EP0835134A1 - Immunoconjugues comprenant des fragments monocatenaires de regions variables d'anticorps anti-cd-19 - Google Patents
Immunoconjugues comprenant des fragments monocatenaires de regions variables d'anticorps anti-cd-19Info
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- EP0835134A1 EP0835134A1 EP96916466A EP96916466A EP0835134A1 EP 0835134 A1 EP0835134 A1 EP 0835134A1 EP 96916466 A EP96916466 A EP 96916466A EP 96916466 A EP96916466 A EP 96916466A EP 0835134 A1 EP0835134 A1 EP 0835134A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
- A61K47/6867—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of a blood cancer
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3061—Blood cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- CD antigens directed against cell surface molecules defined by cluster differentiation (CD) antigens represent a unique opportunity for the development of therapeutic reagents.
- Certain CD antigen expression is highly restricted to specific lineage lymphohematopoietic cells and, over the past several years, antibodies directed against lymphoid-specific CD antigens have been used to develop treatments that were effective either in vitro or in animal models (Ghetie et ah, 1988; Uckun et al., 1986; Myers et al., 1991; Jansen et al., 1992).
- intact antibodies and antibody-toxin conjugates have several disadvantages that limit their efficiency.
- Fragments of some toxins are then translocated across the membrane of this organelle.
- Other immunotoxins for example,ricin
- Other immunotoxins are routed further to the trans-Golgi network, where a minority undergo translocation to the cytoplasm.
- cytoplasm most are routed to lysosomes, where they are degraded.
- the toxins used clinically act either to adenosine diphosphate (ADP)-ribosylate elongation factor 2 (for example, Pseudomonas exotoxin (PE)) or to inactivate the 60S ribosomal subunit so that it has a decreased capacity to bind elongation factor 2 (for example, ricin).
- ADP adenosine diphosphate
- PE Pseudomonas exotoxin
- the immunoconjugate should be specific and should not react with normal tissues. Binding to tissues that do not express antigen can be reduced by removal of the nonspecific natural cell-binding subunits or domains of the toxin.
- plant glycoprotein toxins contain mannose oligosaccharides that bind to cells of the reticuleondothelial system and, in some cases, also contain fucose residues that are recognized by the receptors on hepatocytes, deglycosylation of plant toxins may be required to avoid rapid clearance and potential cytotoxic effects on these cells, (ii) The linkage of the toxin to the antibody should not impair the capacity of the antibody to bind antigen, (iii) The immunotoxin must be internalized into endosomic vesicles.
- toxins directed by monoclonal antibodies to surface receptors that are normally internalized may be more active than those directed toward noninternalizing cell surface molecules,
- the active component of the toxin must translocate into the cytoplasm.
- the first generation of heterobifunctional cross- linkers used to bind the toxin to the monoclonal antibody generated disulfide bonds that were unstable in vivo. This problem was solved in part by the synthesis of more stable cross-linkers, which used phenyl or methyl groups, or both, adjacent to the disulfide bond to restrict access to the bond.
- an immunotoxin is initially assessed by measuring its ability to kill cells with target antigens on their surfaces. Because toxins act within the cells, receptors and other surface proteins that naturally enter cells by endocytosis usually make good targets for immunotoxins, but surface proteins that are fixed on the cell surface do not. However, if several antibodies recognizing different epitopes on the same cell surface protein are available, it is useful to test them all, because some, perhaps by producing a conformational change in the target protein's structure, may induce its internalization or direct its intracellular routing to an appropriate location for toxin translocation (May et al., 1991; Press et al., 1988).
- the immunotoxin contains a form of PE or ricin in which the binding of the toxin moiety to its receptor, although weakened by chemical modification, still occurs and promotes internalization since toxin receptors are efficiently internalized (Willingham et al., 1987; Lambert et al, 1991; Colombatti et al, 1986).
- immunotoxins have been developed and approved for human trials. Two different kinds of trials have been conducted. The first involves the ex vivo addition of immunotoxins to harvested bone marrow to eliminate contaminating tumor cells before reinfusion in patients undergoing autologous bone marrow transplantation. A variety of antibodies, linked to ricin or ricin A chain, including anti-CD5 and anti- CD?, have been used for this purpose (Uckun et al, 1990b). The second kind of trial involves the parenteral administration of immunotoxins, either regionally (such as the peritoneal cavity) or systematically, to patients with cancer. These have been primarily Phase 1 and 2 trials in patients in which conventional treatments have failed, and the patients have a large tumor burden.
- the maturation of human BCPs into functional B lymphocytes represents a developmentally programmed multi-step process, which is accompanied by a cascade of somatic immunoglobuiin gene rearrangements (Korsmeyer et al, 1981), as well as a coordinated acquisition and loss of B-lineage differentiation antigens (Nadler).
- B-lineage differentiation antigens have been identified on B-lineage cells not including the surface immunoglobulins (slg), major histocompatibility (MHC) antigens, or the receptor proteins for defined cytokines.
- slg surface immunoglobulins
- MHC major histocompatibility
- Many of the B-lineage differentiation antigens represent functionally important surface receptors on developing B-lineage cells, and their expression is regulated by different external signals (Knapp et al, 1989a; Clark et al, 1989; Zola, 1987).
- T-lineage differentiation antigens including CD2, CD4, CD8, and CD18/LFA-1 function as cell-surface bound ligands (CD2 for LFA- 3, CD4 for class II MGC, CD8 for class I MHC, CD18/LFA-1 for I-CAM- l/gp80).
- B-lineage antigens such as CD23 and CD40
- CD19, CD22, and B7 antigens are members of the Ig supergene family (Knapp et al, 1989b; Stamenkovic, 1988; Stamenkovic, 1990; Freeman et al, 1989).
- CD21 has been identified as the C3d receptor as well as a receptor for Epstein-Barr virus (EBV) (Knapp et al, 1989b).
- CD19 shows homology to proteins encoded by the int-1 oncogene and by EBV (Stamenkovic, 1988).
- CD19 has been proposed as a bridging molecule important for transduction of slg-mediated signals in mature B cells (Pesando et al, 1989; Carter et al, 1990).
- CD19 as a signal- transducing subunit and CD21 as a ligand-binding subunit linking the B cell to the complement system have been reported to form a complex on the surface of B cells which may be involved in the slg-dependent activation.
- CD19 the function of the CD19 molecule is not dependent on the presence of slg or CD21 because CD19 ligation results in stimulation of phosphoinositide turnover (Uckun et al, 1989) and calcium mobilization in sIg-CD21-BCP populations and modulates their proliferative activity (Uckun et al, 1988; Ledbetter et al, 1988).
- CD22 displays a high degree of homology to the myelin-associated glycoprotein (MAG), a neuronal surface adhesion molecule mediating cell-to-cell interactions between B cells and monocytes (Stamenkovic, 1990. Furthermore, CD22 may also be important for transduction of slg- mediated signals (Pezzutto et al, 1988).
- MAG myelin-associated glycoprotein
- CD22 may also be important for transduction of slg- mediated signals (Pezzutto et al, 1988).
- CD28 T-cell activation antigen which is another member of the Ig superfamily (Linsley et al, 1990).
- CD28-B7 mediated adhesion between activated B cells and T cells might be important for T-cell regulation of antigen-specific B-cell responses.
- Monoclonal antibodies have largely been applied clinically to the diagnosis and therapy of cancer and the modulation of the immune response to produce immunosuppression for treatment of autoimmune and graft versus host diseases (GVHD) and for prevention of allograft rejection.
- Human monoclonal antibodies have also been applied clinically against cytomegalovirus, Varicella zoster virus, and the various specific serotypes of Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneu moniae.
- Antibodies or their fragments can also be genetically engineered to have more rapid clearance. This is desirable when a monoclonal antibody is conjugated to a radionuclide for use in radioimmunoscanning.
- Fab antigen-binding fragment
- F(ab') 2 single chain Fv fragments of monoclonal antibodies have survival half-lives of less than 5 hours. Rapid turnover can also be accomplished by the deletion of the CH2 domain as demonstrated for an antibody reactive with the disaloganglioside GD2 expressed on human tumors of neuroectodermal origin (Mueller et al, 1990).
- the CD19 antigen which is found on mature B cells but not on plasma cells, has proven to be a very useful target for development of immunoconjugates because most lymphomas and B lineage leukemias express this differentiation marker (Uckun et al, 1990a).
- Anti-CD19 immunoconjugates have relied on the chemical conjugation of the antibody and a modified catalytic toxin such as the A chain of ricin (Ghetie et al, 1988) or pokeweed antiviral protein (Uckun et al, 1986; Myers et al, 1991).
- a modified catalytic toxin such as the A chain of ricin (Ghetie et al, 1988) or pokeweed antiviral protein (Uckun et al, 1986; Myers et al, 1991).
- a modified catalytic toxin such as the A chain of ricin (Ghetie et al, 1988) or pokeweed antiviral protein (Uckun et al,
- the approach is to obtain bone marrow from a patient in remission (preferably in the first remission) and to freeze it. If the patient subsequently relapses, the patient is then subjected to "supralethal" therapy with X-irradiation and or chemotherapy in order to eradicate the tumor. The patient is then rescued from death by infusion of his own bone marrow.
- Immunoconjugates may be utilized for ex vivo purging of neoplastic cells from patient bone marrow grafts. These autologous grafts are reintroduced into leukemic patients after aggressive supra lethal chemotherapy and irradiation. The objective of all strategies is to deplete neoplastic cells while leaving unharmed the pluripotent hematopoietic stem cells which repopulate the patient's marrow after reinfusion. Intact immunoconjugates selectively eliminate antigen-positive targets without endangering engraftment and without causing intoxication.
