阅读说明：本技术 精确制导的多功能治疗抗体 (Precision-guided multifunctional therapeutic antibodies ) 是由 管永军 于 2018-06-07 设计创作，主要内容包括：本发明涉及新型多特异性抗体,其包含亲和力精确微调后的低亲和力单结合域蛋白片段的协同组合来选择性靶向含双靶标的肿瘤细胞及其在治疗中的用途,例如在肿瘤精准免疫治疗中的用途。(The present invention relates to novel multispecific antibodies comprising a synergistic combination of low affinity single binding domain protein fragments following precise fine-tuning of affinity to selectively target tumor cells containing dual targets and their use in therapy, e.g., in precise immunotherapy of tumors.)

1. An engineered bispecific antibody comprising: (1) a first chain comprising a first antigen binding domain that binds a first target and has about 10^-5～10^-8Affinity of M; (2) a second strand comprising a second antigen-binding domain that binds a second target and has about 10^-5～10^-8Affinity of M; wherein the first antibodyA antigen-binding domain linked to the N-terminus of the first constant region of the heavy chain of the bispecific antibody, wherein the second antigen-binding domain is linked to the N-terminus of the light chain of the bispecific antibody, wherein the first and second targets are both co-localized on a target cell; and wherein said bispecific antibody is capable of reaching about 10^-9～10^-12M to selectively bind to cells containing the dual targets, but not to single-target cells expressing only the first target or the second target.

2. The antibody of claim 1, wherein the first and second targets are selected from tumor targets, disease-specific receptors, and immunoregulatory functional targets.

3. The antibody of claim 2, wherein the tumor target is selected from the group consisting of Her2, CEA, ROR2, TROP2, mGluR1, EGFR, and the like.

4. The antibody of claim 2, wherein the checkpoint receptor is selected from the group consisting of PD-L1, CD47, L AG3, CD59, and Tim3, among others.

5. An engineered trispecific antibody comprising: (1) a first chain comprising a first antigen binding domain that binds a first target, having about 10^-5～10^-8Affinity of M; (2) a second chain comprising a second antigen-binding domain and a third antigen-binding domain, the second antigen-binding domain binding to a second target, having about 10^-5～10^-8M, the third antigen binding domain binds to a third target, has an affinity of about 10^-5～10^-8Affinity of M; wherein the first antigen-binding domain is linked to the N-terminus of a first constant region of a heavy chain of the trispecific antibody, wherein the second antigen-binding domain is linked to the N-terminus of a light chain of the trispecific antibody, wherein the first and second targets are both co-localized on the same target cell; and wherein the trispecific antibody is capable of up to about 10^-9～10^-12M to selectively bind to cells containing the dual target, but not to single target cells expressing only the first target or the second target; wherein the third antigen binding domain is linked to the C-terminus of the light chain of the trispecific antibody; and wherein the third target is an effector function target or a regulator; and wherein the third antigen binding domain is effective to mediate effector cell function or regulatory factor function to the target cell.

6. The antibody of claim 5, wherein the third effector function target is selected from the group consisting of CD3, CD16a, and CD59, and the like.

7. The antibody of claim 2, wherein the heavy chain comprises any one of the following sequences: seq ID No.1, Seq ID No.2, Seq ID No.3, Seq ID No.4, Seq ID No.5, Seq ID No.6, Seq ID No.7, Seq ID No.8, Seq ID No.9, Seq ID No.10, Seq ID No.11, Seq ID No.2, Seq ID No.28, Seq ID No.29, Seq ID No.32, Seq ID No.33, Seq ID No.36, Seq ID No.37, Seq ID No.40, Seq ID No.41, Seq ID No.60, Seq ID No.61, Seq ID No.66, Seq ID No.67, Seq ID No.68, and Seq ID No. 69.

8. The antibody of claim 2, wherein the light chain comprises any one of the following sequences: seq ID No.1, Seq ID No.2, Seq ID No.3, Seq ID No.4, Seq ID No.5, Seq ID No.6, Seq ID No.7, Seq ID No.8, Seq ID No.9, Seq ID No.10, Seq ID No.11, Seq ID No.12, Seq ID No.30, Seq ID No.31, Seq ID No.34, Seq ID No.35, Seq ID No.38, Seq ID No.39, Seq ID No.42, and Seq ID No.43.

9. The antibody of claim 6, wherein the light chain comprises any one of the following sequences: seq ID No.44, Seq ID No.45, Seq ID No.46, Seq ID No.47, Seq ID No.48, Seq ID No.49, Seq ID No.50, Seq ID No.51, Seq ID No.52, Seq ID No.53, Seq ID No.54, Seq ID No.55, Seq ID No.56, Seq ID No.57, Seq ID No.58, Seq ID No.59, Seq ID No.62, Seq ID No.63, Seq ID No.64 and Seq ID No. 65.

10. The antibody of claim 2, wherein (1) the heavy chain comprises any one of the following sequences: SEQ ID No.1, No.2, No.3, No.4, No.36, No.37, No.40, No. 41; (2) the light chain comprises any one of the following sequences: SEQ ID No.5, No.6, No.7, No.8, No.38, No.39, No.42, No.43, wherein the antibody binds to Her2 and CD47 double positive target cells.

11. The antibody of claim 2, wherein (1) the heavy chain comprises any one of the sequences of SEQ ID Nos. 5, 6, 7, 8, 28, 29, 32, 33 and (2) the light chain comprises any one of the sequences of SEQ ID Nos. 9, 10, 11, 12, 30, 31, 34, 35, wherein the antibody binds to both PD-L1 and CD47 target cells.

12. The antibody of claim 6, wherein (1) the heavy chain comprises any one of the sequences of SEQ ID Nos. 1,2, 3, 4, 36, 37, 40, 41; (2) the light chain comprises any one of the sequences SEQ ID No.5, No.6, No.7, No.8, No.52, No.53, No.54, No.55, No.56, No.57, No.58, No.59, the antibody binds to Her2 and CD47 double positive target cells.

13. The antibody of claim 6, wherein (1) the heavy chain comprises any one of the sequences of SEQ ID Nos. 5, 6, 7, 8, 28, 29, 32, 33 and (2) the light chain comprises any one of the sequences of SEQ ID Nos. 9, 10, 11, 12, 44, 45, 46, 47, 48, 49, 50, 51, wherein the antibody binds to both PD-L1 and CD47 positive target cells.

14. The antibody of any one of claims 1-13 for use in the manufacture of a medicament for treating cancer or a disorder associated therewith.

15. Use of an antibody according to any one of claims 1 to 13 in the manufacture of a medicament for the treatment of cancer or a disorder associated therewith.

Technical Field

The present invention relates to novel bispecific and multispecific antibodies comprising a synergistic combination of low affinity single binding domain fragments with precisely tailored affinities and their use in therapy, e.g., in precision immunotherapy.

Background

Monoclonal antibodies (mabs) have broad diagnostic and therapeutic potential in clinical practice against cancer and other diseases. Monoclonal antibodies play an important role in cancer immunotherapy, either in naked form or in linked form to cytotoxic agents such as radioisotopes, drugs, toxins or prodrug converting enzymes. These methods have gained positive application and have been successful at different levels of development and clinical success. Naked mabs may induce cytotoxic effects by binding to cell surface proteins that are overexpressed on cancer cells, thus achieving clinical therapeutic effects. Studies have shown that these therapeutic effects are achieved by neutralizing toxins or pathogens, controlling programmed cell death (apoptosis), or inducing innate and active immune responses against targets to control disease.

Since the 1975 Cesar Milstein and Georges j.f. kohler invented monoclonal antibody technology, due to its unique property of specifically targeting and mediating immune effector functions, antibodies were developed as drugs for targeted anti-disease immunotherapy. Currently, there are over 60 approved antibody-based biopharmaceuticals, with annual sales in excess of $ 5000 billion worldwide. The current successful use of first generation antibody drugs has prompted the development of the pharmaceutical industry and greatly improved public health. In addition to the development of antibody drugs directed against novel targets, the development of improved combination therapies and innovative bispecific antibodies is also the direction of development of future antibody drugs.

Currently, there are many anti-tumor antibody drugs in the clinic, including Rituxan (1997), Herceptin (1998), Mylotarg (2000), Campath (2001), Zevalin (2002), Bexxer (2003), Avastin (2004), Erbitux (2004), Vectibix (2006), Arzerra (2009), Benlysta (2011), yrevo (2011), Adcetris (2011), Perjeta (2012), kadcycla (2013), Opdivo (2014), Keytruda (2014), Tecentriq (2016). these antibodies are targeted primarily to EGFR, Her2, CD20 or recently to CT L a, PD1 or PD-L1, etc.

Bispecific antibodies are antibodies with dual epitope binding specificities, wherein one specificity is the ability to bind a first epitope or target and the second specificity is the ability to bind a second epitope or target.

In some embodiments, such bispecific antibodies are potentially valuable molecules for immunotherapy. For example, a bispecific antibody can crosslink cytotoxic effector T cells with target cells, thereby killing the target cells. Although many bispecific antibodies have shown in vitro effectiveness, few are clinically approved for use as therapeutic agents. A bispecific antibody, Catumaxomab (trade name Removab), was approved in europe in 2009. One of the reasons for the slow development of bispecific antibodies as therapeutics is the difficulty to produce them in sufficient purity and quantity.

Bispecific antibodies can be produced by chemical cross-linking, hybridoma or transfectoma, or by disulfide bond exchange at the hinge of two different fabs'. The first method results in a non-uniform and uncertain product. The second method requires the purification of the bispecific antibody from a number of hybrid antibody by-products, which may interfere with the cross-linking activity of the cells. The disulfide exchange method is basically applicable only to F (ab')2 and is therefore limited by the sensitivity of monoclonal antibodies to cleavage by enzymatic digestion. In addition, since Fab's have little affinity for each other, very high protein concentrations are required for disulfide bond formation between Fab'. By modifying one Fab' before oxidizing the other using the Ellman reagent, the disulfide exchange process has been improved, thereby reducing the incidence of homodimerization. However, even with this improvement, it is difficult to produce heterodimeric F (ab')2 in yields of greater than 50%.

However, safety issues, low response rates and limited success are common reality issues for current antibody drugs. These disadvantages arise from off-target effects on normal tissues/cells, as the epitopes or targets of antibodies are typically derived from self-antigens, inhibitory microenvironments of immune effector cells, and unintended Fc-mediated effector functions, among others. Thus, there is an urgent need for improved methods for the efficient production of bispecific antibodies and other similar compounds in high purity and safer designs.

Disclosure of Invention

In one aspect, the invention provides an engineered bispecific antibody comprising: (1) a first chain comprising a first antigen binding domain that binds a first target and has about 10^-5～10^-8Affinity of M; (2) a second strand comprising a second antigen-binding domain that binds a second target and has about 10^-5～10^-8Affinity of M; wherein the first antigen-binding domain is linked to the N-terminus of the first constant region of the heavy chain of the bispecific antibody, wherein the second antigen-binding domain is linked to the N-terminus of the light chain of the bispecific antibody, wherein the first and second targets are both co-localized on a target cell; and wherein said bispecific antibody is capable of reaching about 10^-9～10^-12The cooperative affinity of M selectively binds to target cells containing the dual target, rather than to single target cells expressing only the first target or the second target.

In certain aspects, the first target and the second target are selected from the group consisting of a tumor target, a disease-specific receptor, and an immunomodulatory functional target.

In certain aspects, the tumor target is selected from Her2, CEA, ROR2, TROP2, mGluR1, EGFR, and the like.

In certain aspects, the checkpoint receptor is selected from PD-L1, CD47, L AG3, CD59, Tim3, and the like.

In certain aspects, the light chain comprises any one of the following sequences: seq ID No.1, Seq ID No.2, Seq ID No.3, Seq ID No.4, Seq ID No.5, Seq ID No.6, Seq ID No.7, Seq ID No.8, Seq ID No.9, Seq ID No.10, Seq ID No.11, Seq ID No.12, Seq ID No.30, Seq ID No.31, Seq ID No.34, Seq ID No.35, Seq ID No.38, Seq ID No.39, Seq ID No.42, and Seq ID No.43.

