Patent Application
20210269525
This disclosure relates to anti-CD3e (T-cell surface glycoprotein CD3 epsilon chain) antibodies and uses thereof.
Autoimmune processes are related to defects in immunologic tolerance, a state of immune system unresponsiveness to an antigen. Tolerance is maintained by multiple mechanisms including deletion, anergy, and active cellular regulation and strategies to induce immune tolerance are being developed for the treatment of autoimmunity.
CD3 (cluster of differentiation 3) is a T cell co-receptor that is involved in activating both the cytotoxic T cell (CD8+ naive T cells) and also T helper cells (CD4+ naive T cells). Depending on the conditions used, antibodies against CD3 can either stimulate T cells to divide or inhibit the development of effector functions such as cytotoxicity. Anti-CD3 antibody therapy has a demonstrated potential in the context of treating autoimmune diseases. However, the efficacy of anti-CD3 therapy has been limited by in vivo toxicities. A well-known anti-CD3 antibody, OKT3, is used routinely in clinical therapy of transplant rejection but is known to mediate dramatic cytokine release in vivo, leading to a “flu-like” syndrome. This effect has been identified with a humoral response against the OKT3 molecule as well as a release of pro-inflammatory cytokines such as TNF-α. These physiological toxicities restrict the dosage regimens available to patients with anti-CD3 therapy and limit the overall efficacy of anti-CD3 treatment of autoimmune disease. There is a current need for anti-CD3 therapies with less toxicity and more options for different anti-CD3 therapies.
This disclosure relates to anti-CD3e antibodies, antigen-binding fragment thereof, and the uses thereof.
In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof that binds to CD3e (T-cell surface glycoprotein CD3 epsilon chain) comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence, wherein the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following:
(1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, 3, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively;
(2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7, 8, 9, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12, respectively.
In some embodiments, the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3 respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.
In some embodiments, the antibody or antigen-binding fragment is a bispecific antibody.
In some embodiments, the antibody or antigen-binding fragment specifically binds to human CD3e.
In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFV).
In one aspect, the disclosure also provides a nucleic acid comprising a polynucleotide encoding a polypeptide comprising:
(1) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 25, 26, 27, or 36, binds to CD3e;
(2) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 21, 22, 23, 24, or 35, binds to CD3e;
(3) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 32, 33, 34, or 38, binds to CD3e;
(4) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 28, 29, 30, 31, or 37, binds to CD3e.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.
In some embodiments, the VH when paired with a VL specifically binds to human CD3e, or the VL when paired with a VH specifically binds to human CD3e.
In some embodiments, the immunoglobulin heavy chain or the fragment thereof is a humanized immunoglobulin heavy chain or a fragment thereof. In some embodiments, the immunoglobulin light chain or the fragment thereof is a humanized immunoglobulin light chain or a fragment thereof.
In some embodiments, the nucleic acid encodes a bispecific antibody. In some embodiments, the nucleic acid encodes a single-chain variable fragment (scFv).
In some embodiments, the nucleic acid is cDNA.
In another aspect, the disclosure also provides a vector comprising one or more of the nucleic acids as described herein. In one aspect, the disclosure also relates to a vector comprising two of the nucleic acids as described herein, wherein the vector encodes the VL region and the VH region that together bind to CD3e.
In one aspect, the disclosure further relates to a pair of vectors, wherein each vector comprises one of the nucleic acids as described herein, wherein together the pair of vectors encodes the VL region and the VH region that together bind to CD3e.
In one aspect, the disclosure provides a cell comprising the vector as described herein, or the pair of vectors as described herein.
In some embodiments, the cell is a CHO cell.
In some embodiments, the cell comprises one or more of the nucleic acids as described herein. In some embodiments, the cell comprises two of the nucleic acids as described herein.
In some embodiments, the two nucleic acids together encode the VL region and the VH region that together bind to CD3e.
In one aspect, the discourse provides methods of producing an antibody or an antigen-binding fragment thereof. The methods involve (a) culturing the cell as described herein under conditions sufficient for the cell to produce the antibody or the antigen-binding fragment; and (b) collecting the antibody or the antigen-binding fragment produced by the cell.
In one aspect, the disclosure also provides an antibody or antigen-binding fragment thereof that binds to CD3e comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90% identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 21, 22, 23, 24, or 35, and the selected VL sequence is SEQ ID NO: 25, 26, 27, or 36; (2) the selected VH sequence is SEQ ID NO: 28, 29, 30, 31, or 37, and the selected VL sequence is SEQ ID NO: 32, 33, 34, or 38.
In some embodiments, the VH comprises the sequence of SEQ ID NO: 21 and the VL comprises the sequence of SEQ ID NO: 25. In some embodiments, the VH comprises the sequence of SEQ ID NO: 28 and the VL comprises the sequence of SEQ ID NO: 32.
In some embodiments, the antibody or antigen-binding fragment specifically binds to human CD3e.
In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.
In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFV).
In one aspect, the disclosure also provides an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof as described herein covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.
In another aspect, the disclosure further provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described herein, and a pharmaceutically acceptable carrier. In one aspect, the disclosure also provides a pharmaceutical composition comprising the antibody drug conjugate as described herein, and a pharmaceutically acceptable carrier.
In one aspect, the disclosure also provides methods of decreasing immune response in a subject. The methods involve administering an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein, or the antibody-drug conjugate as described herein to the subject.
In some embodiments, the subject has a graft-versus-host disease. In some embodiments, the subject has type I diabetes. In some embodiments, the subject has arthritis, Crohn's disease, or ulcerative colitis.
In one aspect, the disclosure is related to methods of treating an autoimmune disease in a subject. The methods involve administering an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein, or the antibody-drug conjugate as described herein to the subject.
In some embodiments, the subject has type I diabetes, arthritis, Crohn's disease, or ulcerative colitis.
In one aspect, the disclosure relates to methods of treating a subject having cancer. The methods involve administering a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof as described herein to the subject.
In some embodiments, the antibody or antigen-binding fragment is a bispecific antibody, and the bispecific antibody also specifically binds to a tumor associated antigen. In some embodiments, the tumor associated antigen is CD19, CD20, PSA, Glypican 3, Her2, CD123, Ep-CAM, CD66e, PSMA, CD371, or VEGFR2.
In some embodiments, the cancer is breast cancer, prostate cancer, or hematologic malignancy.
In one aspect, the disclosure is also related to methods of decreasing the rate of tumor growth. The methods involve contacting a tumor cell with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein.
In some embodiments, the antibody or antigen-binding fragment is a bispecific antibody, and the bispecific antibody also specifically binds to a tumor associated antigen.
In one aspect, the disclosure is also related to methods of killing a tumor cell. The methods involve contacting a tumor cell with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof as described herein.
In some embodiments, the antibody or antigen-binding fragment is a bispecific antibody, and the bispecific antibody also specifically binds to a tumor associated antigen.
As used herein, the term “antibody” refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementary determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bi-specific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody can contain an Fc region of a human antibody. The term antibody also includes derivatives, e.g., bi-specific antibodies, single-chain antibodies, diabodies, linear antibodies, and multi-specific antibodies formed from antibody fragments.
As used herein, the term “antigen-binding fragment” refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain or a variable domain of light chain). Non-limiting examples of antibody fragments include, e.g., Fab, Fab′, F(ab′)2, and Fv fragments.
As used herein, the term “human antibody” refers to an antibody that is encoded by an endogenous nucleic acid (e.g., rearranged human immunoglobulin heavy or light chain locus) present in a human. In some embodiments, a human antibody is collected from a human or produced in a human cell culture (e.g., human hybridoma cells). In some embodiments, a human antibody is produced in a non-human cell (e.g., a mouse or hamster cell line). In some embodiments, a human antibody is produced in a bacterial or yeast cell. In some embodiments, a human antibody is produced in a transgenic non-human animal (e.g., a bovine) containing an unrearranged or rearranged human immunoglobulin locus (e.g., heavy or light chain human immunoglobulin locus).
As used herein, the term “chimeric antibody” refers to an antibody that contains a sequence present in at least two different antibodies (e.g., antibodies from two different mammalian species such as a human and a mouse antibody). A non-limiting example of a chimeric antibody is an antibody containing the variable domain sequences (e.g., all or part of a light chain and/or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody and the constant domains of a human antibody. Additional examples of chimeric antibodies are described herein and are known in the art.
As used herein, the term “humanized antibody” refers to a non-human antibody which contains minimal sequence derived from a non-human (e.g., mouse) immunoglobulin and contains sequences derived from a human immunoglobulin. In non-limiting examples, humanized antibodies are human antibodies (recipient antibody) in which hypervariable (e.g., CDR) region residues of the recipient antibody are replaced by hypervariable (e.g., CDR) region residues from a non-human antibody (e.g., a donor antibody), e.g., a mouse, rat, or rabbit antibody, having the desired specificity, affinity, and capacity. In some embodiments, the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues. In some embodiments, humanized antibodies may contain residues which are not found in the recipient antibody or in the donor antibody. These modifications can be made to further refine antibody performance. In some embodiments, the humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-human (e.g., mouse) immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin. The humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc), typically, that of a human immunoglobulin. Humanized antibodies can be produced using molecular biology methods known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.
As used herein, the term “single-chain antibody” refers to a single polypeptide that contains at least two immunoglobulin variable domains (e.g., a variable domain of a mammalian immunoglobulin heavy chain or light chain) that is capable of specifically binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.
As used herein, the term “multimeric antibody” refers to an antibody that contains four or more (e.g., six, eight, or ten) immunoglobulin variable domains. In some embodiments, the multimeric antibody is able to crosslink one target molecule (e.g., CD3e) to at least one second target molecule (e.g., Her2, Glypican 3) on the surface of a mammalian cell (e.g., a tumor cell).
As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided. Veterinary and non-veterinary applications are contemplated by the present invention. Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old). In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkey, chimpanzee, gorilla, and the like), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pig, miniature pig), equine, canine, feline, bovine, and other domestic, farm, and zoo animals.
As used herein, the term “cancer” refers to cells having the capacity for autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include cancerous growths, e.g., tumors; oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. Also included are malignancies of the various organ systems, such as respiratory, cardiovascular, renal, reproductive, hematological, neurological, hepatic, gastrointestinal, and endocrine systems; as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, and cancer of the small intestine. Cancer that is “naturally arising” includes any cancer that is not experimentally induced by implantation of cancer cells into a subject, and includes, for example, spontaneously arising cancer, cancer caused by exposure of a patient to a carcinogen(s), cancer resulting from insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancer caused by infections, e.g., viral infections. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation. The term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin. A hematopoietic neoplastic disorder can arise from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.
As used herein, when referring to an antibody, the phrases “specifically binding” and “specifically binds” mean that the antibody interacts with its target molecule (e.g., CD3e) preferably to other molecules, because the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the target molecule; in other words, the reagent is recognizing and binding to molecules that include a specific structure rather than to all molecules in general. An antibody that specifically binds to the target molecule may be referred to as a target-specific antibody. For example, an antibody that specifically binds to a CD3e molecule may be referred to as a CD3e-specific antibody or an anti-CD3e antibody.
As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to polymers of amino acids of any length of at least two amino acids.
As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid sequence” are used interchangeably herein to refer to polymers of nucleotides of any length of at least two nucleotides, and include, without limitation, DNA, RNA, DNA/RNA hybrids, and modifications thereof.
