents, or only dual binding of monoclonal anti-T cell antibody and anti-cancer antibody complex or conjugate, or even two monoclonal antibodies connected with beads or biotin-binding beads.
(18) In accordance with the invention the targeted cells include activated allogeneic donor lymphocytes, haploidentical or unrelated fully mismatched off-the-shelf activated killer cells including T cells, NK cells (not excluding NKT cells) as killers of targeted cancer cells. Activation of anti-cancer effector cells can be accomplished by cytokine activation ex vivo prior to cell infusion, followed by continuous in vivo activation of killer cells while they last until being rejected using low dose IL-2 or other cytokines to maximize anti-cancer activity for a few more days until being rejected.
(19) The antibodies included in the complexes and conjugates of the invention are directed against all killer cells (T cells, NK cells and NKT cells) while recruiting antigen presenting cells (dendritic cells and macrophages) via F(ab)2 or Fc domains. IMAC is applied to target cancer-specific or cancer-associated cell-surface antigens, or for elimination of any other undesirable cells or infectious pathogen, depending on the type of clinical condition that needs to be treated.
(20) Any anti-cancer antibody, anti-angiogenic antibody, antibody that can activate the immune system of the patient, or down-regulate suppressor cells, antibody that subject cancer cells to the immune system by elimination of protective cancer's microenvironment (e.g. MSCs and antibodies against adhesion receptors and chemokine receptors), or antibody against any cancer associated target known in the art, can be used in the IMAC of the invention, including commercially available antibodies or new monoclonal antibodies that are currently under development.
(21) The connection of the antibodies may be performed by any means known in the art, for example, by using avidin-biotin non-covalent binding, or by linkers forming covalent or non-covalent binding. The antibodies may be also conjugated using nanoparticles which may be biodegradable, magnetic and/or positively charged and/or comprise additional targeting, binding or activating moieties to improve anti-cancer potential as long as both monoclonal antibodies will jointly and simultaneously target cancer cells.
(22) The biotin may be coupled to the antibodies by any connection known in the art, including but not limited to amide bond, thioether bond, disulfide bond, hydrazone bond, and azido bond. Different antibodies in the immunoglobulin complex may be biotinylated using different connection methods.
(23) Any avidin molecule or avidin derivative capable of binding non-covalently at least two biotins, preferably four biotin binding sites, can be used according to the present invention. This includes natural, modified or genetically produced multivalent avidin molecule. According to some embodiments, the avidin used to conjugate the biotinylated monoclonal antibodies is selected from the group consisting of egg-white avidin, streptavidin and NeutrAvidin (a deglycosylated version of avidin or even dual affinity avidin).
(24) The dissociation constant of avidin-biotin is in the range of 10.sup.15M. As such it forms exceedingly strong non-covalent bonds. The avidin molecule has four binding sites thereby it is capable of binding together up to four biotinylated antibodies. Any side chain or free group on the antibody may be used to bind a biotin moiety using any method known in the art. According to some embodiments, antibodies are readily biotinylated via their lysine molecules. The number of biotin molecules on each of the labeled antibodies is carefully monitored in order to avoid excess biotinylation that may harm the antibody's binding activity.
(25) Complexation of different biotinylated monoclonal antibodies may be accomplished by non-covalent binding to avidin (NeutrAvidin and streptavidin), a molecule that can bind 4 similar or different biotinylated monoclonal antibodies. The clinical use of avidin-biotin complexes is not toxic and already approved for different indications, especially for cancer diagnostic and therapy with radiolabeled antibodies (24). Complexes of monoclonal antibodies are also available with biodegradable, nanoparticles such as immunomagnetic beads.
(26) Immunotherapeutic Monoclonal Antibody Complexes according to the present invention may be created together with nanoparticles as carriers. Such nanoparticles may be biodegradable polymeric scaffolds and/or immunomagnetic beads. Commercially available nanoparticle immunomagnetic beads connected to monoclonal antibodies, that are approved for intravenous use, may be also used according to the present invention. As such, the binding of the monoclonal antibodies to the tumor could be induced by non-covalent binding of two or more monoclonal antibodies to the beads.
(27) According to some embodiments, antibody complexes comprising magnetic beads are used to select only activated NK cells and therefore eliminate or avoid any potential risk of cytokine release syndrome mediated primarily by T cells.
(28) According to some embodiments, the nanoparticles or beads used to complex the antibodies are positively charged in order to better attract to negatively charged cancer cells.
(29) The IMAC comprises for example 2 to 4 biotinylated monoclonal antibodies, or 3 biotinylated monoclonal antibodies and one additional biotinylated anti-cancer agent, some directed against antigens expressed on the malignant cells (or any other cell or infectious agent that needs to be eliminated) and some against effector cells of the immune system or antigen presenting cells (T cells, NK cells, NKT cells, dendritic cells & macrophages), complexed via an avidin moiety. The different antibodies are complexed non-covalently by Biotin-Avidin (NeutrAvidin or StreptAvidin) linkage or otherwise conjugated covalently. The antibodies may be also conjugated with microparticles such as beads that may be according to some embodiments, biodegradable and/or magnetic. In addition to monoclonal antibodies, avidin can be used to bind other biotinylated compounds that can amplify the efficacy of anti-cancer immunotherapy (e.g., biotinylated IL-2 or 4-1BB, etc.) or even biotinylated chemotherapy molecules or radioactive isotopes or oncolytic virus, etc.
(30) According to some embodiments, IMAC can be constructed using chemical linkers that can bind at least two monoclonal antibodies and optionally an anti-cancer agent and/or an immuno-activator (e.g., cytotoxic, radioactive, immune activators such as IL-2 or 4-1BB, etc.).
(31) IMAC can be used primarily for immunotherapy against cancer by combining eradication of cancer cells including cancer stem cells resistant to available anti-cancer modalities by activated mismatched donor lymphocytes, however, due to in vivo activation of donor lymphocytes with IL-2, also patient's own immune system cells may be activated and add to induction of anti-cancer immunity followed by induction of anti-cancer vaccination by patient's long-lasting memory T cells. Cancer Immunotherapy with IMAC is mediated by the capacity of IMAC to bind killer cells and/or antigen presenting cells via F(ab)2 or Fc portion of each of the monoclonal antibodies complexed in the IMAC to activate autologous, haploidentical or unrelated off-the-shelf intentionally mismatched unrelated lymphocytes (T cells, NK cells & NKT cells). Killing of otherwise resistant malignant cells, including cancer stem cells that are a priori resistant to all available anti-cancer modalities, can be accomplished by mismatched activated donor lymphocytes that induce effective anti-cancer effects based on the most effective mechanism of rejection of any MHC mismatched target cells that starts immediately following infusion and lasts until mandatory rejection of intentionally mismatched donor lymphocytes. In contrast, immunotherapy with IMAC based activation of patient's own immune system cells as long as IMAC persists results in induction of anti-cancer immunity and memory T cells that are not expected to be rejected.
(32) Killing of cancer cells can also be accomplished using IMAC that consists of radiolabeled biotinylated monoclonal antibodies or biotinylated antibodies anti-cancer chemotherapeutic agents complexed with avidin too. The therapeutic use of avidin is not hampered by anti-avidin antibodies thus encouraging its further use for different clinical indications (25). For example, using radiolabeled somatostatin analogues or radiolabeled biotin complexed with other biotinylated monoclonal antibodies with avidin can be used for treatment of neuroendocrine pancreatic cancer (26). For example, .sup.90Y-labelled biotin can be targeted to breast cancer cells by conjugation with biotinylated monoclonal antibodies against Her2/neu and also additional antibodies to target breast cancer and antigen presenting cells that can be triggered by cancer antigens released in the process of cytotoxicity (27). As such, in parallel with elimination of breast cancer metastases, as one example, anti-cancer immunity can also be established against residual or recurrent disease. Alternatively, antibodies complexed with biodegradable and/or immunomagnetic nanoparticles can be used, wherein such nanoparticles are optionally positively charged and/or include other anti-cancer properties.
(33) Since induction of long-lasting anti-cancer immunity that can control residual malignant cells or recurrent disease is established by treatment with IMAC, the method can be used for intentional induction of anti-cancer immunity instead of the conventional preparation of dendritic cells pulsed with cancer antigens that may not be available, as diagrammatically described in
(34) Binding of IMAC complex using avidin, can also result in selective and more effective anti-cancer immunotherapy by elimination of inhibitory cells (regulatory T cells, checkpoint inhibitors, myeloid-derived suppressor cells and mesenchymal stromal cells) that can impair development of effective anti-cancer immunotherapy on the one hand, and protect the microenvironment of cancer metastases from an attack by the immune system, in parallel with induction of anti-cancer effects.
(35) As such, antibody-complexes or antibody-conjugates according to the present invention may be designed to target not only cancer cells but also other undesired cells such as cancer-supporting blood vessels in addition to cells involved in suppressive microenvironment (e.g., regulatory T cells, checkpoint inhibitors, myeloid-derived suppressor cells, tumor associated macrophages and mesenchymal stromal cells that convey resistance at the tumor microenvironment).
(36) In addition to treatment of cancer, IMAC according to the present invention may also be utilized for treatment of non-malignant indications, e.g. for elimination of normal hematopoietic cells as part of the conditioning in preparation for autologous or allogeneic stem cell transplantation for treatment malignant and non-malignant diseases (e.g. autoimmune diseases, etc.) and also for elimination of undesirable genetically abnormal bone marrow stem cells in preparation for allogeneic stem cell transplantation for genetic diseases using monoclonal antibodies against hematopoietic stem cells (e.g., anti-CD34 or CD133). Accordingly, IMAC could be used for elimination of normal hematopoietic stem cells (using for example biotinylated monoclonal anti-CD34 or anti-AC133), to induce bone marrow myeloablation, thus replacing the need for hazardous myeloablative chemotherapy and/or total body irradiation, or using anti-lymphocyte antibodies to eliminate transiently patient's immune system indicated in preparation for stem cell transplantation for treatment of hematologic malignancies, or for induction of mixed chimerism aiming for induction of transplantation tolerance for safer durable organ transplantation while avoiding the use of life-long immunosuppressive treatment to prevent allograft rejection.
(37) In principle, IMAC could also potentially be used for elimination of infectious pathogens or cells infected with infectious agents using relevant antibodies (e.g., anti-HIV antibodies).
(38) In addition to systemic administration, IMAC can also be injected into the primary tumor that cannot be surgically removed or into any visible easily accessible tumor metastasis alone or in combination with additional agents that can amplify anti-cancer immunity to induce local anti-cancer cytotoxicity followed by systemic anti-cancer vaccination against remote metastases, inducing the so called abscopal effect as featured in
(39) The present invention also provides the possibility of rapid design and manufacture of personalized anti-cancer agents against a broad spectrum of clinical indications at the patient's bedside using off-the-shelf activated donor lymphocytes and off-the-shelf panel of biotinylated monoclonal antibodies. The use of avidin based IMAC, readily available activated donor lymphocytes and relevant biotinylated antibodies, allow to prepare suitable anti-cancer treatment a soon as indicated with no technical delay.
(40) According to some embodiments, commercial immunomagnetic beads (approved for injection in vivo) used for positive or negative selection of cells, conjugated with relevant monoclonal antibodies against any desirable cell of the immune system, can be also connected covalently or non-covalently with more than one antibody to form IMAC and used for cancer immunotherapy.
(41) According to certain embodiments, the immunomagnetic beads are selected from the group consisting of anti-biotin microbeads, immunomagnetic beads, CliniMACS CD1c (BDCA-1), CliniMACS CD4, CliniMACS CD8, CliniMACS CD14, CliniMACS CD25, CliniMACS CD34, CliniMACS CD56, CliniMACS CD133 and CliniMACS CD304 (BDCA-4).
(42) Subcutaneous injection of low dose IL-2 may be sufficient for continuous activation of mismatched donor lymphocytes in vivo following infusion. Higher doses of IL-2 can also be used when attempting activation of patient's own lymphocytes that may be activated together with IMAC application. In a preferred embodiment, IL-2 (or another activating cytokine or agent), can also be administered prior to administration of the IMAC when using freshly obtained unmanipulated donor lymphocytes, MHC identical, haploidentical or unrelated and fully mismatched for treatment of patients in centers where the use of in vitro processed lymphocytes is unapproved, being considered advanced therapeutic medicinal product (ATMP), in order to induce in vivo activation of allogeneic anti-cancer effector cells in case it is mandatory to avoid the use of a procedure considered ATMP.
(43) Although the use of IMAC is primarily designed to eliminate resistant cancer cells and induce long-lasting anti-cancer immunity, the principles behind the IMAC invention can also be used as anti-cancer vaccine as shown diagrammatically in
(44) The therapeutic effects of IMAC may be further enhanced by using IMAC after systemic down-regulation of all known inhibitors of the immune responses such as regulatory T cells, CTLA-4, PD-1/PDL-1, myeloid-derived suppressor cells (MDSC), mesenchymal stem cells (MSC) inflammatory cytokines. Alternatively, IMAC may be used for suppression of checkpoint inhibitors (e.g. using biotinylated ipilimumab and nivolumab or pembrolizumab), or against MSCs (e.g. biotinylated monoclonal antibodies against CD90, CD73, or CD 105) or against MDSC (e.g. against CD14).
