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HK1169318B - Targeted immunoconjugates - Google Patents

Targeted immunoconjugates Download PDF

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Publication number
HK1169318B
HK1169318B HK12110035.2A HK12110035A HK1169318B HK 1169318 B HK1169318 B HK 1169318B HK 12110035 A HK12110035 A HK 12110035A HK 1169318 B HK1169318 B HK 1169318B
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HK
Hong Kong
Prior art keywords
seq
immunoconjugate
sequence
variable region
chain variable
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HK12110035.2A
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Chinese (zh)
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HK1169318A1 (en
Inventor
Ralf Hosse
Ekkehard Moessner
Michela Silacci-Melkko
Pablo Umana
Original Assignee
罗切格利卡特公司
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Priority claimed from PCT/EP2010/061810 external-priority patent/WO2011020783A2/en
Publication of HK1169318A1 publication Critical patent/HK1169318A1/en
Publication of HK1169318B publication Critical patent/HK1169318B/en

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Description

Targeted immunoconjugates
Technical Field
In general, the invention relates to antigen-specific immunoconjugates for selectively delivering effector moieties that affect cellular activity. In addition, the invention relates to nucleic acid molecules encoding such immunoconjugates and vectors and host cells comprising such nucleic acid molecules. The invention further relates to methods for producing the immunoconjugates of the invention, and methods of using these immunoconjugates in the treatment of disease.
Background
Selective destruction of individual cells or specific cell types is often desirable in many clinical settings. For example, the primary goal of cancer therapy is to specifically destroy tumor cells, while leaving healthy cells and tissues intact and undamaged. Many signal transduction pathways in cells are associated with cell survival and/or death. Thus, direct delivery of factors to pathways involved in cell survival or death may be used to facilitate maintenance or destruction of cells.
Cytokines are cell signaling molecules involved in the regulation of the immune system. When used in cancer therapy, cytokines may act as immune modulators, which have anti-tumor effects, and which may increase the immunogenicity of some types of tumors. However, rapid blood clearance and lack of tumor specificity require systemic administration of high doses of cytokines to achieve cytokine concentrations sufficient to activate an immune response or have an anti-tumor effect at the tumor site. These high levels of systemic cytokines can lead to severe toxicity and adverse reactions.
One way to deliver a factor of a signal transduction pathway, such as a cytokine, to a specific site in the body (e.g., a tumor or tumor microenvironment) is to conjugate the factor with an immunoglobulin specific for that site. Early strategies were directed to delivering factors of signal transduction pathways, such as cytokines, to specific sites in the body, including immunoglobulin heavy chains conjugated to various cytokines, including lymphotoxin, tumor necrosis factor-alpha (TNF- α), interleukin-2 (IL-2), and granulocyte macrophage colony stimulating factor (GM-CSF). Immunoglobulin heavy chains are either chemically conjugated to cytokines or immunoglobulin-cytokine conjugates are expressed as fusion proteins. See Nakamura k. and Kubo, a. cancer Supplement 80: 2650-2655 (1997); jun, l, et al, chi.med.j.113: 151-153 (2000); and beckerj.c. et al, proc.natl.acad.sci.usa 93: 7826-7831(1996). Researchers have observed that not only can they target cytokines to specific sites in the body, they can also take advantage of the fact that monoclonal antibodies have a longer serum half-life than most other proteins. The ability of immunoglobulin-cytokine fusion proteins to maximize immunostimulatory activity at the tumor site at lower doses while keeping systemic side effects to a minimum, due to the systemic toxicity associated with high doses of certain unconjugated cytokines, i.e., IL-2, led researchers to believe that immunoglobulin-cytokine immunoconjugates are the best therapeutic agents. However, one of the major limitations in the clinical utility of immunoglobulins as delivery agents is their inadequate uptake and poor distribution in tumors, due in part to the large size of the immunoglobulin molecule. See Xiang, j, et al, immunol. cell biol.72: 275-285(1994). In addition, it has been suggested that immunoglobulin-cytokine immunoconjugates can activate complement and interact with Fc receptors. This inherent immunoglobulin characteristic has been viewed disadvantageously because therapeutic immunoconjugates can target Fc receptor-expressing cells rather than the preferred antigen-bearing cells.
One approach to overcome these problems is to use engineered immunoglobulin fragments. Many studies have detailed the characteristics of immunoglobulin fragment-cytokine immunoconjugates. See Savage, p, et al, br.j. cancer 67: 304-310 (1993); harville, e.t. and Morrison s.l., immunotechnol.1: 95-105 (1995); and Yang j, et al, mol.immunol.32: 873-881(1995). In general, there are two common immunoglobulin fragment-cytokine fusion protein constructs, F (ab') expressed in mammalian cells2Cytokines and scFv-cytokines expressed in E.coli (Escherichia coli). See Xiang, j.hum.antibodies 9: 23-36(1999). Both the tumor binding reactivity of the immunoglobulin parent molecule and the functional activity of cytokines are maintained in most of these types of immunoconjugates. Recent preclinical studies have shown that these fusion proteins are able to target cytokines to tumors expressing tumor-associated antigens in vivo and inhibit both primary and metastatic tumors in animal models of immunocompetence.
Examples of immunoglobulin fragment-cytokine immunoconjugates include scFv-IL-2 immunoconjugates as set forth in PCT publication WO2001/062298A 2; immunoglobulin heavy chain fragment-GM-CSF immunoconjugates as set forth in U.S. patent No.5,650,150; immunoconjugates as set forth in PCT publication WO2006/119897a2, in which the first subunit of scFv-IL-12 shares only a disulfide bond with the second subunit of IL-12-scFv, and immunoconjugates as described in PCT publication WO 99/29732a2, in which the first subunit of Ig heavy chain fragment-IL-12 shares only a disulfide bond with the second subunit of Ig heavy chain fragment-IL-12. While these second generation immunoconjugates have some improved properties compared to the first generation immunoglobulin-cytokine conjugates, the development of more and even safer specific therapeutic agents is desirable for greater efficiency against tumor cells and for a reduction in the number and severity of side effects (e.g., toxicity, destruction of non-tumor cells, etc.) of these products. In addition, it is desirable to identify ways to further stabilize immunoconjugates while maintaining acceptable levels of therapeutic activity.
The present invention provides immunoconjugates exhibiting improved efficacy, high specificity of action, reduced toxicity and improved stability in blood relative to known immunoconjugates.
Summary of The Invention
One aspect of the invention relates to immunoconjugates exhibiting improved efficacy, high specificity of action, reduced toxicity and improved stability in blood compared to known immunoconjugates. The immunoconjugates of the invention can be used to selectively deliver an effector moiety to a target site in the body. In another embodiment, the immunoconjugate delivers a cytokine to the target site, wherein the cytokine may exert a local biological effect, such as a local inflammatory response, stimulation of T cell growth and activation, and/or activation of B and/or NK cells.
One aspect of the invention relates to an immunoconjugate comprising at least a first effector moiety and at least a first and a second antigen binding moiety independently selected from the group of: fv and Fab, wherein a first effector moiety shares an amino-or carboxy-terminal peptide bond with a first antigen-binding moiety, and a second antigen-binding moiety shares an amino-or carboxy-terminal peptide bond with i) said first effector moiety or ii) said first antigen-binding moiety
Another aspect of the invention is an immunoconjugate comprising at least a first single-chain effector moiety linked at its amino-terminal amino acid to one or more scFv molecules, and wherein the first single-chain effector moiety is linked at its carboxy-terminal amino acid to one or more scFv molecules.
Another aspect of the invention is an immunoconjugate comprising at least a first single chain effector moiety and first and second antigen binding moieties, wherein each of the first and second antigen binding moieties comprises an scFv molecule linked at its carboxy-terminal amino acid to a constant region comprising an immunoglobulin constant domain independently selected from the group consisting of: IgG CH1, IgG ck and IgE CH4, and wherein the first antigen binding moiety is linked at its constant region carboxy-terminal amino acid to the amino-terminal amino acid of one of the effector moieties, and wherein the first and second antigen binding moieties are covalently linked via at least one disulfide bond.
Another aspect of the invention is an immunoconjugate comprising at least a first single chain effector moiety and first and second antigen binding moieties, wherein each of the first and second antigen binding moieties comprises an scFv molecule linked at its carboxy-terminal amino acid to an IgG1 CH3 domain, and wherein the first antigen binding moiety is linked at its carboxy-terminal amino acid to an amino-terminal amino acid of one of the effector moieties, and wherein the first and second antigen binding moieties are covalently linked via at least one disulfide bond.
Another aspect of the invention relates to an immunoconjugate comprising a first and a second single chain effector moiety and a first and a second antigen binding moiety, wherein each antigen binding moiety comprises a Fab molecule linked at its heavy or light chain carboxy-terminal amino acid to an IgG1 CH3 domain, and wherein each IgG1 CH3 domain is linked at its carboxy-terminal amino acid to an amino-terminal amino acid of one of the effector moieties, and wherein the first and second antigen binding moieties are covalently linked via at least one disulfide bond.
In one embodiment, the immunoconjugate comprises the polypeptide sequence of SEQ ID NO: 95. in another embodiment, the immunoconjugate comprises the polypeptide sequence of SEQ ID NO: 104. in another embodiment, the immunoconjugate comprises the polypeptide sequence of SEQ ID NO: 105. in another embodiment, the immunoconjugate comprises the polypeptide sequence of SEQ ID NO: 106. in another embodiment, the immunoconjugate comprises the polypeptide sequence of SEQ ID NO: 107. in another embodiment, the immunoconjugate comprises the polypeptide sequence of SEQ ID NO: 96. in yet another embodiment, the immunoconjugate comprises the polypeptide sequence of SEQ id no: 96 and a polypeptide sequence selected from the group consisting of: SEQ ID NO: 95 and 104-107. In another embodiment, the immunoconjugate comprises a polypeptide having a sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of seq id no: SEQ ID NO: 95. 96 and 104-107.
In one embodiment, the immunoconjugate comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 108, a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In another embodiment, the immunoconjugate comprises a polypeptide consisting of the polynucleotide sequence of SEQ ID NO: 108, or a pharmaceutically acceptable salt thereof. In one embodiment, the immunoconjugate comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 117 at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence encoding the polypeptide. In another embodiment, the immunoconjugate comprises a polypeptide consisting of the polynucleotide sequence of SEQ ID NO: 117 to seq id no. In one embodiment, the immunoconjugate comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 118, or a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In another embodiment, the immunoconjugate comprises a polypeptide consisting of the polynucleotide sequence of SEQ ID NO: 118, or a pharmaceutically acceptable salt thereof. In one embodiment, the immunoconjugate comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 119, or a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In another embodiment, the immunoconjugate comprises a polypeptide consisting of the polynucleotide sequence of SEQ ID NO: 119, or a pharmaceutically acceptable salt thereof. In one embodiment, the immunoconjugate comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 120, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide sequence encoded by the polynucleotide sequence. In another embodiment, the immunoconjugate comprises a polypeptide consisting of the polynucleotide sequence of SEQ ID NO: 120, or a pharmaceutically acceptable salt thereof. In one embodiment, the immunoconjugate comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 109, a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In another embodiment, the immunoconjugate comprises a polypeptide consisting of the polynucleotide sequence of SEQ ID NO: 109, or a pharmaceutically acceptable salt thereof.
In one embodiment, the immunoconjugate comprises a sequence identical to the sequence of SEQ ID NO: 13 or SEQ id no: 15, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical heavy chain variable region. In another embodiment, the immunoconjugate comprises a sequence identical to the sequence of SEQ ID NO: 9 or SEQ ID NO: 11, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the light chain variable region sequence. In yet another embodiment, the immunoconjugate comprises a sequence identical to the sequence of SEQ ID NO: 13 or SEQ ID NO: 15 and a heavy chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 9 or SEQ ID NO: 11, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the light chain variable region sequence.
In one embodiment, the immunoconjugate comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16, a heavy chain variable region sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In another embodiment, the immunoconjugate comprises a polypeptide consisting of the polynucleotide sequence of SEQ ID NO: 14 or SEQ ID NO: 16, or a heavy chain variable region sequence encoded by seq id no. In one embodiment, the immunoconjugate comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 12, a light chain variable region sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In another embodiment, the immunoconjugate comprises a polypeptide consisting of the polynucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 12, or a light chain variable region sequence encoded by seq id no.
In one embodiment, the immunoconjugate comprises a sequence identical to the sequence of SEQ ID NO: 99 a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In another embodiment, the immunoconjugate comprises a sequence identical to the sequence of SEQ ID NO: 100 or SEQ ID NO: 215 is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In another embodiment, the immunoconjugate comprises a sequence identical to the sequence of SEQ ID NO: 101 or SEQ ID NO: 235 is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In yet another embodiment, the immunoconjugate comprises a sequence that hybridizes to seq id NO: 100 and a polypeptide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 101 at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In yet another embodiment, the immunoconjugate comprises a sequence identical to the sequence of SEQ ID NO: 215 and a polypeptide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 235 is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical.
In one embodiment, the immunoconjugate comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 112, or a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In another embodiment, the immunoconjugate comprises a polypeptide consisting of the polynucleotide sequence of SEQ ID NO: 112, or a pharmaceutically acceptable salt thereof. In one embodiment, the immunoconjugate comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 113 or SEQ ID NO: 216, or a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In another embodiment, the immunoconjugate comprises a polypeptide consisting of the polynucleotide sequence of SEQ ID NO: 113 or SEQ ID NO: 216, or a pharmaceutically acceptable salt thereof. In one embodiment, the immunoconjugate comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 114 or SEQ ID NO: 236, or a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In another embodiment, the immunoconjugate comprises a polypeptide consisting of the polynucleotide sequence of SEQ ID NO: 114 or SEQ ID NO: 236.
In one embodiment, the immunoconjugate comprises a heavy chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of seq id no: SEQ ID NO: 7, SEQ ID NO: 179, SEQ ID NO: 183, SEQ ID NO: 187, SEQ id no: 191, SEQ ID NO: 195, SEQ ID NO: 199, SEQ ID NO: 203 and SEQ ID NO: 207. in another embodiment, the immunoconjugate comprises a light chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of seq id no: SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 177, SEQ ID NO: 181, SEQ id no: 185, SEQ ID NO: 189, SEQ ID NO: 193, SEQ ID NO: 197, SEQ ID NO: 201 and SEQ ID NO: 205. in yet another embodiment, the immunoconjugate comprises a heavy chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of seq id no: SEQ ID NO: 7, SEQ ID NO: 179, SEQ ID NO: 183, SEQ ID NO: 187, SEQ ID NO: 191, SEQ ID NO: 195, SEQ ID NO: 199, SEQ ID NO: 203 and SEQ ID NO: 207 and a light chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of seq id no: SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 177, SEQ ID NO: 181, SEQ ID NO: 185, SEQ ID NO: 189, SEQ ID NO: 193, SEQ ID NO: 197, SEQ ID NO: 201 and SEQ ID NO: 205.
In one embodiment, the immunoconjugate comprises a heavy chain variable region sequence encoded by a polynucleotide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of seq id no: SEQ ID NO: 8, SEQ ID NO: 180, SEQ ID NO: 184, SEQ ID NO: 188, SEQ ID NO: 192, SEQ ID NO: 196, SEQ ID NO: 200, SEQ ID NO: 204 and SEQ ID NO: 208. in another embodiment, the immunoconjugate comprises a heavy chain variable region sequence encoded by a polynucleotide sequence selected from the group consisting of seq id no: SEQ ID NO: 8, SEQ ID NO: 180, SEQ id no: 184, SEQ ID NO: 188, SEQ ID NO: 192, SEQ ID NO: 196, SEQ ID NO: 200, SEQ ID NO: 204 and SEQ ID NO: 208. in one embodiment, the immunoconjugate comprises a light chain variable region sequence encoded by a polynucleotide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of seq id no: SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 178, SEQ ID NO: 182, SEQ ID NO: 186, SEQ ID NO: 190, SEQ ID NO: 194, SEQ ID NO: 198, SEQ ID NO: 202 and SEQ ID NO: 206. in another embodiment, the immunoconjugate comprises a light chain variable region sequence encoded by a polynucleotide sequence selected from the group consisting of seq id no: SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 178, SEQ ID NO: 182, SEQ ID NO: 186, SEQ ID NO: 190, SEQ ID NO: 194, SEQ ID NO: 198, seq id NO: 202 and SEQ ID NO: 206.
In one embodiment, the immunoconjugate comprises a polypeptide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of seq id no: SEQ ID NO: 239. SEQ ID NO: 241 and SEQ ID NO: 243. in another embodiment, the conjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of seq id no: SEQ ID NO: 245. SEQ ID NO: 247 and SEQ ID NO: 249. in yet another embodiment, the immunoconjugate comprises a polypeptide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of seq id no: SEQ ID NO: 239. SEQ ID NO: 241 and SEQ ID NO: 243, and a polypeptide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of seq id no: SEQ ID NO: 245. SEQ ID NO: 247 and SEQ ID NO: 249.
in one embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of seq id no: SEQ ID NO: 240. SEQ ID NO: 242 and SEQ ID NO: 244. in another embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence selected from the group consisting of seq id no: SEQ ID NO: 240. SEQ ID NO: 242 and SEQ ID NO: 244. in one embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of seq id no: SEQ ID NO: 246. SEQ ID NO: 248 and SEQ ID NO: 250. in another embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence selected from the group consisting of seq id no: SEQ ID NO: 246. SEQ ID NO: 248 and SEQ ID NO: 250.
In one embodiment, the immunoconjugate comprises a heavy chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of seq id no: SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 139, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 155, SEQ ID NO: 159, SEQ ID NO: 163, SEQ ID NO: 167, SEQ ID NO: 171 and SEQ ID NO: 175. in another embodiment, the immunoconjugate comprises a light chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of seq id no: SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 121, SEQ ID NO: 125, SEQ ID NO: 129, SEQ ID NO: 133, seq id NO: 137, SEQ ID NO: 141, SEQ ID NO: 145, SEQ ID NO: 149, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 165, SEQ ID NO: 169 and SEQ ID NO: 173. in yet another embodiment, the immunoconjugate comprises a heavy chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of seq id no: SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 123, SEQ id no: 127, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 139, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 155, SEQ ID NO: 159, SEQ ID NO: 163, SEQ ID NO: 167, SEQ ID NO: 171 and SEQ ID NO: 175, a light chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of seq id no: SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 121, SEQ ID NO: 125, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 137, SEQ ID NO: 141, SEQ ID NO: 145, SEQ ID NO: 149, SEQ ID NO: 153, seq id NO: 157, SEQ ID NO: 161, SEQ ID NO: 165, SEQ ID NO: 169 and SEQ ID NO: 173.
In one embodiment, the immunoconjugate comprises a heavy chain variable region sequence encoded by a polynucleotide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of seq id no: SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO: 124, SEQ id no: 128, SEQ ID NO: 132, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 144, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 168, SEQ ID NO: 172 and SEQ ID NO: 176. in another embodiment, the immunoconjugate comprises a heavy chain variable region sequence encoded by a polynucleotide sequence selected from the group consisting of seq id no: SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 144, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 168, SEQ ID NO: 172 and SEQ ID NO: 176. in one embodiment, the immunoconjugate comprises a light chain variable region sequence encoded by a polynucleotide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of seq id no: SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO: 122, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 134, SEQ ID NO: 138, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, SEQ ID NO: 154, SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 170 and SEQ ID NO: 174. in another embodiment, the immunoconjugate comprises a light chain variable region sequence encoded by a polynucleotide sequence selected from the group consisting of seq id no: SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO: 122, SEQ ID NO: 126, SEQ ID NO: 130, SEQ id no: 134, SEQ ID NO: 138, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, SEQ ID NO: 154, SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 170 and SEQ ID NO: 174.
In one embodiment, the immunoconjugate comprises a polypeptide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of seq id no: SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225 and SEQ ID NO: 227. in another embodiment, the immunoconjugate comprises a polypeptide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of seq id no: SEQ ID NO: 229, SEQ ID NO: 231. SEQ ID NO: 233 and SEQ ID NO: 237. in yet another embodiment, the immunoconjugate comprises a polypeptide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of seq id no: SEQ ID NO: 211. SEQ ID NO: 219 and SEQ ID NO: 221, and a sequence identical to SEQ ID NO: 231 a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of seq id no: SEQ ID NO: 209. SEQ ID NO: 223. SEQ ID NO: 225 and SEQ ID NO: 227, and a sequence identical to SEQ ID NO: 229 is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In yet another embodiment, the immunoconjugate comprises a sequence identical to the sequence of SEQ ID NO: 213, and a polypeptide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 233 is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In yet another embodiment, the immunoconjugate comprises a sequence identical to the sequence of SEQ ID NO: 217, and a polypeptide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 237 a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical.
In one embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of seq id no: SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226 and SEQ ID NO: 228. in another embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence selected from the group consisting of seq id no: SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, seq id NO: 226 and SEQ ID NO: 228. in one embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of seq id no: SEQ ID NO: 230. SEQ ID NO: 232. SEQ ID NO: 234 and SEQ ID NO: 238. in another embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence selected from the group consisting of seq id no: SEQ ID NO: 230. SEQ ID NO: 232. SEQ ID NO: 234 and SEQ ID NO: 238.
In one embodiment, the immunoconjugate comprises a sequence identical to the sequence of SEQ ID NO: 257 or SEQ ID NO: 261 are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In another embodiment, the immunoconjugate comprises a sequence that hybridizes to seq id NO: 259 or SEQ ID NO: 271 light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In yet another embodiment, the immunoconjugate comprises a sequence identical to the sequence of SEQ ID NO: 257 or SEQ ID NO: 261, and a heavy chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 259 or SEQ ID NO: 271 light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical.
In one embodiment, the immunoconjugate comprises a polypeptide sequence consisting of a sequence identical to the sequence of SEQ ID NO: 258 or SEQ ID NO: 262, a heavy chain variable region sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In another embodiment, the immunoconjugate comprises a polypeptide consisting of the polynucleotide sequence of SEQ ID NO: 258 or SEQ ID NO: 262, or a heavy chain variable region sequence encoded by seq id no. In one embodiment, the immunoconjugate comprises a polypeptide sequence consisting of a sequence identical to the sequence of SEQ ID NO: 260 or SEQ ID NO: 272, and a light chain variable region sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In another embodiment, the immunoconjugate comprises a polypeptide consisting of the polynucleotide sequence of SEQ ID NO: 260 or SEQ ID NO: 272.
In one embodiment, the immunoconjugate comprises a sequence identical to the sequence of SEQ ID NO: 251 or SEQ ID NO: 255 is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In another embodiment, the immunoconjugate comprises a sequence identical to the sequence of SEQ ID NO: 253 or SEQ ID NO: 265 is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In yet another embodiment, the immunoconjugate comprises a sequence that hybridizes to seq id NO: 251 or SEQ ID NO: 255, and a polypeptide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 253 or SEQ ID NO: 265 is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical.
In one embodiment, the immunoconjugate comprises a polypeptide sequence consisting of a sequence identical to the sequence of SEQ ID NO: 252 or SEQ ID NO: 256 at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In another embodiment, the immunoconjugate comprises a polypeptide consisting of the polynucleotide sequence of SEQ ID NO: 252 or SEQ ID NO: 256, or a pharmaceutically acceptable salt thereof. In one embodiment, the immunoconjugate comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 254 or SEQ ID NO: 266, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide sequence encoded by the polynucleotide sequence. In another embodiment, the immunoconjugate comprises a polypeptide consisting of the polynucleotide sequence of SEQ ID NO: 254 or SEQ ID NO: 266, or a pharmaceutically acceptable salt thereof.
In another embodiment, the immunoconjugate comprises at least one effector moiety, wherein said effector moiety is a cytokine. In a specific embodiment, the effector moiety is a cytokine selected from the group consisting of: interleukin-2 (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF), interferon-alpha (INF-alpha), interleukin-12 (IL-12), interleukin-8 (IL-8), macrophage inflammatory protein-1 alpha (MIP-1 alpha), macrophage inflammatory protein-1 beta (MIP-1 beta), and transforming growth factor-beta (TGF-beta). In another embodiment, at least one antigen binding moiety is specific for one of the following antigenic determinants: the extra domain b of fibronectin (edb), the a1 domain of tenascin (TNC-a1), the a2 domain of tenascin (TNC-a2), Fibroblast Activation Protein (FAP); and Melanoma Chondroitin Sulfate Proteoglycan (MCSP).
In another embodiment, the immunoconjugates of the invention have a dissociation constant (K) that is at least about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 fold greater than the dissociation constant of the control effector moietyD) Binding to the effector module receptor. In another embodiment, the immunoconjugate inhibits tumor volume increase in vivo by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more at the end of the administration period. In another embodiment, the immunoconjugate upon administration to a mammal in need thereof extends survival of a mammal having a malignancy by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% relative to an effector moiety in a control effector moiety or a "diabody" immunoconjugate (immunoconjugate) molecule.
Another aspect of the invention relates to an isolated polynucleotide encoding an immunoconjugate of the invention, or a fragment thereof. Another aspect of the invention relates to an expression vector comprising an expression cassette comprising a polynucleotide sequence of the invention.
Another aspect of the invention relates to a host cell expressing the immunoconjugate of the invention or a fragment thereof.
Another aspect of the invention relates to a method for producing an immunoconjugate of the invention or a fragment thereof, wherein the method comprises culturing a host cell transformed with an expression vector encoding the immunoconjugate or fragment of the invention under conditions suitable for its expression.
Another aspect of the invention relates to a method for promoting proliferation and differentiation in activated T lymphocyte cells, comprising contacting activated T lymphocyte cells with an effective amount of an immunoconjugate of the invention.
Another aspect of the invention relates to a method for promoting proliferation and differentiation in activated B lymphocyte cells, comprising contacting activated B lymphocyte cells with an effective amount of an immunoconjugate of the invention.
Another aspect of the invention relates to a method for promoting proliferation and differentiation in Natural Killer (NK) cells comprising contacting the NK cells with an effective amount of an immunoconjugate of the invention.
Another aspect of the invention relates to a method for promoting proliferation and differentiation in granulocytes, monocytes or dendritic cells comprising contacting the granulocytes, monocytes or dendritic cells with an effective amount of an immunoconjugate of the invention.
Another aspect of the present invention relates to a method for promoting differentiation of Cytotoxic T Lymphocytes (CTLs), comprising contacting T lymphocytes with an effective amount of the immunoconjugate of the invention.
Another aspect of the invention relates to a method for inhibiting viral replication comprising contacting a virus-infected cell with an effective amount of an immunoconjugate of the invention.
Another aspect of the invention relates to a method for upregulating major histocompatibility complex i (mhc i) expression, comprising contacting a target cell with an effective amount of an immunoconjugate of the invention.
Another aspect of the invention relates to a method for inducing cell death comprising administering to a target cell an effective amount of an immunoconjugate of the invention.
Another aspect of the invention relates to a method for inducing chemotaxis in a target cell comprising administering to the target cell an effective amount of an immunoconjugate of the invention.
Another aspect of the invention relates to a method for treating a disease in an individual comprising the step of administering to the individual a therapeutically effective amount of a composition comprising an immunoconjugate of the invention and a pharmaceutically acceptable carrier.
Brief Description of Drawings
FIG. 1: schematic representation of various immunoconjugate fusion formats. All constructs in fig. 1 comprise two antibody scFv fragments (in an antigen binding moiety) and one or two cytokine molecules linked to it (as effector moieties). Panels a to E show the different connections and stoichiometries of the antigen binding and effector modules. Panel A) depicts a "diabody" -IL-2 fusion. "diabodies" are non-covalently assembled from two identical polypeptide chains. Panel B shows an immunoconjugate comprising the heavy chain of a Fab molecule fused at its carboxy-terminus to a cytokine, which is in turn fused at its carboxy-terminus to a second Fab heavy chain. Co-expressing the light chain with the heavy chain Fab-cytokine-heavy chain Fab polypeptide to form an immunoconjugate. Alternatively, the two light chains may be fused to a cytokine and the heavy chains co-expressed. In panel C, the two Fab heavy chains are fused directly to each other. The cytokine shares an amino-terminal peptide bond with the heavy chain of the second antigen binding module. The two molecular forms of panels B and C can be altered such that the Fab chain is replaced with an scFv fragment, as shown in panels D and E.
FIG. 2: schematic representation of other immunoconjugates comprising two antigen binding moieties and at least one or more effector moieties. Panel a shows a Fab molecule fused via its carboxy terminus to an IgG CH3 domain. To achieve covalent antigen binding module homodimerization, an artificial disulfide bond (immunoconjugate on the right in panel a) can be introduced at the carboxy terminus of the IgG CH3 domain. The IgG1 CH3 domain shown in panel a may be replaced with an IgE CH4 domain. The Fab module in panel A was replaced with the scFv fragment in panel B. For the panel C immunoconjugates, the native IgG hinge is fused at the C-terminus of the Fab molecule. Because the carboxy terminal region of the hinge may impose some geometric constraints on the assembly of a constant domain fused to the C-terminus of the IgG hinge region, an artificial linker may be introduced between the carboxy terminal region of the hinge and the amino terminus of the IgG CH3 domain. It is also possible to introduce a hinge region between the scFv fragment and the immunoglobulin constant domain, as shown in panel D. In panels a to D, IgG1 CH3 or IgE CH4 domains were used to homodimerize the constructs. Panel E depicts an immunoconjugate in which dimerization is achieved via a CH 1/ck heterodimerization interaction. The immunoconjugate of panel D may have one or two cytokines per immunoconjugate.
FIG. 3 presents the results of efficacy experiments with two different interleukin-2 immunoconjugate molecular forms specific for tumor stroma. F9 teratomas were injected subcutaneously into 129SvEv mice and tumor size was measured using calipers. The "diabody" -IL-2 molecule was compared to Fab-interleukin-2-Fab (Fab-IL2-Fab) immunoconjugates at two different concentrations, where the concentrations reflect a similar number of immunoconjugate molecules. In FIG. 3, the Fab-IL2-Fab immunoconjugate is labeled "Fab-L19", the unconjugated interleukin-2 control is labeled "Unconj rIL-2", and the "diabody" -IL-2 molecule is labeled "diabody". The L19 antibody directed against the extra domain b (edb) of fibronectin was used to generate the antigen binding moiety in both diabodies and Fab-L19 immunoconjugates. The amount of immunoconjugate injected per mouse (in μ g) is indicated in the figure.
FIG. 4 presents the results of survival experiments with two different interleukin-2 immunoconjugate molecular forms specific for tumor stroma. The human gastric tumor cell line LS174T was injected intrasplenically into SCID-beige mice. In FIG. 4, the Fab-IL2-Fab immunoconjugate is labeled "Fab-L19", the unconjugated interleukin-2 control is labeled "Unconj rIL-2", and the "diabody" -IL-2 molecule is labeled "diabody". anti-EDB antibody L19 was used to generate antigen binding moieties in both diabodies and Fab-L19 immunoconjugates. The amount of immunoconjugate injected per mouse (in μ g) is indicated in the figure and reflects the same number of immunoconjugate molecules.
Figure 5 shows immunohistochemical images of human uterine tissue at 100X and 400X magnification. The 2B10 variable region generated by the method described in example 3 binds to the a2 domain of human tenascin (tenascin) (TNC-a 2). The 2B10 variable region in the Fab fragment was fused to a FLAG fragment (SHD2B 10-FLAG). Healthy and cancerous human uterine tissue samples were prepared for immunohistochemical staining. Subsequently, the samples were incubated with SHD2B10-FLAG Fab fragment. The sample was then washed and incubated with a fluorescent antibody specific for the FLAG epitope. Cancerous tissue samples exhibit higher expression levels of TNC-a2 compared to healthy tissue samples.