Unless otherwise defined, 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. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
The present disclosure provides examples of antibodies, antigen-binding fragment thereof, that bind to CD3e (T-cell surface glycoprotein CD3 epsilon chain; also known as CD3ε or T-Cell Surface Antigen T3/Leu-4 Epsilon Chain).
Cluster of differentiation 3 (CD3) is a multimeric protein complex, known historically as the T3 complex, and is composed of four distinct polypeptide chains; epsilon (ε), gamma (γ), delta (δ) and zeta (ζ), that assemble and function as three pairs of dimers (εγ, εδ, ζζ). The CD3 complex serves as a T cell co-receptor that associates noncovalently with the T cell receptor (TCR). The CD3 protein complex is a defining feature of the T cell lineage, therefore anti-CD3 antibodies can be used effectively as T cell markers.
Ligation of the TCR/CD3 results in activation of src and syk family PTKs associated with the intracellular CD3 domains that then activate PLC and Ras-dependent pathways. However, signaling via the TCR complex is not a linear event starting at the receptor and ending in the nucleus. Instead, there appears to be complex feedback and feedforward regulation at each step.
Because CD3 is required for T cell activation, drugs (often monoclonal antibodies) that target it are being investigated as immunosuppressant therapies (e.g., otelixizumab) for graft vs host disease, and various autoimmune diseases (e.g., arthritis, type 1 diabetes). CD3e (or CD3c) is a non-glycosylated polypeptide chain of 20 kDa.
Therapeutic anti-CD3e antibodies bind to the epsilon chain of the CD3/TCR complex that characterizes T lymphocytes. Several nonmutually exclusive mechanisms have been proposed to explain the therapeutic effect of anti-CD3e antibodies. After a short lasting capping of the CD3 complex, the CD3/T-cell receptor complex disappears from the cell surface by internalization or shedding, a process called antigenic modulation that renders T cells temporarily blind to their cognate antigens. Anti-CD3e antibody-induced signaling can also preferentially induce anergy or apoptosis in activated T cells while sparing Tregs. Heterogeneity of TCR expression by different T-cell subsets might explain the differential effect of anti-CD3e antibody on effector versus regulatory or naïve T cells.
The tolerogenic function of anti-CD3e antibodyis independent of effector functions that are linked to the Fc region of the antibody, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP), as F(ab′)2 fragments are sufficient for tolerance induction. It has been shown that T cells become rapidly activated in response to intravenous anti-CD3e antibody as measured by increased expression of CD69 and CD25 and serum concentrations of TGF-β and IFN-γ briefly after injection, even when using nonmitogenic anti-CD3e antibody. The direct effects of anti-CD3e antibody on T cells (capping, antigenic modulation, induction of apoptosis and anergy) are all short-term and are gone after clearance of the antibody from the circulation. Yet, the pharmacological effects mediated by anti-CD3E antibody therapy can be long lasting, indicating that additional and more durable mechanisms are involved in anti-CD3e antibody mediated tolerance. Evidence suggests a link between anti-CD3e antibody-induced apoptosis, phagocytosis of the resulting apoptotic bodies by macrophages and a subsequent increase of TGF-β. TGF-β plays an essential role in regulating immune responses and the production of TGF-β is crucial for the therapeutic effect of anti-CD3e antibody. TGF-β has pleiotropic effects on the adaptive immunity, including induction of adaptive FoxP3+ Tregs, inhibition of T-cell activation and proliferation and blocking dendritic cell maturation, and all these outcomes are observed after anti-CD3e antibody mediated tolerance induction. Indeed, it has been demonstrated that anti-CD3e antibody therapy increases TGF-β dependent Tregs, renders effector T cells more susceptible to TGF-β mediated regulation and confers a tolerogenic phenotype to dendritic cells.
A detailed description of CD3e, and the use of anti-CD3e antibodies to treat various diseases are described, e.g., in Smith-Garvin, et al. “T cell activation.” Annual review of immunology 27 (2009): 591-619; Kuhn, et al. “Therapeutic anti-CD3 monoclonal antibodies: from bench to bedside.” Immunotherapy 8.8 (2016): 889-906; US 20060275292; and US 20070292416; each of which is incorporated by reference in its entirety.
Furthermore, bispecific antibodies that target CD3 and an antigen on a tumor cell (e.g., CD19, Glypican 3, Her2) can redetect T cells to tumor cells. Because T cells can be redirected to tumor cells regardless of the specificity of T cell receptors, these bispecific antibodies are considered efficacious especially for less immunogenic tumors lacking enough neoantigens. Its clinical efficacy has been exemplified by blinatumomab, a bispecific T cell engager targeting CD19 and CD3, which has shown marked clinical responses against hematological malignancies.
A detailed description of bispecific antibodies target CD3 and an antigen on a tumor cell are described, e.g., in Ishiguro, et al. “An anti-glypican 3/CD3 bispecific T cell—redirecting antibody for treatment of solid tumors.” Science translational medicine 9.410 (2017); Gao, Y., et al. “Efficient inhibition of multidrug-resistant human tumors with a recombinant bispecific anti-P-glycoprotein×anti-CD3 diabody.” Leukemia 18.3 (2004): 513; Topp, et al. “Safety and activity of blinatumomab for adult patients with relapsed or refractory B-precursor acute lymphoblastic leukaemia: a multicentre, single-arm, phase 2 study.” The Lancet Oncology 16.1 (2015): 57-66; US20160355588; US20170210819 each of which is incorporated herein by reference in its entirety.
The present disclosure provides several anti-CD3e antibodies (including bispecific antibodies), antigen-binding fragments thereof, and methods of using these anti-CD3e antibodies and antigen-binding fragments to inhibit immune response, treat autoimmune diseases, and methods of using bispecific antibodies to inhibit tumor growth and to treat cancers.
The present disclosure provides anti-CD3e antibodies and antigen-binding fragments thereof. In general, antibodies (also called immunoglobulins) are made up of two classes of polypeptide chains, light chains and heavy chains. A non-limiting antibody of the present disclosure can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains. The heavy chain of the antibody can be of any isotype including IgM, IgG, IgE, IgA, or IgD or sub-isotype including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain. An antibody can comprise two identical copies of a light chain and two identical copies of a heavy chain. The heavy chains, which each contain one variable domain (or variable region, VH) and multiple constant domains (or constant regions), bind to one another via disulfide bonding within their constant domains to form the “stem” of the antibody. The light chains, which each contain one variable domain (or variable region, VL) and one constant domain (or constant region), each bind to one heavy chain via disulfide binding. The variable region of each light chain is aligned with the variable region of the heavy chain to which it is bound. The variable regions of both the light chains and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FR).
These hypervariable regions, known as the complementary determining regions (CDRs), form loops that comprise the principle antigen binding surface of the antibody. The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding region.
Methods for identifying the CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known, and a number of definitions of the CDRs are commonly used. The Kabat definition is based on sequence variability, and the Chothia definition is based on the location of the structural loop regions. These methods and definitions are described in, e.g., Martin, “Protein sequence and structure analysis of antibody variable domains,” Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan, et al. “Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains,” Molecular immunology 45.14 (2008): 3832-3839; Wu, T. T. and Kabat, E. A. (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203:121-53 (1991); Morea et al., Biophys Chem. 68(1-3):9-16 (October 1997); Morea et al., J Mol Biol. 275(2):269-94 (January 1998); Chothia et al., Nature 342(6252):877-83 (December 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007); each of which is incorporated herein by reference in its entirety. Unless specifically indicated in the present disclosure, Kabat numbering is used in the present disclosure as a default.
The CDRs are important for recognizing an epitope of an antigen. As used herein, an “epitope” is the smallest portion of a target molecule capable of being specifically bound by the antigen binding domain of an antibody. The minimal size of an epitope may be about three, four, five, six, or seven amino acids, but these amino acids need not be in a consecutive linear sequence of the antigen's primary structure, as the epitope may depend on an antigen's three-dimensional configuration based on the antigen's secondary and tertiary structure.
In some embodiments, the antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. The sequences and differences of the IgG subclasses are known in the art, and are described, e.g., in Vidarsson, et al, “IgG subclasses and allotypes: from structure to effector functions.” Frontiers in immunology 5 (2014); Irani, et al. “Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases.” Molecular immunology 67.2 (2015): 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is incorporated herein by reference in its entirety.
The antibody can also be an immunoglobulin molecule that is derived from any species (e.g., human, rodent, mouse, camelid). Antibodies disclosed herein also include, but are not limited to, polyclonal, monoclonal, monospecific, polyspecific antibodies, and chimeric antibodies that include an immunoglobulin binding domain fused to another polypeptide. The term “antigen binding domain” or “antigen binding fragment” is a portion of an antibody that retains specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specific binding to an epitope on the intact antibody's target molecule. It includes, e.g., Fab, Fab′, F(ab′)2, and variants of these fragments. Thus, in some embodiments, an antibody or an antigen binding fragment thereof can be, e.g., a scFv, a Fv, a Fd, a dAb, a bispecific antibody, a bispecific scFv, a diabody, a linear antibody, a single-chain antibody molecule, a multi-specific antibody formed from antibody fragments, and any polypeptide that includes a binding domain which is, or is homologous to, an antibody binding domain. Non-limiting examples of antigen binding domains include, e.g., the heavy chain and/or light chain CDRs of an intact antibody, the heavy and/or light chain variable regions of an intact antibody, full length heavy or light chains of an intact antibody, or an individual CDR from either the heavy chain or the light chain of an intact antibody.
In some embodiments, the antigen binding fragment can form a part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor are fusions of single-chain variable fragments (scFv) as described herein, fused to CD3-zeta transmembrane- and endodomain. In some embodiments, the chimeric antigen receptor also comprises intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor comprises multiple signaling domains, e.g., CD3z-CD28-41BB or CD3z-CD28-OX40, to increase potency. Thus, in one aspect, the disclosure further provides cells (e.g., T cells) that express the chimeric antigen receptors as described herein.
In some embodiments, the scFV comprises one heavy chain variable domain, and one light chain variable domain. In some embodiments, the scFV comprises two heavy chain variable domains, and two light chain variable domains.
The disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to CD3e. The antibodies and antigen-binding fragments described herein are capable of binding to CD3e. The disclosure provides e.g., mouse anti-CD3e antibodies 25-10A4 (“10A4”), 30-1B1 (“1B1”), the chimeric antibodies thereof, and the humanized antibodies thereof.
The CDR sequences for 10A4, and 10A4 derived antibodies (e.g., humanized antibodies) include CDRs of the heavy chain variable domain, SEQ ID NOs: 1-3, and CDRs of the light chain variable domain, SEQ ID NOs: 4-6 as defined by Kabat numbering. The CDRs can also be defined by Chothia system. Under the Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 13, 14, 3 and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 4-6.
Similarly, the CDR sequences for 1B1, and 1B1 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 7-9, and CDRs of the light chain variable domain, SEQ ID NOs: 10-12, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 15, 16, 9, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 10-12.
The amino acid sequences for heavy chain variable regions and light variable regions of the humanized antibodies are also provided. As there are different ways to humanize a mouse antibody (e.g., a sequence can be modified with different amino acid substitutions), the heavy chain and the light chain of an antibody can have more than one version of humanized sequences. The amino acid sequences for the heavy chain variable regions of humanized 10A4 antibody are set forth in SEQ ID NOs: 21-24. The amino acid sequences for the light chain variable regions of humanized 10A4 antibody are set forth in SEQ ID NOs: 25-27. Any of these heavy chain variable region sequences (SEQ ID NO:21-24) can be paired with any of these light chain variable region sequences (SEQ ID NO: 25-27).