(45) According to yet additional embodiments, IMAC can be used for safer and more effective myeloablative/immunosuppressive conditioning in preparation for autologous or allogeneic stem cell transplantation for treatment of cancer and genetic disorders. Alemtuzumab-based IMAC can also be used to eliminate self-reactive immune system cells in patients with life-threatening autoimmune diseases. Using IMAC, optimal immunosuppression and myeloablation can be accomplished combining the use of biotinylated alemtuzumab (anti-CD52 monoclonal antibodies that can target both T cells and B cells) and biotinylated antibodies against CD34 or 133 for elimination of hematopoietic stem cells. Following transplantation of T cell depleted autologous or allogeneic hematopoietic stem cells, newly derived T cells can be easily tolerized similarly to induction of self-tolerance in utero, or to induction of neonatal tolerance, thus resulting in re-induction of self-tolerance in patients with autoimmune diseases or in recipients with cancer or genetic disorders, respectively. Effective elimination of donor's immune system by targeting all T cells and B cells may be indicated for rescue of life-threatening GVHD following unsuccessful allogeneic stem cell transplantation.
(46) The use of lymphoablative/immunosuppressive IMAC may be also indicated for induction of transplantation tolerance to cadaveric or living related donor's bone marrow and organ allografts. Co-transplantation of donor's bone marrow cells at the time of harvesting any donor's organ results in life long transplantation tolerance and the use of IMAC for intensive immunosuppression can facilitate engraftment while preventing the risk of graft-vs-host disease (GVHD). Induction of transplantation tolerance to organ allografts is yet unmet need and considered the holy grail of organ transplantation in order to avoid the risks of mandatory life-long immunosuppressive treatment that is currently unavoidable.
(47) In contrast to the use of conventional bispecific antibodies against a limited number of cancers, the use of IMAC provides a large number of patients with a broad spectrum of malignant disorders including all hematologic malignancies and metastatic solid tumors, an opportunity to benefit from readily available and constantly growing number of immunoglobulin complexes and conjugates. These immunoglobulin complexes and conjugates are composed, in some embodiments, of approved monoclonal antibodies available in the market, in an easy, fast, inexpensive and tailor-made methods. In contrast to bispecific antibodies capable of binding to cancer or killer cells with a single domain via a single chain Fab, treatment with IMAC is designed to induce more robust and higher affinity and stronger avidity by binding via two binding domains of each F(ab)2 of at least two monoclonal antibodies directed against both cancer cells and anti-cancer effector cells simultaneously, thus doubling the number of binding domains of each antibody and with the potential to attack different cancer antigens on the same malignant cells simultaneously. According to some embodiments of the invention, the multi-potent antibodies complexed with a larger number of anti-cancer effector cells via avidin molecule can bind up to 4 different biotinylated monoclonal antibodies. According to some embodiments of the invention, the multi-potent antibodies complexed with a larger number of anti-cancer effector cells via avidin molecule can bind three monoclonal antibodies with one additional biotinylated anti-cancer agent.
(48) The IMAC of the invention provides the possibility of personalized treatment by designing disease-specific complexes or conjugates of antibodies using relevant antibodies from readily available sources and against many types of antigens. The antibodies are selected from available sources based on target expression on the same cancer cells, cancer-supporting blood vessels, and/or cancer protective microenvironment. In addition, the treatment methods of the invention make it possible to induce effective anti-cancer vaccination by production of memory cells against residual or recurrent disease.
(49) The antibody complexes or conjugates of the invention can be used for treatment of different types of cancer inexpensively at an optimal timing with no need to depend on long-term cultures using a GMP facility as used for preparation of CAR-T cells or tumor infiltrating lymphocytes (TIL) that is not available in most oncology centers and rarely if ever result in cure, certainly not long-lasting immunity. The IMAC of the invention could be applied in parallel with in vivo activation of donor and patient's lymphocytes using administration of IL-2 with no prior cell processing ex vivo prior to cell infusion in centers where the use of ex vivo cell culturing is considered ATMP and not approved by local regulatory authorities. In vivo activation of donor lymphocytes makes it possible using freshly obtained MHC compatible, haploidentical or fully mismatched unrelated mononuclear cells mixed with IMAC for in vivo induction of anti-cancer immunotherapy which can be further enhanced by concomitant administration of activating cytokines.
(50) Targeting killer cells exclusively against cancer cells by IMAC prevents undesirable reactivity against patient's normal (non-malignant) cells, even when using intentionally mismatched donor lymphocytes, yet minimizing toxicity and the risk of GVHD-like effects because of consistent rejection of mismatched lymphocytes in every non-immunosuppressed recipient.
(51) According to some embodiments, the binding mechanism used for connection of the antibodies in the complex is avidin-biotin and therefore, conjugates or complexes of invention may be composed of 2, 3 or 4 different antibodies. According to other embodiments, the different antibodies are conjugated to polymeric scaffolds or nanoparticles or beads which may be magnetic, positively charged and/or biodegradable.
(52) The antibodies of the IMAC can target one or more cell surface antigens, and also one or more killer cells simultaneously, thus maximizing the binding and consequently the anti-cancer effects of killer cells on the one hand, followed by induction of anti-cancer vaccination, on the other hand. It may be possible to attack cancer cells by combination of monoclonal antibodies, each targeted against a different antigen expressed on the malignant cells (e.g. anti-CD20 and anti-CD19 antibodies against B cell malignancies, anti-Her2/neu antibody and anti-EGFR antibodies against breast cancer cells, anti-GD2 antibodies and anti-EGFR against many types of solid tumors, etc.), or treating the patient with two different IMACs each directed against different cancer surface antigens and each targeting different types of T cells (e.g., using anti-CD3 and anti-CD28 monoclonal antibodies, or other combinations of antibodies against T cells). Simultaneous targeting of cancer cells with more anti-cancer killer cells and with more subsets of T cells will improve the efficacy of elimination of multi-drug resistant cancer cells.
(53) According to some embodiments, the antibodies are anti-T cell antibodies such as anti-CD3, anti-CD28, anti-CD2, anti-CD52 (which could also target malignant B cells), anti-CD4, anti-CD8, anti-IL-2, and/or antibodies against NK cells such as anti-HNK-1, anti-CD16 (Leu11), anti-VEGF, etc.
(54) According to some embodiments, the antibody is selected from the group consisting of Abagovomab, Abituzumab, Abrilumab, Actoxumab, Adalimumab, Adecatumumab, Aducanumab, Afasevikumab, Afutuzumab, ALD518, Alemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab, Anetumab ravtansine, Anifrolumab, Anrukinzumab (IMA-638), Apolizumab, Ascrinvacumab, Aselizumab, Atezolizumab, Atinumab, Atlizumab (tocilizumab), Atorolimumab, Avelumab, Bapineuzumab, Basiliximab, Bavituximab, Begelomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Bimagrumab, Bimekizumab, Bivatuzumab mertansine, Bleselumab, Blontuvetmab, Blosozumab, Bococizumab, Brazikumab, Brentuximab vedotin, Briakinumab, Brodalumab, Brolucizumab, Brontictuzumab, Burosumab, Cabiralizumab, Canakinumab, Cantuzumab mertansine, Cantuzumab ravtansine, Caplacizumab, Capromab pendetide, Carlumab, Carotuximab, cBR96-doxorubicin immunoconjugate, Cedelizumab, Cergutuzumab amunaleukin, Cetuximab, Ch.14.18, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumab tetraxetan, Codrituzumab, Coltuximab ravtansine, Conatumumab, Concizumab, Crenezumab, Crotedumab, CR6261, Dacetuzumab, Daclizumab, Dalotuzumab, Dapirolizumab pegol, Daratumumab, Dectrekumab, Demcizumab, Denintuzumab mafodotin, Denosumab, Depatuxizumab mafodotin, Derlotuximab biotin, Detumomab, Dinutuximab, Diridavumab, Domagrozumab, Drozitumab, Duligotumab, Dupilumab, Durvalumab, Dusigitumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Eldelumab, Elgemtumab, Elotuzumab, Elsilimomab, Emactuzumab, Emibetuzumab, Emicizumab, Enavatuzumab, Enfortumab vedotin, Enlimomab pegol, Enoblituzumab, Enokizumab, Enoticumab, Ensituximab, Epitumomab cituxetan, Epratuzumab, Erenumab, Etaracizumab, Etrolizumab, Evinacumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, Felvizumab, Fezakinumab, Fibatuzumab, Ficlatuzumab, Figitumumab, Firivumab, Flanvotumab, Fletikumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galcanezumab, Galiximab, Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumab ozogamicin, Gevokizumab, Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, Guselkumab, Ibalizumab, Ibritumomab tiuxetan, Icrucumab, Idarucizumab, IMAB362, Imalumab, Imciromab, Imgatuzumab, Inclacumab, Indatuximab ravtansine, Indusatumab vedotin, Inebilizumab, Infliximab, Intetumumab, Inolimomab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Isatuximab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lampalizumab, Lanadelumab, Landogrozumab, Laprituximab emtansine, Lebrikizumab, Lemalesomab, Lendalizumab, Lenzilumab, Lerdelimumab, Lexatumumab, Libivirumab, Lifastuzumab vedotin, Ligelizumab, Lilotomab satetraxetan, Lintuzumab, Lirilumab, Lodelcizumab, Lokivetmab, Lorvotuzumab mertansine, Lucatumumab, Lulizumab pegol, Lumiliximab, Lumretuzumab, MABpl, Mapatumumab, Margetuximab, Mavrilimumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mirvetuximab soravtansine, Mitumomab, Mogamulizumab, Monalizumab, Morolimumab, Motavizumab, Moxetumomab pasudotox, Muromonab-CD3, Namilumab, Naratuximab emtansine, Narnatumab, Natalizumab, Navicixizumab, Navivumab, Nebacumab, Necitumumab, Nemolizumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab, Obiltoxaximab, Obinutuzumab, Ocaratuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab, Ontuxizumab, Opicinumab, Oregovomab, Orticumab, Otelixizumab, Otlertuzumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Pamrevlumab, Vectibix, Pankomab, Panobacumab, Parsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab, Patritumab, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab, Plozalizumab, Pogalizumab, Polatuzumab vedotin, Ponezumab, Prezalizumab, Priliximab, Pritoxaximab, Pritumumab, PRO 140, Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ralpancizumab, Ramucirumab, Raxibacumab, Refanezumab, Regavirumab, Reslizumab, Rilotumumab, Rinucumab, Risankizumab, Rituximab, Rivabazumab pegol, Robatumumab, Roledumab, Romosozumab, Rontalizumab, Rovalpituzumab tesirine, Rovelizumab, Ruplizumab, Sacituzumab govitecan, Samalizumab, Sapelizumab, Sarilumab, Satumomab pendetide, Secukinumab, Seribantumab, Setoxaximab, Sevirumab, Sibrotuzumab, SGN-CD19A, SGN-CD33A, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Sofituzumab vedotin, Solanezumab, Sonepcizumab, Sontuzumab, Stamulumab, Suvizumab, Tabalumab, Tacatuzumab tetraxetan, Talizumab, Tamtuvetmab, Tanezumab, Taplitumomab paptox, Tarextumab, Tefibazumab, Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, Tesidolumab, Tetulomab, Tezepelumab, TGN1412, Ticilimumab (tremelimumab), Tildrakizumab, Tigatuzumab, Timolumab, Tisotumab vedotin, TNX-650, Tocilizumab (atlizumab), Toralizumab, Tosatoxumab, Tositumomab, Tovetumab, Tralokinumab, Trastuzumab, Trastuzumab emtansine, Tregalizumab, Tremelimumab, Trevogrumab, Tucotuzumab celmoleukin, Tuvirumab, Ublituximab, Ulocuplumab, Urelumab, Urtoxazumab, Ustekinumab, Utomilumab, Vadastuximab talirine, Vandortuzumab vedotin, Vantictumab, Vanucizumab, Vapaliximab, Varlilumab, Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Visilizumab, Vobarilizumab, Volociximab, Vorsetuzumab mafodotin, Votumumab, Xentuzumab, Zalutumumab, Zanolimumab, Zatuximab, Ziralimumab, Zolimomab aritox, and any combination thereof. Each possibility represents a separate embodiment of the invention.