Figure 6 shows TNC-a2 expression levels in various human tissue samples in% immunofluorescence surface area. Various human tissue samples from healthy individuals and cancer patients were incubated with SHD2B10-FLAG Fab fragment as depicted in figure 5.
Figure 7 shows Fibroblast Activation Protein (FAP) expression levels in various human tissue samples in% immunofluorescence surface area. Various human tissue samples from healthy individuals and cancer patients were incubated with a commercial antibody against FAP (Abcam). The top portion of each bar on the graph represents tumor expression of FAP, while the bottom portion of each bar on the graph represents normal FAP expression.
Figure 8 presents BIACORE data showing the affinity of the known IgG antibody L19 for EDB.
Figure 9 presents BIACORE data showing affinity for Fab-IL-2-Fab immunoconjugates specific for EDB. The Fab fragment in the immunoconjugate was derived from the L19 antibody.
Figure 10 presents BIACORE data showing affinity for EDB-specific "diabody" -IL2 fusion protein. Diabody scFv fragments were derived from the L19 antibody. "diabody" -IL2 fusion protein comprises an 8 amino acid linker between the scFv fragment and the IL-2 molecule.
Figure 11 presents BIACORE data showing affinity for EDB-specific "diabody" -IL2 fusion protein. Diabody scFv fragments were derived from the L19 antibody. "diabody" -IL2 fusion protein comprises a 12 amino acid linker between the scFv fragment and the IL-2 molecule.
Figure 12 presents BIACORE data showing the affinity of a known IgG antibody F16 for the immobilised tenascin domain a1(TNC-a 1). FIG. 12 also presents BIACORE data showing the affinity of the Fab fragment of the F16 antibody for TNC-A1. The calculated dissociation constants (K) for F16IgG and Fab molecules are indicated in the figureD)。
FIG. 13 presents BIACORE data showing the affinity of IL-2 for the immobilized IL-2 receptor. Heterodimerization of the β and γ chains of IL-2R is achieved by fusing the corresponding chains to "protrusion-into-hole" variants of the human IgG1Fc moiety, as described in Merchant, a.m. et al, nat.biotech.16: 677-. K calculated from BIACORE data is indicated in the figure DThe value is obtained.
FIG. 14 presents BIACORE data showing the affinity of the "diabody" -IL-2 fusion protein for TNC-A1 and the IL-2 receptor. The scFv molecules in the antibody were derived from the F16 antibody. K calculated from BIACORE data is indicated in the figureDThe value is obtained.
FIG. 15 presents BIACORE data showing the affinity of Fab-IL-2-Fab immunoconjugates for TNC-A1 and the IL-2 receptor. The Fab molecules in the immunoconjugate are derived from the F16 antibody. K calculated from BIACORE data is indicated in the figureDThe value is obtained.
FIG. 16 presents BIACORE data showing the affinity of scFv-IL-2-scFv immunoconjugates for TNC-A1 and the IL-2 receptor. The scFv molecules in the immunoconjugate are derived from the F16 antibody. K calculated from BIACORE data is indicated in the figureDThe value is obtained.
FIG. 17 is K obtained from the BIACORE study presented in FIGS. 12-16DSummary of values.
Figure 18 presents the results of efficacy experiments comparing the "diabody" -IL-2 molecule targeting the EDB domain of fibronectin with the Fab-interleukin-2-Fab immunoconjugate targeting tenascin C A2 domain (labeled "Fab-SH 2B 10" comprising the heavy and light chain variable regions of SEQ ID NOs 3 and 7, respectively). In FIG. 18, the unconjugated interleukin-2 control label is "Unconj rIL-2" and the "diabody" -IL-2 molecule is labeled "L19 diabody". anti-EDB antibody L19 was used to generate the antigen binding moiety in the diabody immunoconjugate. Teratoma cell line F9 was injected subcutaneously into 129 line immunocompetent mice. The amount of immunoconjugate injected per mouse (in μ g) is indicated in the figure. Treatment was started on day 6 and a total of 5 injections were administered until day 11 of the experiment.
Figure 19 shows V anti-FAP, or anti-tenascin C, Fab-IL2-Fab immunoconjugates (using 3F2, 3D9, 4B3 (anti-FAP), 2F11 and 2B10 constructs (anti-tenascin C)) compared to unconjugated human IL-2HAnd VLSequence generation) induction of NK-92 cell proliferation. Cell proliferation was measured using the CellTiter Glo system after two days of incubation.
Figure 20 presents the results of an ELISA assay measuring the induction of IFN- γ production by various immunoconjugates containing interleukin 12 compared to the unconjugated cytokine, or an immunoconjugate containing the p35 and p40 domains of IL-12 in separate molecules. Panel a shows the results on fibronectin coated plates. Panel B shows the results in solution.
Figure 21 shows a Surface Plasmon Resonance (SPR) -based kinetic analysis of affinity matured anti-FAP Fab fragments. Processing kinetic datasets are presented for clone 19G1 binding to human (hu) FAP (a) and murine (mu) FAP (B), for clone 20G8 binding to hu FAP (C), mu FAP (D) and for clone 4B9 binding to hu FAP (E) and mu FAP (F). The smooth line represents the global fit of the data to the 1: 1 interaction model.
Figure 22 shows SPR-based kinetic analysis of affinity matured anti-FAP Fab fragments. The processing kinetic dataset was presented for clone 5B8 binding to hu FAP (a) and mu FAP (B), for clone 5F1 binding to hu FAP (C), mu FAP (D) and for clone 14B3 binding to hu FAP (E) and mu FAP (F). The smooth line represents the global fit of the data to the 1: 1 interaction model.
Figure 23 shows SPR-based kinetic analysis of affinity matured anti-FAP Fab fragments. The processing kinetic dataset was presented for clone 16F1 binding to hu FAP (a) and mu FAP (B), for clone 16F8 binding to hu FAP (C), mu FAP (D) and for clone O3C9 binding to hu FAP (E) and mu FAP (F). The smooth line represents the global fit of the data to the 1: 1 interaction model.
Figure 24 shows SPR-based kinetic analysis of affinity matured anti-FAP Fab fragments. Processed kinetic datasets are presented for clone O2D7 binding to hu FAP (a) and mu FAP (B), for clone 28H1 binding to hu FAP (C), mu FAP (D), cyno FAP (E), and for clone 22A3 binding to hu FAP (F), mu FAP (G), and cynomolgus monkey (cyno) FAP (H). The smooth line represents the global fit of the data to the 1: 1 interaction model.
Figure 25 shows SPR-based kinetic analysis of affinity matured anti-FAP Fab fragments. Processed kinetic data sets were presented for clone 29B11, which binds hu FAP (a), mu FAP (B), cyno FAP (C), and for clone 23C10, which binds FAP (d), mu FAP (E), and cyno FAP (F). The smooth line represents the global fit of the data to the 1: 1 interaction model.
Figure 26 shows SPR-based kinetic analysis of affinity matured anti-TNC A2Fab fragments that bind to human (hu) TNC A2. The processing kinetic data sets were presented for clone 2B10_ C3B6(a), clone 2B10_6a12(B), clone 2B10_ C3a6(C), clone 2B10_ O7D8(D), clone 2B10_ O1F7(E), and clone 2B10_6H10 (F). The smooth line represents the global fit of the data to the 1: 1 interaction model.
FIG. 27 gives an overview of the three purification steps performed to purify the 3F 2-based Fab-IL 2-Fab.
FIG. 28 shows the results from purification of a 3F 2-based Fab-IL2-Fab (A and B) and 4G 8-based Fab-IL2-Fab (C and D). (A, C) 4-12% Bis-Tris and 3-8% Tris acetate SDS-PAGE and fractions and final products during the purification protocol. (B, D) analysis after three applied purification steps was performed by size exclusion chromatography.
Figure 29 shows the results from purification of 2B10 Fab-IL2-Fab immunoconjugate. (A) 4-12% Bis-Tris SDS-PAGE and fractions and final products during the purification protocol. B) The analysis after the three applied purification steps was performed by size exclusion chromatography.
FIG. 30 shows stability assessment of Fab-IL2-Fab based on anti-fibronectin EDB L19. L19Fab-IL2-Fab was formulated at a concentration of 6.3mg/ml in 20mM histidine hydrochloride, 140mM NaCl, pH6.0 and stored at room temperature and 4 ℃ for 4 weeks. Samples were analyzed weekly for (a) concentration by UV spectroscopy (after centrifugation to precipitate potential precipitate material) and by analysis for (B) aggregate content by size exclusion chromatography.
Figure 31 shows SPR-based kinetic analysis of FAP-targeting 3F2 Fab-IL2-Fab immunoconjugates against human, murine and cynomolgus (cyno) FAP and human IL-2 receptor- β/γ (IL2R bg), as determined by surface plasmon resonance. The smooth line represents the global fit of the data to the 1: 1 interaction model.
Figure 32 shows SPR-based kinetic analysis of FAP-targeting 4G8Fab-IL2-Fab immunoconjugates for human, murine and cynomolgus (cyno) FAP, as determined by surface plasmon resonance. The smooth line represents the global fit of the data to the 1: 1 interaction model.
Figure 33 shows SPR-based kinetic analysis of FAP-targeting 4G8Fab-IL2-Fab constructs directed against human and murine IL-2 receptor β/γ and α chains, as determined by surface plasmon resonance. The smooth line represents the global fit of the data to the two-state reaction model.
Figure 34 shows SPR-based kinetic analysis of FAP targeting 3D9 Fab-IL2-Fab constructs against human, murine and cynomolgus (cyno) FAP and human IL-2 receptor- β/γ (IL2R bg), as determined by surface plasmon resonance. The smooth line represents the global fit of the data to the 1: 1 interaction model.
Figure 35 shows SPR-based kinetic analysis of TNC a 2-targeting 2B10Fab-IL2-Fab constructs against human, murine and cynomolgus (cyno) chimeric TNC a2 fusion proteins and human IL-2 receptor- β/γ (IL2R bg), as determined by surface plasmon resonance. The smooth line represents the global fit of the data to the 1: 1 interaction model.
FIG. 36 shows the efficacy of targeted IL-2Fab-IL2-Fab immunoconjugates recognizing TNC A2(2B10) or FAP (3F2 and 4G8) in inducing NK92 cell proliferation compared to IL-2(Proleukin) and the L19 diabody recognizing fibronectin-EDB. The x-axis is normalized with respect to the number of IL-2 molecules, since the diabody has two IL-2 effector modules, whereas the Fab-IL2-Fab construct contains only one IL-2 effector module. Cell proliferation was measured after 2 days of incubation using the CellTiter Glo system.
FIG. 37 shows STAT5 phosphorylation induction due to IL-2 mediated IL-2 receptor signaling following FAP-targeted 4G8-based IL-2Fab-IL2-Fab immunoconjugates (FAP-targeted 4G8-based IL-2Fab-IL2-Fab immunoconjugates) in solution with FAP-targeted 4G8-based IL-2Fab-IL2-Fab immunoconjugates that recognize FAP on different effector cell populations from human PBMC, including (A) CD56 PBMC+NK cells, (B) CD4+CD25-CD127+Helper T cells, (C) CD3+，CD8+Cytotoxic T cells and (D) CD4+CD25+FOXP3+Regulatory T cells (Tregs).
Figure 38 shows the efficacy of targeting IL-2Fab-IL2-Fab immunoconjugates recognizing TNC a1(2F11), TNC a2(2B10) or FAP (3F2, 4B3 and 3D9) in inducing IFN- γ release and NK92 cell proliferation compared to IL-2 when the immunoconjugate is present in solution or immobilized via FAP or TNC a2 coated on microtiter plates.
FIG. 39 presents the results of survival experiments with two different IL-2 immunoconjugate molecular forms specific for tumor stroma. The human colon tumor cell line LS174T was injected intrasplenically into SCID mice. The TNCA 2-targeted 2B10 Fab-IL2-Fab immunoconjugate was labeled "SH 2B 10", the unconjugated IL-2 control was labeled "Proleukin", and the fibronectin EDB-targeted diabody-IL-2 molecule was labeled "diabody". The amount of immunoconjugate injected per mouse (in μ g) is indicated in the figure and reflects the same number of immunoconjugate molecules.
FIG. 40 presents the results of survival experiments with two different IL-2 immunoconjugate molecular forms specific for tumor stroma. The human renal cell line ACHN was injected intrarenally into SCID mice. The FAP-targeting 3F2 or 4G8Fab-IL2-Fab immunoconjugates are labeled "FAP-3F 2" and "FAP-4G 8", the unconjugated IL-2 control label is "Proleukin", and the fibronectin EDB-targeting diabody-IL-2 molecule is labeled "diabody". The amount of immunoconjugate injected per mouse (in μ g) is indicated in the figure and reflects the same number of immunoconjugate molecules.
FIG. 41 presents the results of survival experiments with two different IL-2 immunoconjugate molecular forms specific for tumor stroma. The human NSCLC cell line aj549i.v was injected into SCID mice. The TNC A2 targeting 2B10 Fab-IL2-Fab immunoconjugate was labeled "2B 10", and the fibronectin EDB targeting diabody-IL-2 molecule was labeled "diabody". The amount of immunoconjugate injected per mouse (in μ g) is indicated in the figure and reflects the same number of immunoconjugate molecules.
Figure 42 presents (a) an overview of the purification protocol for Fab-GM-CSF-Fab immunoconjugates with L19 (fibronectin extracellular domain-B binding agent) as Fab, and (B) SDS-PAGE (reducing, non-reducing) of the purified Fab-GM-CSF-Fab immunoconjugates.
FIG. 43 presents the results of a GM-CSF dependent proliferation assay comparing the effect of GM-CSF on TF-T cells with purified Fab-GM-CSF-Fab immunoconjugates having L19 (a fibronectin extracellular domain-B binding agent) as the Fab.
Figure 44 presents (a) an overview of the purification protocol for Fab-IL12-Fab immunoconjugates with 4G8(FAP binder) as Fab, and (B) SDS-PAGE (reducing, non-reducing) of the purified Fab-IL12-Fab immunoconjugates.
Figure 45 presents results of an assay testing IL-12 induced IFN- γ release comparing the effect of IL-12 and purified Fab-IL12-Fab immunoconjugates with 4G8(FAP binding agent) as Fab, using PBMCs isolated from fresh human blood of healthy donors.
Figure 46 presents (a) an overview of the purification protocol for Fab-IFN α 2-Fab immunoconjugates with L19 (fibronectin extracellular domain-B binding agent) as Fab, and (B) SDS-PAGE (reducing, non-reducing) of the purified Fab-IFN α 2-Fab immunoconjugates.
Figure 47 presents the results of assays testing IFN- α -induced inhibition of proliferation of (a) Jurkat T cells and (B) a549 tumor cells comparing the effect of IFN- α (Roferon a, Roche) and purified Fab-IFN α 2-Fab immunoconjugates with L19 (fibronectin extracellular domain-B binding agent) as fabs.
FIG. 48 shows (A) elution profiles from purified MCSP targeting MHLG-based Fab-IL2-Fab and (B) results from analytical characterization of the same Fab-IL2-Fab by SDS-PAGE (NuPAGE Novex Bis-Tris mini-gel, Invitrogen, MOPS running buffer, reducing and non-reducing).
FIG. 49 shows (A) elution profiles from purified MCSP targeting a Fab-IL2-Fab based on MHLG1 and (B) results from analytical characterization of the same Fab-IL2-Fab by SDS-PAGE (NuPAGE Novex Bis-Tris mini-gel, Invitrogen, MOPS running buffer, reducing and non-reducing).
Figure 50 presents the results of an assay using IL-2 starved NK92 cells testing IL-2 induced IFN- γ release comparing the effect of purified Fab-IL2-Fab immunoconjugates with 4G8(FAP binder) as Fab with purified Fab-IL2-Fab immunoconjugates with MHLG KV9(MCSP binder) as Fab.
Figure 51 presents the results of an assay using IL-2 starved NK92 cells testing for IL-2 induced IFN- γ release comparing the effect of purified Fab-IL2-Fab immunoconjugates with 4G8(FAP binder) as Fab with purified Fab-IL12-Fab immunoconjugates with MHLG1 KV9(MCSP binder) as Fab.
Figure 52 shows binding of MCSP targeted MHLG1 KV9 Fab-IL2-Fab immunoconjugates to Colo38 cells as determined by flow cytometry. Secondary antibody alone or cells alone are shown as negative controls.
Figure 53 presents an overview of (a) the purification protocol for 2B10 Fab-IL2-Fab immunoconjugate with 2B10(TNC a2 binding agent) as Fab, and (B) SDS-PAGE (reducing, non-reducing) of the purified 2B10 Fab-IL2-Fab immunoconjugate.
Detailed Description
Definition of
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, nucleic acid chemistry and hybridization described below are those well known and commonly employed in the art. Standard techniques and procedures are generally performed in accordance with conventional methods in the art and various general references (see generally, Sambrook et al Molecular Cloning: Laboratory Manual, 2 nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated herein by reference) provided throughout this document.
As used herein, the term "immunoconjugate" refers to a polypeptide molecule comprising at least one effector moiety and at least one antigen binding moiety. In one embodiment, the immunoconjugate comprises at least one single-chain effector moiety and at least two antigen-binding moieties. The antigen binding molecule can be linked to the effector moiety through a variety of interactions and in a variety of conformations, as described herein.
As used herein, the term "effector moiety" refers to a polypeptide, such as a protein or glycoprotein, that affects cellular activity, e.g., via signal transduction or other cellular pathways. Thus, the effector modules of the present invention may be associated with receptor-mediated signaling that relays signals from outside the cell membrane to modulate responses in cells carrying one or more of the effector module receptors. In one embodiment, the effector moiety may elicit a cytotoxic response in a cell that carries one or more effector moiety receptors. In another embodiment, the effector moiety may elicit a proliferative response in a cell carrying one or more effector moiety receptors. In another embodiment, the effector moiety may initiate differentiation in a cell carrying the receptor of the effector moiety. In another embodiment, the effector moiety may alter the expression (i.e., up-regulate or down-regulate) of an endogenous cellular protein in a cell carrying the receptor of the effector moiety. Non-limiting examples of effector modules include cytokines, growth factors, hormones, enzymes, substrates, and cofactors. The effector moiety may be combined with the antigen binding moiety in a variety of configurations to form an immunoconjugate.
As used herein, the term "cytokine" refers to a molecule that mediates and/or modulates a biological or cellular function or process (e.g., immunity, inflammation, and hematopoiesis). As used herein, the term "cytokine" includes "lymphokines", "chemokines", "monokines" and "interleukins". Examples of useful cytokines include, but are not limited to, GM-CSF, IL-1 α, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IFN- α, IFN- β, IFN- γ, MIP-1 α, MIP-1 β, TGF- β, TNF- α, and TNF- β.
As used herein, the term "single-chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In one embodiment, the effector module is a single-chain effector module. Non-limiting examples of single-chain effector modules include cytokines, growth factors, hormones, enzymes, substrates, and cofactors. Where the effector moiety is a cytokine and the cytokine of interest is normally found in multimers in nature, each subunit of the multimeric cytokine is sequentially encoded by a single chain of the effector moiety. Thus, non-limiting examples of useful single-chain effector moieties include GM-CSF, IL-1 α, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IFN- α, IFN- β, IFN- γ, MIP-1 α, MIP-1 β, TGF- β, TNF- α, and TNF- β.
As used herein, the term "control effector moiety" refers to an unconjugated effector moiety. For example, in comparing an IL-2 immunoconjugate of the invention to a control effector moiety, the control effector moiety is free, unconjugated IL-2. Likewise, for example, in comparing the IL-12 immunoconjugates of the invention to a control effector moiety, the control effector moiety is free, unconjugated IL-12 (e.g., present as a heterodimeric protein, wherein the p40 and p35 subunits share only disulfide bonds)).
As used herein, the term "effector moiety receptor" refers to a polypeptide molecule capable of specifically binding an effector moiety. For example, in the case of IL-2 is an effector moiety, the effector moiety receptor that binds IL-2 (e.g., an immunoconjugate comprising IL-2) is the IL-2 receptor. Similarly, for example in the IL-12 is immune conjugates of effector module in the case of the effector module, the effector module receptor is IL-12 receptor. In the case where an effector moiety specifically binds more than one receptor, all receptors that specifically bind the effector moiety are "effector moiety receptors" for the effector moiety.
As used herein, the term "antigen-binding moiety" refers to a polypeptide molecule that specifically binds an antigenic determinant. In one embodiment, the antigen binding module is capable of directing the entity to which it is attached (e.g., the effector module or the second antigen binding module) to a target site, such as a particular type of tumor cell or tumor stroma that carries an antigenic determinant. Antigen binding moieties include antibodies and fragments thereof, as further defined herein. By "specific binding" is meant that binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. In one embodiment, the immunoconjugate comprises at least one, typically two or more antigen binding moieties with a constant region, as further defined herein and known in the art. Useful heavy chain constant regions include any of 5 isoforms: α, γ, or μ. Useful light chain constant regions include any of the 2 isoforms: κ and λ.
As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule to which an antigen-binding moiety binds to form an antigen-binding moiety-antigen complex (e.g., a continuous segment of amino acids or a conformational structure composed of different regions of discrete amino acids).
As used herein, the term "control antigen binding moiety" refers to an antigen binding moiety that is free of other antigen binding moieties and effector moieties when it would be present. For example, in comparing the Fab-IL2-Fab immunoconjugate of the invention to a control antigen binding moiety, the control antigen binding moiety is a free Fab, wherein both the Fab-IL2-Fab immunoconjugate and the free Fab molecule specifically bind to the same antigenic determinant.
As used herein, the terms "first" and "second" in the context of antigen binding modules, effector modules, and the like are used to facilitate distinction when more than one module of each type is present. The use of these terms is not intended to confer a particular order or orientation to the immunoconjugate unless specifically so stated.
Where a term used and/or accepted in the art has two or more definitions, as used herein, the definition of the term is intended to include all such meanings unless expressly stated to the contrary. One specific example is the use of the term "complementarity determining regions" ("CDRs") to describe non-contiguous antigen binding sites found within the variable regions of both heavy and light polypeptides. This particular region has been identified by Kabat et al, U.S. Dept. of Health and Human Services, "Sequences of Proteins of immunological interest" (1983) and by Chothia et al, J.mol.biol.196: 901-917(1987) (which are incorporated herein by reference), where this definition includes overlapping or subsets of amino acid residues when compared to each other. However, any definition refers to the application of the CDRs of an antibody or variant thereof is intended to be within the scope of that term, as defined and used herein. Suitable amino acid residues encompassing the CDRs as defined by each of the references cited above are listed below in table I for comparison. The precise number of residues covering a particular CDR will vary depending on the sequence and size of the CDR. In view of the variable region amino acid sequence of an antibody, one skilled in the art can routinely determine which residues make up a particular CDR.
Table 1: CDR definition1
CDR Kabat Chothia AbM2
VH CDR1 31-35 26-32 26-35
VH CDR2 50-65 52-58 50-58
VH CDR3 95-102 95-102 95-102
VL CDR1 24-34 26-32 24-34
VL CDR2 50-56 50-52 50-56
VL CDR3 89-97 91-96 89-97
1The numbering of all CDR definitions in Table 1 follows the numbering convention set forth by Kabat et al (see below).
2"AbM" with the lower case letter "b" as used in table 1 refers to the CDRs as defined by the "AbM" antibody modeling software of Oxford Molecular.
Kabat et al also define a numbering system that can be applied to the variable domain sequences of any antibody. One of ordinary skill in the art can explicitly include this system of "Kabat numbering" into any variable domain sequence without relying on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al, U.S. Dept. of Health and Human Services, "Sequence of proteins of Immunological Interest" (1983). Unless otherwise specified, reference to the numbering of a particular amino acid residue position in an antigen binding module of the invention is according to the Kabat numbering system. The polypeptide sequences of the sequence Listing (i.e., SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 96, 97, etc.) are not numbered according to the Kabat numbering system. However, it is well within the ordinary skill of those in the art to convert the sequence numbering of the sequence listing to Kabat numbering.
Immunoconjugates
An immunoconjugate is a polypeptide molecule comprising at least one effector moiety and at least one antigen binding moiety. In one embodiment, the effector module is a single-chain effector module. In one embodiment, the immunoconjugate comprises at least two antigen binding moieties. The antigen binding modules and effector modules of the invention include those described in detail above and below and in the accompanying drawings. The antigen binding moiety of the immunoconjugate may be directed against a variety of target molecules (e.g., antigenic determinants on protein molecules expressed on tumor cells or tumor stroma). Non-limiting examples of antigen binding moieties are described herein. In one embodiment, at least one antigen binding moiety is directed against an antigenic determinant of one or more polypeptides represented in table 5 below. In particular, in contrast to known immunoconjugates of different structure targeting the same antigenic determinant and carrying the same effector moiety, the immunoconjugates of the invention typically exhibit one or more of the following properties: high specificity of action, reduced toxicity and/or improved stability.
In one embodiment, the immunoconjugate comprises at least a first effector moiety and at least first and second antigen binding moieties. In a preferred embodiment, the first effector moiety is a single-chain effector moiety. In a preferred embodiment, the first and second antigen binding moieties are uniquely selected from the group consisting of: fv and Fab. In a particular embodiment, the first effector moiety shares an amino-or carboxy-terminal peptide bond with the first antigen-binding moiety, and the second antigen-binding moiety shares an amino-or carboxy-terminal peptide bond with either i) the first effector moiety or ii) the first antigen-binding moiety. In another embodiment, the immunoconjugate consists essentially of a first single-chain effector moiety and first and second antigen-binding moieties.
In one embodiment, the first effector moiety shares a carboxy-terminal peptide bond with the first antigen-binding moiety and further shares an amino-terminal peptide bond with the second antigen-binding moiety. In another embodiment, the first antigen binding moiety shares a carboxy-terminal peptide bond with the first effector moiety, preferably a single-chain effector moiety, and further shares an amino-terminal peptide bond with the second antigen binding moiety. In another embodiment, the first antigen binding moiety shares an amino-terminal peptide bond with the first effector moiety, preferably a single-chain effector moiety, and further shares a carboxy-terminal peptide with the second antigen binding moiety.
In one embodiment, the effector moiety, preferably the single-chain effector moiety, shares a carboxy-terminal peptide bond with the first heavy chain variable region and further shares an amino-terminal peptide bond with the second heavy chain variable region. In another embodiment, the effector moiety, preferably the single chain effector moiety, shares a carboxy-terminal peptide bond with the first light chain variable region and further shares an amino-terminal peptide with the second light chain variable region. In another embodiment, the first heavy or light chain variable region is linked to the first effector moiety, preferably a single chain effector moiety, by a carboxy-terminal peptide bond and is further linked to the second heavy or light chain variable region by an amino-terminal peptide bond. In another embodiment, the first heavy or light chain variable region is linked to the first effector moiety, preferably a single chain effector moiety, by an amino-terminal peptide bond and further linked to the second heavy or light chain variable region by a carboxy-terminal peptide bond.
In one embodiment, the effector moiety, preferably the single chain effector moiety, shares a carboxy-terminal peptide bond with the first Fab heavy or light chain and further shares an amino-terminal peptide bond with the second Fab heavy or light chain. In another embodiment, the first Fab heavy or light chain shares a carboxy-terminal peptide bond with the first single chain effector moiety and further shares an amino-terminal peptide bond with the second Fab heavy or light chain. In other embodiments, the first Fab heavy or light chain shares an amino-terminal peptide bond with the first single chain effector moiety and further shares a carboxy-terminal peptide bond with the second Fab heavy or light chain.
In one embodiment, the immunoconjugate comprises at least a first effector moiety sharing an amino-terminal peptide bond with one or more scFv molecules, and wherein the first effector moiety further shares a carboxy-terminal peptide bond with the one or more scFv molecules. In a preferred embodiment, the effector moiety is a single-chain effector moiety.
In another embodiment, the immunoconjugate comprises at least a first effector moiety, preferably a single chain effector moiety, and a first and a second antigen binding moiety, wherein each antigen binding moiety comprises an scFv molecule linked at its carboxy-terminal amino acid to a constant region comprising an immunoglobulin constant domain, and wherein the first antigen binding moiety is linked at its carboxy-terminal amino acid to the amino-terminal amino acid of the first effector moiety, and wherein the first and second antigen binding moieties are covalently linked via at least one disulfide bond. In a preferred embodiment, the constant regions are independently selected from the group consisting of: IgG CH1, IgG CH2, IgG CH3, IgG ck, IgG C λ, and IgE CH4 domains. In one embodiment, the immunoglobulin domain of the first antigen binding moiety is covalently linked to the immunoglobulin domain of the second antigen binding moiety via a disulfide bond. In one embodiment, the at least one disulfide bond is at the carboxy terminus of the immunoglobulin domains of the first and second antigen binding moieties. In another embodiment, at least one disulfide bond is located amino-terminal to the immunoglobulin domains of the first and second antigen binding moieties. In another embodiment, at least two disulfide bonds are located amino-terminal to the immunoglobulin domains of the first and second antigen binding moieties.
In a specific embodiment, the immunoconjugate comprises a first and a second antigen binding moiety, each of which comprises an scFv molecule linked at its carboxy-terminal amino acid to a constant region comprising an IgG CH1 domain, wherein the first antigen binding moiety is linked at its carboxy-terminal amino acid to the amino-terminal amino acid of a first effector moiety, preferably a single-chain effector moiety, and wherein the first and second antigen binding moieties are covalently linked via at least one disulfide bond. The second antigen binding moiety of the immunoconjugate may be further linked at its carboxy-terminal amino acid to the amino-terminal amino acid of the second effector moiety. In one embodiment, the second effector module is a single-chain effector module.
In a specific embodiment, the immunoconjugate comprises a first and a second antigen binding moiety, each comprising an scFv molecule linked at its carboxy-terminal amino acid to a constant region comprising an IgG ck domain, wherein the first antigen binding moiety is linked at its carboxy-terminal amino acid to the amino-terminal amino acid of a first effector moiety, preferably a single chain effector moiety, and wherein the first and second antigen binding moieties are covalently linked via at least one disulfide bond. The second antigen binding moiety of the immunoconjugate may be further linked at its carboxy-terminal amino acid to the amino-terminal amino acid of the second effector moiety. In one embodiment, the second effector module is a single-chain effector module.
In another specific embodiment, the immunoconjugate comprises a first and a second antigen binding moiety, each of which comprises an scFv molecule linked at its carboxy-terminal amino acid to a constant region comprising an IgE CH4 domain, wherein the first antigen binding moiety is linked at its carboxy-terminal amino acid to the amino-terminal amino acid of a first effector moiety, preferably a single chain effector moiety, and wherein the first and second antigen binding moieties are covalently linked via at least one disulfide bond. The second antigen binding moiety of the immunoconjugate may be further linked at its carboxy-terminal amino acid to the amino-terminal amino acid of the second effector moiety. In one embodiment, the second effector module is a single-chain effector module.
In another specific embodiment, the immunoconjugate comprises a first and a second antigen binding moiety, each comprising an scFv molecule joined at its carboxy-terminal amino acid to the IgE CH3 domain, wherein the first antigen binding moiety is joined at its carboxy-terminal amino acid to the amino-terminal amino acid of a first effector moiety, preferably a single chain effector moiety, and wherein the first and second antigen binding moieties are covalently linked via at least one disulfide bond. The second antigen binding moiety of the immunoconjugate may be further linked at its carboxy-terminal amino acid to the amino-terminal amino acid of the second effector moiety. In one embodiment, the second effector module is a single-chain effector module.