Similarly, the amino acid sequences for the heavy chain variable region of humanized 1B1 antibody are set forth in SEQ ID NOs: 28-31. The amino acid sequences for the light chain variable region of humanized 1B1 antibody are set forth in SEQ ID NOs: 32-34. Any of these heavy chain variable region sequences (SEQ ID NO: 28-31) can be paired with any of these light chain variable region sequences (SEQ ID NO:32-34).
As used herein, the term “humanization percentage” means the percentage identity of the heavy chain or light chain variable region sequence as compared to human antibody sequences in International Immunogenetics Information System (IMGT) database. The top hit means that the heavy chain or light chain variable region sequence is closer to a particular species than to other species. For example, top hit to human means that the sequence is closer to human than to other species. Top hit to human and Macaca fascicularis means that the sequence has the same percentage identity to the human sequence and the Macaca fascicularis sequence, and these percentages identities are highest as compared to the sequences of other species. In some embodiments, humanization percentage is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%. A detailed description regarding how to determine humanization percentage and how to determine top hits is known in the art, and is described, e.g., in Jones, et al. “The INNs and outs of antibody nonproprietary names.” MAbs. Vol. 8. No. 1. Taylor & Francis, 2016, which is incorporated herein by reference in its entirety. A high humanization percentage often has various advantages, e.g., more safe and more effective in humans, more likely to be tolerated by a human subject, and/or less likely to have side effects.
Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three heavy chain variable region CDRs selected from the group of SEQ ID NOs: 1-3, SEQ ID NOs: 7-9, SEQ ID NOs: 13, 14, 3, and SEQ ID NOs: 15, 16, 9; and/or one, two, or three light chain variable region CDRs selected from the group of SEQ ID NOs: 4-6, and SEQ ID NOs 10-12.
In some embodiments, the antibodies can have a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR3 amino acid sequence, and a light chain variable region (VL) comprising CDRs 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR3 amino acid sequence. The selected VH CDRs 1, 2, 3 amino acid sequences and the selected VL CDRs, 1, 2, 3 amino acid sequences are shown in
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 1 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 3 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 7 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 9 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 13 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 14 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO:3 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO:15 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO:16 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO:9 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 4 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 5 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 6 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 10 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 12 with zero, one or two amino acid insertions, deletions, or substitutions.
The insertions, deletions, and substitutions can be within the CDR sequence, or at one or both terminal ends of the CDR sequence.
The disclosure also provides antibodies or antigen-binding fragments thereof that bind to CD3e. The antibodies or antigen-binding fragments thereof contain a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 21, 22, 23, 24, or 35, and the selected VL sequence is SEQ ID NO: 25, 26, 27, or 36. In some embodiments, the selected VH sequence is SEQ ID NO: 28, 29, 30, 31, or 37 and the selected VL sequence is SEQ ID NO: 32, 33, 34, or 38.
To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90%, 95%, or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For purposes of the present disclosure, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
The disclosure also provides nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin light chain. The immunoglobulin heavy chain or immunoglobulin light chain comprises CDRs as shown in
The anti-CD3e antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multi-specific (e.g., bi-specific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal, monoclonal, multi-specific (multimeric, e.g., bi-specific), human antibodies, chimeric antibodies (e.g., human-mouse chimera), single-chain antibodies, intracellularly-made antibodies (i.e., intrabodies), and antigen-binding fragments thereof. The antibodies or antigen-binding fragments thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.
Fragments of antibodies are suitable for use in the methods provided so long as they retain the desired affinity and specificity of the full-length antibody. Thus, a fragment of an antibody that binds to CD3e will retain an ability to bind to CD3e. An Fv fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can have the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site.
Single-chain Fv or (scFv) antibody fragments comprise the VH and VL domains (or regions) of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.
The Fab fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CH1) of the heavy chain. F(ab′)2 antibody fragments comprise a pair of Fab fragments which are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.
Diabodies are small antibody fragments with two antigen-binding sites, which fragments comprise a VH connected to a VL in the same polypeptide chain (VH and VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
Antibodies and antibody fragments of the present disclosure can be modified in the Fc region to provide desired effector functions or serum half-life.
Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.
Alternatively, antibody homodimers may be formed through chemical linkage techniques known in the art. For example, heterobifunctional crosslinking agents including, but not limited to SMCC (succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate) and SATA (N-succinimidyl S-acethylthio-acetate) can be used to form antibody multimers. An exemplary protocol for the formation of antibody homodimers is described in Ghetie et al. (Proc. Natl. Acad. Sci. U.S.A. 94: 7509-7514, 1997). Antibody homodimers can be converted to Fab′2 homodimers through digestion with pepsin. Another way to form antibody homodimers is through the use of the autophilic T15 peptide described in Zhao et al. (J. Immunol. 25:396-404, 2002).
In some embodiments, the multi-specific antibody is a bi-specific antibody. Bi-specific antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture. For example, the interface can contain at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. This method is described, e.g., in WO 96/27011, which is incorporated by reference in its entirety.
Bi-specific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Heteroconjugate antibodies can also be made using any convenient cross-linking methods. Suitable cross-linking agents and cross-linking techniques are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.
Methods for generating bi-specific antibodies from antibody fragments are also known in the art. For example, bi-specific antibodies can be prepared using chemical linkage. Brennan et al. (Science 229:81, 1985) describes a procedure where intact antibodies are proteolytically cleaved to generate F(ab′)2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab′ TNB derivatives is then reconverted to the Fab′ thiol by reduction with mercaptoethylamine, and is mixed with an equimolar amount of another Fab′ TNB derivative to form the bi-specific antibody.
Any of the antibodies or antigen-binding fragments described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or in solution). Non-limiting examples of stabilizing molecules include: a polymer (e.g., a polyethylene glycol) or a protein (e.g., serum albumin, such as human serum albumin). The conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of an antibody or an antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in a human).
In some embodiments, the antibodies or antigen-binding fragments described herein can be conjugated to a therapeutic agent. The antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can covalently or non-covalently bind to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoids such as DM-1 and DM-4, dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs).
The antibodies or antigen-binding fragments thereof described herein can bind to CD3e.
In some embodiments, by binding to CD3e, the antibody can inhibit CD3 signaling pathway. Thus, in some embodiments, the antibodies or antigen-binding fragments thereof as described herein are CD3 antagonist. In some embodiments, the antibodies or antigen-binding fragments thereof are CD3 agonist. In some embodiments, the antibodies or antigen-binding fragments thereof can downregulate immune response, promote immune tolerance, induce internalization or shedding of T cell receptor, induce anergy or apoptosis in activated T cells, increase expression of CD69 and/or CD25, increase the expression of TGF-β, induce induction of adaptive FoxP3+ Tregs, inhibit T-cell activation and proliferation, or block dendritic cell maturation. In some embodiments, the antibodies or antigen-binding fragments thereof can upregulate immune response.
In some embodiments, the antibodies or antigen-binding fragments thereof as described herein can decrease the activity or number of T cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the antibodies or antigen-binding fragments thereof as described herein can increase immune response, activity or number of T cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or 20 folds.
In some implementations, the antibody (or antigen-binding fragments thereof) specifically binds to CD3e (e.g., human CD3e, monkey CD3e, mouse CD3e, and/or chimeric CD3e) with a dissociation rate (koff) of less than 0.1 s−1, less than 0.01 s−1, less than 0.001 s−1, less than 0.0001 s−1, or less than 0.00001 s−1. In some embodiments, the dissociation rate (koff) is greater than 0.01 s−1, greater than 0.001 s−1, greater than 0.0001 s−1, greater than 0.00001 s−1, or greater than 0.000001 s−1.
In some embodiments, kinetic association rates (kon) is greater than 1×102/Ms, greater than 1×103/Ms, greater than 1×104/Ms, greater than 1×105/Ms, or greater than 1×106/Ms. In some embodiments, kinetic association rates (kon) is less than 1×105/Ms, less than 1×106/Ms, or less than 1×107/Ms.
Affinities can be deduced from the quotient of the kinetic rate constants (KD=koff/kon). In some embodiments, KD is less than 1×10−6M, less than 1×10−7M, less than 1×10−8M, less than 1×10−9M, or less than 1×10−10 M. In some embodiments, the KD is less than 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1×10−7M, greater than 1×10−8M, greater than 1×10−9M, greater than 1×10−10 M, greater than 1×10−11M, or greater than 1×10−12M.
In some embodiments, the antibody (or antigen-binding fragments thereof) binds to human CD3e with KD less than or equal to about 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, or 0.7 nM.
General techniques for measuring the affinity of an antibody for an antigen include, e.g., ELISA, RIA, and surface plasmon resonance (SPR). In some embodiments, the antibody binds to human CD3e (SEQ ID NO: 17), monkey CD3e (e.g., rhesus macaque (Macaca mulatta) CD3e (SEQ ID NO: 19), or Macaca fascicularis CD3e (SEQ ID NO: 39)), chimeric CD3e (SEQ ID NO: 20), and/or mouse CD3e (SEQ ID NO: 18). In some embodiments, the antibody does not bind to human CD3e (SEQ ID NO: 17), monkey CD3e (e.g., rhesus macaque CD3e (SEQ ID NO: 19), or Macaca fascicularis CD3e (SEQ ID NO: 39)), chimeric CD3e (SEQ ID NO: 20), and/or mouse CD3e (SEQ ID NO: 18).
In some embodiments, the antibody (or antigen-binding fragments thereof) binds to monkey CD3e with KD less than or equal to about 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM.
In some embodiments, the antibody (or antigen-binding fragments thereof) binds to human PBMC, monkey PBMC, or mouse PBMC. In some embodiments, the antibody (or antigen-binding fragments thereof) does not bind to human PBMC, monkey PBMC, or mouse PBMC.
In some embodiments, the antibodies have a relatively high expression efficiency. For example, the expression efficiency for the antibodies described herein can be at least 10%, 20%, 30%, 40%, 50%, or 100% higher than an reference antibody (e.g., a mouse antibody, a chimeric antibody, or Yervoy) under the same conditions. In some embodiments, the concentration of the antibodies from the supernatant (e.g., as measured by the methods described in examples) can be at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65 ug/ml. In some embodiments, the concentration of the antibodies from the culture medium (e.g., as measured by the methods described in examples) can be at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 ug/ml.
In some embodiments, thermal stabilities are determined. The antibodies or antigen binding fragments as described herein can have a Tm (e.g., as determined by FACS, differential scanning fluorimetry, or thermofluor assay) greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95° C.
As IgG can be described as a multi-domain protein, the melting curve sometimes shows two transitions, with a first denaturation temperature, Tm D1, and a second denaturation temperature Tm D2. The presence of these two peaks often indicate the denaturation of the Fc domains (Tm D1) and Fab domains (Tm D2), respectively. When there are two peaks, Tm usually refers to Tm D2. Thus, in some embodiments, the antibodies or antigen binding fragments as described herein has a Tm D1 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95° C. In some embodiments, the antibodies or antigen binding fragments as described herein has a Tm D2 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95° C.
In some embodiments, Tm, Tm D1, Tm D2 are less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95° C.
In some embodiments, the antibodies or antigen-binding fragments thereof as described herein are CD3 antagonist. In some embodiments, the antibodies or antigen binding fragments decrease CD3 signal transduction in T cells.
In some embodiments, the antibodies or antigen binding fragments increase CD3 signal transduction in T cells.
