WO2000030680A1 - Tumor antigen-specific antibody-gp39 chimeric protein constructs - Google Patents
Tumor antigen-specific antibody-gp39 chimeric protein constructs Download PDFInfo
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- WO2000030680A1 WO2000030680A1 PCT/US1999/027654 US9927654W WO0030680A1 WO 2000030680 A1 WO2000030680 A1 WO 2000030680A1 US 9927654 W US9927654 W US 9927654W WO 0030680 A1 WO0030680 A1 WO 0030680A1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- the present invention relates to novel chimeric proteins which comprise a tumor antigen-specific monoclonal antibody or some fragment thereof at the amino terminus fused to an immunostimulatory ligand at the carboxyl terminus.
- Such chimeric proteins serve to enhance anti-tumor immune responses at the site of the tumor by stimulating endogenous leukocytes which express receptor for the immunostimulatory ligand portion of the chimeric protein on their cell surface.
- Tumor-associated antigens are also expressed on normal cells to varying degrees, generally during different stages of differentiation. Therefore, these antigens are also known as "differentiation antigens" (Fundamental Immunology, 3 rd edition, W. Paul, ed., Raven Press, New York, 1993, Ch. 32). Despite the fact that TAAs are also expressed on normal cells to some extent, studies suggest that differences in expression levels between normal and malignant cells can often be enough to favor a therapeutic response (Oettgen, H.F. and Old, L.J., The History of Cancer Immunotherapy; DeVita et al. eds.; Biologic Therapy of Cancer, Lippincott, Philadelphia, 1991, pp. 87-119).
- tumor-associated antigens A variety of tumor-associated antigens (TAA) have been described as summarized in U.S. Patent No. 5,766,588, herein incorporated by reference in its entirety.
- assays for identifying tumor associated antigens and tumor specific antigens are known in the art, as described in U.S. Patent No. 5,763,164, also incorporated by reference in its entirety. Identification of tumor-specific and tumor-associated antigens will enable the identification and isolation of monoclonal antibodies specific for these antigens using well-established techniques in the art. However, it is generally the case that tumor specific antibodies will not in and of themselves exert sufficient antitumor effects to make them useful in cancer immunotherapy. Indeed, despite the variety of tumor- associated antigens which have now been identified, tumor cells remain poorly immunogenic.
- cytokine genes have been introduced into tumor cells to produce vaccines having varying degrees of effect on both tumorigenicity and immunogenicity .
- Tumor cells have been modified with genes for interleukin-2 (IL- 2) (Porgador, A., et al., Int. J. Cancer, 53:471-477 [1993]); interferon-alpha, (IFN- alpha)(Porgador, A., et al., Int. Immunol., 150: 1458-1570 [1993]); granulocyte- macrophage colony stimulating factor (GM-CSF) (Dranoff, G., et al., Proc. Nat. Acad. Sci.
- IL-2 interleukin-2
- IFN- alpha interferon-alpha
- GM-CSF granulocyte- macrophage colony stimulating factor
- tumor cells are killed in vitro by a process involving antibody coating, opsonization and either phagocytosis by macrophages or antibody-dependent cell-mediated cytotoxicity in the presence of macrophages, natural killer cells or neutrophils.
- TNF produced by macrophages has been shown to be responsible for the cytotoxic effects observed in vitro (Urban et al. 1986, Proc. Natl. Acad, Sci USA, 83: 5233-5237). Corresponding effects in vivo, however, have not been readily achieved.
- the immune system is capable of producing two types of antigen- specific responses to foreign antigens.
- Cell-mediated immunity is the term used to refer to effector functions of the immune system mediated by T lymphocytes.
- Humoral immunity is the term used to refer to production of antigen-specific antibodies by B lymphocytes. It has long been appreciated that the development of humoral immunity against most antigens requires not only antibody-producing B lymphocytes but also the involvement of helper T (hereinafter Th) lymphocytes. (Mitchison, Eur. J.
- B cell activation involves an obligatory interaction between cell surface molecules on B cells and Th cells. 5 Such an interaction is further supported by the observation that isolated plasma membranes of activated T cells can provide helper functions necessary for B cell activation. (Brian, Proc. Natl. Acad. Sci. USA, 85:564-568 (1988); Hodgkin et al., J. Immunol, 145:2025-2034 (1990); Noelle et al., J. Immunol, 146: 1118-1124 (1991)). l o The process by which T cells help B cells to differentiate has been divided into two distinct phases; the inductive and effector phases (Vitetta et al., Adv.
- T cell helper function can be Ag-independent and MHC-nonrestricted (Clement et al., J. Immunol, 132:740 (1984); Hirohata et al., J. Immunol, 140:3736 (1988); Whalen et al., J. Immunol, 143:1715 (1988)).