In certain aspects, the heavy chain of the above antibody comprises any one of the following sequences: SEQ ID No.1, No.2, No.3, No.4, No.36, No.37, No.40, No. 41; the light chain of the above antibody comprises any one of the following sequences: SEQ ID No.5, No.6, No.7, No.8, No.38, No.39, No.42, No.43, wherein the antibody binds to Her2 and CD47 double positive target cells.

In some aspects, the heavy chain of the above antibody comprises any one of the sequences SEQ ID Nos. 5, 6, 7, 8, 28, 29, 32, 33 the light chain of the above antibody comprises any one of the sequences SEQ ID Nos. 9, 10, 11, 12, 30, 31, 34, 35, wherein the antibody binds to a target cell that is double positive for PD-L1 and CD 47.

In another aspect, the invention provides an engineered trispecific antibody comprising: (1) a first chain comprising a first antigen binding domain that binds a first target, having about 10^-5～10^-8Affinity of M; (2) a second chain comprising a second antigen-binding domain and a third antigen-binding domain, the second antigen-binding domain binding to a second target, having about 10^-5～10^-8M, the third antigen binding domain binds to a third target, has an affinity of about 10^-5～10^-8Affinity of M; wherein the first antigen-binding domain is linked to the N-terminus of a first constant region of a heavy chain of the trispecific antibody, wherein the second antigen-binding domain is linked to the N-terminus of a light chain of the trispecific antibody, wherein the first and second targets are both co-localized on the same target cell; and wherein the trispecific antibody is capable of up to about 10^-9～10^-12The cooperative affinity of M selectively binds to target cells containing the dual target, rather than to single target cells expressing only the first target or the second target. Wherein the third antigen binding domain is linked C-terminal to the light chain of the trispecific antibody; and wherein the third target is an effector function target or a regulatory factor target; and wherein the third antigen binding domain is effective to mediate effector cell function or regulatory factor function to the target cell.

In certain aspects, the first target and the second target are selected from the group consisting of a tumor target, a disease-specific receptor, and an immunomodulatory functional target.

In certain aspects, the tumor target is selected from Her2, CEA, ROR2, TROP2, mGluR1, and EGFR.

In certain aspects, the checkpoint receptor is selected from PD-L1, CD47, L AG3, CD59, and Tim 3.

In certain aspects, the third target is selected from CD3, CD16a, and CD 59.

In certain aspects, the heavy chain comprises any one of the following sequences: seq ID No.1, Seq ID No.2, Seq ID No.3, Seq ID No.4, Seq ID No.5, Seq ID No.6, Seq ID No.7, Seq ID No.8, Seq ID No.9, Seq ID No.10, Seq ID No.11, Seq ID No.2, Seq ID No.28, Seq ID No.29, Seq ID No.32, Seq ID No.33, Seq ID No.36, Seq ID No.37, Seq ID No.40, Seq ID No.41, Seq ID No.60, Seq ID No.61, Seq ID No.66, Seq ID No.67, Seq ID No.68, and Seq ID No. 69.

In some aspects, the light chain comprises any one of the following sequences: seq ID No.44, Seq ID No.45, Seq ID No.46, Seq ID No.47, Seq ID No.48, Seq ID No.49, Seq ID No.50, Seq ID No.51, Seq ID No.52, Seq ID No.53, Seq ID No.54, Seq ID No.55, Seq ID No.56, Seq ID No.57, Seq ID No.58, Seq ID No.59, Seq ID No.62, Seq ID No.63, Seq ID No.64, and Seq ID No. 65.

In certain aspects, the heavy chain of the above antibody comprises any one of the following sequences: SEQ ID No.1, No.2, No.3, No.4, No.36, No.37, No.40, No. 41. The light chain of the above antibody comprises any one of the following sequences: SEQ ID Nos. 5, 6, 7, 8, 52, 53, 54, 55, 56, 57, 58 and 59. Wherein the antibody binds to Her2 and CD47 double positive target cells.

In certain aspects, the heavy chain of the above antibody comprises any one of SEQ ID Nos. 5, 6, 7, 8, 28, 29, 32, 33 and the light chain of the above antibody comprises any one of SEQ ID Nos. 9, 10, 11, 12, 44, 45, 46, 47, 48, 49, 50, 51, wherein the antibody binds to a target cell that is double positive for PD-L1 and CD 47.

In another aspect, the invention provides an antibody as described above for use in the manufacture of a medicament for the treatment of cancer or a disorder associated therewith.

In another aspect, the invention provides a method of treating cancer or a condition associated therewith, comprising administering to a human a therapeutically effective dose of the antibody described above.

In another aspect, the invention provides methods of treating a subject in need of treatment with an antibody provided by the invention.

In some embodiments, the subject may produce a sustained response after cessation of treatment.

In some embodiments, immunotherapy is performed continuously and intermittently.

In some embodiments, the individual has colorectal cancer, melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, pancreatic cancer, a hematologic malignancy, or renal cell carcinoma.

In some embodiments, wherein the antibody is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, inhalation, intrathecally, intraventricularly or intranasally.

In some embodiments, the therapeutic combination or pharmaceutical composition of the invention further comprises an effective dose of an additional therapeutic agent, such as an anti-cancer agent.

In some embodiments, the anti-cancer agent is an anti-metabolite, an inhibitor of topoisomerase I and II, an alkylating agent, a microtubule inhibitor, an anti-androgen agent, a GNRh modulator, or a mixture thereof.

In some embodiments, the additional therapeutic agent is a chemotherapeutic agent selected from tamoxifen, raloxifene, anastrozole, exemestane, letrozole, imastanib, paclitaxel, cyclophosphamide, lovastatin, minox, gemcitabine, cytarabine, 5-fluorouracil, methotrexate, docetaxel, goserelin, vincristine, vinblastine, nocodazole, teniposide, etoposide, gemcitabine, epothilone, vinorelbine, camptothecin, daunorubicin, actinomycin D, mitoxantrone, acridine, doxorubicin, epirubicin, or idarubicin.

In another aspect, the present invention provides a method for treating a disease in a subject in need of such treatment, the method comprising administering to the subject a therapeutic or pharmaceutical composition provided herein.

In some embodiments, the disease condition is a tumor.

In some embodiments, the disease condition comprises abnormal cell proliferation.

In some embodiments, the abnormal cell proliferation comprises a precancerous lesion. In some embodiments, the abnormal proliferation is of cancer cells.

In some embodiments, the cancer is selected from: breast cancer, colorectal cancer, diffuse large B-cell lymphoma, endometrial cancer, follicular lymphoma, gastric cancer, glioblastoma, head and neck cancer, hepatocellular cancer, lung cancer, melanoma, multiple myeloma, ovarian cancer, pancreatic cancer, prostate cancer, and renal cell carcinoma.

In another aspect, the invention provides a kit comprising a therapeutic composition provided by the invention, together with instructions.

Drawings

The novel features believed characteristic of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are incorporated, and the accompanying drawings of which:

figure 1 depicts a precisely directed multifunctional therapeutic antibody (GCT Ab). It has the following characteristics: (1) safe paired binding with precise fine-tuning of affinity of the dual-target binding domain; (2) designing bi-specific and tri-specific antibodies with cooperative target and effector functions; (3) the bivalent nature of each targeting binding domain in the multifunctional antibody; (4) an Fc region with durable PK and standard production IgG antibody formats and no unexpected deleterious effector functions. These combinations of features determine GCT Ab as a highly potent antibody drug.

FIGS. 2A-F depict single binding domain based Fab and IgG antibody formats. A: a monovalent bispecific antibody fragment. B: a bivalent bispecific antibody (DBB Ab). C: a monovalent trispecific antibody fragment. D: a monovalent tetraspecific antibody fragment. E: a monovalent trispecific antibody-albumin drug. F: a precisely directed multifunctional therapeutic antibody (GCT Ab).

Figures 3A and 3B depict the precise fine-tuning of affinity for safer specific targeting. 3A: synergistic binding of TCR and CD4/CD8 co-receptors to MHC peptides. 3B: the delicate association of a pair of binding domains in GCT can safely mediate the killing of tumor cells without affecting normal cells.

Figure 4 depicts bispecific GCT antibodies ABP366 and ABP336, left panel depicts GCT antibody ABP366, comprising an engineered single domain antibody to HER2 linked to the N-terminus of CH1 by a linker, and an engineered SIRPa single binding domain to CD47 linked to the N-terminus of CK by a linker, Fc of IgG1 remains wild-type, right panel depicts GCT antibody ABP336, comprising an engineered SIRPa single binding domain to CD47 linked to the N-terminus of CH1 by a linker, and an engineered PD-1 single binding domain to PD-L1 linked to the N-terminus of CK by a linker, P329G-L a L a mutant to IgG1 is used to retain long PK while eliminating the potential detrimental effects of Fc effector function.

Figures 5A-5D show the results of E L ISA binding of bispecific GCT antibodies to HER2 and CD 47.5A shows the parent individual binding domain antibody to HER2, while 5B shows the parent individual binding domain antibody to CD 47.

5C and 5D show the GCT antibodies AbD066 and AbD068-1 binding to HER2 and CD47, respectively.

Figures 6A and 6B show cell surface staining results of bispecific GCT antibodies AbD066 and AbD068-1 against HER2 and CD47, respectively, detected by flow cytometry. The upper left panels of 6A and 6B show the percentage of positive cells, while the upper right panels of 6A and 6B show the Median Fluorescence Intensity (MFI) of the stained cell population. The lower panels of FIGS. 6A and 6B show the results of overlapping histogram staining for a panel of 4 cell populations (black filled: control cells; open dashed line: HER2+ single positive cells; open solid line: CD47+ single positive cells; light dashed line: CD47+ HER2+ double positive cells).

Figure 7 shows the results of E L ISA binding of bispecific GCT antibodies against PD-L1 and CD47 the upper panel shows the parent individual binding domain antibody against PD-L1, the middle and lower panels show the GCT antibodies AbD036 and AbD 037. hac binding to PD-L1 and CD47, respectively, the high affinity antibody against PD-L1 and the high affinity antibody against CD47 for CV1, were used as positive controls.

FIGS. 8A and 8B show cell surface staining results of bispecific GCT antibodies to AbD036 and AbD037, detected by flow cytometry, against PD-L1 and CD47, respectively, the upper left panels of 8A and 8B show the percentage of positive cells, while the upper right panels of 8A and 8B show the Median Fluorescence Intensity (MFI) of the stained cell populations, the lower panels of 8A and 8B show overlapping histogram staining results for a set of 4 cell populations (black filled: control cells; open dashed line: PD-L1 single positive cells; open solid line: CD47 single positive cells; light dashed line: CD47 and PD-L1 double positive cells).

Detailed Description

Aspects of the invention are described below and illustrated by way of example. It should be understood that numerous specific details, relationships, and methods are set forth below to provide a full understanding of the invention. One of ordinary skill in the art may practice the invention without one or more of the specific details or other methods. The present invention is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and/or concurrently with other acts or events.

Moreover, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, the use of "including," having, "" and "or variations thereof in the description and/or in the claims is intended to be inclusive in a manner similar to the term" comprising.

"about" means within an acceptable error range for the particular value, as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. For example, "about" can mean within 1 or greater than 1 standard deviation, as practiced in the art. Alternatively, "about" may mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and still more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within one order of magnitude of the value, preferably within 5 times the value, more preferably within 2 times the value. Where a particular value is described in the application and claims, unless otherwise stated, it is assumed that "about" indicates that the particular value is within an acceptable error range.