In some embodiments, the antibodies or antigen binding fragments have a functional Fc region. In some embodiments, effector function of a functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, effector function of a functional Fc region is phagocytosis. In some embodiments, effector function of a functional Fc region is ADCC and/or phagocytosis. In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.
In some embodiments, the antibodies or antigen binding fragments (e.g., an IgG antibody or a bispecific antibody) do not have a functional Fc region. For example, the antibodies or antigen binding fragments are Fab, Fab′, F(ab′)2, and Fv fragments. In some embodiments, the Fc region has LALA mutations (L234A and L235A mutations in EU numbering), or LALA-PG mutations (L234A, L235A, P329G muations in EU numbering).
In some embodiments, the antibodies or antigen binding fragments can stimulate T cells in a subject (e.g., increase the total number of T cells, activate T cells, and increase the expression of CD69 or CD25 in T cells). The antibodies or antigen binding fragments can be administered to a subject, and the cells from the blood or the spleen can be collected. In some embodiments, after the stimulation, more than 10%, 20%, 30%, 40%, 50%, or 60% of T cells express CD69. In some embodiments, after the stimulation, more than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% of T cells expression CD25. In some embodiments, the expression of CD69 or CD25 in a T cell can increase at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1 fold, 2 folds, 5 folds, or 10 folds.
In some embodiments, the antibodies or antigen binding fragments can activate NF-κB pathway or NFAT (Nuclear factor of activated T-cells) pathway. In some embodiments, the antibodies or antigen binding fragments can induce at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1 fold, 2 folds, 5 folds, 10 folds, 100 folds, or 1000 folds increase of the NF-κB pathway or NFAT pathway. In some embodiments, EC50 for stimulating T cells (e.g., as measured by the methods as described in the examples) is less than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, or 20 ug/ml.
In some embodiments, the antibodies or antigen binding fragments can increase serum concentrations of TGF-β and/or IFN-γ. In some embodiments, the antibodies or antigen binding fragments can induce at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1 fold, 2 folds, 5 folds, 10 folds, 100 folds, or 1000 folds increase of the serum concentrations of TGF-β and/or IFN-γ.
In some embodiments, the antibodies or antigen binding fragments does not cause dramatic cytokine release in vivo, and/or does not lead to a “flu-like” syndrome in a subject.
An isolated fragment of human CD3e can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be raised in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of an antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species to be immunized. Animals can be injected with the antigenic peptide or protein more than one time (e.g., twice, three times, or four times).
The full-length polypeptide or protein can be used or, alternatively, antigenic peptide fragments thereof can be used as immunogens. The antigenic peptide of a protein comprises at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of CD3e and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein. As described above, the full length sequence of human CD3e is known in the art (SEQ ID NO: 17).
An immunogen typically is used to prepare antibodies by immunizing a suitable subject (e.g., human or transgenic animal expressing at least one human immunoglobulin locus). An appropriate immunogenic preparation can contain, for example, a recombinantly-expressed or a chemically-synthesized polypeptide (e.g., a fragment of human CD3e). The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulatory agent.
Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a CD3e polypeptide, or an antigenic peptide thereof (e.g., part of CD3e) as an immunogen. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme-linked immunosorbent assay (ELISA) using the immobilized CD3e polypeptide or peptide. If desired, the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A of protein G chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the specific antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al. (Nature 256:495-497, 1975), the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985), or trioma techniques. The technology for producing hybridomas is well known (see, generally, Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, N.Y.). Hybridoma cells producing a monoclonal antibody are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest, e.g., using a standard ELISA assay.
Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding a human, humanized, or chimeric antibody, or antigen-binding fragment thereof described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acids sequences that make-up the antigen-binding site of the antibody or an antigen-binding domain. In a population of such variants, some antibodies or antigen-binding fragments will have increased affinity for the target protein, e.g., CD3e. Any combination of deletions, insertions, and/or combinations can be made to arrive at an antibody or antigen-binding fragment thereof that has increased binding affinity for the target. The amino acid changes introduced into the antibody or antigen-binding fragment can also alter or introduce new post-translational modifications into the antibody or antigen-binding fragment, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence such that a different sugar is attached by enzymes present in a cell), or introducing new glycosylation sites.
Antibodies disclosed herein can be derived from any species of animal, including mammals. Non-limiting examples of native antibodies include antibodies derived from humans, primates, e.g., monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chicken, goats, and rodents (e.g., rats, mice, hamsters and rabbits), including transgenic rodents genetically engineered to produce human antibodies.
Human and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequence as those derived from) human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs.
A humanized antibody, typically has a human framework (FR) grafted with non-human CDRs. Thus, a humanized antibody has one or more amino acid sequence introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed by e.g., substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. These methods are described in e.g., Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988); each of which is incorporated by reference herein in its entirety. Accordingly, “humanized” antibodies are chimeric antibodies wherein substantially less than an intact human V domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically mouse antibodies in which some CDR residues and some FR residues are substituted by residues from analogous sites in human antibodies.
The choice of human VH and VL domains to be used in making the humanized antibodies is very important for reducing immunogenicity. According to the so-called “best-fit” method, the sequence of the V domain of a mouse antibody is screened against the entire library of known human-domain sequences. The human sequence which is closest to that of the mouse is then accepted as the human FR for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)).
It is further important that antibodies be humanized with retention of high specificity and affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
Ordinarily, amino acid sequence variants of the human, humanized, or chimeric anti-CD3e antibody will contain an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% percent identity with a sequence present in the light or heavy chain of the original antibody.
Identity or homology with respect to an original sequence is usually the percentage of amino acid residues present within the candidate sequence that are identical with a sequence present within the human, humanized, or chimeric anti-CD3e antibody or fragment, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
Additional modifications to the anti-CD3e antibodies or antigen-binding fragments can be made. For example, a cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have any increased half-life in vitro and/or in vivo. Homodimeric antibodies with increased half-life in vitro and/or in vivo can also be prepared using heterobifunctional cross-linkers as described, for example, in Wolff et al. (Cancer Res. 53:2560-2565, 1993). Alternatively, an antibody can be engineered which has dual Fc regions (see, for example, Stevenson et al., Anti-CancerDrug Design 3:219-230, 1989).
In some embodiments, a covalent modification can be made to the anti-CD3e antibody or antigen-binding fragment thereof. These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatization agent that is capable of reacting with selected side chains or the N- or C-terminal residues.
In some embodiments, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering); however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody can be further engineered to replace the Asparagine at position 297 with Alanine (N297A).
In some embodiments, to facilitate production efficiency by avoiding Fab-arm exchange, the Fc region of the antibodies was further engineered to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P). A detailed description regarding S228 mutation is described, e.g., in Silva et al. “The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation.” Journal of Biological Chemistry 290.9 (2015): 5462-5469, which is incorporated by reference in its entirety.
The present disclosure also provides recombinant vectors (e.g., an expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein), host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide), and the production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.
As used herein, a “vector” is any construct capable of delivering one or more polynucleotide(s) of interest to a host cell when the vector is introduced to the host cell. An “expression vector” is capable of delivering and expressing the one or more polynucleotide(s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and/or a poly-A tail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.
A vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and/or transduction (e.g., with recombinant virus). Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant virus), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.
In some implementations, a polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus, or may use a replication defective virus. In the latter case, viral propagation generally will occur only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. N.Y. Acad Sci. 569:86-103; Flexner et al., 1990, Vaccine, 8:17-21; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner-Biotechniques, 6:616-627, 1988; Rosenfeld et al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be “naked,” as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749, and Cohen, 1993, Science, 259:1691-1692. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads that are efficiently transported into the cells.
For expression, the DNA insert comprising an antibody-encoding or polypeptide-encoding polynucleotide disclosed herein can be operatively linked to an appropriate promoter (e.g., a heterologous promoter), such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled artisan. The expression constructs can further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
As indicated, the expression vectors can include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Appropriate culture mediums and conditions for the host cells described herein are known in the art.
Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
Non-limiting bacterial promoters suitable for use include the E. coli lad and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter.
In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y, and Grant et al., Methods Enzymol., 153: 516-544 (1997).
Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.
Transcription of DNA encoding an antibody of the present disclosure by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide or they may be heterologous signals.
The polypeptide (e.g., antibody) can be expressed in a modified form, such as a fusion protein (e.g., a GST-fusion) or with a histidine-tag, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
The antibodies or antigen-binding fragments thereof of the present disclosure can be used for various therapeutic purposes.
In one aspect, the disclosure provides methods for treating, preventing, or reducing the risk of developing disorders associated with an abnormal or unwanted immune response, e.g., an autoimmune disorder, e.g., by affecting the functional properties of the circulating CD3+ T cells (e.g., reducing their proliferative capacity) or by inducing regulatory cells. These autoimmune disorders include, but are not limited to, Alopecia areata, lupus, ankylosing spondylitis, Meniere's disease, antiphospholipid syndrome, mixed connective tissue disease, autoimmune Addison's disease, multiple sclerosis, autoimmune hemolytic anemia, myasthenia gravis, autoimmune hepatitis, pemphigus vulgaris, Behcet's disease, pernicious anemia, bullous pemphigoid, polyarthritis nodosa, cardiomyopathy, polychondritis, celiac sprue-dermatitis, polyglandular syndromes, chronic fatigue syndrome (CFIDS), polymyalgia rheumatica, chronic inflammatory demyelinating, polymyositis and dermatomyositis, chronic inflammatory polyneuropathy, primary agammaglobulinemia, Churg-Strauss syndrome, primary biliary cirrhosis, cicatricial pemphigoid, psoriasis, CREST syndrome, Raynaud's phenomenon, cold agglutinin disease, Reiter's syndrome, Crohn's disease, Rheumatic fever, discoid lupus, rheumatoid arthritis, Cryoglobulinemia sarcoidosis, fibromyalgia, scleroderma, Grave's disease, Sjogren's syndrome, Guillain-Barre, stiff-man syndrome, Hashimoto's thyroiditis, Takayasu arteritis, idiopathic pulmonary fibrosis, temporal arteritis/giant cell arteritis, idiopathic thrombocytopenia purpura (ITP), ulcerative colitis, IgA nephropathy, uveitis, diabetes (e.g., Type I), vasculitis, lichen planus, and vitiligo. The anti-CD3e antibodies or antigen-binding fragments thereof can also be administered to a subject to treat, prevent, or reduce the risk of developing disorders associated with an abnormal or unwanted immune response associated with cell, tissue or organ transplantation, e.g., renal, hepatic, and cardiac transplantation, e.g., graft versus host disease (GVHD), or to prevent allograft rejection. In some embodiments, the subject has Crohn's disease, ulcerative colitis or type 1 diabetes.
In some embodiments, the treatment can reduce T cell proliferation by about at least 20%. In some embodiments, T cell proliferation is reduced by at least about 30%, about 40%, about 50%, about 60%, about 70% about 80%, or about 90% from pre-treatment levels. In addition, concentrations of IL-10 and/or TGF-β, or levels of cells secreting these cytokines, can be measured in the peripheral blood, e.g., using an enzyme-linked immunosorbent assay (ELISA) or a cell-based assay such as FACS scanning, to monitor the induction of tolerance. In some embodiments, the antibodies or antigen binding fragments can increase levels of cells secreting IL-10 and/or TGF-β as measured in the peripheral blood by about 20% or more. In some embodiments, levels of cells secreting IL-10 and/or TGF-β as measured in the peripheral blood are increased by at least about 60%, 70%, 80%, 90%, or 100%, e.g., doubled.