- terminal B cell differentiation requires both contact- and
- lymphokine-mediated stimuli from T cells intermediate stages of B cell differentiation can be induced by activated T cell surfaces in the absence of secreted factors (Crow et al., J. Exp. Med., 164: 1760 (1986); Brian, Proc. Natl Acad. Sci., USA, 85:564 (1988); Sekita et al., Eur. J. Immunol, 18:1405 (1988); Hodgkin et al., J. Immunol, 145:2025 (1990); Noelle et al., FASEB J, 5:2770
- a cell surface molecule, CD40 has been identified on immature and mature B lymphocytes which, when crosslinked by antibodies, induces B cell proliferation.
- Valle et al. Eur. J. Immunol, 19: 1463- 1467 (1989); Gordon et al., J. Immunol, 140:1425-1430 (1988); Gruder et al., J. Immunol, 142:4144-4152 (1989).
- CD40 has been molecularly cloned and characterized (Stamenkovic et al., EMBO J., 8:1403-1410 (1989)). CD40 is expressed on B cells, interdigitating dendritic cells, macrophages, follicular dendritic cells, and thymic epithelium (Clark, Tissue Antigens 36:33 (1990); Alderson et al., J. Exp. Med., 178:669 (1993); Galy et al., J. Immunol 142:772 (1992)).
- Human CD40 is a type I membrane protein of 50 kDa and belongs to the nerve growth factor receptor family (Hollenbaugh et al., Immunol Rev., 138:23 (1994)). Signaling through CD40 in the presence of IL-10 induces IgA, IgM and IgG production, indicating that isotype switching is regulated through these interactions. The interaction between CD40 and its ligand results in a primed state of the B cell, rendering it receptive to subsequent signals.
- a ligand for CD40, g ⁇ 39 (also called CD40 ligand or CD40L) has recently been molecularly cloned and characterized (Armitage et al., Nature, 357:80-82 (1992); Lederman et al., J. Exp. Med., 175:1091-1101 (1992); Hollenbaugh et al., EMBO J., 11:4313-4319 (1992)).
- the gp39 protein is expressed on activated, but not resting, CD4 + Th cells.
- gp39 is a type II membrane protein and is part of a new gene super family which includes TNF- ⁇ , TNF- ⁇ and the ligands for FAS, CD27, CD30 and 4-1BB.
- gp39 + T cells produce IL-2, IL-4 and IFN- ⁇ (Van der Eetwegh et al., J. Exp. Med., 178:1555 (1993)).
- HIM hyper-IgM syndrome
- anti-gp39 has been shown to prevent formation of memory B cells and germinal centers in mouse spleen (Foy et al., J. Exp. Med., 180:157 (1994)).
- CD40 ligation has also been shown to play an important role in IL2-R expression, IL-12 production and B7.1 expression by activated B cells (Nishioka and Lipsky, 1994, j. Immunol. 153: 1027).
- CD40 ligation on dendritic cells stimulates IL-12 production (Koch et al., 1996, J. Exp. Med. 184(2) :741-746) and enhances surface expression of ICAM-1 and B7.1 (Cella et al, 1996, J. Exp. Med. 184(2):747-752), all of which are important contributors to Th 1 -type, CTL-mediated immune responses (Heufler et al, 1996, Eur. J. Immunol, 26:659).
- gp39-CD40 interaction appears to play a role in both cell-mediated immunity and antibody-specific immune responses, and is likely to enhance tumor specific responses in cancers which respond to either cell-mediated or humoral immune mechanisms.
- the present invention addresses the deficiencies of the prior art by providing chimeric protein constructs which contain a tumor antigen-specific antibody binding domain (or TAA-specific antibody) fused to the CD40 binding domain of the gp39 ligand.
- chimeric proteins provide effective and convenient therapeutic reagents which will enhance immune responses toward a wide variety of cancers for which a tumor-specific or tumor-associated antigen may be identified.
- Chimeric proteins containing an antibody component fused to other types of molecules have previously been described and are known in the art.
- Antibody fusions have been generated to deliver cells, cytotoxins, or drugs to specific sites.
- An important use has been to deliver host cytotoxic cells, such as natural killer or cytotoxic T cells, to specific cellular targets. (Staerz et al. Nature, 314:628 (1985); Songilvilai, et al, Clin. Exp. Immunol, 79:315 (1990)).
- Another important use has been to deliver cytotoxic proteins to specific cellular targets. (Raso et al. Cancer Res., 41:2073 (1981); Nissan et al, Cytotechnology, 4:59 (1990)).
- Antibodies have also been fused to toxic molecules for the purpose of delivering them specifically to cancer cells.
- U.S. Patent No. 5,756,699 herein incorporated by reference in its entirety, provides a thorough review of the state of art concerning immunotoxins, whereby the variable region of an antibody gene is fused to the gene for a bacterial toxin. It was hoped that such reagents could be used to target tumor cells, however, such fusion proteins have been shown to be immunogenic and toxic in animals.