(55) According to some embodiments, the antibody is against a target selected from the group consisting of: 1-40--amyloid, 4-1BB (CD137), 5AC, activated F9, F10, activin receptor-like kinase 1, ACVR2B, adenocarcinoma antigen, AGS-22M6, alpha-fetoprotein, angiopoietin 2, angiopoietin 3, anthrax toxin, AOC3 (VAP-1), B7-H3, Bacillus anthracis anthrax, BAFF, beta amyloid, B-lymphoma cell, C5, CA-125, CA-125 (imitation), calcitonin, Canis lupus familiaris IL31, carbonic anhydrase 9 (CA-IX), cardiac myosin, CCL11 (eotaxin-1), CCR2, CCR4, CCR5, CD11, CD18, CD125, CD140a, CD147 (basigin), CD15, CD152, CD154 (CD40L), CD19, CD2, CD20, CD200, CD22, CD23 (IgE receptor), CD25 (a chain of IL-2 receptor), CD27, CD4, CD6, CD28, CD3, CD3 epsilon, CD30 (TNFRSF8), CD33, CD37, CD38, CD4, CD40, CD44 v6, CD5, CD51, CD52, CD56, CD6, CD70, CD74, CD79B, CD80, CEA, CEA-related antigen, CFD, CGRP, ch4D5, CLDN18.2, Clostridium difficile, clumping factor A, coagulation factor III, CSF1R, CSF2, CTGF, CTLA-4, CXCR4 (CD184), cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, DLL3, DLL4, DPP4, DR5, E. coli shiga toxin type-1, E. coli shiga toxin type-2, EGFL7, EGFR, endoglin, endotoxin, EpCAM, ephrin receptor A3, episialin, ERBB3 (HER3), F protein of respiratory syncytial virus, FAP, FGF 23, fibronectin extra domain-B, folate hydrolase, folate receptor alpha, Frizzled receptor, GCGR, GD2 ganglioside, GDF-8, glypican 3, GMCSF receptor -chain, GPNMB, growth differentiation factor 8, GUCY2C, hemagglutinin, hepatitis B surface antigen, HER1, HER2/neu, HGF, HHGFR, histone complex, HIV-1, HLA-DR, HNGF, human beta-amyloid, human TNF, ICAM-1 (CD54), ICOSL, IFN-, IFN-, IgE, IgE Fc region, IGF-1 receptor (CD221), IGF1, IGF2, IGHE, IL 17A, IL 17A and IL 17F, IL 20, IL-1, IL-12, IL-23, IL-13, IL-17, IL17A and IL17F, ILIA, IL-1, IL2, IL-22, IL23, IL23A, IL31RA, IL-4, IL-5, IL-6, IL-6 receptor, IL9, ILGF2, influenza A virus hemagglutinin HA, integrin 4, integrin 47, integrin 51, integrin 77, integrin v3, interferon gamma-induced protein, interferon receptor, interferon / receptor, ITGA2 (CD49b), kallikrein, KIR2D, KLRC1, Lewis-Y antigen, LFA-1 (CD11a), LINGO-1, lipoteichoic acid, LOXL2, L-selectin (CD62L), LTA, MCP-1, mesothelin, MIF, MS4A1, MSLN, MUC1, mucin CanAg, myelin-associated glycoprotein, myostatin, NCA-90 (granulocyte antigen), neural apoptosis-regulated proteinase 1, NGF, N-glycolylneuraminic acid, NOGO-A, Notch 1, Notch receptor, NRP1, Oryctolagus cuniculus, OX-40, oxLDL, PCSK9, PD-1, PDCD1, PDGF-R a, phosphate-sodium co-transporter, phosphatidylserine, platelet-derived growth factor receptor beta, prostatic carcinoma cells, Pseudomonas aeruginosa, Pseudomonas aeruginosa type III secretion system, rabies virus glycoprotein, RANKL, respiratory syncytial virus, RHD, Rhesus factor, RON, RTN4, scatter factor receptor kinase, sclerostin, SDC1, selectin P, SLAMF7, SOST, sphingosine-1-phosphate, Staphylococcus aureus, STEAP1, TAG-72, TEM1, tenascin C, TFPI, TGF beta 1, TGF beta 2, TGF-, TNFR superfamily member 4, TNF-, TRAIL-R1, TRAIL-R2, TSLP, tumor antigen CTAA16.88, tumor specific glycosylation of MUC1, tumor-associated calcium signal transducer 2, TWEAK receptor, TYRP1 (glycoprotein 75), VEGF, VEGF-A, VEGFR-1, VEGFR2, vimentin, VWF, and any combination thereof. Each possibility represents a separate embodiment of the invention.
(56) In contrast to simple, readily available and inexpensive treatment of cancer using IMAC, other available cell-mediated modalities against cancer such as tumor infiltrating lymphocytes (TIL) and particularly the chimeric antigen receptors (CAR-T) that are complicated, time consuming, laborious and expensive, and are based on enrichment of lymphocytes that sometimes cannot be accomplished because of prior chemotherapy or radiation. In addition, such cellular procedures are effective against a limited number of cancer types and even then, cure is rare if ever, and therefore, following induction of complete remission of certain hematologic malignancies by CAR-T cells, often an allogeneic stem cell transplantation is indicated, a hazardous and expensive procedure, in an attempt to eliminate residual malignant cells.
(57) In sharp contrast to the CAR-T procedures currently considered the most effective cell-mediated anti-cancer immunotherapy, IMAC is likely to be much more effective for cure of many different types of cancer as soon as indicated in many more oncology centers and at a lower cost. Whereas CAR-T treatment depends on availability of GMP facility, team experienced in gene therapy and cell cultures, several weeks needed for expansion of the number of T cells that may be suppressed by prior anti-cancer treatment and certainly too expensive to serve every patient with cancer in need, the IMAC technology is provides more practical solution for a larger number of patients in need. IMAC is a much simpler treatment method that can be applied as soon as indicated for treatment a broader spectrum of cancers, using off-the-shelf components, or applicable following 4-6 days of culturing if used with freshly obtained haploidentical donor lymphocytes. Treatment using IMAC that induces more effective anti-cancer cytotoxicity based on the use of activated mismatched donor lymphocytes will also provide induction of anti-cancer vaccination not yet available by TIL or CAR-T cells at a reasonable cost affordable for every patient in need.
(58) Without wishing to be bound by theory, the anti-cancer immunotherapy of the invention that comprises disease-specific IMAC has the following advantages as compared with currently available bispecific antibodies, CAR-T cells or TIL technology.
(59) Cancer killing capacity of intentionally mismatched pre-activated killer cells, readily available off-the-shelf, or freshly prepared mononuclear donor lymphocytes obtained by apheresis with no need for prior ex vivo activation, or using donor lymphocytes, containing both T cells, NK cells and NKT cells, pre-activated ex vivo and also following infusion in vivo, are likely to be much more effective than using patient's own lymphocytes that are a priori tolerant to cancer. In contrast, alternative cell-based therapeutic procedures based on the use of bispecific antibodies, TIL or CAR-T cells focus on targeting patient's own lymphocytes against cancer cells. We have already documented that cure (>35 years disease-free survival) of otherwise malignancies resistant to supra-lethal myeloablative conditioning can be accomplished with alloreactive donor lymphocytes, suggesting the feasibility to eradicate the last cancer cell by alloreactive donor lymphocytes (2-5). In contrast, immunotherapy by CAR-T frequently fails to eradicate all malignant cells (28) and therefore it is usually followed by allogeneic stem cell transplantation. Similarly, immunotherapy with TIL alone rarely if ever results in complete cure.
(60) In addition to the most potent anti-cancer effects that can be mediated spontaneously by mismatched donor lymphocytes against MHC incompatible cancer cells, anti-cancer effects of donor (as well as patient's) lymphocytes can be further activated ex vivo and/or in vivo with cytokines (e.g. IL-2; IL-12; IL-15; IL-17 etc.) or any other factors or antibodies-activating T cell (e.g. anti-OX40, co-stimulatory anti-CD28 or ICOS; or targeting CD69, CD25, CD71, etc.), or using intentionally mismatched NK cells by engagement of CD16, CD56, NKG2D, GM-CSF, IFN gamma, or TNF alpha, etc., likely to induce much more powerful eradication of multi-drug resistant cancer cells and cancer stem cells.
(61) Anti-cancer cytotoxic activity by combined activity of different killer cells, and against different targets on the cell surface of cancer cells can be much more effective than anti-cancer effects mediated by bispecific antibodies, TIL or CAR-T cells. intentionally mismatched donor T cells and NK cells, attacking different targets on cancer cells simultaneously, is likely to be much more effective than cytotoxicity induced against one cell-surface antigen induced by antigen-specific autologous T cells induced by bispecific antibodies, TIL or CAR-T cells acting alone. As was already documented in pre-clinical animal models and cumulative clinical experience, allogeneic lymphocytes, especially if intentionally mismatched can eliminate the last cancer cell and result in cure (2-5, 7, 8). Unfortunately, the majority of patients treated with CAR-T are not cured (28) and this is why allogeneic stem cell transplantation is recommended following successful remission induction with CAR-T in most oncology centers.
(62) Killing of cancer cells using IMAC can be targeted by intentionally mismatched NK cells alone while avoiding the use of T cells that are the cause of CRS. NK cell-mediated anti-cancer cytotoxicity may result in less or no CRS at all, as compared with frequent and potentially hazardous CRS that results following treatment with CAR-T, and which can result in severe or occasionally fatal outcome. A better safety profile anticipated by treatment with IMAC as compared with CAR-T is a major potential advantage of using IMAC.
(63) The use of the compounds and methods of the invention makes it possible to extend the spectrum of patients with different types of cancer in need with eligible clinical indication for cell therapy, and that can benefit from a large number of existing monoclonal antibodies against cancer-associated cell surface antigens (e.g. CD10; CD19; CD20; CD38; Her2/neu; GD2; MUC1; CA-125; CEA; VEGF; EGFR) or against T cells, NK cells and antigen presenting cells (dendritic cells and macrophages). In contrast, immunotherapy with bispecific antibodies or CAR-T cells is restricted by a limited presence of suitable vectors against target antigens.
(64) Recent studies have indicated that at the time of cancer progression additional events may develop in a large proportion of patients: (1) tumor-associated cell surface antigen may internalize or no longer exist (e.g., CD19 or CD20 in B cell malignancies); and (2) PD-1 and PD-L1 may be upregulated, as examples, thus turning off the anti-cancer capacity of CAR-T treatment mediated by T cells. In contrast, anti-cancer activity of avidin-based IMAC may be less affected by disappearance of one cell surface antigen because killing can be directed against more than one target antigen and besides, the killing is mediated against the mismatched target cells.
(65) Whereas bispecific antibodies, TIL or CAR-T are directed against cancer antigens, the anti-cancer efficacy may be partially blocked by different mechanisms that can down-regulate anti-cancer effects (e.g., regulatory T cells; checkpoint inhibitors; myeloid-derived suppressor cells; tumor associated macrophages and infiltrating mesenchymal stromal cells) using avidin-based IMAC makes it possible to combine effective cancer cytotoxicity in parallel with down-regulation of some of the suppressive mechanisms.
(66) Clinical application of anti-cancer immunotherapy using IMAC can be prepared instantaneously, as soon as indicated, consequently at an optimal stage of minimal residual disease and at optimal clinical condition of the patient, in sharp contrast with the delay of treatment with CAR-T cells or TIL technology, both depend on time needed for culturing T cells in an approved GMP facility, associated with major logistics limited to few highly equipped centers with GMP facilities with sufficient experience using long-term cell cultures and gene therapy. Besides, expanding sufficient number of patient's lymphocytes is not always successful, especially following prior treatment with chemotherapy and radiation therapy.
(67) Due to technical considerations and prohibitive high cost of treatment with TIL and CAR-T cells (currently estimated at $100,000 to 450,000/patient) such treatments are not available for the large majority of patients in need.
(68) Moreover, the IMAC technology that can be easily assembled with disease-specific components to match nearly every patient with cancer in need, seems to be the only available treatment that combines most successful anti-cancer effects followed by induction of anti-cancer vaccination. Accordingly, when applied at the stage of minimal residual disease, there is a good chance that successful clinical application of IMAC can result in cure of otherwise resistant hematologic malignancies and certain metastatic solid tumors.
(69) Without wishing to be bound by theory, the use of IMAC for induction of anti-cancer vaccination has the following advantages as compared with conventional dendritic cell vaccines:
(70) Conventional anti-cancer vaccines are based on in vitro preparation of dendritic cells in a well-established GMP facility. This is an ATMP procedure that is laborious, expensive and requires apheresis of the patient. Preparation of such vaccines takes 7-10 days and can be accomplished by skilled technicians working in an approved GMP facility. Moreover, until to date there is no evidence that conventional anti-cancer vaccines can result in cure so most oncologists almost never recommend patients in need to consider anti-cancer vaccination.
(71) Conventional dendritic cells vaccines depend on availability of tumor-cells, tumor cell lysates or tumor-specific peptides that may or may not be available, whereas anti-cancer vaccination based on IMAC is likely to occur in vivo, even if no cancer samples are available and cancer-specific antigens unknown.
(72) Conventional vaccines are based on pulsing dendritic cells with antigens derived from the primary tumor or sometimes from tumor metastases obtained ahead of time and cryopreserved. It is well known that tumor cells mutate constantly both spontaneously and especially following exposure to anti-cancer chemotherapy. As such, phenotype, tumor-associated antigens and cancer-specific expression of cell surface antigens mutate and change constantly. Therefore, a vaccine based on sensitization of patient's immune system cells against the available original tumor sample cryopreserved at the time of diagnosis or following surgery may no longer be relevant against cancer metastases developing at a later stage. In contrast, a vaccine based on in vivo sensitization against patient's current cancer antigens, accomplished by the use of IMAC, by patient's in vivo anti-cancer activity is likely to be much more effective than preparation of anti-cancer vaccine using a small number of antigen presenting cells in the laboratory against cancer antigens that may no longer exist.