- Autologous marrow may be purged of residual leukemia cells without destroying hematopoietic stem cells by the use of immunoconjugates either in vivo or ex vivo.
- Ex vivo treatment with immunoconjugates has been shown to eliminate most T or B cells present in human marrow without damaging the ability of the marrow to reconstitute lethally irradiated recipients. While the efficiency of immunoconjugates to kill "the last" leukemic cells still remains an issue the even greater efficiency of radiolabeled immunoconjugates should greatly increase the chances of successful treatment.
- Radiolabeled Immunoconjugates It has been reported that an immunotoxin can specifically eliminate more than 99.99% of clonogenic leukemic T cells even in the presence of excess human bone marrow. The use of a radiolabeled immunotoxin should eliminate even more leukemic T cells, possibly at a rate of greater than 5 logs or 99.999%, indicating that the radiolabeled immunotoxin may be extremely useful for the ex vivo elimination of leukemic cells in autologous BMT.
- Radiolabeled monoclonal antibodies have been developed as alternative immunoconjugates for delivery of a cytotoxic effector to target cells and for radioimaging (Schlom; Kozak et al, 1985). These species possess potential to compensate for the observed shortcomings of immunotoxins. Toxin conjugates do not pass easily from the endosome to the cytosol. Furthermore, the toxins are immunogenic and thus provide only a short therapeutic window before the development of antibodies directed toward the toxin. Radioimmunodetection with the use of radiolabeled monoclonal antibodies, most often with monoclonal antibodies to carcinoembryonic antigen, is widely used to complement other approaches for tumor detection.
- F(ab') 2 and Fab fragments are preferred for imaging because both targeting and blood clearance are most rapid, which reduces the background. Tumors as small as 0.5 cm, which are sometimes missed by other radiological methods, can be imaged with antibodies or antibody fragments labeled with suitable radionuclides.
- radiolabeled monoclonal antibody conjugates for therapy is that with the appropriate choice of radionuclide, radiolabeled monoclonal antibodies can kill cells from a distance of several cell diameters and may therefore kill antigen-negative cells adjacent to antigen-expressing cells. Furthermore, the radiolabeled antibody need not be internalized to kill the tumor cell. Such techniques are exemplified in the teachings of U.S. Patent No. 4,831,122 to Buschbaum et al, incorporated herein by reference. In a radiolabeled monoclonal antibody, the radionuclide must be tightly linked to the antibody either directly or by a bifunctional chelate.
- a monoclonal antibody-chelate complex For a monoclonal antibody-chelate complex to be effective, it must meet criteria in addition to those that are true for all monoclonal antibodies: (i) the chelating agent coupled to the monoclonal antibody should not compromise antibody specificity; (ii) the chelation and radiolabeling procedure should not alter the distribution and catabolism of the monoclonal antibody; and (iii) the bifunctional chelate should not permit elution and thus premature release of the radiolabeled metal in vivo. Failure to fulfill this last requirement has led to unacceptable toxicity and reduced efficacy. There are a number of suitable ⁇ -, ⁇ -, and y-emitting radionuclides.
- Isotopes emitting ⁇ particles although superior to y- emitting radionuclides, are not optimal because their low linear energy transfer released over a relatively long distance results in inefficient local killing of target cells coupled with toxicity to distant normal tissues. Nevertheless, ⁇ -emitting radionuclides such as 1311, 90 ⁇ , i88R e , and
- 67Cu have been useful in immunotherapy.
- hepatoma- bearing patients have been successfully treated with I3ii-l a beled antibodies to ferritin (Order, 1985).
- 9o ⁇ -l a beled antibodies to ferritin combined with autologous marrow transplantation resulted in complete remissions in four of eight patients with Hodgkin's disease (Order, 1985).
- 90Y-labeled anti-Tac was effective in prolonging the survival of cardiac allografts and xenografts in a subhuman primate model (Kozak et al, 1989).
- 9o ⁇ -i a beled anti-Tac was evaluated for the treatment of patients with HTLV-I-associated, Tac-expressing ATL.
- doses used 5 and 10 miCi per patient
- no toxicity was observed in five of six patients studied; modest granulocytopenia and thrombocytopenia were observed in one patient.
- Five of these six patients underwent a sustained partial or complete remission after 9o ⁇ -i a beled anti-Tac therapy.
- the target CD19 antigen a 95 kDa B lineage restricted phosphoglycoprotein, is not expressed on life-maintaining non- hematopoietic tissues, normal hematopoietic progenitor cells, or most immature normal B-lineage lymphoid progenitor cells, but it is expressed by virtually 100% of B lineage ALLs.
- the present invention provides an isolated and purified polynucleotide encoding a single chain variable region polypeptide that binds to a CD19 antigen.
- the isolated and purified polynucleotide of the invention encodes a polypeptide that has a molecular weight of approximately 28 kDa. More preferably, the polynucleotide of the invention encodes a polypeptide that binds to a CD19 antigen with a K a of at least 1 x 10 9 M-i.
- Also provided by the invention is a process for preparing an isolated and purified polynucleotide encoding a single chain variable region polypeptide that binds to a CD19 antigen comprising the steps of (a) isolating RNA from hybridomas producing monoclonal antibodies to CD19 antigen; (b) transcribing isolated RNA to cDNA; (c) amplifying separate cDNA sequences encoding a heavy and a light variable region of the monoclonal antibody by a polymerase chain reaction; (d) cloning of separate amplification products encoding the heavy and the light variable regions, respectively, into vector constructs; (e) cloning of DNA encoding the heavy and the light variable regions, wherein the separate sequences are joined through a linker nucleotide sequence, into a vector construct; and (f) digesting the clones with appropriate restriction endonucleases.
- the process of the invention utilizes a linker sequence that is the polynucleotide sequence described by SEQ ID NO: 7.
- the present invention also contemplates an isolated and purified polynucleotide preparable by a process comprising the steps of (a) isolating RNA from hybridomas producing monoclonal antibodies to CD19 antigen; (b) transcribing isolated RNA to cDNA; (c) amplifying separate cDNA sequences encoding a heavy and a light variable region of the monoclonal antibody by a polymerase chain reaction; (d) cloning of separate amplification products encoding the heavy and the light variable regions, respectively, into vector constructs; (e) cloning of DNA encoding the heavy and the light variable regions, wherein the separate sequences are joined through a linker nucleotide sequence, into a vector construct; and (f) digesting the clones with appropriate restriction endonucleases.
- the present invention provides an isolated and purified polynucleotide prepared by a process comprising the steps of (a) isolating RNA from hybridomas producing monoclonal antibodies to CD19 antigen; (b) transcribing isolated RNA to cDNA; (c) amplifying separate cDNA sequences encoding a heavy and a light variable region of the monoclonal antibody by a polymerase chain reaction; (d) cloning of separate amplification products encoding the heavy and the light variable regions, respectively, into vector constructs; (e) cloning of DNA encoding the heavy and the light variable regions, wherein the separate sequences are joined through a linker nucleotide sequence, into a vector construct; and (f) digesting the clones with appropriate restriction endonucleases.
- the process of the invention utilizes a linker sequence that is the polynucleotide sequence described by SEQ ID NO: 7.
- the isolated and purified polynucleotide of the claimed invention encodes a polypeptide comprising an amino acid residue sequence according to SEQ ID NO:20, 22 or 22. More preferably, the isolated and purified polynucleotide of the invention comprises a nucleotide sequence according to SEQ ID NO:23, 24 or 25.
- the present invention provides an isolated and purified polynucleotide comprising a nucleotide base sequence that is identical or complimentary to a segment of at least 10 contiguous nucleotide bases of SEQ ID NO:23, 24 or 25, wherein the polynucleotide hybridizes to a polynucleotide that encodes a single chain variable region polypeptide that binds to a CD19 antigen.
- the polynucleotide of this embodiment of the invention encodes a polypeptide that has a molecular weight of approximately 28 kDa. More preferably still, the encoded polypeptide binds to a CD19 antigen with a K a of at least 1 x 109 M-i.
- this embodiment of the invention provides an isolated and purified polynucleotide comprising a nucleotide base sequence that is identical or complimentary to a segment of 25 or 50 or 100 contiguous nucleotide bases of SEQ ID NO:23, 24 or 25, wherein the polynucleotide hybridizes to a polynucleotide that encodes a single chain variable region polypeptide that binds to a CD19 antigen.
- the present invention contemplates an isolated and purified single chain variable region polypeptide that binds to a CD19 antigen.
- the polypeptide of this embodiment has a molecular weight of approximately 28 kDa. More preferably, the polypeptide binds to a CD19 antigen with a K a of at least 1 x 10 9 M- . More preferably still, the polypeptide comprises an amino acid residue sequence according to SEQ ID NO:20, 21 or 22.
- the isolated and purified polypeptide of the claimed invention is further modified by the site specific insertion of a cysteine residue at the C-terminus of the polypeptide.
- a dimer of an isolated and purified single chain variable region polypeptide wherein the dimer is prepared by linking a first polypeptide modified by the site specific insertion of a cysteine residue at the C-terminus of the polypeptide with a second polypeptide modified by the site specific insertion of a cysteine residue at the C-terminus of the polypeptide, the linking accomplished through a disulfide bond between a C-terminus cysteine residue on each polypeptide.
- the present invention provides a process for preparing an isolated and purified single chain variable region polypeptide that binds to a CD19 antigen comprising the steps of (a) cloning a DNA sequence that encodes the polypeptide into an expression vector; (b) transforming E. coli cells with the expression vector; and (c) maintaining the transformed cells under biological conditions sufficient for expression of the polypeptide.
- the process of this embodiment uses E. coli cells from a BL21(DE3) strain.