- Challida et al. disclose a B7.1 -antibody fusion protein specific for the tumor-associated antigen HER2/neu. However, in contrast to the recombinant DNA constructs of the present invention, the construct disclosed in Challida et al.
- PLAP is a protein that activates phospholipase A, a lipolytic enzyme which hydrolyzes the 2-acyl fatty acid ester of glycerophospholipids. This hydrolysis releases arachidonic acid which is converted into a number of biologically active compounds called eicosanoids.
- PLAP has been postulated to be involved in the inflammatory cascade in certain biological settings, and induces eicosanoid release and stimulation of joint inflammation, related to rheumatoid arthritis.
- the present invention pertains to dual function chimeric proteins comprising both an antigen binding domain and a receptor ligand binding domain, wherein said receptor ligand is involved in immune cell modulation and stimulation.
- Such proteins may be constructed by linking or bonding together two protein or protein fragments, or may be synthesized from recombinant DNA constructs as fusion proteins.
- the chimeric proteins of the present invention are synthesized from recombinant DNA constructs.
- a construct comprises a nucleic acid encoding at least a heavy chain variable region binding domain of a disease antigen-specific antibody fused to a nucleic acid encoding at least a binding portion of an immunostimulatory ligand such that expression of said nucleic acid molecule yields a fusion protein having the heavy chain antibody variable region domain at its amino terminus and the binding portion of the immunostimulatory ligand at its carboxyl terminus.
- a chimeric protein is formed comprising at the very least an Fv fragment of the antibody fused to the binding domain of an immunostimulatory ligand.
- a nucleic acid molecule according to the present invention may also comprise a nucleic acid encoding at least one antibody constant region.
- a construct with one heavy chain constant region will result in a fusion protein wherein the binding portion of the immunostimulatory ligand is fused to a Fab fragment of the disease antigen-specific antibody.
- a construct with at least part of the second constant region of the antibody will include sequences encoding the cysteine residue involved in heavy chain disulfide bridge formation, and will result in a fusion protein wherein the binding portion of the immunostimulatory ligand is fused to an F(ab') 2 fragment of the disease antigen-specific antibody (See Figure 1). Fusion proteins comprising full length antibody proteins are also included.
- a nucleic acid molecule according to the present invention may also comprise a nucleic acid encoding a light chain antibody or antibody fragment 5 fused with said nucleic acid encoding said antibody variable region.
- the nucleic acid encoding said light chain antibody or antibody fragment is typically fused to said nucleic acid encoding said antibody variable region at the 5' end in such a way that upon expression, the light chain and heavy chain variable regions associate to form an antigen binding pocket (See Figure ID).
- Proper o association and folding of the two antibody fragments typically requires a flexible peptide linker, which is encoded by a nucleic acid present between the light chain and heavy chain coding regions in the nucleic acid construct.
- the chimeric proteins of the present invention are specifically designed to enhance immune responses by cells of the immune system in the vicinity of 5 diseased cells by providing a dual function binding protein that binds to both an antigen on the surface of the diseased cell and to a receptor on the surface of an immune cell.
- the diseased cell antigen may be a tumor antigen or a viral antigen, or any antigen that is specifically expressed on the particular cells targeted for enhanced immune responses.
- the immune cell receptor may be any receptor specifically expressed on immune cells, but is preferably one which stimulates immune cell responses upon ligand interaction, such as cytokine production, costimulatory molecule expression, APC function or T helper cell stimulation.
- the immunostimulatory ligand is a CD40 ligand, and is most preferably gp39 or the 5 receptor binding portion thereof.
- the two binding portions of the dual function chimeric protein may optionally be connected by a linker peptide of variable length, which is encoded by an additional, in-frame coding region in the nucleic acid constructs of the present invention.
- the necessity of such a linker peptide will depend on the nature of the targeted antigen, i.e., its location and accessibility, the receptor for the immunostimulatory ligand, and the particular constraints of tertiary protein structure for each particular immunostimulatory ligand and antigen binding domain.
- the present invention also encompasses chimeric proteins encoded by the nucleic acid constructs described above, as well as chimeric proteins that can be constructed by binding together the two separate protein domains, i.e., using bifunctional chelators or other linking proteins.
- Pharmaceutical compositions comprising the chimeric proteins of the present invention in a pharmaceutically acceptable carrier are also included. It has been demonstrated by Shopes (J. Immunol, 148(9) :2918-2922, 1992) that "tail-to-tail" dimeric IgG-IgG dimers having tetravalent binding could be generated through the formation of a disulfide linkage between individual heavy chains on the Ig molecule. A similar approach was used by Caron et al (J. Exp.
- the present invention also includes vectors and host cells comprising the nucleic acid molecules described above.