1. Definitions and abbreviations

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 to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic and nucleic acid chemistry, and hybridization are those well known and commonly employed in the art. Nucleic acid and peptide synthesis using standard techniques. The techniques and procedures are generally performed according to conventional methods in the art and various general references provided throughout this document. The nomenclature used herein and the laboratory procedures in analytical chemistry and organic compositions described below are those well known and commonly employed in the art. Chemical synthesis and chemical analysis use standard techniques or modifications thereof.

While various features of the invention may be described in the context of a single embodiment, these features may also be provided separately or in any suitable combination. Conversely, although the invention may be described in the context of separate embodiments for clarity, the invention may also be implemented in a single embodiment.

Reference in the specification to "some embodiments," "one embodiment," or "other embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the disclosure.

As used herein, ranges and amounts can be expressed as "about" a particular value or range, "about" also includes precise amounts, and thus "about 5 μ L" means "about 5 μ L," and can also be expressed as "5 μ L.

"Polypeptides", "peptides" and "proteins" are used interchangeably herein to refer to linear amino acid residues linked to each other by peptide bonds, including proteins, polypeptides, oligopeptides, peptides and fragments thereof, which proteins may be composed of naturally occurring amino acids and/or synthetic (e.g., modified or non-naturally occurring) amino acids.

"nucleic acid" in the present invention refers to DNA or RNA, or a molecule containing deoxy and/or ribonucleotides. Nucleic acids can be naturally occurring or synthetic, and include analogs of naturally occurring polynucleotides in which one or more nucleotides are modified to naturally occurring nucleotides.

"conjugation" and "linkage" generally refer to a chemical bond, either covalent or non-covalent, that associates one molecule with the proximal end of a second molecule.

"isolated" is intended to mean that the compound is separated from all or some of the components with which it is associated in nature. "isolated" also refers to the state in which a compound is separated from all or some of its accompanying components during manufacture (e.g., chemical synthesis, recombinant expression, culture medium, etc.).

"purified" is intended to mean that the compound of interest has been isolated from nature or a component accompanying it during manufacture and provided in an enriched form.

"potency" as used in the context of the compounds of the present invention refers to the ability of the compound to exhibit a desired activity.

"concentration" as used in some molecules, such as peptide fragments, refers to the molecular weight present in a given volume. In some embodiments, the concentration of molecules, given in molar concentration, refers to the number of moles of molecules present in a given volume of solution.

"antigen" and "epitope" refer to the portion of a molecule (e.g., a polypeptide) that is specifically recognized by a component of the immune system, such as an antibody. As used herein, "antigen" encompasses an antigenic epitope, e.g., an antigenic fragment that is an antigenic epitope.

"antibodies" include polyclonal antibodies and monoclonal antibodies, which can be of any class of interest (e.g., IgG, IgM and subclasses thereof), as well as hybrid antibodies, modified antibodies, F (ab')2 fragments, F (ab) molecules, Fv fragments, single chain fragments (scFv) displayed on phage, single chain antibodies, single domain antibodies, diabodies, chimeric antibodies, humanized antibodies, and fragments thereof. In some embodiments, a fragment of an antibody may be a functional fragment that exhibits the immunological binding characteristics of the parent antibody molecule. The antibodies of the invention may be detectably labeled with, for example, a radioisotope, an enzyme that produces a detectable product, a fluorescent protein, and the like. Detectable labels used in vivo imaging are of interest. The antibody may be further conjugated to other moieties, such as cytotoxic or other molecules, members of specific binding pairs, and the like.

It is known that typical antibody building blocks, especially full-length antibody building blocks, comprise a tetramer, each tetramer being composed of two identical pairs of polypeptide chains, each pair having one "light chain" (about 25kD) and one "heavy chain" (about 50-70kD), the N-terminus of each chain having a variable region of about 100 to 110 or more amino acids, primarily responsible for antigen recognition, variable light chain (V L) and variable heavy chain (VH) refer to the variable regions of these light and heavy chains, respectively.

In an antibody molecule, the three hypervariable regions of the light and heavy chains are arranged relative to one another in three-dimensional space to form an antigen-binding "surface" that mediates recognition and binding of a target antigen.

Antibodies and fragments thereof of the invention include bispecific antibodies and fragments thereof. Bispecific antibodies are similar to a single antibody (or antibody fragment), but have two different antigen binding sites or domains. Bispecific antibodies have binding specificities for at least two different epitopes. Bispecific antibodies and fragments can also be in the form of xenogenous antibodies. A xenogenous antibody is two or more antibodies or antibody binding fragments (e.g., fabs) linked together, each antibody or fragment having a different specificity.

The invention also provides antibody conjugates. Conjugates include any of the antibodies and reagents of the invention. The agent may be selected from a therapeutic agent, an imaging agent, a labeling agent, or an agent useful for therapeutic and/or labeling purposes.

The strength or affinity of an immunological binding interaction between an antibody (or fragment thereof) and a particular antigen (or epitope) can be expressed in terms of the dissociation constant (Kd) of the interaction, where a smaller Kd represents a greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art, which require measuring the rate of formation and dissociation of an antigen binding site/antigen complex, which is dependent on the concentration of the complex partner, the affinity of the interaction, and geometric parameters that affect the rate in both directions. Thus, both the "on rate constant" (Kon) and the "off rate constant" (Koff) can be determined by calculating the concentration and the actual rate of binding and dissociation. The ratio of Koff/Kon removes all affinity-independent parameters and is therefore equal to the equilibrium dissociation constant Kd (see Davies et al, Ann. Rev. biochem.1990,59: 439. 15473).

In describing the characteristics of an antibody, "specific binding of an antibody" or "antigen-specific antibody" refers to the ability of an antibody to preferentially bind a particular antigen present in a mixture of different antigens. In some embodiments, specific binding (or "target" and "non-target" antigens) to suitable and unsuitable antigens in the sample is distinguished, and in some embodiments, binding to the target antigen is more than about 10-fold to 100-fold or even more (e.g., more than 1000-fold or 10,000-fold) than to the non-target antigen. In some embodiments, when the antibody and antigen are specifically bound in an antibody-antigen complex, the affinity between the antigen and the antibody is characterized by a KD (dissociation constant) of less than 10^-6M, less than 10^-7M, less than 10^-8M, less than 10^-9M, less than 10^-9M, less than 10^-11M, or less than about 10^-12M。

"monoclonal antibody" refers to an antibody composition having a homogeneous population of antibodies. Monoclonal antibodies include intact antibody molecules, as well as Fab molecules, F (ab')2 fragments, Fv fragments, single chain fragments displayed on phage (scFv), fusion proteins consisting of the antigen-binding portion of an antibody and a non-antibody protein, and other molecules with the immunological binding characteristics of the parent monoclonal antibody molecule. Methods for making and screening polyclonal and monoclonal antibodies are known in the art.

"derivative" and "variant" refer to, but are not limited to, any compound or antibody having a structure or sequence derived from the compounds and antibodies of the invention and which structure/sequence is sufficiently similar to and based on the similarity of those disclosed herein. One skilled in the art can expect "derivatives" and "variants" to have the same or similar activity and utility as the claimed and/or referenced compound or antibody, and thus may also be referred to as "functional equivalents". Modifications to obtain a "derivative" or "variant" include, for example, by addition, deletion and/or substitution of one or more amino acid residues. A functional equivalent or a fragment of a functional equivalent may have one or more conservative amino acid substitutions. "conservative amino acid substitution" refers to the replacement of an amino acid by another amino acid having similar properties to the original amino acid. Conserved amino acid groups are known in the art.

Conservative substitutions may be introduced at any position of the preferred preselected peptide or fragment thereof. Non-conservative substitutions may also be introduced at any one or more positions, particularly but not exclusively non-conservative substitutions. Non-conservative substitutions result in fragments of functionally equivalent peptides that differ substantially in polarity, charge, and/or steric bulk, while retaining the function of the derivative or variant fragment.

The "percent sequence similarity" is determined by comparing two optimally aligned sequences over a comparison window, in which a portion of the polynucleotide or polypeptide sequence may have additions or deletions (e.g., gaps) as compared to the reference sequence (which is not added or deleted). The percentage of sequence similarity can be determined by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison to calculate the percentage and multiplying the result by 100.

A percentage of "identity" or "similarity" in two or more nucleic acids or polypeptide sequences refers to two or more identical sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides (e.g., 60% similarity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% similarity over the entire polypeptide sequence or a particular region of a single polypeptide), that have the greatest correspondence when compared and aligned over a comparison window or using one of the following sequence comparison algorithms or specified regions detected by manual alignment and visual inspection. These sequences are said to be "substantially identical". This definition also refers to the complement of the detection sequence. Optionally, similarity exists over a region of at least about 5 to 50 nucleotides or polypeptide sequences in length, or more preferably over a region of 100 to 500 or more than 1000 nucleotides or polypeptide sequences in length.

For sequence comparison, one sequence is typically used as a reference sequence, which is compared to the test sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters may be used, or other parameters may be specified. The sequence comparison algorithm then calculates the percent sequence similarity of the test sequence relative to the reference sequence based on the program parameters.

As used herein, a "comparison window" compares a sequence selected from the full-length sequence or a sequence of from 20 to 600, from about 50 to about 200, or from about 100 to about 150 amino acids or nucleotides, after optimal alignment with a reference sequence, to compare the sequence to the reference sequence in the same number of consecutive positions.

One example of an algorithm for determining sequence identity and percent sequence similarity is the B L AST and B L AST 2.0 algorithms described in Altschul et al, Nuc. acids Res.25: 3389-.

The term "cell of interest" or "target cell" as used herein interchangeably refers to one or more cells intended to modulate one or more signaling pathways. In some embodiments, the target cell includes, but is not limited to, a cancer cell. In certain other embodiments, the target cells include immune effector cells, such as natural killer cells, T cells, dendritic cells, and macrophages.

As used herein, "cancer cell" refers to a cell that exhibits a neoplastic cell phenotype, which may be characterized by one or more of the following: abnormal cell growth, abnormal cell proliferation, loss of density-dependent growth inhibition, the potential for anchorage-independent growth, the ability to promote tumor growth and/or development in an immunocompromised non-human animal model, and/or appropriate signs of any cellular transformation. "cancer cells" are used interchangeably herein with "tumor cells" and include solid tumors, semi-solid tumors, primary tumors, metastatic tumors, and the like.

In the description of a disease or condition, "treatment" refers to improving a symptom associated with the condition afflicting an individual, where improvement in the broad sense refers to at least reducing a parameter, such as a symptom associated with the disease being treated (e.g., cancer). Treatment also includes completely inhibiting (e.g., preventing) the occurrence or cessation (e.g., termination) of a pathological condition or symptom associated therewith, termination such that the host no longer suffers from the condition or at least no longer characterizes the symptom of the condition. Thus, the treatment includes: (1) prevention, i.e., reducing the risk of development of clinical symptoms, includes disabling the development of clinical symptoms, e.g., preventing the disease from developing into a harmful state; (2) inhibiting, i.e., arresting the development or further development of clinical symptoms, e.g., reducing or completely inhibiting active disease, e.g., reducing tumor burden, such reduction may include elimination of detectable cancer cells, or protection from disease caused by bacterial infection, including elimination and/or (3) alleviation of detectable bacteria, cells, i.e., causing regression of clinical symptoms.

An "effective amount" of a composition of the present invention means an amount that is not lethal but sufficient to provide the desired utility of the composition. For example, an effective amount of an antibody (active, potent or functional) results in a significant and substantial change in the level of signaling pathway activity, including down-regulation and up-regulation of the signaling pathway as compared to an antibody without the use of an antibody or a control (null or nonfunctional) antibody, in order to elicit a favorable response in the cell of interest (the target cell), such as modulation of a signaling pathway. Changes in the level of activity of a signaling pathway can be measured by a variety of methods known in the art. In another example, an effective amount is an amount that reduces, eliminates, or alleviates symptoms associated with a disease (e.g., cancer) in order to elicit a beneficial response in a subject for treating the disease, such as cancer metastasis, elimination of cancer cells, and the like. As will be appreciated by those of ordinary skill in the art, the exact amount required will vary from individual to individual, depending upon the species and age, the severity of the condition or disease being treated, the particular composition used, the mode of administration, and the like. Thus, it is not possible to specify an exact "effective amount," but one of ordinary skill in the art can determine an appropriate effective amount using only routine experimentation.