In some embodiments, the antibodies or antigen binding fragments can increase the total number of regulatory T cells, e.g., by about 50%, 100%, 200%, 300% or more. In some embodiments, the antibodies or antigen binding fragments can increase the activity of regulatory T cells, e.g., by about 50%, 100%, 200%, 300% or more.
Furthermore, because bispecific antibodies can bind to CD3 and a tumor associated antigen, and bridge the activation molecule CD3 to the tumor associated antigen, normal T-cell-receptor/MHC interactions are bypassed and cytotoxicity is dictated by the bispecific antibody specificity (bispecific antibody mediated T lymphocyte retargeting). Therefore, cytotoxicity occurs irrespective of T cell receptor specificity and irrespective of the degree of MHC expression/peptide presentation by the tumor cell. Thus, bispecific antibodies can also be used to treat tumors. As used herein, the term “tumor associated antigen” refers to antigens that are expressed on cancer cell surfaces. These antigens can be used to identify tumor cells. Normal cells rarely express tumor associated antigens or only expression them at a very low level. Some exemplary tumor associated antigens include, e.g., CD19, CD20, PSA, Her2, CD123, Ep-CAM, CD66e, PSMA, CD371, and VEGFR2. PSA are primarily expressed on prostate cancer cells, and Her2 are primarily expressed on breast cancer cells.
Thus, in one aspect, the disclosure provides methods for treating a cancer in a subject, methods of reducing the rate of the increase of volume of a tumor in a subject over time, methods of reducing the risk of developing a metastasis, or methods of reducing the risk of developing an additional metastasis in a subject. In some embodiments, the treatment can halt, slow, retard, or inhibit progression of a cancer. In some embodiments, the treatment can result in the reduction of in the number, severity, and/or duration of one or more symptoms of the cancer in a subject.
In one aspect, the disclosure features methods that include administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof (e.g., a bispecific antibody) disclosed herein to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, a cancer), e.g., breast cancer (e.g., triple-negative breast cancer), carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematologic malignancy (e.g., Hodgkin's lymphoma, leukemia). In some embodiments, the cancer is hematologic malignancy, breast cancer, or prostate cancer.
In some embodiments, the compositions and methods disclosed herein can be used for treatment of patients at risk for a cancer. Patients with cancer can be identified with various methods known in the art.
In some embodiments, the antibody has a tumor growth inhibition percentage (TGI %) that is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the antibody has a tumor growth inhibition percentage that is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The TGI % can be determined, e.g., at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the treatment starts, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the treatment starts. As used herein, the tumor growth inhibition percentage (TGI %) is calculated using the following formula:
TGI (%)=[1−(Ti−T0)/(Vi−V0)]×100
Ti is the average tumor volume in the treatment group on day i. T0 is the average tumor volume in the treatment group on day zero. Vi is the average tumor volume in the control group on day i. V0 is the average tumor volume in the control group on day zero.
In some embodiments, the antibody can induce immune tolerance, thus promoting tumor growth. Thus, the tumor growth in an animal model can be used as an effective indicator for immune tolerance.
As used herein, by an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., an autoimmune disease or a cancer. An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the antibody, antigen binding fragment, antibody-encoding polynucleotide, vector comprising the polynucleotide, and/or compositions thereof is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.
An effective amount can be administered in one or more administrations. By way of example, an effective amount of an antibody or an antigen binding fragment is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay progression of an autoimmune disease or a cancer in a patient or is an amount sufficient to ameliorate, stop, stabilize, reverse, slow and/or delay proliferation of a cell (e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line)) in vitro. As is understood in the art, an effective amount of an antibody or antigen binding fragment may vary, depending on, inter alia, patient history as well as other factors such as the type (and/or dosage) of antibody used.
Effective amounts and schedules for administering the antibodies, antibody-encoding polynucleotides, and/or compositions disclosed herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the antibodies, antibody-encoding polynucleotides, and/or compositions disclosed herein, the route of administration, the particular type of antibodies, antibody-encoding polynucleotides, antigen binding fragments, and/or compositions disclosed herein used and other drugs being administered to the mammal. Guidance in selecting appropriate doses for antibody or antigen binding fragment can be found in the literature on therapeutic uses of antibodies and antigen binding fragments, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., 1985, ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York, 1977, pp. 365-389.
A typical daily dosage of an effective amount of an antibody is 0.01 mg/kg to 100 mg/kg. In some embodiments, the dosage can be less than 100 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, or 0.1 mg/kg. In some embodiments, the dosage can be greater than 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, 0.1 mg/kg, 0.05 mg/kg, or 0.01 mg/kg. In some embodiments, the dosage is about 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.9 mg/kg, 0.8 mg/kg, 0.7 mg/kg, 0.6 mg/kg, 0.5 mg/kg, 0.4 mg/kg, 0.3 mg/kg, 0.2 mg/kg, or 0.1 mg/kg.
In any of the methods described herein, the at least one antibody, antigen-binding fragment thereof, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, or pharmaceutical compositions described herein) and, optionally, at least one additional therapeutic agent can be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day). In some embodiments, at least two different antibodies and/or antigen-binding fragments are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition). In some embodiments, the at least one antibody or antigen-binding fragment and the at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, the at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is administered in a sustained-release oral formulation.
In some embodiments, the one or more additional therapeutic agents can be administered to the subject prior to, or after administering the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein). In some embodiments, the one or more additional therapeutic agents and the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) are administered to the subject such that there is an overlap in the bioactive period of the one or more additional therapeutic agents and the at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) in the subject.
In some embodiments, the subject can be administered the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or following the effectiveness of treatment (e.g., the observation of at least one symptom of cancer). As described herein, a skilled medical professional can also change the identity and number (e.g., increase or decrease) of antibodies or antigen-binding antibody fragments (and/or one or more additional therapeutic agents) administered to the subject and can also adjust (e.g., increase or decrease) the dosage or frequency of administration of at least one antibody or antigen-binding antibody fragment (and/or one or more additional therapeutic agents) to the subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art).
In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of B-Raf, an EGFR inhibitor, an inhibitor of a MEK, an inhibitor of ERK, an inhibitor of K-Ras, an inhibitor of c-Met, an inhibitor of anaplastic lymphoma kinase (ALK), an inhibitor of a phosphatidylinositol 3-kinase (PI3K), an inhibitor of an Akt, an inhibitor of mTOR, a dual PI3K/mTOR inhibitor, an inhibitor of Bruton's tyrosine kinase (BTK), and an inhibitor of Isocitrate dehydrogenase 1 (IDH1) and/or Isocitrate dehydrogenase 2 (IDH2). In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2,3-dioxygenase-1) (IDO1) (e.g., epacadostat).
In some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of HER3, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2, an inhibitor of CHK1, an inhibitor of activated hedgehog signaling pathway, and an agent that selectively degrades the estrogen receptor.
In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, an Hsp90 inhibitor, Ad-GM-CSF, Temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.
In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a treatment targeting CX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, a treatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and a Selectin agonist.
In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the subject.
In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or an anti-GITR antibody.
Also provided herein are pharmaceutical compositions that contain at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described herein. Two or more (e.g., two, three, or four) of any of the antibodies or antigen-binding fragments described herein can be present in a pharmaceutical composition in any combination. The pharmaceutical compositions may be formulated in any manner known in the art.
Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., oral, intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The compositions can include a sterile diluent (e.g., sterile water or saline), a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfate, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates, or phosphates, and isotonic agents, such as sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride), or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Pat. No. 4,522,811). Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations), proper fluidity can be maintained by, for example, the use of a coating, such as lecithin, or a surfactant. Absorption of the antibody or antigen-binding fragment thereof can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).
Compositions containing one or more of any of the antibodies or antigen-binding fragments described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage).
Toxicity and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). One can, for example, determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population): the therapeutic index being the ratio of LD50:ED50. Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to minimize potential damage (i.e., reduce unwanted side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.
Data obtained from cell culture assays and animal studies can be used in formulating an appropriate dosage of any given agent for use in a subject (e.g., a human). A therapeutically effective amount of the one or more (e.g., one, two, three, or four) antibodies or antigen-binding fragments thereof (e.g., any of the antibodies or antibody fragments described herein) will be an amount that treats the disease in a subject (e.g., kills cancer cells) in a subject (e.g., a human subject identified as having cancer), or a subject identified as being at risk of developing the disease (e.g., a subject who has previously developed cancer but now has been cured), decreases the severity, frequency, and/or duration of one or more symptoms of a disease in a subject (e.g., a human). The effectiveness and dosing of any of the antibodies or antigen-binding fragments described herein can be determined by a health care professional or veterinary professional using methods known in the art, as well as by the observation of one or more symptoms of disease in a subject (e.g., a human). Certain factors may influence the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and the presence of other diseases).
Exemplary doses include milligram or microgram amounts of any of the antibodies or antigen-binding fragments described herein per kilogram of the subject's weight (e.g., about 1 μg/kg to about 500 mg/kg; about 100 μg/kg to about 500 mg/kg; about 100 μg/kg to about 50 mg/kg; about 10 μg/kg to about 5 mg/kg; about 10 μg/kg to about 0.5 mg/kg; or about 1 μg/kg to about 50 μg/kg). While these doses cover a broad range, one of ordinary skill in the art will understand that therapeutic agents, including antibodies and antigen-binding fragments thereof, vary in their potency, and effective amounts can be determined by methods known in the art. Typically, relatively low doses are administered at first, and the attending health care professional or veterinary professional (in the case of therapeutic application) or a researcher (when still working at the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the antibody or antibody fragment in vivo.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. The disclosure also provides methods of manufacturing the antibodies or antigen binding fragments thereof for various uses as described herein.
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
To generate mouse antibodies against human CD3e (hCD3e; SEQ ID NO: 17), 6-8 weeks old female BALB/c mice were immunized with human CD3e. Anti-hCD3e antibodies were collected by the methods as described below and shown in
6-8 weeks old female BALB/c mice were immunized with His-tagged human CD3e proteins at 20 μg/mouse at a concentration of 100 μg/ml. The His-tagged human CD3e proteins were emulsified with adjuvant and injected at four positions on the back of the mice. For the first subcutaneous (s.c.) injection, the diluted antigen was emulsified with Complete Freund's Adjuvant (CFA) in equal volume. In the following subcutaneous injections, the protein was emulsified with Incomplete Freund's Adjuvant (IFA) in equal volume. Three days after the third injection or the booster immunization, blood (serum) was collected and analyzed for antibody titer using ELISA.
In another experiment, 6-8 weeks old female BALB/c mice were immunized by injecting the expression plasmid encoding human CD3e into the mice. The plasmids encoding the antigen were injected into the tibialis anterior muscle (intramuscular injection; i.m. injection) of the mice by using gene guns at the concentration of 1000 μg/ulat 60 μg per mouse. At least four injections were performed with at least 14 days between two injections. Blood (serum) was collected seven days after the last immunization and the serum was tested for antibody titer by ELISA.
Procedures to enhance immunization were also performed at least fourteen days after the previous immunization (either by injecting the plasmid or by injecting the proteins). Jurkat cells were intravenously injected into the mice through tail veins. Spleen was then collected four days after the injection.
Spleen tissues were grinded. Spleen cells were first selected by CD3e Microbeads and Anti-Mouse IgM Microbeads, and then fused with SP2/0 cells. The cells were then plated in 96-well plates with hypoxanthine-aminopterin-thymidine (HAT) medium.