- Host cells may be prokaryotic or eukaryotic depending on the purpose for expressing the vector. For instance, prokaryotic cells will be more convenient for multiplying, isolating and maintaining vectors comprising the nucleic acid, whereas eukaryotic cells may be required for expression and isolation of properly folded and active protein 5 depending on the antigen binding and ligand binding domains chosen for the nucleic acid construct. Host cells may also contain the nucleic acids of the present invention integrated into the chromosome. Any type of vector may be used depending upon the particular host cell chosen for expression of the nucleic acid, including prokaryotic and eukaryotic vectors, viral or phage vectors, etc.
- Host cells which express the chimeric proteins of the present invention may be used in a method of making the chimeric proteins comprising expressing the nucleic acid molecule of the invention in a host cell and isolating the resulting chimeric protein.
- the particular method of protein purification will depend upon the particular expression system used as well as the nature of the chimeric protein.
- chimeric proteins having an antigen binding domain may be isolated and purified using columns made from the target antigen.
- purification columns may be designed using the immune cell receptor which interacts with the ligand binding domain.
- Classical methods of protein purification including DSFF or CM chromatography may also be used depending on
- the chimeric proteins of the present invention may be used in a method of enhancing disease antigen-specific antibody responses in a subject who expresses the relevant disease antigen. Such a method comprises administering the chimeric
- disease antigen-specific or systemic immune responses i.e., cytokine production, costimulatory molecule expression, APC function, helper and cytotoxic T cell stimulation, etc.
- Such enhanced immune response may also provide a method of treating a disease in a patient in need of such treatment, where such treatment may prevent, alleviate or cure the particular disease.
- Diseases which particularly benefit from the method of the present invention include cancer and viral infections such as AIDS.
- kits comprising the chimeric proteins in sterile form whereby the proteins may be easily administrated to a patient are also encompassed in the present invention.
- kits comprising the nucleic acid molecules, vectors and appropriate host cells are also included. Columns for purifying the resulting chimeric protein may also be included.
- Figure 2 A diagrammatic representation of the anti-tumor immune response generated by a TAA specific monoclonal antibody-gp39 chimeric protein construct.
- a “nucleic acid molecule” may be DNA or RNA, and may contain modified nucleotide bases so long as such modified bases do not inhibit expression of the encoded chimeric protein.
- a “heavy chain variable region binding domain” is the most minimal portion of an antibody heavy chain variable region capable of association with the corresponding light chain of the antibody and subsequent antibody recognition.
- a “disease antigen” may be any antigen specifically expressed by, or shown to be preferentially associated with, a diseased cell.
- a “diseased cell” may be a tumor, cancer, or malignant cell, or a virally-infected cell.
- a tumor antigen may be tumor-specific, i.e., only expressed by tumor cells, or tumor-associated (TAA), i.e., also expressed by normal cells but perhaps at a different time or at a different level.
- TAA tumor-associated
- a disease antigen for the purposes of the present invention must be expressed on the surface of a diseased cell, either by virtue of its own secretory signals, or in context with an MHC molecule.
- a disease antigen may also be expressed on the surface of a phagocytic cell, i.e., a macrophage, following phagocytosis, for instance in context with an MHC molecule.
- An "antibody” according to the present invention may be from any species so long as it is specific for the targeted antigen.
- the antibody may be derived from a humanized antibody or other antibody gene which has been genetically engineered.
- the "antibody” may be an antibody fragment, i.e., an Fv, FAB, F(ab') or F(ab) 2 fragment, the structure of all of which are known in the art as described in U.S. Patent No. 5,648,237, herein incorporated by reference.
- fused means that the two nucleic acids are fused in such a way that a single protein molecule or peptide chain is formed upon expression of the fused nucleic acid construct.
- a "binding portion of an immunostimulatory ligand” means the minimal region of any ligand which interacts with or binds to the immunostimulatory receptor molecule which is targeted by the chimeric protein of the present invention.
- An "immunostimulatory receptor molecule” is any receptor on the surface of an immune cell, the ligation or binding of which stimulates an immune response from the cell on which it is expressed.
- the immunostimulatory ligand is any molecule which interacts with and binds to CD40, i.e., a CD40 monoclonal antibody or fragment thereof, gp39, or any other molecule which is demonstrated to bind specifically or particularly to CD40.
- a “fusion protein” is a chimeric protein resulting from expression of a nucleic acid sequence which encodes peptide sequences derived or designed from more than one native protein sequence.
- the term “derived” indicates the sequences were designed from the native protein sequence, but may contain amino acid substitutions which do not inhibit, or perhaps even increase, the stability or functional capabilities of the chimeric protein. Such amino acid substitutions may be a necessary result of nucleotide base changes required for the formation of restriction endonuclease cleavage sites during construction of the recombinant DNA construct.