As used herein, "pharmaceutically acceptable excipient" refers to any suitable substance that provides a pharmaceutically acceptable compound for administration of a compound of interest to a subject. The "pharmaceutically acceptable excipient" may include what is referred to as a pharmaceutically acceptable diluent, a pharmaceutically acceptable additive, and a pharmaceutically acceptable carrier.

An "individual" or "subject" encompasses humans, mammals, and other animals. "individual" or "subject" are used interchangeably herein and refer to any mammalian subject to which an antibody or fragment thereof of the invention is administered.

Certain embodiments feature bispecific antibodies, antigen-binding fragments, or recombinant proteins thereof, which are capable of modulating the activity of one or more signaling pathways in a cell of interest. Modulation of one or more signaling pathways may result in changes in certain target cell behaviors, such as stimulation or reduction of cell proliferation, cell growth, cell differentiation, cell survival, cell secretion, adhesion regulation, and/or cell motility.

As used herein, "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the compounds of the present invention and is not biologically or otherwise undesirable. In some cases, the compounds of the present invention are capable of forming acid and/or basic salts due to the presence of amino and/or carboxyl groups or groups similar thereto (e.g., phenol or hydroxyamidic acid). Pharmaceutically acceptable acid-derived salts can be formed with inorganic and organic acids. Inorganic acids from which salts may be derived include, for example, hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like. Organic acids from which salts can be derived include, for example, acetic, propionic, glycolic, pyruvic, oxalic, maleic, malonic, succinic, fumaric, tartaric, citric, benzoic, cinnamic, mandelic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicylic, and the like. Pharmaceutically acceptable base-derived salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; particularly preferred are ammonium, potassium, sodium, calcium and magnesium salts. Organic bases from which salts can be derived include, for example, primary, secondary and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, particularly isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, basic or acidic moiety, by conventional chemical methods. Such salts can generally be prepared by reacting the free acid forms of these compounds with a stoichiometric amount of the appropriate base (e.g., Na, Ca, Mg, or K hydroxides, carbonates, bicarbonates, and the like), or by reacting the free bases. These compounds have the appropriate acid form in stoichiometric amounts. Such reactions are generally carried out in water or an organic solvent or a mixture of both. Generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred where feasible. A list of other suitable salts can be found, for example, in Remington's Pharmaceutical Sciences, 20 th edition, Mack Publishing Company, Easton, Pa. (1985), which is incorporated herein by reference.

As used herein, "pharmaceutically acceptable carrier/excipient" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial and antifungal agents), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavorants, dyes, and like materials and combinations thereof known to those of ordinary skill in the art (e.g., Remington's Pharmaceutical Sciences,18th ed. mack Printing Company,1990, pp.1289-1329), incorporated herein by reference. It is currently contemplated that any conventional carrier will be used in therapeutic or pharmaceutical compositions unless incompatible with the active ingredient.

As used herein, "therapeutic combination" or "combination" refers to a combination of one or more active drug substances, i.e., compounds having therapeutic utility. Typically each such compound in the therapeutic combination of the invention will be present in a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier. As part of this protocol, the compounds of the therapeutic combinations of the present invention may be administered simultaneously or separately.

2. Pharmaceutical composition

The engineered antibodies of the invention comprise two single chain fragments, e.g., one light chain (domain 1 and C L) and one heavy chain (domain 2 and CH1), each of which recognizes a different antigen with relatively low affinity, e.g., less than 10^-8M, preferably 10^-5M to 10^-7M. the two chains are linked by a constant region of each chain, e.g. the linkage of C L and CH 1.

Although each single strand has a low affinity, e.g. 10^-5M to 10^-8M, but the two single-stranded fragments bind synergistically with much higher affinity, e.g., 10^-9M to 10^-12M。

In one aspect, the present invention provides an innovative multispecific antibody drug platform for precisely directed multifunctional therapeutic antibodies (GCT abs) with the objective of greatly improving the safety, efficacy and effectiveness of antibody immunotherapy. As shown in fig. 1, the present invention includes the following features: (1) bispecific antibodies minimize off-target effects and select binding fragments for each target of fine-tuned binding affinity. (2) The efficacy is significantly improved by novel trispecific antibodies directed against antibody binding fragments of disease specific targets, immune modulatory functional targets and defined effector functional targets; (3) efficacy is enhanced by innovatively designing IgG formats with multiple single domain binding fragments and the bivalent nature of each binding domain, as well as durable PK and standard antibody production characteristics.

The design of a selection of a pair of antibody binding fragments to safely fine-tune binding affinity for each target mimics the interaction between the TCR complex and MHC complex from the theory of the human natural immune control mechanism (Alberti, s.a. high affinity T cell receptor. The affinity of TCRs for MHC peptides is fine-tuned to a safe range during T cell development and maturation, does not interact adversely with normal MHC without exogenous peptides, and can efficiently recognize specific MHC peptide complexes by synergistic binding of CD4/CD8 to MHC. Although the affinity of CD4/CD8 to MHC was 10⁴～10⁶M^-1(Davies,D.R.,Padlan,E.A.&Sheriff, S.Antibody-antigen complexes.annual review of biochemistry 59,439-473(1990)), and TCR affinity for MHC peptides at 10⁵～10⁶M^-1(Matsui, K., et al. L ow affinity interaction of peptide-MHC complexes with T cell receptors. science 254,1788-1791 (1991)). T cells can safely and effectively recognize specific MHC peptide complexes on target cells by synergistic binding (Alberti, S.A. high affinity T cell receptorWith the control mechanism (fig. 2A), our invention selects a pair of disease-specific targets and a pair of binding domains for targets with lower affinity to the individual, so they will only loosely bind to the targets on normal tissue cells and can effectively selectively bind to dual-target disease cells by means of an additive and/or synergistic effect through a bispecific binding format, as shown in fig. 2B.

In another aspect, the invention provides a method comprising controlled Fc function design (e.g. P329G L a L a-Fc (WO2012130831 a1) without all Fc mediated effector functions) to avoid the potential for uncontrolled/unexpected adverse effector function effects while retaining affinity (PK) for FcRn with long half-life and protein a binding for standardized production.

In another aspect, the present invention provides novel trispecific multifunctional antibodies directed against disease specific targets, immunoregulatory functional targets and defined effector functional targets to substantially improve the efficacy of a drug.

In another aspect, the present invention provides a novel design of an antibody format with bivalent nature of multiple single domain binding fragments and each binding domain and durable PK and standard antibody production characteristics (fig. 1 and 3F). a pair of disease specific binding domains are linked to CH1 and C L, respectively, each binding domain having intact function. this single binding domain based bispecific antibody design can be used alone as Fab format of monovalent bispecific antibody fragments or as intact IgG antibody format of bivalent bispecific antibody (DBBAb) (fig. 3A and 3B). furthermore, a third binding domain for effector function targeting is linked at the C-terminus of C L to direct effector cells to the disease site.

A: single binding domain based Fab and IgG antibody formats。

The present invention provides a variety of antibodies, such as Fab and IgG antibodies, based on a combination of single binding domains.

A1: monovalent bispecific antibodies

In one aspect, the invention provides an engineered monovalent bispecific antibody comprising: (1) a first chain comprising a first antigen-binding single domain that binds to a first target and is linked to the N-terminus of CH1 of the Fab heavy chain with an affinity of about 10^-5～10^-8M, preferably 10^-5～10^-7A second strand comprising a second antigen-binding single domain linked to the N-terminus of C L of the light chain (kappa or lambda strand), binding a second target, and having about 10^-5～10^-8Second affinity of M, preferably 10^-5～10^-7M。

In some embodiments, the engineered antibody has only two single chains, e.g., one light chain and one heavy chain, covalently linked after co-transfection of the two genes in the expression cassette into the expression cell system. Fig. 2A shows an example.

Typically, as provided herein, the first antigen is a disease-specific target and the second antigen is an immunomodulatory functional target associated with the same disease.

A2: dual bivalent bispecific antibody

In another aspect, the invention provides an engineered bibivalent bispecific antibody (DBB) antibody comprising (1) a first chain comprising a first antigen-binding single domain linked to the N-terminus of CH1 of an IgG heavy chain that binds a first target, and having about 10^-5～10^-8M, a first affinity, (2) a second strand comprising a second antigen-binding single domain linked to the N-terminus of C L of the light chain (kappa or lambda strand), binding a second target, and having about 10^-5～10^-8A second affinity for M; (3) (iii) a third strand identical to the first strand, and (iv) a fourth strand identical to the second strand, wherein the first strand is linked to the second strand to form a first arm, the third strand is linked to the fourth strand to form a second arm, and the first arm and the second arm are linked by IgG Fc dimerization. An example is shown in fig. 2B.

In some embodiments, the engineered antibody has a total of four chains, e.g., two light chains (each comprising a binding domain and one C L) and two heavy chains (each comprising a binding domain and one CH 1). the two light chains have the same sequence and the two heavy chains have the same sequence.

Typically, as provided herein, the first antigen is a disease-specific target and the second antigen is an immunomodulatory functional target associated with the same disease.

A3: monovalent trispecific antibodies

In another aspect, the invention provides an engineered monovalent trispecific antibody comprising: (1) a first chain comprising a first antigen-binding single domain attached to the N-terminus of CH1 of the Fab heavy chain that binds to a first target and has a first affinity of about 10^-5～10^-8M, preferably 10^-5～10^-7M, (2) a second chain comprising a second antigen-binding single domain linked to the N-terminus of C L of the light chain (kappa or lambda chain), binding a second target, and having about 10^-5～10^-8Second affinity of M, preferably 10^-5～10^-7M, and a third antigen-binding single domain linked to the C-terminus of C L of the light chain (kappa or lambda chain) that binds a third antigen and has a molecular weight of about 10^-5～10^-7A second affinity of M. FIG. 2C showsAn example is shown.

In some embodiments, the engineered antibody has only two chains, e.g., one light chain and one heavy chain covalently linked by the Fab constant regions of CH1 and C L1.

Typically, the first antigen is a disease-specific target, the second antigen is an immunomodulatory functional target associated with the same disease, and the third antigen is an effector functional target.

A4: monovalent tetraspecific antibodies

In one aspect, the invention provides an engineered monovalent tetraspecific antibody comprising: (1) a first chain comprising a first antigen binding single domain linked to the N-terminus of CH1 of the Fab heavy chain, which binds to a first target, having about 10^-5～10^-8A first affinity for M; a fourth antigen binding single domain linked to the C-terminus of CH1 of the Fab heavy chain, binding a fourth target, having about 10^-5～10^-8M, a fourth affinity, (2) a second chain comprising a second antigen-binding single domain linked to the N-terminus of C L of the light chain (kappa or lambda chain), which binds a second target, and also has about 10^-5～10^-8A second affinity of M, and a third antigen-binding single domain linked to the C-terminus of C L of the light chain (kappa or lambda chain) that binds a third target, having about 10^-5～10^-8A third affinity of M. Fig. 2D shows an example.

Typically, the first antigen is a disease-specific target, the second antigen is a disease-associated target for immunomodulatory function, the third antigen is a target for effector function, and the fourth antigen is a target for a fourth function, such as albumin or other target. After binding, the in vivo half-life of the antibody can be safely extended.