(73) To summarize the advantages of the invention, avidin-based complexes enables the use of combination of 2 to 4 different biotinylated monoclonal antibodies targeting lymphocytes against one or more cell surface cancer-associated antigens simultaneously, or 3 biotinylated monoclonal antibodies with one additional biotinylated antibodies anti-cancer agent, or 2 biotinylated monoclonal antibodies against 1 or 2 cancer-associated antigens, binding 2 different killer cells (either T cells guided by 2 different anti-T cells cell antibodies (e.g. CD4 & CD8) or 2 NK killers (e.g. using anti-CD56 & anti-CD16)), or any other biotinylated anti-cancer treatment (e.g., radiolabeled agent or chemotherapy, or antibody-drug candidate) and possibly additional cancer activating molecules, targeted against cancer cell surface antigens combined with intentionally mismatched nave or activated effector cells of the immune system, or bound in vivo with patient's own anti-cancer effector cells and antigen presenting cells. Both intentionally mismatched killer cells or patient's own cells can be activated prior to and following cell infusion. For treatment of very resistant cancer, two different IMAC complexes or conjugates may be used jointly against multiple available cancer-associated antigens, attacking resistant cancer cells simultaneously. The use of intentionally mismatched killer cells represents the most effective approach for killing of multi-drug resistant cancer cells, including otherwise resistant cancer stem cells.
(74) Attacking simultaneously several antigens on cancer cells by killer cells guided by more than one monoclonal antibody, is likely to be much more cytotoxic compared with targeting only one cancer cell surface antigen (e.g., attacking malignant B cells by both anti-CD20 and anti-CD19; or attacking breast cancer cells by both anti-Her2/neu and anti-EGFR simultaneously). Using conjugates, with limited number of binding sites, cancer immunotherapy may be attempted by combining one conjugate against one cell-surface antigen and another against another cell-surface antigen of the same cancer cell. The method of the invention will also result in induction of long-lasting anti-cancer vaccination induced by patient's own memory T cells.
(75) IMAC is based on the use of readily available and clinically approved monoclonal antibodies for treatment of cancer and cytokines for activation of T cells, NK cells, NKT cells and antigen presenting cells, with suitable conjugates or complexes that can be easily prepared as indicated for each patient in need on a fully personalized basis. As such, IMAC provides an option to treat a large number of different malignancies using a broad spectrum of commercially available monoclonal antibodies complexed using an avidin molecule or conjugates prepared using previously approved linkers. Taken together, the same treatment that will result in elimination of cancer cells will also induce automatic vaccination against recurrent disease by induction of long-lasting resistance against residual or recurrent disease. Using avidin injected directly into cancer tissue, followed by intravenous injection of biotinylated monoclonal antibodies, a similar procedure can be used for simply, fast and most effective induction of personalized anti-cancer vaccination.
(76) For treatment of minimal residual disease, IMAC can applied with no prior conditioning or using minimally immunosuppressive conditioning (e.g. using low dose cyclophosphamide or combination of fludarabine and cyclophosphamide), thus suppressing regulatory T cells that are particularly sensitive to low dose cyclophosphamide, and also extending the duration of donor lymphocytes and enhancing the contribution of newly host derived immune system cells by optimizing the so-called homeostatic proliferation.
(77) The IMAC procedure can be applied using freshly obtained or off-the-shelf unrelated killer cells prepared from circulating blood lymphocytes, lymph node lymphocytes or even expanded NK cells derived from unrelated fully mismatched lymphocytes, haploidentical or MHC compatible lymphocytes freshly activated prior to cell infusion or readily available ex vivo activated prior to cryopreservation as readily available for immediate use off-the-shelf. Further activation of intentionally mismatched killer cells, both T, NK and NKT cells, can be easily activated in vivo by administration of well-tolerated low-dose IL-2 (for activation of T cells & NK cells) and using KRN7000 (for activating of NKT cells) for a few days until rejection will eliminate all mismatched donor lymphocytes. Consistent rejection of intentionally mismatched killer cells guarantees no risk from GVHD-like toxicity.
(78) In principle, as indicated above, the IMAC technology could be useful for elimination of undesirable non-malignant cells as well as infective agents based on similar principles used for elimination of cancer cell followed by induction of residual long-lasting immunity.
(79) The efficacy of the IMAC technology of the present invention for complete elimination of cancer cells was confirmed by results of experiments summarized in
(80) The results of the in vivo experiments suggested that selective killing of targeted cancer cells could be accomplished by IMAC while avoiding any cytotoxicity against normal patient's tissues before all allogeneic killer cells will be rejected and that long lasting anti-cancer immunity was mediated by activation of recipient's immune system cells connected and activated by IMAC following rejection of all malignant melanoma cells. Indeed, rejection of mismatched lymphocytes leaves behind the IMAC skeleton, with monoclonal antibodies ready to be connected to cancer antigens and recipient/patient's own T cells.
(81) Taken together, due to the capacity of IL-2 activated mismatched killer cells to eliminate all cancer cells when applied against a lethal challenge of cancer cells and induce long-lasting anti-cancer vaccination, treatment with IMAC is likely to result in cure especially if applied against minimal residual disease following conventional treatment. On the other hand, due to intentional use of mismatched donor lymphocytes such killer cells are always rejected within a week or so, thus avoiding the risk of GVHD-like toxicity. In case the recipient may be partially immunosuppressed by prior anti-cancer treatment, IL-2 activated donor lymphocytes can be exposed to sublethal radiation that blocks proliferation of T cells and retains the cytotoxic capacity of NK cells (21).
(82) In addition to direct elimination of otherwise resistant malignant cells the present invention provides, according to an aspect, a method of vaccination and induction of long-lasting anti-cancer immunity, the method comprising an intra-tumor injection of avidin, optionally using ultrasound or CT guided needle and administering in-vivo biotinylated monoclonal antibodies together with intentionally mismatched cytokine activated donor killer lymphocytes (T & NK cells), first resulting in in situ lysis of cancer cells, followed by influx of patient's own lymphocytes and antigen presenting cells following rejection of donor lymphocytes. As such, systemic immunity can be established against untreated remote metastases (abscopal effect).
(83) The present invention thus also provides a method of induction of anti-cancer vaccination, the method comprising direct (intratumor) injection of avidin into the primary tumor or visible easily accessible metastatic lesion, followed by injection of biotinylated monoclonal antibodies alone or with additional biotinylated immune activating agents (e.g., IL-2 or 4-1BB) or any other anti-cancer modality (e.g., chemotherapy or radiolabeled molecule or biotinylated oncolytic virus). According to this method, the avidin complex or an equivalent conjugate, binds to biotinylated monoclonal antibodies and/or any of the aforementioned agents, thus resulting in in-vivo local cytotoxicity to cancer cells and local recruitment of antigen presenting cells, using the treated cancer lesion as in situ vaccine, even if no cancer-associated antigen is evident and even if the full phenotype of the type of cancer to be treated cannot be fully defined, thus creating fully personalized in situ anti-cancer vaccine in vivo.
(84) According to some embodiments, the present invention provides a method for vaccination of a subject having cancer with no known cancer-associated antigen, the method comprises injection of cancer cells together with IMAC containing anti-T cells and/or NK cells to attract patient's own T cells, NK cells and antigen presenting cells to the lymphatic system, where optimal anti-cancer vaccination can occur. According to some embodiments, the vaccination method comprises injecting of the complex or conjugate proximally to the draining lymph nodes. According to some embodiments, sensitization of patients T cells is performed by concomitant injection of GM-CSF.
(85) For induction of anti-cancer vaccination there are several treatment options available, ideally based on the use of mismatched activated donor lymphocytes attached to biotinylated antibodies monoclonal antibodies that can selectively target cancer lesion containing avidin, followed by in situ activation of anti-cancer immunity by patient's immune system after rejection of mismatched donor cells by interaction with IMAC as documented in
(86) Induction of anti-cancer vaccination should be combined with systemic activation of patient's own immune system (e.g., using checkpoint inhibitors; combinations of IL-2 and alpha interferon, or any other immune stimulating agents) to maximize an immune response against the targeted cancer lesion and towards more effective activation of patient's own immune system against cancer. Systemic activation of patient's immune system with intra-lesional, in situ activation of T cells and dendritic cells can result in induction of helper and cytotoxic T cells and also memory T cells for elimination of residual malignant cells and for protection against recurrent disease.
(87) According to some embodiments, the method comprises administration of the immunoglobulin complex or conjugate against residual malignant cells following conventional anti-cancer modalities or after autologous or allogeneic stem cell transplantation, based on the cumulative experience confirming that activated allogeneic lymphocytes, particularly if mismatched, can eliminate all malignant cells and result in cure, as confirmed in pre-clinical animal models of murine leukemia/lymphoma and in clinical trials, as well as confirmation that donor lymphocyte infusion (DLI) can be used to eliminate residual malignant cells after maximally tolerated myeloablative conditioning rescued with allogeneic stem cell transplantation (17-20, 29-31).
(88) Any method of treating cancer, using the pharmaceutical compositions of the present invention, may be part of a regiment of cancer treatment, including but not limited to chemotherapy, immunotherapy, biotherapy, radiation, bone-marrow transplantation and surgery.
(89) Anti-cancer cell-mediated immunotherapy according to the present invention may be also supported by administration of an anti-inflammatory agent that suppresses upregulation of cancer genes by inflammation as well as controls fever and malaise that may result by intensive immune system activation.
(90) The following examples are presented in order to fully illustrate some embodiments of the invention. They should, in no way be construed as limiting the scope of the invention.
EXAMPLES
(91) Compounds and Methods
(92) Biotinylated antibodies are commercially available.
(93) Biotinylation of other monoclonal antibodies is performed by binding biotin to lysine residues using the N-hydroxysuccinimide or p-nitrophenyl esters or other activated esters, as described for example in Methods in Enzymology vol. 184, pages 3-746 (1990), Edited by Meir Wilchek and Edward A. Bayer.
Avidin molecules to be used include but are not limited to egg-white Avidin, StreptAvidin, NeutrAvidin, Neutralight Avidin, NitroAvidin, Caped Avidin, Avidins from bacteria, and/or genetically engineered and modified Avidins.
To increase the number of antibodies on the Avidins, the Avidin is crosslinked or polymerized using methods known in the art.
Covalently crosslinking and polymerize of antibodies is performed using cleavable and non-cleavable crosslinking reagents known in the art. Exemplified IMAC structure and binding is shown in
Example 1: Biotinylation of Monoclonal Antibodies
(94) An exemplary method for biotinylation of monoclonal antibodies free amine group of a Lysine residue in the antibody's sequence is utilizes as described above, and also in Mao S Y. (2010), Biotinylation of Antibodies. In: Oliver C. and Jamur M. (eds) Immunocytochemical Methods and Protocols. Methods in Molecular Biology (Methods and Protocols), vol 588. Humana Press. Biotinylation of clinical grade commercially available monoclonal antibody is carried using methods known in the art or using commercially available biotinylated monoclonal antibodies against cancer associated antigens (e.g., CD20, CD19, CD38, GD2, Her2/neu or against any other known cancer associated cell-surface antigen), or against any undesirable normal or abnormal cells. Clinical grade NeutrAvidin or StreptAvidin or any other Avidin described above is used to connect biotin-containing antibodies.
Example 2: Producing Conjugated IMACs by a Variety of Biotinylated Monoclonal Antibodies
(95) A biotinylated monoclonal antibody against CD20 (e.g., Rituximab, MabThera) and a second biotinylated monoclonal anti-CD3 antibody are mixed in equal concentrations, aiming for 50:50 binding to NeutrAvidin to create an IMAC that can be suitable for treatment of any CD20 positive B cell malignancy, acute or chronic lymphocytic leukemia or NHL.
(96) Such IMAC can be mixed with donor lymphocytes containing both T cells and NK cells in vivo or with donor lymphocytes pre-activated ex vivo for a minimum of 4-5 days with an activating agent such as IL-2.
Example 3: Confirming Superior Anti-Cancer Cytotoxicity of Mismatched Activated Killer Cells Targeted by IMAC Complexed with Biotinylated Anti-CD3 & Anti-CD20 Monoclonal Antibodies to Avidin Against Multiple Myeloma and Ramos Lymphoma Cells Even at Very Low Effector:Target Cell Ratio
(97) The objective of this study was to evaluate the efficacy of avidin-based IMAC targeting against different types of malignant cells.