- this embodiment of the invention provides an isolated and purified single chain variable region polypeptide that binds to a CD19 antigen, wherein the polypeptide is preparable by a process comprising the steps of (a) cloning a DNA sequence that encodes the polypeptide into an expression vector; (b) transforming E. coli cells with the expression vector; and (c) maintaining the transformed cells under biological conditions sufficient for expression of the polypeptide.
- this aspect of the invention provides an isolated and purified single chain variable region polypeptide that binds to a CD19 antigen, wherein the polypeptide is prepared by a process comprising the steps of (a) cloning a DNA sequence that encodes the polypeptide into an expression vector; (b) transforming E. coli cells with the expression vector; and (c) maintaining the transformed cells under biological conditions sufficient for expression of the polypeptide.
- the present invention contemplates a polypeptide prepared as described immediately above, wherein the polypeptide is further modified by the site specific insertion of a cysteine residue at the C-terminus of the polypeptide.
- This aspect also provides a dimer of an isolated and purified single chain variable region polypeptide, wherein the dimer is prepared by linking a first polypeptide modified by the site specific insertion of a cysteine residue at the C-terminus of the polypeptide with a second polypeptide modified by the site specific insertion of a cysteine residue at the C-terminus of the polypeptide, the linking accomplished through a disulfide bond between a C-terminus cysteine residue on each polypeptide.
- polypeptide comprising an amino acid residue sequence of from five to sixty contiguous amino acid residues identical to any five to sixty contiguous amino acid residues of the polypeptide as defined by SEQ ID NO: 20, 21 or 22, wherein the polypeptide retains an ability to bind to a CD19 antigen with a K a of at least 1 x 109 M-i.
- the claimed invention provides an immunoconjugate for the treatment of cancer comprising a single chain variable region polypeptide that binds to a CD19 antigen, wherein the polypeptide is linked to at least one cytotoxic agent.
- the cancer susceptible to treatment with the immunoconjugate of the invention is a B-cell leukemia.
- the immunoconjugate of this embodiment of the invention comprises an amino acid residue sequence according to SEQ ID NO: 20, 21 or 22.
- the cytotoxic agent of the immunoconjugate of the invention is selected from the group consisting of single chain, double chain, and multiple chain toxins.
- the cytotoxic agent of the immunoconjugate of the invention is selected from the group consisting of beta-emitting metallic radionuclides, alpha emitters, and gamma emitters.
- the present invention provides an immunoconjugate for the treatment of cancer comprising a polypeptide that is a dimer of an isolated and purified single chain variable region polypeptide, wherein the dimer is prepared by linking a first polypeptide modified by the site specific insertion of a cysteine residue at the C- terminus of the polypeptide with a second polypeptide modified by the site specific insertion of a cysteine residue at the C-terminus of the polypeptide, the linking accomplished through a disulfide bond between a C-terminus cysteine residue on each polypeptide. and wherein the polypeptide is linked to at least one cytotoxic agent.
- this immunoconjugate of the invention is efficacious for the treatment of B- cell leukemia.
- the cytotoxic agent of this immunoconjugate is selected from the group consisting of single chain, double chain, and multiple chain toxins.
- the cytotoxic agent is a radionuclide selected from the group consisting of beta-emitting metallic radionuclides, alpha emitters, and gamma emitters.
- the immunoconjugate comprises both a toxin and a radionuclide.
- the present invention also contemplates a process for preparing an immunoconjugate comprising a single chain variable region polypeptide that binds to a CD19 antigen, wherein the process comprises the steps of (1) preparing the polypeptide according to a method comprising the steps of (a) cloning a DNA sequence that encodes the polypeptide into an expression vector; (b) transforming E. coli cells with the expression vector; and (c) maintaining the transformed cells under biological conditions sufficient for expression of the polypeptide; (2) providing a suitable toxin; and (3) conjugating the polypeptide to the toxin.
- the process also further comprises the step of labelling the immunoconjugate with a radionuclide.
- the toxin used in the process of the invention is selected from the group consisting of single chain, double chain, and multiple chain toxins.
- the radionuclide used in the claimed process is selected from the group consisting of beta- emitting metallic radionuclides, alpha emitters, and gamma emitters.
- the polypeptide of the immunoconjugate comprises an amino acid residue sequence according to SEQ ID NO:20, 21 or 22.
- the present invention provides an immunoconjugate for the treatment of cancer comprising a single chain variable region polypeptide that binds to a CD19 antigen, wherein the immunoconjugate is preparable by a process comprising the steps of (1) preparing the polypeptide according to a method comprising the steps of (a) cloning a DNA sequence that encodes the polypeptide into an expression vector; (b) transforming E. coli cells with the expression vector; and (c) maintaining the transformed cells under biological conditions sufficient for expression of the polypeptide; (2) providing a suitable toxin; and (3) conjugating the polypeptide to the toxin.
- the immunoconjugate for the treatment of cancer comprising a single chain variable region polypeptide that binds to a CD19 antigen is prepared by a process comprising the steps of (1) preparing the polypeptide according to a method comprising the steps of (a) cloning a DNA sequence that encodes the polypeptide into an expression vector; (b) transforming E. coli cells with the expression vector; and (c) maintaining the transformed cells under biological conditions sufficient for expression of the polypeptide; (2) providing a suitable toxin; and (3) conjugating the polypeptide to the toxin.
- the immunoconjugate described immediately above wherein the polypeptide of the immunoconjugate is linked to at least one cytotoxic agent.
- the cytotoxic agent is selected from the group consisting of single chain, double chain, and multiple chain toxins or, alternatively, the cytotoxic agent is a radionuclide selected from the group consisting of beta-emitting metallic radionuclides, alpha emitters, and gamma emitters, or the immunoconjugate comprises one of each type of cytotoxic agent. Such an immunoconjugate is contemplated to be efficacious in the treatment of B-cell leukemia.
- the present invention further contemplates an additional embodiment of a method for the treatment of cancer comprising the steps of (a) selecting a patient evidencing symptoms of a B-cell cancer, wherein the cancer is selected from the group consisting of leukemia and B-cell lymphoma; (b) administering to the patient, in a biocompatible dosage form, a therapeutically effective amount of an immunoconjugate, prepared according to a process comprising the steps of (1) preparing a polypeptide according to a method comprising the steps of (a) cloning a DNA sequence that encodes the polypeptide into an expression vector; (b) transforming E.
- the coli cells with the expression vector; and (c) maintaining the transformed cells under biological conditions sufficient for expression of the polypeptide; (2) providing a suitable toxin; (3) conjugating the polypeptide to the toxin; and (4) labelling the immunoconjugate with a radionuclide.
- the radionuclide with which the immunoconjugate is labelled is 13 H.
- the present invention provides for a method for the treatment of cancer comprising the steps of (a) selecting a patient evidencing symptoms of a B-cell cancer, wherein the cancer is selected from the group consisting of leukemia and B-cell lymphoma; and (b) administering to the patient, in a biocompatible dosage form, a therapeutically effective amount of an immunoconjugate comprising a polypeptide that is a dimer of an isolated and purified single chain variable region polypeptide, wherein the dimer is prepared by linking a first polypeptide modified by the site specific insertion of a cysteine residue at the C-terminus of the polypeptide with a second polypeptide modified by the site specific insertion of a cysteine residue at the C-terminus of the polypeptide, the linking accomplished through a disulfide bond between a C-terminus cysteine residue on each polypeptide. and wherein the polypeptide is linked to a toxin and labelled with a radionuclide.
- Figure 1 Cloning strategy for development of anti CD19 scFv.
- the variable domain of the heavy chain and the linker which encodes (G S) 3 were ligated into Bluescript K5 plasmid at Xhol and SacI sites.
- Variable domains of the light chain were inserted into Sstl and Bglll sites following the linker.
- the pERT vector which was constructed by modifying pET3b was used as the expression vector for scFv.
- the nucleotides between Ndcl and Xhol sites of pERT encode four amino acids which are part of the FR1 of VH but not included in the PCR products of VH -
- the scFv encoding fragment was cloned into the pERT vector at Xhol and Bgl II sites. Positive clones were identified by restriction enzyme analysis and DNA sequencing.
- Figure 2 Comparison of the DNA sequence of the different variable regions from the heavy and light chains (in two panels).
- A Heavy chain sequence.
- B Light chain sequence.
- lower case letters are n nucleotide additions and they flank the germline encoded DH gene sequences.
- Capitol letters indicate primers used in PCR.
- Figure 3 Amino acid sequence alignment of the variable heavy and light chain regions from the three different hybridomas: B43, 25C1 and BLY3 (in two panels).
- Lane 1 Molecular weight markers (97, 66, 45, 31, 21 KD); Lane 2, Uninduced cells; Lane 3, Induced cells; Lane 4, Sonicated supernatant; Lane 5, Detergent-solubilized supernatant; Lane 6, Pellet; Lane 7, Pellet purified by Q sepharose.
- the X axis represents binding of FITC labelled class I antibody
- Y axis represents binding of phycoerythrin labelled CD19 antibody.
- Panel A negative control
- panel B positive control
- panel C specific blocking with FVS191
- panel D specific blocking with FVS192.
- Results are plotted with molecules/cell on horizontal axis and molecule L per cell mole on vertical axis.
- the derived K a is 2 X 109.
- scFvs Single chain fragments have been developed to overcome the problems associated with intact antibodies.
- scFvs contain only the variable regions from the heavy and light chains and have a molecular mass of approximately 28 kDa compared to that of the intact antibody of 150 kDa.
- many scFvs expressed in bacteria are insoluble, difficult to refold, and their ability to retain binding to the antigen of interest is highly variable.