- a nucleic acid encoding a "compatible" antibody light chain or light chain fragment contains a variable region domain which, upon expression, contributes to the formation of the antigen binding pocket.
- This nucleic acid molecule may be fused in frame to the N terminus of the heavy chain recombinant DNA construct by a sequence of nucleotides which encode a flexible peptide linker.
- This flexible peptide linker is designed with an appropriate length and sequence such that the light chain and heavy chain regions of the antibody may take on the native conformation of the antigen binding pocket.
- a "subject who expresses a disease antigen” or a “patient” may be either human or animal.
- a patient may be "in need of treatment” without actually demonstrating physical symptoms of disease so long as the patient expresses the disease antigen on a diseased cell.
- treatment encompasses any therapeutic regimen where the aim is to prevent, alleviate or cure the particular disease, and may be accomplished if the disease progression is merely slowed or symptoms are alleviated for any period of time no matter how brief.
- Disease antigen-specific responses may include cytokine production, costimulatory molecule expression, APC function, T helper cell stimulation, the infiltration of immune cells to the site of the diseased antigen or cell, such as, infiltration by T cells, B cells, macrophages and other lymphocytes.
- the immunostimulatory moieties in the fusion molecules of the invention can also cause or modulate, for example, the activation of lymphocyte cells, the expression of lymphocyte-specific compounds, the elaboration of antibodies, the enhancement of phagocytosis by phagocytes, and the enhancement of tumor cell lysis, or any immune cell response which is a specific result of the binding or ligation of the target immunostimulatory receptor.
- variable regions of both heavy and light chains show considerable variability in structure and amino acid composition from one antibody molecule to another, whereas the constant regions show little variability.
- the term "variable” as used in this specification refers to the diverse nature of the amino acid sequences of the antibody heavy and light chain variable regions.
- Each antibody recognizes and binds antigen through the binding site defined by the association of the heavy and light chain variable regions into an F[V ]area.
- the light-chain variable region V[L ]and the heavy-chain variable region V[H ]of a particular antibody molecule have specific amino acid sequences that allow the antigen-binding site to assume a conformation that binds to the antigen epitope recognized by that particular antibody.
- variable regions are found regions in which the amino acid sequence is extremely variable from one antibody to another.
- Three of these so-called “hypervariable” regions or “complementarity-determining regions” (CDR's) are found in each of the light and heavy chains.
- the three CDR's from a light chain and the three CDR's from a corresponding heavy chain form the antigen-binding site.
- the Chimeric Protein Construct is found in each of the light and heavy chains.
- the chimeric proteins of the invention be constructed according to one of the following basic forms.
- the first form comprises an antigen binding, i.e., the variable region from an antibody heavy chain fused at its C terminus to an immunoeffector protein moiety.
- the protein may optionally contain a peptide spacer between the two functional domains, so that the structure will generally be: NH2-V[H]-immunoeffector moiety-COOH or NH2-V[H]- spacer-immunoeffector moiety-COOH.
- the construct may optionally contain the variable region from the corresponding light chain antibody fused to the N- terminus, preferably by a flexible peptide linker, such that the basic form will generally be: NH2-V[L]-spacer-V[H]-immunoeffector moiety-COOH.
- the protein may also comprise constant region domains from both the light and heavy chains of the antibody, such that varying portions of a single chain antibody are fused to the N-terminus of the immunostimulatory ligand (See Figure 1).
- Linkers used in the fusion constructs of the invention can be any of those linkers known or used in the art. Skilled artisans will be able to determine the appropriate linker to be used for a particular construct. It is preferred that the linkers utilized in constructing the antigen-binding fusion proteins of the invention are between 0 and 50 amino acids in length. In some cases it may be necessary to separate the antigen-binding part of a fusion protein from the immunoeffector part of the fusion protein by a peptide spacer, in order to preserve both activities of the fusion protein. It is also preferred that the spacers are between 0 and 50 amino acids in length.
- Fv is defined to be a covalently or noncovalently-associated heavy and light chain heterodimer which does not contain constant domains.
- Fab' may be defined as a polypeptide comprising a heterodimer of the variable domain and the first constant domain of an antibody heavy chain, plus the variable domain and constant domain of an antibody light chain, plus at least one additional amino acid residue at the carboxyl terminus of the heavy chain C[H]1 domain including one or more cysteine residues.
- F(ab')2 antibody fragments are pairs of Fab' antibody fragments which are linked by a covalent bond(s).
- the Fab' heavy chain may include a hinge region. This may be any desired hinge amino acid sequence. Alternatively the hinge may be entirely omitted in favor of a single cysteine residue or, preferably a short (about 1-10 residues) cysteine-containing polypeptide. In certain applications, a common naturally occurring antibody hinge sequence (cysteine followed by two prolines and then another cysteine) is used; this sequence is found in the hinge of human IgGl molecules (E. A. Kabat, et al, Sequences of Proteins of Immunological Interest, 3rd edition (National Institutes of Health, Bethesda, Md, 1987)). The hinge region may also be selected from another desired antibody class or isotype.