(98) Test System
(99) Part I: Cell Lines:
(100) Six human hematological malignant cell lines, all obtained from American Type Culture Collection (ATCC), Manassas, VA, USA:
(101) Multiple Myeloma (MM):
(102) U266B1 [U266] (ATCC TIB-196) B lymphocyte, Plasmacytoma; Myeloma RPMI 8226 (ATCC CCL-155) B lymphocyte, Plasmacytoma; Myeloma MM.1R (ATCC CRL-2975) B lymphoblast, Multiple myeloma
Lymphoma/Leukemia: Raji (ATCC CCL-86) B lymphocyte, Burkitt's lymphoma Ramos (RA 1) (ATCC CRL-1596) B lymphocyte, Burkitt's lymphoma Daudi (ATCC CCL-213) B lymphoblast, Burkitt's lymphoma
Antibodies for Part I: CD20 Monoclonal Antibody (2H7), Biotin, eBioscience, 100 g (Thermo Fisher, Waltham, MA USA, Catalog #13-0209-82) CD38 Monoclonal Antibody (HIT2), Biotin, eBioscience, 100 g (Thermo Fisher, Waltham, MA USA, Catalog #13-0389-82) Mouse IgG2b kappa Isotype Control (eBMG2b), Biotin, eBioscience, 500 g (Thermo Fisher, Waltham, MA USA, Catalog #13-4732-85) Mouse IgG1 kappa Isotype Control (P3.6.2.8.1), Biotin, eBioscience, 500 g (Thermo Fisher, Waltham, MA USA, Catalog #13-4714-85) Fluorescein (FITC)-conjugated IgG Fraction Monoclonal Mouse Anti-Biotin, 500 g (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA, Catalog #200-092-211)
Antibodies for Part II: CD20 Monoclonal Antibody (2H7), Biotin, eBioscience, 100 g (Thermo Fisher, Waltham, MA USA, Catalog #13-0209-82) CD38 Monoclonal Antibody (HIT2), Biotin, eBioscience, 100 g (Thermo Fisher, Waltham, MA USA, Catalog #13-0389-82) CD3 Monoclonal Antibody (OKT3), Biotin, eBioscience, 100 g (Thermo Fisher, Waltham, MA USA, Catalog #13-0037-82) CD4 Monoclonal Antibody (RPA-T4), Biotin, eBioscience, 100 g (Thermo Fisher, Waltham, MA USA, Catalog #13-0049-82) CD8 Monoclonal Antibody (MEM-31), Biotin, 100 g (Thermo Fisher, Waltham, MA USA, Catalog #MA1-19484) NeutrAvidin Protein, 10 mg (Thermo Fisher, Waltham, MA USA, Catalog #31000) Proleukin 18 MIU (Human IL-2): Powder for solution for injection or infusion
Flow Cytometry Antibodies of Part I: Anti-human CD3 PE (Biogems, Westlake Village, CA USA, Cat #05131-60-100) Anti-human CD8a PerCP-Cyanine5.5 (Biogems, Westlake Village, CA USA, Cat #10121-70-100) Anti-human CD4 APC (Biogems, Westlake Village, CA USA, Cat #06111-80-100) Anti-human CD20 FITC (Invitrogen, Carlsbad, CA, United States, Cat #MA1-19613) Anti-human CD38 FITC (Invitrogen, Carlsbad, CA, United States, Cat #MA1-12112) Viobility 405/452 (Miltenyi Biotech, Bergisch Gladbach, Germany, Cat #130-109-816)
Cell Culture Materials RPMI-1640 medium (Biological Industries, Beit-HaEmak, Israel, Catalogue #01-100-1A) Fetal Bovine Serum (FBS, Biological Industries, Beit-HaEmak, Israel, Catalogue #04-007-1A) Penicillin-Streptomycin solution (Biological Industries, Beit-HaEmak, Israel, Catalogue #03-031-1B). PhosphateBuffered Saline (PBS, Biological Industries, Beit-HaEmak, Israel, Catalogue #02-023-1A) -mercaptoethanol (Sigma Aldrich, Rehovot, Israel, Catalogue #M6250). Trypan Blue (Sigma Aldrich, Rehovot, Israel, Catalogue #T8154).
Binding Test:
(103) Cells were maintained according to Pharmaseed's SOPs 400, 401, 402 and 403, and manufacturer's instructions. On the day of cell staining with the antibodies, each cell line was counted and seeded at 110.sup.6 cells/well in a 96 U shape plate. The plate was centrifuged (1500 rpm, 5 minutes, 4 C.) and the cells were resuspended with FACS buffer/Isotype control/Antibody. The cells were then incubated on ice for 30 minutes. The plates were centrifuged (1500 rpm, 5 minutes, 4 C.), medium was discarded, and the cells were resuspended with secondary antibody at 100 L/well. The cells were incubated on ice for 30 minutes. Then, the plates were centrifuged (1500 rpm, 5 minutes, 4 C.), medium was discarded, and the cells were resuspended with 200 L flow cytometry buffer and CD20 and CD38 expression was analyzed by FACS. One Multiple Myeloma and one Lymphoma/Leukemia cell line with the highest expression of CD38 and CD20, respectively, was chosen for PART II.
(104) Part II Efficacy Study
(105) PBMCs Isolation and Preparation for Killing:
(106) Human venous blood (10-15 mL) was collected to heparinized vials and mixed well by gently inverting the tube several times. The blood was diluted 2 with PBS. Histopaque was added to a 50 mL centrifuge tube in half of the final blood:PBS volume. The blood was gently layered on the top of Histopaque using a 1 mL auto pipette. The layering was done very slowly that blood and Histopaque stayed as two different layers. The tubes were centrifuged (without any delay) at 400g for 30 min in +20 C. in a swing-out bucket without break. The whitish buffy coat (PBMCs) formed in the interphase between Histopaque and medium was aspirated without any delay. The cells were washed (centrifuged in 800 g for 10 min in +20 C.) twice with 10 mL of PBS. PBMCs were incubated at 210.sup.6 cells/mL with Recombinant human IL-2 (6,000 IU/ml) for 6 days in PBMCs killer medium.
Assay Set Up (Day of End of Incubation Period of PBMCs with IL-2): MM (RPMI2886) and Lymphoma/Leukemia (Ramos) cell lines were maintained according to Pharmaseed's SOPs 400, 401, 402 and 403, and manufacturer's instructions. The cells were collected, washed in PBS, counted and adjusted to 0.210.sup.6 cells/mL in PBS, each. The cells were added to wells at 100 L (20,000 cells/well) Plates were centrifuged, PBS was discarded and 50 L Ab/Ab-avidin was added. The plates were incubated on ice for 1 h. PBMCs were centrifuged (1,200 rpm, 15 minutes), medium was removed, and the cells were washed once again with PBS. PBMCs were resuspended in 1 ml PBMCs culture medium and counted with trypan blue. PBMCs were adjusted to 0.410.sup.6 cells/mL, 210.sup.6 cells/mL, and 810.sup.6 cells/mL, and added at 50 L, yielding 20,000 PBMCs/well, 100,000 PBMCs/well and 400,000 PBMCs/well, respectively. The plated were incubated in 37 C. 5% CO.sub.2 for 24 h.
Cell Counting and Flow Cytometry Analysis At the end of incubation period, cells were counted with Trypan blue (one well/replicate). Multiple Myeloma plates were stained with CD38, CD3, CD4, CD8 Abs, and viability dye. Lymphoma plates were stained with CD20, CD3, CD4, CD8 Abs, and viability dye. Samples were analyzed by FACS.
Formulation of the Test Items and Vehicle
Cell Lines Culture Medium:
(107) RPMI-1640 medium supplemented with 10% FBS, and 1% Penicillin-Streptomycin solution.
(108) Flow Cytometry Buffer:
(109) PBS supplemented with 2% FBS.
(110) PBMCs Culture Medium:
(111) RPMI-1640 medium supplemented with 10% FBS, 0.05 M -mercaptoethanol, and 1% Penicillin-Streptomycin solution.
(112) PBMCs Killer Medium:
(113) RPMI-1640 medium supplemented with 10% autologous heat inactivated serum, 0.05 M -mercaptoethanol, and 1% Penicillin-Streptomycin solution.
(114) Antibodies' Working Concentrations for Part I (in Flow Cytometry Buffer):
(115) CD38 Monoclonal Antibody (0.5 g/test/100 L) Mouse IgG1 kappa Isotype Control (0.25 g/test/100 L) CD20 Monoclonal Antibody (0.25 g/test/100 L) Mouse IgG2b kappa Isotype Control (0.125 g/test/100 L) FITCMouse Anti-Biotin (1 L/test/100 L
Antibodies' Working Concentrations for Part II (in Flow Cytometry Buffer): Anti-human CD3 PE (5 l/test/100 L) Anti-human CD8a PerCP-Cyanine5.5 (5 l/test/100 L) Anti-human CD4 APC (5 l/test/100 L) Anti-human CD20 FITC (5 l/test/100 L) Anti-human CD38 FITC (5 l/test/100 L) Viobility 405/452 (1 l/test/100 L)
NeutrAvidin Protein:
(116) NeutrAvidin Protein was reconstituted with 1 ml ultrapure water+9 ml PBS. The specific activity for biotin binding was approximately 14 g/mg of protein.
(117) Mm Ab Complex:
(118) 2.8 g CD38 Ab (5.6 l)+2.8 g CD3 Ab (5.6 l)+2.8 g CD4 Ab (5.6 l)+2.8 g CD8 Ab (2.8 l) in 800 l PBS.
MM Avidin-Ab Complex: 5.25 g CD38 Ab (10.5 l)+5.25 g CD3 Ab (10.5 l)+5.25 g CD4 Ab (10.5 l)+5.25 g CD8 Ab (5.25 l) in 1500 l PBS.
Lymphoma/Leukemia Ab Complex: 2.8 g CD20 Ab (5.6 l)+2.8 g CD3 Ab (5.6 l)+2.8 g CD4 Ab (5.6 l)+2.8 g CD8 Ab (2.8 l) in 800 l PBS.
Lymphoma/Leukemia Avidin-Ab Complex: 5.25 g CD20 Ab (10.5 l)+5.25 g CD3 Ab (10.5 l)+5.25 g CD4 Ab (10.5 l)+5.25 g CD8 Ab (5.25 l) in 1500 l PBS.
Results
Part I: Binding Test
(119) As seen in Table 1, among all three Lymphoma cell lines, Ramos exhibited the highest CD20 expression (and also CD38 expression). Among all three Multiple myeloma cell lines, RPMI8226 exhibited the highest CD38 expression. These cell lines were chosen for part II.
(120) TABLE-US-00001 TABLE 1 Part I flow cytometry analysis* CD20 CD38 Cell type Cell line NS CD20 CD20 iso CD38 CD38 iso expression expression Lymphoma Daudi 86 15148 621 32623 10183 24.4 3.2 Raji 96 8391 201 26559 237 41.7 112.1 Ramos 72 12163 147 26233 188 82.7 139.5 Multiple RPM18226 142 379 232 13008 254 1.6 51.2 Myeloma U2661B 101 180 174 6691 199 1.0 33.6 MM1R 111 198 174 3807 186 1.1 20.5 *CD20 MFI/CD20 isotype MFI = CD20 expression, CD38 MFI/CD38 isotype MFI = CD38 expression.
Part II: Efficacy Study
As seen in
As seen in
Summary and Conclusions
(121) As shown in
(122) As can be seen in
(123) As can be seen in
Example 4: Confirming Superior Anti-Cancer Cytotoxicity of Mismatched Activated Killer Cells Targeted by IMAC Complexed with Biotinylated Anti-CD3 & Anti-CD20 Monoclonal Antibodies to Avidin Against Daudi & Ramos Lymphoma Cells Even at Very Low Effector:Target Cells Ratio
(124) The objective of this study was to evaluate the efficacy of avidin-based IMAC targeting against different types of lymphoma cell lines expressing CD20.
(125) Test System
(126) Cell Lines
(127) Two human malignant lymphoma cell lines, obtained from American Type Culture Collection (ATCC), Manassas, VA, USA: Ramos (RA 1) (ATCC CRL-1596) B lymphocyte, Burkitt's lymphoma (American) Daudi (ATCC CCL-213) B lymphoblast, Burkitt's lymphoma
Antibodies and Proteins for Binding Assay CD20 Monoclonal Antibody (2H7), Biotin, eBioscience, 100 g (Thermo Fisher, Waltham, MA USA, Catalog #13-0209-82) CD3 Monoclonal Antibody (OKT3), Biotin, eBioscience, 100 g (Thermo Fisher, Waltham, MA USA, Catalog #13-0037-82) CD4 Monoclonal Antibody (RPA-T4), Biotin, eBioscience, 100 g (Thermo Fisher, Waltham, MA USA, Catalog #13-0049-82) CD8 Monoclonal Antibody (MEM-31), Biotin, 100 g (Thermo Fisher, Waltham, MA USA, Catalog #MA1-19484) NeutrAvidin Protein, 10 mg (Thermo Fisher, Waltham, MA USA, Catalog #31000) Proleukin 18 MIU (Human IL-2): Powder for solution for injection or infusion
Flow Cytometry Reagents: Anti-human CD3 PE (Biogems, Westlake Village, CA USA, Cat #05131-60-100) Anti-human CD56 Anti-Human CD56 (NCAM) (Biogems, Westlake Village, CA USA, Cat #08631-50-100) Viobility 405/452 (Miltenyi Biotech, Bergisch Gladbach, Germany, Cat #130-109-816) Vybrant DiD Cell-Labeling Solution (Thermo Fisher, Waltham, MA USA, Cat #V22887) PFA 4% (for cell fixation after staining)
Cell Culture Material RPMI-1640 medium (Biological Industries, Beit-HaEmak, Israel, Catalogue #01-100-1A) Fetal Bovine Serum (FBS, Biological Industries, Beit-HaEmak, Israel, Catalogue #04-027-1A) BIOTARGET serum free-medium (Biological Industries, Beit-HaEmak, Israel, Catalogue #05-080-1A) L-Glutamine (Biological Industries, Beit-HaEmak, Israel, Catalogue #03-020-1B) Penicillin-Streptomycin solution (Biological Industries, Beit-HaEmak, Israel, Catalogue #03-031-1B). PhosphateBuffered Saline (PBS, Biological Industries, Beit-HaEmak, Israel, Catalogue #02-023-1A) -mercaptoethanol (Sigma Aldrich, Rehovot, Israel, Catalogue #M6250). Trypan Blue (Sigma Aldrich, Rehovot, Israel, Catalogue #T8154).