In another embodiment, the immunoconjugate comprises a first and a second effector moiety, and a first and a second antigen binding moiety, wherein each antigen binding moiety comprises a Fab molecule linked at its heavy or light chain carboxy-terminal amino acid to an IgG1CH3 domain, and wherein each IgG1CH3 domain is linked at its respective carboxy-terminal amino acid to an amino-terminal amino acid of one of the effector moieties, and wherein the first and second antigen binding moieties are covalently linked via at least one disulfide bond. In a preferred embodiment, the first and/or second effector modules are single-chain effector modules. In yet another embodiment, the IgG1CH3 domains of the antigen binding moiety may be linked by disulfide bonds. In another embodiment, at least one disulfide bond is located at the carboxy terminus of the IgG1CH3 domain of the first and second antigen binding moieties. In another embodiment, at least one disulfide bond is located amino-terminal to the IgG1CH3 domain of the first and second antigen binding moieties. In another embodiment, at least two disulfide bonds are located amino-terminal to the IgG1CH3 domains of the first and second antigen binding moieties.
In another embodiment, the immunoconjugate comprises one or more proteolytic cleavage sites located between the effector moiety and the antigen binding moiety.
The components of the immunoconjugate (e.g., antigen binding moiety and/or effector moiety) may be linked directly or via various linkers (e.g., peptide linkers comprising one or more amino acids, typically about 2-10 amino acids) described herein or known in the art.
In a specific embodiment, the immunoconjugate has improved stability in solution, in particular compared to known immunoconjugate preparations. In one embodiment, the immunoconjugate has a dissociation constant (K) at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 fold lower than the dissociation constant of the control antigen binding moietyD) Binding to an antigenic determinant. In a more specific embodiment, the immunoconjugate binds to the K of the moiety in contrast to the control antigenDK about 10 times lowerDBinding to an antigenic determinant. In one embodiment, the immunoconjugate has a K of less than about 10nM, less than about 1nM, or less than about 0.1nMDBinding to an antigenic determinant.
In another embodiment, the immunoconjugate has superior safety compared to known immunoconjugate preparations. Preferably, the immunoconjugate causes fewer and less severe side effects, such as toxicity, destruction of non-tumor cells, and the like. The reduction in side effects can be attributed to the reduced binding affinity of the immunoconjugates of the invention for the effector moiety receptors. In one embodiment, the immunoconjugate is conjugated to control the K of the effector moiety DA K at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 times greaterDBinding to the effector module receptor. In a more specific embodiment, the immunoconjugate is conjugated to control the K of the effector moietyDK about 2 times greaterDBinding to the effector module receptor. In another embodiment, the immunoconjugate is conjugated to a control effector moiety with a KDK about 10 times greaterDBinding to the effector module receptor. In another embodiment, the immunoconjugate is conjugated to a K that is greater than the K of the corresponding effector moiety in a "diabody" immunoconjugate moleculeDK at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greaterDBinding to the effector module receptor. In another embodiment, the immunoconjugate is in the form of a "diabody" immunoconjugate moleculeDissociation constant K of the corresponding effector module is about 10 times greaterDBinding to the effector module receptor.
In another embodiment, the immunoconjugate has superior efficacy, particularly compared to known immunoconjugate preparations. In one embodiment, the immunoconjugate is better able to inhibit tumor volume increase in vivo and/or is better able to prolong survival in a mammal with a malignancy. In one embodiment, by the end of an administration period of about at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, the immunoconjugate inhibits an increase in tumor volume in vivo by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In one embodiment, the immunoconjugate inhibits tumor volume increase in vivo by at least 50%, 55%, 60%, 65%, 70%, or 75% by the end of the 13 day administration period. In another embodiment, the immunoconjugate, when administered to a mammal in need thereof, extends survival of a mammal having a malignancy by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% relative to a control effector moiety. In another embodiment, the immunoconjugate, when administered to a mammal in need thereof, extends survival of the mammal having the malignancy by at least 30%, 32%, or 35% relative to a control effector moiety. In another embodiment, the immunoconjugate, when administered to a mammal in need thereof, extends survival of the mammal having the malignancy by at least about 30% relative to a control effector moiety. In another embodiment, the immunoconjugate, when administered to a mammal in need thereof, extends survival of a mammal having a malignancy by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% relative to the effector moiety in the "diabody" immunoconjugate molecule. In another embodiment, the immunoconjugate, when administered to a mammal in need thereof, extends survival of the mammal having a malignancy by at least 30%, 32%, or 35% relative to the effector moiety in the "diabody" immunoconjugate molecule. In another embodiment, the immunoconjugate, when administered to a mammal in need thereof, extends survival of a mammal having a malignancy by about 30% relative to the effector moiety in a "diabody" immunoconjugate molecule. In another embodiment, the immunoconjugate, when administered to a mammal in need thereof, extends survival of a mammal having a malignancy by at least 5%, 10% or 15% relative to an effector moiety in a control effector template or "diabody" immunoconjugate molecule.
Antigen binding modules
The antigen binding moiety of the immunoconjugates of the invention is generally a specific type of polypeptide molecule that binds to a specific epitope and is capable of directing the entity to which it is attached (e.g., an effector template or a second antigen binding moiety) to a target site, such as a tumor cell or tumor stroma that carries the epitope. The immunoconjugate may bind to an antigenic determinant found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, free in blood serum, and/or in the extracellular matrix (ECM).
Non-limiting examples of tumor antigens include MAGE, MART-1/Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin b, colorectal-associated antigen (CRC) -C017-1A/GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2 and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor/CD 3-zeta chain, MAGE-tumor antigen family (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A8, MAGE-A9, MAGE-A3626, MAGE-A3683, and MAG, MAGE-A12, MAGE-Xp2(MAGE-B2), MAGE-Xp3(MAGE-B3), MAGE-Xp4(MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-tumor antigen family (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, NYT 4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, alpha-, beta-and gamma-catenin, prct 120, gp-protein, Pmel-7, Pmel-27, Pmel-protein, The cell lining proteins, connexin 37, Ig-idiotypes, P15, gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus proteins, the Smad tumor antigen family, lmp-1, P1A, EBV encoded nuclear antigen (EBNA) -1, brain glycogen phosphorylase, SSX-1, SSX-2(HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7 and c-erbB-2.
Non-limiting examples of viral antigens include influenza hemagglutinin, Epstein Barr virus LMP-1, hepatitis C virus E2 glycoprotein, HIV gp160, and HIV gp 120.
Non-limiting examples of ECM antigens include cohesin glycan, heparanase, integrin, osteopontin, Link, cadherin, laminin EGF type, lectin, fibronectin, Notch, tenascin, and matriptase.
The immunoconjugates of the invention can bind to the following specific non-limiting examples of cell surface antigens: FAP, Her2, EGFR, CD2(T cell surface antigen), CD3 (TCR-binding heteromultimer), CD22(B cell receptor), CD23 (low affinity IgE receptor), CD25(IL-2 receptor alpha chain), CD30 (cytokine receptor), CD33 (myeloid cell surface antigen), CD40 (tumor necrosis factor receptor), IL-6R (IL6 receptor), CD20, MCSP and PDGF beta R (beta platelet-derived growth factor receptor).
In one embodiment, the immunoconjugates of the invention comprise two or more antigen binding moieties, wherein each of these antigen binding moieties specifically binds to the same antigenic determinant. In another embodiment, the immunoconjugates of the invention comprise two or more antigen binding moieties, wherein each of these antigen binding moieties specifically binds to a different antigenic determinant.
The antigen binding moiety may be any type of antibody or fragment thereof that retains specific binding to an antigenic determinant. Antibody fragments include, but are not limited to, VHFragment, VLFragments, Fab fragments, F (ab')2Fragments, scFv fragments, Fv fragments, minibodies, diabodies, triabodies and tetrabodies (see, for example, Hudson and Souriau, Nature Med.9: 129-.
In one embodiment, the immunoconjugate comprises at least one, typically two or more antigen binding moieties specific for the extra domain b (edb) of fibronectin. In another embodiment, the immunoconjugate comprises at least one, typically two or more antigen binding moieties that can compete with monoclonal antibody L19 for binding to an EDB epitope. See, for example, PCT publication WO2007/128563A1 (which is incorporated herein by reference in its entirety). In yet another embodiment, the immunoconjugate comprises a polypeptide sequence in which a first Fab heavy chain derived from the L19 monoclonal antibody shares a carboxy-terminal peptide bond with an IL-2 molecule, which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain derived from the L19 monoclonal antibody. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence in which a first Fab heavy chain derived from the L19 monoclonal antibody shares a carboxy-terminal peptide bond with an IL-12 molecule, which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain derived from the L19 monoclonal antibody. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence in which a first Fab heavy chain derived from the L19 monoclonal antibody shares a carboxy-terminal peptide bond with an IFN α molecule, which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain derived from the L19 monoclonal antibody. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence in which a first Fab heavy chain derived from the L19 monoclonal antibody shares a carboxy-terminal peptide bond with a GM-CSF molecule, which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain derived from the L19 monoclonal antibody. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence in which a first scFv derived from the L19 monoclonal antibody shares a carboxy-terminal peptide bond with an IL-12 molecule, which in turn shares a carboxy-terminal peptide bond with a second scFv derived from the L19 monoclonal antibody. In a more specific embodiment, the immunoconjugate comprises the polypeptide sequence of SEQ ID NO: 95 or a variant thereof which retains functionality. In another embodiment, the immunoconjugate comprises a Fab light chain derived from the L19 monoclonal antibody. In a more specific embodiment, the immunoconjugate comprises a sequence identical to SEQ ID NO: 96, or a variant thereof that retains functionality, is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In yet another embodiment, the immunoconjugate comprises a sequence identical to SEQ ID NO: 95 and SEQ ID NO: 96 at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical two polypeptide sequences or functional retaining variants thereof. In a more specific embodiment, the immunoconjugate comprises a sequence identical to SEQ ID NO: 104 or a variant thereof that retains functionality, or a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In yet another embodiment, the immunoconjugate comprises a sequence identical to SEQ ID NO: 104 and SEQ ID NO: 96 at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical two polypeptide sequences or functional retaining variants thereof. In a more specific embodiment, the immunoconjugate comprises a sequence identical to SEQ ID NO: 105, or a variant thereof that retains functionality, is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In yet another embodiment, the immunoconjugate comprises a sequence identical to SEQ ID NO: 105 and SEQ ID NO: 96 at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical two polypeptide sequences or functional retaining variants thereof. In a more specific embodiment, the immunoconjugate comprises a sequence identical to SEQ ID NO: 106, or a variant thereof that retains functionality, is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In yet another embodiment, the immunoconjugate comprises a sequence identical to SEQ id no: 106 and SEQ ID NO: 96 at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical two polypeptide sequences or functional retaining variants thereof. In a more specific embodiment, the immunoconjugate comprises a sequence identical to SEQ ID NO: 107 or a variant thereof that retains functionality, or a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In yet another embodiment, the immunoconjugate comprises a sequence identical to SEQ ID NO: 107 and SEQ ID NO: 96 at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical two polypeptide sequences or functional retaining variants thereof. In another specific embodiment, the polypeptides are covalently linked, for example by disulfide bonds.
In one embodiment, the immunoconjugate of the invention comprises at least one, typically two or more antigen binding moieties which are specific for the a1 domain of tenascin (TNC-a 1). In another embodiment, the immunoconjugate comprises at least one, typically two or more antigen binding moieties that can compete with monoclonal antibody F16 for binding to the TNC-a1 epitope. See, for example, PCT publication WO 2007/128563a1 (which is incorporated herein by reference in its entirety). In one embodiment, the immunoconjugate comprises at least one, typically two or more antigen binding moieties, which is specific for the a1 and/or a4 domain of tenascin (TNC-a1 or TNC-a4 or TNC-a1/a 4). In another embodiment, the immunoconjugate comprises a polypeptide sequence in which a first Fab heavy chain specific for the a1 domain of tenascin shares a carboxy-terminal peptide bond with an IL-2 molecule, IL-12 molecule, IFN α molecule, or GM-CSF molecule, which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain specific for the a1 domain of tenascin. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence in which a first Fab heavy chain specific for the a1 domain of tenascin shares a carboxy-terminal peptide bond with an IL-2 molecule which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain specific for the a1 domain of tenascin. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence in which a first scFv specific for the a1 domain of tenascin shares a carboxy-terminal peptide bond with an IL-2 molecule, which in turn shares a carboxy-terminal peptide bond with a second scFv specific for the a1 domain of tenascin. In a specific embodiment, the antigen binding moiety of the immunoconjugate comprises a sequence that is identical to SEQ ID NO: 13 or SEQ ID NO: 15, or a variant thereof that retains functionality, is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain variable region sequence. In another specific embodiment, the antigen binding moiety of the immunoconjugate comprises a sequence that is identical to SEQ ID NO: 9 or SEQ ID NO: 11, or a variant thereof that retains functionality, is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In a more specific embodiment, the antigen binding moiety of the immunoconjugate comprises a sequence that is identical to SEQ ID NO: 13 or SEQ ID NO: 15, or a variant thereof that retains functionality, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain variable region sequence of SEQ ID NO: 9 or SEQ ID NO: 11, or a variant thereof that retains functionality, is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In another specific embodiment, the heavy chain variable region sequence of the antigen binding moiety of the immunoconjugate consists of a sequence identical to SEQ ID NO: 14 or SEQ id no: 16, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence. In yet another specific embodiment, the heavy chain variable region sequence of the antigen binding moiety of the immunoconjugate is encoded by the polynucleotide sequence of SEQ ID NO: 14 or SEQ ID NO: and 16, coding. In another specific embodiment, the light chain variable region sequence of the antigen binding moiety of the immunoconjugate consists of a sequence identical to SEQ ID NO: 10 or SEQ ID NO: 12, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the other. In yet another specific embodiment, the light chain variable region sequence of the antigen binding moiety of the immunoconjugate is encoded by the polynucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: and 12, coding. In a specific embodiment, the immunoconjugate comprises a peptide that is identical to SEQ id no: 99 or a variant thereof that retains functionality, or a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In another specific embodiment, the immunoconjugate of the invention comprises a sequence identical to SEQ ID NO: 100 or SEQ ID NO: 215, or a variant thereof that retains functionality, is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In yet another specific embodiment, the immunoconjugate of the invention comprises a peptide that is identical to SEQ id no: 101 or SEQ ID NO: 235 or a variant thereof that retains functionality, is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In a more specific embodiment, the immunoconjugate of the invention comprises a sequence identical to SEQ ID NO: 100 and SEQ ID NO: 101 at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or a functionally retained variant thereof. In another specific embodiment, the immunoconjugate of the invention comprises a sequence identical to SEQ ID NO: 215 and SEQ ID NO: 235 or a variant thereof that retains functionality, which is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In a specific embodiment, the immunoconjugate comprises a polypeptide consisting of a sequence identical to SEQ ID NO: 112, or a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In another specific embodiment, the immunoconjugate comprises a polypeptide consisting of the polynucleotide sequence of seq id NO: 112, or a pharmaceutically acceptable salt thereof. In another specific embodiment, the immunoconjugate comprises a polypeptide consisting of a sequence identical to SEQ ID NO: 113 or SEQ ID NO: 216, or a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In yet another specific embodiment, the immunoconjugate comprises a polypeptide consisting of the polynucleotide sequence of SEQ ID NO: 113 or SEQ ID NO: 216, or a pharmaceutically acceptable salt thereof. In another specific embodiment, the immunoconjugate comprises a polypeptide consisting of a sequence that binds to seq id NO: 114 or SEQ ID NO: 236, or a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In yet another embodiment, the immunoconjugate comprises a polypeptide consisting of the polynucleotide sequence of SEQ ID NO: 114 or SEQ ID NO: 236.
In one embodiment, the immunoconjugate comprises at least one, typically two or more antigen binding moieties which are specific for the a2 domain of tenascin (TNC-a 2). In another embodiment, the immunoconjugate comprises a polypeptide sequence in which a first Fab heavy chain specific for the a2 domain of tenascin shares a carboxy-terminal peptide bond with an IL-2 molecule, IL-12 molecule, IFN α molecule, or GM-CSF molecule, which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain specific for the a2 domain of tenascin. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence in which a first Fab heavy chain specific for the a2 domain of tenascin shares a carboxy-terminal peptide bond with an IL-2 molecule which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain specific for the a2 domain of tenascin. In a specific embodiment, the antigen-binding moiety of the immunoconjugate comprises a heavy chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of seq id no: SEQ ID NO: 7. SEQ ID NO: 179. SEQ ID NO: 183. SEQ ID NO: 187. SEQ ID NO: 191. SEQ ID NO: 195. SEQ ID NO: 199. SEQ ID NO: 203 and SEQ ID NO: 207, or a variant thereof that retains functionality. In another specific embodiment, the antigen-binding moiety of the immunoconjugate comprises a light chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of seq id no: SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 177. SEQ ID NO: 181. SEQ ID NO: 185. SEQ ID NO: 189. SEQ ID NO: 193. SEQ ID NO: 197. SEQ ID NO: 201 and SEQ ID NO: 205, or a variant thereof that retains functionality. In a more specific embodiment, the antigen binding moiety of the immunoconjugate comprises a heavy chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of seq id no: SEQ ID NO: 7. SEQ ID NO: 179. SEQ ID NO: 183. SEQ ID NO: 187. SEQ ID NO: 191. SEQ ID NO: 195. SEQ ID NO: 199. SEQ ID NO: 203 and SEQ ID NO: 207, or a variant thereof that retains functionality, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of seq id no: SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 177. SEQ ID NO: 181. SEQ ID NO: 185. SEQ ID NO: 189. SEQ ID NO: 193. SEQ ID NO: 197. SEQ ID NO: 201 and SEQ ID NO: 205, or a variant thereof that retains functionality. In another specific embodiment, the heavy chain variable region sequence of the antigen binding moiety of the immunoconjugate is encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of seq id no: SEQ ID NO: 8. SEQ ID NO: 180. SEQ ID NO: 184. SEQ ID NO: 188. SEQ ID NO: 192. SEQ ID NO: 196. SEQ ID NO: 200. SEQ ID NO: 204 and SEQ ID NO: 208. in yet another specific embodiment, the heavy chain variable region sequence of the antigen binding moiety of the immunoconjugate is encoded by a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 8. SEQ ID NO: 180. SEQ ID NO: 184. SEQ ID NO: 188. SEQ ID NO: 192. SEQ ID NO: 196. SEQ ID NO: 200. SEQ ID NO: 204 and SEQ ID NO: 208. in another specific embodiment, the light chain variable region sequence of the antigen binding moiety of the immunoconjugate is encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of seq id no: SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 178. SEQ ID NO: 182. SEQ ID NO: 186. SEQ ID NO: 190. SEQ ID NO: 194. SEQ ID NO: 198. SEQ ID NO: 202 and SEQ ID NO: 206. in yet another specific embodiment, the light chain variable region sequence of the antigen binding moiety of the immunoconjugate is encoded by a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 178. SEQ ID NO: 182. SEQ ID NO: 186. SEQ ID NO: 190. SEQ ID NO: 194. SEQ ID NO: 198. SEQ ID NO: 202 and SEQ ID NO: 206. in a specific embodiment, the immunoconjugate of the invention comprises a residue sequence that hybridizes to a sequence selected from SEQ ID NOs: 239. SEQ ID NO: 241 and SEQ ID NO: 243, or a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or a variant thereof that retains functionality. In another specific embodiment, the immunoconjugate of the invention comprises a residue sequence that hybridizes to a sequence selected from SEQ ID NOs: 245. SEQ ID NO: 247 and SEQ ID NO: 249, or a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or a variant thereof that retains functionality. In a more specific embodiment, the immunoconjugate of the invention comprises a residue sequence that hybridizes to a sequence selected from SEQ ID NOs: 239. SEQ ID NO: 241 and SEQ ID NO: 243, or a variant thereof that retains functionality, and a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 245. SEQ ID NO: 247 and SEQ ID NO: 249, or a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or a variant thereof that retains functionality. In another specific embodiment, the immunoconjugate of the invention comprises a sequence identical to SEQ ID NO: 239 and SEQ ID NO: 247 or SEQ ID NO: 249, or a variant thereof which retains functionality, is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In yet another specific embodiment, the immunoconjugate of the invention comprises a sequence identical to SEQ ID NO: 241 and SEQ ID NO: 245 or SEQ ID NO: 247 at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or a variant thereof that retains functionality. In another specific embodiment, the immunoconjugate of the invention comprises a sequence identical to SEQ ID NO: 243 and SEQ ID NO: 245 at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or a variant thereof that retains functionality. In a specific embodiment, the immunoconjugate comprises a polypeptide sequence consisting of a sequence identical to a sequence selected from SEQ ID NOs: 240. SEQ ID NO: 242 and SEQ ID NO: 244, or a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence selected from the group of seq id no: SEQ ID NO: 240. SEQ ID NO: 242 and SEQ ID NO: 244. in another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of seq id no: SEQ ID NO: 246. SEQ ID NO: 248 and SEQ ID NO: 250. in yet another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence selected from the group of seq id no: SEQ ID NO: 246. SEQ ID NO: 248 and SEQ ID NO: 250.
In one embodiment, the immunoconjugate comprises at least one, typically two or more antigen binding moieties that are specific for Fibroblast Activation Protein (FAP). In another embodiment, the immunoconjugate comprises a polypeptide sequence in which a first Fab heavy chain specific for FAP shares a carboxy-terminal peptide bond with an IL-2 molecule, IL-12 molecule, IFN α molecule, or GM-CSF molecule, which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain specific for FAP. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence in which a first Fab heavy chain specific for FAP shares a carboxy-terminal peptide bond with an IL-2 molecule, which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain specific for FAP. In another embodiment, the immunoconjugate comprises a polypeptide sequence in which a first Fab heavy chain specific for FAP shares a carboxy-terminal peptide bond with an IL-12 molecule, which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain specific for FAP. In a specific embodiment, the antigen-binding moiety of the immunoconjugate comprises a heavy chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of seq id no: SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 131, SEQ ID NO: 135, seq id NO: 139, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 155, SEQ ID NO: 159, SEQ ID NO: 163, SEQ ID NO: 167, SEQ ID NO: 171 and SEQ ID NO: 175, or a variant thereof that retains functionality. In another specific embodiment, the antigen-binding moiety of the immunoconjugate comprises a light chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of seq id no: SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 121, SEQ ID NO: 125, SEQ ID NO: 129, SEQ id no: 133, SEQ ID NO: 137, SEQ ID NO: 141, SEQ ID NO: 145, SEQ ID NO: 149, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 165, SEQ ID NO: 169 and SEQ ID NO: 173, or a variant thereof that retains functionality. In a more specific embodiment, the antigen binding moiety of the immunoconjugate comprises a heavy chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of seq id no: SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 31, seq id NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 139, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 155, SEQ ID NO: 159, SEQ ID NO: 163, SEQ ID NO: 167, SEQ ID NO: 171 and SEQ ID NO: 175, or a variant thereof that retains functionality, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of seq id no: SEQ ID NO: 17, SEQ ID NO: 19, SEQ id no: 23, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ id no: 121, SEQ ID NO: 125, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 137, SEQ ID NO: 141, SEQ ID NO: 145, SEQ ID NO: 149, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 165, SEQ ID NO: 169 and SEQ ID NO: 173, or a variant thereof that retains functionality, in another specific embodiment, the heavy chain variable region sequence of the antigen binding moiety of the immunoconjugate is encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group consisting of seq id no: SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, SEQ id no: 90, SEQ ID NO: 94, SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 144, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 168, SEQ ID NO: 172 and SEQ ID NO: 176. in yet another specific embodiment, the heavy chain variable region sequence of the antigen binding moiety of the immunoconjugate is encoded by a polynucleotide sequence selected from the group of seq id no: SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO: 124, SEQ ID NO: 128, SEQ id no: 132, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 144, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 168, SEQ ID NO: 172 and SEQ ID NO: 176. in another specific embodiment, the light chain variable region sequence of the antigen binding moiety of the immunoconjugate is encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of seq id no: SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO: 122, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 134, SEQ ID NO: 138, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, SEQ ID NO: 154, SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 170 and SEQ ID NO: 174. in yet another specific embodiment, the light chain variable region sequence of the antigen binding moiety of the immunoconjugate is encoded by a polynucleotide sequence selected from the group of seq id no: SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO: 122, SEQ id no: 126, SEQ ID NO: 130, SEQ ID NO: 134, SEQ ID NO: 138, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, SEQ ID NO: 154, SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 170 and SEQ ID NO: 174. in another specific embodiment, the immunoconjugate of the invention comprises a polypeptide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of seq id no: SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225 and SEQ ID NO: 227, or a variant thereof that retains functionality. In yet another specific embodiment, the immunoconjugate of the invention comprises a polypeptide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of seq id no: SEQ ID NO: 229. SEQ ID NO: 231. SEQ ID NO: 233 and SEQ ID NO: 237 or a variant thereof which retains functionality. In a more specific embodiment, the immunoconjugate of the invention comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of seq id no: SEQ ID NO: 211. SEQ ID NO: 219 and SEQ ID NO: 221 or a functional-retaining variant thereof, and a variant of SEQ ID NO: 231 at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical polypeptide sequence or a functionally retained variant thereof. In another specific embodiment, the immunoconjugate of the invention comprises a polypeptide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of seq id no: SEQ ID NO: 209. SEQ ID NO: 223. SEQ ID NO: 225 and SEQ ID NO: 227 or a functional-retaining variant thereof, and a polypeptide that differs from SEQ ID NO: 229, or a variant thereof that retains functionality, is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In a particular embodiment, the immunoconjugate of the invention comprises a sequence identical to SEQ ID NO: 213 and SEQ ID NO: 233 two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate of the invention comprises a sequence identical to SEQ ID NO: 217 and SEQ ID NO: 237 two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate of the invention comprises a sequence identical to SEQ ID NO: 221 and SEQ ID NO: 231 two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate of the invention comprises a sequence identical to SEQ ID NO: 223 and SEQ ID NO: 229 two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate of the invention comprises a sequence identical to SEQ ID NO: 225 and SEQ ID NO: 229 two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate of the invention comprises a sequence identical to SEQ ID NO: 227 and SEQ ID NO: 229 two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or variants thereof that retain functionality. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of seq id no: SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226 and SEQ ID NO: 228. in yet another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence selected from the group of seq id no: SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226 and SEQ ID NO: 228. in another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of seq id no: SEQ ID NO: 230. SEQ ID NO: 232. SEQ ID NO: 234 and SEQ ID NO: 238. in yet another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence selected from the group of seq id no: SEQ ID NO: 230. SEQ ID NO: 232. SEQ ID NO: 234 and SEQ ID NO: 238.
In one embodiment, the immunoconjugate comprises at least one, typically two or more antigen binding moieties that are specific for Melanoma Chondroitin Sulfate Proteoglycan (MCSP). In another embodiment, the immunoconjugate comprises a polypeptide sequence in which a first Fab heavy chain specific for MCSP shares a carboxy-terminal peptide bond with an IL-2 molecule, an IL-12 molecule, an IFN α molecule, or a GM-CSF molecule, which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain specific for MCSP. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence in which a first Fab heavy chain specific for MCSP shares a carboxy-terminal peptide bond with an IL-2 molecule, which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain specific for MCSP. In a specific embodiment, the antigen binding moiety of the immunoconjugate comprises a sequence that is identical to the sequence of SEQ ID NO: 257 or SEQ ID NO: 261, or a variant thereof that retains functionality, is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In another specific embodiment, the antigen binding moiety of the immunoconjugate comprises a sequence that is identical to the sequence of SEQ id no: 259 or SEQ ID NO: 271 light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical, or a variant thereof that retains functionality. In a more specific embodiment, the antigen binding moiety of the immunoconjugate comprises a sequence that is identical to the sequence of SEQ ID NO: 257 or SEQ ID NO: 261, or a variant thereof that retains functionality, and a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 259 or SEQ ID NO: 271 light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical, or a variant thereof that retains functionality. In a more specific embodiment, the antigen binding moiety of the immunoconjugate comprises a sequence that is identical to the sequence of SEQ ID NO: 257, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain variable region sequence of SEQ ID NO: 259 at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical light chain variable region sequence. In another specific embodiment, the antigen binding moiety of the immunoconjugate comprises a sequence that is identical to the sequence of SEQ ID NO: 261, and a heavy chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 259 at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical light chain variable region sequence. In another specific embodiment, the heavy chain variable region sequence of the antigen binding moiety of the immunoconjugate consists of a sequence identical to the sequence of SEQ ID NO: 258 or SEQ ID NO: 262 at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In yet another specific embodiment, the heavy chain variable region sequence of the antigen binding moiety of the immunoconjugate is encoded by the polynucleotide sequence of SEQ ID NO: 258 or SEQ ID NO: 262 encoding. In another specific embodiment, the light chain variable region sequence of the antigen binding moiety of the immunoconjugate consists of a sequence identical to the sequence of SEQ ID NO: 260 or SEQ ID NO: 272 are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In yet another specific embodiment, the light chain variable region sequence of the antigen binding moiety of the immunoconjugate is encoded by the polynucleotide sequence of SEQ ID NO: 260 or SEQ ID NO: 272 are encoded. In a specific embodiment, the immunoconjugate of the invention comprises a peptide that is identical to SEQ id no: 251 or SEQ ID NO: 255, or a variant thereof that retains functionality, is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In another specific embodiment, the immunoconjugate of the invention comprises a sequence identical to SEQ ID NO: 253 or SEQ ID NO: 265, or a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or a variant thereof that retains functionality. In a more specific embodiment, the immunoconjugate of the invention comprises a sequence identical to SEQ ID NO: 251 or SEQ ID NO: 255, or a variant thereof that retains functionality, and a polypeptide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ id no: 253 or SEQ ID NO: 265, or a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or a variant thereof that retains functionality. In another specific embodiment, the immunoconjugate of the invention comprises a sequence identical to SEQ ID NO: 251 or a variant thereof that retains functionality, and a polypeptide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 253, or a variant thereof that retains functionality, is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In another specific embodiment, the immunoconjugate of the invention comprises a sequence identical to SEQ ID NO: 255 or a variant thereof that retains functionality, and a polypeptide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 253, or a variant thereof that retains functionality, is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence consisting of a sequence identical to SEQ ID NO: 252 or SEQ ID NO: 256 at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In yet another specific embodiment, the immunoconjugate comprises a polypeptide consisting of the polynucleotide sequence of SEQ ID NO: 252 or SEQ ID NO: 256, or a pharmaceutically acceptable salt thereof. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence consisting of a sequence identical to SEQ ID NO: 254 or SEQ ID NO: 266, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide sequence encoded by the polynucleotide sequence. In yet another specific embodiment, the immunoconjugate comprises a polypeptide consisting of the polynucleotide sequence of SEQ ID NO: 254 or SEQ ID NO: 266, or a pharmaceutically acceptable salt thereof.
In one embodiment, the antigen binding moiety comprises at least a variable region capable of binding an antigenic determinant. Non-limiting variable regions useful in the present invention may be of murine, primate, or human origin. The human variable region may be derived from a human monoclonal antibody generated by a hybridoma method. Human myeloma and mouse-human heteromyeloma cell lines used to produce human monoclonal antibodies have been described by, for example, Kozbor et al, J immunol.133: 3001-3005(1984) and Brodeur et al, Monoclonal Antibody production techniques and Applications, pp 51-63 (Marcel Dekker, Inc., NewYork, 1987). Human variable regions can also be generated by transgenic animals (e.g., mice) that are capable of generating a repertoire of human antibodies after immunization without endogenous immunoglobulin production. For example, antibody heavy chain joining regions (J) in chimeric and germline mutant mice have been describedH) Homozygous deletion of the gene results in complete inhibition of endogenous antibody production. Transfer of a collection of human germline immunoglobulin genes in such germline mutant mice results in human antibody production following antigen challenge. See, e.g., Jakobovits et al, Nature 362: 255-258(1993).