- scFvs developed from three hybridomas that produce antibodies that bind to the CD19 antigen of B cells have been cloned and expressed. Polynucleotides and Methods of the Invention.
- the present invention provides an isolated and purified polynucleotide encoding a single chain variable region polypeptide that binds to a CD19 antigen.
- the isolated and purified polynucleotide of the invention encodes a polypeptide that has a molecular weight of approximately 28 kDa. More preferably, the polynucleotide the invention encodes a polypeptide that binds to a CD19 antigen with a K a of at least 1 x 10 9 M-i.
- the term "polynucleotide” means a sequence of nucleotides connected by phosphodiester linkages.
- Also provided by the invention is a process for preparing an isolated and purified polynucleotide encoding a single chain variable region polypeptide that binds to a CD19 antigen comprising the steps of (a) isolating RNA from hybridomas producing monoclonal antibodies to CD19 antigen; (b) transcribing isolated RNA to cDNA; (c) amplifying separate cDNA sequences encoding a heavy and a light variable region of the monoclonal antibody by a polymerase chain reaction; (d) cloning of separate amplification products encoding the heavy and the light variable regions, respectively, into vector constructs; (e) cloning of DNA encoding the heavy and the light variable regions, wherein the separate sequences are joined through a linker nucleotide sequence, into a vector construct; and (f) digesting the clones with appropriate restriction endonucleases.
- the process of the invention utilizes a linker sequence that is the polynucleotide
- the present invention also contemplates an isolated and purified polynucleotide preparable by a process comprising the steps of (a) isolating RNA from hybridomas producing monoclonal antibodies to CD19 antigen; (b) transcribing isolated RNA to cDNA; (c) amplifying separate cDNA sequences encoding a heavy and a light variable region of the monoclonal antibody by a polymerase chain reaction; (d) cloning of separate amplification products encoding the heavy and the light variable regions, respectively, into vector constructs; (e) cloning of DNA encoding the heavy and the light variable regions, wherein the separate sequences are joined through a linker nucleotide sequence, into a vector construct; and (f) digesting the clones with appropriate restriction endonucleases.
- the present invention provides an isolated and purified polynucleotide prepared by a process comprising the steps of (a) isolating RNA from hybridomas producing monoclonal antibodies to CD19 antigen; (b) transcribing isolated RNA to cDNA; (c) amplifying separate cDNA sequences encoding a heavy and a light variable region of the monoclonal antibody by a polymerase chain reaction; (d) cloning of separate amplification products encoding the heavy and the light variable regions, respectively, into vector constructs; (e) cloning of DNA encoding the heavy and the light variable regions, wherein the separate sequences are joined through a linker nucleotide sequence, into a vector construct; and (f) digesting the clones with appropriate restriction endonucleases.
- the process of the invention utilizes a linker sequence that is the polynucleotide sequence described by SEQ ID NO: 7. More preferably, the isolated and purified polynucleotide of the claimed invention encodes a polypeptide comprising an amino acid residue sequence according to SEQ ID NO:20, 21 or 22. More preferably still, the isolated and purified polynucleotide of the invention comprises a nucleotide sequence according to SEQ ID NO:23, 24 or 25.
- the present invention provides an isolated and purified polynucleotide comprising a nucleotide base sequence that is identical or complimentary to a segment of at least 10 contiguous nucleotide bases of SEQ ID NO:23, 24 or 25, wherein the polynucleotide hybridizes to a polynucleotide that encodes a single chain variable region polypeptide that binds to a CD19 antigen.
- the polynucleotide of this embodiment of the invention encodes a polypeptide that has a molecular weight of approximately 28 kDa. More preferably still, the encoded polypeptide binds to a CD19 antigen with a K a of at least 1 x
- this embodiment of the invention provides an isolated and purified polynucleotide comprising a nucleotide base sequence that is identical or complimentary to a segment of 25 or 50 or 100 contiguous nucleotide bases of SEQ ID NO:23, 24 or 25, wherein the polynucleotide hybridizes to a polynucleotide that encodes a single chain variable region polypeptide that binds to a CD19 antigen. Polypeptides and Methods of the Invention.
- the scFv polypeptides developed from three hybridomas were expressed at high levels in bacteria. No instability of the protein, as determined by examination of Coomassie stained SDS-PAGE gels, was noted over the period of induction (3 hrs.) and all clones produced approximately the same quantities of protein. However, the ability of the scFv from each of these clones to bind to the target antigen varied greatly. Although the BLy3 and B43 hybridomas produced heavy chain and light chain variable proteins that were from the same family, only the protein produced from the B43 clone (FVS191) was able to show any ability to bind to the CD19 protein.
- the present invention contemplates an isolated and purified single chain variable region polypeptide that binds to a CD19 antigen.
- the polypeptide of this embodiment has a molecular weight of approximately 28 kDa.
- the polypeptide binds to a CD19 antigen with a K a of at least 1 x 109 M- 1 . More preferably still, the polypeptide comprises an amino acid residue sequence according to SEQ ID NO:20, 21 or 22.
- the isolated and purified polypeptide of the claimed invention is further modified by the site specific insertion of a cysteine residue at the C-terminus of the polypeptide.
- a dimer of an isolated and purified single chain variable region polypeptide wherein the dimer is prepared by linking a first polypeptide modified by the site specific insertion of a cysteine residue at the C-terminus of the polypeptide with a second polypeptide modified by the site specific insertion of a cysteine residue at the C-terminus of the polypeptide, the linking accomplished through a disulfide bond between a C-terminus cysteine residue on each polypeptide.
- polypeptide means a polymer of amino acids connected by amide linkages, wherein the number of amino acid residues can range from about 5 to about one million. Preferably, a polypeptide has from about 10 to about 1000 amino acid residues and, even more preferably from about 20 to about 500 amino residues. Thus, as used herein, a polypeptide includes what is often referred to in the art as an oligopeptide (5-10 amino acid residues), a polypeptide (11-100 amino acid residues) and a protein (>100 amino acid residues).
- a polypeptide encoded by an encoding region can undergo post-translational modification to form conjugates with carbohydrates, lipids, nucleic acids and the like to form glycopolypeptides (e.g., glycoproteins), lipopolypeptides (e.g. lipoproteins) and other like conjugates.
- glycopolypeptides e.g., glycoproteins
- lipopolypeptides e.g. lipoproteins
- Polypeptides are disclosed herein as amino acid residue sequences.
- amino acid residue sequences are denominated by either a single letter or a three letter code as indicated in Table 1 below.
- Modifications and changes may be made in the structure of a polypeptide of the present invention and still obtain a molecule having like characteristics.
- certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide's biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a polypeptide with like properties.
- the hydropathic index of amino acids can be considered.
- the importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art (Doolittle, et al. 1982). It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics.
- Amino Acid Index Amino Acid Index isoleucine (+4.5) tryptophan (-0.9) valine (+4.2) tyrosine (-1.3) leucine (+3.8) proline (-1.6) phenylalanine (+2.8) histidine (-3-2) cysteine (+2.5) glutamate (-3.5) methionine (+1.9) glutamine (-3.5) alanine (+1.8) aspartate (-3.5) glycine (-0.4) asparagine (-3.5) threonine (-0.7) lysine (-3-9) serine (-0.8) arginine (-4.5)
- the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, for example, enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid may be substituted by another amino acid having a similar hydropathic index and still obtain a biologically functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within +0.5 are even more particularly preferred.
- substitution of like amino acids can also be made on the basis of hydrophilicity, particularly where the biologically functionally equivalent peptide or polypeptide thereby created is intended for use in immunological embodiments.
- U.S. Patent 4,554,101 incorporated herein by reference, states that the greatest local average hydrophilicity of a polypeptide, as governed by the hydrophilicity of its adjacent amino acids, correlate with its immunogenicity and antigenicity, i.e. with a biological property of the polypeptide.
- hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 + 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5 ⁇ 1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (- 1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
- amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent, polypeptide.
- substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ _0.5 are even more particularly preferred.
- amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
- substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
- the present invention thus contemplates functional equivalents of the claimed polypeptides.
- the present invention provides a process for preparing an isolated and purified single chain variable region polypeptide that binds to a CD19 antigen comprising the steps of (a) cloning a DNA sequence that encodes the polypeptide into an expression vector; (b) transforming E. coli cells with the expression vector; and (c) maintaining the transformed cells under biological conditions sufficient for expression of the polypeptide.
- the process of this embodiment uses E. coli cells from a BL21(DE3) strain.
- this embodiment of the invention provides an isolated and purified single chain variable region polypeptide that binds to a CD19 antigen, wherein the polypeptide is preparable by a process comprising the steps of (a) cloning a DNA sequence that encodes the polypeptide into an expression vector; (b) transforming E. coli cells with the expression vector; and (c) maintaining the transformed cells under biological conditions sufficient for expression of the polypeptide.
- this aspect of the invention provides an isolated and purified single chain variable region polypeptide that binds to a CD19 antigen, wherein the polypeptide is prepared by a process comprising the steps of (a) cloning a DNA sequence that encodes the polypeptide into an expression vector; (b) transforming E. coli cells with the expression vector; and (c) maintaining the transformed cells under biological conditions sufficient for expression of the polypeptide.
- the present invention contemplates a polypeptide prepared as described immediately above, wherein the polypeptide is further modified by the site specific insertion of a cysteine residue at the C-terminus of the polypeptide.
- This aspect also provides a dimer of an isolated and purified single chain variable region polypeptide, wherein the dimer is prepared by linking a first polypeptide modified by the site specific insertion of a cysteine residue at the C-terminus of the polypeptide with a second polypeptide modified by the site specific insertion of a cysteine residue at the C-terminus of the polypeptide, the linking accomplished through a disulfide bond between a C-terminus cysteine residue on each polypeptide.