Experimental Design
PBMC Isolation and Preparation of Killer Cells Human Venous blood (10-15 mL) from a healthy donor were collected to heparinized vials and mixed well by gently inverting the tube several times. The blood was diluted 2 with PBS. Histopaque was added to a 50 mL centrifuge tube into half of the final blood:PBS volume. The blood was gently layered on the top of Histopaque using a 1 mL auto pipette. The layering was done very slowly that blood and Histopaque stayed as two different layers. The tubes were centrifuged (without any delay) at 400g for 30 min at +20 C. in a swing-out bucket without break. The whitish buffy coat (PBMCs) formed in the interphase between Histopaque and medium was aspirated without any delay. The cells were washed (centrifuged in 800g for 10 min at +20 C.) twice with 10 mL of PBS. PBMCs were incubated at a concentration of 210.sup.6 cells/mL with Recombinant human IL-2 (6,000 l/ml) for six days.
Target Cells Labeling (One Day Prior to End of Incubation Period of PBMCs with IL-2): Each target cell line was suspended at a density of 106/mL in serum-free RPMI-1640 medium. Cell-labeling solution was added to the cells (5 L per mL of cell suspension). Cells were mixed well, and incubated for 20 minutes at 37 C. The labeled cells were centrifuged at 1500 rpm for 5 minutes, at room temperature. The supernatant was removed, and the cells were gently resuspended in warm culture medium. This procedure was then repeated two more times.
Assay Set Up (Day of End of Incubation Period of PBMCs with IL-2): Lymphoma/Leukemia cell lines were maintained according to Pharmaseed's SOPs No. 400, 401, 402 and 403, and manufacturer's instructions. The cells were collected, washed in PBS, counted and adjusted to a concentration of 0.210.sup.6 cells/mL in PBS, each. The cells were added to wells at 100 L (20,000 cells/well). Plates were centrifuged, PBS was discarded and 50 L Ab/Ab-avidin was added to wells. The plates were incubated on ice for 1 h. Plates were centrifuged, Ab/Ab-avidin was discarded and PBMCs or medium only were added. PBMCs were centrifuged (1,200 rpm, 15 minutes, at RT), medium was removed, and the cells were washed once again with PBS. PBMCs were resuspended in 1 mL PBMCs culture medium and counted with trypan blue. PBMCs were adjusted to 1010.sup.5 cells/mL, 210.sup.5 cells/mL, and 110.sup.5 cells/mL, and added at 100 L to indicated wells according to scheme in Table 1, yielding 100,000 PBMCs/well, 20,000 PBMCs/well and 10,000 PBMCs/well, respectively. The plates were incubated in 37 C. 5% CO.sub.2 for 2 h and 24 h (two plates for each time point).
Flow Cytometry Analysis At the end of each incubation period, the plates were centrifuged, washed with PBS, and stained with CD3, CD56, and viability dye. Each plate was fixated with PFA 4% and stored at 4 C. until FACS analysis (up to two days later). Samples were analyzed by FACS for target cell viability at each treatment.
Formulations: Cell lines culture medium: RPMI-1640 medium supplemented with 10% FBS, and 1% Penicillin-Streptomycin solution. Flow cytometry buffer: PBS supplemented with 2% FBS. Antibodies' working concentrations (in flow cytometry buffer): Anti-human CD56 FITC (5 L/test/100 Anti-human CD3 PE (5 L/test/100 Viobility 405/452 (1 L/test/100 PBMCs culture medium for killer cell formation: RPMI-1640 medium supplemented with 10% autologous serum (heat inactivated), 0.05 M -mercaptoethanol, and 1% Penicillin-Streptomycin solution. PBMCs culture medium for incubation with Target cells: Serum free medium supplemented with, 0.05 M -mercaptoethanol, and 1% Penicillin-Streptomycin solution. NeutrAvidin Protein: NeutrAvidin Protein was reconstituted with 1 mL ultrapure water+9 mL PBS. The specific activity for biotin binding is approximately 14 g/mg of protein. CD20+CD3+CD4+CD8 complex (40 samples): 7.7 g CD20 Ab+7.7 g CD3 Ab+7.7 g CD4 Ab+7.7 g CD8 Ab in 2.2 mL PBS. CD20+CD3+CD4+CD8+Avidin complex (40 samples): 7.7 g CD20 Ab+7.7 g CD3 Ab+7.7 g CD4 Ab+7.7 g CD8 Ab in 2.2 mL suspended NeutrAvidin. CD20+CD3 complex (40 samples): 15.4 g CD20 Ab+15.4 g CD3 Ab in 2.2 mL PBS. CD20+CD3+Avidin complex (40 samples): 15.4 g CD20 Ab+15.4 g CD3 Ab in 2.2 mL suspended NeutrAvidin. CD20 complex (40 samples): 30.8 g CD20 Ab in 2.2 mL PBS. CD20+Avidin complex (40 samples): 30.8 g CD20 Ab in 2.2 mL suspended NeutrAvidin. Avidin complex (40 samples): 2.2 mL suspended NeutrAvidin.
Results
The results of the experiments are shown in
The cytotoxic efficacy of biotinylated antibodies against CD20 and T cells (equal mixture of anti-CD3, anti-CD4 & anti-CD8) attached to IL-2 activated lymphocytes containing T cells and NK cells complexed with avidin or reacting alone against CD20 positive lymphoma cells was determined at two incubation time points (2 and 24 hours) at very low effector:target cells ratio of 0.5:1, 1:1 and 5:1.
(128)
(129)
(130)
(131)
(132)
(133)
(134)
(135)
(136) Summary and Conclusions
(137) As can be seen in
Example 5: Investigating the Use of IMAC Against Daudi Cells Targeting IL-2 Activated Lymphocytes with Monoclonal Antibody Against CD28
(138) The objective of this study was twofold: [1] investigate the efficacy of anti-cancer cytotoxicity when IL-2 activated killer cells are guided by agonistic monoclonal anti-CD28 antibody instead of a mixture of classical anti-T cells antibodies (including CD3, CD4 & CD8); [2] compare the efficacy of 1-step anti-cancer cytotoxicity against Daudi cells by IMAC consisting of avidin linked to killer cells by biotinylated monoclonal antibodies against CD20 and CD28, compared with a 2-step procedure incubating anti-CD20 with Daudi cells first, followed by adding IL-2 activated lymphocytes linked to biotinylated anti-CD28 monoclonal antibody attached to avidin.
(139) Test System
(140) Cell Line
(141) Daudi (ATCC CCL-213) B lymphoblast, Burkitt's lymphoma
Antibodies and Proteins for Binding Assay CD28 Biotinylated (supplied by Sponsor) Human CD20 (Rituximab) Biotinylated MAb (R&D, FAB9575B-100) NeutrAvidin Protein, 10 mg (Thermo Fisher, Waltham, MA USA, Catalog #31000) Proleukin 18 MIU (Human IL-2): Powder for solution for injection or infusion.
Flow Cytometry Reagents: Viobility 405/452 (Miltenyi Biotech, Bergisch Gladbach, Germany, Cat #130-109-816) Vybrant DiD Cell-Labeling Solution (Thermo Fisher, Waltham, MA USA, Cat #V22887) PFA 4%
Cell-Culture Materials RPMI-1640 medium (Biological Industries, Beit-HaEmek, Israel, Catalogue #01-100-1A). Fetal Bovine Serum (FBS, Biological Industries, Beit-HaEmek, Israel, Catalogue #04-01A). BIOTARGET serum free-medium (Biological Industries, Beit-HaEmek, Israel, Catalogue #05-080-1A). L-Glutamine (Biological Industries, Beit-HaEmek, Israel, Catalogue #03-020-1B). Penicillin-Streptomycin solution (Biological Industries, Beit-HaEmek, Israel, Catalogue #03-031-1B). PhosphateBuffered Saline (PBS, Biological Industries, Beit-HaEmek, Israel, Catalogue #02-023-1A). -mercaptoethanol (Sigma Aldrich, Rehovot, Israel, Catalogue #M6250). Trypan Blue (Sigma Aldrich, Rehovot, Israel, Catalogue #T8154).
Experimental Design
PBMCs Isolation and Preparation of Killer Cells Human venous blood (10-15 mL) from a healthy donor was collected to heparinized vials and mixed well by gently inverting the tube several times. The blood was diluted 2 with PBS. Histopaque was added to a 50 mL centrifuge tube into half of the final blood:PBS volume. The blood was gently layered on the top of Histopaque using a 1 mL auto pipette. The layering will be done very slowly in order that blood and Histopaque will stay as two different layers. The tubes were centrifuged (without any delay) at 400g for 30 min at +20 C. in a swing-out bucket without break. The whitish buffy coat (PBMCs) formed in the interphase between Histopaque and medium were aspirated without any delay. The cells were washed (centrifuged in 800g for 10 min at +20 C.) twice with 10 mL of PBS. PBMCs were reconstituted in serum free media and counted. The cells were incubated at a concentration of 210.sup.6 cells/mL with recombinant human IL-2 (6,000 IU/ml) for six days.
Target Cells Labeling (One Day Prior to End of Incubation Period of PBMCs with IL-2) Daudi cells were suspended at a density of 10.sup.6/mL in serum-free RPMI-1640 medium. Cell-labeling solution was added to the cells (5 L per mL of cell suspension). The cells mixed well, and incubated for 20 minutes at 37 C. The labeled cells were centrifuged at 1500 rpm for 5 minutes, at room temperature. The supernatant was removed, and the cells were gently resuspended in warm culture medium. This procedure was then repeated two more times.
Assay Set Up (Day of End of Incubation Period of PBMCs with IL-2): PBMCs were centrifuged (1200 rpm, 15 minutes, at RT), medium was removed, and the cells were washed once again with PBS. PBMCs were resuspended in 1 mL PBS and counted with trypan blue. PBMCs were adjusted to 410.sup.5 cells/mL, 2010.sup.5 cells/mL, 4010.sup.5 cells/mL, and 8010.sup.5 cells/mL, and added at 50 L to the wells, yielding 20,000 PBMCs/well, 100,000 PBMCs/well, 200,000 PBMCs/well, and 400,000 PBMCs/well respectively. Plate was centrifuged; PBS was discarded and 50 L PBS/Ab/Ab-avidin/avidin was added to indicated wells. The plate was incubated on ice for 1 h. Plate was centrifuged, PBS/Ab/Ab-avidin/avidin was discarded and Daudi cells (target cells) or medium only was added to the wells. The cells were collected, washed in PBS, counted, and adjusted to a concentration of 210.sup.5 cells/mL in PBS, each. The cells were added to wells at 100 L (20,000 cells/well). The plate was then incubated in 37 C. 5% CO.sub.2 for 6 h.
Flow Cytometry Analysis At the end of incubation period, the plate was centrifuged (1500 rpm, 5 minutes, 4 C.), washed with PBS, and stained with viability dye. The plate was fixated with Transcription Factor Fix/Perm reagent (Transcription Factor Fix/Perm Concentrate diluted 4 in Transcription Factor Fix/Perm Diluent) and stored at 4 C. until FACS analysis (up to two days later). Samples were analyzed by FACS for target cell viability.
Formulations Cell lines culture medium: RPMI-1640 medium supplemented with 10% FBS, and 1% Penicillin-Streptomycin solution. Flow cytometry buffer: PBS supplemented with 2% FBS. Antibodies' working concentrations (in flow cytometry buffer): Viobility 405/452 (1 l/test/100 L) PBMCs culture medium for killer cell formation: RPMI-1640 medium supplemented with 10% autologous serum (heat inactivated), 0.05 M -mercaptoethanol, and 1% Penicillin-Streptomycin solution. PBMCs culture medium for incubation with Target cells: Serum free medium supplemented with: 0.05 M -mercaptoethanol and 1% Penicillin-Streptomycin solution. NeutrAvidin Protein: NeutrAvidin Protein is reconstituted with 1 ml ultrapure water+9 mL PBS. The specific activity for biotin binding is approximately 14 g/mg of protein. CD20+CD28 complex (12 samples): 5.25 g CD20 Ab+5.25 g CD28 Ab in 0.75 mL PBS. CD20+CD28+Avidin complex (12 samples): 5.25 g CD20 Ab+5.25 g CD28 Ab in 0.75 mL suspended NeutrAvidin. Avidin complex (12 samples): 0.75 mL suspended NeutrAvidin.