Alternatively, phage display can be used to generate human antibodies and human variable regions in vitro from, for example, a repertoire of immunoglobulin variable (V) domain genes from non-immunized donors. (McCafferty et al, Nature 348: 552-554 (1990)). In one example of this technique, antibody V domain genes are cloned in-frame into the major or minor coat protein genes of filamentous phage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle.
Because the filamentous particle contains a phage genome of single-stranded DNA copies, selection based on the functional properties of the antibody/antibody fragment also results in selection of genes encoding antibodies/antibody fragments that exhibit those properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats. For a review of phage display formats, see Hoogenboom et al, Nucleic Acids Res.19: 4133-4137(1991). Several sources of V gene segments can be used for phage display. Clackson et al isolated a panel of different antibodies from a small random combinatorial library of V genes derived from the spleen of immunized miceAn oxazolone antibody. See Clackson et al, Nature 352: 624-628(1991). A complete set of V genes from non-immunized human donors can be constructed and antibodies to a panel of different antigens (including self-antigens) can be isolated, essentially following Marks et al, j.mol.bio.222: 581-597 (1991). In the natural immune response, antibody genes accumulate mutations at a high rate (somatic hypermutations). Some of the changes introduced confer higher affinity and B cells displaying high affinity surface immunoglobulins preferentially replicate and differentiate during subsequent antigen challenge. This natural process can be simulated by employing a technique known as "chain shuffling". See Marks et al, Biotech.10: 779-783(1992). In this method, the affinity of a "primary" human antibody or variable region obtained by phage display can be improved by sequential replacement of heavy and light chain V region genes with a full set of naturally occurring V domain gene variants (repertoires) obtained from non-immunized donors. This technique allows the generation of antibodies and variable regions with affinities in the nM range. Strategies for generating very large repertoires of phage antibodies have been described by Waterhouse et al, Nucl. acids Res.21: 2265-2266(1993), and Griffith et al, J.cell.Bio.120: 885-896(1993) have reported the isolation of high affinity human antibodies directly from such large phage libraries. Gene shuffling can also be used to derive human antibodies and variable regions from rodent antibodies, where the human antibodies or variable regions have similar affinity and specificity to the starting rodent antibody or variable region. According to this method, which is also known as "epitope imprinting", heavy or light chain V domain genes of rodent antibodies obtained by phage display technology are replaced with a repertoire of human V domain genes, creating a rodent-human chimera. Selection with antigen results in the isolation of human variable regions that restore a functional antigen binding site, i.e. the selection of epitope-determining (imprints) partners. Upon repeating this process to replace the remaining rodent V domains, human antibodies are obtained (see PCT publication WO 93/06213). Unlike traditional humanization of rodent antibodies, epitopic imprinting techniques provide fully human antibodies or variable regions that are free of framework or CDR residues of rodent origin.
Variable regions that can be used also include murine variable region sequences that have been primatized or humanized or primate variable region sequences that have been humanized. As used herein, the term "humanized" refers to antigen binding module variable region sequences derived from non-human antibodies, e.g., murine antibodies, that retain or substantially retain the antigen binding properties of the parent molecule, but which are less immunogenic in humans. This can be achieved by a variety of methods, including (a) grafting only non-human CDRs onto human framework regions with or without retention of critical framework residues (e.g., those important for retaining good antigen binding affinity or antigen function), and (b) "masking" the non-human variable regions with human-like moieties by replacing surface residues. Such methods are described by Jones et al, Morrison et al, proc.natl.acad.sci., 81: 6851-6855 (1984); morrison and Oi, adv.immunol., 44: 65-92 (1988); verhoeyen et al, Science, 239: 1534 — 1536 (1988); padlan, molec. immun., 28: 489-498 (1991); padlan, molec. immun., 31 (3): 169, 217(1994), all of which are hereby incorporated by reference in their entirety. There are typically 3 complementarity determining regions, or CDRs, (CDR1, CDR2, and CDR3) in each of the heavy and light chain variable regions of an antibody, which are flanked by four framework subregions (i.e., FR1, FR2, FR3, and FR4) in each of the heavy and light chain variable domains of an antibody: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. A discussion of antibodies having humanized variable regions can be found in U.S. Pat. No.6,632,927 and published U.S. application No.2003/0175269, etc., both of which are incorporated herein by reference in their entirety.
Similarly, as used herein, the term "primatized" is used to refer to antigen binding module variable regions derived from a non-primate antibody, e.g., a murine antibody, which retains or substantially retains the antigen binding properties of the parent molecule, but which is less immunogenic in primates.
The choice of human variable domains (both light and heavy) in generating the humanized antigen binding modules is very important for reducing antigenicity. The rodent antigen binding module variable region sequences are screened against an entire library of known human variable region sequences according to the so-called "best-fit" method. The human Framework Regions (FR) that received the rodent-proximal human sequence as the humanized antigen-binding moiety were then received (Sims et al, J.Immunol., 151: 2296 (1993); Chothia et al, J.mol.biol., 196: 901 (1987)). Another method of selecting human framework sequences is to compare the sequences of each individual subregion, or some combination of individual subregions (e.g., FR1 and FR2), of a fully rodent framework (i.e., FR1, FR2, FR3 and FR4) against a library of known human variable region sequences (e.g., as determined by Kabat numbering) corresponding to the framework subregions and select the human sequence of each subregion, or combination, that is closest to the rodent (U.S. patent application publication No.2003/0040606a 1). Another approach uses specific framework regions derived from the consensus sequence of all human antibodies of a specific subset of light or heavy chains. The same framework can be used for several different humanized antigen binding modules (Carter et al, Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al, J.Immunol., 151: 2623 (1993)).
In general, the antigen binding moiety of the immunoconjugates of the invention retain high affinity for a particular antigenic determinant and other favorable biological properties. Thus, humanized variable regions are prepared by analyzing the parent sequence and various conceptual humanized products using three-dimensional models of the parent and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs are available which exemplify and display the possible three-dimensional conformational structures of selected candidate immunoglobulin variable region sequences. Studies on these displays allow analysis of the likely role of residues in the functioning of candidate immunoglobulin variable region sequences, i.e., the analysis of residues that affect the ability of a candidate variable region sequence to bind its antigen. In this way, FR residues can be selected from the recipient and import sequences and combined to achieve a desired antigen binding module characteristic, such as increased affinity for the target antigen. In general, hypervariable region residues are directly and most substantially involved in the effect on antigen binding.
In another embodiment, the antigen binding molecules of the invention are engineered to have enhanced binding affinity according to methods disclosed, for example, in U.S. patent application publication No.2004/0132066, the entire contents of which are incorporated herein by reference. The ability of the immunoconjugates of the invention to bind to an effector module receptor or a specific epitope can be measured via enzyme-linked immunosorbent assays (ELISAs) or other techniques familiar to those skilled in the art, such as surface plasmon resonance techniques (analyzed on the BIACORE T100 system) (Liljeblad et al, Glyco.J.17: 323-containing 329(2000)), and conventional binding assays (Heeley, R.P., Endocr.Res.28: 217-containing 229 (2002)).
Effector module
Generally, the effector moiety used in the present invention is a polypeptide that affects the activity of a cell, e.g. via a signal transduction pathway. Thus, the effector moiety of the immunoconjugates of the invention may be associated with receptor-mediated signaling that relays signals from outside the cell membrane to modulate intracellular responses. For example, the effector moiety of the immunoconjugate may be a cytokine. In a particular embodiment, the effector moiety is a single-chain effector moiety, as defined herein. In one embodiment, the one or more effector moieties, typically single chain effector moieties, of the immunoconjugates of the invention are cytokines selected from the group consisting of: IL-2, GM-CSF, IFN-alpha and IL-12. In another embodiment, the one or more single-chain effector templates of the immunoconjugate are cytokines selected from the group of: IL-8, MIP-1 alpha, MIP-1 beta and TGF-beta.
In one embodiment, the effector moiety, preferably the single chain effector moiety, of the immunoconjugate is IL-2. In a specific embodiment, the IL-2 effector moiety may elicit one or more cellular responses selected from the group consisting of: proliferation in activated T lymphocytes, differentiation in activated T lymphocytes, cytotoxic T Cell (CTL) activity, proliferation in activated B cells, differentiation in activated B cells, proliferation in Natural Killer (NK) cells, differentiation in NK cells, and NK/lymphocyte-activated killer (LAK) anti-tumor cytotoxicity. In one embodiment, the effector moiety, preferably the single chain effector moiety, of the immunoconjugate is GM-CSF. In a specific embodiment, the GM-CSF effector moiety may initiate proliferation and/or differentiation in granulocytes, monocytes or dendritic cells. In one embodiment, the effector moiety, preferably the single chain effector moiety, of the immunoconjugate is IFN- α. In a particular embodiment, the IFN- α effector module may elicit one or more cellular responses selected from the group consisting of: inhibit viral replication and up-regulate major histocompatibility complex i (mhc i) expression in virus-infected cells. In another specific embodiment, the IFN α effector moiety can inhibit proliferation in a tumor cell. In one embodiment, the effector moiety, preferably a single chain effector moiety, of the immunoconjugate is IL-12. In a specific embodiment, the IL-12 effector module can elicit one or more cellular responses selected from the group consisting of: proliferation in NK cells, differentiation in NK cells, proliferation in T cells and differentiation in T cells. In one embodiment, the effector moiety, preferably the single chain effector moiety, of the immunoconjugate is IL-8. In a specific embodiment, the IL-8 effector module may elicit chemotaxis in neutrophils. In one embodiment, the effector moiety, preferably the single chain effector moiety, of the immunoconjugate is MIP-1 α. In a specific embodiment, the MIP-1 α effector moiety can elicit chemotaxis in monocytes and T-lymphocytes. In one embodiment, the effector moiety, preferably the single chain effector moiety, of the immunoconjugate is MIP-1 β. In a specific embodiment, the MIP-1 β effector module can elicit chemotaxis in monocytes and T-lymphocytes. In one embodiment, the effector moiety, preferably the single chain effector moiety, of the immunoconjugate is TGF- β. In a particular embodiment, the TGF- β effector moiety may elicit one or more cellular responses selected from the group consisting of: chemotaxis in monocytes, chemotaxis in macrophages, upregulation of IL-1 expression in activated macrophages, and upregulation of IgA expression in activated B cells.
Immunoconjugates polypeptides and polynucleotides
The immunoconjugates of the invention comprise polypeptides and fragments thereof. As used herein, the term "polypeptide" is intended to encompass both a single "polypeptide" and a plurality of "polypeptides" and refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids, and does not refer to a particular length of the product. As such, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to one or more chains of two or more amino acids, are encompassed within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the product of post-expression modification of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. The polypeptides may be derived from natural biological sources or produced by recombinant techniques, but need not be translated from a specified nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.
The polypeptides of the invention may have a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, although they need not have such a structure. Polypeptides having a defined three-dimensional structure are said to be folded, rather than possessing a defined three-dimensional structure, and polypeptides that can adopt a number of different conformations are said to be unfolded.
An "isolated" polypeptide or variant or derivative thereof means a polypeptide that is not in its natural environment. No specific level of purification is required. For example, an isolated polypeptide may be removed from its natural or native environment. For the purposes of the present invention, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.
Also included as polypeptides of the invention are derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof. In reference to a polypeptide of the present invention, the terms "variant", "derivative" and "analogue" include any polypeptide that retains at least some of the biological, antigenic, or immunogenic properties of the corresponding native polypeptide. Variants of the polypeptides of the invention include polypeptides having an altered amino acid sequence due to amino acid substitution, deletion, or insertion. Variants may be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be generated using mutagenesis techniques known in the art. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions. The polypeptide derivatives of the invention are polypeptides that have been altered to exhibit other characteristics not found on the native polypeptide. Examples include fusion proteins. Variant polypeptides may also be referred to herein as "polypeptide analogs". As used herein, a "derivative" of a polypeptide refers to a polypeptide having one or more residues chemically derivatized by reaction of a functional side chain group. Also included as "derivatives" are those peptides that contain one or more naturally occurring amino acid derivatives of the 20 standard amino acids. For example, proline may be replaced by 4-hydroxyproline; lysine can be replaced by 5-hydroxylysine; 3-methylhistidine can be used for replacing histidine; homoserine can be substituted for serine; and replacement of lysine with ornithine.
Alternatively, recombinant variants encoding these same or similar polypeptides may be synthesized or selected by exploiting "redundancy" in the genetic code. Various codon substitutions may be introduced, such as generating silent changes in various restriction sites to optimize cloning of the plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system. Mutations in the polynucleotide sequence may be reflected in the domain of the polypeptide or other peptides added to the polypeptide to modify the properties of any part of the polypeptide, altering characteristics such as ligand binding affinity, interchain affinity, or degradation/turnover rate.
Preferably, an amino acid "substitution" is the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., a conservative amino acid substitution. "conservative" amino acid substitutions may be made on the basis of the polar, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Preferably, an "insertion" or "deletion" ranges from about 1 to about 20 amino acids, more preferably, 1 to 10 amino acids. The variation allowed can be determined experimentally by using recombinant DNA techniques to systematically generate amino acid insertions, deletions, or substitutions in a polypeptide molecule and assaying the resulting recombinant variants for activity.
A polypeptide having an amino acid sequence that is at least, e.g., 95% "identical" to a query amino acid sequence of the present invention means that the subject polypeptide has an amino acid sequence that is identical to the query sequence, except that the subject polypeptide sequence may contain up to 5 amino acid changes per 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to the query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. These changes to the reference sequence can occur at the amino-or carboxy-terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
Indeed, known computer programs can be used routinely to determine whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, percent, 97%, 98%, or 99% identical to a reference polypeptide. A solution based on Brutlag et al, comp.appl.biosci.6: 237-245(1990) to determine the preferred method for determining the best overall match between a query sequence (a sequence of the invention) and a subject sequence (also known as global sequence alignment). In sequence alignment, the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The results of the global sequence alignment are in percent identity. Preferred parameters used in the FASTDB amino acid ratio are: the matrix is PAM0, k-tuple is 2, mismatch penalty is 1, ligation penalty is 20, randomization group length is 0, retention score is 1, window size is sequence length, gap penalty is 5, gap size penalty-0.05, window size is 500 or the length of the subject amino acid sequence (whichever is shorter).
If the subject sequence is shorter than the query sequence due to N or C terminal deletions, not due to internal deletions, manual corrections must be made to the results. This is because the FASTDB program does not consider N-and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N and C termini, the percent identity is corrected by calculating the number of residues of the query sequence at the N and C termini of the subject sequence that do not match/align with the corresponding subject residues as a percentage of the total bases of the query sequence relative to the query sequence. The results of the FASTDB sequence alignment are used to determine whether residues are matched/aligned. This percentage is then subtracted from the percent identity calculated by the FASTDB program described above using the specified parameters to arrive at the final percent identity score. This final percent identity score is used for purposes of the present invention. To manually adjust the percent identity score, only residues at the N-and C-termini of the subject sequence and not matched/aligned with the query sequence are considered. That is, only the query residue positions outside the most distal N-and C-terminal residues of the subject sequence.
For example, a subject sequence of 90 amino acid residues is aligned with a query sequence of 100 residues to determine percent identity. Deletions occurred at the N-terminus of the subject sequence, and thus, FASTDB alignments did not show a match/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues accounted for 10% of the sequence (number of unmatched N and C terminal residues/total number of residues in the query sequence), thus 10% was subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues are perfectly matched, the final percent identity would be 90%. In another example, a subject sequence of 90 residues is compared to a query sequence of 100 residues. This time, the deletion is an internal deletion, and thus there are no residues at the N-or C-terminus of the subject sequence that match/align with the query. In this case, the percent identity calculated by FASTDB was not corrected manually. Again, only residue positions outside the N-and C-terminal ends of the subject sequence (as exhibited in the FASTDB alignment) and not matched/aligned with the query sequence are corrected manually. No other manual corrections are made for the purposes of the present invention.
The polypeptides of the invention include those that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences listed in tables 3 and 4 below, including functional fragments or variants thereof. The invention also encompasses polypeptides comprising the sequences of table 3 or 4 and having conservative amino acid substitutions.
The polypeptides of the invention may be encoded by a single polynucleotide. Alternatively, it may be encoded by multiple (e.g., two or more) polynucleotides, thereby co-expressing the polypeptide. Polypeptides co-expressed from multiple polynucleotides may be combined to form a functional immunoconjugate via, for example, disulfide bonds or other means. For example, the heavy chain portion of the antigen binding module may be encoded by a different polynucleotide than the immunoconjugate portion comprising the light chain portion of the antigen binding module and the effector module. Upon co-expression, the heavy chain polypeptide will bind to the light chain polypeptide to form an antigen binding module. Alternatively, in another example, the light chain portion of the antigen binding moiety may be encoded by a different polynucleotide than the immunoconjugate portion comprising the heavy chain portion of the antigen binding moiety and the effector moiety.
Generally, the immunoconjugates and fragments thereof of the invention are encoded by polynucleotides. The term "polynucleotide" is intended to encompass both single and multiple nucleic acids, and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), virus-derived RNA, or plasmid DNA (pDNA). Polynucleotides may comprise conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, such as found in Peptide Nucleic Acids (PNAs)). The term "nucleic acid" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. An "isolated" nucleic acid or polynucleotide means a nucleic acid molecule, DNA or RNA that has been removed from its natural environment. For example, for the purposes of the present invention, a recombinant polynucleotide encoding a therapeutic polypeptide contained in a vector is considered to be isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partial or substantial) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the invention, as well as the positive and negative strand forms, and double-stranded forms, of pestiviruses disclosed herein.
Isolated polynucleotides or nucleic acids according to the invention further include synthetically produced such molecules. In addition, the polynucleotide or nucleic acid may be or may comprise regulatory elements such as a promoter, ribosome binding site, or transcription terminator.
As used herein, a "coding region" is the portion of a nucleic acid that consists of codons that are translated into amino acids. Although the "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered part of the coding region (if present), but any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, 5 'and 3' untranslated regions, etc., are not part of the coding region. The two or more coding regions of the invention may be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. In addition, any vector may contain a single coding region, or may contain two or more coding regions, e.g., a vector of the invention may encode one or more multiple proteins that are separated post-translationally or co-translationally into the final protein via proteolytic cleavage. In addition, the vectors, polynucleotides, or nucleic acids of the invention may encode a heterologous coding region, fused or unfused with a first or second nucleic acid encoding an immunoconjugate of the invention, or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs such as secretory signal peptides or heterologous functional domains.
In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid encoding a polypeptide may typically comprise a promoter and/or other transcriptional or translational control elements in operable combination with one or more coding regions. Operably linked means that the coding region of the gene product (e.g., a polypeptide) is now linked to one or more regulatory sequences in a manner such that expression of the gene product is under the influence or control of the regulatory sequences. Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in transcription of mRNA encoding the desired gene product, and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region will be operably associated with a nucleic acid encoding a polypeptide if the promoter is capable of effecting transcription of the nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of DNA only in predetermined cells. In addition to promoters, other transcriptional control elements, such as enhancers, operators, repressors, and transcriptional termination signals may be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein.
Various transcriptional control regions are known to those skilled in the art. These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (e.g., the immediate early promoter, along with intron a), simian virus 40 (e.g., the early promoter), and retroviruses (such as, for example, Rous (Rous) sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes, such as actin, heat shock proteins, bovine growth hormone, and rabbit β -globin, and other sequences capable of controlling gene expression in eukaryotic cells. Other suitable transcriptional control regions include tissue-specific promoters and enhancers and lymphokine-inducible promoters (e.g., interferon or interleukin-inducible promoters).
Similarly, many translational control elements are known to those of ordinary skill in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (in particular internal ribosome entry sites, or IRES, also known as CITE sequences).
In other embodiments, the polynucleotide of the invention is RNA, e.g., in the form of messenger RNA (mRNA). The RNA of the present invention may be single-stranded or double-stranded.
The polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions encoding secretion or signal peptides. The secretion or signal peptide directs the secretion of a polypeptide encoded by a polynucleotide of the invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory signal sequence that is cleaved from the mature protein once export of the growing protein chain through the rough endoplasmic reticulum has been initiated. One of ordinary skill in the art will recognize that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the intact or "full-length" polypeptide to produce the secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide, such as an immunoglobulin heavy or light chain signal peptide, or a functional derivative of such a sequence, is used that retains the ability to direct secretion of the polypeptide to which it is operably bound. Alternatively, a heterologous mammalian signal peptide, or functional derivative thereof, may be used. For example, the wild-type leader sequence may be replaced with the leader sequence of human Tissue Plasminogen Activator (TPA) or mouse β -glucuronidase.
The term "expression cassette" refers to a recombinantly or synthetically produced polynucleotide having a series of defined nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of the expression vector includes the nucleic acid sequence to be transcribed, a promoter, and the like. In one embodiment, the expression cassette of the invention comprises a polynucleotide sequence encoding the immunoconjugate of the invention, or a fragment thereof.
The term "expression vector" is synonymous with "expression construct" and refers to a DNA molecule used to introduce a specific gene to which a target cell is operably bound and direct expression. The expression vector of the present invention comprises an expression cassette. Expression vectors allow transcription of large amounts of stable mRNA. Once the expression vector is inside the target cell, the cellular transcription and/or translation machinery produces the ribonucleic acid molecule or protein encoded by the gene. In one embodiment, the expression vector of the invention comprises an expression cassette comprising a polynucleotide sequence encoding the immunoconjugate of the invention or a fragment thereof.
The term "artificial" refers to a synthetic, or non-host cell derived composition, e.g., a chemically synthesized oligonucleotide.
A nucleic acid or polynucleotide having a nucleotide sequence that is at least, e.g., 95% "identical" to a reference nucleotide sequence of the present invention means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or up to 5% of the number of nucleotides of the total nucleotides in the reference sequence may be inserted into the reference sequence.
Indeed, known computer programs can be used routinely to determine whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, percent, 97%, 98%, or 99% identical to a nucleotide sequence or polypeptide sequence of the present invention. A solution based on Brutlag et al, comp.appl.biosci.6: 237-245(1990) to determine the preferred method for determining the best overall match between a query sequence (a sequence of the invention) and a subject sequence (also known as global sequence alignment). In sequence alignment, both the query and subject sequences are DNA sequences. RNA sequences can be compared by converting U to T. The results of the global sequence alignment are in percent identity. Preferred parameters used in performing FASTDB alignments on DNA sequences to calculate percent identity are: the matrix is unary, k-tuple is 4, mismatch penalty is 1, ligation penalty-30, randomization group length is 0, cut-off score is 1, gap penalty is 5, gap size penalty is 0.05, window size is 500 or the length of the subject nucleotide sequence (whichever is shorter).
If the subject sequence is shorter than the query sequence due to 5 'or 3' deletions, not due to internal deletions, manual corrections must be made to the results. This is because the FASTDB program does not consider the 5 'and 3' truncations of the subject sequence when calculating the global percent identity. For subject sequences truncated at the 5 'or 3' end, the percent identity is corrected by calculating the number of mismatched/aligned bases of the query sequence at the 5 'and 3' ends of the subject sequence as a percentage of the total bases of the query sequence relative to the query sequence. The result of the FASTDB sequence alignment is used to determine whether the nucleotides are matched/aligned. This percentage is then subtracted from the percent identity calculated by the FASTDB program described above using the specified parameters to arrive at the final percent identity score. This corrected score is used for purposes of the present invention. To manually adjust the percent identity score, only bases outside the 5 'and 3' bases of the subject sequence (as demonstrated by FASTDB alignment) and that do not match/align with the query sequence are calculated.
For example, a 90 base subject sequence is aligned with a 100 base query sequence to determine percent identity. Deletion occurs at the 5 'end of the subject sequence, and thus, FASTDB alignment does not show matching/alignment of the first 10 bases of the 5' end. The 10 unpaired bases make up 10% of the sequence (number of mismatched 5 'and 3' end bases/total number of bases in the query sequence), thus 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases are perfectly matched, the final percent identity would be 90%. In another example, a subject sequence of 90 bases is compared to a query sequence of 100 bases. This time, the deletion is an internal deletion, such that there are no bases that are 5 'or 3' to the subject sequence and that do not match/align with the query. In this case, the percent identity calculated by FASTDB was not corrected manually. Again, only bases that are 5 'and 3' to the subject sequence and that do not match/align with the query sequence are corrected manually. No other manual corrections are made for the purposes of the present invention.
Polynucleotides of the invention include those that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences listed in tables 6 and 8 below, including functional fragments or variants thereof. The polynucleotide may be expressed as a single polynucleotide encoding the entire immunoconjugate or as multiple (e.g., two or more) polynucleotides that are co-expressed. The polypeptides encoded by the co-expressed polynucleotides may be joined via, for example, disulfide bonds or other means to form functional immunoconjugates. For example, the heavy chain portion of the antigen binding module may be encoded by a different polynucleotide than the immunoconjugate portion comprising the light chain portion of the antigen binding module and the effector module. Upon co-expression, the heavy chain polypeptide will bind to the light chain polypeptide to form an antigen binding module. Alternatively, in another example, the light chain portion of the antigen binding moiety may be encoded by a different polynucleotide than the immunoconjugate portion comprising the heavy chain portion of the antigen binding moiety and the effector moiety.
In a particular embodiment, an isolated polynucleotide of the invention encodes an immunoconjugate fragment comprising at least one effector moiety, preferably a single chain effector moiety, and at least one, preferably two or more antigen binding moieties, wherein a first effector moiety shares an amino or carboxy terminal peptide bond with a first antigen binding moiety and a second antigen binding moiety shares an amino or carboxy terminal peptide bond with the first effector moiety or the first antigen binding moiety. In a preferred embodiment, the antigen binding moieties are independently selected from the group consisting of: fv and Fab. In another specific embodiment, the polynucleotide encodes one of the heavy chain and the effector moiety of both of the antigen binding moieties. In another specific embodiment, the polynucleotide encodes one of the light chain of both of the antigen binding modules and the effector module. In another specific embodiment, the polynucleotide encodes a light chain from one of the antigen binding modules, a heavy chain from the second antigen binding module and one of the effector modules.
In another specific embodiment, the isolated polynucleotide of the invention encodes a fragment of an immunoconjugate, wherein the polynucleotide encodes the heavy chains of the two Fab molecules and an effector moiety, preferably a single chain effector moiety. In another specific embodiment, the isolated polynucleotide of the invention encodes a fragment of an immunoconjugate, wherein the polynucleotide encodes the light chains of the two Fab molecules and an effector moiety, preferably a single chain effector moiety. In another specific embodiment, the isolated polynucleotide of the invention encodes a fragment of an immunoconjugate, wherein the polynucleotide encodes the heavy chain of one Fab molecule, the light chain of a second Fab molecule, and an effector moiety, preferably a single chain effector moiety.
In one embodiment, the isolated polynucleotide of the invention encodes an immunoconjugate comprising at least one effector moiety, preferably a single chain effector moiety, linked at its amino and carboxy-terminal amino acids to one or more scFv molecules.
In one embodiment, the isolated polynucleotide of the invention encodes an immunoconjugate fragment comprising at least one effector moiety, preferably a single chain effector moiety, and at least first and second antigen binding moieties, wherein each antigen binding moiety comprises an scFv molecule linked at its carboxy-terminal amino acid to a constant region comprising an immunoglobulin constant domain independently selected from the group consisting of: IgG1CH1, IgG ck and IgE CH4, and wherein one of the antigen binding moieties is linked at its constant region carboxy-terminal amino acid to the amino-terminal amino acid of one of the effector moieties, and wherein the first and second antigen binding moieties are covalently linked via a disulfide bond. In yet another embodiment, the polynucleotide of the invention encodes one of the antigen binding modules and an effector module, preferably a single chain effector module.
In one embodiment, the isolated polynucleotide of the invention encodes an immunoconjugate fragment comprising first and second effector modules and two antigen binding modules, wherein each antigen binding module comprises an scFv molecule linked at its carboxy-terminal amino acid to a constant region comprising an immunoglobulin constant domain, and wherein one of the antigen binding modules is linked at its carboxy-terminal amino acid to an amino-terminal amino acid of one of the effector modules, and wherein the second antigen binding module is linked at its carboxy-terminal amino acid to an amino-terminal amino acid of the second effector module, and wherein the first and second antigen binding modules are covalently linked via a disulfide bond. In a preferred embodiment, the first and/or second effector modules are single-chain effector modules. In a preferred embodiment, the constant domains are independently selected from the group consisting of: IgG1 CH1, IgG ck, and IgE CH 4. In yet another embodiment, a polynucleotide of the invention encodes one of the antigen binding modules and one of the effector modules.
In one embodiment, the isolated polynucleotide of the invention encodes an immunoconjugate fragment comprising at least one effector moiety, preferably a single chain effector moiety, and at least first and second antigen binding moieties, wherein each antigen binding moiety comprises an scFv molecule joined at its carboxy-terminal amino acid to an IgG CH3 domain, and wherein one of the antigen binding moieties is joined at its carboxy-terminal amino acid to an amino-terminal amino acid of one of the effector moieties, and wherein the first and second antigen binding moieties are covalently linked via a disulfide bond. In yet another embodiment, the polynucleotide of the invention encodes one of the antigen binding modules and an effector module, preferably a single chain effector module.
In one embodiment, the isolated polynucleotide of the invention encodes an immunoconjugate fragment comprising two effector modules and two antigen binding modules, wherein each antigen binding module comprises an scFv molecule joined at its carboxy-terminal amino acid to an IgG CH3 domain, and wherein one of the antigen binding modules is joined at its carboxy-terminal amino acid to the amino-terminal amino acid of one of the effector modules, and wherein the second antigen binding module is joined at its carboxy-terminal amino acid to the amino-terminal amino acid of the second effector module, and wherein the first and second antigen binding modules are covalently linked via a disulfide bond. In a preferred embodiment, the first and/or second effector modules are single-chain effector modules. In yet another embodiment, the polynucleotide of the invention encodes one of the antigen binding modules and one of the effector modules, preferably a single chain effector module.
In one embodiment, the isolated polynucleotide of the invention encodes an immunoconjugate fragment comprising two effector modules and two antigen binding modules, wherein each antigen binding module comprises a Fab molecule linked at its heavy or light chain carboxy-terminal amino acid to an IgG1 CH3 domain, and wherein each IgG1 CH3 domain is linked at its carboxy-terminal amino acid to an amino-terminal amino acid of one of the effector modules, and wherein the first and second antigen binding modules are covalently linked via a disulfide bond. In a preferred embodiment, the first and/or second effector modules are single-chain effector modules. In yet another embodiment, the polynucleotide of the invention comprises a sequence encoding a heavy chain variable region of one of the antigen binding modules and one of the effector modules, preferably a single chain module. In yet another embodiment, the polynucleotide of the invention comprises a sequence encoding a light chain variable region of one of the antigen binding modules and one of the effector modules, preferably a single chain effector module.