- antibodies may be "humanized” (i.e., if the subject is a human).
- a humanized antibody is generally understood to be an immunoglobulin amino acid sequence variant or fragment thereof which is capable of binding to a predetermined antigen and which comprises a FR (framework) region having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immunoglobulin or a sequence engineered to bind to a preselected antigen.
- the chimeric protein constructs of the present invention comprise an immunostimulatory ligand binding domain.
- a preferred immunostimulatory ligand is gp39, which has been shown to bind to CD40 on the surface of antigen presenting cells and thereby enhance immune responses.
- the chimeric proteins of the present invention may contain any portion of gp39 or other immunostimulatory ligand so long as that portion is capable of interacting with or binding to its cognate receptor, and the chimeric protein as a whole is able to effectuate an immune response stemming from ligation of this cognate receptor.
- binding portions may be identified using any assay known in the art for measuring the binding capabilities or affinities of peptide fragments of a ligand to the corresponding receptor.
- peptide fragments may be synthesized according to the known sequence of gp39, and used in a competitive binding assay with gp39+ T cells.
- peptide display libraries may be generated using techniques known in the art and screened for the capability to bind CD40+ cells.
- the entire extracellular portion of the gp39 molecule may also be used.
- the immunoeffector domain may lack ligand binding activity individually so long as it is capable of assembly into a functional binding domain when expressed as a portion of the chimeric antigen-binding protein.
- chimeric proteins of the present invention are preferably produced by recombinant DNA techniques.
- chimeric proteins are encoded by nucleic acid fusion constructs, which are cloned into expression vectors and expressed in appropriate host cells for the production of the chimeric protein.
- host cells may be prokaryotic or eukaryotic.
- U.S. Patent No. 5,468,237 herein incorporated by reference in its entirety, many have had success with antibody expression and isolation using prokaryotic expression systems.
- U.S. Patent No. 5,648,237 discloses cloning vectors and methods for expressing antibodies and antibody fragments in E. coli, whereby the antibodies are secreted into and isolated from the periplasmic space of the microorganism. Also provided are methods for effecting covalent bond formation at the hinge region following antibody isolation.
- the vector disclosed in U.S. Patent No. 5,648,237 is a dicistronic expression vector whereby the light chain and heavy chain fragments are each under the control of an inducible bacterial promoter.
- the redox environment in the bacterial periplasmic space apparently favors disulfide bond formation between light and heavy chains but not between the hinge cysteine residues.
- Others have had success in expressing heavy and light chains as a single polypeptide chain in bacteria.
- Ig fusion proteins may be expressed in prokaryotic cells using the vectors described therein, depending on the immunoeffector moiety of the chimeric protein, i.e., the importance of glycosylation and proper protein secretion in maintaining binding function of the ligand, the chimeric proteins of the present invention may also be expressed using a eukaryotic, baculovirus or retrovirus expression system.
- Treatment encompasses administration of compounds prophylactically to prevent or suppress an undesired condition, and therapeutic administration to eliminate or reduce the extent or symptoms of the condition.
- Treatment according to the invention may be for a human or an animal so long as there are cells in said human or animal which express the target antigen. Treatment may be by systemic administration to the subject or by local application to an affected site.
- compositions of the present invention i.e., compositions comprising a chimeric protein of the present invention
- the chimeric proteins may be administered to a subject either singly or in combination with other chimeric proteins of the invention, or other known reagents commonly used in treatment regiments for the particular disease of interest.
- the proteins may also be used in therapy in conjunction with other anti-cancer or anti-viral drugs and biologicals, or in conjunction with other immune modulating therapy including bone marrow or lymphocyte transplants or medications.
- acceptable is defined as being compatible with other ingredients of the formulation and not injurious to the subject or other non-target cells.
- These carriers include those well known to practitioners in the art as suitable for oral, rectal, nasal, topical, buccal, sublingual, vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, and intradermal) administration.
- the compounds according to the invention may also be used in the manufacture of pharmaceuticals for the treatment or prophylaxis of cancer and viral infections.
- Such formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use.
- sterile liquid carrier for example, water for injections, immediately prior to use.
- Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
- the mode of administration and dosage of protein chosen should be adequate to result in effective circulating levels of the chimeric protein.
- dosages for intravenous administration will generally range from 0.001 mg/kg to about 10 mg/kg body weight of the patient, to be administered over several days or weeks by daily infusions depending on the patient's tolerance.
- an "effective amount" of the composition is such as to produce the desired effect in a patient which can be monitored using several end-points known to those skilled in the art. For example, such effects could be monitored in terms of a therapeutic effect, e.g., alleviation of some symptom associated with the disease being treated, or further evidence suggesting enhanced immune response in the targeted area. These methods are by no means all-inclusive, and further methods to suit the specific application will be apparent to the ordinary skilled artisan.