Results and Conclusions
(142) As can be seen in
(143) Comparing the efficacy of cytotoxicity induced by 1-step and 2-step IMAC indicated that both 1-step and 2-step anti-Daudi cytotoxicity were effective, yet 1-step cytotoxicity with IMAC attacking Daudi cells with both anti-CD20 and anti-CD28 complexed with avidin was statistically significantly better that 2-step approach. These results indicate the importance of avidin as an anchor for antibody complex formation favoring dual simultaneous attack of cancer cells by monoclonal antibodies linked to avidin.
(144) Furthermore, the results of the experiments summarized in
Example 6: Optimizing the Anti-Cancer Effects of Mismatched Donor Lymphocytes by Maximal Pre-Treatment Activation of Donor Lymphocytes
(145) The purpose of the following experiments was to find out the most effective approach for stimulation of all effector cells that can participate in elimination of cancer cells. Human peripheral blood mononuclear cells (PBMC) were isolated as previously described and activated with interleukin 2 (IL-2) alone, phytohemagglutinin (PHA) alone, +-GalactosylCeramide (KRN7000) alone, KRN7000+PHA, IL-2+KRN7000, and IL-2+KRN7000+PHA in comparison with untreated cells.
(146) Experimental Design
(147) PBMCs Isolation
(148) Human Venous blood (20-30 mL) from a healthy donor was collected to heparinized vials and mixed well by gently inverting the tube several times. In addition, approximately 10 mL blood was collected into serum collection tubes. The blood was diluted 2 with PBS. Ficoll-Paque was added at 15 mL max into a 50 mL centrifuge tube (1 tube per 8 mL blood collected was required). The blood:PBS was gently layered on the top of Ficoll-Paque using a 1 mL auto pipette at 1:1 ratio with the Ficoll-Paque. The layering was done very slowly so that blood and Ficoll-Paque will stay as two different layers. The tubes were centrifuged (without any delay) at 400 g for 30 min at +20 C. in a swing-out bucket without break. First, the upper plasma layer was collected and dispensed. Then, the whitish buffy coat (PBMCs) formed in the interphase between Ficoll-Paque and plasma was aspirated (and divided between several 50 mL tubes-per each 10 mL blood collected, use 1 tube). The cells were washed (centrifuged in 400 g for 10 min at +20 C.) twice with 10 mL of PBS+2% FBS. Finally, the cells were resuspended with 10 mL PBS+2% FBS and counted using trypan blue. The PBMCs were centrifuged and adjusted to 110.sup.7 cells/mL in ice cold freezing medium and placed in a cooled Mr. Frosty container (filled with isopropanol). The container was placed in 80 C. for 24 hours and then the cells were moved to a liquid nitrogen container.
Heat Inactivated Serum Preparation: The serum vials were placed in RT for at least 30 minutes. Next, they were centrifuged at 4100 rpm, for 10 minutes, at 4 C. The serum was then collected into a 15 mL tube and placed in 57 C. bath for 15 minutes. After heat inactivation, it was aliquoted into 1 mL aliquots, and frozen at 80 C.
Main Assay Procedure: PBMCs were thawed for 1 minutes in 37 C. bath and transferred immediately into 50 mL tube filled with PBS. The tube was centrifuged at 300 g, 5 minutes, and the cells were resuspended with 1 mL activation medium and counted (diluted 1:10 and 1:2 with TB). The cells were adjusted to 210.sup.6 cells/mL with activation medium and placed in 24-well plate at 0.5 mL/well. The cells were then activated.
Flow Cytometry Analysis:
(149) At the end of incubation, PBMCs from each well were collected into Eppendorf tube, centrifuged at 300g for 5 min in 4 C., washed once with 1 mL PBS, resuspended in 0.1 mL PBS and placed in a well of V shape 96 well plate. The plate was centrifuged, and the cells were resuspended with 100 L diluted viability dye. The plate was incubated 30 min in RT (in dark). The plate was centrifuged at 300g for 5 min in 4 C., and cells were resuspended with 100 L surface markers cocktail. The plate was incubated 30 min on ice (in dark). The plate was centrifuged at 300g for 5 min in 4 C. and resuspended in 0.1 ml of the Transcription Factor Fixation/Permeabilization working solution and mixed thoroughly. The plate was incubated 30 min on ice (in dark). The plate was centrifuged, and Fixation Buffer was discarded. The plate was washed twice with 0.1 mL 1 Permeabilization working solution (centrifuged at 300g for 5 min in RT). The plate was resuspended in 50 L of 1 Permeabilization working solution +FoxP3 antibody. The plate was incubated for 30 minutes at RT (in dark). The plate was centrifuged (300 g, 5 minutes, 4 C.) and washed twice with 0.1 mL 1 Permeabilization working solution. Finally, cells were resuspended in 150 L FACS buffer for flow cytometric analysis.
Material and Formulations: Flow cytometry buffer: PBS (Biological Industries, Cat #01-023-1A) supplemented with 2% FBS (Biological Industries, Cat #04-127-1A). Activation medium: RPMI-1640 medium (Biological Industries, Cat #01-100-1A) supplemented with 10% autologous serum (heat inactivated), 0.05 mM -mercaptoethanol (Sigma, Cat #M6250) and 1% Penicillin-Streptomycin solution (Biological Industries, Cat #03-031-1B). PBMCs freezing medium: 90% FBS (Biological Industries, Cat #04-127-1A) and 10% DMSO (Sigma, Catalogue #D4540). Ficoll-Paque PREMIUM (GH Healthcare, Catalogue #17-5442-02). PHA (Sigma, Cat #L1668) was supplied at a stock concentration of 5 mg/mL. It was first diluted in activation medium 1:10 yielding 500 g/mL. Thereafter 50 from 500 g/mL stock was added to the cells and diluted 1:100 yielding final concentration of 5 g/mL. Proleukin 18 MIU (Human IL-2) is stored at a stock of 1810.sup.6 units/mL. First it was diluted 1:30 in activation medium yielding 0.610.sup.6 units/mL. Thereafter 5 l from 0.610.sup.6 units/mL stock was added to cell and diluted 1:100 yielding final concentration of 6000 units/mL. KRN7000 (0.2 mg/mL) dissolved in DMSO at 1 mg/mL (200 L DMSO was added to 0.2 mg powder). First it was diluted 1:10 and 1:100 in activation medium yielding 100 and 10 g/mL. Thereafter 5 L from these two stocks was added to cells and diluted 1:100 yielding final concentrations of 1000 and 100 ng/mL.
Flow Cytometry Antibodies Viability dye: LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen, Cat #L34966) Dilute in PBS 1:1000. Anti-Human CD3-BV421 (BD Bioscience, Cat #563798). 5 L/100 L FACS buffer (1:20). Anti-Human CD4-PerCP-Cyanine5.5 (Biogems, Catalogue #06121-70). 5 L/100 L FACS buffer (1:20). Anti-Human CD45-PE (Biogems, Catalogue #07151-60). 5 L/100 L FACS buffer (1:20). Anti-Human FOXP3-Alexa Fluor 647 (Biogems, Catalogue #07151-60). 5 L/100 L FACS buffer (1:20). Anti-Human CD56-FITC (Biogems, Catalogue #08631-50). 5 L/100 L FACS buffer (1:20). Permeabilization Buffer Xl: Permeabilization Buffer X10 (Peprotech, cat #92110-00-150) will be diluted with distilled water to 1 prior to use. Transcription Factor Fixation/Permeabilization working solution: will be prepare by mixing one part of the concentrate solution (cat #92550-00) with 3 parts of the diluent (cat #92160-00).
Results and Conclusions
(150) Results of different methods for proliferation of CD45 positive mononuclear blood lymphocytes, CD3+ cells including CD3+CD56 T cells & CD3+CD56+ NKT cells, CD56+ NK cells, FOXP3+ regulatory T cells and consequently CD3+CD56+ NKT cells are shown in
(151) The highest number of effector cells can be obtained by culturing IL-2 activated PBMC at 6,000 IU/ml in combination with 100 ng/ml KRN7000 and 5 g/ml PHA. Under the culture conditions combining IL-2, KRN7000 and PHA the number of activated T cells was the highest observed. In addition, the number of regulatory T cells was doubled as compared with lymphocytes cultured with IL-2 alone, so regulatory T cells could potentially better control any possible untoward cytokine reaction. The highest number of NK cells was also obtained by culturing PBMC with IL-2, 100-1,000 ng/ml KRN7000 and PHA. Mismatched NK cells exert fast and most effective cytotoxicity against MHC mismatched target cells.
(152) Interestingly, adding KRN7000 to lymphocytes activated with IL-2 with or without PHA increased the number of NKT cells by 30% as compared with cells treated with IL-2 alone. Accordingly, NKT cells could also amplify the anticipated anti-cancer effects induced by donor lymphocytes activated by IL-2.
Example 7: Phase 1 Clinical Trial Using IMAC Alone Based on an Attempt to Activate Spontaneously Patient's Own Immune System In Vivo Against B Cell Malignant Diseases (CLL, ALL or NHL) and/or Multiple Myeloma
(153) IMAC comprising equal amounts of biotinylated anti-CD20 and anti-CD3 monoclonal antibodies connected through NeutrAvidin, or StreptAvidin is tested in 3 cohorts of patients with intractable B cell malignancies (CLL, ALL or NHL) for each dose of IMAC. Treatment with IMAC comprises graded increments of IMAC administered intravenously, starting with 1 g and doubling the dose pending no restrictive CRS, allowing for no more than grade II toxicity in 2 of 3 patients. The maximally tolerated dose (MTD) is defined according to methods known in the art (for example references 22, 23) and Common Terminology Criteria for Adverse Events (CTCAE).
(154) Similarly, a study is accomplished using IMAC composed of anti-CD38 and anti-CD3 (or any other monoclonal anti-T cell antibodies).
(155) Initially an intra-patient IMAC dose finding will be carried out to check if any risk may be involved by injection of IMAC and for finding the maximal tolerated dose (MTD) of IMAC that will used in subsequent studies. Dose escalation will be carried out as follows: The first 3 patients will be treated with 1 g IMAC administered intravenously. Pending lack of CRS in all 3 patients, IMAC dose will be escalated to 2 g. Pending lack of CRS in all 3 patients, IMAC dose will be escalated to 4 g. Pending lack of CRS in all 3 patients, IMAC dose will be escalated to 8 g. Pending lack of CRS in all 3 patients, IMAC dose will be escalated to 16 g. Pending lack of CRS in all 3 patients, IMAC dose will be escalated to 32 g. Pending lack of CRS in all 3 patients, IMAC dose will be escalated to 64 g. Pending lack of CRS in all 3 patients, IMAC dose will be escalated to 128 g, etc. Escalation will be discontinued in any cohort if MTD will be reached in 2 of 3 patients.
Example 8: Phase 1 Clinical Trial in Patients with B Cell Malignant Diseases (CLL, ALL or NHL) and/or Multiple Myeloma Based on the Use of IMAC Followed by In Vivo Activation of Patient's Lymphocytes with Low-Dose IL-2
(156) The same procedures are done as described in Example 7, using the safe IMAC MTD as discovered in Example 7, followed by in vivo activation of patient's immune system with low dose IL-2 (10.sup.6 IU/day) administered subcutaneously for 5 days to investigate if IL-2 activation in vivo in the presence of an optimal safe dose of IMAC induces any serious side effects or CRS due to activating patient's own T cells (in case of any signs of CRS, the dose of IMAC will be reduced to a safer level).
Example 9: Phase 1 Clinical Trial in Patients with B Cell Malignant Diseases (CLL, ALL or NHL) and/or Multiple Myeloma Based on the Use of IMAC Followed by In Vivo Infusion of Haploidentical or Unrelated Donor Lymphocytes
(157) The same procedures are done as described in Example 7, using the safe IMAC MTD as discovered in Example 8. In case of any serious side effects or CRS due to activating donor's own T cells, the dose of IMAC or the number of donor lymphocytes will be reduced to a safer level or alternatively subsequent treatment will be based on the use intentionally mismatched donor's NK cells, Donor NK cells can be obtained by deletion of CD3-positive T cells or much simpler by exposure of donor's cells to ionizing radiation (600 cGy) that eliminates proliferation of T cells but retains non-replicating NK function (21, 32). The starting number of donor lymphocytes will consist of 510.sup.7 cells/kg. In case of CRS, the next patient will be treated with a lower dose of donor lymphocytes of 510.sup.6 cells/kg
Example 10: Phase 1 Clinical Trial in Patients with B Cell Malignant Diseases (CLL, ALL or NHL) and/or Multiple Myeloma Based on the Use of IMAC Followed by In Vivo Infusion of Haploidentical or Unrelated Donor Lymphocytes Activated In Vivo with Low-Dose IL-2
(158) The same procedures will be done as described in Example 9, using the safe doses of IMAC and IL-2 as will be discovered in Example 9. The treatment will consist of safer doses of donor lymphocytes and IL-2 as will be defined in Example 9. Assuming that the starting number of donor lymphocytes will consist of 510.sup.7 cells/kg. In case of CRS discovered in Example 7, the next patient will be treated with a lower dose of donor lymphocytes of 510.sup.6 treated in vivo with IL-2 (110.sup.6 IU/day, unless the dose of IL-2 will be reduced in Example 7 to 0.510.sup.6IU/day). If CRS will develop in the 1.sup.st patient, the 2.sup.nd patient will be treated with a full dose of donor cells, 510.sup.7/kg and full dose of IL-2 (110.sup.6IU/day) using donor's NK cells. Donor NK cells can be obtained by deletion of CD3-positive T cells or much simpler by exposure of donor's cells to ionizing radiation (600 cGy) that eliminates proliferation of T cells but retains non-replicating NK function. The assumption is that 70-80% of donor lymphocytes are CD3-positive.