- polypeptide comprising an amino acid residue sequence of from five to sixty contiguous amino acid residues identical to any five to sixty contiguous amino acid residues of the polypeptide as defined by SEQ ID NO: 20, 21 or 22, wherein the polypeptide retains an ability to bind to a CD19 antigen with a K a of at least 1 x 10 9 M-i.
- Immunoconjugates and Methods of the Invention the claimed invention provides an immunoconjugate for the treatment of cancer comprising a single chain variable region polypeptide that binds to a CD19 antigen, wherein the polypeptide is linked to at least one cytotoxic agent.
- the cancer susceptible to treatment with the immunoconjugate of the invention is a B-cell leukemia.
- the immunoconjugate of this embodiment of the invention comprises an amino acid residue sequence according to SEQ ID NO: 20, 21 or 22.
- the cytotoxic agent of the immunoconjugate of the invention is selected from the group consisting of single chain, double chain, and multiple chain toxins.
- the cytotoxic agent of the immunoconjugate of the invention is selected from the group consisting of beta-emitting metallic radionuclides, alpha emitters, and gamma emitters. Toxins
- a structural similarity in plant and bacterial toxins inhibits protein synthesis they are usually heterodimers made of a polypeptide chain (B chain) that binds the toxin to target cells and a second chain (A chain) that displays enzymatic activity (Olsnes et al., 1982).
- B chain polypeptide chain
- a chain second chain
- the two chains are linked by a disulfide bond.
- Diphtheria toxin is a slight exception in that a single proteolytic cleavage is required to generate an A and a B chain (Collier et al, 1971) that are also disulfide bonded.
- a and B chains of abrin and ricin can be interchanged to produce hybrid molecules of relatively high toxicity (Olsnes et al, 1982; Olsnes et al, 1984).
- These observations suggest significant conservation in function and structure. Whether the structural conservation is at the three-dimensional level only or reflects primary amino acid sequence homologies remains to be determined.
- plant toxins composed of A chains only, e.g., gelonin (Stirpe et al, 1980) and pokeweed antiviral protein (PAP) (Olsnes et al, 1982; Barbieri et al, 1982). These A chains function in the same way as the A chains of intact toxins.
- the present invention provides an immunoconjugate for the treatment of cancer comprising a polypeptide that is a dimer of an isolated and purified single chain variable region polypeptide, wherein the dimer is prepared by linking a first polypeptide modified by the site specific insertion of a cysteine residue at the C- terminus of the polypeptide with a second polypeptide modified by the site specific insertion of a cysteine residue at the C-terminus of the polypeptide, the linking accomplished through a disulfide bond between a C-terminus cysteine residue on each polypeptide. and wherein the polypeptide is linked to at least one cytotoxic agent.
- this immunoconjugate of the invention is efficacious for the treatment of B- cell leukemia.
- the cytotoxic agent of this immunoconjugate is selected from the group consisting of single chain, double chain, and multiple chain toxins.
- the cytotoxic agent is a radionuclide selected from the group consisting of beta-emitting metallic radionuclides, alpha emitters, and gamma emitters.
- the immunoconjugate comprises both a toxin and a radionuclide.
- the present invention also contemplates a process for preparing an immunoconjugate comprising a single chain variable region polypeptide that binds to a CD19 antigen, wherein the process comprises the steps of (1) preparing the polypeptide according to a method comprising the steps of (a) cloning a DNA sequence that encodes the polypeptide into an expression vector; (b) transforming E. coli cells with the expression vector; and (c) maintaining the transformed cells under biological conditions sufficient for expression of the polypeptide; (2) providing a suitable toxin; and (3) conjugating the polypeptide to the toxin.
- the process also further comprises the step of labelling the immunoconjugate with a radionuclide.
- the toxin used in the process of the invention is selected from the group consisting of single chain, double chain, and multiple chain toxins.
- the radionuclide used in the claimed process is selected from the group consisting of beta- emitting metallic radionuclides, alpha emitters, and gamma emitters.
- the polypeptide of the immunoconjugate comprises an amino acid residue sequence according to SEQ ID NO:20, 21 or 22.
- the toxins which are usable in the practice of the claimed invention encompass all toxins used in the production of immunotoxins.
- the toxins include heterodimers made of a polypeptide chain (B chain) that binds the toxin to target cells via a sugar on the surface and a second chain (A chain) that displays enzymatic activity.
- the two chains are typically linked by a disulfide bond.
- Examples of two chain toxins are ricin, abrin, modeccin, diphtheria toxin and viscumin.
- single chain toxins i.e. toxins composed of A chains only, e.g., gelonin, pseudomonas aeruginosa Exotoxin A, and amanitin may also be utilized.
- single chain toxins are hemitoxins which are also usable in this invention. They include pokeweed antiviral protein (PAP), saporin and memordin. Other useful single chain toxins include the A-chain fragments of the two chain toxins. A chain toxins with multiple B chains such as Shigella toxin are also usable in the invention.
- PAP pokeweed antiviral protein
- saporin saporin
- memordin Other useful single chain toxins include the A-chain fragments of the two chain toxins.
- a chain toxins with multiple B chains such as Shigella toxin are also usable in the invention.
- 2-chain toxins refers to toxins formed from two chains
- single chain toxins refers to both toxin obtained by cleaving 2- chain toxins as well as toxins having only one chain.
- a preferred toxin is ricin, a toxin lectin extracted from the seeds of Ricinus communis, which contains an enzymatic and protein synthesis inhibiting A chain and a B chain which contains galactose binding site(s).
- Ricin is extremely toxic and it has been calculated that a single molecule of ricin in the cytosol will kill a cell. Ricin may be obtained and purified by the procedures described in U.S. Pat. No. 4,340,535, the disclosure of which is incorporated herein by reference.
- the present invention provides an immunoconjugate for the treatment of cancer comprising a single chain variable region polypeptide that binds to a CD19 antigen, wherein the immunoconjugate is preparable by a process comprising the steps of (1) preparing the polypeptide according to a method comprising the steps of (a) cloning a DNA sequence that encodes the polypeptide into an expression vector; (b) transforming E. coli cells with the expression vector; and (c) maintaining the transformed cells under biological conditions sufficient for expression of the polypeptide; (2) providing a suitable toxin; and (3) conjugating the polypeptide to the toxin.
- the immunoconjugate for the treatment of cancer comprising a single chain variable region polypeptide that binds to a CD19 antigen is prepared by a process comprising the steps of (1) preparing the polypeptide according to a method comprising the steps of (a) cloning a DNA sequence that encodes the polypeptide into an expression vector; (b) transforming E. coli cells with the expression vector; and (c) maintaining the transformed cells under biological conditions sufficient for expression of the polypeptide; (2) providing a suitable toxin; and (3) conjugating the polypeptide to the toxin.
- One general method of preparing immunotoxins is to use a thiol- containing heterobifunctional crosslinker, e.g., SPDP, which attacks primary amino groups on the antibody and by disulfide exchange can attach either the SH-containing A chain or the SPDP-derivatized holotoxin to the antibody (Cumber et al, 1984; Carlsson et al, 1978). If the disulfide exchange is carried out at neutral pH a relatively stable disulfide bond is formed and the conjugate remains intact when incubated with fresh mouse serum in vitro.
- SPDP thiol- containing heterobifunctional crosslinker
- the nature of the linkage between the A chain and the antibody or fragment is of critical important in determining toxicity. If the bond cannot be broken readily in an endosome/phagolysosome (Jansen et al, 1982; Ramakrishnan et al, 1984), e.g., a stable thioether bond, then toxicity is virtually abolished (Jansen et al, 1982). In contrast, if the bond is highly unstable, then the conjugate may dissociate either before it reaches the target cell or, perhaps, prematurely within the target cell. In the latter case, the A chain may be degraded before translocation can occur.
- the immunoconjugate described above wherein the polypeptide of the immunoconjugate is linked to at least one cytotoxic agent.
- the cytotoxic agent is selected from the group consisting of single chain, double chain, and multiple chain toxins or, alternatively, the cytotoxic agent is a radionuclide selected from the group consisting of beta-emitting metallic radionuclides, alpha emitters, and gamma emitters, or the immunoconjugate comprises one of each type of cytotoxic agent.
- cytotoxic agent is selected from the group consisting of single chain, double chain, and multiple chain toxins or, alternatively, the cytotoxic agent is a radionuclide selected from the group consisting of beta-emitting metallic radionuclides, alpha emitters, and gamma emitters, or the immunoconjugate comprises one of each type of cytotoxic agent.
- Such an immunoconjugate is contemplated to be efficacious in the treatment of B-cell leukemia.
- radionuclides used include gamma-emitters, positron-emitters, and X-ray emitters, while beta emitters and alpha emitters may also be used for therapy.
- Suitable radionuclides for forming the immunoconjugate of the invention include
- the present invention further contemplates an additional embodiment of a method for the treatment of cancer comprising the steps of (a) selecting a patient evidencing symptoms of a B-cell cancer, wherein the cancer is selected from the group consisting of leukemia and B-cell lymphoma; (b) administering to the patient, in a biocompatible dosage form, a therapeutically effective amount of an immunoconjugate, prepared according to a process comprising the steps of (1) preparing a polypeptide according to a method comprising the steps of (a) cloning a DNA sequence that encodes the polypeptide into an expression vector; (b) transforming E.
- the coli cells with the expression vector; and (c) maintaining the transformed cells under biological conditions sufficient for expression of the polypeptide; (2) providing a suitable toxin; (3) conjugating the polypeptide to the toxin; and (4) labelling the immunoconjugate with a radionuclide.
- the radionuclide with which the immunoconjugate is labelled is J32I.