A5: monovalent trispecific antibody-albumin drugs

In one aspect, the invention provides an engineered monovalent trispecific antibody-albumin conjugate comprising: (1) a first chain comprising a first antigen-binding single domain attached to the N-terminus of CH1 of the Fab heavy chain that binds to a first target and has a first affinity of about 10^-5～10^-8M; and with Fab heavy chainsA C-terminally linked fourth protein fragment of CH1 that can extend the in vivo half-life of a functional protein (2) a second chain comprising a second antigen-binding single domain linked to the N-terminus of C L of a light chain (kappa or lambda chain), that binds to a second target, and that has about 10^-5～10^-8A second affinity for M and (3) a third antigen-binding single domain linked to the C-terminus of C L of the light chain (kappa or lambda chain), which binds a third antigen, and has a molecular weight of about 10^-5～10^-8A third affinity of M. Fig. 2E shows an example.

Typically, the first antigen is a disease-specific target, the second antigen is a disease-associated target for immune-regulatory function, the third antigen is a target for effector function, and the fourth fragment is albumin or other target that, when bound, can safely extend the in vivo half-life of the antibody.

A6: precision-guided multifunctional therapeutic antibody (GCT Ab)

In one aspect, the invention provides an engineered, precision-directed multifunctional therapeutic antibody comprising (1) a first chain comprising a first antigen-binding single domain linked to the N-terminus of CH1 of an IgG heavy chain, binding a first target, and having about 10^-5～10^-8M, a first affinity, (2) a second strand comprising a second antigen-binding single domain linked to the N-terminus of C L of the light chain (kappa or lambda strand), binding a second target, and having about 10^-5～10^-8A second affinity of M, and a third antigen-binding single domain linked to the C-terminus of C L of the light chain (kappa or lambda chain), which binds a third antigen, and has about 10^-5～10^-8A third affinity of M. (3) A third strand identical to the first strand; (4) a fourth strand identical to the second strand, wherein the first strand is linked to the second strand to form a first arm, the third strand is linked to the fourth strand to form a second arm, and wherein the first arm and the second arm are linked by IgG Fc dimerization. (5) The modified Fc region, except for long half-life for FcRn binding, lacks all effector functions mediated by Fc. Fig. 2F shows an example.

Typically, the first antigen is a disease-specific target, the second antigen is a disease-associated immunoregulatory function target, and the third antigen is an effector function target, Fc if an IgG Fc comprising a P329G-L a L a modification.

In some embodiments, the first strand and the third strand have the same sequence.

In some embodiments, the first antigen binding domain and the third antigen binding domain have the same sequence.

In some embodiments, the second strand and the fourth strand have the same sequence.

In some embodiments, each of the first affinity, the second affinity, the third affinity, or the fourth affinity is less than 10 when applied^-8M, e.g. 10^-5～10^-8M, preferably about 10^-5～10^-7M。

B: disease-specific targets

Typically, the first antigen is a disease-specific target.

The disease-specific target may be a tumor target (e.g., Her2, Jamnani, f.r. et al, T cell expression VHH-directed oligomeric genetic Her2 antigen receptors: towardswstem-directed oligomeric T cell therapy. biochemica et biomedical acta 1840, 378. 386(2014), Even-Desrumeaux, k. fountain, p. Secq., v. body, D. Chames, p. single-domain antigens: a versatilities and rich source for branched antigenic polypeptides, molecular biology systems 8, 236385 (e.g., WO 236378 k) new antigens (e.g., t.r.t. r.t. r.r.p.m.t. 23 r.r.p.m.n.p.m.n.p.t. n.r.t. 3. r.g., t.r. r. t. r.p. n. t. receptor, T. c.t. 23. r.g., t. 3. r. T. g., T. r. g., T. wo. 35, T. g. fr.p. t. 23. T. 3. g. r. T. 3. g., T. 3. g., r. 3. r. 3. g. r. g., r. c. r. 3. c. (r. 3).

In some embodiments, the disease-specific target is selected from a disease marker, cytokine, or chemokine in table 1, or a target in table 2.

Table 1: target list

TABLE 2

In some embodiments, the disease-specific target is selected from antigens that are overexpressed in cancer cells, including intercellular adhesion molecule 1(ICAM-1), ephrin a-type receptor 2(EphA2), ephrin a-type receptor 3(EphA3), ephrin a-type receptor 4(EphA4), or activated leukocyte adhesion molecule (a L CAM).

In some embodiments, the disease-specific target is selected from the group consisting of cancer-or tumor-associated guide antigens, including CD30, CD33, PSMA, mesothelin, CD44, CD73, CD38, mucin 1 cell surface-associated (MUC1), mucin 2 oligomeric mucgel-forming (MUC2), and MUC16 (CA-125).

In some embodiments, the disease-specific target is selected from CD30, CD33, carcinoembryonic antigen (CEA), mesothelin, cathepsin G, CD44, CD73, CD38, Mucl, Muc2, Mucl 6, preferentially expressed melanoma antigen (PRAME), CD52, EpCAM, CEA, gpA33, mucin, tumor-associated glycoprotein 72(TAG-72), carbonic anhydrase IX, PSMA, folate binding protein, ganglioside, L ewis-Y, immature laminin receptor, BING-4, calcium-activated chloride channel 2(CaCC), gp100, synovial sarcoma X breakpoint 2(SSX-2), or SAP-I.

In some embodiments, the disease-specific target is selected from CD30, CD33, lung embryo tension antigen (CEA), mesothelin, cathepsin G, CD44, CD73, CD38, Mucl, MucI6, preferentially expressed melanoma antigen (PRAME), CD52, EpCAM, CEA, gpA33, mucin, TAG-72, carbonic anhydrase IX, PSMA, folate binding protein, ganglioside or L ewis-Y, ICAM-1, EphA2 or a L CAM.

C. Immunoregulation function target

The target of immune modulatory function can be a checkpoint receptor (e.g., PD-L1 (patents WO 2017020801-PAMPH-866 and Zhang, F et al structural basis of a novel PD-L1 nanobody for immune checkpoint. cell discovery3,17004(2017), 8) or a regulatory cytokine receptor, among others.

In some embodiments, the immunomodulatory functional target is selected from one of the receptors provided in table 1.

In some embodiments, the immunoregulatory functional target is associated with an NK cell activation or inhibition pathway and is selected from CD16, CD38, NKG2D, NKG2A, NKp46, or a killer cell immunoglobulin-like receptor (KIR).

In some embodiments, the immunomodulatory functional target is associated with a checkpoint inhibition pathway (which may be active in T cells, NK cells, or the complement system) and is selected from, but not limited to, PD1, CT L a4, CD47, CD59, and Tim 3.

D. Effector function target

Typically, the third antigen is an effector function target.

Defined effector function targets may be T cell markers such as CD3 (patent WO2010037838), NK cells (e.g. CD16, Behar, G et al, Isolation and characterization of anti-fcgamma iii (CD16) collagen single-domain antibodies which are active natural killer cells, design & selection: PEDS21,1-10(2008)), macrophages such as CD47(US8377448B2) and the like. Pairing effector cells with disease-specific targets can direct effector cells to the site of disease with the help of blocking inhibitory immunoregulatory targets to mediate a potent effect on the disease target. Furthermore, the paired, fine-tuned affinity of the effector function targeting domain to the blocking inhibitory immunoregulatory target may also improve the safety of effector targeting as described above.

In some embodiments, the effector function target is selected from one of the receptors in table 1.

E. Single domain antibody binding fragments

In general, the antibodies provided herein comprise a plurality of single domain antigen binding fragments.

Single domain antibodies can be obtained by modifying known antibodies to selected targets, antigens or epitopes by direct screening methods known in the art for the desired antigen.

V_HOr V_LThe binding domain may be derived from any single domain binding source, including but not limited to animal sources (camel, alpaca, engineered mouse/rat, human Ig transgenic mouse/rat, etc.), engineered heavy chain-only antibody repertoires, engineered light chain-only antibody repertoires, humanized antibody binding domains, or the design of known receptors and ligands by binding domains of soluble factors to selected targets, antigens or epitopes, etc.

Most antibodies have a nanomolar (10)^-7To 10^-9) KD values within the range. The KD values for high affinity antibodies are typically in the picomolar range (10)^-9To 10^-11) Internal, but very high affinity antibodies are in the low picomolar range (10)^-11To 10^-12) And (4) the following steps.

Single domain antibodies with lower affinity can be generated by fine-tuning existing antibodies, for example, by altering one or more amino acid sequences, thereby changing the affinity to a desired range, but still retaining specificity.

The single domains of the invention are capable of specific binding to a target. "target" or "marker" in the context of the present invention refers to any entity capable of specifically binding to a particular target therapeutic agent, such as Her 2/Neu. In some embodiments, the target is specifically associated with one or more specific cell or tissue types. In some embodiments, the target is specifically associated with one or more specific disease states. In some embodiments, the target is specifically associated with one or more specific developmental stages. For example, the expression level of a cell type specific marker in that cell type is typically at least 2-fold higher than in a reference cell population. In some embodiments, the cell-type specific marker is at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 50-fold, at least 100-fold, or at least 1000-fold greater than its average expression in the control population. Detection or measurement of cell type specific markers can distinguish one or more cell types of interest from many, most, or all other cell types. As described herein, in some embodiments, a target may comprise a protein, a carbohydrate, a lipid, and/or a nucleic acid.

"specific binding" or "preferential binding" in the context of the present invention means that the binding between two binding partners (e.g., between a targeting moiety and its binding partner) is selective for both binding partners and can be distinguished from unwanted or non-specific interactions, for example the ability of an antigen binding moiety to bind a specific epitope can be determined by enzyme-linked immunosorbent assay (E L ISA) or other techniques familiar to those skilled in the art surface plasmon resonance techniques (analyzed on a BIAcore instrument) (L iljebold et al, Glyco J17, 323-329(2000)) and traditional binding assays (Heeley, Endocr Res 28,217-229 (2002)). the "anti [ antigen ] antibody" and the "antibody that binds to [ antigen ] refer to antibodies that are capable of binding to the corresponding antigen with sufficient affinity such that the antibody targets the antigen for use as a diagnostic and/or therapeutic agent.

In some embodiments, the dissociation constant (KD) for antigen binding is less than 100 μ M, less than 10 μ M, less than 1 μ M, less than 100nM, less than 10nM, less than 1nM, less than 0.1nM, less than 0.01 nM. Or less than 0.001nM (e.g., 10)^-4M or less, e.g. 10^-4M to 10^-12M, e.g. 10^-9M to 10^-13M), preferably 10^-5M to 10^-8M。

In some embodiments, the targeted therapeutic comprises an antibody or functional fragment thereof. In certain specific embodiments, the target is a tumor marker. In some embodiments, a tumor marker is an antigen present in a tumor that is not present in normal organs, tissues, and/or cells. In some embodiments, a tumor marker is an antigen that is more prevalent in a tumor than in a normal organ, tissue, and/or cell. In some embodiments, the tumor marker is an antigen that is more prevalent in malignant cancer cells than in normal cells.

In the present invention, "tumor antigen" refers to an antigenic substance produced in tumor cells, i.e., it triggers an immune response in a host, a normal protein in a human body is not antigenic due to self-tolerance, in the process, cytotoxic T lymphocytes (CT L) produced by self-reaction and B lymphocytes producing autoantibodies are knocked out in the primary lymphoid tissue (BM) at the "center" and secondary lymphoid tissue (T cells are mainly thymus, B cells are spleen/lymph nodes) is knocked out in the peripheral blood.

In some embodiments, the target is preferentially expressed in tumor tissue and/or cells as compared to normal tissue and/or cells.

In some embodiments of the invention, the marker is a tumor marker. The marker may be a polypeptide that is expressed at a higher level at division than in non-dividing cells. For example, Her-2/neu (also known as ErbB-2) is a member of the EGF receptor family and is expressed on the cell surface of tumors associated with breast cancer. Another example is the peptide named F3, which is a suitable targeting agent to direct nanoparticles to nucleolin (Porkka et al, 2002, proc.natl.acad.sci., USA,99: 7444; and Christian et al, 2003, j.cell biol.,163: 871.) it has been shown that targeting particles comprising nanoparticles and an a10 aptamer (which specifically binds PSMA) are capable of specifically and efficiently delivering docetaxel to prostate cancer tumors.