In another embodiment, the invention relates to an isolated polynucleotide encoding an immunoconjugate or fragment thereof, wherein the polynucleotide comprises a sequence encoding a variable region sequence as shown in table 3 below. In another embodiment, the invention relates to an isolated polynucleotide encoding an immunoconjugate or fragment thereof, wherein the polynucleotide comprises a sequence encoding a polypeptide sequence as shown in table 4. In another embodiment, the invention further relates to an isolated nucleic acid encoding an immunoconjugate or fragment thereof, wherein the nucleic acid comprises a sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence shown in tables 6 and 8 below. In another embodiment, the invention relates to an isolated nucleic acid encoding an immunoconjugate or fragment thereof, wherein the nucleic acid comprises a nucleic acid sequence as shown in tables 6 and 8. In another embodiment, the invention relates to an isolated nucleic acid encoding an immunoconjugate or fragment thereof, wherein the nucleic acid comprises a sequence encoding a variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence in table 3. In another embodiment, the invention relates to an isolated nucleic acid encoding an immunoconjugate or fragment thereof, wherein the nucleic acid comprises a sequence encoding a polypeptide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence in table 4. The invention encompasses an isolated nucleic acid encoding an immunoconjugate or fragment thereof, wherein the nucleic acid comprises a sequence encoding a variable region sequence described in table 3 with conservative amino acid substitutions. The invention also encompasses an isolated nucleic acid encoding an immunoconjugate of the invention or a fragment thereof, wherein the nucleic acid comprises a sequence encoding a polypeptide sequence set forth in table 4 with conservative amino acid substitutions.
Table 2.
Table 3.
Table 4.
Table 5.
Table 6.
Table 7.
Table 8.
Host cell
As used herein, the term "host cell" refers to any kind of cellular system that can be engineered to become an immunoconjugate of the invention, or a fragment thereof. In one embodiment, the host cell is engineered to allow for the production of the immunoconjugate fragments. Host cells include cultured cells, for example, mammalian cultured cells such as CHO cells, HEK, BHK cells, NS0 cells, Sp2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, per.c6 cells or hybridoma cells, yeast cells, insect cells, bacterial cells, plant cells, and the like, and also cells contained within transgenic animals, transgenic plants or cultured plants or animal tissues. In one embodiment, the host cell of the invention comprises an expression vector comprising a polynucleotide sequence encoding the immunoconjugate of the invention, or a fragment thereof. The host cells of the invention may be eukaryotic or prokaryotic.
Purification of immunoconjugate polypeptides and fragments thereof
The immunoconjugates of the invention or fragments thereof can be purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions for purifying a particular protein will depend in part on factors such as electrostatic charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those skilled in the art.
For affinity chromatography purification, a matrix with protein a or protein G can be used. Alternatively, for affinity chromatography purificationAny antibody that specifically binds to the single chain effector moiety of the immunoconjugate may be used. For antibody production, various host animals, including but not limited to rabbits, mice, rats, etc., can be immunized by injection with the immunoconjugate of the invention, or fragment thereof. The immunoconjugates can be attached to a suitable carrier, such as Bovine Serum Albumin (BSA), by means of side chain functional groups or linkers attached to side chain functional groups. Depending on the host species, various adjuvants may be used to enhance the immunological response, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, Pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpetHemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacillus calmette guerin) and corynebacterium parvum (corynebacterium parvum). Thus, one embodiment includes a method for producing an immunoconjugate of the invention by culturing a host cell comprising an expression vector comprising a polynucleotide encoding an immunoconjugate of the invention, or a fragment thereof, under conditions suitable for its expression.
Methods of using immunoconjugates
The immunoconjugates of the invention can be used to target specific antigenic determinants and elicit targets and recruit various cellular responses in cells. The immunoconjugates of the invention are also useful as diagnostic reagents. The binding of the immunoconjugate to the antigenic determinant can be readily detected by using a secondary antibody specific for the effector moiety. In one embodiment, the secondary antibody and immunoconjugate facilitate detection of binding of the immunoconjugate to an antigenic determinant located on the surface of a cell or tissue.
In some embodiments, an effective amount of an immunoconjugate of the invention is administered to a cell. In other embodiments, a therapeutically effective amount of an immunoconjugate of the invention is administered to an individual to treat a disease. As used herein, the term "effective amount" is defined as the amount of an immunoconjugate of the invention necessary to cause a physiological change in the cell or tissue to which it is administered. As used herein, the term "therapeutically effective amount" is defined as the amount of an immunoconjugate of the invention that eliminates, reduces, delays, minimizes or prevents the adverse effects of disease.
The immunoconjugates of the invention can be administered to a subject as such or in the form of a pharmaceutical composition. In one embodiment, the disease is a proliferative disorder, such as cancer. Non-limiting examples of proliferative disorders such as cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and renal cancer. Other cell proliferation disorders that can be treated using the immunoconjugates of the invention include, but are not limited to, neoplasms located in: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testis, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thoracic region, and urogenital system. Also included are pre-cancerous conditions or lesions and cancer metastases. Similarly, other cell proliferation disorders can also be treated by the immunoconjugates of the invention. Examples of such cell proliferative disorders include, but are not limited to: hyperpropylglobulinemia, lymphoproliferative disorders, pathoproteinemia, purpura, sarcoidosis, sezary syndrome, waldenstrom's Macroglobulinemia, Gaucher's disease, histiocytosis (histiocytosis), and any other cell proliferative disorder located in the organ systems listed above in addition to neovascularization. In another embodiment, the disease involves autoimmunity, transplant rejection, post-traumatic immune response, and infectious disease (e.g., HIV). More specifically, the compounds can be used in the treatment of immune cell mediated disorders, including lymphomas; immunoconjugates are used in cells involved in autoimmunity, transplant rejection, graft versus host disease, ischemia, and stroke. The skilled artisan will readily recognize that in many cases, immunoconjugates do not provide a cure, but may provide only partial benefit. In some embodiments, physiological changes with some benefit are also considered therapeutically beneficial. As such, in some embodiments, the amount of immunoconjugate that provides the physiological change is considered to be an "effective amount" or a "therapeutically effective amount".
The subject, patient, or individual in need of treatment is typically a mammal, more particularly a human.
Compositions, formulations, dosages and routes of administration
The pharmaceutical compositions of the invention comprise an effective amount of one or more immunoconjugates dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as, for example, a human, when appropriate. The preparation of pharmaceutical compositions containing at least one immunoconjugate and optionally other active ingredients, according to the present disclosure, will be known to those skilled in the art, as exemplified by Remington's pharmaceutical Sciences, 18 th edition Mack Printing Company, 1990 (which is incorporated herein by reference). Further, for animal (e.g., human) administration, it is understood that the formulation should meet sterility, pyrogenicity, general safety and purity standards, as required by the FDA office of biological standards or corresponding authorities in other countries.
As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coating materials, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, fragrances, dyes, and the like, and combinations thereof, as would be known to one of ordinary skill in the art (see, e.g., Remington's pharmaceutical sciences, 18 th edition Mack Printing Company, 1990, pages 1289-1329, incorporated herein by reference). Unless any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.
The immunoconjugate may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it needs to be sterile for the route of administration, such as injection. The invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intraperitoneally, intrasplenically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, by inhalation (e.g., aerosol inhalation), by injection, infusion, continuous infusion, local perfusion directly bathing target cells, via a catheter, by lavage, in an emulsion, in a lipid composition (e.g., liposomes), or by other methods or any combination of the foregoing, as would be known to one of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18 th edition macprinting Company, 1990, which is incorporated herein by reference). Polypeptide molecules such as the immunoconjugates of the invention are most commonly administered using parenteral administration, particularly intravenous injection.
The actual dosage amount of the composition of the present invention administered to a subject may be determined by physical and physiological factors such as body weight, severity of the condition, type of disease being treated, previous or concurrent therapeutic intervention, patient's self-morbidity, and according to the route of administration. In any event, the practitioner responsible for administration will determine the concentration of active ingredient in the composition and the appropriate dosage for the individual subject.
In certain embodiments, the pharmaceutical composition may comprise, for example, at least about 0.1% of an immunoconjugate of the invention. In other embodiments, the immunoconjugate may comprise between about 2% to about 75% of the unit weight, or about 25% to about 60%, e.g., any range therein. In other non-limiting examples, the dose may further comprise about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derived therefrom. In non-limiting examples from the derivable ranges for the numbers listed herein, ranges of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 micrograms/kg/body weight to about 500 milligrams/kg/body weight, etc., can be administered based on the numbers described above.
In any case, the composition may comprise various antioxidants that retard the oxidation of one or more components. In addition, the prevention of the action of microorganisms may be caused by preservatives, such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.
The immunoconjugates can be formulated into compositions in free base, neutral or salt form. Pharmaceutically acceptable salts include acid addition salts, for example, those formed with the free amino groups of the proteinaceous component, or with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric or mandelic acid. Salts with free carboxyl groups can also be formed from inorganic bases such as, for example, sodium hydroxide, potassium, ammonium, calcium or iron; or organic bases such as isopropylamine, trimethylamine, histidine or procaine.
In embodiments where the composition is in liquid form, the carrier can be a solvent or dispersion medium, including, but not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycols, and the like), lipids (e.g., triglycerides, vegetable oils, liposomes), and combinations thereof. May be prepared, for example, by using a coating material, such as lecithin; maintaining a desired particle size by dispersion in a carrier such as, for example, a liquid polyol or lipid; by using surfactants such as, for example, hydroxypropyl cellulose; or a combination of such methods to maintain suitable fluidity. In many cases, it may be preferred to include isotonic agents, such as, for example, sugars, sodium chloride, or combinations thereof, and/or buffering agents to maintain a physiologically acceptable pH.
In other embodiments, eye drops, nasal solutions or sprays, aerosols or inhalants may be used in the present invention. Generally, such compositions are designed to be compatible with the target tissue type. In one non-limiting example, a nasal solution is generally an aqueous solution designed for administration in drops or spray to the nasal passages. Nasal solutions are prepared so that they resemble nasal secretions in many respects, thereby maintaining normal ciliary action. As such, in some embodiments, the aqueous nasal solution is generally isotonic or slightly buffered to maintain a pH of about 5.5 to about 6.5. In addition, antimicrobial preservatives similar to those used in ophthalmic preparations, pharmaceuticals, or suitable pharmaceutical stabilizers may be included in the formulations, if desired. For example, various commercial nasal formulations are known and contain medicaments such as antibiotics or antihistamines.
In certain embodiments, the immunoconjugate is prepared for administration by a route such as oral ingestion. In these embodiments, the solid composition may comprise, for example, a solution, a suspension, an emulsion, a tablet, a pill, a capsule (e.g., a hard or soft shell gelatin capsule), a sustained release formulation, a buccal composition, a lozenge, an elixir, a suspension, a syrup, a fast dissolving tablet, or a combination thereof. The oral composition may be incorporated directly into a dietary food. Preferred carriers for oral administration comprise inert diluents, assimilable edible carriers, or a combination thereof. In other aspects of the invention, the oral compositions may be prepared as syrups or elixirs. A syrup or elixir may contain, for example, at least one active agent, sweetening agent, preservative, flavoring agent, dye, preservative, or a combination thereof.
In certain embodiments, the oral composition may comprise one or more binders, excipients, disintegrants, lubricants, flavorants, and combinations thereof. In certain embodiments, the composition may comprise one or more of the following: a binder such as, for example, gum tragacanth, gum acacia, corn starch, gelatin or a combination thereof; excipients such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or combinations thereof; a disintegrant such as, for example, corn starch, potato starch, alginic acid, or a combination thereof; lubricants, such as, for example, magnesium stearate; a sweetening agent such as, for example, sucrose, lactose, saccharin or combinations thereof; flavoring agents such as, for example, peppermint, oil of wintergreen, cherry flavoring, orange flavoring, and the like; or a combination of the foregoing. Where the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a carrier, such as a liquid carrier. Various other materials may be present as coating materials or otherwise modifying the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar or both.
Other formulations suitable for other modes of administration include suppositories. Suppositories are solid dosage forms of various weights and shapes intended for insertion into the rectum, vagina or urethra, usually with the addition of a drug. After insertion, the suppository softens, melts or dissolves in the luminal fluid. Generally, for suppositories, conventional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, active ingredients in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.
Sterile injectable solutions are prepared by incorporating the immunoconjugate of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains a basic dispersion medium and/or other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsions, the preferred methods of preparation are vacuum drying or freeze-drying techniques which yield a powder containing the active ingredient plus any additional desired ingredient from its liquid medium which has previously been subjected to sterile filtration. If necessary, the liquid medium should be suitably buffered and the liquid diluent first given isotonic followed by injection of sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as a solvent to produce extremely rapid penetration is contemplated, delivering high concentrations of active agent to a small area.
The compositions must be stable under the conditions of manufacture and storage and preserved/preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept to a minimum, at a safe level, for example, less than 0.5ng/mg protein.
In particular embodiments, delayed absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin, or combinations thereof.
Pharmaceutical compositions comprising the immunoconjugates of the invention may be manufactured by means of conventional mixing, dissolving, granulating, drage-making, levigating, emulsifying, encapsulating (encapsulating) or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations which can be used pharmaceutically. Suitable formulations depend on the route of administration chosen.
For topical administration, the immunoconjugates of the invention can be formulated as solutions, gels, ointments, creams, suspensions, and the like, as is well known in the art.
Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneal injection, and those designed for transdermal, transmucosal, inhalation, oral, or pulmonary administration.
For injection, the immunoconjugates of the invention can be formulated in aqueous solutions, preferably in biologically compatible buffers such as Hanks 'solution, Ringer's solution, or physiological saline buffer. The solution may contain formulating agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the immunoconjugate may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the immunoconjugate can be readily formulated by combining it with a pharmaceutically acceptable carrier as is well known in the art. Such carriers enable the immunoconjugates of the invention to be formulated as tablets, pills, lozenges, capsules, liquids, gels, syrups, ointments, suppositories, and the like, for oral ingestion by a patient to be treated. For oral solid formulations, such as, for example, powders, capsules and tablets, suitable excipients include fillers such as sugars, for example lactose, sucrose, mannitol and sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); a granulating agent; and a binder. If desired, disintegrating agents such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate may be added.
If desired, the solid dosage form may be sugar-coated or enteric-coated using standard techniques.
For oral liquid preparations such as, for example, suspensions, elixirs and solutions, suitable carriers, excipients or diluents include water, glycols, oils, alcohols and the like. In addition, aromatic, antiseptic, colorant, etc. may be added.
For buccal administration, the immunoconjugate may take the form of a tablet, lozenge, or the like formulated in conventional manner.
For administration by inhalation, the immunoconjugates for use according to the invention are conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer with the aid of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of gelatin (cartridges) for use in an inhaler or insufflator may be formulated containing a powder mix of the immunoconjugate and a suitable powder base such as lactose or starch.
The immunoconjugates may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the immunoconjugates can also be formulated as depot preparations (depotpreperation). Such long acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the immunoconjugates can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt.
Alternatively, other drug delivery systems may be employed. Liposomes and emulsions are well known examples of delivery vehicles that can be used to deliver the immunoconjugates of the invention. Certain organic solvents such as dimethylsulfoxide can also be employed, albeit usually at the expense of greater toxicity. In addition, the immunoconjugates can be delivered using a sustained release system, such as a semipermeable matrix of a solid polymer containing the therapeutic agent. Various sustained release materials have been established and are well known to those skilled in the art. Depending on its chemical nature, sustained release capsules can release the immunoconjugate for several weeks up to over 100 days. Other strategies for immunoconjugate stabilization may be employed, depending on the chemical nature and biological stability of the immunoconjugate.
Because the immunoconjugates of the invention can contain charged side chains or termini, they can be included in any of the formulations described above as free acids or bases or as pharmaceutically acceptable salts. Pharmaceutically acceptable salts are those salts that substantially retain the biological activity of the free base and are prepared by reaction with a mineral acid. Pharmaceutically acceptable salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms.
Generally, the immunoconjugates of the invention will be used in an amount effective to achieve the intended purpose. For use in treating or preventing a disease condition, the immunoconjugates of the invention or pharmaceutical compositions thereof are administered or applied in a therapeutically effective amount. A therapeutically effective amount is an amount effective to ameliorate or prevent symptoms, or prolong survival of the treated patient. Determining a therapeutically effective amount is well within the ability of those skilled in the art, particularly in light of the detailed disclosure provided herein.
For systemic administration, the therapeutically effective dose can initially be assessed in an in vitro assay. For example, the dose can be formulated in animal models to achieve an IC that includes as determined in cell culture50The circulating concentration range of (c). Such information can be used to more accurately determine useful doses in humans.
Initial doses may also be assessed from in vivo data, such as animal models, using techniques well known in the art. Administration to humans can be readily optimized by one of ordinary skill in the art based on animal data.
The dosage and time interval may be adjusted individually to provide plasma levels of the immunoconjugate sufficient to maintain the therapeutic effect. Common patient doses for administration by injection range from about 0.1 to 50 mg/kg/day, usually about 0.5 to 1 mg/kg/day. Therapeutically effective serum levels can be achieved by administering multiple doses per day.
In the case of topical administration or selective uptake, the effective local concentration of the immunoconjugate may be independent of the plasma concentration. One skilled in the art would be able to optimize therapeutically effective topical dosages without undue experimentation.
The amount of immunoconjugate administered will, of course, depend on the subject being treated, the weight of the subject, the severity of the affliction, the mode of administration, and the judgment of the prescribing physician.
Intermittent repeat therapy may be performed when symptoms are detectable or even when symptoms are not detectable. Therapy may be provided alone or in combination with other drugs. In the case of autoimmune disorders, drugs that can be used in combination with the immunoconjugates of the invention include, but are not limited to, steroidal and non-steroidal anti-inflammatory agents.
Toxins
A therapeutically effective dose of the immunoconjugates described herein will generally provide therapeutic benefit without causing substantial toxicity. The toxicity of the immunoconjugate can be determined by standard pharmaceutical procedures in cell culture or experimental animals, e.g., by determining the LD50(50% lethal dose of population) or LD100(a dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index. In one embodiment, the immunoconjugate exhibits a high therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic, e.g., for use in humans. Preferably, the dose of the immunoconjugate described herein lies within a circulating concentration range, which includes an effective dose with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be selected by each physician, taking into account the condition of the patient. (see, e.g., Fingl et al, 1975, in: The pharmaceutical Basis of Therapeutics, Chapter 1, page 1) (which is hereby incorporated by reference in its entirety).
Other Agents and treatments
It is contemplated that other agents may be used in combination with the present invention to improve the therapeutic efficacy of the treatment. These additional agents include immunomodulatory agents, agents that affect upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, cell adhesion inhibitors, or agents that increase the sensitivity of hyperproliferative cells to inducers of apoptosis. Immunomodulatory agents include tumor necrosis factor; interferons α, β, and γ; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1 β, MCP-1, RANTES and other chemokines. It is further contemplated that upregulation of cell surface receptors or their ligands such as Fas/Fas ligand, DR4 or DR5/TRAIL will enhance the apoptosis-inducing capabilities of the present invention by establishing autocrine or paracrine effects on hyperproliferative cells. The increased intercellular signaling achieved by increasing the number of GAP junctions increases the anti-hyperproliferative effect on the adjacent hyperproliferative cell population. In other embodiments, cytostatic or differentiating agents may be used in combination with the present invention to improve the anti-hyperproliferative efficacy of the treatment. Inhibitors of cell adhesion are contemplated to improve the efficacy of the invention. Examples of cell adhesion inhibitors are Focal Adhesion Kinase (FAK) inhibitors and Lovastatin (Lovastatin). It is further contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as antibody C225, may be used in combination with the present invention to improve therapeutic efficacy.
Hormone therapy may also be used with the present invention or in combination with any of the other cancer therapies previously described. The use of hormones to reduce the levels or block the effects of certain hormones, such as testosterone or estrogen, may be employed in the treatment of certain cancers, such as breast, prostate, ovarian, or cervical cancer. This treatment is often used in combination with at least one other cancer therapy, either as a treatment option or to reduce the risk of metastasis.
The immunoconjugates of the invention can also be administered with chemotherapy, radiation therapy, or other immunotherapy. The anti-cancer agent for such combination therapy may for example be selected from the group of: microtubule-disrupting drugs (e.g., vinca alkaloids such as vinblastine or vincristine, taxanes such as Docetaxel (Docetaxel) or paclitaxel (paclitaxel), epothilones (epothilones) such as ixabepilone (ixabepilone)), antimetabolites (e.g., antifolates such as methotrexate or aminopterin, antipurines such as fludarabine (flarabareine), 6-mercaptopurine or 6-thioguanine, antipyrimidines such as 5-fluorouracil, capecitabine (capecitabine) or gemcitabine (gemcitabine), hydroxyurea (hydroxyurea), topoisomerase inhibitors (e.g., camptothecin (camptothecin), irinotecan (irinotecan), topotecan (topotecan), or podophyllotoxin (podophyllotoxin) such as etoposide), DNA intercalators (e.g., Docetaxel), doxorubicin (daunorubicin), erythromycin (bleomycin), e.g., epothilone (epothilone), epothilone (epothilone), e.g., etoposide (etoposide), DNA intercalator, Chlorambucil (chlorembucil), nitrosoureas such as carmustine or nimustine (nimustine), streptozocin (streptozocin), busulfan (busufan), cisplatin (cisclin), oxaliplatin (oxaliplatin), tritylamine (triethylelelamine), dacarbazine (dacarbazine), hormonal therapy (e.g., glucocorticoids, aromatase inhibitors such as tamoxifen (tamoxifene), antiandrogens (antiandrogens) such as flutamide, gonadotropin-releasing hormone (rh) analogs such as leuprolide (leuprolide), antibiotics, kinase inhibitors (e.g., erlotinib), gefitinib (gefitinib), receptor antagonists (e.g., antibodies targeting cell surface receptors known to promote carcinogenesis and tumor growth), enzyme inhibitors (e.g., cyclin dependent kinase inhibitors (CDK), cyclin dependent inhibitors (CDK) such as CDK (CDK), and norgestimatinib (noctamide), and certain inhibitors (e.g., leuprolide), and the like, Leucovorin, retinoids, activators of tumor cell apoptosis, and anti-angiogenic agents.
Examples
Example 1
Antibody-mediated delivery of cytokines was first initiated by harville e.t. and Morrison s.l. immunotech.1 (2): 95-105(1995) in practiceThe construct has two antigen binding modules for tumor antigen targeting and two cytokine modules for induction of lymphocyte activation (an IL-2 molecule fused to the C-terminus of each heavy chain of IgG 3). Cytokines generally have a higher affinity for their receptors. Molecules carrying two cytokine units may have even higher affinity for cytokine receptors due to avidity (or multivalent) effects. Thus, such molecules can readily activate lymphocytes in the bloodstream even before tumor targeting occurs. This effect may not be desirable for the patient. In contrast, an immunoconjugate molecule carrying only one cytokine module and two or more targeting domains is less likely to activate lymphocytes in circulation, and the entire immunoconjugate molecule can be directed to a tumor where lymphocyte activation can occur at a slower rate. Thus, the molecules as depicted in FIGS. 1 and 2 were constructed using interleukin-2 (IL-2) as a model cytokine. All molecules generated were bivalent for tumor antigens and bivalent or monovalent for IL-2 cytokines, as indicated in the figure. The L19 antibody was used as an example to compare affinity against antigen for two different immunoconjugate formats. For reference, this antibody was cloned into the human IgG1 format, diabody format (FIG. 1A), and Fab-IL2-Fab fusion (FIG. 1B). Testing a variable within the diabody format such that V HAnd VLThe linker peptide in between is 8 or 12 amino acids in length. The purified antigen fibronectin Extra Domain B (EDB) was immobilized on BIACORE chips and the antibody fusion construct was used as a soluble analyte for affinity determination. The results of this experiment are shown in figures 8 to 11. IgG is considered to be an ideal case for bivalent binding events. Here, an affinity constant of 260pM was observed. The Fab-IL2-Fab fusion construct gave an affinity of 310pM, which was essentially identical to IgG. The two variants of the diabody (shown in figures 10 and 11) have a measured affinity of 270pM and 360pM, respectively. Thus, all of these constructs have similar affinities for antigens. Similar constructs based on the F16 antibody sequence were used to address affinity for antigen and IL-2 receptor (Brack, S.S. et al, Clin. Canc. Res.12 (10): 3200-3208 (2006)). FIG. 12 shows the TNC-A1 domainBIACORE sensorgrams of F16IgG and its corresponding monovalent Fab fragments when immobilized on BIACORE chips. Under these particular experimental conditions, the IgG molecules showed an affinity constant of 2.6nM, whereas the Fab molecules showed an affinity constant of 50 nM. Here, the affinity increase contributing to the bivalency is 20-fold. Diabodies, Fab-IL2-Fab and scFv-IL2-scFv immunoconjugates showed affinities for antigen of 5nM, 4.8nM and 12nM, respectively. Thus, all of these constructs have an affinity for antigen that more closely resembles the bivalent character of an IgG molecule than the monovalent properties of the Fab fragment.
The difference is more pronounced when the affinity towards the IL-2 receptor is considered. To investigate IL-2 receptor binding affinity, tools were generated that allowed expression of the heterodimeric IL-2 receptor; the beta chain of the IL-2 receptor is fused to an Fc molecule that is engineered to heterodimerize (Fc (overhang)) using the "protrusion-into-hole" technique (Merchant, A.M. et al, nat. Biotech.16: 677-681 (1998)). The gamma chain of the IL-2 receptor is then fused to an Fc (hole) variant that heterodimerizes with Fc (bulge), and this heterodimeric Fc-fusion protein is then used as a substrate to analyze IL-2/IL-2 receptor interactions. FIG. 13 shows BIACORE sensorgrams for commercial IL-2(Proleukin) as an analyte in the case of immobilized IL-2 receptor. The measured affinity of about 0.5nM is consistent with previously published values. The affinities of the various constructs for the IL-2 receptor are summarized in figure 17. An important result observed is the difference between the diabody (F16 dia IL2), which is bivalent in terms of cytokines, and the Fab-IL2-Fab molecule, which carries only one IL-2 moiety. The IL-2 receptor binding affinity (0.8nM) of the diabody is similar to that (0.5nM) of unconjugated IL-2(Proleukin), although the diabody is bivalent and IL-2 is monovalent. The Fab-IL2-Fab fusion had almost 10-fold reduced IL-2 receptor binding affinity compared to the diabody, which is reflected in the induction of reduced NK92 cell proliferative performance, as shown in figure 19.
Example 2
Construction of Universal Fab libraries
A universal antibody library in Fab format was constructed based on human germline genes using the following V-domain pairings: vk3_20 kappa light chain and VH3_23 heavy chain (for the DP47-3 library) and Vk1_17 kappa light chain and VH1_69 heavy chain (for the DP88-3 library). See table 2. Both libraries were randomized in the CDR3 of the light chain (L3) and CDR3 of the heavy chain (H3) and assembled from 3 fragments each by Splicing of Overlap Extension (SOE) PCR. Fragment 1 contained the 5 'end of the antibody gene including randomized L3, fragment 2 was a central constant fragment spanning from L3 to H3, and fragment 3 contained randomized H3 and the 3' portion of the antibody gene.
The following primer combinations were used to generate library fragments of the DP47-3 library: segment 1(LMB3-LibL1b _ new), segment 2(MS63-MS64) and segment 3(Lib 2H-fdseqlong). See table 9. The following primer combinations were used to generate library fragments of the DP88-3 library: segment 1(LMB3-RJH _ LIB3), segment 2(RJH31-RJH32) and segment 3(LIB88_ 2-fdsolong). See table 10.
Table 9.
Table 10.
The PCR protocol used to generate the library fragments included: initial denaturation at 94 ℃ for 5 minutes; 25 cycles of 1 minute at 94 ℃, 1 minute at 58 ℃, and 1 minute at 72 ℃; and end extension at 72 ℃ for 10 minutes. For assembly, an equimolar ratio of 3 fragments was used as template. The assembly PCR protocol included: initial denaturation at 94 ℃ for 3 min; and 5 cycles of 30 seconds at 94 ℃, 1 minute at 58 ℃ and 2 minutes at 72 ℃. At this stage, primers complementary to sequences outside of fragments 1-3 were added and an additional 20 cycles were performed followed by 10 min end extension at 72 ℃.
After assembly of a sufficient amount of the full-length randomized Fab construct, the Fab construct was digested with NcoI/NotI (for the DP47-3 library) and with NcoI/NheI (for the DP88-3 library), together with a similarly treated phagemid-receiving vector. For the DP47-3 library, 22.8. mu.g of the Fab library was ligated with 16.2. mu.g of the phagemid vector. For the DP88-3 library, 30.6. mu.g of the Fab library was ligated with 30.6. mu.g of the phagemid vector.
68 transformations (for the DP47-3 library) and 64 transformations (for the DP88-3 library) were performed using the purified ligate to obtain a final library size of 4.2X 10, respectively10(for DP47-3) and 3.3X 109(for DP 88-3).
Phagemid particles exhibiting the Fab library were rescued and purified by PEG/NaCl purification for selection.
Example 3
Selection of anti-TNC A2 clone 2B10
Selection was performed against human TNC-A2 expressed in E.coli, subcloned 5' of the avi tag and the 6 x his tag. See SEQ ID NO in table 5: 57. the antigen is biotinylated in vivo after expression. The selection has been carried out in solution according to the following scheme: (i) about 10 of library DP88-312Individual phagemid particles and 100nM biotinylated human TNC a2 bound for 0.5 hours in a total volume of 1 ml; (ii) by adding 5.4X 10 7The streptavidin-coated magnetic beads capture biotinylated human TNC-a2 and attached phage for 10 minutes; (iii) the beads were washed with 5x1ml PBS/Tween20 and 5x1ml PBS; (iv) the phage particles were eluted by adding 1mL of 100mM TEA (triethylamine) for 10 minutes and neutralized by adding 500. mu.L of 1M Tris/HCl pH 7.4; and (v) reinfection of log phase E.coli TG1 cells with the helper phage VCSM13 followed by PEG/NaCl precipitation of phagemid particles for use in subsequent selection rounds.
Selection was performed in 3 rounds using a constant antigen concentration of 100 nM. In round 2, the antigen was performed on neutravidin (neutravidin) plates instead of streptavidin beads: and (4) capturing the phage complex. Specific binders were identified by ELISA as follows: 100 μ l of 100nM biotinylated human TNC-A2 was coated in each well of the neutravidin plate.
Fab-containing bacterial supernatants were added and bound Fab was detected via its Flag tag by using an anti-Flag/HRP secondary antibody. Once identified, clone 2B10 was expressed as bacteria in a 0.5 liter culture volume, affinity purified, and further characterized by SPR analysis using BIACORE T100. See SEQ id no: 3 and 7.
Example 4
Selection of anti-TNC A1/A4 clone 2F11
Selection was performed against human TNC a1 expressed in e.coli, subcloned 5' of the avi tag and the 6 x his tag. See SEQ ID NO in table 5: 59. the antigen is biotinylated in vivo after expression. The selection has been carried out in solution according to the following scheme: (i) about 10 of library DP47-312Individual phagemid particles and 100nM biotinylated human TNC-a1 bound for 0.5 hours in a total volume of 1 ml; (ii) by adding 5.4X 107The streptavidin-coated magnetic beads capture biotinylated human TNC-a1 and attached phage for 10 minutes; (iii) the beads were washed with 5x1ml PBS/Tween20 and 5x1ml PBS; (iv) the phage particles were eluted by adding 1mL of 100mM TEA (triethylamine) for 10 minutes and neutralized by adding 500. mu.L of 1M Tris/HCl pH 7.4; and (v) reinfection of log phase E.coli TG1 cells with the helper phage VCSM13 followed by PEG/NaCl precipitation of phagemid particles for use in subsequent selection rounds.
Selection was performed in 3 rounds using a constant antigen concentration of 100 nM. In round 2, the antigens were performed on neutravidin plates instead of streptavidin beads: and (4) capturing the phage complex.