- the present invention provides for a method for the treatment of cancer comprising the steps of (a) selecting a patient evidencing symptoms of a B-cell cancer, wherein the cancer is selected from the group consisting of leukemia and B-cell lymphoma; and (b) administering to the patient, in a biocompatible dosage form, a therapeutically effective amount of an immunoconjugate comprising a polypeptide that is a dimer of an isolated and purified single chain variable region polypeptide, wherein the dimer is prepared by linking a first polypeptide modified by the site specific insertion of a cysteine residue at the C-terminus of the polypeptide with a second polypeptide modified by the site specific insertion of a cysteine residue at the C-terminus of the polypeptide, the linking accomplished through a disulfide bond between a C-terminus cysteine residue on each polypeptide, and wherein the polypeptide is linked to a toxin and labelled with a radionuclide.
- V__ and V variable regions
- B43 produced by F. Uckun (Uckun et al, 1986), SJ25C1, produced by S. Pieper, and BLY3, produced by S. Poppema (Knapp et al, 1989b). All were maintained in RPMI 1640 supplemented with 10% fetal calf serum.
- RNA was isolated by the method of Chomczynski and Sacchi (Chomczynski, 1987) and either used directly for RT-PCR or further purified by oligo dT column chromatograph. By way of example, and without limitation, the following protocol describes isolation of RNA from 100 mg of rat mammary tissue according to the method referenced above.
- the tissue was minced on ice and homogenized (at room temperature) with 1 ml of solution D in a glass-Teflon homogenizer and subsequently transferred to a 4-ml polypropylene tube. Sequentially, 0.1 ml of 2M sodium acetate, pH 4, 1 ml of phenol (water saturated), and 0.2 ml of chloroform-isoamyl alcohol mixture (49:1) were added to the homogenate, with thorough mixing by inversion after the addition of each reagent. The final suspension was shaken vigorously for 10 s and cooled on ice for 15 min. Samples were centrifuged at 10,000g for 20 min. at 4°C.
- RNA was present in the aqueous phase whereas DNA and proteins were present in the interphase and phenol phase.
- the aqueous phase was transferred to a fresh tube, mixed with 1 ml of isopropanol, and then placed at -20°C for at least 1 h to precipitate RNA. Sedimentation at 10,000g for 20 min. was again performed and the resulting RNA pellet was dissolved in 0.3 ml of solution D, transferred into a 1.5-ml Eppendorf tube, and precipitated with 1 vol of isopropanol at -20°C for 1 h. After centrifugation in an Eppendorf centrifuge for 10 min.
- RNA pellet was resuspended in 75% ethanol, sedimented, vacuum dried (15 min.), and dissolved in 50 ⁇ l 0.5% SDS at 65°C for 10 min.
- the RNA preparation could be used for poly(A)+ selection by oligo (dT) chromatography, Northern blot analysis, and dot blot hybridization. Isopropanol precipitation can be replaced by precipitation with a double volume of ethanol.
- RNA reverse transcription of the isolated RNA was performed according to the recommendations of the manufacturer (Life Technologies) using random hexamers and was performed in a 50 microliter reaction volume with 1-2 micrograms of polyadenylated RNA or 5-10 micrograms of total RNA. Approximately 10 microliters of the reverse transcribed material was used for the polymerase chain reaction using one pair of the several different primers listed in Table 1.
- the primers Z221 and Z222 anneal to the constant regions of heavy and light chains, respectively, and were only used for isolating clones for verification of sequence but not for the production of variable regions that were subsequently used in the creation of the scFv.
- the cycle parameters were 1 cycle of 94°C for 5' before the addition of the TaqI polymerase then 30 cycles of 94° C 1' 30", 54°C 1' 30", 72°C 1', followed by 1 cycle of 94°C 1'30", 54°C 2'30", 72°C 10'.
- the PCR products were cloned either after treatment with Klenow into Smal digested pBluescript or directly using the pCRI vector (Invitrogen) which has compatible T overhangs.
- Clones were identified based on the size of inserts (approximately 350bp for the VL gene and 450bp for the VH gene) and were confirmed by sequencing using standard dideoxynucleotide chain termination techniques (Sequenase, US Biochemicals). At least three different clones from three different PCR reactions were sequenced for each variable region to confirm the absence of any mutations induced by Taq polymerase before clones were used for the creation of scFV.
- Fig. 2 and Fig. 3 The DNA and the predicted amino acid sequences of the clones of the variable regions from the three hybridomas are shown in Fig. 2 and Fig. 3. As discussed in Materials and Methods, at least three clones from three independent PCR reactions were sequenced to ensure that no Taq- introduced mutations were present within the clones that were used for the scFv development. All heavy chain variable regions from the three hybridomas were from the J558 family which includes approximately 50% of all mouse heavy chain variable region genes (Brön et al, 1984). Although clone 25CI uses JH , clones of B43 and BLy3 use JH4. As expected, the B43 and Bly3 clones differed most within the CDR3 region due to N region differences.
- the linker used in these studies was (Gly 4 Ser) 3 as previously described (Huston et al, 1988).
- the scFvs were created by ligation of the linker region oligonucleotides (Table 3) using the strategy outlined in FIG. 1.
- Heavy chain variable region was mixed simultaneously with linker and Bluescript to obtain the V ⁇ -linker construct shown in FIG. 1.
- Clones that contained the heavy chain variable region were digested with Xhol and BsfEII. Success of the procedure was confirmed by sequencing. Clones that contained the heavy chain variable region and the linker were then digested with SstI and Bglll and ligated to gel purified light chain variable region that was digested with the same enzymes.
- Clones were identified by the appearance of appropriately sized restriction endonuclease fragments and finally by nucleotide sequence analysis. scFvs were then digested with Xhol and Bglll and gel purified before ligation into the pERT expression vector.
- VK B1865 GAAGATCTACGI ⁇ A ⁇ TCCAGCTTGGTCCC
- the primers Z221, 222, 407 and 462 are based on sequemces from Huse et al. (1989).
- the primers B1867 adn 1865 are based on primer sequences from Orlandi et al. (1989).
- the * denotes primers that were used for the generation of clones used only for sequencing.
- the oligonucleotides used for the liner are based on the thses developed by Huston et al.
- the vector used to express the scFv in these studies was developed using the pET3b plasmid established by Studier et al. (Studier et al, 1990). This plasmid vector was developed for cloning and expressing target DNAs under control of a T7 promoter and designated pET vectors (plasmid for expression by T7 RNA polymerase) (Rosenberg et al, 1987). These vectors contain a T7 promoter inserted into the BamHI site of the multi-copy plasmid pBR322 in the orientation that transcription is directed counterclockwise, opposite to that from the TET promoter. In the absence of T7 RNA polymerase, transcription of target DNAs by E.
- coli RNA polymerase is low enough that very toxic genes can be cloned in these vectors. However, some expression can be detected, so it is possible that an occasional gene may be too toxic to be cloned in them.
- Most of the pET vectors described confer resistance to ampicillin. In such vectors, the bla gene is oriented so that it will be expressed from the T7 promoter along with the target gene. However, in the pET-9 series of vectors, the bla gene has been replaced by kan gene in the opposite orientation. In these vectors, the only coding sequence transcribed from the T7 promoter is that of the target gene.
- the T7 promoter in the pET vectors is derived from the ⁇ lO promoter, one of six strong promoters in T7 DNA that have the identical nucleotide sequence from positions -17 to +6, where +1 is the position of the first nucleotide of the RNA transcribed from the promoter.
- the ⁇ lO promoter fragments carried by the vectors all begin at bp -23 and continue to bp +2, +3, +26, and +96 or beyond.
- Some of the vectors also contain a transcription termination signal or an RNase III cleavage site downstream of the cloning site for the target DNA.
- pET3b was modified to allow for the cloning and expression of the constructs of the present invention by ligating an oligonucleotide that coded for the first four amino acids (LESG) that are commonly found at the amino terminus of the heavy chain variable region to the vector that was digested with Ndel and EcoRI.
- This oligonucleotide also contained sequences for recognition sites for Xhol, Bglll, BamHI, and EcoRI allowing for the cloning of the scFv into the vector at the Xhol and Bglll sites with the possibility of cloning other potentially therapeutic genes in the future (Table 3).
- the supernatant was discarded and the pellet was resuspended by sonication in 1/20 of the original culture volume in 10 mM Tris-HCl pH 8.0 and 5 mM EDTA. After resuspension was complete the mixture was digested with 0.2% lysozyme (Sigma) for a minimum of lhour. Finally, 1/3 volume of 10% sodium deoxycholate was added and the mixture was incubated at room temperature for 1 hour before centrifugation at 30,000 x g for 30 minutes. The pellet was washed three times in water by resuspending the pellet by sonication and centrifugation as described above.
- Pellets were either stored at -20° C or dissolved in 0.1M Tris-HCl ph 8.0, 6M guanidine HCl, 0.3M DTE, 2mM EDTA at room temperature for a minimum of 2hours. Refolding of the denatured scFv was performed according to the method of Buchner et. al. (15).
- the protein concentration was measured using the Bradford assay and the solution was then rapidly diluted at least 100 fold to a concentration of 30 ug/ml protein in 0.1M Tris-HCl pH8.0, 0.5M L- arginine, 8mM GSSG and 2mM EDTA. After a minimum of 12 hours, at 10° C the refolded protein was concentrated using an Amicon spiral concentrator and spin concentrator before being chromatographed on Q Sepharose and finally Superose 75. As judged by the presence of a single peak on Superose chromatography and Coomassie stain of SDS-Page gels, protein was pure. If the concentration was too low for use in experiments the protein was concentrated by Amicon spin concentrators. Concentration of the protein was determined using the Bradford assay (BioRad) with bovine serum albumin as a standard.