Antibodies or other drugs that specifically target these tumor targets specifically interfere with and modulate signaling pathways of tumor cell biological behavior, directly modulate or block signaling pathways to inhibit tumor cell growth or induce apoptosis. To date, dozens of target drugs for clinical research and treatment of solid tumors or hematologic malignancies, and many targeted drugs for hematologic malignancies, have been approved.

In some embodiments, the tumor antigen (or tumor target) is selected from: CD2, CD19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, and CD 137.

In some embodiments, the tumor antigen (or tumor target) is selected from the group consisting of 4-1BB, 5T, AGS-5, AGS-16, angiopoietin 2, B7.1, B7.2, B7, B7H, B7H, B7H, BT-062, BT A, CAIX, carcinoembryonic antigen, CT A, Cripto, EDB, ErbB, ErbB, ErbB, EGF 07, EpCAM, EphA, EphA, EphB, FAP, fibronectin, folate saponin 3, GD, glucocorticoid-induced tumor necrosis factor receptor (GITR), gp100, gpA, GPNMB, ICOS, IGF1, integrin, KIR, TtltTtT transition "= L &lTtTtTtTtTtTtTtTtTtT-3, Wis Y antigen, mesothelin, C-MET, MN, MUAC 1, MULAT 1 =" L &L & -gTtT-3, GAV-3, VEGFR-R-5, VEGFR-1, VEGFR-5, VEGFR-16, VEGFR-1, VEGFR-A, VEGFR-14, VEGFR, VE.

An "immunoglobulin" or "antibody" in the context of the present invention refers to a full-length (i.e., naturally occurring or formed by the process of recombination of normal immunoglobulin gene fragments) immunoglobulin molecule (e.g., an IgG antibody) or an immunologically active (i.e., specifically binding) portion of an immunoglobulin. An immunoglobulin molecule, such as an antibody fragment, may be coupled to or derived from an antibody or antibody fragment within the scope of the claimed subject matter. Such antibodies include IgG1, IgG2, IgG3, IgG4 (and IgG4 subtypes), and IgA isotypes.

The "antibody" in the present invention is used in the broadest sense and encompasses a variety of antibody structures as long as they exhibit the desired antigen binding activity and comprise an Fc region or a region equivalent to an Fc region of an immunoglobulin, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments. The present invention uses "full length antibody", "intact antibody" and "whole antibody" interchangeably refer to an antibody having a structure substantially similar to a native antibody or having a heavy chain comprising an Fc region as defined herein.

For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains, which are disulfide-bonded, each heavy chain having, from N-terminus to C-terminus, a variable region (VH), also known as a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2 and CH3), also known as heavy chain constant regions.

An "antibody fragment" of the invention refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab ', Fab ' -SH, F (ab ')2, diabodies, linear antibodies, single chain antibody molecules (e.g., scFv), single domain antibodies, and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Hudson et al, Nat Med 9, 129-. For reviews on scFv fragments see Pliickthun, in The Pharmacology of Monoclonal Antibodies, vol.113, Roscnburg and Moore eds., Springer-Verlag, New York, pp.269-315(1994), see also WO 93/16185 and U.S. Pat. Nos. 5,571,894 and 5,587,458. See U.S. Pat. No.5,869,046 for a discussion of Fab and F (ab')2 fragments that salvage receptor binding epitope residues and have increased half-life in vivo. Diabodies are antibody fragments with two antigen binding sites that can be bivalent or bispecific. For example EP 404,097, WO 1993/01161; hudson et al, Nat Med 9, 129-; nat Med 9: 129-134(2003). See Hollinger et al, Proc Natl Acad Sci USA 90, 6444-. Tri-and tetrabodies are also described in Hudson et al Nat Med 9, 129-134 (2003). Single domain antibodies are antibody fragments that comprise all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody. In some embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No.6,248,516B 1). As described herein, antibody fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production of recombinant host cells (e.g., E.coli or phage).

An antigen binding domain may be provided by, for example, one or more antibody variable domains (also referred to as antibody variable regions or single domain antibodies or domain antibodies). in particular, an antigen binding domain comprises an antibody light chain variable region (V L) and an antibody heavy chain variable region (VH). the antigen binding domain may be provided, for example, by a soluble domain of a receptor or ligand, e.g., soluble PD-1 domain bound PD-L1/L2 or soluble SIRPA domain bound CD 47.

The variable domains of the heavy and light chains of natural antibodies (VH and V L, respectively) typically have similar structures, each domain comprising four conserved Framework Regions (FRs) and three hypervariable regions (HVRs), see, e.g., Kindt et al, KubyImmunology, 6 th edition, w.h.freeman and co., page 91 (2007). a single VH or V L domain may be sufficient to confer antigen-binding specificity.

Generally, a naturally occurring four-chain antibody comprises six HVRs, three of the VH (HI, H2, H3) and three of the V L (L I, L2, L3). generally, HVRs comprise amino acid residues from the hypervariable loops and/or from Complementarity Determining Regions (CDRs) which have the highest sequence variability and are involved in antigen recognition.in addition to CDR1 in the VH, the CDRs generally comprise amino acid residues forming hypervariable loops.

The antibody of the invention may be a chimeric antibody, a humanized antibody, a human antibody or an antibody fusion protein.

"chimeric antibody" in the present invention refers to a recombinant protein comprising the variable regions of the heavy and light chains of an antibody, including the Complementarity Determining Regions (CDRs) of an antibody derived from one species, preferably a rodent antibody, more preferably a murine antibody. The constant domains of antibodies are derived from the constant domains of human antibodies. For veterinary applications, the constant domains of the chimeric antibodies may be derived from domains of other species, such as a sub-human primate, cat or dog.

"humanized antibodies" of the invention refer to recombinant proteins derived from the CDRs of an antibody of one species, e.g., the transfer of a rodent antibody from the heavy and light chain variable domains of a rodent antibody into the human heavy and light chain variable domains. The constant domains of the antibody molecule are derived from the constant domains of human antibodies. In some embodiments, specific residues of the framework regions of the humanized antibody, particularly those contacting or near the CDR sequences, can be modified, e.g., replaced with corresponding residues from the original rodent, sub-human primate, or other antibody.

In this technique, elements of human heavy and light chain gene loci are introduced into mouse strains derived from a targeted disrupted embryonic stem cell line containing endogenous heavy and light chain gene loci, transgenic mice can synthesize human antibodies specific for human antigens, and these mice can be used to produce hybridomas secreting human antibodies.A method for obtaining human antibodies from transgenic mice was described in 1994 Green et al Nature Genet.7:13, 1994L onberg et al Nature 368:856, and 1992 Taylor et al int.Immun.6: 579. also whole human antibodies can be constructed by genetic or chromosomal transfection methods and phage display techniques, all of which are known in the art.Cafferty et al Nature: 553, the humanized donor genes are expressed in vitro by the expression of the phage coat gene, the expression of which results in the expression of the recombinant DNA, and the expression of the filamentous phage display gene fragment of this phage 5, the expression of which results in the expression of the recombinant phage display of the recombinant DNA, and the expression of the filamentous phage display of the functional expression of the recombinant DNA fragment of the phage, such as a filamentous phage display protein, and its expression of the filamentous phage display protein, such as described in vitro phage 5-expressing the chimeric antibody.

An "antibody fusion protein" in the context of the present invention refers to a recombinantly produced antigen-binding molecule in which two or more identical or different natural antibodies, single-chain antibodies, or antibody fragments with identical or different specificities are linked together. The fusion protein comprises at least one specific binding site. The valence of the fusion protein indicates the total number of binding arms or binding sites of the fusion protein to the antigen or epitope; i.e., mono-, di-, tri-, or polyvalent. The multivalency of an antibody fusion protein means that it can take advantage of multiple interactions with antigen binding, thereby increasing the affinity of binding to a certain antigen or to a different antigen. Specificity indicates how many different types of antigens or epitopes the antibody fusion protein is capable of binding; i.e., monospecific, bispecific, trispecific, and multispecific. According to the above definition, a natural antibody (e.g. IgG) is bivalent because it has two binding arms, but it is monospecific because it binds to one type of antigen or epitope. Monospecific multivalent fusion proteins have more than one binding site for the same antigen or epitope. For example, a monospecific diabody is a fusion protein with two binding sites reactive with the same antigen. The fusion protein may comprise multivalent or multispecific combinations of different antibody components or multiple copies of the same antibody component. The fusion protein may further comprise a therapeutic agent.

"target" or "label" in the context of the present invention refers to any entity capable of specifically binding to a particular targeting moiety. In some embodiments, the target is specifically associated with one or more specific cell or tissue types. In some embodiments, the target is specifically associated with one or more specific disease states. In some embodiments, the target is specifically associated with one or more specific developmental stages. For example, the cell type-specific marker is typically expressed at a level at least 2-fold higher in that cell type than in a control cell population. In some embodiments, the cell-type specific marker is at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 50-fold, at least 100-fold, or at least 1000-fold greater than its average expression in the control population. Detection or measurement of cell type specific markers can distinguish one or more cell types of interest from many, most, or all other cell types. As described herein, in some embodiments, a target may comprise a protein, a carbohydrate, a lipid, and/or a nucleic acid.

A substance is considered "targeted" if it specifically binds to a nucleic acid targeting moiety. In some embodiments, the nucleic acid targeting moiety specifically binds to the target under stringent conditions. A complex or compound of the invention comprising a targeting moiety is considered to be "targeted" if the targeting moiety specifically binds to the target, thereby delivering the entire complex or compound composition to a particular organ, tissue, cell, extracellular matrix component, and/or intracellular compartment.

In some embodiments, the antibodies of the invention comprise single domain antibodies or fragments that specifically bind to one or more targets (e.g., antigens) associated with an organ, tissue, cell, extracellular matrix component, and/or intracellular compartment. In some embodiments, the compound comprises a targeting moiety that specifically binds to a target associated with a particular organ or organ system. In some embodiments, the compounds of the invention comprise a nuclear targeting moiety that specifically binds one or more intracellular targets (e.g., organelles, intracellular proteins). In some embodiments, the compound comprises a targeting moiety that specifically binds to a target associated with the diseased organ, tissue, cell, extracellular matrix component, and/or intracellular compartment. In some embodiments, the compound comprises a targeting moiety that specifically binds to a target associated with a particular cell type (e.g., endothelial cells, cancer cells, malignant cells, prostate cancer cells, etc.).

In some embodiments, the antibodies of the invention comprise a domain antibody or fragment that has target binding specific for one or more specific tissue types (e.g., liver tissue versus prostate tissue). In some embodiments, the compounds of the invention comprise a domain that binds to a target specific for one or more particular cell types (e.g., T cell versus B cell). In some embodiments, the antibodies of the invention comprise a target-binding domain specific for one or more specific disease states (e.g., tumor cells versus healthy cells). In some embodiments, the compounds of the invention comprise a targeting moiety that binds to a target specific for one or more particular developmental stages (e.g., stem cells versus differentiated cells).

In some embodiments, the target may be a marker that is associated exclusively or predominantly with one or several cell types, one or several diseases and/or one or several developmental stages. The level of expression of a cell-type specific marker in that cell type is typically at least 2-fold higher than in a control cell population, which may consist of, for example, a mixture comprising roughly equal numbers of multiple cells (e.g., 5-10 or more) in different tissues or organs. In some embodiments, the cell-type specific marker is at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 50-fold, at least 100-fold, or at least 1000-fold greater than its average expression in the control population. Detection or measurement of cell type specific markers can distinguish one or more cell types of interest from many, most, or all other cell types.