Primary screening of the hybridoma supernatant in the 96-well plates was performed using Fluorescence-Activated Cell Sorting (FACS) pursuant to standard procedures. Chinese hamster ovary (CHO) cells were added to 96-well plates (2×104 cells per well) before the screening. 50 μl of supernatant was used. The antibodies that were used in experiments were
(1) Fluorescein (FITC)-conjugated AffiniPure F(ab)2 Fragment Goat Anti-Mouse IgG, Fcγ Fragment Specific, and
(2) Alexa Fluor® 647-conjugated AffiniPure F(ab)2 Fragment Goat Anti-Human IgG, Fcγ Fragment Specific.
Sub-cloning was performed using ClonePix2. In short, the positive wells identified during the primary screening were transferred to semisolid medium, and IgG positive clones were identified and tested. FITC anti-mouse IgG Fc antibody was used.
1×106 positive hybridoma cells were injected intraperitoneally to B-NDG® mice (Beijing Biocytogen, Beijing, China; Catalog number: B-CM-002). Monoclonal antibodies were produced by growing hybridoma cells within the peritoneal cavity of the mouse. The hybridoma cells multiplied and produced ascites fluid in the abdomens of the mice. The fluid contained a high concentration of antibody which can be harvested for later use.
Antibodies in ascites fluid were purified using GE AKTA protein chromatography (GE Healthcare, Chicago, Ill., United States). 25-10A4 (“10A4”), and 30-1B1 (“1B1”), 16-3G3, 18-1H11, 18-2F3, 16-2D4, 18-2F4, 30-1F11, 26-2E3, 30-2E7, 30-3D7, 30-3H6, 34-5B8, 34-6A9, 34-6D8, 26-6E5, 30-10F1, and several other antibodies were among the mouse antibodies produced by the methods described above.
The VH, VL and CDR regions for some of the antibodies were determined. The heavy chain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3 amino acid sequences of 10A4 are shown in SEQ ID NOs: 1-6 (Kabat numbering) or SEQ ID NOs: 13, 14, 3, 4, 5, 6 (Chothia numbering).
The heavy chain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3 amino acid sequences of 1B1 are shown in SEQ ID NOs: 7-12 (Kabat numbering) or SEQ ID NOs: 15, 16, 9, 10, 11, 12 (Chothia numbering).
The starting point for humanization was the mouse antibodies (e.g., 10A4 and 1B1). The amino acid sequences for the heavy chain variable region and the light chain variable region of these mouse antibodies were determined.
Four humanized heavy chain variable region variants (SEQ ID NOs: 21-24) and three humanized light chain variable region variants (SEQ ID NOs: 25-27) for 10A4 were constructed, containing different modifications or substitutions.
Four humanized heavy chain variable region variants (SEQ ID NOs: 28-31) and three humanized light chain variable region variants (SEQ ID NOs: 32-34) for 1B1 were constructed, containing different modifications or substitutions.
These humanized heavy chain variable region variants can be combined with any of the light chain variable region variants based on the same mouse antibody. For example, 10A4-H1 (SEQ ID NO:21) can be combined with any humanized light chain variable region variant based on the same mouse antibody 10A4 (e.g., 10A4-K3 (SEQ ID NO:27)), and the antibody is labeled accordingly (e.g., 10A4-H1K3).
Chimeric antibodies based on 10A4 and 1B1 were also made. Among them, C3E-25-10A4-mHvKv-IgG1-LALA (“25-10A4-mHvKv-IgG1-LALA”) have heavy chain variable region from 10A4 (SEQ ID NO: 35), light chain variable region from 10A4 (SEQ ID NO: 36), and human IgG1 constant regions with LALA (L234A/L235A) mutations. Similarly, C3E-30-1B1-mHvKv-IgG1-LALA (“30-1B1-mHvKv-IgG1-LALA”) have heavy chain variable region from 1B1 (SEQ ID NO: 37), light chain variable region from 1B1 (SEQ ID NO: 38), and human IgG1 constant regions with LALA (L234A/L235A) mutations.
Experiments were performed to test the efficacy of anti-hCD3e antibodies in vivo. A humanized CD3e mouse model was generated. The humanized CD3e mouse model was engineered to express a chimeric CD3e protein (SEQ ID NO: 20) wherein a part of the extracellular region of the mouse CD3e protein was replaced with the corresponding human CD3e extracellular region. The amino acid residues 1-126 of this chimeric CD3e (SEQ ID NO: 20) were from human CD3e (SEQ ID NO: 17). The humanized mouse model (B-hCD3e) provides a new tool for testing new therapeutic treatments in a clinical setting by significantly decreasing the difference between clinical outcome in human and in ordinary mice expressing mouse CD3e.
1 μg or 5 μg of anti-mCD3 antibody was injected intraperitoneally to the mice. Saline water with equal volumes were injected into the mice as negative controls, and Teplizumab was injected into the mice for comparison purpose. The spleens were collected 24 hours after the injection, and the spleen samples were grinded. The samples were then passed through 70 μm cell mesh. The filtered cell suspensions were centrifuged and the supernatants were discarded. Erythrocyte lysis solution was added to the sample, which was lysed for 5 min and neutralized with PBS solution. The solution was centrifuged again and the supernatants were discarded. The cells were washed with PBS and stained with PE labeled anti-mCD25 antibody (mCD25 PE), APC labeled anti-mCD69 antibody (mCD69 APC), and PerCP/Cy5.5 anti-mouse TCR β chain (mTcR β PerCP).
Flow cytometry was performed. The expression of CD25 and CD69 indicated the activation of T cells. The results are shown in
The results indicated that 10A4 have better effects in terms of stimulating T cells than 16-3G3, 18-1H11, 18-2F3, and 16-2D4. For CD69 expression in T cells, the effects of 10A4 were comparable to Teplizumab. With respect to CD25 expression in T cells, 10A4 was more effective than Teplizumab.
Experiments were performed to determine whether anti-hCD3e antibodies can bind to human PBMC.
PBMC cells from two different human subjects were collected and labeled as PBMC-1 and PBMC-2. After being thawed, the cells were cultured for 3-4 hours. The medium was then discarded, and the cells were resuspended in phosphate-buffered saline (PBS). The cell concentration was then adjusted to 2×106/25 μL PBS.
A 96-well plate was prepared. 25 ul of cell suspension was added to each well. Anti-hCD3 antibodies 16-2D4, 16-3G3, 18-1H11, 18-2F3, 25-10A4, 18-2F4 were diluted to 20 ug/ml (2×) in PBS, and were added to the 96-well plate. For controls, 25 ul PBS was added. The cells and the antibodies were incubated for 15 minutes at 4° C. 185 ul PBS was then added to each well. After centrifuge, the residues were discarded. The samples were washed again. 25 ul of PBS was then added to each well to resuspend cells.
AF647-anti-mIgG was then added to each well. The cells and the antibodies were incubated for 15 minutes at 4° C. 185 ul PBS was then added to each well. After centrifuge, the residues were discarded. The samples were washed again. 25 ul of PBS was then added to each well to resuspend the cells.
14 ul of PerCP anit-hTcRβ (Biolegend, Cat #306724) and 14 ul of FITC anti-hCD19 (Biolegend, Cat #302206) were added to 350 ul PBS, and mixed. 25 ul of the mixture was then added to each well. The cells and the antibodies were incubated for 15 minutes at 4° C. 185 ul PBS was then added to each well. After centrifuge, the residues were discarded. The samples were washed again by PBS. 300 ul of PBS was then added to each well to resuspend the cells.
FACS was performed. The samples were processed according to the table below.
The results are shown in
Experiments were performed to determine whether anti-hCD3e antibodies can bind to monkey PBMC.
PBMC cells from Macaca fascicularis were collected. After being thawed, the cells were cultured for a few hours and suspended in a culture medium. The medium was then discarded, and the cells were resuspended in phosphate-buffered saline (PBS).
A 96-well plate was prepared. 25 ul of cell suspension was added to each well. Anti-hCD3 antibodies 25-10A4 and Teplizumab were diluted to 20 ug/ml (2×) in PBS. 25 ul of 25-10A4 and 25 ul of Teplizumab were added to the 96-well plate. The cells and the antibodies were incubated for 15 minutes at 4° C. 185 ul PBS was then added to each well. After centrifuge at 2000 rpm for 5 minutes, the medium was discarded. The samples were washed again. 25 ul of PBS was then added to resuspend cells.
AF647-anti-mIgG or AF647-anti-hIgG was added to appropriate wells. The cells and the antibodies were incubated for 15 minutes at 4° C. 185 ul PBS was then added to each well. After centrifuge, the residues were discarded. The samples were washed again. 25 ul of PBS was then added to resuspend cells.
200 ul of PBS was then added to each well. FACS was performed. All samples were processed according to the table below.
The results are shown in
Binding assays were performed to determine whether the anti-hCD3e antibodies can bind to Jurkat cells (immortalized cell line of human T cells).
The anti-hCD3e antibodies were collected from mouse ascites fluid and purified by chromatography. 25 μl of Jurkat and Raji cells (1:1) were added to wells in a 96-well plate. The purified antibodies were titrated to final concentrations of 10, 1, 0.1, 0.01, and 0.001 μg/ml. The titrated antibodies were added to each well at 25 μl per well at 4° C. and incubated for 30 minutes.
After being washed with phosphate-buffered saline (PBS) twice, 50 μl of anti-mouse IgG Fc-FITC (diluted at 1:100) was added to each well. The cells with the antibodies were incubated at 4° C. for 30 minutes, followed by PBS wash. The signals for FITC were determined by flow cytometry.
The results showed that 16-3G3, 18-1H11, 18-2F3, and 16-2D4 can effectively bind to Jurkat cells at 10 μg/ml. But there were no clear binding signals for these antibodies at 1, 0.1, 0.01, and 0.001 μg/ml. In addition, 18-2F4 cannot bind to Jurkat cells at any of the tested concentrations. The percentages of cells that were positive for anti-mouse IgG Fc-FITC binding are shown below, with a higher percentage indicates more effective binding of the anti-hCD3e antibodies to the Jurkat cells.
The results indicate that 25-10A4 have stronger binding affinities with Jurkat cells as compared to 16-3G3, 18-1H11, 18-2F3, 16-2D4, and 18-2F4.
Experiments were performed to determine whether the anti-hCD3e antibodies can activate Jurkat-GFP cells (SBI-System Biosciences, Cat #TR850A-1) in vitro. Expression of GFP (up to 30-fold over background) in these cells only occurs in the presence of active NF-κB signaling.
The anti-hCD3e antibodies were collected from mouse ascites fluid and purified by chromatography. The purified antibodies were titrated to final concentrations of 100, 10, 1, 0.1, and 0.01 μg/ml. 200 μl of anti-hCD3e antibodies with different concentrations were added to wells in a 96-well plate.
3×104 Jurkat-GFP cells were added to each well. After being incubated at 37° C. for 24 hours, the cells were transferred a new 96-well plate. After being washed by PBS, the GFP signals were analyzed by flow cytometry.
The results show that 25-10A4 and Teplizumab have stronger effects than 16-2D4, 16-3G3, 18-2F3, and 18-1H11 in terms of activating Jurkat cells. The tested antibodies are listed in the table below.
Experiments were performed to determine whether the anti-hCD3e antibodies can activate Jurkat-Luc-NFAT cells (Promega, Cat #J1601) in vitro.
The anti-hCD3e antibodies were collected from mouse ascites fluid and purified by chromatography. The purified antibodies were titrated to appropriate concentrations.
4×104 CHO-FcγRIIB cells were added to the wells and were incubated at 37° C. overnight. The 96-well plate was centrifuged and the supernatant was discarded.