All binding reactions were supplemented with 100nM non-biotinylated human IgG CH3 constant domain containing a carboxy-terminal avi tag and a 6 × his tag to compete for unwanted clones recognizing the antigen tag.
In the first screening step, specific binders were identified by ELISA as follows: 100 μ l of 100nM biotinylated human TNC-A1 was coated in each neutravidin plate. Fab-containing bacterial supernatants were added and Fab specifically binding to human TNC-a1 was detected via its Flag tag by using an anti-Flag/HRP secondary antibody.
In the second screening step, the above ELISA was also repeated using murine TNC-a1, human TNCA4 and mu TNCA4 as antigens to determine cross-reactivity. All antigens contained the same avi-tag and 6 × his-tag at their C-terminus and were biotinylated in vivo. See table 5 for SEQ ID NO: 50 and 61.
Once identified, clone 2F11 was expressed as bacteria in a 0.5 liter culture volume, affinity purified, and further characterized by SPR analysis using BIACORE T100. See table 3 for SEQ ID NO: 9 and 13.
Example 5
Selection of anti-FAP clones (Primary selection)
Selection was performed against the extracellular domain of human or murine Fibroblast Activation Protein (FAP), cloned upstream of polylysine and the 6 × his tag. See table 5 for SEQ ID NO: 53 and 55. Before selection, antigens were coated into immune tubes at a concentration of 10. mu.g/ml or 5. mu.g/ml, depending on the selection round. Selection was performed according to the following protocol: (i) about 10 of library DP47-3 12Individual phagemid particles bind to immobilized human or murine FAP for 2 hours; (ii) the immune tubes were washed with 5X 5mL PBS/Tween20 and 5X 5mL PBS; (iii) the phage particles were eluted by adding 1mL of 100mM TEA (triethylamine) for 10 minutes and neutralized by adding 500. mu.L of 1M Tris/HCl pH 7.4; and (v) reinfection of log phase E.coli TG1 cells with helper phage VCSM13Staining followed by PEG/NaCl precipitation of the phagemid particles for use in subsequent selection rounds.
Selection has been performed in 3 or 4 rounds using human FAP at reduced antigen concentrations and in some cases 5ug/ml murine FAP in the final selection round. Specific binders were defined as signals 5 x higher than background and identified by ELISA. NUNC Maxisorp plates were coated with 10ug/ml human or murine FAP, followed by addition of Fab-containing bacterial supernatant and detection of specifically bound Fab via its Flag tag by using an anti-Flag/HRP secondary antibody.
ELISA positive clones were expressed as 1ml culture bacteria in 96-well format and supernatants were subjected to kinetic screening experiments using BIACORE T100.
Example 6
Construction of anti-FAP affinity maturation library
3 affinity matured libraries were constructed based on pre-selected antibodies from the preliminary anti-FAP selection. More precisely, they are based on (i) anti-human FAP clone 2D9 (library a.m.fap2d9) (see SEQ ID NOs 67 and 69 of table 3), (ii) anti-murine FAP clone 4B8 (library a.m.fap4B8) (see SEQ ID NOs 71 and 73 of table 3) and (iii) cross-reactive clones 7a1, 13B2, 13C2, 13E8, 14C10 and 17a11 (library a.m.fappool) (see SEQ ID NOs 75 and 77 of table 3, which correspond to the 7a1 variable region sequence; SEQ ID NOs 79 and 81 of table 3, which correspond to the 13C2 variable region sequence; SEQ ID NOs 83 and 85, which correspond to the 13E8 variable region sequence; SEQ ID NOs 87 and 89, which correspond to the 14C10 variable region sequence; and SEQ ID NOs 91 and 93, which correspond to the 13 a11 variable region sequence).
Each of these libraries consisted of two sub-libraries randomized in CDR1 and CDR2 of the light chain (L1/L2) or CDR1 and CDR2 of the heavy chain (H1/H2), respectively. These sub-libraries were pooled after transformation. Each of these sub-libraries was constructed by four subsequent amplification and assembly steps.
For the L1/L2 library, the amplification and assembly protocol included: (i) amplification of fragment 1(LMB3-DPK22_ CDR1_ rand _ ba _ opt) and fragment 2(DPK22_ CDR1_ fo-DPK22_ Ck _ BsiWI _ ba); (ii) assembling fragments 1 and 2 using the outer primers LMB3 and DPK22_ Ck _ BsiWI _ ba to create a template for fragment 3; (iii) fragment 3(LMB3-DPK22_ CDR2_ rand _ ba) and fragment 4(DPK22_ CDR2_ fo-DPK22_ Ck _ BsiWI _ ba) were amplified; and (iv) final assembly of fragments 3 and 4 using the same outer primers as above. See table 11 for primer sequences.
Table 11.
Bold: 60% of the original bases and 40% randomization to M
Underlining: 60% of the original bases and 40% randomization to N
For the H1/H2 library, the amplification and assembly protocol included: (i) amplifying segment 1(RJH53-DP47_ CDR1_ rand _ ba _ opt) and segment 2(DP47_ CDR1_ fo-MS 52); (ii) assembling fragments 1 and 2 using the outer primers RJH53 and MS52 to create a template for fragment 3; (iii) amplifying segment 3(RJH53-DP47_ CDR2_ rand _ ba) and segment 4(DP47_ CDR2_ fo-MS 52); and (iv) final assembly of fragments 3 and 4 using the same outer primers as above. See table 12 for primer sequences.
Table 12.
Bold: 60% of the original bases and 40% randomization to M
Underlining: 60% of the original bases and 40% randomization to N
The final assembly product has been digested with NcoI/BsiWI (L1/L2 sub-library for a.m.fap2d9 and a.m.fap4b 8), with MunI and NheI (H1/H2 sub-library for a.m.fap2d9 and a.m.fap4b 8) and with NcoI/BamHI (L1/L2 library for a.m.fap pool) and with BspEI/PstI (H1/H2 library for a.m.fap pool), respectively, together with similarly treated recipient vectors based on an equimolar mixture of plasmid preparations of clones 2D9, 4B8 or clones 7a1, 13B2, 13C2, 13E8, 14C10 and 17a11, respectively. The following amounts of digested randomized (partial) V-domain and digested recipient vector were ligated for the corresponding library (μ g V-domain/μ g vector): an a.m.FAP2D9L1/L2 sub-library (5.7/21.5), an a.m.FAP2D9H1/H2 sub-library (4.1/15.5), an a.m.FAP4B8L1/L2 sub-library (6.5/24.5), an a.m.FAP4B8H1/H2 sub-library (5.7/21.5), an a.m.FAPPoolL1/L2 sub-library (4.4/20), and an a.m.FAPPool H1/H2 sub-library (3.4/15.5).
Purified L1/L2 and H1/H2 sublibrary ligations were pooled and used for 60 transformations (for each of 3 affinity maturation libraries) to obtain a final library size of 6.2X 10 9(for a.m. fap2d9), 9.9 × 109(for a.m.FAP4B8) and 2.2X 109(for a.m. fappool).
Phagemid particles displaying these Fab libraries were rescued and purified by PEG/NaCl purification for secondary selection.
Example 7
Selection of affinity matured anti-FAP clones
Selection was performed against the extracellular domain of human or murine Fibroblast Activation Protein (FAP), cloned 5' of polylysine and a 6 × his tag. See table 5 for SEQ ID NO: 53 and 55. Before selection, the antigen was coated into the immune tubes at a concentration of 10. mu.g/mL, 5. mu.g/mL or 0.2. mu.g/mL depending on the library and round of selection. Selection was performed according to the following protocol: (i) about 10 of library a.m.fap2d9, a.m.fap4b8 or a.m.fappoo12Individual phagemid particles bind to immobilized human or murine FAP for 2 hours; (ii) wash the immune tubes with 10-20 × 5mL PBS/Tween20 and 10-20 × 5mL PBS (from library and selection round); (iii) the phage particles were eluted by adding 1mL of 100mM TEA (triethylamine) for 10 min and passingAdd 500. mu.L of 1M Tris/HCl pH 7.4 to neutralize; and (v) reinfection of log phase E.coli TG1 cells with the helper phage VCSM13 followed by PEG/NaCl precipitation of phagemid particles for use in subsequent selection rounds.
Selection was performed in 2 rounds and individually for each of the 3 libraries of regulatory conditions. In detail, the selection parameters are: FAP2d9 (5 μ g/mL for round 1 and 20 washes total, 1 μ g/mL for round 2 and 30 washes total), FAP4b8 (1 μ g/mL for round 1 and 30 washes total, 0.2 μ g/mL for round 2 and 40 washes total) and a m fapopool (5 μ g/mL for round 1 and 30 washes total, 5 μ g/mL for round 2 and 30 washes total). Specific binders were defined as signals 5 x higher than background and identified by ELISA. NUNCMaxisorp plates were coated with 1 μ g/mL or 0.2 μ g/mL human or murine FAP, followed by addition of Fab-containing bacterial supernatant and detection of specifically bound fabs via their Flag tags by using anti-Flag/HRP secondary antibodies.
ELISA positive clones were expressed as 1ml culture bacteria in 96-well format and supernatants were subjected to kinetic screening experiments using BIACORE T100.
Example 8
Efficacy studies of different forms of targeted IL-2
Efficacy experiments were performed using two different interleukin-2 immunoconjugate molecular forms specific for tumor stroma. F9 teratomas were injected subcutaneously into 129SvEv mice and tumor size was measured using calipers. The "diabody" -IL-2 molecule was compared to Fab-interleukin-2-Fab (Fab-IL2-Fab) immunoconjugates at two different concentrations, where the concentrations reflect a similar number of immunoconjugate molecules. The results are shown in fig. 3. The Fab-IL2-Fab immunoconjugate showed significant tumor growth inhibition and was better than the two different concentrations of the diabody form and better than the control.
Mice treated with two different interleukin-2 immunoconjugate molecular forms specific for tumor stroma were also examined for survival. The human gastric tumor cell line LS174T was injected intrasplenically into SCID-beige mice. The "diabody" -IL-2 molecule is compared to Fab-IL-2-Fab immunoconjugates at two different concentrations, where the concentrations reflect a similar number of immunoconjugate molecules. The results are shown in fig. 4. The Fab-IL-2-ab form resulted in a higher percentage survival compared to the diabody form and the control.
Example 9
Recombinant DNA technology
The DNA is manipulated using standard methods, such as those described in Sambrook, j, et al, Molecular cloning: a laboratory manual; cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions.
For general information on the nucleotide sequences of human immunoglobulin light and heavy chains see: kabat, E.A. et al, (1991) Sequences of Proteins of Immunological Interest, fifth edition, NIH publication No. 91-3242.
DNA sequencing
The DNA sequence was determined by double-strand sequencing.
Gene synthesis
The desired gene segments were prepared from synthetic oligonucleotides and PCR products by automated gene synthesis from Geneart AG (Regensburg, Germany). The gene segment flanked by single restriction endonuclease cleavage sites was cloned into the pGA18(ampR) plasmid. Plasmid DNA was purified from transformed bacteria and the concentration was determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. Gene segments are designed with appropriate restriction sites to allow subcloning into the corresponding expression vector. All constructs were designed with a 5' DNA sequence encoding a leader peptide that targets a protein secreted in eukaryotic cells. Exemplary leader peptides and polynucleotide sequences encoding them are given in tables 13 and 14, respectively.
Table 13.
Leader sequence for secretion: polypeptide sequence
Polypeptide sequence SEQ ID NO
MDWTWRILFLVAAATGAHS 273
MDMRVPAQLLGLLLLWFPGARC 276
MGWSCIILFLVATATGVHS 278
Table 14.
Leader sequence for secretion: polynucleotide sequences
Preparation of immunoconjugates
The resulting DNA sequences were subcloned into mammalian expression vectors (one for the light chain and one for the heavy chain/fusion protein) under the control of the MPSV promoter and upstream of the synthetic poly a site, each vector carrying the EBV OriP sequence.
The immunoconjugates as used in the examples below were generated by co-transfecting exponentially growing HEK293-EBNA cells with a mammalian expression vector by using calcium phosphate transfection. Alternatively, HEK293 cells grown in suspension were transfected with expression vectors via Polyethylenimine (PEI), or a collection of stably transfected CHO cells was used. While FAP-targeting Fab-IL2-Fab constructs based on 3F2 and 4G8 can be purified by affinity chromatography using a protein a matrix, TNC a2 targeting Fab-IL2-Fab constructs based on 2B10 must be purified by affinity chromatography on a protein G matrix.
Briefly, TNC a2 targeting 2B10 Fab-IL2-Fab was purified from the supernatant by an affinity step (protein G) followed by size exclusion chromatography (Superdex200, GE Healthcare). The protein G column was equilibrated in 20mM sodium phosphate, 20mM sodium citrate, pH7.5, the supernatant was loaded, and the column was washed with 20mM sodium phosphate, 20mM sodium citrate, pH 7.5. Fab-IL2-Fab was eluted with 8.8mM formic acid pH 3. The eluted fractions were combined and purified by size exclusion chromatography in the final formulation buffer (polish): 25mM potassium phosphate, 125mM sodium chloride, 100mM glycine, pH 6.7. Fig. 53 shows exemplary results from purification and analysis.
FAP-targeted 3F2 Fab-IL2-Fab or 4G8 Fab-IL2-Fab was purified by a similar method comprising an affinity step (protein a) followed by size exclusion chromatography (Superdex 200, GE Healthcare). The protein A column was equilibrated in 20mM sodium phosphate, 20mM sodium citrate pH7.5, the supernatant was loaded, and the column was washed with 20mM sodium phosphate, 20mM sodium citrate, 500mM sodium chloride, pH7.5, followed by 13.3mM sodium phosphate, 20mM sodium citrate, 500mM sodium chloride, pH 5.45. Optionally, a third wash is performed with 10mM MES, 50mM NaCl pH5. Fab-IL2-Fab was eluted with 20mM sodium citrate, 100mM sodium chloride, 100mM glycine, pH 3. The eluted fractions were combined and refined in the final formulation buffer by size exclusion chromatography: 25mM potassium phosphate, 125mM sodium chloride, 100mM glycine, pH 6.7.
Example 10
Construction of additional anti-FAP affinity maturation libraries (based on clones 3F2, 3D9, 4G8, 4B3 and 2C6)
Four individual affinity maturation libraries were constructed based on preselected cross-reactive antibodies from the first affinity maturation activity of the anti-FAP antibody, clones 3F2, 3D9, 4G8, 4B3, and 2C6 (see SEQ ID NOs: 17 and 21 of table 3, which correspond to 3F2 variable region sequences; SEQ ID NOs: 23 and 25 of table 3, which correspond to 3D9 variable region sequences; SEQ ID NOs: 33 and 35 of table 3, which correspond to 4B3 variable region sequences; SEQ ID NOs: 41 and 43 of table 3, which correspond to 2C6 variable region sequences). More precisely, the four libraries were based on 1) anti-FAP clones 3F2, 4G8 and 4B3 (randomized VH library in CDR1 and 2 of the variable heavy chain, i.e. H1/H2 library), 2) anti-FAP clones 3D9 and 2C6 (randomized VL library in CDR1 and 2 of the variable light chain, i.e. L1/L2 library), 3) anti-FAP clone 3F2 (L3 library with soft randomization in CDR3 of the light chain, i.e. L3 library) and 4) anti-FAP clone 3F2 (H3 library with soft randomization in CDR3, i.e. H3 library). For the L1/L2 and H1/H2 libraries, the first two libraries were constructed in exactly the same manner as outlined in the first affinity maturation activity against the FAP antibody, respectively. In contrast, for the L3 and H3 affinity maturation libraries based on clone 3F2, two new primers were used to introduce the L3 of the parental clone (AM-3F 2-DPK 22-L3-ba: CACTTTGGTCCCCTGGCCGAACGT) CGGGGGAAGCA TAATACCCTGCTGACAGTAATACACTGC, where the underlined bases are 60% of the given base and 40% of the mixture N (mixture of four nucleotides A, C, G and T)) and H3(AM _3F2_ DP47_ H3_ fo: GGCCGTATATTACTGTGCG AA GGG TGGTT GGT GGT TTTAC TACTGGGGCCAAGGAAC, where the underlined bases are 60% given base and 40% mixture N, the italicized bases are 60% given base and 40% G, and the italicized underlined bases are 60% given base and 40% mixture K (mixture of two nucleotides G and T)). Library sizes were as follows: H1/H2 library (1.13X 10)10) L1/L2 library (5.6X 10)9) L3 library (2.3X 10)10) And H3 library (2.64X 10)10)。
Example 11
Selection of affinity matured anti-FAP clones
Selection was performed for the extracellular domain of human and murine Fibroblast Activation Protein (FAP) cloned upstream of the 6 x-lysine and 6x-his tags (see SEQ ID NOs: 53 and 55 of Table 5). Before selection, antigens were coated into immune tubes at a concentration of 1. mu.g/ml, 0.2. mu.g/ml or 0.02. mu.g/ml, depending on the library and round of selection. Selection and ELISA-based screening were performed as described for the first affinity maturation activity of the anti-FAP antibody. Secondary screening was performed using a ProteOn XPR36 biosensor (Biorad) and affinity purified Fab preparations were analyzed on the same instrument to determine kinetic rate constants and affinities. The following affinity matured clones were identified: 19G1 (see SEQ ID NOS: 121 and 123 of Table 3), 20G8 (see SEQ ID NOS: 125 and 127 of Table 3), 4B9 (see SEQ ID NOS: 129 and 131 of Table 3), 5B8 (see SEQ ID NOS: 133 and 135 of Table 3), 5F1 (see SEQ ID NOS: 137 and 139 of Table 3), 14B3 (see SEQ ID NOS: 141 and 143 of Table 3), 16F1 (see SEQ ID NOS: 145 and 147 of Table 3), 16F8 (see SEQ ID NOS: 149 and 151 of Table 3), O3C9 (see SEQ ID NOS: 153 and 155 of Table 3), 22A3 (see SEQ ID NOS: 165 and 167 of Table 3) and 29B11 (see SEQ ID NOS: 169 and 171 of Table 3) (all clones selected from the H1/H2 library and derived from clone 3F 2), O2D 2 (see SEQ ID NO: 7D 3 (see SEQ ID NO: 163 and 2) (see SEQ ID NO: 175 and 175) of Table 3) and clone No. 26 and 2 (see SEQ ID NO: 175) of Table 3) (all clones selected from the H638 and 175 and III) of Table 3) (these two clones were selected from the H1/H2 library and derived from the parental clone 4G 8).
Figures 21-25 show surface plasmon resonance sensorgrams of fabs against FAP for selected affinity maturation, while table 15 gives the corresponding affinities. The selected fabs span the high affinity range of the pM to nM range and are cross-reactive for human (hu) and murine (mu) FAP and cynomolgus (cyno) FAP as determined for the selected clones. The affinity matured anti-FAP Fab was converted to the Fab-IL2-Fab form. Binding specificity was shown by the lack of binding to DPPIV, which is a similar homologue of FAP expressed on HEK293 or CHO cells.
Table 15.
Affinity matured anti-FAP antibodies as kinetic equilibrium constants (K) for Fab fragments (monovalent binding)D) Summary of
Example 12
Construction of an affinity maturation library against TNC A2 (based on clone 2B10)
Affinity maturation libraries were constructed based on pre-selected antibodies from the preliminary TNC a2 selection. More precisely, it is based on the parent clone 2B10 and consists of two sub-libraries: 1) a VL sub-library randomized in the light chain CDR1 and CDR2(L1/L2) and 2) a VH sub-library randomized in the heavy chain CDR1 and CDR2 (H1/H2). These sub-libraries were pooled after transformation. Each of these sub-libraries was constructed by four subsequent amplification and assembly steps. For the L1/L2 library: 1) amplifying segment 1(LMB3-AM _ Vk1A30_ L1_ ba) and segment 2(RJH50(Vk1A30_ L1/L2_ fo) -RJH51(Vk1A30_ BsiWI _ ba)), 2) assembling segments 1 and 2 using external primers LMB3 and RJH51(Vk1A30_ BsiWI _ ba) to create templates, 3) amplifying segment 3(LMB3-AM _ Vk1A30_ L2_ ba) and segment 4(RJH52(Vk1A30_ L2/L3) - RJH51(Vk1a30_ BsiWI _ ba)), and 4) final assembly of fragments 3 and 4 using the same external primers as above. For the H1/H2 library: 1) amplifying segment 1(RJH53-AM _ DP88_ H1_ ba _ opt) and segment 2(RJH54(DP88_ H1/H2_ fo) -MS52), 2) assembling segments 1 and 2 using external primers RJH53 and MS52 to create templates, 3) amplifying segment 3(RJH53-AM _ DP88_ H2_ ba) and segment 4(RJH55(DP88_ H2H3_ fo) -MS52), and 4) finally assembling segments 3 and 4 using the same external primers as above. The final assembly product has been digested with NcoI/BsiWI (for VL sub-libraries) and MunI and NheI (for VH sub-libraries) and cloned in a similarly digested recipient vector. Library size Generation 1.16X 1010Individual clones.
Table 16.
Underlining: 60% of the original bases and 40% randomization to V
Bold: 60% of the original bases and 40% randomization to N
Table 17.
Underlined: 60% of the original bases and 40% randomization to V
Bold: 60% of the original bases and 40% randomization to N
Example 13
Selection of affinity matured anti-TNC A2 clones
Selection was performed against human TNC A2 expressed in E.coli cloned upstream of the avi tag and the 6 × his tag (see SEQ ID NO: 57 of Table 5). The antigen is biotinylated in vivo after expression. The selection in solution has been performed using reduced concentrations of human TNC a2 in the range of 100 to 2nM, as described for the preliminary TNC a2 selection. After identification of affinity matured clones by ELISA, secondary screening was performed using a ProteOn XPR36 biosensor (Biorad) and affinity purified Fab preparations were analyzed on the same instrument to determine kinetic rate constants and affinities. The following affinity matured clones were identified: 2B10_ O1F7 (see SEQ ID NOS: 201 and 203 of Table 3), 2B10_6H10 (see SEQ ID NOS: 205 and 207 of Table 3), 2B10_ C3A6 (see SEQ ID NOS: 185 and 187 of Table 3), 2B10_ D1A2 (see SEQ ID NOS: 189 and 191 of Table 3) and 2B10_ O7D8 (see SEQ ID NOS: 197 and 199 of Table 3), all of which are derived from a VL sub-library, and 2B10_ C3B6 (see SEQ ID NOS: 177 and 179 of Table 3) and 2B10_6A12 (see SEQ ID NOS: 181 and 183 of Table 3), both of which are derived from a VH sub-library. Furthermore, for clone 2B10_ D1A2, the V32D mutant was generated (see SEQ ID NOS: 193 and 195 in Table 3) (numbering according to Kabat).
Figure 26 shows the surface plasmon resonance sensorgram for Fab against TNC a2 for selected affinity maturation, while table 18 gives the corresponding affinities. Selected fabs span the high affinity range in the pM range.
Table 18.
Kinetic equilibrium constant (K) of affinity matured anti-TNC A2 antibody as Fab fragment (monovalent binding)D) A summary of (1).
Example 14
Preparation of Fab-IL2-Fab constructs based on 2B10, 3F2 and 4G8
An alternative purification method (in addition to that described in example 9) was developed for 2B10, 3F2 and 4G8 based Fab-IL2-Fab constructs. Although Fab-IL2-Fab constructs based on 3F2 and 4G8 could be purified by affinity chromatography using a protein A matrix (e.g., MabSelectSure), Fab-IL2-Fab constructs based on 2B10 must be purified by affinity chromatography on a protein G matrix. The purification procedure is based on the following four steps:
1. affinity chromatography with MabSelect Sure or protein G
2. Maintaining low pH for retroviral inactivation
3. Anion exchange chromatography-CaptoQ chromatography to remove DNA
4. Cation exchange chromatography-SP Sepharose FF chromatography to remove aggregates
To remove small scale aggregates, size exclusion chromatography on a Superdex 200 column (GEHealthcare) may alternatively be used.
Subsequently, an example of a purification protocol was given for the 3F 2-based Fab-IL 2-Fab. In a first step, supernatants from transient PEI-transfected HEK293 cells in Freestyle medium (Invitrogen) were adjusted to pH7 and applied to a MabSelect protein A column (GE Healthcare) with 100mM NaPO4250mM NaCl pH7, and eluted with 8.8mM sodium formate pH 3. Selected fractions were replaced in wash buffer and applied to CaptoQ column (GE Healthcare) using 10mm napo440mM NaCl pH6.5, and eluted with 2M NaCl. The flow through was adjusted to pH5 and applied to a SP Sepharose FF column (GE Healthcare), washed with 25mM sodium acetate, 25mM NaCl, pH5, and eluted with 25mM sodium acetate, 300mM NaCl, pH 5. The fractions were exchanged into the final formulation buffer. Figure 27 shows an overview of the purification procedure.
The purified 3F 2-based Fab-IL2-Fab was pure after purification (fig. 28A) and contained no aggregates (fig. 28B). The described purification protocol was applied to 4G 8-based Fab-IL 2-Fab. The 4G 8-based Fab-IL2-Fab behaves similarly to the 3F 2-based Fab-IL 2-Fab. The purified material was pure after purification and contained no aggregates (fig. 28, C-D).
The Fab-IL2-Fab form, which had 2B10(TNC A2 binding agent) as the Fab fragment, was purified. In a first step, the supernatant of transient PEI transfected HEK293 cells from Freestyle medium (Invitrogen) was adjusted to pH7 and applied to a protein G column (GE Healthcare) with 100mM NaPO4250mM NaCl pH7, and eluted with 8.8mM sodium formate pH 3. Selected fractions were replaced in wash buffer and applied to CaptoQ column (GE Healthcare) using 10mm napo440mM NaCl pH6.5, and eluted with 2M NaCl. The flow through was adjusted to pH5 and applied to a SP Sepharose FF column (GE Healthcare), washed with 25mM sodium acetate, 25mM NaCl, pH5, and eluted with 25mM sodium acetate, 300mM NaCl, pH 5. The fractions were exchanged into the final formulation buffer.
FIG. 29 shows results from (A) analytical characterization of products by SDS-PAGE (NuPAGE Novex Bis-Tris mini-gel, Invitrogen, MOPS running buffer, reducing and non-reducing) and (B) analysis of products after each of the three purification steps by size exclusion chromatography. 2.3% aggregates were detected in the final product.
Example 15
Stability test
Stability testing was performed on a Fab-IL2-Fab format with the fibronectin ectodomain-B binding agent L19 as a Fab fragment. For stability testing, the Fab-IL2-Fab construct was purified by protein a affinity chromatography and elution step at pH3, followed by size exclusion chromatography on a Superdex 200 column (GE Healthcare), as described. Three different buffers were tested and 20mM histidine hydrochloride, 140mM NaCl, pH6.0 was identified as a suitable buffer. Subsequently, L19Fab-IL2-Fab was formulated at a concentration of 6.3mg/ml in 20mM histidine hydrochloride, 140mM NaCl, pH6.0 and stored at room temperature and 4 ℃ for 4 weeks. Fig. 30 shows exemplary stability data: the probes were analyzed weekly for concentration by UV spectroscopy (fig. 30A) (after centrifugation to precipitate potential precipitated material) and for aggregate content by size exclusion chromatography on a Superdex 200 column (fig. 30B). The results show that no aggregation and degradation occurred when the constructs were stored at 4 ℃ or at room temperature for 28 days and at a concentration of 6mg/ml after freeze/thaw cycles. These data show that the Fab-IL2-Fab format is highly stable and behaves comparably to IgG antibodies.
Example 16
FAP-Targeted Fab-IL2-Fab binding affinity by surface plasmon resonance (Biacore)
The binding affinity of the three FAP targeting constructs, 3F2 Fab-IL2-Fab, 4G8 Fab-IL2-Fab and 3D9 Fab-IL2-Fab, was determined by surface plasmon resonance.
To determine FAP binding, FAP was captured by an immobilized anti-His antibody (Penta His, Qiagen #34660) and the construct was used as the analyte. The assay temperature was 25 ℃ and the Fab-IL2-Fab construct was diluted 1: 5 from 10nM to 3.2 pM. The following measurement parameters were applied: the binding time was 180 seconds, the dissociation 900 seconds, and the flow 90. mu.l/min. The chip was regenerated for 60 seconds with 10mM glycine pH 2. The curve was fitted with a 1: 1 model to obtain KDValue (Rmax local, RI ═ 0).
To determine the affinity for the IL-2 receptor (IL-2R) chain, the beta and gamma chains (b/g; protrusion-into-pocket construct) or the alpha chain (a) of IL-2R were immobilized on a chip and the Fab-IL2-Fab construct was used as analyte. The analysis temperature was 25 ℃. Fab-IL2-Fab constructs 3F2 and 3D9 were diluted 1: 2 from 25nM to 0.78nM, and the following measurement parameters were applied: the binding time was 100 seconds, dissociation 180 seconds, flow 90. mu.l/min. The chip was regenerated for 20 seconds with 10mM glycine pH 1.5. The Fab-IL2-Fab construct 4G8 was diluted from 100nM to 3.125nM and the following measurement parameters were applied: the binding time was 180 seconds, the dissociation 180 seconds, and the flow 40. mu.l/min. With 3M MgCl 2Regeneration is carried out for 30 seconds.
Table 19 gives a summary of the binding affinities of the 3F2Fab-IL2-Fab, 4G8Fab-IL2-Fab and 3D9Fab-IL2-Fab immunoconjugates. Picomolar affinity values reached the limit of Biacore detection. Figures 31-34 show the corresponding Biacore sensorgrams and affinities.
Table 19.
Kinetic equilibrium constants (K) for FAP-targeting Fab-IL2-Fab constructs directed against FAP and IL-2 receptors from different species as determined by surface plasmon resonanceD) Summary of
Example 17
TNC A2 targeting Fab-IL2-Fab binding affinity by surface plasmon resonance (Biacore)
To determine TNC a2 binding, biotinylated antigens (TNC fn5-a1-a2-A3 domains, fused together, expressed in e.coli, although fn5 and A3 domains are always of human origin, a1 and a2 domains are human, murine or cynomolgus) were immobilized on streptavidin chips and an immunoconjugate construct was used as the analyte. The analysis temperature was 25 ℃. Fab-IL2-Fab was diluted 1: 2 from 25nM to 0.39nM and the following measurement parameters were applied: the binding time was 180 seconds, the dissociation 180 seconds, and the flow 50. mu.l/min. Regeneration was carried out with 10mM glycine pH1.5 for 60 seconds. The curve was fitted with a 1: 1 model to obtain K DThe value is obtained. As negative controls, TNC domains 1 to 8(TNC 1-8HEK) produced in HEK cells were used.
To determine the affinity for the beta and gamma chains (b/g; protrusion-into-pocket constructs) of the IL-2 receptor (IL-2R), the IL-2R construct was immobilized on a chip and a Fab-IL2-Fab immunoconjugate was used as the analyte. The analysis temperature was 25 ℃. Fab-IL2-Fab immunoconjugates were diluted 1: 2 from 25nM to 0.78nM, and the following measurement parameters were applied: integration time 100 seconds, solutionFrom 180 seconds, flow 90. mu.l/min. Regeneration was completed with 10mM glycine pH1.5 for 20 seconds. The Fab-IL2-Fab construct was diluted from 100nM to 3.125nM and the following measurement parameters were applied: the binding time was 180 seconds, the dissociation 180 seconds, and the flow 40. mu.l/min. With 3M MgCl2Regeneration is carried out for 30 seconds. Table 20 gives a summary of the binding affinities of the 2B10 Fab-IL2-Fab immunoconjugates, and figure 35 shows the corresponding Biacore sensorgrams and affinities.
Table 20.