Example 11: Phase 1 Clinical Trial in Patients with B Cell Malignant Diseases (CLL, ALL or NHL) and/or Multiple Myeloma Based on the Use of IMAC Followed by Infusion of Haploidentical or Unrelated Donor Lymphocytes T & NK Cells Activated Ex Vivo with IL-2 Prior to Cell Infusion and In Vivo Following Cell Infusion with Low-Dose IL-2
(159) The safe protocol defined in Example 10 will serve as the basis for an identical protocol based on infusion of ex vivo activated donor lymphocytes for 5 days. The safe number of donor lymphocytes containing both T and NK cells or using T cell depleted NK cells will be used and activated in vivo with low dose IL-2 (110.sup.6 IU/day if well tolerated or reduced dose of 0.510.sup.6 IU/day if associated with toxicity or CRS).
Example 12: Phase 1 Clinical Trial in Patients with High-Risk Metastatic Solid Tumors Based on the Use of IMAC Followed by Infusion of Haploidentical or Unrelated Donor Lymphocytes Activated Ex Vivo and/or In Vivo with IL-2
(160) Pending successful results of phase 1 studies as shown in Examples #7-10, several future studies will be designed for treatment of patients with otherwise incurable metastatic solid tumors. Successful Phase 2 and phase 3 will consist of the basis for using the IMAC technology for patients with high-risk cancer at the stage of minimal residual disease, aiming for cure.
(161) The details of the design of any future clinical trial depend on information obtained from the pilot clinical trials described in examples 7-11 and considering potential efficacy that may be suggested even in the course of a phase I clinical trial in view of the exquisite anti-cancer efficacy anticipated. In any event, the goal is to focus on IMAC procedures, both for eradication of cancer and for induction of anti-cancer immunity, that could be applied on a much larger scale, provided that the IMAC treatment will be well tolerated, with no more than transient grade II toxicity.
Example 13: Additional Studies to Improve the Anticipated Therapeutic Effects of IMAC
(162) Following determining safe and optimal IMAC concentrations used for in vivo activation of patient's own immune system, donor's immune system and readily available killer cells of activated intentionally mismatched donor lymphocytes, additional procedures will be considered in order to improve the anticipated anti-cancer effects of IMAC, as follows: 1. Start IMAC therapy as early as possible at the stage visible or measurable minimal residual disease that can be accomplished in most patients with cancer, hematological malignancies and solid tumors, following conventional first line treatment since this may represent the best window of opportunity to accomplish cure, when patient's general condition and immune system are still well preserved. Existence of visible or measurable disease by PET/CT imaging, or comparing circulating tumor cells (CTC) or circulating tumor DNA (ctDNA) before and after IMAC treatment will be essential in order to prove clinical efficacy before considering large-scale prospective randomized clinical studies in hematopoietic malignancies and high-risk solid tumors. 2. Her2/neu positive metastatic breast cancer, for example, targeting killer cells with anti-Her2/neu monoclonal antibodies (Herceptin) is an example of a disease category suitable for a pilot clinical trial for investigation of the role of IMAC as a model of metastatic solid tumors.
REFERENCES
(163) 1. Slavin S, Or R, Kapelushnik Y, Drakos P, Ackerstein A, Vourka-Karussis U, Weiss L, Nagler A: Immunotherapy of minimal residual disease in conjunction with autologous and allogeneic bone marrow transplantation (BMT). Leukemia 6: 164-166, 1992. 2. Slavin S, Naparstek E, Nagler A, Ackerstein A, Samuel S, Kapelushnik J, Brautbar C, Or R. Allogeneic cell therapy with donor peripheral blood cells and recombinant human interleukin-2 to treat leukemia relapse post allogeneic bone marrow transplantation. Blood 1996; 87:2195-2204. 3. Slavin S, Naparstek E, Nagler A, Ackerstein A, Kapelushnik Y, Or R: Allogeneic cell therapy for relapsed leukemia following bone marrow transplantation with donor peripheral blood lymphocytes. Exp Hematol. 1995; 23:1553-1562. 4. Slavin S, Nagler A, Naparstek E, Kapelushnik Y, Aker M, Cividalli G, Varadi G, Kirschbaum M, Ackerstein A, Samuel S, Ben-Tal O, Eldor A, Or R. Nonmyeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and non-malignant hematologic diseases. Blood 1998; 91(3): 756-763. 5. Or R, Ackerstein A, Nagler A, Amar A, Naparstek E, Varadi G, Kapelushnik J, Samuel S, Pugatsch T, Brautbar C, Slavin S. Allogeneic cell-mediated and cytokine-activated immunotherapy for malignant lymphoma at the stage of minimal residual disease after autologous stem cell transplantation. J. Immunother 1998; 21(6):447-53. 6. Slavin S. Cancer immunotherapy with alloreactive lymphocytes. N. Engl. J. Med. 2000 (Sep. 14); 343:802-803. 7. Nagler A, Ackerstein A, Or R, Naparstek E, Slavin S. Adoptive immunotherapy with haploidentical allogeneic peripheral blood lymphocytes (PBL) following autologous bone marrow transplantation (ABMT). Exp Hematol 2000:28 (11); 1225-1231. 8. Slavin S. Immunotherapy of Cancer with Alloreactive Lymphocytes (Review). Lancet Oncol 2001; 2: 491-98. 9. Nicola Daniele, Maria Cristina Scerpa, Maurizio Caniglia, Chiara Ciammetti, Cecilia Rossi, Maria Ester Bernardo, Franco Locatelli, Giancarlo Isacchi, and Francesco Zinno. Overview of T-cell depletion in haploidentical stem cell transplantation. Blood Transfus. 2012 July; 10(3): 264-272. 10. Luznik L, O'Donnell P V, Symons H J, Chen A R, Leffell M S, Zahurak M et al. HLA-haploidentical bone marrow transplantation for hematologic malignancies using nonmyeloablative conditioning and high-dose, posttransplantation cyclophosphamide. Biol Blood Marrow Transplant 2008; 14: 641-650. 11. Strhlein M A, Siegel R, Jger M, Lindhofer H, Jauch K W, Heiss M M. Induction of antitumor immunity by trifunctional antibodies in patients with peritoneal carcinomatosis. J Exp Clin Cancer Res. 2009 Feb. 14; 28:18. 12. Eissler N, Ruf P, Mysliwietz J, Lindhofer H, Mocikat R. Trifunctional bispecific antibodies induce tumor-specific T cells and elicit a vaccination effect. Cancer Res. 2012 Aug. 15; 72(16):3958-66. 13. Morecki S, Lindhofer H, Yacovlev E, Gelfand Y, Slavin S. Use of trifunctional bispecific antibodies to prevent graft versus host disease induced by allogeneic lymphocytes. Blood. 2006; 107:1564-1569. 14. Morecki S, Lindhofer H, Yacovlev E, Gelfand Y, Ruf P, Slavin S. Induction of long-lasting antitumor immunity by concomitant cell therapy with allogeneic lymphocytes and trifunctional bispecific antibody. Exp Hematol. 2008 August; 36(8):997-1003. 15. U.S. Pat. No. 8,066,989 16. U.S. Pat. No. 6,551,592 17. Cohen P, Vourka-Karussis U, Weiss L, Slavin S. Spontaneous and IL-2 induced anti-leukemic and anti-host effects against tumor- and host-specific alloantigens. J Immunol 1993; 151:4803-4810. 18. Slavin S, Shapira M Y, Morecki S, Samuel S, Ackerstein A, Gelfand Y, Resnick I, Bitan M, Or R. Immunotherapy for resistant hematologic malignancies using matched or mismatched rIL-2 activated donor lymphocytes positively selected for CD56+ after allogeneic stem cell transplantation for allogeneic cell therapy without GVHD. 45th ASH Annual Meeting. San Diego, California, USA. Dec. 6-9, 2003. Blood 2003; 102 (11):Abs #5329 p. 400b. 19. Slavin S. Allogeneic cell-mediated immunotherapy at the stage of minimal residual disease following high-dose chemotherapy supported by autologous stem cell transplantation. Acta Haematologica. 2005; 114:214-220. 20. Slavin S, Ackerstein A, Or R, Shapira M Y, Gesundheit B, Askenasy N, Morecki S. Immunotherapy in high-risk chemotherapy-resistant patients with metastatic solid tumors and hematological malignancies using intentionally mismatched donor lymphocytes activated with rIL-2: a phase I study. Cancer Immunol Immunother. 2010 October; 59(10):1511-9. 21. Tsirigotis P, Resnick I B, Kapsimalli V, Dray L, Psarra E, Samuel S, Spyridonidis A, Konsta E, Vikentiou M, Or R, Slavin S, Shapira M Y. Irradiated mononuclear cells express significant in vitro cytotoxic activity: promise for in vivo clinical efficacy of irradiated mismatched donor lymphocytes infusion. Immunotherapy. 2014 April; 6(4):409-17. 22. Morecki S, Lindhofer H, Yacovlev E, Gelfand Y, Slavin S. Use of trifunctional bispecific antibodies to prevent graft versus host disease induced by allogeneic lymphocytes. Blood. 2006; 107:1564-1569. 23. Morecki S, Lindhofer H, Yacovlev E, Gelfand Y, Ruf P, Slavin S. Induction of long-lasting antitumor immunity by concomitant cell therapy with allogeneic lymphocytes and trifunctional bispecific antibody. Exp Hematol. 2008 August; 36(8):997-1003. 24. Goldenberg D M, Cancer Therapy with Radiolabeled Antibodies. CRC Press, 1995. 25. Petronzelli F, Pelliccia A, Anastasi A M, Lindstedt R, Manganello S, Ferrari L E, Albertoni C, Leoni B, Rosi A, D'Alessio V, Deiana K, Paganelli G, De Santis R. Therapeutic use of avidin is not hampered by antiavidin antibodies in humans. Cancer Biother Radiopharm. 2010 October; 25(5):563-70. 26. Muros M A, Varsaysky M, Iglesias Rozas P, Valdivia J, Delgado J R, Forrer F, Bodei L, Paganelli G. Outcome of treating advanced neuroendocrine tumours with radiolabelled somatostatin analogues. Clin Transl Oncol. 2009 January; 11(1):48-53. 27. Paganelli G, De Cicco C, Ferrari M E, Carbone G, Pagani G, Leonardi M C, Cremonesi M, Ferrari A, Pacifici M, Di Dia A, De Santis R, Galimberti V, Luini A, Orecchia R, Zurrida S, Veronesi U. Intraoperative avidination for radionuclide treatment as a radiotherapy boost in breast cancer: results of a phase II study with (90)Y-labeled biotin. Eur J Nucl Med Mol Imaging. 2010 February; 37(2):203-11. 28. Park J H, Rivire I, Gonen M, Wang X, Snchal B, Curran K J, Sauter C, Wang Y, Santomasso B, Mead E, Roshal M, Maslak P, Davila M, Brentjens R J, Sadelain M. Long-Term Follow-up of CD19 CAR Therapy in Acute Lymphoblastic Leukemia. N Engl J Med. 2018 Feb. 1; 378(5):449-459. 29. Panigrahi S, Yacovlev E, Gelfand Y, Schuger L, Slavin S, Morecki S. Intraportal and systemic allogeneic cell therapy in a murine model of hepatic metastatic breast cancer. Cytokines, Cellular & Molecular Therapy. 2002; 7(3):99-106. 30. Morecki S, Yacovlev E, Gelfand Y, Vilensky A, Slavin S. Allogeneic vs syngeneic killer splenocytes as effector cells for induction of graft vs tumor effect. Biology of Blood & Marrow Transplantation. 2004; 10(1):40-48. 31. Ackerstein, a, Morecki S, Gelfand Y, Samuel S, Or R, Slavin S. Outpatient non-myeloablative cell-mediated immunotherapy with intentionally mismatched allogeneic lymphocytes with rIL-2 for patients with metastatic solid tumors. 45th ASH Annual Meeting. San Diego, California, USA. Dec. 6-9, 2003. Blood 2003; 102 (11):Abs #5366 p. 409b. 32. Zarcone D, Tilden A B, Lane V G, Grossi C E. Radiation sensitivity of rating and activated nonspecific cytotoxic cells of T lineage and NK lineage. Blood, 73: 1615-1621, 1989.