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- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU16317/00A AU1631700A (en) | 1998-11-23 | 1999-11-23 | Tumor antigen-specific antibody-gp39 chimeric protein constructs |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10960798P | 1998-11-23 | 1998-11-23 | |
US60/109,607 | 1998-11-23 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2000030680A1 true WO2000030680A1 (en) | 2000-06-02 |
Family
ID=22328583
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1999/027654 WO2000030680A1 (en) | 1998-11-23 | 1999-11-23 | Tumor antigen-specific antibody-gp39 chimeric protein constructs |
Country Status (2)
Country | Link |
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AU (1) | AU1631700A (en) |
WO (1) | WO2000030680A1 (en) |
Cited By (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008081008A1 (en) | 2007-01-05 | 2008-07-10 | University Of Zurich | Method of providing disease-specific binding molecules and targets |
WO2008110372A1 (en) | 2007-03-13 | 2008-09-18 | University Of Zurich | Monoclonal human tumor-specific antibody |
WO2010069603A1 (en) | 2008-12-19 | 2010-06-24 | Neurimmune Therapeutics Ag | Human anti-alpha-synuclein autoantibodies |
DE202011103324U1 (en) | 2011-07-12 | 2012-01-02 | Nekonal S.A.R.L. | Therapeutic anti-TIRC7 antibodies for use in immune and other diseases |
WO2012049570A1 (en) | 2010-10-11 | 2012-04-19 | Panima Pharmaceuticals Ag | Human anti-tau antibodies |
WO2012080518A1 (en) | 2010-12-17 | 2012-06-21 | Neurimmune Holding Ag | Human anti-sod1 antibodies |
EP2527369A2 (en) | 2007-09-13 | 2012-11-28 | University Of Zurich Prorektorat Forschung | Monoclonal amyloid beta (abeta)-specific antibody and uses thereof |
WO2012177972A1 (en) | 2011-06-23 | 2012-12-27 | Biogen Idec International Neuroscience Gmbh | Anti-alpha synuclein binding molecules |
WO2013041962A1 (en) | 2011-09-19 | 2013-03-28 | Axon Neuroscience Se | Protein-based therapy and diagnosis of tau-mediated pathology in alzheimer's disease |
WO2013093122A2 (en) | 2011-12-23 | 2013-06-27 | Phenoquest Ag | Antibodies for the treatment and diagnosis of affective and anxiety disorders |
WO2014102399A1 (en) | 2012-12-31 | 2014-07-03 | Neurimmune Holding Ag | Recombinant human antibodies for therapy and prevention of polyomavirus-related diseases |
WO2016050822A2 (en) | 2014-09-30 | 2016-04-07 | Neurimmune Holding Ag | Human-derived anti-dipeptide repeats (dprs) antibody |
EP3269740A1 (en) | 2016-07-13 | 2018-01-17 | Mabimmune Diagnostics AG | Novel anti-fibroblast activation protein (fap) binding agents and uses thereof |
WO2020167912A1 (en) | 2019-02-13 | 2020-08-20 | The Brigham And Women's Hospital, Inc. | Anti-peripheral lymph node addressin antibodies and uses thereof |
EP3792278A2 (en) | 2012-12-21 | 2021-03-17 | Biogen MA Inc. | Human anti-tau antibodies |
US10973826B2 (en) | 2015-10-29 | 2021-04-13 | Novartis Ag | Antibody conjugates comprising toll-like receptor agonist |
WO2022153212A1 (en) | 2021-01-13 | 2022-07-21 | Axon Neuroscience Se | Antibodies neutralizing sars-cov-2 |
Citations (3)
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WO1997029781A1 (en) * | 1996-02-15 | 1997-08-21 | Immunex Corporation | Methods and compositions for modulating an immune response |
US5747024A (en) * | 1993-03-08 | 1998-05-05 | Immunex Corporation | Vaccine adjuvant comprising interleukin-15 |
US5767260A (en) * | 1994-10-13 | 1998-06-16 | Enzon Inc. | Antigen-binding fusion proteins |
-
1999
- 1999-11-23 WO PCT/US1999/027654 patent/WO2000030680A1/en active Application Filing
- 1999-11-23 AU AU16317/00A patent/AU1631700A/en not_active Abandoned
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5747024A (en) * | 1993-03-08 | 1998-05-05 | Immunex Corporation | Vaccine adjuvant comprising interleukin-15 |
US5767260A (en) * | 1994-10-13 | 1998-06-16 | Enzon Inc. | Antigen-binding fusion proteins |
WO1997029781A1 (en) * | 1996-02-15 | 1997-08-21 | Immunex Corporation | Methods and compositions for modulating an immune response |