50 μl Jurkat-Luc-NFAT cells were then added the 96-well plate (5×104 cells/well). 25 μl of anti-hCD3e antibodies with different concentrations were also added to wells in the 96-well plate. After being incubated at 37° C. for 6 hours, 75 μl of luciferase assay reagents were added and incubated for 5-10 minutes. The fluorescence signals were measured.
The results are shown in
Experiments were performed to determine whether chimeric anti-hCD3e antibodies can bind to human PBMC.
PBMC cells from human subjects were collected. After being thawed, the cells were suspended in PBS. 50 ul of cell suspension was added to each well in a 96-well plate.
The anti-hCD3 antibodies were collected from CHO-S cells. The tested antibodies are listed in the table below.
The antibodies were then added to the 96-well plate and were incubated at 4° C. for 30 minutes. 100 ul PBS was added, and the plate was centrifuged at 2000 rpm for 7 minutes.
50 μL of anti-hTcRβ-PerCP antibodies (1:70) and anti-human IgG Fc-Alexa Fluor 647 (1:500) were added, and incubate for 30 min at 4° C.
150 μL of PBS was added to each well, and then the plate was centrifuged at 2000 rpm for 7 min. The supernatant was then removed. The cells were then resuspended in 200 μL PBS, and were analyzed by flow cytometry.
The results show that not all chimeric anti-hCD3e antibodies can effectively bind to human PMBC (
Experiments were performed to determine whether chimeric anti-hCD3e antibodies can bind to monkey PBMC.
PBMC cells from Macaca fascicularis were thawed, the cells were suspended in 1 ml PBS. 50 ul of cell suspension was added to each well in a 96-well plate.
Anti-hCD3 antibodies were added to the 96-well plate and were incubated at 4° C. for 30 minutes. 100 ul PBS was added, and the plate was centrifuged at 2000 rpm for 7 minutes.
50 μL of anti-hTcRβ-PerCP antibodies (1:70) and anti-human IgG Fc-Alexa Fluor 647 (1:500) were added, and incubate for 30 min at 4° C.
150 μL of PBS was added to each well, and then the plate was centrifuged at 2000 rpm for 7 min. The supernatant was then removed. The cells were then resuspended in 200 μL PBS, and were analyzed by flow cytometry.
The results show that not all chimeric anti-hCD3e antibodies can effectively bind to monkey PMBC (
The results are summarized below.
The binding affinity of the anti-hCD3e antibodies were measured using surface plasmon resonance (SPR) using Biacore (Biacore, INC, Piscataway N.J.) T200 biosensor equipped with pre-immobilized Protein A sensor chips (Protein A (Lot:10260138)-5).
Anti-hCD3e antibodies were collected from CHO-S cells. The antibodies (1:20) were injected into Biacore T200 biosensor at 10 μL/min for 35 seconds to achieve to a desired protein density. Histidine-tagged human CD3e proteins (hCD3e-His) with different concentrations (100 nM 0.390625 nM) were then injected to the system at 30 μL/min for 150 seconds. Dissociation was monitored for 600 seconds. The chip was regenerated after the last injection of each titration with Glycine (pH 2.0, 30 μL/min for 5 seconds).
Kinetic association rates (kon) and dissociation rates (koff) were obtained simultaneously by fitting the data globally to a 1:1 Langmuir binding model. Affinities were deduced from the quotient of the kinetic rate constants (KD=koff/kon).
Experiments were also performed to test the binding affinities with rCD3e (rhesus macaque, or Macaca mulatta). Anti-hCD3e antibodies (1:20) were injected into Biacore T200 biosensor at 10 μL/min for 30 seconds to achieve to a desired protein density. Histidine-tagged rCD3e proteins (rCD3e-His) with different concentrations (100 nM 0.390625 nM) were then injected to the system at 30 μL/min for 150 seconds. Dissociation was monitored for 450 seconds. The chip was regenerated after the last injection of each titration with Glycine (pH 2.0, 30 μL/min for 12 seconds).
Kinetic association rates (kon) and dissociation rates (koff) were obtained simultaneously by fitting the data globally to a 1:1 Langmuir binding model. Affinities were deduced from the quotient of the kinetic rate constants (KD=koff/kon).
In order to test the anti-CD3e antibodies in vivo and to predict the effects of these antibodies, the anti-CD3e antibodies were tested for their effect on tumor growth in vivo in a model of colon carcinoma. MC-38 cancer tumor cells (colon adenocarcinoma cell) were injected subcutaneously in C57BL/6 mice. When the tumors in the mice reached a volume of 100˜150 mm3, the mice were randomly placed into different groups based on the volume of the tumor (five mice in each group).
The mice were then injected with physiological saline(PS) (G1), anti-mPD-1 antibody (RMP1-14) (10 mg/kg) (G2), 25-10A4 (2 mg/kg) (G3), Teplizumab (2 mg/kg) (G4), and anti-mCD3e antibody (BioXcell, Cat #BE0001-1) (2 mg/kg) (G5) by intraperitoneal administration. The antibody was given on the first day and the fourth day of each week for 3 weeks (6 injections in total).
The length of the long axis and the short axis of the tumor were measured and the volume of the tumor was calculated as 0.5×(long axis)×(short axis). The weight of the mice was also measured before the injection, when the mice were placed into different groups (before the first antibody injection), twice a week during the antibody injection period, and before euthanization.
The tumor growth inhibition percentage (TGI %) was calculated using the following formula: TGI (%)=[1−(Ti−T0)/(Vi−V0)]×100. Ti is the average tumor volume in the treatment group on day i. T0 is the average tumor volume in the treatment group on day zero. Vi is the average tumor volume in the control group on day i. V0 is the average tumor volume in the control group on day zero.
The weight of mice in different groups were similar at the end of the experiment (
The tumor size in G2 group (anti-mPD-1 antibody) did not increase. The tumor size in G1 group, G3 group (25-10A4), G4 group (Teplizumab) continued to increase. The results suggest that anti-hCD3e antibody had no obvious impact on the tumor size as compared to the control group. However, In G5 (anti-mCD3e) group, the tumor sizes were actually greater than the tumor size in control group. The results indicated that 25-10A4 and Teplizumab had no clear effect in wildtype mice, and anti-mCD3e antibody can induce immune tolerance.
In order to test the anti-hCD3e antibodies in vivo and to predict the effects of these antibodies in human, a humanized CD3e mouse model was generated. The humanized CD3e mouse model was engineered to express a chimeric CD3e protein (SEQ ID NO: 20) wherein a part of the extracellular region of the mouse CD3e protein was replaced with the corresponding human CD3e extracellular region. The amino acid residues 1-126 of this chimeric CD3e (SEQ ID NO: 20) were from human CD3e (SEQ ID NO: 17).
The anti-hCD3e antibodies were tested for their effect on tumor growth in vivo in a model of colon carcinoma. MC-38 cancer tumor cells (colon adenocarcinoma cell) were injected subcutaneously in B-hCD3e mice. When the tumors in the mice reached a volume of 100˜150 mm3, the mice were randomly placed into different groups based on the volume of the tumor (five mice in each group).
The mice were then injected with physiological saline (PS) (G1), anti-mPD-1 antibody (RMP1-14) (10 mg/kg) (G2), 25-10A4 (2 mg/kg) (G3), and Teplizumab (2 mg/kg) (G4) by intraperitoneal administration. The antibody was given on the first day and the fourth day of each week for 3 weeks (6 injections in total).
The length of the long axis and the short axis of the tumor were measured and the volume of the tumor was calculated as 0.5×(long axis)×(short axis). The weight of the mice was also measured before the injection, when the mice were placed into different groups (before the first antibody injection), twice a week during the antibody injection period, and before euthanization.
The weight of the mice was monitored during the entire treatment period. The weight of mice in different groups were similar (
The tumor size in G2 group (anti-mPD-1 antibody) did not increase. The tumor size in G1 group continued to increase. However, In G3 (25-10A4) and G4 (Teplizumab) groups, the tumor sizes were actually greater than the tumor size in control group. The results indicated that 25-10A4 and Teplizumab effectively inhibited the immune response in humanized CD3e mice. The tumor growth can indicate the degree of immune tolerance.
Experiments were performed to characterize humanized 10A4 antibodies with different combinations of humanized heavy chain variable region variants (SEQ ID NOs: 21-24) and humanized light chain variable region variants (SEQ ID NOs: 25-27).
The humanized anti-hCD3 antibodies were collected from 24-well plates after culturing CHO-S cells with vectors encoding the heavy chain and light chain of humanized antibodies for 3 days. The total amount of the antibodies in the culture plates was measured. The table below shows the total amount of antibodies (μg/ml) in the supernatant from the cultured cells. The percentages under the humanized heavy chain variants and the humanized light chain variants indicate humanization percentages. The result suggests that H3K1, H3K2, H3K3, H4K2 and H4K3 had relatively higher expression efficiency. Among them, H3K2 had the highest expression efficiency.
The anti-hCD3 antibodies were also collected from 250 ml culture medium in flasks. The total amount of the antibodies was measured after culturing the transfected CHO-S cells for 3 days. The table below shows the total amount of antibodies (μg/ml) that were collected. The result indicates that H1K1, H1K2, H1K3, H3K1, H3K2, H3K3, H4K1, H4K2 and H4K3 had relatively higher expression efficiency. Among them, H3K3 had the highest expression efficiency. Anti-CTLA4 antibody Yervoy was also tested for comparison purpose. The amount of Yervoy was 20˜50 μg/ml under the same conditions.
Binding Activities with Human CD3e
Experiments were performed to determine whether humanized anti-hCD3e antibodies can bind to human PBMC.
PBMC cells were collected from a human subject. After being thawed, the cells were cultured for 3-4 hours. The medium was then discarded, and the cells were resuspended in phosphate-buffered saline (PBS). The cell concentration was then adjusted to 2×106/25 μl PBS.
A 96-well plate was prepared. 25 ul of cell suspension was added to each well. Anti-hCD3e antibodies with different combinations of humanized 10A4 heavy chain variants and humanized 10A4 light chain variants were diluted to 20 ug/ml (2×) in PBS, and were added to the 96-well plate. The cells and the antibodies were incubated. After centrifuge, the residues were discarded. The samples were washed again. 25 ul of PBS was then added to each well to resuspend cells.
Anti-human IgG Fc-Alexa Fluror647 (1:500) and anit-hTcRβ PerCP (1:30) were then added to each well. The cells and the antibodies were incubated and then the cells were washed again by PBS. 300 ul of PBS was then added to each well to resuspend the cells. FACS was performed. The results are shown in
The binding affinities of the anti-hCD3e antibodies to human CD3e were further measured using surface plasmon resonance (SPR) using Biacore (Biacore, INC, Piscataway N.J.) by the methods as described in the examples. The results are summarized below:
The results show that these humanized 10A4 antibodies with different combinations of humanized 10A4 heavy chain variants and humanized 10A4 light chain variants have high binding affinities with human CD3e.
Binding Activities with Monkey CD3e
Experiments were performed to determine whether humanized anti-hCD3e antibodies can bind to monkey PBMC.
PBMC cells from Macaca fascicularis were collected. After being thawed, the cells were cultured for a few hours and suspended in a culture medium. The medium was then discarded, and the cells were resuspended in phosphate-buffered saline (PBS).
A 96-well plate was prepared. 25 ul of cell suspension was added to each well. Anti-hCD3 antibodies were added to the 96-well plate. The cells and the antibodies were incubated for 15 minutes at 4° C. 185 ul PBS was then added to each well. After centrifuge at 2000 rpm for 5 minutes, the medium was discarded. The samples were washed again. 25 ul of PBS was then added to resuspend cells.