Kinetic equilibrium constants (K) for TNC A2-targeted Fab-IL2-Fab constructs against TNC A2 and IL-2 receptor-beta/gamma from different species as determined by surface plasmon resonanceD) Summary of
Example 18
Biological activity assay with Targeted IL-2 Fab-IL2-Fab immunoconjugates
The biological activity of the targeting IL-2Fab-IL2-Fab immunoconjugates was studied in several cell assays compared to IL-2 (Proleukin).
Induction of NK92 cell proliferation
The potential of targeting IL-2Fab-IL2-Fab molecules recognizing TNC a2(2B10) or FAP (3F2 and 4G8) to induce NK92 cell proliferation was investigated compared to IL-2(Proleukin) and IL-2L19 diabodies recognizing fibronectin-EDB.
2 μ g/ml human tenascin, FAP or fibronectin were coated overnight at 4 ℃ in 96-well flat-bottom ELISA plates. After blocking the plates, the Fab-IL2-Fab construct or diabody was titrated into the plates and incubated for 90 minutes at Room Temperature (RT) for binding. After vigorous washing to remove unbound constructs, IL-2 starved NK92 cells (10000 cells/well) were added). As a positive control, Proleukin was added to some wells in solution. Cells were incubated at 37 ℃ in a humidified incubator (with 5% CO)2) After 2 days of incubation, cells were lysed to determine proliferation by ATP measurement using the CellTiter Glo kit (Promega).
The results in FIG. 36 show that all Fab-IL2-Fab constructs are able to activate IL-2R signaling on NK92 cells and stimulate their proliferation. Due to the reduced binding affinity of the IL-2R β/γ heterodimer required for signaling, the efficacy of inducing cell growth is reduced by about 10-fold or more compared to IL-2 (Proleukin). However, the overall efficacy of the higher dose was retained and comparable to IL-2 (Proleukin).
Induction of STAT5 phosphorylation
In another experiment, we tested in solution different effector cell populations, including A) CD56 from human PBMC+NK cells, B) CD4+CD25-CD127+Helper T cell, C) CD3+，CD8+Cytotoxic T cells and D) CD4+CD25+FOXP3+STAT5 phosphorylation induction due to IL-2 mediated IL-2 receptor signaling after incubation with IL-2 Fab-IL2-Fab molecules (based on 4G8) that recognize FAP was tested on regulatory T cells (Tregs).
PBMCs isolated from healthy donor blood were treated with different concentrations of Proleukin or Fab-IL2-Fab for 20 minutes before they were fixed/permeabilized and stained with anti-phosphostat 5 antibody (Becton Dickinson) according to the supplier's instructions. Following intracellular staining of phosphorylated STAT5 and FOXP3, surface markers (CD3, CD4, CD8, CD56, and CD127) were stained to determine different subpopulations by flow cytometry (FACS Canto II).
The results in fig. 37 confirm the findings from fig. 36 and show that the 4G8Fab-IL2-Fab construct was able to activate IL-2R signaling and induce IL-2R downstream signaling and STAT5 phosphorylation on various IL-2R positive effector cells. The efficacy of inducing phosphorylation of STAT5 is reduced by about 10-fold or more compared to IL-2(Proleukin) due to the reduced binding affinity of the IL-2R β/γ heterodimer required for signaling. However, the overall efficacy of the higher dose was retained and comparable to IL-2 (Proleukin).
IFN-gamma release and proliferation induction in solution and after immobilization
In another experiment, we aimed to mimic what would occur in tumors, where a targeting IL-2 immunoconjugate is bound and immobilized on tumor cells or tumor stroma, and it can activate effector cells. To accomplish this, we performed an IFN- γ release assay with NK92 cells and a proliferation assay with the immunoconjugate in solution or we coated microtiter plates with TNC or FAP antigens such that the targeted IL-2 immunoconjugate was immobilized upon binding to TNC or FAP.
2 μ g/ml human tenascin or FAP were coated overnight at 4 ℃ in 96-well flat-bottom ELISA plates. After blocking the plates, the Fab-IL2-Fab construct was titrated into the plates and incubated for 90 minutes at RT for binding. After vigorous washing to remove unbound constructs, IL-2 starved NK92 cells (10000 cells/well) were added. As a positive control, Proleukin was added to some wells in solution. To determine proliferation, cells were incubated at 37 ℃ in a humidified incubator (with 5% CO)2) After 2 days of incubation, cells were lysed to determine proliferation by ATP measurement using the CellTiter Glo kit (Promega). IFN- γ release was measured in a different method using a human IFN- γ ELISA kit from Becton Dickinson after 24 hours incubation with Fab-IL-2-Fab in the cell supernatant.
The results confirmed that all Fab-IL2-Fab constructs targeting FAP, TNC a1 or TNC a2 were able to activate IL-2R signaling on NK92 cells and induce cell proliferation (fig. 38A) and IFN- γ secretion (fig. 38C) when present in solution. Due to the reduced binding affinity of the IL-2R β/γ heterodimer required for signaling, the efficacy of inducing IFN- γ release was reduced by about 10-fold or more compared to IL-2 (Proleukin). However, the overall efficacy of the higher dose was retained and comparable to IL-2 (Proleukin). If microtiter plates were coated with FAP or TNC and the constructs were immobilized on plates, all Fab-IL2-Fab constructs targeting FAP, TNC a1 or TNC a2 were still able to activate IL-2R signaling on NK92 cells and induce cell growth (fig. 38B) and IFN- γ release (fig. 38D). The difference in efficacy between uncoated IL-2(Proleukin) and the immobilized Fab-IL2-Fab construct was an order of magnitude higher compared to the assays performed in solution, however, the overall efficacy of the higher doses was retained and comparable to IL-2 (Proleukin).
These data strongly support the idea of preparing targeted immunoconjugates with low systemic exposure, but accumulated at the site of the disease where they mediate their function.
In the following examples, we investigated whether these in vitro properties translate into superior in vivo efficacy in xenograft models.
Example 19
In vivo efficacy of targeted Fab-IL2-Fab immunoconjugates against FAP and TNC A2 in xenografts of human tumor cell lines
Targeted Fab-IL2-Fab immunoconjugates against FAP and TNC a2 were tested for anti-tumor efficacy in several xenograft models.
LS174T xenograft model
TNC a2 targeting 2B10Fab-IL2-Fab immunoconjugates were tested in a human colorectal LS174T cell line injected intrasplenically into SCID mice.
LS174T cells (human colon cancer cells) were originally obtained from ECACC (European Collection of Cell cultures) and, after expansion, were deposited in Glycart internal Cell banks. LS174T was cultured in MEM Eagle's medium containing 10% FCS (PAA Laboratories, Austria), 1% Glutamax and 1% MEM non-essential amino acids (Sigma). Cells were incubated at 37 ℃ in a water-saturated atmosphere at 5% CO2Culturing. Intrasplenic injections using passage 15 in vitro were 92.8% viable. A small incision was made in the left abdominal region of anesthetized SCID/beige mice. Injection of 50 microliters (3 x10 in AimV medium) through the abdominal wall just below the splenic tunica 6LS174T cells). The skin wound was closed using a clip (clamp).
Female SCID mice (aged 8-9 weeks at the start of the experiment) (purchased from Taconics, Denmark) were maintained under pathogen-free conditions at a daily period of 12 hours light/12 hours dark according to promised guidelines (GV-Solas; Felassa; TierschG). Experimental study protocol was reviewed and approved by local government (P2008016). After arrival, animals were maintained for one week for adaptation to the new environment and observed. Continuous health monitoring is performed periodically.
Mice were injected intrasplenically with 3x10 on study day 06LS174T cells were randomized and weighed. Mice were injected i.v. with TNC A2-targeted 2B10Fab-IL2-Fab (Fab-IL2-Fab-SH2B10), fibronectin-EDB-targeted L19 IL-2-diabody, or Proleukin for three weeks 1 week after tumor cell injection.
All mice were injected i.v. with 200. mu.l of the appropriate solution. Mice in the vehicle group were injected with PBS, and the treatment group was injected with Fab-IL2-Fab construct, diabody, or Proleukin. To obtain the appropriate amount of immunoconjugate per 200 μ l, the stock solution was diluted with PBS as necessary.
Figure 39 shows the superior efficacy of TNC a 2-targeting 2B10Fab-IL2-Fab immunoconjugates (TNC a2 targeted 2B10Fab-IL2-Fab immunoconjugates) for prolonged median survival compared to naked IL-2(Proleukin) and fibronectin-EDB-targeting IL-2 diabody molecules.
Table 21.
ACHN xenograft model
FAP targeting 3F2 or 4G8 Fab-IL2-Fab immunoconjugates were tested in the human renal cell line ACHN injected intrarenally into SCID mice.
ACHN cells (human renal adenocarcinoma cells) were originally obtained from ATCC (American Type Culture Collection) and were deposited in the Glycart internal cell bank after expansion. ACHN was cultured in DMEM containing 10% FCS. Cells were incubated at 37 ℃ in a water-saturated atmosphere at 5% CO2And (5) culturing. Intrarenal injection was performed using passage 9 in vitro with a viability of 97.7%. Small incisions (2cm) were made in the right flank and abdominal wall of anesthetized SCID mice. 50 μ l (1X 10 in AimV Medium) was injected 2mm under the kidney middle capsule6ACHN cells) cell suspension. The skin wound and abdominal wall were closed using clips.
Female SCID mice (aged 8-9 weeks at the start of the experiment) (purchased from Charles River, Sulzfeld, Germany) were maintained under specific pathogen-free conditions with a daily period of 12 hours of light/12 hours of darkness according to the promised guidelines (GV-Solas; Felasa; tirschg). Experimental study protocol was reviewed and approved by local government (P2008016). After arrival, animals were maintained for one week for adaptation to the new environment and observed. Continuous health monitoring is performed periodically.
Mice were injected intrarenally with 1x10 on study day 06Individual ACHN cells, randomized and weighed. Mice were injected i.v. with Proleukin, L19IL-2 diabody or FAP targeting 4G8 or 3F2Fab-IL2-Fab immunoconjugates for three weeks 1 week after tumor cell injection twice a week.
All mice were injected i.v. with 200. mu.l of the appropriate solution. Mice in the vehicle group were injected with PBS and treatment groups were injected with Proleukin, L19IL-2 diabody or FAP-targeted 4G8 or 3F2Fab-IL2-Fab immunoconjugates. To obtain the appropriate amount of immunoconjugate per 200 μ l, the stock solution was diluted with PBS as necessary.
Figure 40 shows the superior efficacy of FAP-targeted 3F2 and 4G8 Fab-IL2-Fab immunoconjugates for extended median survival compared to naked IL-2(Proleukin) and fibronectin-EDB targeted IL-2 diabody molecules. The 4G 8-based Fab-IL2-Fab (which has a higher affinity for murine FAP) mediated superior efficacy compared to the 3F 2-based Fab-IL 2-Fab.
Table 22.
A549 xenograft model
TNC a2 targeting 2B 10Fab-IL2-Fab immunoconjugates were tested in human NSCLC cell line a549 i.v injected into SCID-human Fc γ RIII transgenic mice.
A549 non-small cell lung cancer cells were originally obtained from ATCC (CCL-185) and were deposited in the Glycart internal cell bank after expansion. Tumor cell lines were grown in DMEM containing 10% FCS (Gibco) at 37 ℃ in a water-saturated atmosphere at 5% CO 2And (5) performing conventional culture. The 2 nd generation was used for transplantation with a viability of 98%. Each animal was 2x106Individual cells were i.v injected into the tail vein in 200. mu.l of Aim V cell culture medium (Gibco)
Female SCID-Fc γ RIII mice (GLYCART-RCC) (aged 8-9 weeks at the start of the experiment) (bred at RCC, Switzerland) were maintained under pathogen-free conditions with a daily period of 12 hours of light/12 hours of darkness according to the promised guidelines (GV-Solas; Felassa; TierschG). Experimental study protocol was reviewed and approved by local government (P2008016). After arrival, animals were maintained for one week for adaptation to the new environment and observed. Continuous health monitoring is performed periodically.
Mice were injected i.v. with 5x10 on study day 06A549 cells, randomizing, andand (5) weighing. Mice were injected i.v. with 2B10 Fab-IL2-Fab or L19IL-2 diabody for three weeks 1 week after tumor cell injection twice a week.
All mice were injected i.v. with 200. mu.l of the appropriate solution. Mice in the vehicle group were injected with PBS and the treatment group was injected with either the 2B10 Fab-IL2-Fab construct or the L19IL-2 diabody. To obtain the appropriate amount of immunoconjugate per 200 μ l, the stock solution was diluted with PBS as necessary.
Figure 41 shows that TNC a2 targeting 2B10 Fab-IL2-Fab immunoconjugates mediate superior efficacy with respect to prolonged median survival compared to fibronectin-EDB targeting IL-2 diabody molecules.
Table 23.
Example 20
Purification of targeting GM-CSF Fab-GM-CSF-Fab immunoconjugates
Initial purification of Fab-GM-CSF-Fab immunoconjugates with L19 (fibronectin ectodomain-B binding agent) as Fab fragment was performed from the supernatant of transiently transfected HEK 293 EBNA cells. Briefly, Fab-GM-CSF-Fab was purified by protein A, followed by size exclusion chromatography. The protein A column was equilibrated in 20mM sodium phosphate, 20mM sodium citrate pH 7.5. The supernatant was loaded and the column was washed first with 20mM sodium phosphate, 20mM sodium citrate pH7.5, followed by 20mM sodium phosphate, 20mM sodium citrate, 100mM sodium chloride, pH 7.5. Targeted GM-CSF was eluted with 20mM sodium citrate, 100mM sodium chloride, 100mM glycine pH3 and subsequently neutralized. For the formulations, the following buffers were applied: 25mM potassium phosphate, 125mM sodium chloride, 100mM glycine pH 6.7.
FIG. 42 shows the elution profile from purification and results from analytical characterization of the product by SDS-PAGE (NuPAGENOVEx Bis-Tris mini-gel, Invitrogen, MOPS running buffer, reducing and non-reducing). The yield was 4.8 mg/L.
Example 21
Biological activity assays with targeting GM-CSF Fab-GM-CSF-Fab immunoconjugates
Subsequently, purified Fab-GM-CSF-Fab immunoconjugates with L19 (fibronectin ectodomain-B binding agent) as Fab were analyzed in a GM-CSF dependent proliferation assay. Briefly, TF-1 cells (which were dependent on GM-CSF for growth) were seeded into 96-well flat-bottom plates at 10000 cells/well after overnight starvation. Human recombinant GM-CSF (Miltenyi #130-093-862) or Fab-GM-CSF-Fab immunoconjugates were titrated into cells in solution. At 37 ℃ in a humidified incubator with 5% CO2After 2 days of proliferation in case (2), cells were lysed and ATP content was measured using the CellTiter Glo assay from Promega. For calculation, cells not treated with GM-CSF were set to 0% growth. The results in FIG. 43 show that Fab-GM-CSF-Fab immunoconjugates induce strong proliferation of TF-1 cells.
Example 22
Purification of Targeted IL-12 Fab-IL12-Fab immunoconjugates
Initial purification of Fab-IL12-Fab immunoconjugates with 4G8(FAP binding agent) as Fab fragment was performed from the supernatant of transiently transfected HEK 293 EBNA cells. Briefly, Fab-IL12-Fab was purified by protein A followed by size exclusion chromatography. The protein A column was equilibrated with 20mM sodium phosphate, 20mM sodium citrate pH 7.5. The supernatant was loaded and the column was washed with 20mM sodium phosphate, 20mM sodium citrate, 500mM sodium chloride pH 7.5. A second wash was performed with 13.3mM sodium phosphate, 20mM sodium citrate, 500mM sodium chloride, pH 5.45. After a third wash with 10mM MES, 50mM sodium chloride pH5, the targeting IL-12 was eluted with 20mM sodium citrate, 100mM sodium chloride, 100mM glycine, pH3 and subsequently neutralized. For the formulations, the following buffers were applied: 25mM potassium phosphate, 125mM sodium chloride, 100mM glycine pH 6.7.
FIG. 44 shows the elution profile from purification and results from analytical characterization of the product by SDS-PAGE (NuPAGENOVEx Bis-Tris mini-gel, Invitrogen, MOPS running buffer, reducing and non-reducing). The yield thereof was found to be 43 mg/L.
Example 23
Biological activity assay with Targeted IL-12 Fab-IL12-Fab immunoconjugates
Subsequently, the effect of IL-12 and purified 4G8Fab-IL12-Fab immunoconjugates was compared by analyzing IL-12 induced IFN- γ release on purified Fab-IL12-Fab immunoconjugates with 4G8(FAP binding agent) as Fab using PBMCs isolated from fresh human blood of healthy donors.
Briefly, PBMCs were isolated from fresh human blood of healthy donors and plated in 96-well U-bottom plates (1.5x 10)5Individual cells/well) were inoculated in AIM V medium. A constant concentration of 10ng/ml hu IL-2(Peprotech) was added to all wells. The Fab-IL12-Fab construct was diluted in culture medium and titrated onto PBMCs. Supernatants were collected after about 20 hours to determine IFN- γ concentrations using hu IFN- γ ELISA kit II from Becton Dickinson (# 550612).
The results in figure 45 show that a) selected amounts of human (hu) IL-2 alone and IL-12 alone were unable to induce significant IFN- γ release from human PBMCs, while the combination of these two cytokines resulted in significant IFN- γ release from PBMCs. B) The Fab-IL-12-Fab construct induces IFN- γ release from human PBMC in a concentration-dependent manner in the presence of 10ng/ml human IL-2.
Example 24
Purification of Targeted IFN-alpha Fab-IFN alpha 2-Fab immunoconjugates
Initial purification of Fab-IFN α 2-Fab immunoconjugates with L19 (fibronectin extracellular domain-B binding agent) as Fab fragment was performed from the supernatant of transiently transfected HEK 293 EBNA cells. Briefly, Fab-IFN α 2-Fab was purified by protein A followed by size exclusion chromatography. The protein A column was equilibrated with 20mM sodium phosphate, 20mM sodium citrate pH 7.5. The supernatant was loaded and the column was washed first with 20mM sodium phosphate, 20mM sodium citrate pH7.5, followed by 20mM sodium phosphate, 20mM sodium citrate, 100mM sodium chloride, pH 7.5. Fab-IFN alpha 2-Fab was eluted with 20mM sodium citrate, 100mM sodium chloride, 100mM glycine pH3 and subsequently neutralized. For the formulations, the following buffers were applied: 25mM potassium phosphate, 125mM sodium chloride, 100mM glycine pH 6.7.
FIG. 46 shows the elution profile from purification and results from analytical characterization of the product by SDS-PAGE (NuPAGENOVEx Bis-Tris mini-gel, Invitrogen, MOPS running buffer, reducing and non-reducing). The yield was 8.4 mg/L.
Example 25
Biological activity assay with IFN-alpha Fab-IFN alpha 2-Fab immunoconjugates
Subsequently, purified Fab-IFN α 2-Fab immunoconjugates with L19 (fibronectin extracellular domain-B binding agent) as Fab were analyzed for IFN- α -induced inhibition of proliferation of Jurkat T cells and a549 tumor cells, comparing the effect of IFN- α (Roferon a, Roche) and purified L19 Fab-IFN α 2-Fab immunoconjugates. Briefly, A549 and Jurkat T cells susceptible to IFN- α -induced proliferation inhibition were seeded at 5000 cells/well (A549) or 10000 cells/well (Jurkat) in 96-well flat-bottom plates. Dilutions of Roferon A (Roche) or Fab-IFN alpha 2-Fab in appropriate cell culture mediaTitrate onto cells in solution. At 37 ℃ in a humidified incubator with 5% CO2After 2 days of proliferation in case (2), cells were lysed and ATP content was measured using the CellTiter Glo assay from Promega. For calculation, cells not treated with IFN- α were set to 0% growth.
The results in figure 47 show that the Fab-IFN α 2-Fab construct inhibited the proliferation of a) Jurkat T cells and B) a549 cells in a concentration-dependent manner comparable to IFN- α (RoferonA).
Example 26
Preparation of MCSP targeting Fab-IL2-Fab immunoconjugates
Humanized anti-MCSP MHLG antibodies, as described in WO 2006/100582 (see in particular example 1 therein), were generated (which is incorporated herein by reference in its entirety) and converted to the Fab-IL2-Fab form (see SEQ ID NOS: 255, 256, 261, 262).
A humanized anti-MCSP MHLG1 antibody was generated as follows: the murine amino acid sequences (light and heavy chains, see below) of anti-MCSP antibody 225.28 were aligned with a collection of human germline antibody V genes and classified according to sequence identity and homology.
225.28 light chain; GenBank accession number CAA65007(SEQ ID 267):
DIELTQSPKFMSTSVGDRVSVTCKASQNVDTNVAWYQQKPGQSPEPLLFSASYRYTGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPLTFGGGTKLEIK
225.28 heavy chain; GenBank accession No. (SEQ ID 268):
QVKLQQSGGGLVQPGGSMKLSCVVSGFTFSNYWMNWVRQSPEKGLEWIAEIRLKSNNFGRYYAESVKGRFTISRDDSKSSAYLQMINLRAEDTGIYYCTSYGNYVGHYFDHWGQGTTVTVSS
potential acceptor sequences were selected based on high overall homology and the presence of correct canonical residues already in the acceptor sequence. The human germline sequence IGHV3-15(IMGT accession number X92216) was selected as acceptor for the heavy chain and the sequence IGKV1-9(IMGT accession number Z00013) was selected as acceptor for the light chain. Humanized constructs were denoted M-KV1 (see SEQ ID NOs: 263, 264, 269, 270), 7(SEQ ID NOs: 265, 266, 271, 272) and 9(SEQ ID NOs: 253, 254, 259, 260) (for the light chain) and MHLG1 (see SEQ ID NOs: 251, 252, 257, 258) (for the heavy chain).
Genes for those designed antibody sequences were generated by conventional PCR techniques and fused with human IgG1 and kappa constant domains to construct expression plasmids.
Antibodies are expressed as IgG or as Fab-IL2-Fab fusion proteins in mammalian cell culture systems, such as HEK or CHO, and purified via protein a and size exclusion chromatography. Comparison of the binding data for the light chain variant M-KV1 with M-KV7 revealed that the proline residue at Kabat position 46 is critical for functional binding to antigen. Two different methods were used to ensure the presence of this amino acid: A) so-called back mutations were introduced into the human framework of IGKV 1-9. And B) in order to avoid the presence of back mutations, the entire framework 2 region (Kabat positions 35 to 49) was replaced by the corresponding region of a human antibody with GenBank entry AAA 17574. This antibody naturally has a proline residue at position 46.
The MCSP targeted MHLG or MHLG1KV9 Fab-IL2-Fab was purified by the method described above (example 9) comprising an affinity step (protein a) followed by size exclusion chromatography (Superdex 200, GE Healthcare). The protein a column was equilibrated in 20mM sodium phosphate, 20mM sodium citrate pH7.5, the supernatant was loaded, and the column was washed with 20mM sodium phosphate, 20mM sodium citrate, 500mM sodium chloride, pH7.5, followed by 13.3mM sodium phosphate, 20mM sodium citrate, 500mM sodium chloride, pH 5.45. Optionally, a third wash with 10mM MES, 50mM NaCl, pH5 is included. Fab-IL2-Fab was eluted with 20mM sodium citrate, 100mM sodium chloride, 100mM glycine, pH 3. The eluted fractions were combined and refined by size exclusion chromatography in the final formulation buffer as follows: 25mM potassium phosphate, 125mM sodium chloride, 100mM glycine pH 6.7.
FIG. 48 (for MHLG Fab-IL2-Fab) and FIG. 49 (for MHLG1 Fab-IL2-Fab) show (A) elution profiles from purification and (B) results from analytical characterization of MCSP targeting MHLG or MHLG1 Fab-IL2-Fab by SDS-PAGE (NuPAGE Novexbis-Tris mini-gel, Invitrogen, MOPS running buffer, reducing and non-reducing).
Example 27
Biological activity assay with MCSP targeting Fab-IL2-Fab immunoconjugates
Subsequently, purified Fab-IL2-Fab immunoconjugates with MHLG KV9 or MHLG1 KV9(MCSP binders) as Fab were analyzed for IL-2 induced IFN- γ release, comparing the effect of purified 4G8Fab-IL2-Fab (FAP binder) and MHLG or MHLG1 Fab-IL2-Fab on NK92 cells.
IL-2 starved NK92 cells (preincubated for 2 hours in the absence of IL-2) were seeded in NK cell culture medium (MEMa + 10% FCS + 10% horse serum +0.1mM 2-mercaptoethanol +0.2mM inositol +0.02mM folate) in 96-well U-plates (105 cells/well). MCSP-targeted Fab-IL-2-Fab immunoconjugates were diluted in NK cell culture media and titrated onto NK92 cells, in direct comparison to FAP-targeted 4G 8-based Fab-IL2-Fab immunoconjugates. Supernatants were collected after 22 to 24 hours to determine IFN-. gamma.concentrations using human IFN-. gamma.ELISA kit II from Becton Dickinson (# 550612).
The results in figure 50 (for MHLG KV9Fab-IL2-Fab) and figure 51 (for MHLG1 KV9Fab-IL2-Fab) show that all Fab-IL2-Fab immunoconjugates targeting MCSP or FAP induce comparable IFN- γ secretion in NK92 cells in a concentration-dependent manner, independent of the antigen-binding moiety used.
Example 28
Cell binding assay with MCSP targeting MHLG1 KV9 Fab-IL2-Fab immunoconjugates
Purified MCSP targeting MHLG1-KV9 Fab-IL2-Fab immunoconjugates were tested for binding to Colo38 melanoma cells expressing human MCSP by flow cytometry. Briefly, cells were harvested, counted, and examined for viability. Cells were conditioned to 1.112 × 10 in PBS/0.1% BSA6One (viable) cell/ml and equally divided into 180. mu.l/well (200' 000 cells/well) in round bottom 96-well plates. Mu.l MHLG1 KV9 Fab-IL2-Fab immunocytokine (at different dilutions) was added to the wells containing the cells and incubated for 30 min at 4 ℃. Subsequently, the cells were harvested by centrifugation (4 min, 400Xg), washed with 150. mu.l/well PBS/0.1% BSA, resuspended, and combined with 12. mu.l/well secondary antibody (FITC-conjugated AffiniPure F (ab')2Fragment goat anti-human F (ab')2(Jackson Immuno Research Lab # 109-. Subsequently, cells were washed in 150 μ l/well PBS/0.1% BSA, followed by a washing step in PBS, collected by centrifugation (4 min, 400Xg), and resuspended with 200 μ l/well PBS/0.1% BSA, which contained Propidium Iodide (PI). Measurements were performed using a FACSCAntoII machine (Software FACS Diva). The results are presented in figure 52, which shows that the MCSP targeted MHLG1 KV9 Fab-IL2-Fab immunoconjugate binds very well to Colo38 cells in a dose dependent manner.
***
It is to be appreciated that the detailed description section, and not the summary and abstract sections, is intended to be used to interpret the claims. The summary and abstract sections may list one or more, but not all exemplary embodiments of the invention, as contemplated by the inventors, and as such, are not intended to limit the invention and the appended claims in any way.
The invention has been described above with the aid of illustrative functional components that perform the specified functions and relationships thereof. Boundaries of such functional elements have been arbitrarily defined herein for convenience of description. Alternate boundaries can be defined such that the specified functions and relationships thereof are appropriately performed.
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.
The breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

Claims (172)

1. An immunoconjugate, comprising:
(a) at least a first single-chain effector moiety, wherein the effector moiety is a cytokine; and
(b) first and second antigen binding moieties, wherein each of the first and second antigen binding moieties is a Fab molecule,
wherein the first antigen binding moiety is linked at its heavy or light chain carboxy-terminal amino acid to the amino-terminal amino acid of the single-chain effector moiety, and wherein the single-chain effector moiety is in turn linked at its carboxy-terminal amino acid to the amino-terminal amino acid of the heavy or light chain of the second antigen binding moiety.
2. The immunoconjugate of claim 1, wherein said immunoconjugate consists essentially of a first effector moiety and first and second antigen binding moieties linked by one or more linker sequences.
3. The immunoconjugate of claim 1 or 2, wherein a proteolytic cleavage site is located between said antigen binding moiety and said effector moiety.
4. The immunoconjugate of claim 1 or 2, wherein the variable regions of said first and second antigen binding moieties are specific for the same antigen.
5. The immunoconjugate of claim 1 or 2, wherein the variable regions of said first and second antigen binding moieties are specific for different antigens.
6. The immunoconjugate of claim 4, wherein said first and second antigen binding moieties are specific for the extra domain B of fibronectin (EDB).
7. The immunoconjugate of claim 5, wherein said first or said second antigen binding moiety is specific for the extra domain B of fibronectin (EDB).
8. The immunoconjugate of claim 6 or claim 7, wherein said one or two antigen binding moieties specific for EDB compete with monoclonal antibody L19 for binding to an epitope of EDB.
9. The immunoconjugate of claim 6 or claim 8, wherein said immunoconjugate comprises the polypeptide sequence of SEQ ID NO 95.
10. The immunoconjugate of claim 6 or claim 8, wherein said immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence having at least 80% identity to SEQ ID NO: 108.
11. The immunoconjugate of claim 10, wherein said immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide sequence of SEQ ID No. 108.
12. The immunoconjugate of claim 6 or claim 8, wherein said immunoconjugate comprises the polypeptide sequence of SEQ ID NO 104.
13. The immunoconjugate of claim 6 or claim 8, wherein said immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence having at least 80% identity to SEQ ID NO: 117.
14. The immunoconjugate of claim 13, wherein said immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide sequence of SEQ ID NO: 117.
15. The immunoconjugate of claim 6 or claim 8, wherein said immunoconjugate comprises the polypeptide sequence of SEQ ID NO 105.
16. The immunoconjugate of claim 6 or claim 8, wherein said immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence having at least 80% identity to SEQ ID NO: 118.
17. The immunoconjugate of claim 16, wherein said immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide sequence of SEQ ID No. 118.
18. The immunoconjugate of claim 6 or claim 8, wherein said immunoconjugate comprises the polypeptide sequence of SEQ ID NO 106.
19. The immunoconjugate of claim 6 or claim 8, wherein said immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence having at least 80% identity to SEQ ID NO: 119.
20. The immunoconjugate of claim 19, wherein said immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide sequence of SEQ ID NO: 119.
21. The immunoconjugate of claim 6 or claim 8, wherein said immunoconjugate comprises the polypeptide sequence of SEQ ID NO 107.
22. The immunoconjugate of claim 6 or claim 8, wherein said immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence having at least 80% identity to SEQ ID NO: 120.
23. The immunoconjugate of claim 22, wherein said immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide sequence of SEQ ID NO 120.
24. The immunoconjugate of claim 6 or claim 8, wherein said immunoconjugate comprises the polypeptide sequence of SEQ ID NO 96.
25. The immunoconjugate of any one of claims 6 to 8, wherein said immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence having at least 80% identity to SEQ ID NO: 109.
26. The immunoconjugate of claim 25, wherein said immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide sequence of SEQ ID No. 109.
27. The immunoconjugate of any one of claims 9-23, wherein said immunoconjugate further comprises the polypeptide sequence of SEQ ID NO: 96.
28. The immunoconjugate of claim 4, wherein said first and second antigen binding moieties are specific for the A1 domain of tenascin (TNC-A1).
29. The immunoconjugate of claim 5, wherein said first or said second antigen binding moiety is specific for the A1 domain of tenascin (TNC-A1).