- FACS analysis was performed on either RS4:11 (Stong et al, 1985) or Bl cell line (Cohen et al, 1991), both of which express CD19 and HLA Class I and carry the 4:11 translocation.
- the RS4:11 cell line was established from bone marrow of a patient with t(4:ll)-associated acute leukemia. Morphological, immunologic, and cytochemical characteristics of RS4:11 cells were found to be consistent with ALL. The cells are strongly positive for TdT. An in-depth analysis of RS4:11 revealed characteristics of both lymphoid and myeloid lineages.
- the cells are rearranged for immunoglobuiin heavy and k-chain genes, providing strong evidence for a commitment to B cell lineage. Although occasional heavy chain gene rearrangements have been noted in T cells and myeloid cells, light chain gene rearrangements have been restricted to the B cell lineage (Arnold et al, 1983; Korsmeyer et al, 1983; Ford et al, 1983).
- B4 is additional support for B lineage classification, since within the hematopoietic system, this antigen is expressed very early in normal B cell ontogeny and is restricted to B lineage cells (Nadler et al, 1983).
- RS4:11 cells bind 1G10, a mAb that reacts with granulocytic cells, some monocytes (Bernstein et al, 1988), and CFU-GM precursor cells (Andrews et al, 1983). Some RS4:11 cells weakly express the gpl70,95/TA-l antigen found on monocytic precursors (Andrews et al, 1983) and peripheral blood monocytes (LeBein et al, 1980). The ultrastructural detection of basophil/mast cell granules and peroxidase activity in a minor population of RS4:11 cells is supportive evidence of myeloid commitment.
- TPA can induce human myeloid and lymphoid leukemic cells to more differentiated phenotypes that are primarily dictated by the differentiative potential of the target cells (Koeffler, 1983; LeBien et al, 1982; Nadler et al, 1982; Nagasawa et al, 1980).
- RS4:11 cells became reactive with TA-1, OKM1, and MCS2, became phagocytic, and showed greatly enhanced NSE activity in a pattern characteristic of monocytic cells.
- the patient's bone marrow sample at diagnosis and relapse contained over 95% malignant cells characterized by the t(4:ll) (q21;q23) chromosomal translocation and biphenotypic expression of lymphoid and myeloid cell markers (often associated with this translocation).
- the cell line was established by incubating leukemic cells (106/mL) in oo-MEM containing 10% heat-inactivated fetal calf serum (FCS). After 8 weeks, the cells were cloned in semisolid methylcellulose and single colonies were isolated and expanded in liquid culture medium. The cell line established this way resembled the donor's leukemic cells. The karyotype of the line showed t (4:11) (q21; a23) in all metaphases. In addition, other chromosomal abnormalities, including trisomy 6, der(l)t(l;8) (p36; ql3), der(10)t(l;10)(qll; pl5), were consistently observed in all metaphases.
- Cytochemical analysis showed a profile of periodic acid Schiff (PAS)-positive, acid phosphatase-positive, nonspecific esterase- positive, and Sudan black-negative staining.
- the leukemic cells lacked T- and B-cell markers (E-, slg-, clg-) and were CD10- and CD20-, but had undergone IgH( ⁇ ) gene rearrangement.
- Flow cytometric analysis showed that Bl cells expressed early pre-B-cell markers such as CD19+ and HLA- DR+.
- HLA-DR is coexpressed with My-9 (CD33), a marker of myeloid lineage on 20% of the cells.
- Other myeloid differentiation markers such as My- 7, Mo-1, and Mo-2, were undetectable on the surface of Bl cells.
- FACS analyses were used to evaluate the scFvs.
- the scFvs from B43 and 25C1 hybridomas which are refered to as FVS191 (Fragment, Variable, Single chain, anti CD29, number 1) and FVS192, respectively, were able to inhibit the binding of FITC labeled 25C1 but not an anti HLA class I monclonal antibody to cells that were CD19+, HLA class 1+ (Fig. 5).
- the scFv derived from BLy3 did not block the binding of the competing antibody.
- binding to target cells could not be detected by biotinylating the scFv developed from this hybridoma and using this material with streptavidin labeled phycoerythrin (data not shown).
- streptavidin labeled phycoerythrin data not shown.
- the failure of BLy3 scFv to bind in these two assays suggests that the protein was not properly folded.
- Iodine labelling of the proteins was accomplished using Iodobeads (Pierce) and the specific activity was determined. Beads were washed with iodination buffer, dried, and added to solution of carrier free Nal25I (1 mCi/100 ⁇ g of protein) and allowed to react for five minutes. The reaction was stopped and the beads were washed. Gel filtration (Pharmacia PD5) was used to remove excess Nal25I. TCA precipitation was carried out followed by determination of specific activity using standard calculations.
- Immunoreactive fractions were subsequently determined (with reagents generally in the range of 0.05). Scatchard analysis was determined using
- FVS191 and FVS192 Due to the ability of FVS191 and FVS192 to specifically bind to cells that express the CD19 antigen we evaluated their affinity. Proteins were iodinated and used for Scatchard analysis as described in Materials and Methods. The results (Fig.6) demonstrated that the FVS191 had an K a of 2x109 M- . Although FVS192 was able to successfully compete with 25C1 binding to the CD19 antigen it did not demonstrate sufficient avidity of binding to be evaluated in Scatchard analysis and its Ka therefore could not be determined.
- Example 4 Formation of dimers of Anti-CD19 Single-Chain Fv.
- Single-chain Fv antibody fragments have the advantage of improved tumor penetration over intact antibody. Dimers of scFv may possess higher binding constants and have potential as diagnostic or therapeutic agents.
- an additional cysteine residue was site-specifically inserted at the C-terminal of the scFv constructs of the present invention to form the scFv-cys.
- the scFv-cys proteins were isolated from bacterial inclusion bodies, reduced with guanidine, and refolded in redox buffer containing DTE and GSSG.
- Q-Sepharose-purified scFv-cys proteins were treated with 2 mM DTT. The DTT was removed using a Pharmacia PD10 column. Disulfide bonds between C-terminal cysteins were formed by air oxidation.
- Leukemia is likely to be successfully treated using radiolabled anti- CD19 scFv because it is radiosensitive and there is ready access of antibody to the marrow space.
- Clinical studies have shown that iodine-labled antiferritin antibodies provided symptomatic relief to 77% patients with refractory Hodgkin disease and produced objective tumor regression in 40% of patients.
- radiolabled anti-CD33 and - 35 antibodies were used in combination with high a dose of cyclophosphamide, an overall of 19% complete remissions and 75% partial remissions were achieved for 210 evaluable patients with hematologic malignancies.
- iodine- labled antibodies The major side effect associated with the use of iodine- labled antibodies was reported to be thrombocytopenia, which occurred more frequently when the dose of iodine used was greater than 200 mCi/patient (see review by Grossbard et al, 1992). Radiolabled antibodies kill target cells by by-stander effect.
- radiolabled antibodies Internalization of radiolabled antibodies is probably not desirable. It has shown that internalized radiolabled antibodies had a much shorter retention time and a faster rate of deiodination, which would dramatically reduce the efficacy of the therapeutic values of the antibodies (Richard et al, 1992).
- the single chain antibodies have the advantages of being small, with relatively high affinity toward the antigens and not being internalized by the target cells.
- FVS 191 and FVS 192 single chain antibody The antibody is expressed in Escherichia coli as inclusion bodies.
- the single chain antibody is labeled with Na 1311 using a Idogen kit (Pierce, Rockford, IL).
- the ratio of Idogen to antibody is adjusted to approximately 100ug:lmg as described by Badger et al. (1985).
- the labeled antibody will be separated from free 1311 by gel filtration.
- the labeling efficiency and specific activity will be determined by cyclic anhydride method (Hantowich et al, 1983).
- a specific activity of l.OCi/g or less should be suitable for the experiments.
- the same amounts of whole monoclonal antibody and Fab of an unrelated antibody should be labeled with 1311 the same way to serve as controls.
- Immunoreactivity is defined as percentage of counts that are able to bind at antigen excess. Briefly, a serial dilution of target cells (CD19+, 10 6 - 7/ml will be incubated with labeled antibody (4-5 ng/ml) for 1 h at RT. Cells are centrifuged and supernatant radioactivity is counted. Immunoreactivity will be determined by Lineweaver-Burk analysis. Avidity of the antibody will be determined by incubation fixed amounts of cells (lOVml) with a serial dilutions of labeled antibody for 1 h at RT. Cells are washed and the cell pellet radioactivity is used to calculate the avidity (association constant and the number of binding site per cell).
- Pharmacokinetic studies are carried out by injecting labeled single chain antibody into a group of 4 BALB/c mice via the tail vein. Blood samples are collected at various time intervals. Radioactivities associated with the blood samples will be determined and T alpha 1/2 and T beta 1/2 of the single chain antibody will be calculated by computer simulation.
- the parental monoclonal antibody is labeled and injected into the mice as described above. Biodistribution is performed with paired labeling, e.g. the single chain antibody will be labeled with 13 1 and the controlled antibody labeled with 125 1.
- anti CD19 scFv, FVS 191 has been successfully labled with 251 an d used in immunochemistry and pharmacokinetics studies.
- this scFv can be readily labled with 1 51 with a specific activity of 2.4 mci/mg.
- the immunoreactivity of the labled antibody was 55%.
- FVS 191 is more resistant to labeling damage than intact antibody.
- Results of Scatchard analysis showed that the affinity of FVS 191 toward CD19 antigen was 7.2 X 10 8 M- . This value is about four fold higher than its parent monoclonal antibody (1.93 X 10 8 M-i), suggesting that scFv may be a better targeting reagent than intact antibody.