Anti-human IgG Fc-Alexa Fluror647 (1:500) and anti-hTcRβ PerCP (1:30) were then added to each well. The cells and the antibodies were incubated for 15 minutes at 4° C. 185 ul PBS was then added to each well. After centrifuge, the residues were discarded. The samples were washed again. 25 ul of PBS was then added to resuspend cells. The results show that that humanized 10A4 antibodies with different combinations of humanized 10A4 heavy chain variants and humanized 10A4 light chain variants can all effectively bind to monkey PBMC (
The binding affinity of the anti-hCD3e antibodies with monkey CD3e (Cynomolgus CD3 epsilon Protein (SEQ ID NO: 39 AA Gln 22-Asp 117), His Tag, Acrobiosystems, Cat #CDE-05226) were also measured using surface plasmon resonance (SPR) using Biacore (Biacore, INC, Piscataway N.J.) by the methods as described in the examples. The results are summarized below:
The results show that these humanized 10A4 antibodies with different combinations of humanized 10A4 heavy chain variants and humanized 10A4 light chain variants have high binding affinities with monkey CD3e. The binding affinities are similar to the chimeric antibodies.
The antibodies were incubated at 60° C. and then were cooled to the room temperature. FACS was performed with cells expressing human CD3e. The results show that these tested antibodies could still bind to these cells as analyzed by FACS. Thus, the results suggest that Tm for these antibodies were greater than 60° C.
Thermofluor assay was also performed using the Protein Thermal Shift™ Dye Kit (Thermo Fisher Scientific) and QuantStudio™ 5 Real Time PCR Systems (Thermo Fisher Scientific). This assay measured thermal stability using a fluorescent dye that binds to hydrophobic patches exposed as the protein unfolds. A temperature ramp of about 1.6° C./s was applied until the temperature reached 25° C., and the temperature ramp of about 0.05° C./s was then applied until the temperature reached 99° C. The Tm was recorded. Some testing antibodies had two Tms. This is because that IgG has a multi-domain structure. In the case that there were two Tms, the second Tm (Tm for Fab) was recorded. The experiments were performed according to the manufacturer's protocol.
The table below summarizes the Tm obtained from FACS and Tm obtained from Protein Thermal Shift™ Dye Kit for several humanized anti-CD3e antibodies. The results show that humanized 10A4 antibodies with different combinations of humanized 10A4 heavy chain variants and humanized 10A4 light chain variants have similar Tm as measured by FACS or Protein Thermal Shift™ Dye Kit.
Experiments were performed to determine whether the anti-hCD3e antibodies can activate Jurkat-Luc-NFAT cells (Promega, Cat #J1601) in vitro.
The anti-hCD3e antibodies were collected from either (1) supernatant of cultured CHO cells on culture plates or (2) CHO cells within a culture medium. The anti-hCD3e antibodies collected from the culture medium were further purified. The purified antibodies were then titrated to appropriate concentrations.
4×104 CHO-FcγRIIB cells were added to the wells and were incubated at 37° C. overnight. The 96-well plate was centrifuged and the supernatant was discarded.
50 μl Jurkat-Luc-NFAT cells were then added the 96-well plate (5×104 cells/well). 25 μl of anti-hCD3e antibodies with different concentrations were also added to wells in the 96-well plate. After being incubated at 37° C. for 6 hours, 75 μl of luciferase assay reagents were added and incubated for 5-10 minutes. The fluorescence signals were measured. The results are shown in the table below.
The results show that these humanized 10A4 with different combinations of heavy chain variants and light chain variants all have in vitro biological activities and can activate Jurkat cells.
In summary, the results in this example show that these humanized 10A4 antibodies with different combinations of heavy chain variants and light chain variants have relatively high binding affinities and thermal stabilities. The antibodies with H2 heavy chains have relatively lower expression efficiency. Because H1, H2, K1, K2, and K3 have relatively high humanization percentages, H1K1, H1K2, H1K3, H2K1, H2K2, and H2K3 are good candidates for further testing in clinical trials.
Experiments were performed to characterize various humanized 1B1 antibodies with different combinations of humanized heavy chain variable region variants (SEQ ID NOs: 28-31) and humanized light chain variable region variants (SEQ ID NOs: 32-34).
The humanized anti-hCD3 antibodies were collected from 24-well plate after culturing CHO-S cells with vectors encoding the heavy chain and light chain of humanized antibodies for 3 days. The total amount of the antibodies in the culture plate was measured. The table below shows the total amount of antibodies (μg/ml) in the supernatant from the cultured cells. The percentages under the humanized heavy chain variants and the humanized light chain variants indicate humanization percentages. The result suggests that H1K2, H2K2, H3K2, H4K2, H1K3, H2K3, H3K3, and H4K3 had relatively higher expression efficiency. Among them, H1K2 had the highest expression efficiency. Anti-CTLA4 antibody Yervoy was also tested for comparison purpose. The amount of Yervoy that was expressed was 28.86 μg/ml under the same conditions.
Binding Activities with Human CD3e
Experiments were performed to determine whether humanized anti-hCD3e antibodies can bind to human PBMC.
PBMC cells were collected from a human subject. After being thawed, the cells were cultured for 3-4 hours. The medium was then discarded, and the cells were resuspended in phosphate-buffered saline (PBS). The cell concentration was then adjusted to 2×106/25 μl PBS.
A 96-well plate was prepared. 25 ul of cell suspension was added to each well. Anti-hCD3e antibodies with different combinations of humanized 1B1 heavy chain variants and humanized 1B1 light chain variants were diluted to 20 ug/ml (2×) in PBS, and were added to the 96-well plate. The cells and the antibodies were incubated. After centrifuge, the residues were discarded. The samples were washed again. 25 ul of PBS was then added to each well to resuspend cells.
Anti-human IgG Fc-Alexa Fluror647 (1:500) and anit-hTcRβ PerCP (1:30) were then added to each well. The cells and the antibodies were incubated and then the cells were washed again by PBS. 300 ul of PBS was then added to each well to resuspend the cells. FACS was performed. The results are shown in
The binding affinity of the anti-hCD3e antibodies with human CD3e were further measured using surface plasmon resonance (SPR) using Biacore (Biacore, INC, Piscataway N.J.) by the methods as described in the examples. The results are summarized below:
The results show that these humanized 1B1 antibodies with different combinations of humanized 1B1 heavy chain variants and humanized 1B1 light chain variants have high binding affinities with human CD3e.
Binding activities with monkey CD3e
Experiments were performed to determine whether humanized anti-hCD3e antibodies can bind to monkey PBMC.
PBMC cells from Macaca fascicularis were collected. After being thawed, the cells were cultured for a few hours and suspended in a culture medium. The medium was then discarded, and the cells were resuspended in phosphate-buffered saline (PBS).
A 96-well plate was prepared. 25 ul of cell suspension was added to each well. Anti-hCD3 antibodies were added to the 96-well plate. The cells and the antibodies were incubated for 15 minutes at 4° C. 185 ul PBS was then added to each well. After centrifuge at 2000 rpm for 5 minutes, the medium was discarded. The samples were washed again. 25 ul of PBS was then added to resuspend cells.
Anti-human IgG Fc-Alexa Fluror647 (1:500) and anti-hTcRβ PerCP (1:30) were then added to each well. The cells and the antibodies were incubated for 15 minutes at 4° C. 185 ul PBS was then added to each well. After centrifuge, the residues were discarded. The samples were washed again. 25 ul of PBS was then added to resuspend cells. The results show that that humanized 1B1 antibodies with different combinations of humanized 1B1 heavy chain variants and humanized 1B1 light chain variants can all effectively bind to monkey PBMC (
The binding affinity of the anti-hCD3e antibodies with monkey CD3e (Cynomolgus CD3 epsilon Protein (SEQ ID NO: 39 AA Gln 22-Asp 117), His Tag, Acrobiosystems, Cat #CDE-05226) were also measured using surface plasmon resonance (SPR) using Biacore (Biacore, INC, Piscataway N.J.) by the methods as described in the examples. The results are summarized below:
The results show that these humanized 1B1 antibodies with different combinations of humanized 1B1 heavy chain variants and humanized 1B1 light chain variants have high binding affinities with monkey CD3e. The binding affinities are similar to the chimeric antibodies.
The antibodies were incubated at 70° C. and then were cooled to the room temperature. FACS was performed with cells expressing human CD3e. The results show that these tested antibodies could still bind to these cells as analyzed by FACS. Thus, the results suggest that Tm for these antibodies were greater than 70° C.
Thermofluor assay was also performed using the Protein Thermal Shift™ Dye Kit (Thermo Fisher Scientific) and QuantStudio™ 5 Real Time PCR Systems (Thermo Fisher Scientific). This assay measured thermal stability using a fluorescent dye that binds to hydrophobic patches exposed as the protein unfolds. A temperature ramp of about 1.6° C./s was applied until the temperature reached 25° C., and the temperature ramp of about 0.05° C./s was then applied until the temperature reached 99° C. The Tm was recorded. Some testing antibodies had two Tms. This is because that IgG has a multi-domain structure. In the case that there were two Tms, the second Tm (Tm for Fab) was recorded. The experiments were performed according to the manufacturer's protocol.
The table below summarizes the Tm obtained from FACS and Tm obtained from Protein Thermal Shift™ Dye Kit for several humanized anti-CD3e antibodies. The results show that humanized 1B1 antibodies with different combinations of humanized 1B1 heavy chain variants and humanized 1B1 light chain variants have similar Tm as measured by FACS or Protein Thermal Shift™ Dye Kit.
Experiments were performed to determine whether the anti-hCD3e antibodies can activate Jurkat-Luc-NFAT cells (Promega, Cat #J1601) in vitro.
The anti-hCD3e antibodies were collected from either (1) supernatant of cultured CHO cells on a culture plate or (2) CHO cells within a culture medium. The anti-hCD3e antibodies collected from the culture medium were further purified. The purified antibodies were then titrated to appropriate concentrations.
4×104 CHO-FcγRIIB cells were added to the wells and were incubated at 37° C. overnight. The 96-well plate was centrifuged and the supernatant was discarded.
50 μl Jurkat-Luc-NFAT cells were then added the 96-well plate (5×104 cells/well). 25 μl of anti-hCD3e antibodies with different concentrations were also added to wells in the 96-well plate. After being incubated at 37° C. for 6 hours, 75 μl of luciferase assay reagents were added and incubated for 5-10 minutes. The fluorescence signals were measured. The results are shown in the table below.
The results show that these humanized 1B1 with different combinations of heavy chain variants and light chain variants all have in vitro biological activities and can activate Jurkat cells to various extent. Among them, 1B1-H1K1-IgG1-LALA and 1B1-H4K1-IgG1-LALA had higher activation efficiency.
In summary, the results in this example show that these humanized 1B1 antibodies with different combinations of heavy chain variants and light chain variants have relatively high binding affinities and thermal stabilities. The antibodies with H3 heavy chains and the antibodies with K1 light chains have relatively lower expression efficiency. Because H1, H2, K1, K2, and K3 have relatively high humanization percentages, H1K1, H1K2, H1K3, H2K1, H2K2, and H2K3 are good candidates for further testing in humans.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2018/093725 | Jun 2018 | CN | national |
This application claims the benefit of international Application No. PCT/CN2018/093725, filed on Jun. 29, 2018. The entire contents of the foregoing are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2019/091928 | 6/19/2019 | WO | 00 |