Cited By (29)
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EP3239175A1 (en) | 2007-01-05 | 2017-11-01 | University of Zurich | Method of providing disease-specific binding molecules and targets |
EP3239174A1 (en) | 2007-01-05 | 2017-11-01 | University of Zurich | Anti-beta-amyloid antibody and uses thereof |
EP2426143A2 (en) | 2007-01-05 | 2012-03-07 | University of Zurich | Method of providing disease-specific binding molecules and targets |
EP2436696A1 (en) | 2007-01-05 | 2012-04-04 | University of Zurich | Method of providing disease-specific binding molecules and targets |
WO2008081008A1 (en) | 2007-01-05 | 2008-07-10 | University Of Zurich | Method of providing disease-specific binding molecules and targets |
WO2008110372A1 (en) | 2007-03-13 | 2008-09-18 | University Of Zurich | Monoclonal human tumor-specific antibody |
EP3159355A1 (en) | 2007-03-13 | 2017-04-26 | Universität Zürich | Monoclonal human tumor-specific antibody |
EP2457928A2 (en) | 2007-03-13 | 2012-05-30 | Universität Zürich | Monoclonal human tumor-specific antibody |
US8519106B2 (en) | 2007-03-13 | 2013-08-27 | University Of Zurich | Monoclonal human tumor-specific antibody |
EP2527369A2 (en) | 2007-09-13 | 2012-11-28 | University Of Zurich Prorektorat Forschung | Monoclonal amyloid beta (abeta)-specific antibody and uses thereof |
EP3521309A1 (en) | 2008-12-19 | 2019-08-07 | Biogen International Neuroscience GmbH | Human anti-alpha-synuclein antibodies |
EP2949666A1 (en) | 2008-12-19 | 2015-12-02 | Biogen Idec International Neuroscience GmbH | Human anti-alpha-synuclein antibodies |
WO2010069603A1 (en) | 2008-12-19 | 2010-06-24 | Neurimmune Therapeutics Ag | Human anti-alpha-synuclein autoantibodies |
WO2012049570A1 (en) | 2010-10-11 | 2012-04-19 | Panima Pharmaceuticals Ag | Human anti-tau antibodies |
EP3628689A1 (en) | 2010-12-17 | 2020-04-01 | Neurimmune Holding AG | Human anti-sod1 antibodies |
WO2012080518A1 (en) | 2010-12-17 | 2012-06-21 | Neurimmune Holding Ag | Human anti-sod1 antibodies |
WO2012177972A1 (en) | 2011-06-23 | 2012-12-27 | Biogen Idec International Neuroscience Gmbh | Anti-alpha synuclein binding molecules |
DE202011103324U1 (en) | 2011-07-12 | 2012-01-02 | Nekonal S.A.R.L. | Therapeutic anti-TIRC7 antibodies for use in immune and other diseases |
EP3275461A1 (en) | 2011-09-19 | 2018-01-31 | Axon Neuroscience SE | Protein-based therapy and diagnosis of tau-mediated pathology in alzheimer's disease field |
WO2013041962A1 (en) | 2011-09-19 | 2013-03-28 | Axon Neuroscience Se | Protein-based therapy and diagnosis of tau-mediated pathology in alzheimer's disease |
WO2013093122A2 (en) | 2011-12-23 | 2013-06-27 | Phenoquest Ag | Antibodies for the treatment and diagnosis of affective and anxiety disorders |
EP3792278A2 (en) | 2012-12-21 | 2021-03-17 | Biogen MA Inc. | Human anti-tau antibodies |
WO2014102399A1 (en) | 2012-12-31 | 2014-07-03 | Neurimmune Holding Ag | Recombinant human antibodies for therapy and prevention of polyomavirus-related diseases |
EP3517545A1 (en) | 2012-12-31 | 2019-07-31 | Neurimmune Holding AG | Recombinant human antibodies for therapy and prevention of polyomavirus-related diseases |
WO2016050822A2 (en) | 2014-09-30 | 2016-04-07 | Neurimmune Holding Ag | Human-derived anti-dipeptide repeats (dprs) antibody |
US10973826B2 (en) | 2015-10-29 | 2021-04-13 | Novartis Ag | Antibody conjugates comprising toll-like receptor agonist |
EP3269740A1 (en) | 2016-07-13 | 2018-01-17 | Mabimmune Diagnostics AG | Novel anti-fibroblast activation protein (fap) binding agents and uses thereof |
WO2020167912A1 (en) | 2019-02-13 | 2020-08-20 | The Brigham And Women's Hospital, Inc. | Anti-peripheral lymph node addressin antibodies and uses thereof |
WO2022153212A1 (en) | 2021-01-13 | 2022-07-21 | Axon Neuroscience Se | Antibodies neutralizing sars-cov-2 |
Also Published As
Publication number | Publication date |
---|---|
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