In some embodiments, the target comprises a protein, a carbohydrate, a lipid, and/or a nucleic acid. In some embodiments, the target comprises a protein and/or characteristic portions thereof, such as tumor markers, integrins, cell surface receptors, transmembrane proteins, intercellular proteins, ion channels, membrane transporters, enzymes, antibodies, chimeric proteins, glycoproteins, and the like. In some embodiments, the target comprises carbohydrates and/or characteristic portions thereof, such as glycoproteins, sugars (e.g., monosaccharides, disaccharides, and polysaccharides), glycocalyx (i.e., a carbohydrate-rich peripheral region of the outer surface of most eukaryotic cells, etc.). In some embodiments, the target comprises a lipid and/or characteristic portion thereof, such as an oil, fatty acid, glyceride, hormone, steroid (e.g., cholesterol and bile acid), vitamin (e.g., vitamin E), phospholipid, sphingolipid, lipoprotein, and the like. In some embodiments, the target comprises a nucleic acid and/or characteristic portions thereof, such as a DNA nucleic acid, an RNA nucleic acid, a modified DNA nucleic acid, a modified RNA nucleic acid, and a nucleic acid comprising any combination of DNA, RNA, modified DNA, and modified RNA.

Exemplary receptors include, but are not limited to, transferrin receptor, L D L receptor, growth factor receptors (e.g., epidermal growth factor receptor family members EGFR, Her2, Her3, Her4) or vascular endothelial growth factor receptors, cytokine receptors, cell adhesion molecules, integrins, selectins, and CD molecules.

In some embodiments, the binding domain specifically binds to a tumor cell or preferentially binds to a tumor cell as compared to a non-tumor cell.

Binding of the target moiety to the tumor cell can be measured using assays known in the art.

In some embodiments, the tumor cell is a carcinoma, sarcoma, lymphoma, myeloma, or central nervous system cancer.

In some embodiments, the binding domain is capable of specifically or preferentially binding to a tumor antigen as compared to a non-tumor antigen.

In certain specific embodiments, the target is a tumor marker. In some embodiments, a tumor marker is an antigen present in a tumor that is not present in normal organs, tissues, and/or cells. In some embodiments, a tumor marker is an antigen that is more prevalent in a tumor than in a normal organ, tissue, and/or cell. In some embodiments, the tumor marker is an antigen that is more prevalent in malignant cancer cells than in normal cells.

In some embodiments, the targeting moiety comprises folic acid or a derivative thereof.

In recent years, research on folic acid has been advanced. Folic acid is a small molecule vitamin essential for cell division. Tumor cells divide abnormally and express folate receptors at high levels on the tumor cell surface to capture sufficient folate to support cell division.

The data show that the expression rate of FR in tumor cells is 20-200 times higher than that of normal cells, the expression rate of FR in various malignant tumors is 82% of ovarian cancer, 66% of non-small cell lung cancer, 64% of renal cancer, 34% of colon cancer and 29% of breast cancer (Xia W, L ow PS, L ate-targeted therapeutics for cancer. J Med chem.2010; 14; 53(19): 6811-24). the expression rate of FA is positively correlated with the malignancy of invasion and metastasis of epithelial tumors, FA enters cells through FR-mediated endocytosis, and FA forms FA complexes with drugs entering the cells through carboxyl thereof, and the FA releases the drugs into cytoplasm under acidic conditions (pH 5).

Clinically, the system can be used to deliver drugs that selectively attack tumor cells. Folic acid has a small molecular weight, is non-immunogenic and highly stable, and is inexpensive to synthesize. More importantly, the chemical coupling between the drug and the carrier is simple, so that the construction of a drug delivery system by using FA as a target molecule has become a hot research point for cancer treatment. Currently, EC145(FA chemotherapeutic drug conjugates) in clinical trials can effectively attack cancer cells (Prible P and Edelman MJ.EC 145: anovel targeted agent for the lung. ExpertOpin. investig. drugs (2012)21:755 Across 761).

In some embodiments, the targeting moiety comprises an extracellular domain (ECD) or a soluble form of PD-1, PD L-1, CT L a4, CD47, BT L a, KIR, TTM3, 4-IBB, and L AG3, full length surface ligand amphiregulin, cytokine, EGF, Ephrin, Epigen, epithelial regulatory protein, IGF, neuregulin, TGF, TRAI L, or VEGF.

In some embodiments, the targeting moiety comprises a Fab, Fab ', F (ab')2, single domain antibody, T and Abs dimer, Fv, scFv, dsFv, ds-scFv, Fd, linear antibody, miniantibody, diabody, bispecific antibody fragment, diabody, triabody, sc diabody, kappa (lamda) antibody, BiTE, DVD-Ig, SIP, SMIP, DART, or an antibody analog comprising one or more CDRs.

In some embodiments, the targeting moiety is an antibody or antibody fragment selected based on the specificity of the antigen expressed on the target cell or target site of interest a variety of tumor-specific or other disease-specific antigens have been identified and antibodies against those antigens have been or are proposed for use in the treatment of such tumors or other diseases antibodies known in the art can be used in the compounds of the invention, particularly in the treatment of diseases associated with the target antigen examples of target antigens (and their associated diseases) that can be targeted by the antibody-linker-drug conjugates of the invention include CD, CD137, CD, 5T, AGS-5, AGS-16, angiopoietin 2, B7.1, B7.2, B7H, BT-tt α, CAIX, carcinoembryonic antigen, CT a, Cripto, ErbB3, VEGFR, ErbB2, egfr, VEGFR-R, egfr-T-receptor, ttt 2, ttt-T-protein, T-protein, T-R, T-protein, T-R-T-5, agc, VEGFR-5, AGS-5, agc, 2, egfr-R-5, egfr, 2, egfr, ttt-R-5, 2, ttt-R-5, ttt-R-.

F. Antibody production

All antibody formats are based on the heavy and light chains of an IgG antibody and can be produced using methods known in the art, typically including the following steps: heavy and light chain gene expression cassettes are constructed, the two genes are integrated into a suitable cell system to produce recombinant antibodies and to produce stable and high-yielding cell clones, which are then fermented by the cells to produce the cGMP final antibody product.

3. Pharmaceutical formulations and administration

The invention further relates to a pharmaceutical formulation comprising a compound of the invention, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

The compounds of the present invention, including pharmaceutically acceptable carriers, such as addition salts or hydrates thereof, may be delivered to a patient using a variety of routes or modes. Suitable routes of administration include, but are not limited to, inhalation, transdermal, oral, rectal, transmucosal, intestinal and parenteral administration, intramuscular, subcutaneous and intravenous injection. Preferably, the compounds of the invention comprising an antibody or antibody fragment as targeting moiety are administered parenterally, more preferably intravenously.

As used herein, "administering" is intended to encompass all means for delivering a compound directly and indirectly to its intended site of action.

The compounds of the present invention or pharmaceutically acceptable salts and/or hydrates thereof may be administered alone in combination with other compounds of the present invention, and/or in admixture with other therapeutic agents. Of course, how a therapeutic agent is selected for co-administration with a compound of the invention will depend in part on the condition being treated.

For example, when administered to a patient suffering from a disease state caused by an autoinducer-dependent organism, the compounds of the invention may be administered in a mixture that typically contains agents useful for treating conditions associated with pain, infection, and other symptoms and side effects. Such agents include, for example, analgesics, antibiotics, and the like.

When administered to a patient undergoing cancer treatment, these compounds may be administered in a mixture with an anti-cancer agent and/or a co-enhancer. The compounds may also be administered in mixtures containing drugs that treat side effects of radiation therapy, such as antiemetics and radioprotectors.

Supplemental enhancers that may be co-administered with the compounds of the invention include, for example, tricyclic antidepressants (e.g., imipramine, desipramine, amitriptyline, clomipramine, trimipramine, doxepin, nortriptyline, protriptyline, amoxapine, and maprotiline); non-tricyclic and anti-depressant drugs (e.g., sertraline, trazodone and citalopram); ca²⁺Antagonists (e.g., verapamil, nifedipine, nitrendipine and carpivastine); amphotericin, triphenyl alcohol analogs (e.g., tamoxifen); antiarrhythmic drugs (such as quinidine); hypotensive agents (e.g., reserpine); thiol depleting agents (e.g., buthionine and sulfenimide) and calcium folinate.

One or more of the active compounds of the present invention may be administered by itself, or in admixture with other pharmaceutical compositions. Wherein the active compound may be mixed with one or more pharmaceutically acceptable carriers, excipients or diluents. Pharmaceutical compositions for use in accordance with the present invention are generally formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Suitable formulations depend on the chosen route of administration.

For mucosal 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 compounds may be formulated simply by combining the active compound with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for oral administration to a patient to be treated. Oral pharmaceutical preparations can be obtained by obtaining solid excipients, optionally grinding the resulting mixture, and processing the mixture, after adding suitable auxiliaries, to obtain tablets or dragee cores. Suitable excipients are especially fillers, for example sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, for example maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, such as cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof, such as sodium alginate.

Dragee cores require suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain acacia, talc, polyvinyl pyrrolidone, carbomer gel, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments can be added to the tablets or dragee coatings in order to identify or characterize different combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Push-fit capsules can contain the active ingredients in admixture with fillers (e.g., lactose), binders (e.g., starches) and/or lubricants (e.g., talc or magnesium stearate) and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, for example fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

Administration by inhalation requires the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas, and the compounds for use in the present invention are conveniently delivered in aerosol form from pressurized packs or a nebulizer. In the case of a pressurized aerosol, the dosage unit may be determined by delivering a measured quantity through a valve. Capsules and cartridges (e.g., of gelatin) containing a powder mix of the compound and a suitable powder base (e.g., lactose or starch) for use in an inhaler or insufflator may be formulated.

The compounds may be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Injection is the preferred method of administration of the compositions of the present invention. Formulations for injection may be presented in unit dosage form, for example in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents, for example suspending, stabilizing and/or dispersing agents may be added, for example cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof (e.g. sodium alginate).

Pharmaceutical preparations for parenteral administration comprise aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils (e.g. sesame oil), or synthetic fatty acid esters (e.g. ethyl oleate or triglycerides), or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, for example sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to allow for the preparation of highly concentrated solutions. For injection, the agent of the present invention may be formulated into an aqueous solution, preferably into a physiologically compatible buffer such as hanks 'solution, ringer's solution or physiological saline buffer.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

The compounds may also be formulated in rectal 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 compounds may also be formulated as depot preparations. Such long acting formulations may be administered by implantation or transdermal delivery (e.g. subcutaneous or intramuscular), intramuscular injection or transdermal patch. The compounds may thus be formulated with suitable polymeric or hydrophobic materials (e.g. an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g. a sparingly soluble salt).

The pharmaceutical compositions may also comprise suitable solid or gel phase carriers or excipients. Such carriers or excipients include calcium carbonate, calcium phosphate, various sugars, starch, cellulose derivatives, gelatin, and polymers (e.g., polyethylene glycol).

Preferred pharmaceutical compositions are compositions formulated for injection, e.g., intravenous injection, and comprise from about 0.01% to about 100% by weight of the compound of the present invention, based on 100% by weight of the total pharmaceutical composition. The drug-ligand conjugate may be an antibody-cytotoxin conjugate in which an antibody has been selected for a particular cancer.

In some embodiments, the pharmaceutical compositions of the present invention further comprise an additional therapeutic agent.

In some embodiments, the additional therapeutic agent is an anti-cancer agent.

In some embodiments, the additional anti-cancer agent is selected from an anti-metabolite, an inhibitor of topoisomerase I and II, an alkylating agent, a microtubule inhibitor, an anti-androgen agent, a GNRh modulator, or a mixture thereof.

Examples include, but are not limited to, gemcitabine, irinotecan, doxorubicin, 5-fluorouracil, cytosine arabinoside ("Ara-C"), cyclophosphamide, tiotropium, busulfan, cytotoxins, TAXO L, methotrexate, cisplatin, melphalan, vinblastine, and carboplatin.

In some embodiments, the second chemotherapeutic agent is selected from tamoxifen, raloxifene, anastrozole, exemestane, letrozole, imastanib, paclitaxel, cyclophosphamide, lovastatin, minoxidil, gemcitabine, cytarabine, 5-fluorouracil, methotrexate, docetaxel, vinblastine, nocodazole, teniposide etoposide, gemcitabine, epothilone, vinorelbine, camptothecin, daunorubicin, actinomycin D, mitoxantrone, an aidine, doxorubicin, epirubicin or idarubicin.