30. The immunoconjugate of claim 28 or 29, wherein said one or two antigen binding moieties specific for TNC-a1 compete with monoclonal antibody F16 for binding to the TNC-a1 epitope.
31. The immunoconjugate of claim 28 or claim 30, wherein said immunoconjugate comprises the polypeptide sequence of SEQ ID NO 99.
32. The immunoconjugate of claim 28 or claim 30, wherein said immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence having at least 80% identity to SEQ ID NO: 112.
33. The immunoconjugate of claim 32, wherein said immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide sequence of SEQ ID No. 112.
34. The immunoconjugate of claim 28 or claim 30, wherein said immunoconjugate comprises the polypeptide sequence of SEQ ID NO: 100.
35. The immunoconjugate of claim 28 or claim 30, wherein said immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence having at least 80% identity to SEQ ID No. 113.
36. The immunoconjugate of claim 35, wherein said immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide sequence of SEQ ID No. 113.
37. The immunoconjugate of any one of claims 28 to 30, wherein said immunoconjugate comprises the polypeptide sequence of SEQ ID No. 101.
38. The immunoconjugate of any one of claims 28 to 30, wherein said immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence having at least 80% identity to SEQ ID NO: 114.
39. The immunoconjugate of claim 38, wherein said immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide sequence of SEQ ID No. 114.
40. The immunoconjugate of any one of claims 34 to 36, wherein said immunoconjugate further comprises the polypeptide sequence of SEQ ID No. 101.
41. The immunoconjugate of claim 28, wherein said immunoconjugate comprises polypeptide sequence SEQ id no: 215.
42. The immunoconjugate of claim 28, wherein said immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence having at least 80% identity to SEQ ID No. 216.
43. The immunoconjugate of claim 42, wherein said immunoconjugate comprises a polypeptide sequence encoded by polynucleotide sequence SEQ ID NO 216.
44. The immunoconjugate of claim 28 or 29, wherein said immunoconjugate comprises the polypeptide sequence of seq id No. 235.
45. The immunoconjugate of claim 28 or 29, wherein said immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence having at least 80% identity to SEQ id No. 236.
46. The immunoconjugate of claim 45, wherein said immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide sequence of SEQ ID NO 236.
47. The immunoconjugate of any one of claims 41 to 43, wherein said immunoconjugate further comprises the polypeptide sequence of SEQ ID NO 235.
48. The immunoconjugate of claim 28 or claim 29, wherein said immunoconjugate comprises a heavy chain variable region sequence of SEQ ID NO:13 or SEQ ID NO: 15.
49. The immunoconjugate of claim 28 or claim 29, wherein said immunoconjugate comprises the light chain variable region sequence of SEQ ID No. 9 or SEQ ID No. 11.
50. The immunoconjugate of claim 48, wherein said immunoconjugate further comprises a light chain variable region sequence of SEQ ID NO 9 or SEQ ID NO 11.
51. The immunoconjugate of claim 48, wherein said heavy chain variable region sequence is encoded by a polynucleotide sequence having at least 80% identity to SEQ ID NO 14 or SEQ ID NO 16.
52. The immunoconjugate of claim 51, wherein said heavy chain variable region sequence is encoded by the polynucleotide sequence of SEQ ID NO 14 or SEQ ID NO 16.
53. The immunoconjugate of claim 49, wherein said light chain variable region sequence is encoded by a polynucleotide sequence having at least 80% identity to SEQ ID NO 10 or SEQ ID NO 12.
54. The immunoconjugate of claim 53, wherein said light chain variable region sequence is encoded by the polynucleotide sequence of SEQ ID NO 10 or SEQ ID NO 12.
55. The immunoconjugate of claim 4, wherein said first and second antigen binding moieties are specific for the A2 domain of tenascin (TNC-A2).
56. The immunoconjugate of claim 5, wherein said first or said second antigen binding moiety is specific for the A2 domain of tenascin (TNC-A2).
57. The immunoconjugate of claim 55 or 56, wherein said immunoconjugate comprises a heavy chain variable region sequence selected from the group of SEQ ID NO: SEQ ID NO 7, SEQ ID NO 179, SEQ ID NO 183, SEQ ID NO 187, SEQ ID NO 191, SEQ ID NO 195, SEQ ID NO 199, SEQ ID NO 203 and SEQ ID NO 207.
58. The immunoconjugate of claim 55 or 56, wherein said immunoconjugate comprises a light chain variable region sequence selected from the group of SEQ ID NO:3, 5, SEQ ID NO; 177, 181, 185, 189, 193, 197, 201 and 205.
59. The immunoconjugate of claim 57, wherein said immunoconjugate further comprises a light chain variable region sequence selected from the group of SEQ ID NO:3, 5, SEQ ID NO; 177, 181, 185, 189, 193, 197, 201, 205.
60. The immunoconjugate of claim 59, wherein said immunoconjugate comprises (i) the heavy chain variable region sequence of SEQ ID NO:7 and the light chain variable region sequence of SEQ ID NO: 5; (ii) the heavy chain variable region sequence of SEQ ID NO 179 and the light chain variable region sequence of SEQ ID NO 177; (iii) the heavy chain variable region sequence of SEQ ID NO:183 and the light chain variable region sequence of SEQ ID NO: 181; (iv) the heavy chain variable region sequence of SEQ ID NO. 187 and the light chain variable region sequence of SEQ ID NO. 185; (v) the heavy chain variable region sequence of SEQ ID NO. 191 and the light chain variable region sequence of SEQ ID NO. 189; (vi) the heavy chain variable region sequence of SEQ ID NO:195 and the light chain variable region sequence of SEQ ID NO: 193; (vii) the heavy chain variable region sequence of SEQ ID NO:199 and the light chain variable region sequence of SEQ ID NO: 197; (viii) the heavy chain variable region sequence of SEQ ID NO. 203 and the light chain variable region sequence of SEQ ID NO. 201; or (ix) the heavy chain variable region sequence of SEQ ID NO:207 and the light chain variable region sequence of SEQ ID NO: 205.
61. The immunoconjugate of claim 60, wherein said immunoconjugate comprises a heavy chain variable region sequence of SEQ ID NO 7 and a light chain variable region sequence of SEQ ID NO 5.
62. The immunoconjugate of claim 57, wherein said heavy chain variable region sequence is encoded by a polynucleotide sequence having at least 80% identity to a sequence selected from the group of SEQ ID NO: SEQ ID NO 8, 180, 184, 188, 192, 196, 200, 204 and 208.
63. The immunoconjugate of claim 62, wherein said heavy chain variable region sequence is encoded by a polynucleotide sequence selected from the group of SEQ ID NO: SEQ ID NO. 8, SEQ ID NO. 180, SEQ ID NO. 184, SEQ ID NO. 188, SEQ ID NO. 192, SEQ ID NO. 196, SEQ ID NO. 200, SEQ ID NO. 204 and SEQ ID NO. 208.
64. The immunoconjugate of claim 58, wherein said light chain variable region sequence is encoded by a polynucleotide sequence having at least 80% identity to a sequence selected from the group of SEQ ID NO: SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 178, SEQ ID NO 182, SEQ ID NO 186, SEQ ID NO 190, SEQ ID NO 194, SEQ ID NO 198, SEQ ID NO 202 and SEQ ID NO 206.
65. The immunoconjugate of claim 64, wherein said light chain variable region sequence is encoded by a polynucleotide sequence selected from the group of SEQ ID NO: SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 178, SEQ ID NO. 182, SEQ ID NO. 186, SEQ ID NO. 190, SEQ ID NO. 194, SEQ ID NO. 198, SEQ ID NO. 202 and SEQ ID NO. 206.
66. The immunoconjugate of claim 55, wherein said immunoconjugate comprises a polypeptide sequence at least 80% identical to a sequence selected from the group of SEQ ID NO: SEQ ID NO 239, 241 and 243.
67. The immunoconjugate of claim 66, wherein said immunoconjugate comprises a polypeptide sequence at least 95% identical to a sequence selected from the group of SEQ ID NO: SEQ ID NO 239, 241 and 243.
68. The immunoconjugate of claim 67, wherein said immunoconjugate comprises a polypeptide sequence identical to a sequence selected from the group of SEQ ID NO: SEQ ID NO 239, 241 and 243.
69. The immunoconjugate of claim 55, wherein said immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide having at least 80% identity to a sequence selected from the group of SEQ ID NO:240, 242 and 244.
70. The immunoconjugate of claim 69, wherein said immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence selected from the group of SEQ ID NO:240, 242 and 244 SEQ ID NO.
71. The immunoconjugate of claim 55 or 56, wherein said immunoconjugate comprises a polypeptide sequence at least 80% identical to a sequence selected from the group of SEQ ID NO:245, 247 and 249.
72. The immunoconjugate of claim 71, wherein said immunoconjugate comprises a polypeptide sequence at least 95% identical to a sequence selected from the group of SEQ ID NO:245, 247 and 249.
73. The immunoconjugate of claim 72, wherein said immunoconjugate comprises a polypeptide sequence identical to a sequence selected from the group of SEQ ID NO:245, 247 and 249.
74. The immunoconjugate of claim 55 or 56, wherein said immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide having at least 80% identity to a sequence selected from the group of SEQ ID NO:246, 248 and 250.
75. The immunoconjugate of claim 74, wherein said immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence selected from the group of SEQ ID NO:246, 248 and 250.
76. The immunoconjugate of any one of claims 66 to 70, wherein said immunoconjugate further comprises a polypeptide sequence at least 80% identical to a sequence selected from the group of SEQ ID NO:245, 247 and 249.
77. The immunoconjugate of claim 76, wherein said immunoconjugate further comprises a polypeptide sequence at least 95% identical to a sequence selected from the group of SEQ ID NO:245, 247 and 249.
78. The immunoconjugate of claim 77, wherein said immunoconjugate further comprises a polypeptide sequence identical to a sequence selected from the group of SEQ ID NO:245, 247 and 249.
79. The immunoconjugate of claim 4, wherein said first and second antigen binding moieties are specific for Fibroblast Activation Protein (FAP).
80. The immunoconjugate of claim 5, wherein said first or said second antigen binding moiety is specific for Fibroblast Activation Protein (FAP).
81. The immunoconjugate of claim 79 or 80, wherein said immunoconjugate comprises a heavy chain variable region sequence selected from the group of SEQ ID NO:21, 25, 27, 31, 35, 39, 43, 47, 51, 69, 73, 77, 81, 85, 89, 93, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171 and 175.
82. The immunoconjugate of claim 79 or 80, wherein said immunoconjugate comprises a light chain variable region sequence selected from the group of SEQ ID NO: SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 23, SEQ ID NO 29, SEQ ID NO 33, SEQ ID NO 37, SEQ ID NO 41, SEQ ID NO 45, SEQ ID NO 49, SEQ ID NO 67, SEQ ID NO 71, SEQ ID NO 75, SEQ ID NO 79, SEQ ID NO 83, SEQ ID NO 87, SEQ ID NO 91, SEQ ID NO 121, SEQ ID NO 125, SEQ ID NO 129, SEQ ID NO 133, SEQ ID NO 137, SEQ ID NO 141, SEQ ID NO 145, SEQ ID NO 149, SEQ ID NO 153, SEQ ID NO 157, SEQ ID NO 161, SEQ ID NO 165, SEQ ID NO 169 and SEQ ID NO 173.
83. The immunoconjugate of claim 81, wherein said immunoconjugate further comprises a light chain variable region sequence selected from the group of SEQ ID NO: SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 23, SEQ ID NO 29, SEQ ID NO 33, SEQ ID NO 37, SEQ ID NO 41, SEQ ID NO 45, SEQ ID NO 49, SEQ ID NO 67, SEQ ID NO 71, SEQ ID NO 75, SEQ ID NO 79, SEQ ID NO 83, SEQ ID NO 87, SEQ ID NO 91, SEQ ID NO 121, SEQ ID NO 125, SEQ ID NO 129, SEQ ID NO 133, SEQ ID NO 137, SEQ ID NO 141, SEQ ID NO 145, SEQ ID NO 149, SEQ ID NO 153, SEQ ID NO 157, SEQ ID NO 161, SEQ ID NO 165, SEQ ID NO 169 and SEQ ID NO 173.
84. The immunoconjugate of claim 83, wherein said immunoconjugate comprises (i) the heavy chain variable region sequence of SEQ ID NO:131 and the light chain variable region sequence of SEQ ID NO: 129; (ii) 163 and 161; (iii) the heavy chain variable region sequence of SEQ ID NO 31 and the light chain variable region sequence of SEQ ID NO 29; (iv) the heavy chain variable region sequence of SEQ ID NO 21 and the light chain variable region sequence of SEQ ID NO 19; (v) the heavy chain variable region sequence of SEQ ID NO 25 and the light chain variable region sequence of SEQ ID NO 23; (vi) the heavy chain variable region sequence of SEQ ID NO 35 and the light chain variable region sequence of SEQ ID NO 33; (vii) the heavy chain variable region sequence of SEQ ID NO 39 and the light chain variable region sequence of SEQ ID NO 37; (viii) the heavy chain variable region sequence of SEQ ID NO 43 and the light chain variable region sequence of SEQ ID NO 41; (iix) the heavy chain variable region sequence of SEQ ID NO:47 and the light chain variable region sequence of SEQ ID NO: 45; (ix) the heavy chain variable region sequence of SEQ ID NO 51 and the light chain variable region sequence of SEQ ID NO 49; (x) The heavy chain variable region sequence of SEQ ID NO:69 and the light chain variable region sequence of SEQ ID NO: 67; (xi) The heavy chain variable region sequence of SEQ ID NO 73 and the light chain variable region sequence of SEQ ID NO 71; (xii) The heavy chain variable region sequence of SEQ ID NO 77 and the light chain variable region sequence of SEQ ID NO 75; (xiii) The heavy chain variable region sequence of SEQ ID NO 81 and the light chain variable region sequence of SEQ ID NO 79; (xiii) The heavy chain variable region sequence of SEQ ID NO 85 and the light chain variable region sequence of SEQ ID NO 83; (xiv) The heavy chain variable region sequence of SEQ ID NO. 89 and the light chain variable region sequence of SEQ ID NO. 87; (xv) The heavy chain variable region sequence of SEQ ID NO 93 and the light chain variable region sequence of SEQ ID NO 91; (xvi) The heavy chain variable region sequence of SEQ ID NO 123 and the light chain variable region sequence of SEQ ID NO 121; (xvii) The heavy chain variable region sequence of SEQ ID NO:127 and the light chain variable region sequence of SEQ ID NO: 125; (xviii) The heavy chain variable region sequence of SEQ ID NO. 135 and the light chain variable region sequence of SEQ ID NO. 133; (iiixx) the heavy chain variable region sequence of SEQ ID NO:139 and the light chain variable region sequence of SEQ ID NO: 137; (ixx) the heavy chain variable region sequence of SEQ ID NO:143 and the light chain variable region sequence of SEQ ID NO: 141; (xx) The heavy chain variable region sequence of SEQ ID NO:147 and the light chain variable region sequence of SEQ ID NO: 145; (xxi) The heavy chain variable region sequence of SEQ ID NO 151 and the light chain variable region sequence of SEQ ID NO 149; (xxii) The heavy chain variable region sequence of SEQ ID NO 155 and the light chain variable region sequence of SEQ ID NO 153; (xxiii) The heavy chain variable region sequence of SEQ ID NO:159 and the light chain variable region sequence of SEQ ID NO: 157; (xxiv) The heavy chain variable region sequence of SEQ ID NO:167 and the light chain variable region sequence of SEQ ID NO: 165; (xxv) The heavy chain variable region sequence of SEQ ID NO 171 and the light chain variable region sequence of SEQ ID NO 169; or (xxvi) the heavy chain variable region sequence of SEQ ID NO:175 and the light chain variable region sequence of SEQ ID NO: 173.
85. The immunoconjugate of claim 84, wherein said immunoconjugate comprises a heavy chain variable region sequence of SEQ ID NO:131 and a light chain variable region sequence of SEQ ID NO: 129.
86. The immunoconjugate of claim 84, wherein said immunoconjugate comprises the heavy chain variable region sequence of SEQ ID NO 163 and the light chain variable region sequence of SEQ ID NO 161.
87. The immunoconjugate of claim 84, wherein said immunoconjugate comprises a heavy chain variable region sequence of SEQ ID NO 31 and a light chain variable region sequence of SEQ ID NO 29.
88. The immunoconjugate of claim 81, wherein said heavy chain variable region sequence is encoded by a polynucleotide sequence having at least 80% identity to a sequence selected from the group of SEQ ID NO: SEQ ID NO. 22, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 32, SEQ ID NO. 36, SEQ ID NO. 40, SEQ ID NO. 44, SEQ ID NO. 48, SEQ ID NO. 52, SEQ ID NO. 70, SEQ ID NO. 74, SEQ ID NO. 78, SEQ ID NO. 82, SEQ ID NO. 86, SEQ ID NO. 90, SEQ ID NO. 94, SEQ ID NO. 124, SEQ ID NO. 128, SEQ ID NO. 132, SEQ ID NO. 136, SEQ ID NO. 140, SEQ ID NO. 144, SEQ ID NO. 148, SEQ ID NO. 152, SEQ ID NO. 156, SEQ ID NO. 160, SEQ ID NO. 164, SEQ ID NO. 168, SEQ ID NO. 172 and SEQ ID NO. 176.
89. The immunoconjugate of claim 88, wherein said heavy chain variable region sequence is encoded by a polynucleotide sequence selected from the group of SEQ ID NO: SEQ ID NO. 22, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 32, SEQ ID NO. 36, SEQ ID NO. 40, SEQ ID NO. 44, SEQ ID NO. 48, SEQ ID NO. 52, SEQ ID NO. 70, SEQ ID NO. 74, SEQ ID NO. 78, SEQ ID NO. 82, SEQ ID NO. 86, SEQ ID NO. 90, SEQ ID NO. 94, SEQ ID NO. 124, SEQ ID NO. 128, SEQ ID NO. 132, SEQ ID NO. 136, SEQ ID NO. 140, SEQ ID NO. 144, SEQ ID NO. 148, SEQ ID NO. 152, SEQ ID NO. 156, SEQ ID NO. 160, SEQ ID NO. 164, SEQ ID NO. 168, SEQ ID NO. 172 and SEQ ID NO. 176.
90. The immunoconjugate of claim 82, wherein said light chain variable region sequence is encoded by a polynucleotide sequence having at least 80% identity to a sequence selected from the group of SEQ ID NO:18, 20, 24, 30, 34, 38, 42, 46, 50, 68, 72, 76, 80, 84, 88, 92, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and 174.
91. The immunoconjugate of claim 90, wherein said light chain variable region sequence is encoded by a polynucleotide sequence selected from the group of SEQ ID NO:18, 20, 24, 30, 34, 38, 42, 46, 50, 68, 72, 76, 80, 84, 88, 92, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and 174.
92. The immunoconjugate of claim 79, wherein said immunoconjugate comprises a polypeptide sequence at least 80% identical to a sequence selected from the group of SEQ ID NO:209, 211, 213, 217, 219, 221, 223, 225 and 227.
93. The immunoconjugate of claim 92, wherein said immunoconjugate comprises a polypeptide sequence at least 95% identical to a sequence selected from the group of seq id no:209, 211, 213, 217, 219, 221, 223, 225 and 227.
94. The immunoconjugate of claim 93, wherein said immunoconjugate comprises a polypeptide sequence identical to a sequence selected from the group of seq id no:209, 211, 213, 217, 219, 221, 223, 225 and 227.
95. The immunoconjugate of claim 79, wherein said immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide having at least 80% identity to a sequence selected from the group of SEQ ID NO:210, 212, 214, 218, 220, 222, 224, 226 and 228.
96. The immunoconjugate of claim 95, wherein said immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence selected from the group of seq id no:210, 212, 214, 218, 220, 222, 224, 226 and 228.
97. The immunoconjugate of claim 79 or 80, wherein said immunoconjugate comprises a polypeptide sequence at least 80% identical to a sequence selected from the group of SEQ ID NO:229, 231, 233 and 237 SEQ ID NO.
98. The immunoconjugate of claim 97, wherein said immunoconjugate comprises a polypeptide sequence at least 95% identical to a sequence selected from the group of seq id no:229 in SEQ ID NO, 231 in SEQ ID NO, 233 in SEQ ID NO and 237 in SEQ ID NO.
99. The immunoconjugate of claim 98, wherein said immunoconjugate comprises a polypeptide sequence identical to a sequence selected from the group of seq id no:229, 231, 233 and 237 SEQ ID NO.
100. The immunoconjugate of claim 79 or 80, wherein said immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence having at least 80% identity to a sequence selected from the group of seq id no:230, 232, 234 and 238.
101. The immunoconjugate of claim 100, wherein said immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence selected from the group of seq id no:230, 232, 234 and 238.
102. The immunoconjugate of claim 79, wherein said immunoconjugate comprises a polypeptide sequence selected from the group consisting of SEQ ID NO 211, SEQ ID NO 219, and SEQ ID NO 221, and polypeptide sequence SEQ ID NO 231.
103. The immunoconjugate of claim 79, wherein said immunoconjugate comprises a polypeptide sequence selected from the group consisting of SEQ ID NO 209, SEQ ID NO 223, SEQ ID NO 225, and SEQ ID NO 227, and polypeptide sequence SEQ ID NO 229.
104. The immunoconjugate of claim 79, wherein said immunoconjugate comprises polypeptide sequence SEQ ID NO 213 and polypeptide sequence SEQ ID NO 233.
105. The immunoconjugate of claim 79, wherein said immunoconjugate comprises polypeptide sequence SEQ ID NO 217 and polypeptide sequence SEQ ID NO 237.
106. The immunoconjugate of claim 4, wherein said first and second antigen binding moieties are specific for Melanoma Chondroitin Sulfate Proteoglycan (MCSP).
107. The immunoconjugate of claim 5, wherein said first or said second antigen binding moiety is specific for Melanoma Chondroitin Sulfate Proteoglycan (MCSP).
108. The immunoconjugate of claim 106 or 107, wherein said immunoconjugate comprises a heavy chain variable region sequence of SEQ ID NO 257 or SEQ ID NO 261.
109. The immunoconjugate of claim 106 or 107, wherein said immunoconjugate comprises a light chain variable region sequence of SEQ ID NO 259 or SEQ ID NO 271.
110. The immunoconjugate of claim 108, wherein said immunoconjugate further comprises a light chain variable region sequence of SEQ ID NO 259 or SEQ ID NO 271.
111. The immunoconjugate of claim 108, wherein said heavy chain variable region sequence is encoded by a polynucleotide sequence having at least 80% identity with sequence SEQ ID No. 258 or SEQ ID No. 262.
112. The immunoconjugate of claim 111, wherein said heavy chain variable region sequence is encoded by the polynucleotide sequence of SEQ ID NO:258 or SEQ ID NO: 262.
113. The immunoconjugate of claim 109, wherein said light chain variable region sequence is encoded by a polynucleotide sequence having at least 80% identity with sequence SEQ ID No. 260 or SEQ ID No. 272.
114. The immunoconjugate of claim 113, wherein said light chain variable region sequence is encoded by the polynucleotide sequence of SEQ ID NO:260 or SEQ ID NO: 272.
115. The immunoconjugate of claim 106, wherein said immunoconjugate comprises a polypeptide sequence at least 80% identical to the sequence of SEQ ID No. 251 or SEQ ID No. 255.
116. The immunoconjugate of claim 115, wherein said immunoconjugate comprises a polypeptide sequence at least 95% identical to the sequence of SEQ ID No. 251 or SEQ ID No. 255.
117. The immunoconjugate of claim 116, wherein said immunoconjugate comprises a polypeptide sequence identical to a sequence of SEQ ID No. 251 or SEQ ID No. 255.
118. The immunoconjugate of claim 106, wherein said immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide having at least 80% identity with SEQ ID NO:252 or SEQ ID NO: 256.
119. The immunoconjugate of claim 118, wherein said immunoconjugate comprises a polypeptide sequence encoded by polynucleotide sequence SEQ ID No. 252 or SEQ ID No. 256.
120. The immunoconjugate of claim 106 or 107, wherein said immunoconjugate comprises a polypeptide sequence at least 80% identical to the sequence of SEQ ID No. 253 or SEQ ID No. 265.
121. The immunoconjugate of claim 120, wherein said immunoconjugate comprises a polypeptide sequence at least 95% identical to the sequence of SEQ ID No. 253 or SEQ ID No. 265.
122. The immunoconjugate of claim 121, wherein said immunoconjugate comprises a polypeptide sequence identical to a sequence of SEQ ID No. 253 or SEQ ID No. 265.
123. The immunoconjugate of claim 106 or 107, wherein said immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide having at least 80% identity with SEQ ID No. 254 or SEQ ID No. 266.
124. The immunoconjugate of claim 123, wherein said immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide sequence of SEQ ID No. 254 or SEQ ID No. 266.
125. The immunoconjugate of any one of claims 115-119, wherein said immunoconjugate further comprises a polypeptide sequence at least 80% identical to a sequence of SEQ ID No. 253 or SEQ ID No. 265.
126. The immunoconjugate of claim 125, wherein said immunoconjugate further comprises a polypeptide sequence at least 95% identical to the sequence of SEQ ID No. 253 or SEQ ID No. 265.
127. The immunoconjugate of claim 126, wherein said immunoconjugate further comprises a polypeptide sequence identical to a sequence of SEQ ID No. 253 or SEQ ID No. 265.
128. The immunoconjugate of any one of claims 1 to 4, wherein the variable regions of said first and second antigen binding moieties are specific for a cell surface antigen of a cancer cell or a virus-infected cell or for an ECM molecule expressed in a tumor.
129. The immunoconjugate of claim 128, wherein said cell surface antigen is a tumor associated antigen or a viral antigen.
130. The immunoconjugate of claim 1 or 2, wherein said immunoconjugate has only one effector moiety.
131. The immunoconjugate of claim 130, wherein said cytokine is selected from the group of: interleukin-2 (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF), interferon-alpha (INF-alpha), and interleukin-12 (IL-12).
132. The immunoconjugate of claim 131, wherein said cytokine is IL-2.
133. The immunoconjugate of claim 131, wherein said cytokine is IL-12.
134. The immunoconjugate of any one of claims 130-133, wherein said cytokine elicits a cytotoxic response by a cell bearing said cytokine receptor.
135. The immunoconjugate of any one of claims 130-133, wherein said cytokine elicits a proliferative response from a cell bearing said cytokine receptor.
136. The immunoconjugate of claim 1, wherein said immunoconjugate has two effector moieties.
137. The immunoconjugate of claim 136, wherein each of said cytokines is independently selected from the group of: IL-2, GM-CSF, INF-alpha and IL-12.
138. The immunoconjugate of claim 137, wherein both of said cytokines are IL-2.
139. The immunoconjugate of claim 137, wherein both of said cytokines are IL-12.
140. The immunoconjugate of any one of claims 136-139, wherein said cytokine elicits a cytotoxic response by a cell bearing said cytokine receptor.
141. The immunoconjugate of any one of claims 136-139, wherein said cytokine elicits a proliferative response from a cell bearing said cytokine receptor.
142. The immunoconjugate of any one of claims 1-141, wherein said immunoconjugate is dissociated in comparison to an unconjugated effector moietyDissociation constant (K) at least 2 times greater thanD) Binding to the effector module receptor.
143. The immunoconjugate of any one of claims 1-142, wherein said immunoconjugate inhibits an increase in tumor volume in vivo by at least 30% at the end of an administration period.
144. An isolated polynucleotide encoding a fragment of the immunoconjugate of claim 1 or 2, wherein said polynucleotide encodes the heavy chain of said first and second antigen binding moieties and said first effector moiety.
145. An isolated polynucleotide encoding a fragment of the immunoconjugate of claim 1 or 2, wherein said polynucleotide encodes the light chain of said first and second antigen binding moieties and said first effector moiety.
146. An isolated polynucleotide encoding a fragment of the immunoconjugate of claim 1 or 2, wherein said polynucleotide encodes one light chain from said first antigen binding moiety, one heavy chain from said second antigen binding moiety, and said first effector moiety.
147. An isolated polynucleotide encoding a fragment of the immunoconjugate of claim 1 or 2, wherein said polynucleotide encodes the heavy chain of both said first and second Fab molecules and said effector moiety.
148. An isolated polynucleotide encoding a fragment of the immunoconjugate of claim 1 or 2, wherein said polynucleotide encodes the light chain of both said first and second Fab molecules and said effector moiety.
149. An isolated polynucleotide encoding a fragment of the immunoconjugate of claim 1 or 2, wherein said polynucleotide encodes one light chain from said first Fab molecule, one heavy chain from said second Fab molecule, and said effector moiety.
150. An expression cassette comprising the polynucleotide sequence of any one of claims 144-149.
151. An expression vector comprising the expression cassette of claim 150.
152. A host cell comprising the expression vector of claim 151.
153. The host cell of claim 152, further defined as a prokaryotic host cell.
154. The host cell of claim 152, further defined as a eukaryotic host cell.
155. A method of producing the immunoconjugate of any one of claims 1-143, wherein the method comprises culturing the host cell of any one of claims 152-154 under conditions suitable for expression of the immunoconjugate.
156. Use of the immunoconjugate of any one of claims 1-143 in the manufacture of a medicament for a method of treating a disease in an individual comprising the step of administering to said individual a therapeutically effective amount of a composition comprising the immunoconjugate.
157. The use of claim 156, wherein the disease is cancer.
158. The use of claim 156 or 157, wherein the immunoconjugate is specific for EDB.
159. The use of claim 156 or 157, wherein the immunoconjugate is specific for TNC-a 1.
160. The use of claim 156 or 157, wherein the immunoconjugate is specific for TNC-a 2.
161. The use of claim 156 or 157, wherein the immunoconjugate is specific for FAP.
162. The use of claim 156 or 157, wherein the immunoconjugate is specific for MCSP.
163. The use of any one of claims 156 to 162, wherein the immunoconjugate comprises IL-2.
164. The use of any one of claims 156 to 163, wherein the composition is administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraprostaticaly, intraperitoneally, intrasplenally, intrarenally, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, by inhalation, by injection, by continuous infusion, by local perfusion bathing target cells directly, via a catheter, by lavage, in an emulsion, or in a lipid composition.
165. The use of any one of claims 156 to 163, wherein the composition is administered topically or by infusion.
166. A composition comprising the immunoconjugate of any one of claims 1-143 and a pharmaceutically acceptable carrier.
167. The immunoconjugate of any one of claims 1-143 or the composition of claim 166, for use in the manufacture of a medicament for treating a disease in a patient in need thereof.
168. The immunoconjugate of claim 167, wherein said disease is cancer.
169. Use of the immunoconjugate of any one of claims 1-143 or the composition of claim 166 for the manufacture of a medicament for treating a disease in a patient in need thereof.
170. The use of claim 169, wherein the disease is cancer.
171. The immunoconjugate of any one of claims 1-143, for use in treating a disease in a patient in need thereof.
172. The immunoconjugate of claim 171, wherein said disease is cancer.
HK12110035.2A 2009-08-17 2010-08-13 Targeted immunoconjugates HK1169318B (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US23458409P 2009-08-17 2009-08-17
US61/234,584 2009-08-17
EP10162410 2010-05-10
EP10162410.4 2010-05-10
PCT/EP2010/061810 WO2011020783A2 (en) 2009-08-17 2010-08-13 Targeted immunoconjugates

Publications (2)

Publication Number Publication Date
HK1169318A1 HK1169318A1 (en) 2013-01-25
HK1169318B true HK1169318B (en) 2016-07-15

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