- the observation that scFv showed higher affinity than its parent intact monoclonal antibody is consistent with the findings of others.
- a mixture of equivalent amounts of specific antibody and control antibody with varied concentrations is injected i.v. into a group of 4 mice with human leukemia xenografts. The animals are sacrificed at 1, 24 and 48 h after the injection. Samples of blood, tumor, lung, spleen and kidney are weighed and counted in a gamma counter. The percentage of injected dose per gram of tissue (%ID/g) for each isotope is calculated. For dose escalation studies a single labeling ( 131 I) will be performed to determine the proper dose range for subsequent animal survival tests. E. Demonstration of Therapeutic Efficacy
- leukemia animal models Two types are used in the experiments — e.g. acute human leukemia (Bl or RS4:11 cell) in SCID mice or human acute leukemia xenograft tumor model in SCID or athymic BALB/c mice.
- the human leukemia SCID model has been well established in this laboratory and should be readily available for the experiments.
- the xenograft tumor model is established by injecting human leukemia cells (4-5 X 10 7 in 0.2 ml PBS) into flanks of the mice as described by Richard et al, 1992).
- a palpable tumor module of 0.5-1.0 cm should be detected 8-10 days after the tumor cell injection.
- a single infusion (i.v) of various concentrations (low, medium and high) of radiolabled antibody is given to a group of 4 animals.
- the same amount of controlled antibody labeled with 1311 are treated the same way.
- the percentage of survival will be recorded up to 50 days.
- regression of tumors will be recorded. The definition of complete, and partial regressions needs to be defined. 1.
- radiolabled antibody should show significant target cell killing effect in comparison to control antibody. Complete or partial tumor regression after radiolabled antibody treatment is expected. Due to its small size, single chain antibody is expected to penetrate the tumor more efficiently and show better results as compared to labeled whole MAb.
- Anti-CD19 antibodies have been effective for the treatment of human B cell leukemias or lymphomas when conjugated to toxins, e.g., ricin or pokeweed antiviral protein (Vitetta, et al, Uckun, et al).
- B cell antibodies other than CD19 e.g., anti-CD29
- anti-CD19 FVS 191 and FVS 192 are not internalized by the cell after binding and thus these scFv should be effective as radioimmunoconjugates which should remain on the cell surface for optimal stability and cell killing.
- the anti-CD19 scFv will have very efficient biodistribution and tissue penetration based on the small size and short half life.
- FVS 191 has a T 1 / 2 of 2.5 minutes in the alpha phase and T1 / 2 of 3.7 hour in the beta phase.
- the rapid clearance combined should allow excellent killing of essentially all B cell leukemias and lymphomas (99% of which bear CD19).
- the small size of the 311 scFv should allow excellent killing in marrow lymph nodes and extromedullary sites which often serve as sanctuaries for leukemia and lymphoma cells.
- MOLECULE TYPE protein
- DESCRIPTION Heavy chain SJ25C1 protein
- Gin Arg Ala Thr lie Ser Cys Lys Ala Ser Gin Ser Val Asp Tyr Asp 20 25 30
- MOLECULE TYPE protein
- DESCRIPTION single chain SJ25C1 protein
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Abstract
L'invention se rapporte à des polynucléotides codant des fragments monocaténaires de régions variables d'un anticorps monoclonal contre CD19 et à leurs procédés de préparation. L'invention se rapporte également à des polypeptides monocaténaires de régions variables, à leurs procédés de préparation, à des polypeptides modifiés en certains sites et à des dimères dérivés de ceux-ci. Un autre aspect de l'invention concerne des immunoconjugués formés entre un polypeptide de l'invention et des agents cytotoxiques, leurs procédés de préparation et leur utilisation dans le traitement du cancer.
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US44340895A | 1995-05-17 | 1995-05-17 | |
US443408 | 1995-05-17 | ||
PCT/US1996/006941 WO1996036360A1 (fr) | 1995-05-17 | 1996-05-15 | Immunoconjugues comprenant des fragments monocatenaires de regions variables d'anticorps anti-cd-19 |
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EP96916466A Withdrawn EP0835134A1 (fr) | 1995-05-17 | 1996-05-15 | Immunoconjugues comprenant des fragments monocatenaires de regions variables d'anticorps anti-cd-19 |
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US7531643B2 (en) | 1997-09-11 | 2009-05-12 | Chugai Seiyaku Kabushiki Kaisha | Monoclonal antibody inducing apoptosis |
DE69909459T2 (de) | 1998-04-21 | 2004-05-27 | Micromet Ag | Cd19xcd3 spezifische polypeptide und deren verwendung |
US7696325B2 (en) | 1999-03-10 | 2010-04-13 | Chugai Seiyaku Kabushiki Kaisha | Polypeptide inducing apoptosis |
DE19946142A1 (de) * | 1999-09-27 | 2001-03-29 | Bundesrepublik Deutschland Let | Gentransfer in humane Lymphocyten mittels retroviraler scFv-Zelltargeting Vektoren |
JP2004502412A (ja) * | 2000-06-08 | 2004-01-29 | ザ センター フォー ブラッド リサーチ インコーポレーティッド | 免疫グロブリンを介した再灌流障害を抑制するための方法および組成物 |
EP2351838A1 (fr) * | 2000-10-20 | 2011-08-03 | Chugai Seiyaku Kabushiki Kaisha | Anticorps agonistiques qui réticuler |
CN1308448C (zh) | 2000-10-20 | 2007-04-04 | 中外制药株式会社 | 低分子化的tpo激动剂抗体 |
WO2002033073A1 (fr) * | 2000-10-20 | 2002-04-25 | Chugai Seiyaku Kabushiki Kaisha | Anticorps agoniste degrade |
AU2002210917B2 (en) * | 2000-10-20 | 2006-05-18 | Chugai Seiyaku Kabushiki Kaisha | Degraded TPO agonist antibody |
JP2004279086A (ja) | 2003-03-13 | 2004-10-07 | Konica Minolta Holdings Inc | 放射線画像変換パネル及び放射線画像変換パネルの製造方法 |
AU2004242846A1 (en) | 2003-05-31 | 2004-12-09 | Micromet Ag | Pharmaceutical compositions comprising bispecific anti-CD3, anti-CD19 antibody constructs for the treatment of B-cell related disorders |
JP4733635B2 (ja) * | 2003-07-31 | 2011-07-27 | イミューノメディクス、インコーポレイテッド | 抗cd19抗体 |
US7902338B2 (en) | 2003-07-31 | 2011-03-08 | Immunomedics, Inc. | Anti-CD19 antibodies |
US8137907B2 (en) | 2005-01-03 | 2012-03-20 | Cold Spring Harbor Laboratory | Orthotopic and genetically tractable non-human animal model for liver cancer and the uses thereof |
US8444973B2 (en) | 2005-02-15 | 2013-05-21 | Duke University | Anti-CD19 antibodies and uses in B cell disorders |
US9493569B2 (en) | 2005-03-31 | 2016-11-15 | Chugai Seiyaku Kabushiki Kaisha | Structural isomers of sc(Fv)2 |
US20070044164A1 (en) | 2005-05-31 | 2007-02-22 | Cold Spring Harbor Laboratory | Methods for producing microRNAs |
EP1908482B1 (fr) | 2005-06-10 | 2017-09-06 | Chugai Seiyaku Kabushiki Kaisha | Stabilisant pour une préparation de protéine contenant de la méglumine et son utilisation |
KR101360671B1 (ko) | 2005-06-10 | 2014-02-07 | 추가이 세이야쿠 가부시키가이샤 | sc(Fv)2를 함유하는 의약조성물 |
KR20080032097A (ko) | 2005-06-20 | 2008-04-14 | 메다렉스, 인코포레이티드 | 씨디19 항체 및 그의 용도 |
ES2402591T3 (es) | 2006-08-14 | 2013-05-07 | Xencor Inc. | Anticuerpos optimizados que seleccionan como diana CD19 |
ES2383710T3 (es) | 2006-09-08 | 2012-06-25 | Medimmune, Llc | Anticuerpos anti-CD19 humanizados y su uso en el tratamiento de tumores, trasplantes y enfermedades autoinmunes |
CL2007003622A1 (es) * | 2006-12-13 | 2009-08-07 | Medarex Inc | Anticuerpo monoclonal humano anti-cd19; composicion que lo comprende; y metodo de inhibicion del crecimiento de celulas tumorales. |
EP2409712A1 (fr) | 2010-07-19 | 2012-01-25 | International-Drug-Development-Biotech | Anticorps Anti-CD19 doté de fonctions ADCC et CDC et d'un profil de glycosylation amélioré |
EP2409993A1 (fr) | 2010-07-19 | 2012-01-25 | International-Drug-Development-Biotech | Anticorps Anti-CD19 doté d'une fonction ADCC et d'un profil de glycosylation amélioré |
WO2014165115A1 (fr) | 2013-03-12 | 2014-10-09 | Decimmune Therapeutics, Inc. | Anticorps humanisés anti-n2 |
CN110945028B (zh) | 2017-07-10 | 2023-09-08 | 国际药物发展生物技术公司 | 用非岩藻糖基化促凋亡抗cd19抗体与抗cd20抗体或化疗剂联合治疗b细胞恶性肿瘤 |
CN110003334B (zh) * | 2019-04-12 | 2023-05-09 | 深圳普瑞金生物药业股份有限公司 | 多肽、cd19单域抗体及其制备方法、核苷酸序列及试剂盒 |
CA3173980A1 (fr) * | 2020-03-03 | 2021-09-10 | Systimmune, Inc. | Anticorps anti-cd19 et leurs methodes d'utilisation et de preparation |
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