4. Reagent kit

In another aspect, the invention provides a kit comprising a therapeutic combination provided by the invention and instructions for using the therapeutic combination. The kit may also comprise a container and optionally one or more vials, tubes, flasks, bottles, or syringes. Other forms of kits will be apparent to those skilled in the art and are within the scope of the invention.

5. Medical use

In another aspect, the present invention provides a method of treating a disease in a subject in need thereof, the method comprising: administering to the subject a therapeutic combination or pharmaceutical composition comprising a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In addition to the compositions and constructions described above, the present invention also provides various uses of the combinations of the invention. Uses of the combinations of the invention include: killing or inhibiting the growth, proliferation or replication of tumor cells or cancer cells, treating cancer, treating a precancerous condition, preventing proliferation of tumor cells or cancer cells, preventing cancer, preventing proliferation of cells expressing autoimmune antibodies. Such uses include administering to an animal (e.g., a mammal or a human) in need thereof an effective amount of a compound of the present invention.

The combinations of the invention are useful for treating a disease such as cancer in a subject such as a human. Combinations and uses for treating tumors by providing a composition and a pharmaceutically effective amount of a composition of the invention to a subject in a pharmaceutically acceptable manner are provided.

More specific examples of cancer include, but are not limited to, lung cancer (small and non-small cell), breast cancer, prostate cancer, carcinoid, bladder cancer, gastric cancer, pancreatic cancer, liver cancer (hepatocellular carcinoma), hepatoblastoma, colorectal cancer, head and neck squamous cell carcinoma, esophageal cancer, ovarian cancer, cervical cancer, endometrial cancer, mesothelioma, melanoma, sarcoma, osteosarcoma, liposarcoma, thyroid cancer, desmoid tumor, acute myelocytic leukemia (AM L), and chronic myelocytic leukemia (CM L).

"inhibition" or "treatment" of the present invention refers to both reduction of therapeutic medication and prophylactic treatment, with the purpose of reducing or preventing a targeted pathological disorder or condition. In one example, the tumor size of a cancer patient is reduced after administration of a compound of the invention. "treating" includes (1) inhibiting the disease in a subject experiencing or exhibiting pathology or symptomatology of the disease, (2) alleviating the suffering of the disease in a subject experiencing or exhibiting pathology or symptomatology of the disease, and/or (3) effecting a significant reduction in any disease in a subject or patient experiencing or exhibiting pathology or symptomatology of the disease. To the extent that the compounds of the present invention inhibit the growth of and kill cancer cells, they have cytostatic and/or cytotoxic effects.

By "therapeutically effective amount" of the present invention is meant an amount of a compound provided herein that is effective to "treat" a disease in a subject or mammal. In the case of cancer, a therapeutically effective amount of the drug may reduce the number of cancer cells, reduce the size of the tumor, inhibit cancer cell invasion into peripheral organs, inhibit tumor metastasis, inhibit tumor growth to some extent, and/or alleviate one or more symptoms associated with the cancer to some extent.

Administration "in combination with" one or more other therapeutic agents includes simultaneous and sequential administration in any order. As used herein, "pharmaceutical combination" refers to a product obtained by mixing or combining active ingredients and includes both fixed and non-fixed combinations of active ingredients. By "fixed combination" is meant that the active ingredients, e.g. the compound of formula (1) and the adjuvant, are both administered to the patient simultaneously in a single entity or dose. By "non-fixed combination" is meant that the active ingredients, e.g., the compound of formula (1) and the adjuvant, are both administered to the patient as separate entities simultaneously, simultaneously or sequentially without specific time constraints, wherein such administration provides effective therapeutic levels of the active ingredients in the patient. The latter also applies to the combination therapy, for example the administration of three or more active ingredients.

In some embodiments, the disease condition is a tumor or cancer. In some embodiments, the cancer or tumor is selected from the group consisting of stomach, colon, rectum, liver, pancreas, lung, breast, cervix, uterus, ovary, testis, bladder, kidney, brain/CNS, head and neck, throat, hodgkin's disease, non-hodgkin's lymphoma, multiple myeloma, leukemia, melanoma, non-melanoma skin cancer, acute lymphocytic leukemia, acute myelogenous leukemia, ewing's sarcoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, wilms' tumor, neuroblastoma, hairy cell leukemia, oral/pharyngeal, esophageal, laryngeal, renal cancer, or lymphoma.

In some embodiments, the disease condition comprises abnormal cell proliferation, such as a precancerous lesion.

The invention is particularly useful for treating cancer and inhibiting tumor cells or proliferation of cancer cells in an animal. Cancer or precancerous conditions, including tumors, metastases, or any disease or disorder characterized by uncontrolled cell growth, can be treated or prevented by administering the drug-ligand complexes of the invention. The compounds deliver an activated moiety to a tumor cell or cancer cell. In some embodiments, the targeting moiety specifically binds or associates with a cancer cell or an antigen associated with a tumor cell. Due to its close proximity to the ligand, the activated moiety, after internalization, can be taken up into the interior of the tumor or cancer cell by, for example, receptor-mediated endocytosis. The antigen may be attached to a tumor cell or a cancer cell, or to an extracellular matrix protein associated with a tumor cell or a cancer cell. Once inside the cell, the linker is hydrolyzed or enzymatically cleaved by tumor cell or cancer cell-associated proteases, thereby releasing the active moiety. The released activated moieties are then free to diffuse and induce or enhance the immune activity of the immune or tumor cells. In another embodiment, the activated moiety is cleaved from the compound tumor microenvironment before the drug penetrates the cells.

Representative examples of precancerous conditions that may be targeted by the compounds of the present invention include: transformation, hyperplasia, dysplasia, colorectal polyps, actinic ketosis, actinic cheilitis, human papilloma virus, leukoplakia, lichen planus and bowen's disease.

Representative examples of cancers or tumors that may be targeted by the compounds of the present invention include: lung cancer, colon cancer, prostate cancer, lymphoma, melanoma, breast cancer, ovarian cancer, testicular cancer, CNS cancer, kidney cancer, pancreatic cancer, stomach cancer, oral cancer, nasal cancer, cervical cancer, and leukemia. It will be apparent to one of ordinary skill that the particular targeting moiety used in the compound may be selected to target the activating moiety to the tumor tissue to be treated with the drug (i.e., selecting a particular targeting agent for a tumor-specific antigen). Examples of such targeting moieties are well known in the art, examples of which include anti-Her 2 for the treatment of breast cancer, anti-CD 20 for lymphoma, anti-PSMA for prostate cancer and anti-CD 30 for lymphoma (including non-hodgkin's lymphoma).

In some embodiments, the abnormal proliferation is of cancer cells.

In some embodiments, the cancer is selected from: breast cancer, colorectal cancer, diffuse large B-cell lymphoma, endometrial cancer, follicular lymphoma, gastric cancer, glioblastoma, head and neck cancer, hepatocellular cancer, lung cancer, melanoma, multiple myeloma, ovarian cancer, pancreatic cancer, prostate cancer, and renal cell carcinoma.

In some embodiments, the present invention provides compounds for killing cells. Administering the compound to the cells at a dose sufficient to kill the cells. In a typical embodiment, the compound is administered to a subject having the cell. In another exemplary embodiment, the administration is for delaying or stopping the growth of a tumor comprising the cell (which may be a tumor cell). To enable the administration to delay growth, the growth rate of the cells should be at least 10% less than the growth rate prior to administration. Preferably, the growth rate will be delayed by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or stopped completely.

In addition, the present invention provides a compound or pharmaceutical composition of the present invention for use as a medicament. The invention also provides a compound or pharmaceutical composition for killing, inhibiting or delaying the proliferation of tumor or cancer cells.

6. Effective dose

Pharmaceutical compositions suitable for use in the present invention include compositions containing a therapeutically effective amount of the active ingredient, such as an amount effective to achieve its intended purpose. The actual effective amount for a particular application depends inter alia on the condition to be treated. Determination of an effective amount is well within the ability of those skilled in the art, especially in light of the detailed disclosure of the present invention.

For any of the compounds described herein, a therapeutically effective amount can first be determined from a cell culture assay. The target plasma concentration is the concentration of the active compound that is capable of inhibiting cell growth or division. In a preferred embodiment, cellular activity is inhibited by at least 25%. Presently preferred target plasma concentrations of active compounds are capable of inducing at least about 30%, 50%, 75% or 90% or even higher inhibition of cellular activity. The percentage inhibition of cellular activity in the patient can be monitored to assess whether the achieved plasma drug concentration is appropriate, and the dose can be adjusted up or down to obtain the desired percentage inhibition.

A therapeutically effective amount for use in humans can also be determined from animal models, as is well known in the art. For example, dosages for humans can be formulated based on circulating concentrations that have been found to be effective in animals. As described above, the dose in humans can be adjusted by monitoring cell inhibition and going up or down.

Therapeutically effective dosages can also be determined from human data by identifying compounds known to have similar pharmacological activity. The dosage administered can be adjusted based on the relative bioavailability and potency of the administered compound as compared to known compounds.

It is within the ability of the ordinarily skilled artisan to adjust the dosage to achieve maximum efficacy in humans based on the methods described above and other methods known in the art.

In the case of topical administration, the systemic circulating concentration of the administered compound is not particularly important. In such cases, the administered compound is brought to a concentration in the local area to achieve the desired result.

Therapeutic amounts of the specific antibodies disclosed herein can also be administered as a component of an immunological composition, either as a single mixture or separately. In some embodiments, the therapeutic amount is an amount that eliminates or reduces tumor burden or prevents or reduces metastatic cell proliferation in the patient. The dosage depends on a number of parameters, including the nature of the tumor, the patient's medical history, the patient's condition, possible co-use with other oncolytic agents, and the method of administration. Methods of administration include injection (e.g., parenteral, subcutaneous, intravenous, intraperitoneal, and the like), and the antibody is provided in a pharmaceutically acceptable, non-toxic carrier, such as water, saline, ringer's solution, dextrose solution, 5% human serum albumin, fixed oil, ethyl oleate, or liposomes. Typical dosages may range from about 0.01 to about 20mg/kg, for example from about 0.1mg/kg to about 10 mg/kg. Other effective methods and dosages of administration can be determined by routine experimentation and are within the scope of the present invention.

When used in a multi-functional therapy, the therapeutically effective amount administered (as disclosed herein) may vary depending on the desired effect and the subject to be treated. For example, the subject may be injected intravenously with at least 1mg/kg (e.g., 1mg/kg to 20mg/kg, 2.5mg/kg to 10mg/kg, or 3.75mg/kg to 5mg/kg) of the antibody agent. The dose may be administered in several doses (e.g. 2, 3 or 4 divided doses per day) or in a single dose.

In the method of combined administration, the agent may be administered simultaneously with the antibody used in the present invention, or may be administered before or after the antibody used in the present invention is administered.

For other modes of administration, the dosage and interval may be adjusted individually to provide plasma levels of the administered compound that are effective for the particular clinical indication being treated. For example, in one embodiment, the compounds of the present invention may be administered in relatively high concentrations multiple times per day. Alternatively, it may be preferable to administer the compounds of the present invention at lower effective concentrations and with less frequent dosing schedules. This advantageously provides a treatment regimen that corresponds to the severity of the individual's disease.

With the teachings provided herein, an effective treatment regimen can be planned that does not cause substantial toxicity, but is entirely effective for treating the clinical symptoms exhibited by a particular patient. The regimen should be carefully selected taking into account factors such as the potency of the compound, the relative bioavailability, the body weight of the patient, the presence and severity of adverse side effects, the preferred mode of administration, and the toxicity profile of the drug selected.

The invention is further illustrated, but not limited, by the following examples which illustrate the preparation of the compounds of the invention.